O C C ' 2010 W orld C ongress - B ook of A bstracts - Oxygen Club of ...

oxyclubcalifornia.org

O C C ' 2010 W orld C ongress - B ook of A bstracts - Oxygen Club of ...

OCC

2010

O 2

OXYGEN CLUB

OF CALIFORNIA

OCC’ 2010 World Congress - Book of Abstracts

OXYGEN CLUB OF CALIFORNIA

ESTABLISHED 1994

2010

OXIDANTS AND ANTIOXIDANTS IN BIOLOGY

TRANSLATIONAL REDOX SCIENCE

CO-SPONSORED BY THE

LINUS PAULING INSTITUTE

BOOK OF ABSTRACTS

17-20 MARCH, 2010

FESS PARKER’S DOUBLE TREE RESORT

SANTA BARBARA, CALIFORNIA


OXIDANTS AND ANTIOXIDANTS IN BIOLOGY

TRANSLATIONAL REDOX SCIENCE

17-20 MARCH 2010

SANTA BARBARA, CALIFORNIA

CONFERENCE

ORGANIZERS

___________________________

Chandan K. Sen

Enrique Cadenas

Dipak K. Das

César G. Fraga

John Maguire

Lester Packer

Junji Yodoi

SCIENTIFIC PROGRAM

ORGANIZERS

____________________________

Bruce N. Ames

Dipak K. Das

César G. Fraga

Thomas K. Hunt

Klaus Kraemer

Periannan Kuppusamy

Lester Packer

Chandan K. Sen

Helmut Sies

Roland Stocker

Hideo Utsumi

José Viña

Junji Yodo

2


CONTENTS

KEYNOTE ADDRESSES ...............................................................................................5

SESSION I

WOUND HEALING................................................................................9

SESSION II

REDOX SIGNALING AND INFLAMMATION.........................................19

SESSION III CARDIOVASCULAR ............................................................................29

SESSION IV TRANSLATIONAL SCIENCE BY MICRONUTRIENTS............................35

SESSION V

REDOX IMAGING................................................................................51

SESSION VI AGING ................................................................................................55

POSTERS ...................................................................................................................61

AUTHOR INDEX ......................................................................................................178


KEYNOTE ADDRESSES


Novel infectious agents in human carcinogenesis:

State and perspectives

Harald zur Hausen

Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280,

69120 Heidelberg, Germany

During the past 30 years up to 21% of the global cancer incidence

has been linked to infectious events, involving specific viral,

bacterial and parasitic agents. Particularly the discovery of a role

of Hepatitis B virus in hepatocellular carcinomas and of high risk

human papillomaviruses (HPV) in cervical, other anogenital and

oropharyngeal cancers triggered novel approaches in cancer prevention

by vaccination. A global application of these vaccines

theoretically could reduce the cancer risk in females by 12-14%, in

males by 4-5%. Mechanisms of cell transformation by infectious

agents will be analyzed. Even in cases where infectious agents act

as direct carcinogens and where their persistence is necessary for

the maintenance of the carcinogenic phenotype, additional modifications

of the host cell genome emerge as necessary events for malignant

proliferation. Considerations will be presented to analyze

even cancers not yet linked to infectious events for a possible involvement

of exogenous agents in their etiology. This involves in

part cancers increased under immunosuppression, but also those

with a reduced or not elevated incidence after immunosuppression.

Malignant tumors arising either in the sequence of other infections

will also be discussed or where those infections appear to exert

protective functions. Finally potential synergistic effects of nutritional

carcinogens with virus infections deserve further attention.

New horizons in HIV/AIDS

Luc Montagnier

World Foundation AIDS Research and Prevention, UNESCO, Paris, France

Combined antiretroviral therapy (ARV) keeps the virus multiplication

to a low level as evidenced by the disappearance of viral RNA

in the blood of HIV infected patients but does not completely eliminate

the virus: there is a viral reservoir which remains untouched even

after long term treatment and regenerates viral multiplication if the

treatment is interrupted.

We have approached the identification of this reservoir by a new

technology based on the detection of electromagnetic signals produced

by some viral and bacterial DNAs, in particular by HIV DNA

sequences. Thus, we have detected a reservoir of HIV DNA in the

blood of all patients treated by ARV, both in the plasma and in the

erythrocyte fraction.

This will constitute a useful biomarker for complementary therapeutics

aimed at eradicating the virus infection such as immunotherapy

and antioxidants.

6

7


SESSION I

WOUND HEALING


Homing to hypoxia: The role of oxygen tension in progenitor

cell trafficking to sites of injury

J. Glotzbach, H. Thangarajah, N. Vial, and G.C. Gurtner

Department of Surgery, Stanford University, Stanford, CA, 94305

The isolation of circulating endothelial progenitor cells (EPCs)

has altered our understanding of how new blood vessels form in

adult tissues (heart, brain, wounds) following injury. While an exact

characterization of the responsible cell(s) remains elusive, it is

clear that a subset of the bone marrow is capable of selectively

trafficking to areas of tissue damage and forming blood vessels de

novo through a cascade of adhesion, migration, proliferation and

tubulization. We have identified the chemokine axis CXCR4/

stromal cell-derived factor-1 (SDF-1) as a critical mediator of

ischemia-specific progenitor trafficking and have demonstrated

that SDF-1 is transcriptionally regulated by tissue oxygen tension

via hypoxia-inducible factor-1 (HIF-1). Since both hypoxia and

SDF-1 are also observed in the bone marrow niche, hypoxia may

be a fundamental stimulus for progenitor cell trafficking and function.

After homing to injured tissue, both bone marrow-derived

and adipose-derived mesenchymal stem cells respond to hypoxia

by adopting a proangiogenic phenotype by proliferating, forming

tubes and secreting vascular growth factors. Alterations in hypoxia

signaling may contribute to the pathophysiology of diseases with

known defects in blood vessel growth, such as diabetes. It appears

that hyperglycemia directly alters the molecular structure and function

of HIF-1 and its cofactors by methylglyoxylation of specific

arginine residues preventing assembly of transcriptional machinery.

This results in both decreased angiogenesis and decreased recruitment

of progenitor cells to areas of ischemia in diabetes. This

suggests that the end manifestations of diabetes may be the result

of impaired hypoxia signaling following injury.

Supported by NIH grants RO1-EB002265 and RO1-AG025016

10

Hyperbaric oxygen therapy and stem cell response in

wound healing

Omaida C. Velazquez

The DeWitt Daughtry Family Department of Surgery, Leonard M.

Miller School of Medicine, University of Miami, Miami, FL, USA

Wound healing occurs because of events in two compartments.

Within the bone marrow, various signaling pathways trigger mobilization

of bone marrow-derived progenitor cells (EPC) and other

stem/progenitor cells involved in the healing cascade. Within the

wound, neovascularization occurs because of local chemical factors

that stimulate adjacent cells (angiogenesis) and because of recruited

circulating EPC that differentiate into vascular channels (vasculogenesis).

Many chemo/cytokines trigger EPC release via induction of metalloproteinase-9

(MMP-9) in bone marrow, and Nitric Oxide ( . NO) has

been linked to this process. We have found that HBO 2 will stimulate

EPC recruitment and augment healing of diabetic wounds in mice. Our

studies demonstrated that HBO 2 increases synthesis of . NO in bone

marrow, increases the concentration of circulating stem cell factor, and

stimulates EPC mobilization. If NOS was inhibited, all of the HBO 2 -

mediated events were blocked. Therefore, HBO 2 appears to stimulate

EPC mobilization by a . NO dependent mechanism. We believe the sequence

of events is as follows: HBO 2 NOS . NO nitrosylation

of MMP9cleavage of membrane-bound SCFSCF prompts EPC

proliferation and mobilizationEPC released into peripheral circulation.

However, EPC homing to areas in need of neovascularization

(such as a wound) does not appear to be specifically impacted by

HBO 2. Homing signals such as Stromal Derived Growth Factor 1 alpha

(SDF-1) appear to be more important for EPC homing. We have determined

that (1) Diabetes results in decreased eNOS phosphorolation

within the bone marrow, thus reducing mobilization of endothelial

progenitor cells (EPCs), (2) Diabetes results in downregulation of

SDF-1 within wounds, thus decreasing EPC homing to wounds, and

(3) these two key diabetes-associated impairments in wound healing

and neovascularization may be reversed by the combination of systemic

hyperoxia (to increase EPC mobilization) and wound SDF-1

injections (to increase EPC homing). Most recently, we have further

determined that SDF-1 may mediate its EPC-homing effects via effects

on specific adhesion molecules.

11


Impaired resolution of wound in diabetes

Sashwati Roy

Comprehensive Wound Center, Department of Surgery,

The Ohio State University Medical Center, Columbus, OH

Acute inflammation resolves by mechanisms that are not fully

understood. Persistence of the inflammatory response (chronic inflammation)

can lead to scarring and loss of organ function. An actively

coordinated program of resolution initiates in the first few

hours after an inflammatory response is triggered by tissue injury.

Resolution of inflammation is enabled if granulocytes are eliminated

via efferocytosis and the tissue mononuclear cell population

(macrophages, m) returns to baseline count and phenotypes. We

observed that compared to non diabetic animals, dead cell clearance

activity (efferocytosis) was markedly impaired in wound

macrophages harvested from diabetic mice. Oxidants produced by

NADPH oxidase are directly involved in the oxidation and externalization

of phosphatidyl serine (PS), a process that is critical for

dead cell recognition. Compromised NADPH oxidase activity was

observed in macrophages harvested from diabetic wounds. Milk fat

globule EGF factor 8 (MFG-E8; also known as lactadehrin) is secreted

by activated m. MFG-E8 acts as a bridging molecule that

is capable of binding to phosphatidyl serine on apoptotic cells as

well as v3 or v5 integrin receptors on m. The presence of

arginine residues renders this protein susceptible to glycation. Using

ELISA and Biacor assays we demonstrate that glyoxal, a product

of glucose oxidation, can glycate MFG-E8. Once glycated the

affinity of MFG-E8 for binding with PS markedly diminished.

Diabetic wound tissue showed presence of glycated MFG-E8.

These data suggest compromised NADPH oxidase activity in diabetic

wound cells result in diminished phosphatidylserine oxidation

impairing the process of dead cell recognition by m. Glycation

of MFG-E8 diabetic macrophages represents another major

mechanism that impairs dead cell recognition and clearance in diabetic

wounds. Such dysfunction in macrophage activity is in direct

conflict with resolution of inflammation resulting in chronic inflammation

noted in diabetic wounds. Supported by RO1 DK 076566 to SR.

Mathematical modeling of the role of oxygen in

non-ischemic and ischemic wound healing

Avner Friedman

Mathematical Bioscience Institute, Ohio State University,

Columbus, OH, USA

It is well known that moderate hypoxia in a wound stimulates

macrophages to produce growth factors, which improves healing.

But moderate hyperoxia also improves healing since it enables

cells to proliferate faster. However severe hypoxia impairs healing,

as does extreme hyperoxia – which is toxic. Thus the role of

oxygen in wound repair is quite sensitive. I will present a mathematical

model which focuses on the role that oxygen plays in cutaneous

wound healing. The model includes the important cells and

chemokines that are involved in the healing process, under both

non-ischemic and ischemic conditions. The predictions of the

model are in excellent agreement with experimental results. The

model can be used as a tool for evaluation of different protocols of

oxygen treatment.

12

13


Ischemic wound healing

Cell based therapies for peripheral vascular disease

Douglas W. Losordo

Feinberg Cardiovascular Research Institute, Northwestern University

School of Medicine, Northwestern Memorial Hospital, Chicago, IL USA

As the population ages and the acute mortality from cardiovascular

disease decreases, a large population of patients is emerging

who have symptomatic chronic ischemic vascular disease, many of

whom remain severely symptomatic despite exhausting conventional

medical therapy and mechanical revascularization. In addition,

mounting evidence suggests that microvascular insufficiency

plays a significant role in the pathophysiology of ischemia. At the

present time, there are no therapies that directly address the needs

of this patient population.

Pre-clinical and early clinical data indicate that a variety of

growth factors and stem/progenitor cells may be employed therapeutically

for repair of ischemic tissue.

Preclinical studies documented the potential therapeutic potency

of endothelial progenitor cells, both as cultured and freshly

isolated cells. Early phase clinical trials using a variety of approaches

have been completed providing data of feasibility, safety

and bioactivity. Later phase trials are under way. Accordingly, the

goal of ischemic tissue repair appears feasible and is being approached

in human clinical trials.

The evolution of the strategy of ischemic tissue repair will require

an ongoing dialogue between clinicians, scientists, regulators

and industry to take full advantage of advances in our understanding

of the biology of these processes and their appropriate application

to patients.

Thomas A. Mustoe

Department of Surgery, Northwestern University Feinberg School of Medicine,

Chicago, IL, USA

Chronic wounds are a multibillion dollar health problem in the

USA affecting millions of patients, falling into three major categories:

pressure sores, diabetic foot ulcers, and venous leg ulcers.

Local wound ischemia and ischemia reperfusion, (as well as the

additive impact of aging, and bacterial biofilm) play a fundamental

role in the pathogenesis. We have utilized clinically relevant

and validated animal models to examine separately the impact of

ischemia, and the altered response in the aged animal, and the effects

of ischemia reperfusion (oxidant injury). The skin unlike

other organs, is subjected to repeated cycles of I-R, and the failure

of an adaptive response to repeated cycles is notable. Classically,

therapeutic strategies for chronic wounds have focused on oxygen

delivery to offset the effects of ischemia, but future strategies will

include strategies to reduced oxidant injury. True breakthroughs

will almost certainly incorporate combination therapy, in recognition

that chronic wounds have a multi factorial pathogenesis.

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15


A novel role for caspase-8 in regulating the

cutaneous wound healing response

Pedro Lee 1 , Stacey Lee 1 , and Colin Jamora 1,2

1 Division of Biological Sciences, Section of Cell and Developmental Biology,

University of California, San Diego; 2 Department of Medicine (Division of

Dermatology), University of California, San Diego School of Medicine

The wound healing response is a complex interplay of cells that

coordinate inflammation, stem cell proliferation and tissue remodeling.

The complex nature of this intercellular communication to

localize the wound healing response specifically to the site of

trauma and to reset itself once tissue repair is complete has been a

challenging problem. We found that the protease caspase-8, which

is well established for facilitating receptor mediated apoptosis,

plays a novel role in regulating all phases of the wound healing response.

Using the conditional deletion of caspase-8 to mimic

wound healing in the skin, we were able to decipher the crosstalk

occurring between epidermal keratinocytes, dermal fibroblasts, and

leukocytes to regulate stem cell proliferation. Among the important

constituents of this extracellular signaling is the cytokine interleukin-1

(IL-1). Epidermal keratinocytes serve as a large reservoir

for IL-1a and the reduction of caspase-8 leads to the formation of

the NALP3 inflammasome, which mediates release of this cytokine

into the extracellular milieu. Multiple responses are elicited

by this cytokine depending upon the type of cell it encounters.

Further dissection of the reciprocal exchange of signals is also providing

insights into how the same processes that occur in the context

of tissue repair are usurped in inflammatory diseases.

Microcirculation in tissue injury and repair

Mark G. Clemens

Department of Biology, College of Liberal Arts and Sciences,

University of North Carolina at Charlotte, Charlotte NC 28223

It is well known that vascular perfusion of tissues is essential to

prevent injury as well as to allow repair following injury. Recent

studies demonstrate that that effect on microvascular control of local

inflammation associated with tissue injury contributes to

mechanisms of both injury and repair. The effects of inflammation

on the integrity of the microcirculation are manifested in accumulation

of inflammatory cells and in altered vasoreactivity as well as

in the control of angiogenesis. All of these manifestations involve

altered function of the vascular endothelium with dysregulation of

the interactions between endothelin and gaseous vascular mediators,

nitric oxide, carbon monoxide and hydrogen sulfide. All four

of these mediators are regulated by inflammation. During the injury

phase, inflammatory stimuli alter vascular endothelial cell

signal transduction mechanisms resulting in impaired perfusion

and oxygen supply with resulting tissue injury. During repair, these

mechanisms also contribute to the regulation of existing vasculature

and to angiogenesis. While nitric oxide mechanisms are

widely recognized, the contributions of carbon monoxide and hydrogen

sulfide (H 2 S) are less well known. CO is made from the

metabolism of heme by heme oxygenase, the inducible form of

which (HO-1) is associated with inflammation. CO modulates the

function of heme proteins such as guanyl cyclase while the other

metabolite of HO-1, bilirubin acts as an antioxidant. H 2 S is produced

by via the metabolism of cysteine by cystathionine gammalyase

(CSE) which can also be induced by inflammation. H 2 S has

been shown to improve ulcer healing and to stimulate angiogenesis.

While the primary action of H 2 S is to activate ATP-activated

potassium (K ATP ) channels in vascular smooth muscle cells, many

of its actions appear to be independent of this mechanism. Taken

together, current data strongly suggest that these three gaseous mediators

work in concert with each other and with vasoconstrictors

such as endothelin to determine microvascular function during tissue

injury and repair.

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SESSION II

REDOX SIGNALING AND INFLAMMATION


Redox regulation of thioredoxin and glutaredoxin systems

Arne Holmgren, Rajib Sengupta, Yatao Du,

Sergio Montano, and Jun Lu

Department of Medical Biochemistry and Biophysics, Karolinska Institutet,

171 777 Stockholm, Sweden

To balance the effects of ROS, thioredoxin and glutaredoxin

systems act together with glutathione peroxidases and peroxiredoxins

and via disulfides ultimately also in redox signaling. Mammalian

Trx1 is an electron donor for ribonucleotide reductase (RNR).

This enzyme comprises R1 + R2 in S-phase, which reduces NDPs

and supplies dNTPs for DNA replication. R1 + p53R2 in postmitotic

cells generates dNTPs for DNA repair and mitochondrial

DNA synthesis. Each dNTP made leaves an active site disulfide in

the R1 subunit of RNR which is reduced by C-terminally located

cysteine (CXXC) residues, the target of Trx and Grx. With R1 +

p53R2, Grx operating by a glutathionylation mechanism is catalytically

more efficient (higher k cat /K M ) than Trx, suggesting a major

function of GSH and Grx in DNA repair and mitochondrial

DNA-synthesis. Trx1 itself is subject to redox regulation by modification

of the three structural SH-groups as well as its involvement

in NO-metabolism as the major S-denitrosylation mechanism

of protein S-nitrosothiols. Grxs catalyze GSH-dependent disulfide

oxidoreductions and deglutationylation of proteins. Human Grx2a

in mitochondria is also present as an inactive dimeric holoenzyme

containing an iron-sulfur cluster (2Fe2S) with GSH as a ligand.

The 2Fe2S clusters of dithiol and monothiol glutaredoxins act to

control apoptosis via the mitochondrial pathway and are involved

in iron metabolism. Both Trx and Grx can be taken up in cells and

have cytoprotective and antiapoptotic effects. A synthetic selenium

drug, ebselen, is a very fast oxidant of reduced Trx and a direct

substrate of mammalian TrxR and a promising drug for oxidative

stress diseases or in treating cancer due to redox imbalance.

Inactivation of peroxiredoxin I by tyrosine phosphorylation at

lipid rafts allows spatially controlled accumulation of H 2 O 2 for

intracellular signaling by growth factor or immune receptors

Sue Goo Rhee and Hyun Ae Woo

Department of Life Science, Division of Life and Pharmaceutical Sciences,

Ewha Woman’s University, 11-1 Daehyun-dong, Seodaemun-ku,

Seoul 120-750, Korea

Despite its inherent toxicity, H 2 O 2 is produced by mammalian

cells as a signaling molecule that oxidizes critical cysteine residues

of effectors such as protein tyrosine phosphatases in response to

activation of cell surface receptors. It has remained unclear, however,

how H 2 O 2 concentrations above the threshold required to

modify effectors are achieved in the presence of the abundant detoxification

enzymes peroxiredoxin (Prx) I and Prx II. We now

show that Prx I associated with lipid rafts is transiently phosphorylated

on tyrosine-194 and thereby inactivated both in cells stimulated

via growth factor or immune receptors in vitro as well as in

those at the margin of healing cutaneous wounds in mice. The localized

inactivation of Prx I thus provides a means for allowing the

transient accumulation of H 2 O 2 around lipid rafts, where signaling

components are concentrated, while preventing the toxic accumulation

of H 2 O 2 elsewhere. In contrast, Prx II was inactivated not by

phosphorylation but rather by hyperoxidation of its catalytic cysteine

and only during sustained oxidative stress.

20

21


Thioredoxin and TBP-2 in redox signaling

Zhe Chen 1 , Eiji Yoshihara 1,2 , Yoshiyuki Matsuo 1 , Junji Yodoi 1

1 Department of Biological Responses, Institute for Virus Research,

Kyoto University; 2 Graduate school of Biostudies, Kyoto University

Human Thioredoxin-1 (TRX) is a 12-kDa oxidoreductase containing

a conserved dithiol-disulfide active site (-CPGC-). It was

first identified from the supernatant of HTLV-I infected T cell line

as adult T-cell leukemia-derived factor. Given the essential role in

the intracellular thiol-reducing system, TRX is known to regulate

multiple biological responses in a redox-dependent manner. We

have identified several binding partners for TRX, including Thioredoxin

Binding Protein-2 (TBP-2/VDUP1/TXNIP), which binds

to the reduced TRX inhibiting the biological activity of TRX in

vivo. TBP-2 is a critical signal transducer in cell growth, lipid and

glucose metabolism, and immune responses. Several lines of evidence

indicate that TBP-2 is a crucial component of TRXdependent

redox regulation. It was suggested that ROS generated

in response to oxidative stresses promotes the formation of intermolecular

disulfide bridge between TRX and its targets, whereas

TBP-2 overexpression abolishes the TRX function. The redox

regulation by TRX/TBP-2 system seems to be a ubiquitous phenomenon

in cellular responses, including mitogen-activated signal

transduction (ERK, p38MAPK), ASK1-mediated cell apoptosis,

FoxO-mediated cellular longevity, and anti-inflammatory immune

responses. Conclusively, here we postulate a redox-regulated signaling

complex consisting of TRX or TRX family proteins, TBP-2,

and other putative signaling components (REDOXISOME), in response

to varieties of stresses. The core concept of redox regulation

is that under stress conditions, redoxisome is formed to activate

the stress signaling. The association/dissociation of redoxisome

components induced by the environmental stresses account

for the complicated but precise regulation of redox signaling.

1. Teshigawara K, et al. Adult T leukemia cells produce a lymphokine that augments interleukin

2 receptor expression. J Mol Cell Immunol 1985;2:17-26.

2. Nishiyama A et al. Identification of thioredoxin-binding protein-2/vitamin D(3) upregulated

protein 1 as a negative regulator of thioredoxin function and expression. J

Biol Chem 1999;274:21645-21650.

3. Dansen et al, Redox-sensitive cysteines bridge p300/CBP-mediated acetylation and

FoxO4 activity. Nat. Chem. Biol 2009:5:664-672

Immunoregulatory role of GIF/MIF, a redox-geared cytokine

Katsuji Sugie 1,2 , Misa Kim-Saijo 1 and Aoi Son 2

1 La Jolla Institute for Allergy and Immunology and 2 Kyoto University Institute

for Virus Research

GIF/MIF (GIF, for brevity) is a 13-kDa polypeptide. Originally

identified in T cell culture supernatants, the function of this

molecule has been controversial. The thioredoxin(TRX) motif in

GIF alluded involvement of oxidoreductase activity in the cytokine

biology. GIF-/- mice displayed an enhanced antibody response to

T-dependent antigens. The mutant mice showed more severe allergic

airway inflammation than wildtype mice. CD4 T cells of

GIF-/- mice secreted elevated amounts of IL-4, the key cytokine

for IgE antibody formation and allergic inflammation. Therefore

GIF suppresses IL-4 production and T-dependent immune responses

especially in allergy. Although GIF does not have the signal

peptide sequence, it becomes secreted. GIF secreted from T

cells is cysteinylated at C60, whereas that in the cytosol is unmodified.

The cysteinylation was essential for GIF to suppress IL-4

production by CD4 T cells. Immunofluorescence studies revealed

that cysteinylated GIF colocalized with ER and Golgi apparatus.

GIF in T cell culture supernatants associated with TRX1, a polypeptide

with the capability to control inflammations. To determine

the receptor for cysteinylated GIF, expression cloning was performed.

One of the molecules identified was peroxiredoxin 1

(Prdx-1). RNA interference experiments showed that knocking

down of Prdx-1 diminished cellular binding of cysteinylated GIF.

A previous study demonstrated that Prdx-1 bound GIF. Experiments

are planned to determine whether cysteinylation is important

for this cytokine to bind Prdx-1 and whether Prdx-1 is involved in

transmitting GIF signals at the cell surface. Altogether, the cytokine

GIF is subject to redox regulation when it becomes secreted,

acquires the immunoregulatory activity and acts on target cells.

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23


NADPH oxidases in the lung: beyond host defense

Albert van der Vliet 1 , Derek Sham 1 , Umadevi Wesley 2 ,

David I. Kasahara 1,3 , and Milena Hristova 1

Departments of 1 Pathology and 2 Microbiology and Molecular Genetics,

College of Medicine, University of Vermont, Burlington, VT 05405, U.S.A.,

3 Department of Environmental Health, Harvard School of Public Health,

Boston, MA 02115

In analogy to the deliberate production of reactive oxygen species (ROS)

by NADPH oxidase in professional phagocytes as a critical component of

their antimicrobial activity, airway epithelial cells express homologs of

NADPH oxidase, Dual Oxidase 1 and 2 (DUOX1/2), that can be activated by

a variety of environmental stresses and bacterial stimuli, and contribute to

maintenance of respiratory tract sterility and epithelial integrity. While several

studies have indicated that epithelial DUOX’s generate apical H 2 O 2 as a

substrate for apical lactoperoxidase in the production of antimicrobial oxidants,

recent studies in our laboratory have also demonstrated a critical role

for DUOX1, the major epithelial DUOX isoform, in epithelial cell migration

and intracellular signaling pathways that mediate epithelial responses to a

variety of environmental or microbial insults by inducing various mediators

of inflammation or tissue repair, such as matrix metalloproteinase-9, interleukin-8

or mucins. These responses appear to involve a common mechanism

that is initiated by cellular release of ATP to activate cell surface purinoceptors,

a widely recognized signaling mechanism in airway epithelial responses

to mechanical injury or microbial infection. Similar recent findings of ATP

release and/or NADPH oxidase activation in stress responses in diverse organisms

indicate their common role in biological response mechanisms to

environmental stress throughout the animal and plant kingdoms. Activation

of epithelial DUOX1 was found to promote the shedding of growth factors

and activation of the epidermal growth factor receptor (EGFR), whereas

DUOX1 also appears to stimulate the non-receptor tyrosine kinase c-Src,

which activates EGFR independent of ligand. Current studies are designed to

identify the proximal H 2 O 2 -dependent signaling events involved in EGFR

activation, using redox proteomic strategies. Abnormal expression and/or

activation of EGFR are important features of various lung diseases such as

asthma and lung cancer, and are associated with deregulated epithelial proliferation,

chronic epithelial wound responses, and mucus metaplasia. In this

regard, intriguing observations of enhanced epithelial DUOX1 expression by

Th2 cytokines and upregulation of lung DUOX1 in a mouse model of allergic

asthma may suggest a contributing role for DUOX1 in airway remodeling

and mucus metaplasia as cardinal features of allergic asthma, and may implicate

DUOX as an alternative potential therapeutic target.

Regeneration of infarcted myocardium with nutritionally

modified cardiac stem cells: Implication for redox signaling

Dipak K. Das

Cardiovascular Research Center, University of Connecticut School of Medicine,

Farmington, CT 06030-1110, USA

A major hitch in the effectiveness of stem cell therapy is the death

of stem cells due to the oxidative environment present in the normal

tissue. Reduction of oxidative stress or maintaining a reduced

environment in the target tissue can enhance the stem cell survival

and can enhance the cardiac regeneration after stem cell therapy.

To study the efficiency of maintaining the reduced tissue environment

via pretreatment with natural antioxidant resveratrol in stem

cell therapy. We pretreated male Sprague Dawley rats with resveratrol

(2.5 mg/kg/day gavaged for 2 weeks). Left anterior descending

coronary artery (LAD) occlusion followed by direct injection

of adult cardiac stem cells stably expressing EGFP on the

border zone of the myocardium through survival surgery. The

prevalent of cardiac reduced environment was seen in resveratrol

treated rat hearts via significantly enhanced redox signaling observed

through the nuclear localization of nuclear factor-E2-related

factor-2 (Nrf2) and redox effector factor-1 (Ref-1) 7 days after

LAD occlusion. Significantly improved cardiac functional parameters

(left ventricular ejection fraction and fractional shortening) and

enhanced stem cell survival and proliferation (expression of cell

proliferation marker Ki67) and differentiation of stem cells towards

the regeneration of the myocardium (expression of EGFP)

was evident 28 days after LAD occlusion in rats treated with resveratrol

compared to control rats. Maintaining a reduced tissue environment

by treatment with resveratrol in rats enhanced the cardiac

regeneration by adult cardiac stem cells via improved cell survival,

proliferation and differentiation leading to improved cardiac

function.

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25


Mitochondria, oxidative stress and cell death

Sten Orrenius, Erik Norberg, Vladimir Gogvadze,

and Boris Zhivotovsky

Institute of Environmental Medicine, Division of Toxicology,

Karolinska Institutet, Stockholm, Sweden

In addition to the long established role of the mitochondria in

energy metabolism, regulation of cell death has emerged as a second

major function of these organelles. This, in turn, seems to be

intimately linked to their role as the major intracellular source of

reactive oxygen species (ROS), which are mainly generated at

Complex I and III of the respiratory chain. Excessive ROS production

can lead to the oxidation of macromolecules and has been implicated

in mtDNA mutations, ageing, and cell death. Although mitochondrial

dysfunction per se can result in ATP depletion and necrosis,

these organelles are also involved in the regulation of apoptotic

cell death by mechanisms that have been conserved through

evolution. Hence, many toxic agents target the mitochondria and

cause the release into the cytosol of cytochrome c and other proapoptotic

proteins, which can trigger caspase activation and other

key events in apoptosis. Cytochrome c release occurs by a two-step

process that is initiated by the dissociation of the hemoprotein from

its binding to cardiolipin in the inner mitochondrial membrane

(IMM). Similarly, Apoptosis Inducing Factor (AIF) must also be

detached from the IMM, before it can be exported from the mitochondria

into the nucleus. This occurs by proteolytic cleavage of

the peptide chain that anchors it to the IMM. AIF cleavage is catalyzed

by mitochondrial calpain and preceded by oxidative modification

of AIF triggered by mitochondrial ROS production. Subsequent

release of cytochrome c and AIF into the cytosol occurs via

pores in the outer mitochondrial membrane formed by proapoptotic

Bcl-2 family proteins, or by Ca 2+ /ROS-induced mitochondrial

permeability transition, although the latter mechanism

might be more closely associated with necrotic cell death. Hence, it

is apparent that mitochondrial ROS generation is critically involved

in the control of both apoptotic and necrotic cell death and

an important mediator of mitochondrial toxicity.

26

Development of high field Overhauser-MRI and its application

to in vivo imaging

Hideo Utsumi 1,2

1 Innovation Center for Medical Redox Navigation

2 Department of Bio-functional Science, Faculty of Pharmaceutical Sciences

Kyushu University, Fukuoka 812-8582

e-mail: utsumi@pch.phar.kyushu-u.ac.jp

Proton Magnetic Resonance Imaging (MRI) has provided significant

clinical utility in the diagnosis of diseases. Overhauser enhanced

MRI (OMRI), which is a double resonance technique, creates

highly-resolved images of free radical distributions in small a-

nimals by enhancing the water proton signal intensity via the O-

verhauser Effect. We developed new-sequence for OMRI and succeeded

in simultaneous dual images by using nitroxyl radicals labeled

with 14 N and 15 N nuclei and changing the external magnetic

field for ESR irradiation in OMRI (Utsumi et al. PNAS 2006),

which could visualize pharmacodynamics, redox reactions and redox

statue of inner and/or outer organs of individual animal.The

large difference of gyromagnetic ratio between electron and proton

spins restricted ESR excitation and the proton detection fields. We

have developed a prototype of OMRI scanner with circulartransport-system,

in which the sample object was transported

between ESR to MR magnets circularly, which were operated at

1.5 T (or 0.4 T) and 20 mT for MR detection and ESR excitation,

respectively. Phantom images of nitroxyl spin-probe were successfully

obtained with the OMRI system. The physical resolution of

the OMRI image for the phantom object was less than 0.2 mm. The

developed OMRI system would have a significant advantage for

imaging in vivo redox state,and also offer significant applicability

to simultaneous assessment of independent redox regulation in

gene-modified animals and disease models (JCBF 2009, PNAS

2009).

27


Lipoic acid reverses the age-related loss of Nrf2-mediated

antioxidant gene transcription

Tory M. Hagen

Linus Pauling Institute, Oregon State University, Corvallis, OR, USA

In order to define the benefits of micronutrients in improving

elder health, we show that the dithiol antioxidant, (R)--lipoic acid

(R-LA), improves hepatic antioxidant capacity, which otherwise

declines with age. R-LA does not achieve this by acting as a dietary

antioxidant; rather, it reverses the age-related loss of endogenous

antioxidants, especially glutathione (GSH). This has led to

the discovery that nuclear levels of NF-E2 related-factor 2 (Nrf2),

a transcription factor that regulates expression of GSHsynthesizing

enzymes, markedly declines with age, but R-LA reverses

this loss. Our current focus is to understand why an agerelated

loss of Nrf2-mediated stress defenses occurs, and the

mechanism(s) how R-LA acts to maintain this vital cellular defense

system. Chromatin immunoprecipitation experiments for the

Nrf2-mediated gene, glutamate-cysteine ligase, show that lower

levels of Nrf2 bind to its Antioxidant Response Element (ARE) in

aged versus young rat livers. We also have evidence showing that

the active Nrf2/bZIP transcriptional complex is converted to a repressive

motif during aging as shown by binding of the repressor,

Bach1, and lower ARE-driven luciferase activity. Furthermore,

Nrf2 occupies an alternate ARE site in livers of old rats, indicating

an age-specific promoter switch. Our results show that the conversion

of Nrf2 binding from its typical ARE site to an alternative one

is not adequate to maintain basal expression of hepatic gcl in old

rats, which provides a possible mechanism for the age-related loss

of glutathione and other Phase II enzymes. However, R-LA improves

Nrf2 action by increasing nuclear tenure of Nrf2 and also

promotes its binding to the alternative ARE sequence. Thus, R-LA

may be part of a unique class of dietary micronutrients that promote

“healthspan” by maintaining vital cellular defenses, which

otherwise decline with age.

SESSION III

CARDIOVASCULAR

28


Rescue of diabetes related impairment of myocardial

angiogenesis: Potential and challenges

1 Samson Mathews Samuel, 1 Mahesh Thirunavukkarasu,

1 Suresh Varma Penumathsa, 1 Lijun Zhan, 2 Gautam Maulik,

and 1 Nilanjana Maulik

1 Molecular Cardiology and Angiogenesis Laboratory, Department of Surgery,

UCONN Health Center, Farmington, CT, USA, 2 Department of Thoracic Surgery,

Harvard Medical School, Boston, MA, USA

Maintaining a balanced redox status in the microenvironment

might aid in alleviating diabetes mediated complications related to

impaired myocardial angiogenesis & ventricular remodeling. The

cells defend themselves against oxidative stress induced damage

using antioxidant proteins among which antiapoptotic & growth

stimulatory thioredoxin-1 (Trx1) plays a critical role. In the present

study we have evaluated the reversal of diabetes mediated impairment

of angiogenesis in myocardial infarction (MI) model of streptozotocin

induced Type I diabetic rats by intramyocardial administration

of adenovirus encoding Trx1 (Ad.Trx1), after MI. The

myocardial function was measured by echocardiography 30 days

after the intervention. The Ad.Trx1 administered group exhibited

reduced fibrosis, oxidative stress and cardiomyocyte and endothelial

cell apoptosis as compared to diabetic MI group along with increased

capillary and arteriolar density. Western blot and immunohistochemical

analysis demonstrated myocardial overexpression of

Trx1, HO-1, VEGF, p38MAPK and decreased p-JNK and

p38MAPK in the Ad.Trx1 treated diabetic group. Alternatively,

we have observed significant reduction in the expression of VEGF

in SnPP (HO-1 enzyme inhibitor) treated non-diabetic and diabetic

animals even after Ad.Trx1 therapy. Echocardiographic analysis after

4 weeks of MI revealed significant improvement in the myocardial

functional parameters such as the ejection fraction, fractional

shortening and E/A ratio in the Ad.Trx1 administered group

as compared to the diabetic MI group. For the first time our novel

approach of Trx1 gene therapy may prove to be a strategic therapeutic

modality in the treatment of diabetes related cardiac failure

by stabilizing the redox status in the myocardium, reducing cell

death, inducing neovessel formation & maturation & reducing ventricular

remodeling after MI.

Inducible nitric oxide-mediated nitroso-redox balance as a key

modulator of tolerance to ischemia/reperfusion injury

in diabetic patients

Hajime Otani

The Second Department of Internal Medicine, Kansai Medical University, 10-15

Fumizono-cho, Moriguchi City, 570-8507 Japan

Although expression of inducible nitric oxide (iNOS) and oxidative

stress are increased in the diabetic heart, the role of iNOSmediated

nitroso-redox balance in the pathophysiology of ischemia/reperfusion

(I/R) injury has been poorly understood. Because

iNOS-derived nitric oxide (NO) is a key player in cardioprotection

against I/R injury in the preconditioned heart, it was hypothesized

that inhibition of iNOS uncoupling enhances tolerance of the diabetic

heart to I/R injury through increased bioavailability of NO.

The present study demonstrated that iNOS-derived superoxide

generation was reduced and nitric oxide bioavailability was increased

by treatment with NOS-cofactor, tetrahydrobiopterin

(BH4), before I/R in the heart isolated from the diabetic rat. This

was associated with reduction of infarct size and improvement of

left ventricular (LV) function after I/R. The cardioprotective effect

of BH4 was abrogated by treatment with a thiol reducing agent

dithiothreitol (DTT) but not a NO-sensitive guanylyl cyclase inhibitor

ODQ, suggesting that iNOS-derived NO-mediated cardioprotection

occurs through the protein S-nitrosylation but not the c-

GMP-dependent signaling pathway in the diabetic heart. Indeed,

protein S-nitrosylation was increased by treatment with BH4 in the

diabetic heart and inhibited by DTT. These results suggest that

iNOS-derived NO plays a crucial role in acquisition of tolerance to

ischemia/reperfusion injury through the protein S-nitrosylation.

Therefore, inhibition of iNOS uncoupling and modulation of

iNOS-mediated nitroso-redox balance may represent a novel

therapeutic target for myocardial protection in diabetic patients.

30

31


Regulation of myocardial growth and death by oxidative stress

Junichi Sadoshima

Department of Cell Biology and Molecular Medicine, Cardiovascular

Research Institute, New Jersey Medical School, NJ, USA

Oxidative stress plays an essential role in regulating growth and

death of cardiac myocytes. Thioredoxin 1 (Trx1) has many protective

functions in the heart, including suppression of pathological

hypertrophy. Trx1 facilitates the reduction of signaling molecules

by cysteine thiol-disulfide exchange, thereby regulating their functions.

Trx1 upregulates DnaJb5, a heat shock protein 40, and forms

a multiple-protein complex with DnaJb5 and class II histone

deacetylases (HDACs), master negative regulators of cardiac hypertrophy.

Trx1 reduces critical cysteine residues in class II

HDACs, thereby inducing nuclear localization of class II HDACs

and inhibiting pathological hypertrophy. The NADPH oxidase

family is a group of membrane-associated proteins producing superoxide

in a wide variety of cell types. We have obtained evidence

that Nox4 plays an important role in mediating both death

and survival of heart cells in response to pressure overload and

ischemia/reperfusion in the heart. Nox4 plays an essential role in

mediating increases in oxidative stress in the failing heart by increasing

superoxide production in mitochondria. In this presentation,

I will discuss the role of Trx1 and Nox4 in regulating growth

and death of cardiac myocytes.

Contribution of cholesterol oxidation products

to the progression of the atherosclerotic lesion

Gabriella Leonarduzzi, Simona Gargiulo, Paola Gamba,

Barbara Sottero, and Giuseppe Poli

Department of Clinical and Biological Sciences, University of Turin,

San Luigi Hospital,, 10043 Orbassano(Turin), Italy

Cholesterol oxidation leads to the formation of a number of 27-

carbon atoms compounds, termed oxysterols, which may either

originate in the blood, cells and tissues, through both enzymatic

and not enzymatic reactions, or derive from the diet. These cholesterol

metabolites have been consistently demonstrated in vitro to

be at least one or two orders of magnitude more reactive than

unoxidized cholesterol, showing remarkable pro-oxidant, proinflammatory,

pro-apoptotic, and pro-fibrogenic effects. Moreover,

we recently observed that in vivo, namely in advanced atherosclerotic

lesions of human carotid, which are characterized by the

presence of several cells of the macrophage lineage, the more cholesterol

accumulates, the higher is the production of oxysterols. In

particular, oxysterols might significantly contribute to the vascular

changes due to atherosclerosis, as they appear involved in various

key steps of this complex process, from endothelial cell dysfunction

and foam cell formation up to fibrotic degeneration of the arterial

wall and vulnerable plaque rupture. As regards the way oxysterols

signal to the nucleus of the cells to exert their biochemical

effects, a cholesterol oxide mixture compatible with that detectable

in human hypercholesterolemic plasma and atherosclerotic lesions

has shown the ability to trigger and sustain a molecular cascade in

which the pivotal role is played by PKC and MEK/ERK phosphorylation.

Because of their very strong pro-inflammatory potential, cholesterol

oxidation products could contribute to the progression of

pathologies other than atherosclerosis, whose hypercholesterolemia

represents a primary risk factor.

32

33


SESSION IV

TRANSLATIONAL SCIENCE BY MICRONUTRIENTS


Colors with functions:

Elucidating the biochemical and molecular basis of

carotenoid metabolism

Johannes von Lintig

Department of Pharmacology, School of Medicine, Case Western Reserve

University, WB333, 10900 Euclid Ave. Cleveland, Ohio 44106-4965, USA

Carotenoids affect a rich variety of physiological functions

in nature and are beneficial for human health, serving as antioxidants

in lipohilic environments and blue light filters in the macula

of human retina. These dietary compounds also serve as precursors

of a unique set of apocarotenoid cleavage products, including retinoids

(vitamin A and its derivatives). Although knowledge about

retinoid biology has tremendously increased, the metabolism of

their parent precursors remains poorly understood. Recently, molecular

players in carotenoid metabolism have been identified and

biochemically characterised. Moreover, mutations in their corresponding

genes impair carotenoid metabolism and induce various

pathologies in animal models. Polymorphisms in these genes alter

carotenoid and retinoid homeostasis in humans as well. This lecture

summarizes our current knowledge about the molecular/biochemical

basis of carotenoid metabolism, and especially, the

physiological role of carotenoids in retinoid-dependent physiological

processes.

Carotene dioxygenase-1 polymorphisms

Georg Lietz

Newcastle University, Human Nutrition Research Centre, School of Agriculture, Food

and Rural Development, Agriculture Building, Newcastle upon Tyne, NE1 7RU, UK

-carotene absorption and/or conversion into retinal is extremely

variable among individuals. The proportion of low responders

to -carotene in well nourished individuals has been estimated

to be 45% [Wang et al, 2004]. Still, the mechanisms underlying

this variability are not well understood, and could arise

from genetic variations in genes involved in the absorption, transport

and/or conversion of carotenoids. Carotenoids are transported

across the intestinal epithelial layer via a facilitated process involving

the scavenger receptor-BI (SR-BI) [During et al, 2005]. A

number of polymorphisms in the SRBI gene have been found to

significantly alter fasting plasma carotenoid concentrations [Borel

et al, 2007]. Once absorbed, pro-vitamin A carotenoids are converted

to retinal via the -carotene 15,15’-monoxygenase

(BCMO1). A first indication that genetic background might influence

the conversion efficiency in humans comes from the discovery

of a very rare missense mutation in the BCMO1 gene, which is

associated with hypovitaminosis A and hypercarotenaemia [Lindqvist

et al, 2007]. However, this rare mutation cannot explain the

high occurrence of the low responder phenotype. Screening of the

total open reading frame of the BCMO1 coding region lead to the

identification of two common non-synonymous SNPs (R267S;

rs12934922 and A379V; rs7501331), with variant allele frequencies

in a Caucasian population of 42% and 24%, respectively [Leung

et al, 2009]. Assessment of the responsiveness to a pharmacological

dose of -carotene in female volunteers confirmed that

carriers of both the A379V and R267S/A379V variant had a reduced

ability to convert -carotene [Leung et al, 2009]. Identification

of novel functional SNPs in key enzyme of carotenoid metabolism

indicates the potential importance of micronutrient-gene

interactions. Furthermore, the effect of the genetic variants in

BCMO1 is modulated by ethnicity, sex and BMI.

Borel P, Moussa M, Reboul E et al. J Nutr; 137:2653-9 (2007).

During A, Dawson HD, Harrison EH. Journal of Nutrition; 135:2305-2312 (2005).

Leung W, Hessel S, Méplan C et al. Faseb J; 23, 1041–1053 (2009).

Lindqvist A., Sharvill J., Sharvill D.E., and Andersson S., J. Nutr., 137, 2346-2350 (2007).

Wang Z., Yin S., Zhao X., Russell R.M., and Tang B., Br. J. Nutr., 91, 121-131 (2004).

36

37


-carotene 15,15’-monooxygenase 1 gene affects

circulating levels of carotenoids

Richard D. Semba,* Luigi Ferrucci, John R. B. Perry,

Amy Matteini, Markus Perola, Toshiko Tanaka, Kaisa Silander,

Neil Rice, David Melzer, Anna Murray, Christie Cluett,

Linda P. Fried, Demetrius Albanes, Anna-Maria Corsi,

Antonio Cherubini, Jack Guralnik, Stefania Bandinelli,

Andrew Singleton, Jarmo Virtamo, Jeremy Walston, and

Timothy M. Frayling

Johns Hopkins University School of Medicine*

and thirteen other institutions in U.S., U.K., Italy, and Finland

Introduction. Low circulating carotenoids have been associated

with increased risk of cardiovascular disease, cancer, and

death, and with age-related declines of muscle strength and physical

performance. Whether genetic variation affects the circulating

levels of carotenoids has not been well studied.

Methods. We conducted a genome-wide association study in

1191 adults from the InCHIANTI study in Tuscany, Italy, to identify

novel common variants associated with circulating carotenoid

levels. The effects were replicated in the Women’s Health and Aging

Studies (n = 615) and the Alpha-Tocopherol, Beta-Carotene

Cancer Prevention Study (n = 2136).

Results. In meta-analyses involving all three studies, the G allele

at rs6564851, near the -carotene 15,15’-monooxygenase 1

(BCMO1) gene, was associated with higher circulating -carotene

(P = 1.6 x 10 -24 ) and -carotene (P = 0.0001) concentrations and

lower lycopene (P = 0.003), zeaxanthin (P = 1.3 x 10 -5 ), and lutein

(P = 7.3 x 10 -15 ) concentrations, with effect sizes ranging from

0.10-0.28 standard deviations per allele. No significant relationship

was found between this genetic variant and plasma retinol.

Conclusions. Variation in the -carotene 15,15’-monooxygenase

1 (BCMO1) gene, which catalyzes the first step in the conversion

of provitamin A carotenoids to vitamin A in the small intestine,

is associated with plasma concentrations of carotenoids.

Determination of 9-cis -carotene and -carotene

in biological samples

Jian Qin, Kyung-Jin Yeum, Elizabeth J. Johnson,

Norman I. Krinsky * , Robert M. Russell, and Guangwen Tang

Jean Mayer USDA-Human Nutrition Research Center on Aging,*Department of

Biochemistry, School of Medicine, Tufts University, Boston, MA 02111

Determination of 9-cis -carotene (-C) and -carotene (-C)

in biological samples may provide crucial information on biological

activities of these carotenoids. However, these carotenoids are

often co-eluted when analyzed using high performance liquid chromatography

(HPLC). There is a need to develop a method for 9-

cis -C and -C quantification. Both 9-cis -C and -C have peak

absorbance at 400 and 450 nm, whereas only 9-cis -C has peak

absorbance at 475 nm. Based on these spectral characters, we

developed an HPLC method to determine the amounts of 9-cis -C

and -C in biological sample by using peak absorbance ratios in

these wavelengths. The absorbance of mixture of 9-cis -C and -

C was monitored at 475 and 400 nm. In which, the 9-cis -C was

quantified by using absorbance value at 475 nm; -C was then calculated

from the absorbance at 400 nm by subtracting the 9-cis -C

absorbance at 400 nm (calculated as 39% of the absorbance of 9-

cis -C at 475 nm). This method was used to determine 9-cis -C

concentrations of American lactating women (n=12) in serum (7.1

± 0.8 nM) and breast milk (1.1 ± 0.2 nM), and -C concentrations

of those lactating women in serum (54.2 ± 7.2 nM) and milk (8.3 ±

1.8 nM). We also find that 9-cis -C concentrations of Korean

women (n=16) in serum (15.6 ± 1.1 nM) and breast adipose tissue

(0.2 ± 0.1 nmol/g), and -C concentrations of those Korean women

in serum (49.0 ± 3.9 nM) and adipose tissue (0.3 ± 0.1 nmol/g).

Considering the significant amounts of 9-cis -C and -C in circulation

and tissues of humans from their dietary intakes, investigations

on these carotenoids and their possible health benefits will be

discussed.

38

39


Control of oxidative phosphorylation by vitamin A illuminates a

fundamental role in mitochondria energy homeostasis

Rebeca Acin-Perez*, Beatrice Hoyos**, William S Blaner***,

Giovanni Manfredi* and Ulrich Hammerling**

*Department of Neurology and Neurobiology, Weill Medical College at

Cornell, New York, NY. ** Immunology Program, Sloan-Kettering Institute for

Cancer Research, New York, NY. *** Department of Human Nutrition, College of

Physicians and Surgeons, Columbia University, New York, NY

Whereas many biological effects of vitamin A are attributable to

its derivative, retinoic acid, its action in signal transduction does not

require prior metabolic conversion but is mediated by vitamin A (retinol)

itself. Building on previous findings that retinol binds protein

kinase C (PKC) with high affinity we discovered an important regulatory

pathway in mitochondria, which depends on the presence of retinol.

This signal pathway is anchored on protein kinase C, is situated

in the intermediate space, and transmits an activating signal to the pyruvate

dehydrogenase (PDH) complex located in the matrix. While intermediaries

of this signal chain remain unidentified, the pyruvate dehydrogenase

kinase, isoform 2 (PDK2), becomes inactivated by dephosphorylation

when PKC is active. Since PDK2 negatively regulates

PDH, the reduced PDK2 activity translates into dephosphorylation

of the E1 regulatory subunit of PDH, increasing acetyl-CoA production,

oxygen consumption and ATP synthesis. The physiological

relevance of vitamin A for mitochondrial energy homeostasis became

apparent in the LRAT null mouse strain rendered completely vitamin

A deficient by dietary deprivation. This regimen resulted in markedly

reduced levels of oxygen consumption of their liver mitochondria,

which were promptly normalized by the injection of vitamin A. The

central role of PKC was established by targeted deletion of the prkcd

gene, whereas the need for binding retinol to the zinc-finger domains

of PKC was deduced from the failure of complementing the prkcd

gene ablation by a mutant form with an engineered deletion of the

retinol binding sites. While the upstream signal that activates PKC is

still unknown, our evidence supports the hat retinol functions as a device

to target oxidation to select cysteine residues of the zinc-finger

domain where local unfolding is the crucial initiation event for enzyme

activation.

Chiu, H-J, Fishman, D.A. and Hammerling, U. Vitamin A depletion casuses oxidative stress,

mitochondrial dysfunction and PARP-1-dependent energy deprivation. FASEB J 2008, 22:3878

Acin-Perez, R., Hoyos, B., Zhao, F., Vinogradov, V., Fischman, D.A., Harris, R.A.,

Leitegs, M., Nuttaporn, W., Blaner, W.S., Manfredi, G., and Hammerling, U. Control of oxidative

phosphorylation by vitamin a illuminates a fundamental role in mitochondrial energy

homoeostasis. FASEB J. 2010, 24: 627

40

Public health aspects of -carotene as an important

vitamin A source for humans

Hans K. Biesalski

Department of Biological Chemistry and Nutrition, University Hohenheim,

Garbenstrasse 30, D 70597 Stuttgart

In developed countries major sources for preformed vitamin A

(e.g., liver and liverproducts) are available but poorly consumed.

As a consequence different groups (young females, pregnant

women, elderly, obese people) with poor vitamin A intake and

subsequent low retinol but normal to high -carotene levels are described.

This raises the question whether the recommendation to

consume a diet rich in -carotene to compensate a low intake of

preformed vitamin A is indeed enough. Depending on the cleavage

ratio of -carotene to vitamin A, which migth be different in different

tissues, the importance of this provitamin as a source for vitamin

A is under discussion. To evaluate the state of the art regarding

the role of -carotene as a source for vitamin A an international

consensus meeting was organized on July 11th 2009. (Participants:

Tilman Grune, Jun Shi Chen, Georg Lietz, Andreu Palou, A.

Catharine Ross, Wilhelm Stahl, Guangweng Tang, David

Thurnham, and Hans K. Biesalski). The experts discussed 17 questions

and reached an agreement formulated in a consensus answer.

This consensus answer is based on published valid data, which

were carefully reviewed by the individual experts and are justified

by background statements. Ascertaining the impact of -carotene

on the total intake of dietary vitamin A is complicated, as the efficiency

of conversion of -carotene to retinol is not a single ratio,

and different conversion factors have been used in various surveys

and governmental recommendations. However, a role of -carotene

in fulfilling the recommended intake for vitamin A is apparent

from a variety of studies. Thus, besides, elucidating the various

functions, the distribution and the uptake of -carotene, the consensus

conference placed special emphasis on the provitamin A

function of -carotene and the role of -carotene in the realization

of the required/recommended total vitamin A intake, in both developed

and developing countries.

41


New classes of mitochondria-targeted antioxidants –

non-radical scavenging inhibitors of lipid peroxidation

Valerian E. Kagan

University of Pittsburgh

Free radical reactions are implicated in the pathogenesis of major

chronic cardiovascular and neurodegenerative diseases, cancer, radiation

injury and aging. It is assumed that oxygen radicals, particularly

superoxide radicals (O 2·–),

hydrogen peroxide (H 2 O 2 ) and hydroxyl

radicals cause oxidative modification of biological molecules and initiation

of free radical chain reactions. Numerous attempts have been

made to utilize free radical scavengers as therapeutic and/or preventive

remedies. The initial optimism, however, faded over the years as

clinical trials showed relatively low efficiency of antioxidants in drug

discovery paradigms. Do peroxidation reactions develop as a random

uncontrolled chain process in tissues normally or in disease conditions?

There is a popular concept of chain breaking (sacrificial) water-soluble

and lipid-soluble antioxidants such as vitamin C, vitamin

E, ubiquinol, etc. Is antioxidant action their only or major function,

and do they act as “random” scavengers of “random” radicals?

Recently, we discovered that during apoptosis, cytochrome c (cyt c),

switches its function from a shuttle of electrons between mitochondrial

complexes III and IV, to that of a peroxidase. We found that this

is due to cyt c’s interactions with cardiolipin (CL). Trans-membrane

migration of CL from the inner to the outer mitochondrial membrane

- catalyzed by scramblase-3 early in apoptosis – is a pre-requisite for

its interaction with cyt c. The new catalytic function of cyt c is specific

towards CL resulting in its oxidation to yield different mono-,

di- and tri- hydroperoxy- and hydroxy- molecular species. We will

present data illustrating the principal differences in action of vitamin

E and vitamin C against random reactions of oxidative stress and enzymatically-controlled

reactions of lipid peroxidation. This will be

followed by demonstration of new possibilities to control lipid peroxidation

reactions by mechanism-based inhibitors of the peroxidase

activity of cyt c/cardiolipin complexes. Finally, the data will be

shown illustrating anti-apoptotic potential of these new classes of inhibitors

of lipid peroxidation in vitro and in vivo.

42

Coenzyme Q in plasma membrane electron transport

Plácido Navas 1 , Carlos Santos-Ocaña 1 , Rafael de Cabo 2 , and

Emilio Siendones 1

1 Centro Andaluz de Biología del Desarrollo, Universidad Pablo de

Olavide-CSIC and Centre for Biomedical Research on Rare Diseases

(CIBERER), ISCIII, E-41013 Sevilla, Spain

2 Laboratory of Experimental Gerontology, National Institute on Aging, NIH,

Baltimore, MD 21224, USA

Coenzyme Q (CoQ) is an essential component of the mitochondrial

respiratory chain, functions on the activation of uncoupling

proteins, the regulation of the permeability transition pore, as

substrate for the dihydro-orotate dehydrogenase, and is substrate of

acyl-CoA dehydrogenase. CoQ is located in the inner mitochondrial

membrane, but also in serum lipoproteins, endo-membranes

and the plasma membrane of eukaryotic cells. CoQ is a mediator in

a plasma membrane redox system (PMRS) involved in membrane

antioxidant protection, in the maintenance of intracellular redox

homeostasis, and regulation of cell signaling. NAD(P)H-dehydrogenases

in PMRS include NADH-cytochrome b 5 reductase encoded

by CB5R3 and NAD(P)H:quinone oxidoreductase 1 encoded

by NQO1. CoQ-dependent PMRS blocks the triggering of the ceramide-dependent

apoptotic program. PMRS is activated by vitamin

E deficiency, calorie restriction, and mitDNA deficiency. The

activity of PMRS also equilibrates the NAD + /NADH ratio important

in aging process. Yeast NQR1, homologous of mammal

CB5R3, regulates both chronological and replicative lifespan. This

gene is induced by calorie restriction in parallel to the activation of

respiration. Overexpression of NQR1 promotes oxygen consumption,

inhibits ethanol production, and maintains NAD + /NADH balance.

We propose that PMRS regulates apoptosis, but also regulates

aging by connecting aerobic metabolism and NAD + /NADH

ratio.

Supported by Spanish FIS Grant PI080500, NIH Grant 1R01AG28125-01A1, and the Intramural

Research Program of the NIA/NIH.

43


ECTO-NOX, coenzyme Q 10 , and superoxide production

in aging

D. James Morré 1,2 , Dorothy M. Morré 1,2 , and Dale Kern 3

1 NOX Technologies, Inc., 1291C Cumberland Avenue and 2 Purdue University,

West Lafayette, IN 47906 and 3 NuSkin International, 75 West Center Street,

Provo, UT 84601

The aging related ECTO-NOX (arNOX) family of superoxidegenerating

oxidases increases with age beginning at ca. 30 y. Shed

forms are present in blood associated with serum LDL and in other

body fluids where they may contribute to action-at-a-distance phenomena

such as skin aging and coronary artery disease. arNOX

proteins are membrane anchored with the catalytic N-termini directed

outward. The shed forms result from proteolytic cleavage

of ca. 30 kDa fragments. At least five different isoforms are indicated.

A distinguishing feature is periodic generation of superoxide

anion estimated from superoxide dismutase-inhibited reduction

of ferricytochrome c. The electron donor for the shed forms is protein

thiols. Competition for cytochrome c reduction by reduction

of CoQ 10 by superoxide is not involved. Compared to H 2 O 2 production

by mitochondria (ca. 0.1 nmoles/min/mg mitochondrial

protein), human buffy coats or epidermal epithelia from subjects

55 to 65 y produce ca. 0.06 nmoles superoxide convertible into

H 2 O 2 per 10 7 cells. For sera, ca. 0.3 nmoles/ml/min results in several

millimoles on a daily basis in the proximity of circulating

lipoproteins. Recent focus has identified safe and effective dietary

(e.g., CoQ 10 ) and topical (AgeLoc, NuSkin Enterprises) means to

inhibit arNOX activity. Both the cell-bound and shed arNOX are

inhibited by CoQ 10 but not by ubiquinol (CoQ 10 H 2 ). As ubiquinones

with side chains shorter than that of CoQ 8 do not inhibit,

polyprenyl side chain involvement is indicated. Data for saliva

and sera have demonstrated dose dependent, sustained but reversible

50% reductions in arNOX activity by oral administration of

CoQ 10 in both male and female subjects.

Coenzyme Q 10 in health and disease

Iain P. Hargreaves

National Hospital, London, UK

A major difficulty encountered when assessing (coenzyme Q 10 )

CoQ 10 status in tissues is the lack of a commercially available nonphysiological

internal standard. To address this problem we have

synthesised a dipropoxy analogue of CoQ 10 , which has enabled the

accurate determination of tissue CoQ 10 status. A decrease in CoQ 10

status has been reported in a number of diseases, however, interest

has focused on the possible role CoQ 10 deficiency may have in the

pathophysiology of the rare adverse effects associated with hydroxy-3-methylglutaryl

coenzyme A (HMG-CoA) reductase inhibitor

(statin) treatment. Currently, there is insufficient evidence

from human studies to link statin therapy unequivocally to pathologically

significant decreased tissue CoQ 10 concentrations. To investigate

involvement of mitochondrial respiratory chain (MRC)

dysfunction in myotoxicity associated with statin treatment, MRC

activity and CoQ 10 status was assessed in two patients experiencing

myopathy following treatment with simvastatin (40mg/day)

and cyclosporin and simvastatin (40mg/day) and itraconazole.

Analysis of skeletal muscle biopsies revealed a decreased CoQ 10

status (77 and 132; reference range: 140 - 580 pmol/mg) and cytochrome

oxidase (complex IV) activity (0.006 and 0.007 reference

range: 0.014 – 0.034). To assess statin treatment in the absence of

possible pharmacological interference primary astrocytes were cultured

with lovastatin (100 mM). Lovastatin treatment resulted in a

decrease in CoQ 10 (97.9 ± 14.9; control: 202.9 ± 18.4 pmol/mg; p

< 0.05), and complex IV activity (0.008 ± 0.001; control: 0.011 ±

0.001; p< 0.05). The effect of rosuvastatin therapy was assessed on

blood mononuclear cell (BMC) CoQ 10 status and mitochondrial

ATP synthesis in children with familial hypercholesterolemia.

Samples were taken at baseline and after rosuvastatin treatment.

Twenty-nine patients were treated with 5, 10 or 20 mg rosuvastatin

for 29 weeks. After this time a significant 32% decrease in BMC

CoQ 10 (p=0.024) was observed. BMC ATP synthesis was comparable

to baseline levels (p=0.60). In conclusion these results indicate

a possible association between statin treatment, decreased

CoQ 10 status and loss of complex IV activity.

44

45


Targeting inflammatory pathways “naturally” for prevention

and therapy of cancer and other chronic diseases

Bharat B. Aggarwal and Bokyung Sung

Cytokine Research Laboratory, Department of Experimental Therapeutics, The

University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA

Chronic infections, obesity, alcohol, tobacco, radiation, environmental

pollutants, and high-calorie diet have been recognized

as major risk factors for the most common types of cancer. All

these risk factors are linked to cancer through oxygen radicalmediated

inflammation. While acute inflammation that persists for

short-term mediates host defense against infections, chronic inflammation

that lasts for long-term can predispose the host to various

chronic illnesses, including cancer. Linkage between cancer

and inflammation is indicated by numerous lines of evidence; first,

transcription factors NF-B and STAT3, two major pathways for

inflammation, are activated by most cancer risk factors; second, an

inflammatory condition precedes most cancers; third, NF-B and

STAT3 are constitutively active in most cancers; fourth, hypoxia

and acidic conditions found in solid tumors activate NF-B; fifth,

chemotherapeutic agents and gamma irradiation activate NF-B

and lead to chemoresistance and radioresistance; sixth, most gene

products linked to inflammation, survival, proliferation, invasion,

angiogenesis, and metastasis are regulated by NF-B and STAT3;

seventh, suppression of NF-B and STAT3 inhibits the proliferation

and invasion of tumors; and eighth, most chemopreventive

agents mediate their effects through inhibition of NF-B and

STAT3 activation pathways. Thus suppression of these proinflammatory

pathways may provide opportunities for both prevention

and treatment of cancer. We will discuss the potential of various

natural products in suppression of inflammatory pathways and

their role in prevention and therapy of cancer and other chronic

diseases.

46

Chemoprevention by pomegranate and green tea

Susanne M. Henning, Piwen Wang, Angela Wong,

Roberto Vicinanza, Lynn, S. Adams, William J. Aronson, and

David Heber

Center for Human Nutrition and Department of Urology, David Geffen School

of Medicine, University of California Los Angeles, CA 90095

Ellagitannins from pomegranate and polyphenols from green

tea have limited bioavailability and are highly metabolized by

colonic microflora. Ellagitannins are converted to gallagic acid

(GA), ellagic acid (EA), urolithin A (UA) and B (UB) and tea

polyphenols to phenolic acids, which can be found in the circulation

and urine. During the absorption process polyphenols and their

metabolites are glucuronidated, sulfated and methylated. These

metabolites exhibit biological activities. Recent findings show that

UB, GA, UA inhibit proliferation in aromatase overexpressing

MCF-7-aro breast cancer cells via aromatase inhibition and estrogen

receptor antagonism, and also inhibit inflammation via NFB.

Conjugation and methylation may alter their tissue distribution and

biologic activities. Methylated and sulfated UB retained the aromatase

inhibitory activity. However, the methylated form of one of

the tea polyphenols, epigallocatechin gallate (EGCG), exhibited

decreased anti-proliferative activity, decreased anti-inflammatory

activity (NFB stimulation) and decreased stimulation of apoptosis

compared to the parent compound in human LNCaP prostate cancer

cells. In a mouse and human tea intervention study we demonstrated

that about 50% of EGCG was present in methylated form in

tissue. EGCG itself is a substrate for methyl transferases such as

catechol-O-methyl transferase (COMT) and 5-cytosine DNA

methyl transferase I (DNMT1). In xenograft tumor tissue in SCID

mice drinking green tea both COMT and DNMT1 gene expression

was inhibited significantly. In cell culture experiments combining

EGCG with another methyl inhibitor such as quercetin decreased

EGCG methylation, decreased DNMT1 gene expression and increased

EGCG uptake. In summary, to perform in vitro experiments

with translational potential, the in vivo metabolism of phytochemicals

will need to be considered carefully.

47


Regulation of antioxidant gene expression by selected

dietary polyphenols

Young-Joon Surh

WCU Department of Molecular Medicine and Biopharmaceutical Sciences,

Graduate School of Convergence Science and Technology and College of

Pharmacy, Seoul National University, Seoul 151-742, South Korea

The low risk of chronic diseases, such as coronary heart disease,

neurodegenerative disorders and certain cancers, observed in some

population groups results from consumption of relatively large

amounts of fruits and vegetables. Health benefits associated with

functional foods and beverages containing a great array of bioactive

phytochemicals or phytonutrients have been extensively investigated

and well-documented. As oxidative stress is implicated in a

wide array of human disorders, antioxidant phytochemicals have

also been frequently included as active ingredients of many functional

foods. The most important botanicals for use as nutracuetical

formulations include tea, soy, pomegranate, grape seeds, pycnogenol,

turmeric, ginkgo biloba, aloe, ginseng, etc. The majority of

bioactive chemical compounds contained in the dietary supplements/nutraceuticals

have antioxidant activities. Nuclear transcription

factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2) plays a

crucial role in regulating induction of antioxidant or cytoprotective

gene expression. Many bioactive substances derived from edible

plants have been found to activate this particular redox-sensitive

transcription factor, thereby potentiating cellular antioxidant or detoxification

capacity. Nutraceutical and functional food markets

are rapidly growing, and there are ample outstanding opportunities

of research for the development of botanicals as dietary supplements

or nutraceuticals.

Resveratrol mimics caloric restriction and

retards aging in the heart

Tomas A. Prolla

Departments of Genetics and Medical Genetics,

University of Wisconsin-Madison, Madison WI 53706, USA

Resveratrol in high doses has been shown to extend lifespan in

some studies in invertebrates and to prevent early mortality in mice

fed a high-fat diet. We fed mice from middle age (14-months) to

old age (30-months) either a control diet, a low dose of resveratrol

(4.9 mg kg/day), or a calorie restricted (CR) diet and examined genome-wide

transcriptional profiles. We report a striking transcriptional

overlap of CR and resveratrol in the heart. Both dietary interventions

inhibit gene expression profiles associated with cardiac

and skeletal muscle aging, and prevent age-related cardiac dysfunction.

A more recent analysis using transcriptional supermarkers

of aging in multiple mouse strains shows that the anti-aging effects

of resveratrol are stronger in the heart, whereas lipoic acid

and Coenzyme Q10 have stronger effects in the brain. Our results

suggest that transcriptional supermarkers of aging can be used to

identify and design tissue-specific CR mimetic anti-aging interventions.

1. Surh, Y.-J. (2003) Cancer chemoprevention with dietary phytochemicals. Nature Reviews

Cancer, 3: 768-780

2 . Surh, Y.-J., Kundu, J.-K., and Na, H.-K. (2008) Nrf2 as a master redox switch in turning

on the cellular signaling Involved in the induction of cytoprotective genes by some

chemopreventive phytochemicals. Planta Med., 74: 1526-1539.

3. Surh, Y.-J., Dong, Z., Cadenas, E., and Packer, L. (2008) Dietary Modulation of Cell

Signaling Pathways, CRC Press-Taylor & Francis

48

49


SESSION IV

REDOX IMAGING


Development of PET/SPECT probes and their application to

in vivo molecular imaging

Hideo Saji

Graduate School of Pharmaceutical Sciences, Kyoto University,

Kyoto 606-8501 Japan

“Molecular imaging” is a recently developed approach that is in

vivo imaging of the spatial/temporal distribution of biological/molecular

biological processes (events) at the cellular/ molecular

level and has been contributing to the promotion of studies on the basis

of life science, biological functions and causes of diseases, genetic/regenerative

medicine. In molecular imaging field, methods using

radiation such as positron emission tomography (PET) and single

photon emission computed tomography (SPECT) play a major role. In

tumors, rapid growth of cells and rapid expansion of the tumor mass

usually leaves the generation of new vasculature lagging behind and

results in the lack of oxygen delivery. This condition is under an unbalanced

redox status, oxidative levels in tumor cells being relatively

higher. In such condition hypoxia-inducible factor-1 (HIF-1) is expressed.

HIF-1 is specifically degraded under normoxia, but stable under

hypoxia. Thus, by utilizing the character of HIF-1, we attempted to

develop PET/SPECT imaging agent for molecular imaging of cancer.

Since the degradation of HIF-1 occurs through protein hydroxylation

in the oxygen-dependent degradation domain (ODD) of HIF-1, it is

expected that probes containing the ODD and that are degraded in a

similar manner as HIF-1 can evaluate HIF-1 activity in vivo. Thus,

we recently developed a protein (POS) in which the ODD is fused to

the protein transduction domain (PTD) and a monomeric streptavidin

(SAV). Furthermore, We synthesized a radiolabeled biotin derivative,

(3- 123/125 I-iodobenzoyl)norbiotinamide ( 123/125 I-IBB) and conjugated

the chimeric protein—PTD-ODD-SAV (POS)—and 123/125 I-IBB to

produce 123/125 I-IBB-POS ( 123/125 I-IPOS). POS was degraded in an

oxygen-dependent manner, and a clear image of the tumor was obtained

at 24 h after the injection of 123 I-labeled-POS. Moreover, The

intratumoral distribution of 125 I-IPOS was heterogeneous and corresponded

to the hypoxic areas.

52

Clinical (EPR) oximetry for improving diagnosis and

treatment in ischemic diseases and tumors and wound healing

Harold M. Swartz

Dartmouth Medical School, HB 7785, Hanover, NH 03755

The technique of Electron Paramagnetic Resonance (EPR)

oximetry has now advanced to the stage where it is being used extensively

in animal experiments and several clinical studies are in

process. This has required the development of special instrumentation

to safely and effectively accommodate human subjects. The

potential advantages of EPR oximetry include that it measures the

actual pO 2 directly in the tissues and these measurements can be

made repeatedly from the same site. Most EPR oximetry approaches

require an initial minimally invasive step to place the

oxygen sensitive materials at the sites of interest, but the subsequent

measurements are completely non-invasive. The studies in

human subjects underway at Dartmouth include

1. Measurements of oxygen in tumors to optimize therapy by

enabling the therapy to be delivered at optimum times and/or

altered to deal with significant hypoxia

2. Measurements of oxygen in tissues at risk in peripheral vascular

disease to characterize the pathophysiological state and

to measure response to therapy

3. Acute and chronic measurements of oxygen in healing

wounds.

In order to make the measurements with paramagnetic materials

that are cleared for use in human subjects, most of the measurements

are made using India ink as the oxygen sensitive material.

We are now developing an implantable resonator that should

enable the measurements to be made with greater sensitivity and at

greater depths. Our technique uses multisite spectroscopy, which

can measure oxygen at several defined site simultaneously. The

time resolution of the measurements can be seconds and the measurements

have been repeated in human subjects from the same site

for more than four years.

53


Imaging of tissue oxygenation:

Novel probes and opportunities

Periannan Kuppusamy

Davis Heart and Lung Research Institute,

The Ohio State University, Columbus, OH 43210, USA

Oxygen is a critical determinant in the prediction of treatment

outcome of several disease including surgical interventions, cancer

therapy, tissue graft, and cell therapy. There is a great need for

methods capable of reliable noninvasive measurement and monitoring

of oxygen concentration in tissues. Although several methods

are utilized to measure oxygen concentration, a suitable technique

for noninvasive and repeated measurements of oxygen in the

same tissue or cells on a temporal scale is warranted. While electrode

techniques have evolved as the standard methods for measurement

of oxygen, they generate analytical artifacts during assay

procedures at the freshly probed sites. Near-infrared and nuclear

magnetic resonance techniques are noninvasive methods. However,

they do not report the absolute values of oxygen concentration,

and lack the resolution of oxygen measurements. Electron

paramagnetic resonance (EPR) oximetry enables reliable and accurate

measurements of concentrations of molecular oxygen. EPR

oximetry can measure directly and at the actual site of interest. The

ability of EPR oximetry to make repeated measurements from localized

sites provides a very important capability that can enable

critical aspects of a number of biomedical applications. We have

developed innovative approaches using oxygen-sensing nano/ microcrystalline

probes to perform noninvasive cellular/tissue oximetry/imaging

in a variety of applications including myocardial

ischemia/reperfusion injury, cellular cardiomyoplasty (cell therapy),

organ transplantation, angiogenesis, cancer therapy, and

wound healing. Current developments in in vivo EPR methodologies

enable a number of potential applications that could be very

significant additions to clinical medicine.

SESSION VI

AGING

54


RasGrf1 deficiency delays aging in mice by mimicking

caloric restriction

José Viña, Consuelo Borrás 1 , Daniel Monleón 2 ,

Raul López-Grueso 1 , Juan Gambini 1 , Leonardo Orlando 3 ,

Federico V. Pallardó 1 , Eugenio Santos 4 , and Jaime Font de Mora 3,5

1 Department of Physiology, School of Medicine, University of Valencia.

2 Instituto de Investigación Sanitaria FIHCUV/Fundación de Investigación

Hospital Clínico Universitario de Valencia. 3 Laboratory of Cellular and

Molecular Biology, Centro de Investigación Príncipe, Valencia. 4 Centro de

Investigación del Cáncer (USAL-CSIC), Univeristy of Salamanca. Spain

Lifespan is determined by environmental as well as genetic

factors. Caloric restriction extends longevity via regulatory proteins

that link metabolism and anti-aging effects. RASGRF1 is a

Ras-guanine nucleotide exchange factor implicated in a variety of

physiological processes including learning and memory and glucose

homeostasis. Mice deficient for RasGrf1 display a remarkable

increase in average and most importantly in maximal lifespan. This

is not due to the role of RAS in cancer because tumor-free survival

was also enhanced in these animals. Biomarkers of aging indicate

that RasGrf1 knockout mice (RasGrf1 -/- ) have a lower biological

age than controls. Aged RasGrf1 -/- displayed better motor coordination

than control mice. Protection against oxidative stress is preserved

in old RasGrf1 -/- . Furthermore, Sirtuin expression is increased

in RasGrf1 -/- animals. Consistent with this, blood metabolic

profiles as revealed by metabolomic analysis resembled those

observed in calorie-restricted animals. In addition, positron emission

tomography reveale that in vivo glucose consumption by the

heart is not altered by aging in the knockout model, indicating that

RasGrf1 deficiency renders tissues resistant to aging. Thus, these

observations link Ras signaling to the beneficial effects of caloric

restriction on lifespan and suggest that RasGrf1 is an evolutionary

conserved gene which could be targeted for the development of

therapies with the potential to delay age-related processes.

Work was supported by grants SAF2008-00270; SAF2006-12470; BFU2007-65803/BFI

from the Spanish Ministry of Education and Science; ISCIII2006-RED13-027 from the

“Red Temática de investigación cooperativa en envejecimiento y fragilidad (RETICEF),

and EU Funded COSTB35. D.M. is funded by the Ramon y Cajal Program of the MEC.

Selective Autophagy on the cellular response to stress

Ana Maria Cuervo

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY

Autophagy is the intracellular process that mediates the digestion

of cellular components in lysosomes. The autophagic system

fulfills two major functions in mammalian cells, serving both as an

alternative source of energy, when nutrients are scarce, and as an

efficient mechanism for the removal of any intracellular damage

structure. Autophagic activity has been described to decline with

age in almost all organisms and tissues, as wells as in several agerelated

disorders. On light of the prevalent functions of this catabolic

process, cells with impaired autophagy are often energetically

compromised and present severe problems in maintenance of

cellular homeostasis and in the response to stress. Our group is interested

in the study of the changes with age in different autophagic

pathways and on the consequences of those changes in the

aging organism. In addition, using both genetic manipulations and

regulated diets, we have been able to prevent the decline in

autophagic activity in old rodents and analyze the beneficial effect

of this intervention both in cellular homeostasis and in their energetic

balance. These models would help us evaluate the importance

of maintaining proper autophagic function until advanced ages,

and, on light of the observed cross-talk among autophagic pathways,

the effect that repairing one autophagic pathway may have

on the functioning of the others.

56

57


Regulation of proteasome-mediated protein degradation

during aging

Tilman Grune

University of Hohenheim, Stuttgart, Germany

Protein oxidation is a permanently ongoing process. Products

of protein oxidation might disturb cellular metabolism and, therefore,

oxidized proteins should be degraded effectively. Therefore,

the bulk of oxidatively modified proteins is selectively recognized

and degraded. This process is catalyzed by the proteasomal system

and it is general accepted today, that the 20S proteasome is degrading

the vast amount of oxidized proteins. On the other hand failure

of an adequate degradation of oxidized proteins is leading to various

side effects, including formation of protein aggregates. Such

protein aggregates are major players in the metabolic changes during

aging of postmitotic cells. Protein aggregates accumulate during

aging in the lysosomal compartment. Located in the lysosomes

they might be involved in adverse metabolic pathways leading to

the apoptosis of senescence cells. Lysosomal accumulated protein

aggregates can not be degraded by lysosomal cathepsins. The uptake

of protein aggregates from the cytosol into the endosomallysosomal-compartment

is mediated most likely by macroautophagy.

Therefore, the understanding of the interaction of the cellular

protein degradation machinery, including proteasomes and

lysosomes, are of increasing importance.

Delaying age-related disease with micronutrients: triage theory

Bruce N. Ames

Children’s Hospital Oakland Research Institute

Oakland, California, USA

An inexpensive intervention could delay the degenerative diseases

accompanying aging, such as cancer, cardiovascular disease,

cognitive decline, stroke, and immune dysfunction. Most of the

world’s population, even in developed countries, has inadequate

intake of one or more micronutrients (~40 essential vitamins, minerals,

fatty acids and amino acids) that a varied and balanced diet

should provide. My triage theory (PNAS 103, 17589, 2006; AJCN

90, 889, 2009) posits that, as a result of recurrent shortages of micronutrients

during evolution, natural selection developed a metabolic

rebalancing response to shortage. The rebalancing favors

micronutrient-dependent proteins needed for short-term survival

while starving those only required for long-term health. Triage

theory predicts that the consequence of moderate shortages of even

a single micronutrient, though insufficient to cause overt clinical

symptoms, will impair functions essential for long-term health.

This impairment will result in insidious damage (e.g. increased

oxidative damage) that, over time, leads to the acceleration of ageassociated

diseases (e.g. increased cancer and brain dysfunction).

As people with modest deficiencies have no overt clinical symptoms,

there has been little incentive to correct these deficiencies,

though this could change if it can be shown that they are resulting

in biochemical changes, e.g. chromosome breaks, that are markers

of increased risk of age-related diseases, e.g. cancer. The considerable

experimental and theoretical support for the triage idea will

be discussed as will a strategy for determining the optimum level

of each micronutrient in humans. The triage theory should help to

put micronutrient nutrition on a firm foundation and lead to preventive

medicine for age-related diseases.

58

59


Endothelial dysfunction in aged humans is related to

oxidative stress and vascular inflammation.

A practical approach from traslational research

Leocadio Rodriguez Mañas

Servicio de Geriatría, Hospital Universitario de Getafe; Carretera de Toledo

Km. 12.5; 28905-Getafe, Madrid, Spain

Vascular diseases are the first cause of mortality and functional deterioration

(including dependency) in Western Societies, reaching their highest

incidences and prevalences in people older than 65 years old. Together to the

classical (Hypertension, Smoking, Diabetes Mellitus, Hypercholesterolemia)

and new cardiovascular risk factors (homocystein, CRP, markers of inflammation),

ageing remains as the principal risk factor. Vascular endothelial

dysfunction has been shown in many animal models of ageing and, since one

decade, there are experimental data showing that endothelial dysfunction is

occurring also during the human aging process. Endothelial dysfunction is

now considered a crucial event in the development of many vasculopathies

and vascular diseases, including ischemic heart disease, stroke and peripheral

artery disease. Moreover, endothelial dysfunction has been shown to be a

prognostic factor in cardiovascular diseases. We investigate the underlying

mechanisms of human endothelial dysfunction accompanying the ageing

process, particularly those related to oxidative stress and inflammation, in the

vasculature of subjects between 13-91 years without cardiovascular disease

or risk factors. In isolated mesenteric microvessels from these subjects, an

age-dependent impairment of the endothelium-dependent relaxations to

bradykinin (BK) was observed. In microvessels from subjects younger than

60 years old, BK-induced relaxation was mostly due (80%) to nitric oxide

(NO) release while the rest was sensitive to cycloxygenase (COX) blockade.

In microvessels from subjects older than 60 years old, this COX-derived

vasodilatation was lost but a COX-derived vasoconstriction occurred, although

the expression of both COX-1 and COX-2 was not altered. Evidence

for age-related vascular oxidant and inflammatory environment was observed,

which could be related to the development of endothelial dysfunction.

It must be underscored that these findings are remarkably similar to

those observed in young people with diabetes or hypertension. These data

raised some questions that we are now approaching: 1) If the effect of ageing

on vascular endothelium in humans is very close to that produced by diabetes

or hypertension, do these classical risk factors induce vascular disease in old

people by adding new damage to endothelial dysfunction or there are other

mechanisms involved? 2) These factors involved in producing endothelial

dysfunction in selected populations, are also present in studies made on more

representative cohorts of old people.

POSTERS

60


Effect of cigarette smoke on redox regulation in

chronic obstructive pulmonary disease

Amit R. Agarwal, Jerome V. Garcia, Harsh Sancheti, and

Enrique Cadenas

Department of Pharmacology and Pharmaceutical Sciences, University of

Southern California, Los Angeles, California 90089, USA

Chronic obstructive pulmonary disease (COPD) is a persistent

obstruction of airways in the lungs due to the narrowing of the passage

ways. Cigarette smoking is responsible for approximately 80-

90% of COPD related deaths. Cigarette smoke contains over 4000

chemicals, including carcinogens, strong electrophiles and other

toxins. The current study was undertaken to determine the role of

cigarette smoke in the development of mitochondrial dysfunction

due to oxidative stress. Rat lung epithelial cell cultures (RLE-6TN

cell line) were used to study the effects of cigarette smoke extract

(CSE), individual constituents of CSE like acrolein and nitric oxide

on cell viability and redox status of the cell. We have adapted a

method to prepare cigarette smoke extract in our laboratory by

smoking 4 cigarettes in 20 ml of serum free F-12 media. The

smoke extract was characterized using Agilent 8453 UV-Visible

Spectrophotometer with a consistent peak at 361 nm. We exposed

cells to 0, 0.5%, 1%, 2.5%, and 5% of CSE for varying periods of

time. Our data indicate that treatment of RLE-6TN cells with doses

less than 2.5% CSE for 4 h lead to an increase in the NADPH/

NADP + ratio with no change in the NADH/NAD + ratio as measured

by HPLC. At 24 h, a decrease in both NADPH/NADP + and

NADH/NAD + ratios, indicate of an initial compensatory response

to CSE challenge.

The effect of sprint training on oxidative stress

and antioxidants

Suhana Aiman 1 , Nor Anita MMN 2 and Zaiton Z 2

1 Faculty of Sports Science and Recreation, Universiti Teknologi MARA, 40450

Shah Alam, Selangor, Malaysia; 2 Department of Physiology, Universiti

Kebangsaan Malaysia Medical Center, Jalan Raja Muda Abdul Aziz, 50300

Kuala Lumpur, Malaysia

Numerous studies have shown that aerobic training reduces

oxidative stress and improves antioxidant status. However, the information

on anaerobic training on oxidative stress and antioxidant

enzymes were scarce. The purpose of this study was to determine

the effect of sprint training on MDA and antioxidant enzyme activities.

Sixteen male subjects participated in this study. They randomly

assigned to training group and control group. Training

group engaged in sprint training for eight weeks (three times per

week). Venous blood samples were taken before and after training.

Plasma malondialdehyde (MDA) was measured as oxidative stress

maker. Antioxidant enzymes analyzed were superoxide dismutase

(SOD), glutathione peroxidase (GPX) and catalase (CAT). There

was no significant difference in MDA was observed after the sprint

training. Compared with the control group, SOD level remains unchanged

following training. While there were significant improvement

of GPX and CAT activity after the training. In conclusion,

this study demonstrates that sprint training does not reduce

the oxidative stress but has enhanced antioxidant defences.

62

63


Zinc and the modulation of transcription factors

STAT1 and STAT3 in fetal rat brain and neuronal cells

Lucila Aimo, Suangsuda Supasai, Yo Omata,

Gerardo G. Mackenzie, and Patricia I. Oteiza

Departments of Nutrition and Environmental Toxicology,

University of California Davis, Davis, CA 95616, USA

We recently observed that zinc is required for cell proliferation and

prevention of neuronal apoptosis. The STAT pathway is involved in

the regulation of neurogenesis, neuronal differentiation and survival.

This work investigated the effects of low zinc availability on the

modulation of transcription factors STAT 1 and STAT3 (STAT1/3) in

neuronal cell cultures and animal models of zinc deficiency. Marginal

zinc diets (MZD) fed to rats throughout gestation caused a deregulation

of STAT1/3 modulation in gestation day 18.5 (GD 18.5) fetal

brains. STAT activation (STAT-DNA binding) in total fetal brain

homogenates was lower (24%) and higher (44%), respectively, in

MZD compared to control animals. In nuclear fractions from fetal

brain, STAT-DNA binding was markedly lower for STAT1 (80%) and

STAT3 (85%) in MZD compared to controls. In human neuroblastoma

IMR-32 cells, the incubation in zinc deficient media for 24 h, leads to

an increased total STAT1 and STAT3 activation. However, similarly

to that observed in GD18.5 fetal brain, zinc deficiency caused a 50%

reduction in nuclear STAT1- and STAT3-DNA binding. This was associated

with a decreased transcription of STAT1/3-driven reporter

genes in zinc deficient cells compared to controls. To investigate the

relevance of STAT1 and STAT3 in IMR-32 cell proliferation and survival,

cells were incubated in the presence of inhibitors of STAT1/3

(AG-490) or STAT3 (Stattic V) activation. Cell viability was significantly

lower after 24h incubation in the presence of the inhibitors

(50% decrease at 20 uM AG490 and 1.3 M Stattic V). This was in

part due to apoptotic cell death as evidenced by caspase-3 activation

and DNA fragmentation. In summary, zinc seems to be necessary for

the physiological regulation of STAT1/3 in the nervous system. Zinc

deficiency decreases nuclear STAT1/3 activation and dependent gene

transcription. An impaired STAT1/3 modulation could contribute to

zinc deficiency-induced neuronal apoptosis.

Supported by UC Davis and NIH (HD01743)

64

Scavenging of superoxide in biological contexts:

the role of ubiquinone, ubiquinol and SOD mimics

Robert F. Anderson 1 , Andrej Maroz 1 , Sujata S. Shinde 1 ,

Robin A.J. Smith 2 , and Michael P. Murphy 3

1 Department of Chemistry, The University of Auckland, Private Bag 92019,

Auckland 1142, New Zealand, 2 Department of Chemistry, Otago University, Box

56, Dunedin 9054, New Zealand, 3 Medical Research Council Dunn Human

Nutrition Unit, Cambridge CB2 0XY, UK

Endogenous ubiquinones (UQ) are essential electron carriers in

the mitochondrial electron transport chain, and the two-electron

reduced ubiquinol form (UQH 2 ) is a chain-breaking antioxidant

which is active against lipid peroxidation within mitochondria. Superoxide,

O .- 2 , is the proximal radical produced during mitochondrial

oxidative stress and its reactions with endogenous UQ/UQH 2

and the exogenous UQ, such as Idebenone and MitoQuinone, depend

on the environment where interaction takes place. We have

used pulse radiolysis to study such reactions in water, and in solvent

systems mimicking the surface and core of the biological

.-

membranes where UQ is preferentially located in vivo. While O 2

is scavenged very rapidly by UQ in all solvents (10 8 -10 9 M -1 s -1 ),

both O .- 2 and the hydroperoxyl radical (HOO . ) react slowly (


Tissue engineering of peri-implant space with stem/progenitor

cells: a novel approach to improve implant-level oxygenation

Mirela Anghelina, Omar I. Butt, Anna Bratasz, Leni Moldovan,

Periannan Kuppusamy, and Nicanor I. Moldovan

Davis Heart and Lung Research Institute, Departments of Internal Medicine and

Biomedical Engineering, Ohio State University, Columbus, OH, 43210

Clinical use of implanted medical devices such as biosensors

and drug delivery devices is limited by the cellular foreign body

reaction and consecutive fibrous encapsulation. Traditionally, attempts

have been made to mitigate the impact of these processes

by preventing their development, with little success so far. Based

on our advances in the understanding of the coupling between angiogenesis

and fibrogenesis in the subcutaneous space, we recently

proposed a different approach: to divert the normal 'woundhealing'

like process from fibrosis to a more stable neovascularization,

using stem/progenitor cells (SPC)-based tissue engineering.

Here we will illustrate this concept with our results obtained by using

mouse bone marrow derived c-kit+/sca-1+ SPC embedded in

Matrigel, implanted in recipients near a nanoporous filter-limited

sensor, which continuously monitors device-level oxygen concentrations

by in vivo EPR oximetry. We show that the presence of

exogenous SPC significantly increases implant oxygenation for at

least 10 weeks, coincident with morphological and immunohistological

markers of abundant neovascularization. We will also

show the current progress in our laboratory to develop novel engineered

implant-tissue interfaces using SPC-containing biomaterials.

Collectively, these advances create new bioengineering solutions

to the impact of foreign body reaction on the functioning of

implants, including those dependent on oxygen availability.

66

Ferritin degradation and increase of labile iron pool is

mediated by JNK1 in diallyl trisulfide treated

prostate cancer cells PC-3

1 Jedrzej Antosiewicz, 1 Alicja Sielicka-Dudzin,

1 Andzelika Borkowska, 1 Magorzata Halon,

Anna Herman-Antosiewicz, and Michal Wozniak

1 Department of Bioenergetics and Physiology of Exercise, 2 Department of

Medical Chemistry, 3 Medical University of Gdansk, Department of Molecular

Biology University of Gdansk Poland

In our previous study, we demonstrated that JNK signaling pathway

regulates ferritin degradation and labile iron pool in prostate cancer

cells treated with diallyl trisulfide (DATS). In the present work we

extended this study to determine which of c-jun kinase is responsible

for ferritin degradation and what the role of iron in DATS-induced cell

death is. Moreover, we hypothesized that JNK1, Itch ligase axis will

be responsible for ferritin degradation. PC-3 prostate cancer cells were

used in this study. In order to prove if kinases JNK participate in ferritin

degradation PC-3 cells were transfected with dominant negative

mutant of JNK1 or JNK2 and then treated with DATS. We observed

that DATS did not induced ferritin degradation in cell transfected with

JNK1-DN. On the other hand DATS induced ferritin degradation in

cells transfected with JNK2-DN. Moreover DATS induced neither

ROS formation nor increase LIP in JNK1 transfected cells however

such increase was observed in vector or in JNK2- transfected cells. In

order to established the role of iron in DATS induced cell death the

cells were pretreated with iron chelators but neither DFO nor tujapicin

had any effect on cell death. In addition cell preloaded with iron compound

hemine had the same sensitivity to DATS. Moreover we observed

that DATS increase activating phosphorylation of E3 ligaze

Itch. The phosphorylation was blocked by JNK inhibitor SP600125.

Cell transfected with JNK2-DN but not with JNK1-DN showed lower

level of Itch phosphorylation. Consistently cells transfected with inactive

form of Itch showed DATS induced ferritin degradation and ROS

formation but were much more resistant to DATS induced cell death.

In conclusion our data suggest that JNK1 regulates ferritin degradation

LIP level and ROS formation in prostate cancer cell but it is not related

to Itch ligase activity.

67


Effect of amifostine, a radiation-protecting drug, on oxygen

concentration of normal tissue measured by EPR oximetry

Kazunori Anzai, Atsuko Matsumoto, Ken-ichiro Matsumoto,

Sushma Manda, Megumi Ueno, and Ikuo Nakanishi

Research Center for Charged Particle Therapy, National Institute of

Radiological Sciences, Chiba 263-8555, Japan

For radiotherapy of cancer, oxygen concentration in the tumor

tissues greatly affects the outcome of the therapy. The harmful effects

of radiation on normal tissues are also modified by the oxygen

concentration. Therefore, it is important to measure tissue

oxygen concentrations in vivo. EPR oximetry is one of useful

techniques for measuring oxygen concentration in vivo. In the present

study, we examined the effect of amifostine, a powerful and

clinically approved radiation-protecting drug, on the oxygen concentration

of a normal tissue by EPR oximetry using lithium octan-butoxy-substituted

naphthalocyanine (LiNc-BuO) as a radical

probe. LiNc-BuO was synthesized according to the literature 1 .

The line width of LiNc-BuO probe in vitro linearly responded to

the oxygen concentration that was changed by using mixture of

various concentrations of oxygen and nitrogen. The micro crystals

of LiNc-BuO was inoculated in the hind leg of mice and the EPR

spectra of the LiNc-BuO were measured with a surface coil attached

to an L-band EPR spectrometer. When amifostine, a radiation-protecting

drug, was administered intraperitoneally or intravenously

to the mice, the line width of the EPR spectra of the probe

decreased more than 60 min and then recovered to a normal level.

Since radiosensitivity of cells is dependent on the oxygen concentration

and hypoxic cells are radio-resistant, the present findings

suggest that the lowering of oxygen concentration of normal tissues

might contribute a part for the radiation-protection by amifostine.

TGRL lipolysis induced gene expression in

human aortic endothelial cells

1,2 Hnin H. Aung, 3 Michael Lame, 4 K. Gohil, 2 Dennis W. Wilson,

and 1 John C. Rutledge

1 Department of Internal Medicine, Division of Endocrinology, Clinical

Nutrition, and Vascular Medicine, University of California, Davis, CA 95616;

2 Department of Pathology, Microbiology and Immunology, University of

California, Davis, CA 95616; 3 Department of Molecular Biosciences, University

of California, Davis, CA 95616; 4 Division of Pulmonary and Critical Care

Medicine, Department of Internal Medicine, University of California, Davis, CA

The endothelium plays an important role in the control of the

vascular system. Elevation of triglyceride-rich lipoproteins

(TGRL) is a known cardiovascular risk factor. In the present study

we investigated the effects of TGRL lipolysis on human aortic endothelial

cells (HAEC) focusing on intracellular pathways and

gene expression. Our previous studies indicate that TGRL lipolysis

products in high concentrations injure HAEC. We hypothesized

that TGRL lipolysis products activate activating transcription factor

3 through the Jun N-terminal kinase (JNK) signaling pathway

and contribute to vascular inflammation in pathogenesis of atherosclerosis

affecting the expression of multiple pro-inflammatory cytokines.

To test this hypothesis, we incubated HAEC with either

TGRL or TGRL lipolysis products for 3 hrs. RNA was isolated

from HAEC after a 3 hr exposure to TGRL or TGRL lipolysis

products and subjected to microarray analysis with subsequent

verification using quantitative real-time PCR. Western blotting and

immunofluorescence were used to study intracellular signaling

pathways. Our results indicate that TGRL lIpolysis products initiate

a stress response in HAEC that has the molecular signature of

inflammation with the induction of activating transcription factor 3

down stream of the JNK pathway. These results support the involvement

of TGRL lipolysis products in endothelial dysfunction

via induction of pro-inflammatory genes.

1 M. Afeworki et al., Free Radic. Biol. Med. 25, 72-78 (1998)

69

68


Fra-2 mediates oxygen-sensitive induction of transforming

growth factor in cardiac fibroblasts

Ali Azad, Savita Khanna, Rebecca Schnitt, Sashwati Roy,

Hidenori Ichijo, and Chandan K. Sen

Davis Heart and Lung Research Institute, Department of Surgery,

The Ohio State University Medical Center, Columbus, OH 43210, USA

In the ischemia-reperfused heart, transforming growth factor

beta (TGF) proteins trigger the differentiation of cardiac fibroblasts

(CF) to myofibroblasts contributing to fibrosis. Previously,

we have reported that perceived hyperoxia (Circ Res 92:264-71), a

sub-lethal reoxygenation shock during IR, induces differentiation

of CF to myofibroblasts at the infarct site. In this study, we sought

to characterize the molecular mechanisms responsible for the O 2 -

sensitive transcriptional induction of TGF in adult murine ventricular

CF and to test the significance of such findings in the infarcted

myocardium in vivo using laser capture microdissection

(LCM). We observed that among the AP_1 family of proteins specifically

Fra-2 mediates oxygen-induced TGF transcription in

adult cardiac fibroblasts. Spatially resolved infarct and non-infarct

tissues were collected at post-IR using LCM. All three isoforms of

TGF (TGF1, 2 and 3) were significantly induced in the myocyte-silent

CF-rich peri-infarct heart tissue as well as in CF exposed

to hyperoxic insult during reperfusion following ischemia.

Reporter studies demonstrated that TGF transcription is hyperoxia

inducible. Deletion of any one or both of the AP-1 binding

sites in the TGF reporter construct resulted in loss of O2-

sensitivity demonstrating that AP-1 confers O 2 -sensitivity to TGF

transcription. Fra-2 and Ask-1 were identified as key mediators of

AP-1 dependent O 2 -sensitive TGF transcription. Knock down of

Fra-2 significantly blunted expression of TGF1 in CF. Knock

down of Ask-1 blunted hyperoxia-induced expression of Fra-2

gene expression and nuclear localization. Collectively, these observations

point towards a central role of Ask-1 and Fra-2 in O 2 -

inducible AP-1 activation and induction of TGF. Taken together,

identification of Fra-2 as an O2-sensitive transcriptional regulator

of inducible TGF expression, positions Fra-2 as an important

player in reoxygenation-induced fibrosis.

Supported by NIH Grant RO1-HL-073087 to CKS

MicroRNA biogenesis complex is dysregulated in

ischemic cutaneous wounds

J. Banerjee, S-R A. Hussain, S. Biswas, Y-C Chan, *J. Jiang,

*T. Schmittgen, S. Khanna, S. Roy, and C.K Sen

Comprehensive Wound Center, Department of Surgery,

*College of Pharmacy. The Ohio State University, Columbus, OH 43210

Background. Drosha, a RNA polymerase III, enzyme, and an associated

protein called DGCR8, are the major components of a multisubunit

protein complex termed microprocessor complex. This complex

plays a critical role in the biogenesis pathway, for processing microRNA

precursor RNAs, converting primary transcripts (pri-miRNA)

to pre-miRNAs. Using conditional epidermal specific deletion of

Dicer, an essential enzyme for the biogenesis of mature miRNA, we

have recently observed that impaired epithelialization is one of the major

causes of attenuated dermal wound healing. Objective. To determine

the role of miR biogenesis pathway in impairment of epithelialization

in ischemic wounds. Results. Among miR biogenesis complex,

DGCR8 was significantly downregulated in ischemic wounds on day 3

compared to non-ischemic wounds. In order to assess the role of

DGCR8 in the epithelialization process, we used RNA interference to

deplete DGCR8 in immortalized human keratinocytes. Knockdown of

DGCR8 invoked a pronounced reduction in keratinocyte migration and

proliferation. Furthermore, miRNA profiling showed that the expression

of 19 miRNAs was downregulated relative to non-ischemic

wound while four miRNAs including miR-210 were upregulated. Recent

studies show that aquaporin 3 (AQP3) serves as facilitator of

keratinocyte migration and proliferation. We observed that compared

to non-ischemic wounds, ischemic wounds showed reduced expression

of AQP3 during the early epithelialization phase of healing. Knockdown

of DGCR8 in human keratinocyte caused marked reduction in

the expression of AQP3. Studies are currently in progress to determine

mechanisms of AQP3 downregulation by miR biogenesis complex.

Conclusions. This is the first study demonstrating that DGCR8, a

member of miR biogenesis complex, is downregulated in ischemic

wounds. Such downregulation results in attenuated keratinocyte migration

and proliferation conceivably via decreased AQP3 expression.

Supported by NIH RO1 GM-069589 and GM-077185.

70

71


Dicer ablation in adult hearts induces oxidative stress,

mitochondrial dysfunction and casues rapid loss of cardiac

function

Jaideep Banerjee, Sashwati Roy, Surya C. Gnyawali,

Savita Khanna, and Chandan K. Sen

Department of Surgery, The Ohio State University, Columbus, OH

Objective. Dicer, an RNAse III endonuclease, plays a key role

in miRNA biogenesis. The short-term effects of dicer deficiency

on the adult heart remain to be elucidated. We hypothesize

that change in the level of crucial miRNAs upon heart-specific

conditional dicer knock-out in adult animals result in rapid loss of

cardiac function caused by mitochondrial dysfunction and oxidative

stress. Methods. Double-transgenic (Myh6-cre/Esr1-Dicer fl/fl )

mice were generated. At 8 weeks of age, vehicle or tamoxifen (20

mg/kg) was injected daily for five days. Heart function was determined

using gated cardiac MRI (11.7T) and echocardiography.

Myocardial miR profiling was performed using a bead array system.

Mitochondrial function was assessed by measurement of respiratory

control ratio (RCR) and membrane depolarization. Tissue

redox status was assessed by EPR studies using nitroxide radical

probe. Results. Significant (p


Triptolide alleviates severe liver injury in mice

by attenuating the oxidative stress

Xiaofeng Bao , Yan Lu , and Pingping Shen

State Key Laboratory of Pharmaceutical Biotechnology,

School of Life Sciences, Nanjing University, Nanjing, 210093, PR China

Triptolide, a diterpene triepoxide, is one of the major components

of most functional extracts of Tripterygium wilfordii Hook

and is known to have various biological effects, including immunosuppressive,

anti-inflammatory, anti-tumor and anti-fertility

functions. We studied the inhibitory effect of triptolide on endotoxemia

(ETM)-induced liver damage in mice model. Hepatic

damage was induced in C57BL/6 mice by lipopolysaccharide

(LPS) and D-galactosamine (D-GalN). Pretreatment with triptolide

decreased the ROS levels, mortality rate and liver injury after

LPS/D-GalN injection. We utilized comprehensive proteomics to

identify alterations in liver protein expression during pretreatment

with triptolide and pretreatment with N-acetylcysteine (NAC) after

LPS/D-GalN injection, Forty four proteins were found to be related

to oxidative stress, mitochondria, metabolism and signal transduction.

Furthermore; Both triptolide and NAC inhibited activation of

JNK and p38, phosphorylation of IB and activation of NF-B.

These results demonstrated that triptolide inhibits the activation of

JNK and p38 by decreasing ROS levels, which in turn inhibits the

hepatic injury. In addition, triptolide prolonged ERK activation,

indicating that ERK had a protective role in LPS/D-GalN-induced

liver damage and triptolide could inhibit the hepatic injury by prolonging

ERK activation. On the contrary, NAC had no effect on

ERK activity. These results show that triptolide could effectively

cure severe liver injury by reducing the oxidative stress in the liver,

thus suggesting its use as an inducer of antioxidative defense system

for treating severe liver injury.

74

Enantioselective synthesis and antioxidant activity of

3-(3,4-dihydroxyphenyl)-glyceric acid – basic monomeric

moiety of a biologically active polyether from

Symphytum asperum and S. caucasicum

Vakhtang Barbakadze 1 , Maia Merlani 1 , Lela Amiranashvili 1 ,

Lali Gogilashvili 1 , Karen Mulkijanyan 1 , Elina Yannakopoulou 2 ,

Kyriakos Papadopoulos 2 , and Bezhan Chankvetadze 3

1 Kutateladze Institute of Pharmacochemistry, 36 P. Sarajishvili str.,

0159 Tbilisi, Georgia; 2 NCSR ‘Demokritos’, Institute of Physical Chemistry,

15310 Athens, Greece; 3 Department of Physical and Analytical Chemistry and

Molecular Recognition and Separation Science Laboratory, School of Exact and

Natural Sciences, Tbilisi State University, Chavchavadze Ave 3, 0179 Tbilisi,

Georgia

The racemic and enantioselective synthesis of a novel glyceric

acid derivative, namely, 2,3-dihydroxy-3-(3,4-dihydroxyphenyl)-

propionic acid was carried out and their antioxidant activities was

determined. The virtually pure enantiomers, (+)-(2R,3S)-2,3-

dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid and (–)-(2S,3

R)-2,3-dihydroxy-3-(3,4-dihydroxyphenyl)-propionic acid were

synthesized for the first time via Sharpless asymmetric dihydroxylation

of trans-caffeic acid derivatives using the enantiocomplementary

catalysts, 1,4-bis(9-O-dihydroquinine)-phthalazine [(DH

Q) 2 -PHAL] and 1,4-bis(9-O-dihydroquinidine)-phthalazine [(DH

QD) 2 -PHAL], respectively. The determination of enantiomeric purity

of the novel chiral glyceric acid derivatives was performed by

high-performance liquid chromatographic (HPLC) techniques on

the stage of their alkylated precursors. It was shown that the novel

racemic glyceric acid derivative as well as its enantiomeric pure

derivatives exhibit strong antioxidant activities against reactive

oxygen species, such as hypochlorite or free radicals such as

DPPH. No significant differences have been noted regarding the

antioxidant activities of pure enantiomers and the racemic monomer.

Their antioxidant activity is about 40-fold higher than that of

the corresponding natural polyether and 3-fold higher of transcaffeic

acid itself. Such pronounced antioxidant properties give

appropriate background for further deep investigation of the biological

activity of this phenolic compound, and it seems promising

to use it either as food additive or medicinal preparation for treatment

of oxidative disorder-related diseases.

75


Allantoin- and pyrrolizidine alkaloids-free wound healing

compositions from Caucasian species of comfrey (Symphytum L.)

Vakhtang Barbakadze, Karen Mulkijanyan, Lali Gogilashvili,

Lela Amiranashvili, Maia Merlani, Zhana Novikova,

Marine Sulakvelidze

Kutateladze Institute of Pharmacochemistry, 36 P. Sarajishvili str.,

0159 Tbilisi, Georgia

Extracts from the Caucasian species of comfrey – Symphytum

asperum and S.caucasicum have been used in folk medicine in the

treatment of fractures and wounds. Extracts contain allantoin,

claimed to be a cell proliferation-stimulating agent responsible for

the wound-healing properties of Symphytum, and hepatotoxic pyrrolizidine

alkaloids which strongly restrict internal use of comfrey

extracts. In the present study we obtained allantoin- and pyrrolizidine

alkaloids-free composition containing crude polysaccharides

and novel biopolymer from S. asperum and S.caucasicum roots –

poly[3-(3,4-dihydroxyphenyl)glyceric acid] (PDPGA), and attempted

to appraise its pharmacological properties in vitro (anticomplementary

and antioxidant) assays and in vivo (mouse excisional

wound and skin burn models). In contrast with polysaccharides

PDPGA exhibited marked antioxidant and anticomplementary

activity, Besides, ointment, containing 2.5% crude polysaccharides

and PDPGA (C) was found to have pronounced wound

healing properties, similar to 2.5% allantoin ointment (A) in excisional

wound model. Secondly, we attempted to evaluate pharmacological

efficacy of ointment (C) in comparison with ointment

(A) in skin burn model. Ointment (C) appeared to be approximately

fourfold active than ointment (A) and twice as active as

combined ointment (CA) containing 1.25% of high-molecular fraction

from comfrey roots and 1.25% of allantoin when treatment of

animals began immediately after burn induction. Significantly

lower (p < 0.01) values of burn area in (C)- than in (A)- and (CA)-

treated animals were observed since day 2 till the end of the experiment.

The obtained results allow to suggest that the wound

healing effect of the investigated compositions results from synergistic

action of its constituents in the second phase of wound healing,

the inflammatory response.

Dynamic regulation of signaling at regions of cell-cell contact

by endoplasmic reticulum-bound protein-tyrosine phosphatase 1B

Ahmed Bettaieb 1 , Ali Kinkhabwala 2 , Carsten Schultz 3 ,

Benjamin Neel 4 , Philippe Bastiaens 2 , and Fawaz Haj 1

1

Department of Nutrition, University of California, Davis, CA 95616, U.S.A.

fghaj@ucdavis.edu, 2 Max Planck Institute of Molecular Physiology,

Otto-Hahn-Str. 11, 44227 Dortmund, Germany, 3 European Molecular Biology

Laboratory, Heidelberg, Germany, 4 Ontario Cancer Institute, Toronto, Ontario,

M5G 1L7, Canada

Tyrosine phosphorylation is critical for signal transduction and

maintaining homeostasis. This process is mediated through the opposing

actions of protein-tyrosine kinases and protein-tyrosine

phosphatases (PTPs). Protein-tyrosine phosphatase 1B (PTP1B) is

a widely expressed PTP that is localized on intracellular membranes

via a hydrophobic C-terminal targeting sequence. A role of

PTP1B in the regulation of many cellular functions has been suggested,

including growth factors, integrin and cadherin signaling.

PTP1B activity can be regulated through a variety of mechanisms

namely cellular localization, oxidation and sumoylation. Previously,

we and others showed that PTP1B dephosphorylates RTKs

such as the epidermal growth factor receptor (EGFR) and insulin

receptor (IR) after receptor endocytosis, as they transit past the endoplasmic

reticulum (ER). However, other studies suggest that

PTP1B can access some plasma membrane-bound substrates. To

delineate the mechanism of PTP1B interaction with such substrates,

we utilized advanced quantitative cellular imaging such as

photoactivation and fluorescence lifetime imaging microscopy in

combination with substrate-trapping and mathematical modeling

approaches. In addition, we developed activity sensors to image

regulation of PTP1B activation in live cells. We report that the ER

extends outwards to lie in close proximity to the PM only at apparently

specialized regions of cell-cell contact, enabling PTP1B to

dephosphorylate key substrates at these sites. Our studies show that

ER-anchored PTP1B plays a dynamic role in regulating signaling

at regions of cell-cell contact and identify PM-proximal subregions

of the ER as an important site for regulating cellular signaling.

76

77


Light distribution on the human retina over extended periods:

Implications for photooxidative damage

R.A. Bone and J.C. Gibert

Department of Physics, Florida International University, Miami, FL 33199

Photoxidative damage has been implicated in the etiology of

age-related macular degeneration (AMD). The purpose of our

study was to measure cumulative light distribution on the human

retina over extended periods. Our hypothesis was that such distributions

would tend to peak in the macula where AMD damage is

most pronounced. Using a head-mounted eye-tracker, we captured

the subject's field of view with a video camera (60 frames/s), superimposed

the gaze position on each frame, and continuously recorded

pupil size. Of fifteen subjects (ages 18-65) who participated,

five, who were familiar with the study, were assigned to a

control group; ten, who were naïve, formed a test group. In phase

1, subjects viewed a repeating sequence of photographic images

projected on a screen. In phase 2, they were seated in front of a

monitor and performed arbitrary computer tasks, and in phase 3,

they moved freely around the lab and exterior of the building. The

control group was specifically instructed to gaze at bright features

in the field of view and, in a second test, at dark features. The rest

of the participants were allowed to gaze freely. Based on the subject’s

gaze position and pupil size, the relative cumulative light

distribution over 5 to 15 min periods was calculated for a 20 o (H)

14 o (V) area of the retina centered on the fovea. The data were

presented as a plot of relative intensity of light versus retinal position.

For the control group, cumulative retinal light distributions

peaked and dipped in the fovea for the tests during which subjects

were instructed to gaze at bright or dark features respectively in the

field of view. The light distributions obtained from the test group

in phase 2, but not in phases 1 and 3, showed a tendency to peak in

the macula. In conclusion, our data so far do not confirm our hypothesis

that the macula receives more light than the periphery under

general viewing conditions. However, the specific task of

working in front of a computer monitor exposed the retina to a

higher illuminance in the macular region. This could result in increased

photooxidative damage with implications for development

of AMD.

78

Endothelial and inducible NO synthases expression in the

lungs of the developing mouse after prenatal administration of

vitamin A

Raquel Carvalho 1 , Maria de Lurdes Pinto 1 , Ana Cláudia Coelho 1 ,

Carlos Gonçalves 2 , and Vasco António Bairos 2

1 Department of Veterinary Sciences and Centre for Studies on Agricultural and

Veterinary Sciences, University of Trás-os-Montes e Alto Douro, 5001-801 Vila

Real, Portugal; 2 Institute of Histology and Embryology, Faculty of Medicine,

University of Coimbra, 3004-504 Coimbra, Portugal

Nitric oxide (NO) is a potent vasodilator shown to play a critical

role in various functions. Recent studies relate the defective lung vascular

development seen in the lungs of animals with respiratory distress

syndrome to eNOS deficiency. Inducible NOS is highly expressed

in late stages of lung fetal development. Vitamin A is crucial

for normal cellular differentiation during fetal development. Our previous

studies showed that maternal administration of vitamin A results

in an enhancement of lung fetal organogenesis, and an increase in vascular

endothelial growth factor (VEGF) pulmonary and plasma levels.

A decrease of eNOS activity, associated to a decrease in VEGF expression

was reported by other authors. It has also been suggested that

NO up-regulates VEGF expression. In order to better understand the

modulating action exerted by the vitamin A upon pulmonary eNOS

and iNOS we have conducted an in vivo study in fetal and neonatal

mice. The lungs were collected daily from the 15th gestational day till

the 3th day of life and processed for routine immunohistochemistry. At

the 16th day of gestation there is a markedly increase of eNOS

expression, which does not occur in any other day under study.

Inversely, at this gestaional day, a decrease in iNOS expression is

observed. Prenatal administration of vitamin A did not alter signicantly

eNOS or iNOS expression, however, there is a trend for an increase of

eNOS expression in the lungs of animals subjected to vitamin A

supplementation. Regarding eNOS expression it is important to notice

that at the 16th day of gestation, the lung undergoes marked vasculogenesis

and angiogenesis, in which eNOS seems to play an essential

role. The fact that vitamin A further stimulates eNOS expression

without altering iNOS could be important to lung’s vascular development,

suggesting a bennefical role of vitamin A in lung maturation by

directly regulating angiogenic factors, such as VEGF and eNOS

expression.

79


Antioxidant enzymes gender differences during mice fetal and

neonatal lung development

Raquel Carvalho, Maria de Lurdes Pinto 1 , Ana Cláudia Coelho 1 ,

Dario Santos 2 , Francisco Peixoto 2 , Carlos Gonçalves 3 , and

Vasco António Bairos 3

1 Department of Veterinary Sciences and Centre for Studies on Agricultural and

Veterinary Sciences, University of Trás-os-Montes e Alto Douro, 5001-801 Vila

Real, Portugal ; 2 Department of Biology, University of Trás-os-Montes e Alto

Douro, 5001-801 Vila Real, Portugal; 3 Institute of Histology and Embryology,

Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal

Sexual dimorphism of the adult lung has recently been described

in rodents. Female mice and rats have smaller, and per body mass,

more alveoli and alveolar surface area than males of their respective

species. These differences are regulated by estrogen receptors and

become apparent by the time of puberty. In humans, respiratory function

of very prematurely born infants is influenced by sex, being

characterized by a female advantage. Since the surfactant system and

the antioxidant enzyme system of the fetal lung have chronologically

similar developmental patterns and given the need of prolonged exposure

to oxygen in male premature infants, we investigated if a sex

difference would be present in antioxidant enzymes of the developing

mice lung. Mice lungs were collected daily from the 15th until the

3th day of life. Catalase, glutathione peroxidase, and superoxide dismutase

were measured by the polarographic method and spectrometry,

respectively. Catalase and glutathione peroxidase are more abundant

in the developing lung, whereas superoxide dismutase is unnoticeable

in all the days studied. Catalase and glutathione peroxidase

are higher near the time of birth and the first days of life. Surprisingly,

a higher expression of catalase and glutathione peroxidase is

registered in males. Regarding catalase, the difference between males

and females reached statistical significance at the 16 th gestational

day, the day of birth and the 3 rd day of life. Glutathione peroxidase

levels are significantly higher in the male’s lung at the 15 th gestational

day and at the 2 nd day of life. Whether these gender differences

in lung’s antixodant enzymes arise from a direct stimulation of their

synthesis or by higher levels of reactive oxygen species in the male’s

lung is yet to be investigated. However, gender differences in lung’s

antioxidant enzymes can cause significant changes in redox balance,

which are frequently associated with lung dysfunction.

Evaluation of the in vivo photoprotective effect and the in vitro

antioxidant activity of topical formulations containing

quercetin

Rubia Casagrande 1,2 ; Sandra R. Georgetti 1,2 ;

Marcela M. Baracat 1,2 , Waldiceu A. Verri Jr 3 ;

Fabiana T.M.C. Vicentini 2 , and Maria José Vieira Fonseca 2

1 Department of Pharmaceutical Science, State University of Londrina, Londrina,

PR; 2 Department of Pharmaceutical Science, Faculty of Pharmaceutical

Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP;

3 Department of Pathology, State University of Londrina, Londrina, PR;

The skin is continuously exposed to a combination of environmental

insults including UV radiation and high oxygen concentration,

which constantly jeopardizes the integrity of cellular oxidizable

structures of the skin. In this regard, antioxidants from natural

products such as quercetin (QC) present novel possibilities for

the treatment and prevention of oxidative stress-mediated skin diseases.

Thus, the development of topical formulations added QC as

well as the correct evaluation of their in vitro and in vivo antioxidant

activities would be very helpful to choose the most adequate

one. Therefore, in the present study it was evaluated in vitro antioxidant

activities of different topical formulations (non-ionic

emulsion with high lipid content and anionic emulsion with low

lipid content) added QC and the in vivo photoprotective effect of

these formulations. In vitro antioxidant activity was evaluated by

ability to inhibit lipid peroxidation, H-donor capability and scavenging

hydroxyl radical effect. The skin oxidative stress was induced

in hairless mice by UVB irradiation and the protective effect

of QC was determinate by cutaneous proteinase secretion/activity

using sodium dodecyl sulphate polyacrylamide gel electrophoresis

substrate-embedded enzymography. Lipid peroxidation inhibition

and DPPH • scavenging assays could be successfully applied in

quality control for the antioxidant activity evaluation QC formulations,

nevertheless, the deoxyribose test was not suitable because

of excipient interference. The dose of 1.23 J/cm 2 induced increase

in the proteinase secretion/activity, which was inhibited by QC

formulations. These results suggest QC as a promising drug for the

treatment of photooxidative skin damages.

80

81


The Tephrosia toxicaria extract inhibits lipid hydroperoxides

formation

Rubia Casagrande 1,2 , Vanessa V.M. Zimermann 1 ,

Renata M. Martinez 1 , Joice Sartorato Sanson 1 , César C. Andrei 3 ,

Isabel C. Moreira 4 , Sandra R. Georgetti 1,2 , Marcela M. Baracat 1,2

1 Department of Pharmaceutical Science, State University of Londrina, Londrina,

PR; 2 Department of Pharmaceutical Science, Faculty of Pharmaceutical

Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP;

3 Department of Chemical, State University of Londrina, Londrina, PR; 4 Federal

Technological University of Parana, COALM/UTFPR, Londrina, PR

The specie Tephrosia toxicaria is a source of flavonoids, which

are known to be antioxidants. However, there are few studies concerning

the antioxidant mechanisms of T. toxicaria. Therefore, it

was evaluated the whether the hexanic fraction (HF) and methanolic

fraction (MF) of T. toxicaria extract inhibits lipid peroxidation

(lipid hydroperoxide formation) by a mechanism independent of

iron as well as the precision of the method. An emulsion of linoleic

acid 10 mM was prepared in tween 20. In 2 ml of emulsion it was

added 20 μl of different concentrations of the samples (extract

fractions). Lipid hydroperoxides were determined before and after

the incubation of the emulsion with samples at 37ºC for 8 h and

readings at 234 nm. The precision was evaluated during 3 days.

The inhibition of lipid hydroperoxides formation by T. toxicaria

was concentration dependent for both fractions. The linearity was

2–40 and 2–20 μg/ml, and IC 50 35,97 and 8,53 μg/ml for HF and

MF, respectively. The inter-day precisions presented the relative

standard deviation value near 10%. These results demonstrate that

both fractions of the extract inhibited the lipid peroxidation and

that the MF present greater antioxidant activity than the HF in this

assay. These data suggest the use of T. toxicaria extracts are promising

sources of antioxidants against oxidative stress.

Hypoxia-sensitive miR-200b regulates angiogenic response of

endothelial cells by targeting Ets-1

Yuk-Cheung Chan, Savita Khanna, Sashwati Roy, and

Chandan K. Sen

Davis Heart & Lung Research Institute, Departments of Surgery,

The Ohio State University Medical Center, Columbus, Ohio, 43210

MicroRNAs (miR) are small RNAs involving in epigenetic

regulation of gene expression. MiR-200b plays a crucial role in

epithelial to mesenchymal transition in various cancer cells.

However, its role in other tumorigenic response such as angiogenesis

remains ambiguous. Here we report first evidence that

endothelial miR-200b responds to hypoxic environment and

regulates angiogenesis via v-ets erythroblastosis virus E26

oncogene homolog 1 (Ets-1). Exposure of human microvascular

endothelial cells (HMECs) to hypoxia or stabilization of HIF1

suppressed miR-200b expression. Over-expression of miR-200b in

HMECs inhibited in vitro angiogenesis, which was accompanied

by down-regulation of matrix-metalloproteinase-1 (MMP-1) and

vascular endothelial growth factor receptor 2 (VEGFR2). In silico

studies revealed that the 3’ untranslated region of Ets-1, a crucial

transcription factor in MMP-1 and VEGFR2 expression, contains

two binding sites for miR-200b. Interestingly, miR-200b overexpression

significantly inhibited both mRNA and protein level of

Ets-1. Knock-down of Ets-1 alone mimicked the impaired angiogenic

response that was observed in miR-200b overexpressed

HMECs. This work provides first evidence demonstrating that hypoxia-induced

down-regulation of miR-200b de-repress Ets-1 expression

in endothelial cells, thus further promoting angiogenesis

under low oxygen environment.

Supported by NIH GM069589 and GM077185.

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ESR spectra of black and red hair and the effect of

vitamin C (ascorbic acid) on the photoinduced free radicals

in the hair

Eduard N. Chikvaidze 1 , Temur V. Gogoladze 2 ,

Alexander Miminoshvili 3 , and Inga Khachatryan 1

1 Department of Exact and Natural Sciences, Iv. Javakhishvili Tbilisi State

University, Tbilisi, Georgia, 2 Tbilisi State Medical University, Tbilisi, Georgia,

3 Sukhumi State University, Tbilisi, Georgia

ESR spectra of melanin’s free radicals in black and red hair

have been investigated. It is shown that the ESR spectrum of black

hair is slightly asymmetric singlet with g=2,003 and H=0,5 mTl.

The ESR spectrum of red hair is different from the spectrum of

black hair and represents the superposition of the singlet of black

hair and triplet. Under the action of visible light (blue with max =

450 nm, green with max = 510 nm, and red with max = 650 nm)

both in black and red hair appear photoinduced free radicals, which

is observed in the increase of the intensity of the ESR spectrum of

those already existing in the hair.

It should be noted that the ESR spectra of red hair from various

donors is different. The antioxidant vitamin C has the different effects

on the ESR spectra of hair. In particular, in the case with

black hair the concentration of photoinduced free radicals is reduced,

whereas in red hair the disappearance of the triplet spectrum

is observed and at the same time the spectrum becomes a singlet

with the ESR parameters of black hair and the intensity of the

spectrum increases sharply.

It is assumed, that antioxidants effective for black hair, may be

ineffective for red hair, and vice versa. Therefore, in each specific

case is necessary to investigate separately the effectiveness of an

antioxidant.

Hypertension is associated with oxidative stress and

exaggerated renal angiotensin II AT1 receptor and diminished

dopamine D1 receptor function in old rats

Gaurav Chugh, Mustafa F. Lokhandwala, and Mohammad Asghar

Heart and Kidney Institute, College of Pharmacy, University of Houston,

Houston, Texas 77204

The process of aging alters physiological functions including

sodium homeostasis in the kidney and is associated with an increase

in blood pressure. Sodium homeostasis and thus blood pressure

is maintained by the balance between angiotensin II type I

(AT1) receptor and dopamine D1 receptor function in the renal

proximal tubules. Although the underlying mechanism for ageassociated

increase in blood pressure is not known, we hypothesized

that an imbalance in renal AT1 receptor and D1 receptor

function during aging would favor elevated blood pressure. Renal

hemodynamic, biochemical, molecular biology and western blotting

methodologies were used in the study. Aged (21-month)

Fischer 344 x Brown Norway rats had higher blood pressure compared

to their younger (3-month) counterparts. The natriuretic effects

of AT1 receptor antagonist, candesartan, were potentiated

whereas those of D1 receptor agonist, SKF 38393, were diminished

in aged rats. The levels of mRNAs of AT1 receptor were increased

and those of D1 receptor were decreased in the renal

proximal tubules of aged compared to younger rats. Markers of

oxidative stress (protein carbonyls and 8-isoprostane) and stressactivated

transcription factors NF-B and Sp3 were up-regulated in

the renal proximal tubules of aged rats.

These results document age-associated oxidative stress in the

renal proximal tubules that involves NF-B and Sp3, and an exaggerated

AT1 receptor and diminished D1 receptor function. These

age-related changes may provide an underlying mechanism for altered

sodium homeostasis and higher blood pressure in aging.

Financial support provided by NIH/NIA AG25056 and AG29904.

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85


Improved function of diabetic wound-site macrophages and

accelerated wound closure in response to oral supplementation

of a fermented papaya preparation

Eric Collard and Sashwati Roy

Comprehensive Wound Center, Department of Surgery, Davis Heart and Lung

Research Institute, The Ohio State University Medical Center,

Columbus, OH 43210, USA

Carica papaya Linn is widely known as a medicinal fruit. To

determine its role in wound healing, we investigated the effect of

standardized fermented papaya preparation (FPP) in adult obese

diabetic (db/db) mice. Oral FPP (0.2g/kg for eight weeks) reduced

blood glucose elevations and improved lipid profile. FPP did not

affect weight gain during the eight week supplementation period.

FPP supplementation, prior to wounding, improved wound closure.

Reactive oxygen species support numerous aspects of wound healing,

and NO generation is known to be compromised in diabetic

wounds. Studies on viable macrophages isolated from the wound

site demonstrated that FPP supplementation enhanced respiratory

burst function, as well as inducible NO production. Furthermore,

diabetic mice supplemented with FPP showed elevated CD68 and

CD31 expression at the wound site, suggesting effective recruitment

of monocytes and an enhanced pro-angiogenic response.

These data provide the first evidence that diabetic wound outcomes

may benefit from FPP supplementation by specifically influencing

the response of wound-site macrophages and subsequent angiogenic

response. The challenge of wound healing in diabetes, and

FPP’s long track-record of safe human consumption warrants testing

of FPP in a clinical setting.

Supported by DK 076566 to SR and in part by a funding from Osato Research Institute,

Japan

gp91 phox produced superoxide radical in classical activated

microglia and contributes to the severity of traumatic brain

injury in mouse

Kenji Dohi 1 , Seiji Shioda 2 , Hirokazu Ohtaki 2 ,

Tomoya Nakamachi 2 , Sachiko Yofu 2 , Kazue Satoh 2 ,

Yuko Mihara 1 , Kazuyuki Miyamoto 1 ,

Shohko Tsunawaki 3 , and Tohru Aruga 1

1 Department of Emergency and Critical Care Medicine, 2 Department of

Anatomy, Showa University School of Medicine, Tokyo, Japan

3Department of Infectious Diseases, National Research Institute for Child

Health and Development, Tokyo, Japan

Traumatic brain injury (TBI) is associated with reactive oxygen

species (ROS), and one of the major enzymatic sources of superoxide

anion production in the brain is NADPH oxidase. gp91 phox

(NOX2), one of the catalytic subunit of NADPH oxidase enzymes

(NOX-es), plays as a key enzyme in various neuronal diseases. We

hypothesized that gp91 phox generates superoxide anion is a major

causative role in TBI. Using gp91 phox knockout (gp91 phox-/- ) and

wild-type (gp91 phox +/+) mice (C57BL), unilateral controlled cortical

impact traumatic brain injury was induced. The expressions and

the roles of gp91 phox after TBI were investigated. We also studied

the contributions of gp91 phox to superoxide anion generation after

TBI using in situ detection of oxidized HEt. In wild type mice,

gp91 phox activated 24 h and 48 h after TBI. gp91 phox expressed

mainly in classically activated microglia like cells and weakly in

astrocyte and neuron after TBI. The contusion area and TUNEL

positive apoptotic like cells were suppressed in gp91 phox-/- mice.

TBI increased peroxynitrite (ONOO - ) expression and superoxide

radical (O 2 - ) production (HEt stains cells) in gp91 phox immunopositive

cells. Gp91 phox containing NADPH oxidase expressed in classically

activated like microglia promotes ROS formation and has

an important roles of brain damages after TBI. Modulations of

gp91 phox -containing NADPH oxidase may provide a new therapeutic

strategy to traumatic brain injury.

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87


Oxidant and antioxidant balances during brain

hypothermia therapy in patients with traumatic brain injury

Kenji Dohi 1 , Kazue Satoh 2 , Kazuyuki Miyamoto 1 ,

Shunsuke Nakamura 1 , Seiji Shioda 2 and Tohru Aruga 1

1 Department of Emergency and Critical Care Medicine, School of Medicine,

Showa University, Tokyo, Japan

2 Department of Anatomy, School of medicine, Showa University, Tokyo, Japan

Oxidative stress has important roles in traumatic brain injury

(TBI). The control of free radical production is one of the targets in

the treatment for prevention from brain damage. Brain hypothermia

therapy (BHT) has neuroprotective effect and is thought to

have the effect on the control of free radical production. On the

other hand, the kinetic of free radical during BHT remains unclear.

The aim of this study is to investigate of the kinetic of free radical

during BHT.

Thirteen severe TBI patients are enrolled. 9 patients were

treated at 33°C (BHT group). Other patients were controlled at

35°C (normothermia group). Reactive oxygen metabolites (ROMs)

were measured in jugular blood samples by the diacron reactive

oxygen metabolites (d-ROM) test. Bio antioxidant potency (BAP)

was measured by the BAP test.

ROM level of BHT group at 33°C was lower than that of normothermia

group (35°C). ROM level during hypothermic phase

was suppressed significantly in BHT group. On the other hand,

BAP level in BHT group was suppressed during hypothermic

phase. ROM levels were increased and BAP levels recovered

slowly along the rewarming.

We demonstrated that BHT suppressed free radical production

in clinical settings. However, BHT also led to suppression of antioxidant

potential. These data supported that the imbalance of oxidant

and antioxidant balance exists in rewarming phase. Supporting

therapies of antioxidant potential in rewarming phase in BHT

may settle some troubles during rewarming phase in BHT.

Indiscriminate ("preventive") supplementation of vitamin E

Yedidya Dotan, Ilya Pinchuk Moshe Leshno and Dov Lichtenberg

Department of Physiology and Pharmacology, Sackler Faculty of Medicine,

Tel-Aviv University, Tel Aviv Israel 69978

For many years, the prevailing concept was that LDL oxidation

plays the central role in atherogenesis. As a consequence, supplementation

of vitamin E, became very popular. Unfortunately,

the major randomized clinical trials yielded disappointing results

and two independent meta-analyses concluded that vitamin E supplementation

increases mortality. This conclusion raised much

criticism, most of it relating to the choice of clinical trials included

in the meta-analyses; the end point (only mortality) and the heterogeneity

of the analyzed clinical trials with respect to both population

and treatment.

In an attempt to bring this controversy to an end, we applied a

Markov Model approach, which is free of most of the limitations

of metaanalysis, to compare vitamin E supplemented cohorts with

non supplemented cohorts. The difference between the two virtual

cohorts was expressed in terms of the composite end point denoted

quality-adjusted life years (QALY).

The main result was that the vitamin E-supplemented virtual

cohort had 0.30 QALY (95% CI 0.21 to 0.39) less than the untreated

virtual cohort. Based on the latter result, we conclude that

indiscriminate supplementation of high dose vitamin E can not be

recommended to the general public. We do not know the mechanisms

responsible for the harmful effects of vitamin E, but recent

studies show that the outcome of vitamin E supplementation may

be negative on the cellular and molecular levels.

Several recent studies, conducted with specific groups of patients,

demonstrated positive outcome. In other words, vitamin E is

a “double-edge sword” that should not be consumed indiscriminately.

Carefully-conducted randomized clinical trials with long

follow-up and well-defined end points must be conducted to define

criteria capable of predicting who is likely to benefit from supplementation

of vitamin E.

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89


Topical application of vitamin E -tocotrienol in a porcine

burn model promotes nerve regeneration and

improves wound healing

J. Driggs, S. Biswas, S. Khanna, S. Roy and C.K. Sen

Comp. Wound Cntr., Dpt. of Surg., Davis Heart & Lung Res. Inst.,

Ohio State University Medical Center, Columbus, Ohio

Scar formation from a full thickness thermal injury (burn) results

in a rough scar devoid of rete ridge formation, poor vasculature

and causes neuropathy due to nerve degeneration. We have recently

reported on neuroprotective properties of -tocotrienol

(TCT) and now aim to test that neuroprotectivity in a porcine burn

model to demonstrate its efficacy. Domestic pigs (n=7) were anesthetized

and clipped for dorsal treatment. One side of the dorsum

received 400 mg of control ointment via careful rubbing, while the

other received 400 mg of TCT. The pigs were rubbed for four subsequent

days, given a two day break then burned with 2.5 cm 2

tungsten rods. HPLC analysis confirmed TCT was present on the

treated side. TCT treated samples expressed a faster rate of leukocyte

infiltration over 28 days, consistent with literature of denervated

wounds slowing chemotaxis. TCT-treated wounds saw a 2.5

fold (p


Mimetic peroxidase activities of uniformed platinum

nanoparticles-apoferritin nanoreactor

Jia Fan, Dongxue Xu, Li Xu, Jing-yan Wei, Yuliang Zhao,

and Guangjun Nie*

CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, National

Center for Nanoscience and Technology of China, Beijing, China; 11

Beiyijie, Zhongguancun, Beijing, China 100190 niegj@nanoctr.cn;

Biomimetic enzymatic activities of nanostructures have been

reported recently. Novel and superb catalytic activities have attributed

to their unique size dimensions and enhanced surface area and

the intrinsic properties of nanostructure compositions. We report

that apoferritin (apoFt) was used to template the synthesis of 1 to 2

nm of biocompatible, non-toxic and stable Pt-nanoclusters (PtapoFt)

with unique and intrinsic peroxidase activities. The PtapoFt

showed both catalase and HRP activities with distinctive enzymatic

properties, namely different response to pH and temperature.

For the catalase activities, the Pt-apoFt showed significant increase

in enzymatic activities with increasing pH and temperature,

two unique features not seen in both enzymes and other nanostructures.

For the HRP-like activities, physiological temperature and

slightly acidic conditions are for optimal HRP activities. These results

firstly demonstrate that a Pt nanocluster possess differential

unique enzymatic activities for different substrates under different

conditions. These nanostructures hold great promise for a wide

range of new applications in biomedicine and environmental science.

H 2 O 2 -induced mitochondrial fission in C 2 C 12 myocytes

Xiying Fan, Rajaa Hussien, and George A. Brooks

Exercise Physiology Laboratory, Department of Integrative Biology,

University of California, Berkeley CA 94720-3140

In skeletal muscle and many other cell types, mitochondria exist

as an elaborate and dynamic network. The balance of continuous

mitochondrial fission and fusion define the morphology of the

mitochondrial reticulum. Environmental stimuli, such as oxidative

stress, can influence fusion and fission rates, resulting in a transformation

of the network’s connectivity. Using confocal laser

scanning microscopy of C 2 C 12 mouse myocytes, we show that

acute exposure to the reactive oxygen species (ROS) hydrogen

peroxide (H 2 O 2 ) induces a slow fragmentation of the mitochondrial

reticulum that is reversible over 24 hours. Although H 2 O 2 rapidly

decomposes in culture medium, the full extent of fragmentation

occurs 5-6 hours post-treatment, suggesting that H 2 O 2 affects mitochondrial

morphology by modulating cellular physiology or activating

redox-sensitive signaling. Supraphysiological (>1 mM)

concentrations of H 2 O 2 are cytotoxic, but lower concentrations

(250 mM) sufficient to induce transient fragmentation do not lower

cell viability. The extent of H 2 O 2 -induced mitochondrial fragmentation

correlates with decreases in inner mitochondrial membrane

potential and maximal respiratory rate, suggesting a possible

mechanism. Because H 2 O 2 is produced in contracting muscle, our

results raise the possibility that ROS generation may contribute to

exercise-induced changes in mitochondrial morphology in vivo.

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93


Anthocyanin metabolites inhibit viability of Caco-2 cells

Sarah C. Forester 1 , Andrew L. Waterhouse 1 , and

Patricia I. Oteiza 2

Department of Viticulture and Enology 1 , and Departments of Nutrition and

Environmental Toxicology 2 University of California, Davis

Anthocyanins are a class of polyphenols abundant in the skins

of red grapes. Gut microflora metabolize anthocyanins to primarily

phenolic acids and aldehydes. These metabolites may explain

the relationship between anthocyanin consumption and reduced incidence

of colon cancer. Previously, gallic acid, 3-O-methyl-gallic

acid, and 2,4,6-trihydroxybenzaldehyde were found to decrease

Caco-2 cell viability more than other anthocyanin metabolites. To

investigate the underlying mechanisms, we have now investigated

the capacity of these three anthocyanin meta-bolites to modulate

the cell cycle, and to induce apoptotic cell death. Caco-2 cells

were incubated for 24-72 h in the presence of 10 to 100 μM gallic

acid, 3-O-methylgallic acid, 2,4,6-trihydroxy-benzaldehyde, and

malvidin-3-glucoside. Malvidin-3-glucoside did not affect cell viability

within in the range of concentrations tested. The aldehyde

only reduced cell viability at the highest treatment concentration

(100 μM). Both gallic acid and 3-O-methylgallic acid induced a

time-dependent decrease in cell viability. After 72 h incubation,

the three metabolites caused an arrest in the G 0 /G 1 phase of the cell

cycle, which would lead to a decrease in cell proliferation. Apoptosis

was monitored as caspase-3 activation and DNA fragmentation.

The three metabolites, at a concentration of 50 μM, activated

caspase-3 (evaluated as PARP cleavage and enzyme activity).

However, only gallic acid and 3-O-methylgallic acid increased the

levels of mono- and oligonucleosomes within the cytoplasm and

caused apoptotic nuclear morphologic changes (Hoechst staining).

3-O-methylgallic acid and gallic acid led to a decrease (67.8 % and

47.0 %, respectively) in NF-B-DNA binding. In conclusion, the

decreased Caco-2 cell viability caused by 3-O-methyl-gallic acid

and gallic acid can occur as a cones-quence of both, the induction

of apoptosis and inhibition of cell proliferation. The inhibition of

transcription factor NF-B, which promotes cell proliferation and

survival, can underlie the observed effects.

Genistein decreases CYP1A1 via inhibiting the bindings of the

arylhydrocarbon receptor nuclear translocator and estrogen

receptor alpha to the xenobiotic response element

Erik B. Froyen 1 and Francene M. Steinberg 1

1 Department of Nutrition, University of California, Davis, Davis, CA 95616

The consumption of soy protein and the associated isoflavones

(genistein and daidzein) have been linked with a decreased risk for

certain cancers. One proposed mechanism for cancer prevention is

through decreasing the phase I cytochrome P450 1A1 (CYP1A1)

enzyme. This study evaluated the hypotheses that dietary soy

isoflavones, genistein and daidzein, will 1) decrease hepatic

CYP1A1 enzyme activity and protein in male and female Swiss

Webster mice; 2) decrease mRNA in mouse liver cells (Hepa-

1c1c7); and 3) inhibit the bindings of the arylhydrocarbon receptor

(AhR), AhR nuclear translocator (ARNT), and estrogen receptor

alpha (ER) to the CYP1A1 xenobiotic response element (XRE).

The mice were fed for 1, 3, 5, or 7 d of one of four treatments: control

(casein AIN-93G), or control supplemented with 1500 mg/kg

diet of flavone (positive control), genistein, or daidzein (all aglycones).

To investigate the molecular mechanisms associated with

CYP1A1 modulation, Hepa-1c1c7 cells were treated with control

[0.1% (v:v) DMSO], 1 mol/L -naphthoflavone, or 5 mol/L genistein,

daidzein, or the daidzein metabolite equol, followed by

TaqMan ® and chromatin immunoprecipitation assays. Genistein

decreased liver CYP1A1 activity and protein in the male and female

mice. No significant differences in mRNA were detected in

the cells following treatment with isoflavones. In support of the

animal results, the cells treated with genistein, but not daidzein and

equol, exhibited decreases in ARNT and ER bindings to the

CYP1A1 XRE. In conclusion, these data provide novel molecular

mechanisms associated with the CYP1A1 decreases in mice treated

with genistein under acute and basal conditions.

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95


Systems biology of human aging -- Network model 2009

John D. Furber 1 and Pat Langley 2

1 Legendary Pharmaceuticals, Gainesville, FL 32604; 2 School of Computing and

Informatics, Arizona State University, Tempe, AZ 85287

The many observable signs of human senescence have been

hypothesized by various researchers to result from several primary

causes. Close inspection of the biochemical and physiological

pathways associated with age-related changes and with the hypothesized

causes reveals several parallel cascades of events that

involve several important interactions and feedback loops. We present

a network diagram to aid in conceptualizing the many processes

and interactions among them, including promising intervention

points for therapy development. This diagram is maintained on

the Web as a reference for researchers and students. Content is updated

as new information comes to light (www.LegendaryPharma.

com/chartbg.html). In addition, several researchers have proposed

to adapt the network model's contents into an interactive website

with links to references and background materials. A symposium

to promote this development was held at Arizona State University,

December 2008; abstracts are at http://circas.asu.edu/ symposia/aging/.

A second symposium was held 8-9 Dec 2009 at the National

Institute on Aging in Baltimore, Maryland.

This network model includes both intracellular and extracellular

processes. It ranges in scale from the molecular to the wholebody

level. Important pathways include:

• Extracellular proteins become damaged.

• Lysosomes accumulate reactive, crosslinked lipofuscin.

• Mitochondrial DNA mutates.

• Lamin-A progerin in nucleus.

• Nuclear envelope pore proteins oxidized.

• Nuclear mutations, telomere shortening, chromosome breaks,

chromatin alterations and epigenetic DNA adducts change gene

expression.

• Oxidized aggregates in cytoplasm.

• Proteasomes.

• Redox poise increases.

• Inflammatory cascades.

• Neuroendocrine and immune systems.

• ER stress.

96

PGE2-mediated induction of oncostatin M production in

human chronic wounds

Kasturi Ganesh 1 , Savita Khanna 1 , Urmila Gnyawali 1 ,

Narashimham L. Parinandi 1 , Krishna Rao Maddipati 2 ,

Gayle M. Gordillo 1 , Chandan K. Sen 1 , and Sashwati Roy 1

1 Comprehensive Wound Center, The Ohio State University, Columbus, OH,

USA, 2 Wayne State University, Detroit, MI, USA

Cytokine screening studies in our laboratory recognized oncostatin

M (OSM), an IL-6 family cytokine produced by activated macrophages,

as being highly abundant in human chronic wound fluid. Prostaglandin

E2 (PGE2) is generated by the sequential metabolism of arachidonic

acid by cyclo-oxygenase and prostaglandin E synthase. LC-

MS studies indicated presence of high levels of PGE2 in human

chronic wound fluid. We hypothesized that PGE2 present in wound

fluid induces OSM production by macrophages. Adult chronic wound

(defined as wound present > 4 weeks) patients (25-80 years old) undergoing

VAC® (negative pressure) therapies of their wounds at the

Comprehensive Wound Center (CWC) were recruited (IRB approved)

for the study. VAC dressing (sponges) were collected. In addition,

paired peripheral blood samples were collected from each patient.

Wound fluid and inflammatory cells were derived from the VAC

dressing by lavaging the wound dressing with saline solution. High

levels of PGE2 and OSM (p


Evaluation of physical-chemical and antioxidant stability and

in vivo efficacy of formulations added soybean extract

Sandra R. Georgetti 1,2 , Rubia Casagrande 1,2 , Marcela M. Baracat 1,2 ,

Waldiceu A. Verri Jr 3 , Fabiana T.M.C. Vicentini 2 ,

and Maria José Vieira Fonseca 2

1 Department of Pharmaceutical Science, State University of Londrina,

Londrina, PR; 2 Department of Pharmaceutical Science, Faculty of

Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao

Preto, SP; 3 Department of Pathology, State University of Londrina, Londrina, PR

The skin is constantly challenged by oxidative stress. Therefore,

topical administration of antioxidants provides an efficient

manner to enrich the endogeneous cutaneous protection system.

However, novel formulations must be stable and active in vivo.

Therefore, the present study was designed to evaluate both physical-chemical

and antioxidant activity stability of different formulations

containing SE as well as in vivo protection against TPAinduced

oxidative stress in the skin of hairless mice (estimation of

H 2 O 2 and lipid peroxidation). A non-ionic emulsion with high lipid

content (F1) and anionic emulsion with low lipid content (F2) containing

or not the SE were stored at 4 C, 30 C/60% relative humidity

(RH), and 40 C/70% RH for 6 months. At pre-determined times

(initial, 1, 2, 3 and 6 months) samples were collected for the

evaluation of physical (pH, centrifugation and globule size), and

functional (inhibition of lipid peroxidation) stability. Both formulations

were stable in all parameters evaluated during the 6 months.

The topical treatment of hairless mice with F1 or F2 containing SE

abolished the TPA-induced generation of H 2 O 2 and lipid peroxidation

(MDA formation). Therefore, present data suggest that formulations

containing SE are promising therapeutic approaches to inhibit

oxidative damages of the skin.

98

Oral tocotrienol is transported to human organs and

particularly helpful for patients waitlisted for liver transplantation

Urmila Gnyawali, Viren Patel, Gayle M. Gordillo, Savita Khanna,

Cameron Rink, Bassel Shneker, Sashwati Roy, Kasturi Ganesh,

J. Layne Moore, Atom Sarkar, Benjamin Sun, David Feldman,

Robert Kirkpatrick, Elmahdi Elkhammas, Emily Klatte,

Michel Miller, Michael Firstenberg and Chandan K. Sen

Davis Heart and Lung Research Institute, Department of Surgery,

The Ohio State University Medical Center

Recent works in our laboratory has established alpha-tocotrienol as

the most potent neuroprotective form of natural vitamin E effective

against stroke. In this study we tested the delivery of mixed tocotrienols

(TCT), taken orally, to major human organs. TCT was detected

in all organs collected (brain, adipose, skin, cardiac muscle, cardiac

valve, liver, and skin) from patients. Results from mixed TCT

supplemented humans were compared with those from a control group

who took tocopherols (TCP). TCT supplementation increased the liver

and adipose tissue levels of , , and - TCT significantly. In the cardiac

muscle, levels of and -TCT increased significantly. TCT supplementation

significantly increased , , and - TCT levels in whole

blood and skin when compared to pre-supplementation (time 0) baseline

values. When observing our liver transplantation patients we

came across a striking finding. The model for end-stage liver disease

(MELD) is used to prioritize cirrhotic liver patients who are waiting

for liver transplantation. When we fitted MELD score to a linear model

both pre- and post-supplementation, 50% (7) of patients on TCT supplementation

(n=14) showed a negative sloping (reduction) MELD

score post supplementation. When we compared that to the tocopherol

supplemented group, only 1 patient out of 5 (20%) showed a negative

sloping (reduction) MELD score. Of the TCT supplemented liver patients

5 out 7 (71%) patients with cirrhosis due to viral hepatitis (hepatitis

B, 1/1 and C, 4/6) had a reduction in MELD score. However, all

the patients with a diagnosis of cryptogenetic cirrhosis (n=3) who were

supplemented with TCT had an increase in MELD score. When all patients

were considered those who were supplemented with TCT had a

statistically significantly lower MELD score slope than those supplemented

with TCP. Thus, TCT is transported to all vital human tissues

studied including the brain. Furthermore, tocotrienol supplementation

may lower MELD score in patients waiting for liver transplantation

providing them with a longer time to wait until an appropriate liver is

available for transplantation. [Supported by NINDS NS42617 to CKS].

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Development of a multi-modal in vivo system to image wound

tissue oxygen and perfusion

Surya Gnyawali 1-2 , Jeff Xu 1-3 , Jiwei Huang 1-3 , Urmila Gnyawali 1 ,

Kun Huang 1-4 , Ronald Xu 1-3 , and Chandan K. Sen 1-2

1 Comprehensive Wound Center, 2 Davis Heart and Lung Research Institute,

3 Department of Biomedical Engineering, 4 Department of Biomedical

Informatics, The Ohio State University, Columbus, OH 43210

Assessment of tissue oxygenation and perfusion is important

for evaluating wound healing/regression and guide followed by

therapeutic processes. However, many existing techniques and

clinical practices are subjective to background bias, tissue heterogeneity,

and inter-patient variations. We sought to develop a multimodal

imaging system for in vivo, non-invasive, real-time quantitative

assessment of tissue oxygen and perfusion. Our imaging system

integrated a broadband light source, a high resolution CCD

camera, a highly sensitive infrared camera and a liquid crystal tunable

filter. A user-friendly software interface was developed to

control all components systematically. Image registration and reconstruction

algorithms were developed for reliable reconstruction

of tissue oxygenation without interferences of blood concentration,

tissue scattering, pigmentation and background absorption. Thermal

images and hyperspectral visual/near infrared images were coregistered.

Validity of the algorithm was tested on a tissuesimulating

heterogeneous phantom. The tissue perfusion was reconstructed

from the measurements of the dynamic changes of

hemoglobin concentration and temperature distribution subjected

to vascular occlusion and reperfusion. Image fusion technique that

co-registered tissue morphology, oxygenation and perfusion maps

was displayed in real-time. Clinical feasibility test for quantitative

imaging of tissue oxygenation and perfusion was also performed

on healthy human subjects. Results show that the imaging system

is powerful to detect tissue oxygenation and perfusion in real-time

with minimal bias introduced by tissue heterogeneity and background

absorption. Images are independent of human skin color.

This imaging system, initially targeted to quantitative assessment

of chronic wounds, will help in translational research and clinics.

Supported by NHLBI R01 GM-077185

100

Tocopherol Transfer Protein (TTP) modulates cigarette smoke

(CS) sensitive lung transcriptome

Kishorchandra Gohil, Saji Oommen, Vihas T. Vasu,

Hnin Hnin Aung, and Carroll E. Cross

Department of Internal Medicine, Division of Pulmonary and Critical Care

Medicine, University of California, Davis, CA 95616

Oxidant and inflammatory processes are implicated in CSinduced

lung diseases. Our primary hypothesis is that lung’s intrinsic

antioxidant status affects the expression of multiple genes that

determine physio-pathological phenotypes of lungs. -Tocopherol

(AT) is suggested to be a potent antioxidant. It is sequestered in

lungs and may be a component of lung’s antioxidant network.

Mice and patients deficient in the TTP gene have very low systemic

AT inspite of feeding a normal diet. In this study, 5 month

old male TTP-deficient (KO) mice and their congenic normal

(WT) mice, n = 5/group, were exposed to either air or CS (60 mg

particulates/m 3 ) for 3 and 10 days. Post-exposure lung tissue was

dissected, RNA extracted, 10 mg from each lung pooled, group

wise, and subjected to GeneChip (430A 2.0) analysis. Differential

analysis of the transcriptomes (~15,000 mRNAs) identified CSsensitive

genes that were modulated by AT-TTP deficiency. AT-

TTP deficiency potentiated CS-induced cyp1a1 and cyp1b1 expression;

two genes driven by aromatic hydrocarbon receptor (AhR).

There was selective potentiation of CS-induced Nrf2-driven gene

battery by AT-TTP deficiency. Lymphocyte and leukocyte specific

genes were the largest gene-clusters modulated by AT-TTP deficiency.

These experiments have uncovered a large network of

lung-tissue genes that are modulated by AT-TTP deficiency. Since

lung-tissue does not express TTP, the data suggest that lung AT is

the primary modulator of these gene-network. Furthermore, the effects

on lymphoid and myeloid specific genes uncovered in this

study may indicate a role for AT in bone-marrow-lung-tissue homeostatic

adaptations under physiological conditions and, when

mice, and possibly humans, are exposed to environmental toxins.

101


Hemangioma growth is nox-4 dependent and inhibited by

oral feeding of blueberry extract

Gayle M. Gordillo 1,2 , Huiqing Fang 1,2 , Sashwati Roy 2,3 ,

and Chandan K. Sen 2,3

1 Division of Plastic Surgery, 2 Laboratory of Molecular Medicine Davis Heart

Lung Research Institute, 3 Division of General Surgery,

The Ohio State University, Columbus, Ohio 43210

Hemangiomas are endothelial cell tumors that are the most

common soft tissue tumor in infants occurring in up to 10% of

Caucasian infants. Using an established murine model, we have

shown that injection of endothelial cells (EOMA) results in endothelial

cell tumor (HE) formation that requires the expression of

oxidant inducible monocyte chemoattractant protein-1 (MCP-1).

We sought to identify the source of oxidants stimulating MCP-1

expression in EOMA cells. NADPH oxidase is the primary source

of oxidant production in endothelial cells. Real-time PCR was used

to screen EOMA cells for all isoforms of gp91, the catalytic

subunit of NADPH oxidase, and nox-4 was identified as the predominant

isoform. Immunohistochemistry studies showed nox-4

expression was strikingly elevated in human HE validating nox-4

as a therapeutic target. Post-transcriptional gene silencing of nox-4

using RNA interference techniques in EOMA cells resulted in decreased

oxidant production, decreased angiogenic capacity on Matrigel

and decreased HE size in vivo. We then sought to determine

whether antioxidant delivery using a nutritional intervention such

as blueberry extract (BBE) was effective in treating HE. BBE (150

ug/ml) was shown to inhibit c-Jun N-terminal kinase activation,

AP-1 and NF-kB DNA binding as well as MCP-1 expression in

EOMA cells. Oral gavage feeding of BBE (20 mg/kg) to mice with

HE resulted in significantly decreased HE size and prolonged survival.

These results indicate that HE formation is driven by nox-4

derived oxidant production and that treatment with BBE may be an

effective alternative intervention.

GMG supported by K08 GM066964

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Concordant changes of activity and the level of mRNA of two

superoxide dismutase isoforms in rat blood cells taking

vitamin E

Maliheh Hajiani 1 , Aboualfazl Golestani 1 , Mehdi Froozandeh 2 ,

Ali Akbar Owji 3 , Shahnaz Khaghani 1 , Naghmeh Ghannadian 1 , and

Parvin Pasalar 1*

1 Department of Biochemistry, Azad University of Jahrom; 2 Depatment of

Biotechnology, Terabit Moddares University, Tehran, Iran; 3 Department of

Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran

Vitamin E is the most important lipid soluble antioxidant. More

recently, it has been proposed as a gene regulator, and its effects

have been observed at the level of gene expression, mRNA stability,

protein translation and protein stability. Aim: To investigate the

effects of vitamin E on activity and expression of the most important

endogenous antioxidant enzyme; superoxide dismutase (SOD),

in rats. Materials and Methods: 28 male Sprauge-Dawly rats were

divided into four groups; the control group and three dosing groups

receiving daily 50 μl intraperitoneal injections of 0, 10, 30, and

100 mg/kg of -tochopherol acetate dissolved in liquid paraffin for

6 weeks. Quantitative real time RT-PCR, western blotting and enzyme

assay were used to assess the level of Cu/Zn- SOD and Mn-

SOD mRNA, protein, and the enzyme activity in blood cells respectively

at weeks 0, 2, 4 and 6 following vit E administrations.

Total antioxidant capacity was also assessed in plasma for all the

mentioned groups at same time intervals. Results: Mn-SOD activity

showed significant increase (P


Dose-dependent modulation of systemic lipid peroxidation and

activity of antioxidant enzymes by vitamin E in the rat

Maliheh Hajiani, Abolfazl Golsetani, Ahmad Shariftabrizi,

Roghieh Rastegar, Seyedmehdi Payabvash, Amirali Hasanzadeh,

Ahmad Reza Dehpoor, and Parvin Pasalar

1 Department of Basic Sciences,Azad University of Jahrom; 2 Department of

Biochemistry , Tehran University of Medical Sciences; 3 Department of

Pharmacology , Tehran University of Medical Sciences

The objective of this work was to examine the time-dependent

pro-oxidant versus antioxidant effect of various doses of vitamin E

used commonly in experimental studies. Erythrocyte activity of

superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase

(CAT) and plasma lipid peroxidation levels were investigated

following biweekly intramuscular administration of 100, 300, 600

mg/kg of vitamin E at a baseline timepoint, and 2, 4, 6 weeks after

initiating treatment.Vitamin E had an antioxidant effect when administered

at low doses over short time period, and increased the

antioxidant enzymes. At higher doses and over long time periods,

it increased the level of lipid peroxidation, and attenuated the activity

of antioxidant enzymes. These results suggest that time dependent

variations in vitamin E effects should be considered in design

and interpretation of experimental antioxidant studies, as well

as during clinical trials

Age-dependent increase in cholesterol efflux, clearance and

catabolism in the aging intact 3xTG mouse model of

Alzheimer's disease

Ryan T. Hamilton § , Amanda J. Compadre § , Julian Lemus § ,

Esosa Agbonwaneten § , Jia Yao § , Ronald W. Irwin § , and

Roberta Díaz Brinton §

§ Department of Pharmacology and Pharmaceutical Sciences, School of

Pharmacy, and Department of Neuroscience,

University of Southern California, Los Angeles, CA 90089

Dysregulation of cholesterol homeostasis is an antecedent to

the development of Alzheimer's pathology. Elevated neuronal

membrane cholesterol induces an aging-dependent production of

amyloid beta that leads to amyloid plaque formation in vivo. This

study proposes that cholesterol trafficking, efflux and catabolism is

increased in the triple transgenic AD (3xTG-AD) model. To test

this hypothesis entorhinal cortex sections (C2) were analyzed by

western blot in intact female non-transgenic (non-TG) and 3xTG-

AD mice. 3xTG-AD mice had elevated levels of 24-S-hydroxylase

(CYP46) and ATP binding cassette protein 1 (ABCA1) at all ages

while non-TG mice had an age-dependent increase in both. Apolipoprotein

E and F increased age-dependently in the 3xTG-AD

while it was unchanged in the non-TG. Cholesterol uptake by

apolipoprotein E-receptor appeared to be unchanged at all ages in

both mice whereas low density lipoprotein-receptor decreased age

dependently in both. Lysosomal trafficking of cholesterol by

Niemman Pick C1 was decreased age dependently in both. Mitochondrial

cholesterol trafficking proteins steroid activated receptor

and peripheral benzodiazopine receptor had a dose dependent increase

with age in the 3xTG-AD while was unchanged in the non-

TG. These findings suggest that cholesterol and lipid efflux, clearance

and catabolism pathways are upregulated while uptake and

lysosomal trafficking are decreased suggesting that cholesterol

steady states appear to be associated with the mitochondrial and

extracellular compartments in the 3xTG-AD mice.

104

105


Protein unfolded low density lipoprotein inhibits

mitochondrial bioenergetics: Implication for the atherogenic

phenotype of coronary artery disease

Ryan T. Hamilton, Juliana Hwang-Levine, Howard N. Hodis , and

Enrique Cadenas

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy,

Atherosclerosis Research Unit Keck School of Medicine, University of

Southern California, Los Angeles, CA 90089, USA

Elevated low-density lipoprotein (LDL) is a risk factor for the

development of atherosclerosis and the unfolded-LDL subfraction

in vivo (LDL – ) is intimately associated with the development of

cardiovascular disease. JNK-2 is implicated in the formation of

foam cells and atherosclerotic lesions and interaction of oxLDL

with CD36 activates JNK-2 phosphorylation. These findings support

a protein unfolded LDL-dependent activation of CD36 and

SR-A, the induction of JNK-2 and a subsequent mitochondrial energy-redox

regulation. We found that protein unfolded LDL decreased

ATP production in BAEC by 61 ± 23%, mitochondrial

respiratory capacity by 31 ± 8%, and maximal anaerobic metabolism

by 25 ± 10% while increased proton leak by 36 ± 12% and

basal anaerobic metabolism by 140 ± 46%. ATP synthase and

PDH activity were decreased by protein unfolded LDL (42 ± 4%

and 47 ± 13%, respectively) and p-JNK-2-treated mitochondria

had concomitant decreases in PDH (52 ± 5%) and ATP synthase

activity (67 ± 19%) while JNK inhibitor blocked this loss in activity

(2± 10% and 11 ± 3%). Protein unfolded LDL decreased the

ATP to ADP ratio by 61 ± 10% and glutathione by 52 ± 13% and

pre-treatment with JNK inhibitor ablated this decrease in

ATP/ADP by 1 ± 5% and GSH by 16 ± 15%. These findings demonstrate

a novel mechanism by which protein unfolded LDL

(LDL – ) impairs mitochondrial bioenergetics as mediated by p-

JNK-2 and implicates a necrotic phenotype (low GSH/GSSG and

low ATP) and the importance of LDL – in the development of a

cardiovascular disease phenotype.

Age-dependent increases in AMPK, JNK, and GSK3

and role for caloric restriction:

Implication for mitochondrial energy substrate control

Ryan T. Hamilton, Fei Yin, and Enrique Cadenas

Department of Pharmacology and Pharmaceutical Sciences, School of

Pharmacy, University of Southern California, Los Angeles, CA 90089, USA

Aging-dependent hypometabolism precedes the development

of Alzheimer’s disease by a decade and is mitochondrialdependent.

Several canonical signaling pathways lead to this hypometabolic

phenotype including age-dependent activation of JNK

and GSK3 phosphorylation while activation of AMPK may provide

a protective mechanism and they all converge on mitochondrial

function. It is known that both GSK3 and JNK phosphorylation

increase with age in Fischer rats and that both pathways inhibit

age-dependently PDH activity. These observations supported

the hypothesis that the interplay between JNK and GSK3 signaling

with AMPK may provide mechanistic insight into Alzheimer’s

pathology and that caloric restriction a known modulator of life

span may shift the balance in this paradigm. It was observed that

there was an age-dependent increase in AMPK protein expression

as well as its phosphorylation but its activation to total AMPK levels

were unchanged. Short-term caloric restriction at 6 months of

age increased AMPK phosphorylation while it decreased AMPK

phosphorylation at 14 months of age and p-AMPK/AMPK was unchanged

at all ages. These findings suggest that AMPK may be

mechanistically involved in neuroprotective functions of agedependent

hypometabolism and that caloric restriction may function

protectively at younger age than older age.

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107


Effects of high-dose B-complex vitamins plus calcium,

magnesium and zinc (Berocca®) on subjective mood and

cognitive performance in healthy males

Crystal F. Haskell 1 , David O. Kennedy 1 , Anthony Watson 1 ,

Rachel Veasey 1 , Jens Lüdemann 2 , Volker Baroth 2 ,

Stephen Beveridge 2 , and Silvia Maggini 2

1 Brain Performance and Nutrition Research Centre, Northumbria University,

UK; 2 Bayer Consumer Care Ltd, Basel, Switzerland

A significant proportion of the general population report supplementing

their diet with one or more vitamins or minerals, with stress,

fatigue or improvement of cognitive performance the most commonly

reported reasons for doing so. In this respect, very few studies have

assessed the relationship between supplementation with vitamins/minerals

and psychological functioning in healthy cohorts of

non-elderly adults. In this randomized, placebo-controlled, doubleblind,

parallel groups trial the effect of a high-dose B-complex vitamin

and mineral supplement (Berocca®) was assessed in 215 males aged

30 to 55 years, who were in full time employment. Participants attended

the laboratory prior to, and on the last day of a 33-day treatment

period. During both visits participants completed the Profile of

Mood States (POMS), Perceived Stress Scale (PSS) and General

Health Questionnaire (GHQ-12). Cognitive performance and task related

modulation of mood/fatigue were assessed with the 60 minute

Cognitive Demand Battery (CDB). In addition, on the final day, participants

completed the Stroop task for 40 minutes whilst engaged in

inclined treadmill walking and subsequent executive function was assessed.

Results show that participants in the vitamin/mineral group had

significant improvements in their ratings on the PSS, GHQ-12 and the

‘vigor’ subscale of the POMS. They also performed better on one (of

three) CDB battery tasks (Serial 3s subtractions) and rated themselves

as less ‘mentally tired’ both pre- and post- completion of the Cognitive

Demand Battery. In conclusion, these findings suggest that healthy

members of the general population may benefit from augmented levels

of vitamins/minerals via direct dietary supplementation. Specifically

supplementation led to improved ratings of stress, mental health, and

vigor, and improved cognitive performance during intense mental

processing.

108

Ameliorative effect of photoilluminated riboflavin onto the

cisplatin-induced oxidative stress in vivo

Iftekhar Hassan 1 , Iyad Ali 2 , Sandesh Chibber 1 , Imrana Naseem 1 *

1 Department of Bichemistry, Faculty of Life Sciences, Aligarh Muslim

University, Aligarh, U.P., India; 2 Department of Biochemistry and Genetics,

Faculty of Medicine, An-Najah National University, Nablus, Palestine.

2 Presenting author

Cisplatin is a widely used anticancer drug against various cancers

and solid tumors. It is documented that it generates reactive

oxygen species leading to its major side effects like nephrotoxicity

and hepatotoxicity which limits its clinical use for long time. Riboflavin

is an essential vitamin found in all forms of life. Being a

well known photosensitizer, it generates reactive oxygen species

(ROS) upon photo-illumination at high concentration which are

known to cause the oxidative damage to cellular biomolecules viz.

proteins, lipids and DNA. This property of riboflavin is utilized in

photodynamic therapy (PDT) and ribophototherapy for the treatment

of various diseases including cancer. We have tried to show

the ameliorative effect of riboflavin onto cisplatin induced oxidative

stress in mice. We treated mice with riboflavin and cisplatin

separately and also in the two combination of cisplatin and riboflavin

in the ratio of 1:0.5 and 1:1 under photoilluminated condition.

After 8 doses of the treatment for a month, the assay of major antioxidant

enzymes (superoxide dismutase, catalase, glutathione reductase,

alkaline phosphatase) and antioxidant proteins (reduced

glutathione, sulfhydryl groups) and glutathione –S-transferase

along with the estimation of liver and kidney function markers

were carried-out of their kidney, liver and serum. Beside these, the

extent of lipid peroxidation and protein oxidation were also studied

in the samples. The level of antioxidant enzymes and the proteins

were significantly compromised in the cisplatin treated group

along with higher extent of lipid peroxidation and protein oxidation

as compared to the control. The pattern was similar but not so

pronounced in the riboflavin treated group. The groups under the

treatment with combination of both cisplatin and riboflavin showed

that all the parameters tended towards the normal levels in a dose

dependent manner with increasing dose of riboflavin. Hence, it can

be hypothesized that riboflavin shows ameliorative effect onto the

cisplatin –induced oxidative stress in vivo.

109


Multiple pathophysiological roles of mitochondrial fission and

fusion upon nanophotodynamic effect of fullerene-induced

mitochondrial oxidative stress

Cheng-En Hsieh 1 and Mei-Jie Jou 2

1

School of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan

2

Department of Physiology and Pharmacology, Chang Gung University,

Kwei-Shan, Tao-Yuan, Taiwan

Mitochondrial fission and fusion machinery possesses vital

function in maintaining the mitochondrial integrity and network,

bioelectrical and chemical connectivity, turnover of mitochondria,

and segregation and protection of mitochondrial DNA (mtDNA).

Substantial evidence indicates that extensive mitochondrial fission

occurs during oxidative insult, a key reason for various diseases

and molecular aging, yet the detailed cellular functions and effects

of such morphological response remain unclear. We investigated

pathophysiological roles of mitochondrial fission and fusion machinery

upon mitochondria-targeted reactive oxygen species

(mROS) generation by the photodynamic effect (PDE) of a new

class of water-soluble nanophotosensitizer carbon 60, tris-malonic

acid carboxyfullerene (C 3 ), coupled with 543 nm laser in wildtype,

fission enhanced and fusion enhanced 143B osteosarcoma

cells. Mitochondrial level of dynamic alterations including morphology,

oxidative strength, lipid peroxidation, m , cardiolipin

(CL), and mCa 2+ were time-lapsed imaged using laser scanning

confocal microscopy coupled with various fluorescent probes including

MitoTracker Green, 2',7'-dichlorofluorescin diacetate

(DCF), C11-BODIPY 581/591 , tetramethyl rhodamine methyl ester

(TMRM), 10-N-nonyl acridine orange (NAO), and rhod-2. Our results

revealed that fission of mitochondria reduced significantly

mROS propagation for less lipid peroxidation and m loss

whereas fusion of mitochondria facilitated mitochondrial destruction.

Whereas, fission of mitochondria accelerated and fusion decreased

m depolarization, CL peroxidation, mCa 2+ overload, and

eventual cell death upon lethal doses of mitochondrial oxidative

insults. Thus, rearrangement of mitochondrial network via fission

or fusion may provide potential diverse protection against or augmentation

towards mitochondrial oxidative stress associated apoptosis.

57% average increase in equine serum nitric oxide levels

following oral administration of multi-nutrient nitric oxide

precursor

Gerald K. Huff, Tim Baum (Avid Labs),

and Tamara Sofi (THE Labs)

Subjects evaluated and treated in Las Vegas, Nevada by Gerald K.

Huff, DVM, 2315 North Decatur, Las Vegas, CA 89108. Testing performed

by Avid Labs, 100 Memorial Drive, Greer, South Carolina

29650. Formulation performed by THE Labs, 27128 Paseo Espada,

San Juan Capistrano, CA 92675

Equine laminitis causes chronic lameness or death in countless

horses every year. Laminitis causes inflammation to the laminae, leading

to rotation or sinking of the coffin bone. If the hoof does not realign,

pain will continue and the horse may be euthanized. USDA

grant funded Veterinary researchers Moore and Eades (Louisiana State

University, school of Veterinary Medicine) believe the initiating factor

in the onset of laminitis is an imbalance in blood flow to laminae

caused by decreased production of nitric oxide (NO) (which normally

relaxes blood vessels and increases blood flow) and increased release

of endothelin-1 (which causes blood vessel contraction and subsequent

decreases in blood flow). This imbalance ultimately leads to decreased

laminar blood flow, laminar swelling, tissue death, and subsequent

separation of the laminae. Studies suggest equine ulcer formation may

be caused by a similar NO deficiency pathology. In this equine study

23 horses were tested for serum NO levels (samples drawn from the

jugular vein) using highly reproducible Elisa Assays in order to establish

baselines. Of these 23 subjects, 10 were tested for serum NO levels

before and one half hour after being orally administered NitrOxide,

a medical food containing amino acids (to include L-arginine), B vitamins,

network antioxidants, flavanoids, trace minerals, and herbs.

Subjects experienced an average 57% increase of NO production over

baseline in serum NO levels. Study graph showing individual subject

levels is available.

Med-Pharmex Animal Health funded this study and generously granted permission to share

study results within the context of scientific conferences. Further studies are being planned

and funded.

110

111


Gender differences of LDL receptor production

in response to statin treatment

Juliana Hwang-Levine 1,2 , Enrique Cadenas 1 , and

Howard N. Hodis 2

1 Department of Pharmacology and Pharmaceutical Sciences, 2 Atherosclerosis

Research Unit, Schools of Pharmacy and Medicine,

University of Southern California

Low-density lipoprotein receptor (LDLr) is a cell surface receptor

that recognizes apoB and mediates endocytosis of cholesterol

ester-rich LDL. An LDLr precursor is synthesized at a molecular

weight of 120 kD; N- and O-linked sugars are added to the

LDLr in the endoplasmic reticulum and Golgi apparatus resulting

in an increase of LDLr weight to 160kD. Mature 160 kD LDLr is

transported to the cell surface to become the active binding site for

the LDL particle removing cholesterol from circulation. This study

was conducted to determine whether LDLr production differs between

genders. Male and female human aortic endothelial cells

(HAEC) were cultured according to the manufacturer protocol.

Cells were replated for 24 h in phenol red-free media prior to simvastatin

(Sim) treatment. LDLr protein levels were determined by

western blot analyses in cell lysates and normalized to -actin.

Female HAEC exhibit a greater 160 kD/120 kD LDLr protein ratio

than male HAEC when cultured in normal media. Treatment with

5 mM Sim increased both the 160 kD and 120 kD proteins in male

and female HAEC with increase in the 160 kD protein greater in

male HAEC than female HAEC. These data indicate for the first

time that expression and production of LDLr in HAEC treated with

statin varies between genders. With statin treatment, there is a substantial

increase in the 160 kD/120 kD LDLr protein ratio in male

HAEC as compared with female HAEC indicating that male cells

are more responsive to statin. These results may in part explain the

differential effect of lipid-lowering therapies on cardiovascular

outcomes between genders. Further exploration of these findings

may lead to improve cardiovascular prevention for women.

Differential regulation of brain mitochondrial function by

clinically relevant progestins

Ronald W. Irwin, Jia Yao, S. S. Ahmed, Ryan T. Hamilton,

Enrique Cadenas, and Roberta Díaz Brinton

Pharmacology & Pharmaceutical Sciences, School of Pharmacy,

University of Southern California, Los Angeles, CA 90089

Mitochondrial dysfunction and reduction of the cerebral metabolic

rate for glucose precede neurodegenerative diseases including

Alzheimer’s disease. Previously, we identified brain mitochondrial

proteomic changes associated with acute estradiol (E2) replacement

that indicated enhanced brain mitochondrial efficiency

as evidenced by increased respiratory control ratio, elevated cytochrome-c

oxidase activity and expression while simultaneously reducing

free radical generation in brain (Nilsen, Irwin, et al., J. Neurosci

2007). We extended these findings to demonstrate E2 and

progesterone (P4) regulate oxidative metabolism in brain mitochondria

(Irwin, et al., Endocrinology 2008). P4 is neuroprotective

whereas the synthetic progestin, medroxyprogesterone acetate

(MPA), most widely prescribed for the treatment of menopausal

symptoms is not neuroprotective (Nilsen & Brinton, Endocrinology

2002). In this current study we used a functional biochemical

approach to identify the mechanism responsible for interactions between

estrogen and progestins. We investigated brain mitochondrial

function of ovariectomized (OVX) 5-month female rats (n =

5) following 24h treatment with MPA, E2, E2 + MPA, vehicle, and

ShamOVX. MPA alone and MPA + E2 resulted in diminished mitochondrial

respiration and ATP generation. Treatment changes in

mitochondrial proteins relative to controls were determined via

immunoblot and enzyme activity assay for tissue lysates of hippocampus,

cortex, cerebellum, and purified whole brain mitochondria.

Progestin-mediated alterations in oxygen utilization were

measured via the Seahorse XF24 metabolic analyzer in primary

cultured neurons and isolated brain mitochondria. The set of mitochondrial

proteins altered by interactions between E2 and MPA included

ATP synthase, PDH, COXIV, MnSOD, and Prdx. We are

currently investigating several clinically relevant progestins in order

to distinguish the negative from the positive neural effects of

hormone therapy.

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113


Red wine polyphenols may influence human space memory

F. Jagla, B. Cimrova, S. Budac, M. Jergelova,

S. Bendzala, and O.Pechanova

Institute of Normal and Pathological Physiology, Slovak Academy of Sciences,

Bratislava, Slovakia

Background: The nitric oxide (NO) release within the brain is

necessary for long–term potentiation, the electrophysiological

events related to synaptic plasticity and learning. The NO synthase

(NOS) inhibitors disrupt the performance of animals in learning

tasks while the NO donors display an antiamnestic action. It seems

that NO may be involved in post-training memory processes in

rats. It was the reason to analyze the influence of a polyphenolic

compound, the NO synthase activator - Provinols TM , upon the human

space memory functions.

Methods: The analysis of the accuracy of visually evoked saccadic

eye movements and of saccades driven by memory information

about the eye landing position was used in healthy volunteers.

The saccades elicited by the visual targets were followed by the

memory-guided saccades. The whole procedure was repeated 2

hours after, following the Provinols administration (4 mg/kg of

body weight). According to the clock face the saccadic landing positions

were randomly selected at 1, 2, 4, 5, 7, 8, 10 and 11 hour.

The EEG spectral powers related to the eye movements were analyzed

as well.

Results and Discussion: The Provinols positively affected

the accuracy of both the saccades. The EEG spectral powers point

to the cortex activation by Provinols. The memory-guided saccade

task comprises perception, memorization and execution of a

saccade. Perception is under control of the network which subserve

also the attention mechanisms. Memorization is controlled by the

dorsolateral prefrontal cortex and programming and generation of a

MGS involve the working memory. It is suggested that Provinols

may affect attentional mechanisms as well.

Partly supported by VEGA grant No. 2/0160/08 and APVV - 0538-07.

114

Effects of multivitamin supplementation (Supradyn®) on

perceived fatigue and cognitive functions during extended

multi-tasking assessment

David O. Kennedy 1 , Crystal F Haskell 1 , Bernadette Robertson 1 ,

Joanne Gee 1 , Rebecca Jones 1 , Jens Lüdemann 2 , Volker Baroth 2 ,

Volker Spitzer 2 , and Silvia Maggini 2

1 Brain Performance and Nutrition Research Centre, Northumbria University,

UK; 2 Bayer Consumer Care Ltd, Basel, Switzerland

Fatigue is one of the generally recognized symptoms of micronutrient

deficiencies. Despite the general consensus that fatigue is

a highly prevalent problem, and can be associated with vitamin/mineral

deficiencies, to date no research has directly investigated

the effect of multi-vitamin/mineral supplementation on levels

of fatigue and cognitive functions. In this placebo controlled,

double-blind, randomized, controlled trial the effect of a commercially

available multi-vitamin/mineral (Supradyn®) was assessed

in a cohort of 220 females aged 25 to 50 years, who reported subjective

fatigue and who were in full time employment or childcare.

Participants attended the laboratory prior to, and on the last day of

the 9 week treatment period. During both visits mood was assessed

with questionnaires. Cognitive performance and task related modulation

of mood/fatigue were also assessed during two discrete 20

minute periods during which participants completed a four module

version of the Multi-Tasking Framework, with mood assessments

before and after completion. The results showed that those in the

vitamin/mineral group exhibited an attenuation of the negative effects

on mood/fatigue of extended task completion. Multi-tasking

for this group was also improved overall in terms of accuracy, and

on two of the modules (Mental Arithmetic and Stroop) in terms of

both faster and more accurate responses. An analysis conducted in

a subsection of the cohort (N = 102) demonstrated significant reductions

in blood levels of homocysteine following the vitamins/mineral

supplement. These findings suggest that healthy

members of the general population that report fatigue may benefit

from augmented levels of vitamins/minerals via direct dietary supplementation.

115


Arachidonic acid pathway as a key target for

neuroprotection by nanomolar -tocotrienol

Savita Khanna 1 , Narasimham L. Parinandi 2 , Sainath R. Kotha 2 ,

Sashwati Roy 1 , Cameron Rink 1 , Douglas Bibus 3 , and

Chandan K. Sen 1

Dorothy M. Davis Heart and Lung Research Institute, 1 Department of Surgery,

2 Department of Internal Medicine, The Ohio State University Medical Center,

Columbus, OH 43210 and 3 The Holman Center for Lipid Research and Lipid

Technologies and The Center for Spirituality and Healing,

University of Minnesota, Austin, MN 55912

Previous studies from our laboratory have identified -

tocotrienol (TCT) as a natural form of vitamin E with potent neuroprotective

properties. At nM concentrations, TCT protects

against a variety of insults e.g. glutamate via inhibition of a 12-Lox

catalyzed arachidonic acid (AA) pathway of neurodegeneration.

Phospholipase A 2 (PLA 2 ) activity mobilizes AA from membrane

phospholipids (PLs). The goals of the current study were to test

whether: (i) PLA 2 is sensitive to glutamate challenge; (ii) PLA 2 is

implicated in glutamate-induced cytotoxicity; and (iii) TCT regulates

glutamate-induced PLA 2 function. HT4 neural cells were

studied in culture. Glutamate (10 mM), at 30 min of treatment, induced

the release of [ 3 H] AA from cells that was significantly attenuated

by calcium chelators (EGTA and BAPTA), cPLA 2 -

specific inhibitor, AACOCF 3 , and TCT (250 nM). Glutamate also

caused the elevation of free polyunsaturated fatty acid (AA and

docosahexaenoic acid) levels and disappearance of PL-esterified

AA in HT4 cells. These responses were significantly attenuated by

TCT. Moreover, glutamate induced a time-dependent (0-30 min)

translocation and enhanced serine phosphorylation of cPLA 2 in a

TCT-inhibitible manner. cPLA 2 -specific inhibitor (AACOCF 3 ),

transient knock-down of cPLA 2 expression by cPLA 2 siRNA transfection,

as well as TCT significantly protected against the glutamate-induced

cytotoxicity in HT4 cells. Results of this study represent

the first evidence demonstrating that glutamate-induces cPLA 2

activation and that PLA 2 is directly implicated in glutamateinduced

cytotoxicity. Importantly, glutamate inducible PLA 2 is

sensitive to nM TCT. Thus, TCT may limit membrane AA mobilization

in response to glutamate insult. Support: NIH Grant NS42617

116

Expression level of peroxiredoxin isoforms

in response to cervical carcinogenesis

Kiyoon Kim 1 , Miran Yu 1 , Seulhee Han 1 , Inkyung Oh 1 ,

Young-Jun Choi 2 , Sungsoo Kim 1 , Kyungsik Yoon 1 ,

Minhyung Jung 2 and Wonchae Choe 1

1) Department of Biochemistry and Molecular biology, Medical Science and

Engineering Research Center for Bioreaction to Reactive Oxygen Species,

Biomedical Science Institute, School of Medicine, Kyunghee University, Seoul

130-701; 2 Department of Obstetrics and Gynecology, School of Medicine,

Kyung Hee University, Seoul 130-701, Korea

Despite considerable progress in understanding the function of

peroxiredoxin (Prx) in cancer, its expression patterns have not been

extensively studied in response to cervical carcinogenesis. We

evaluated the expression of Prx isoforms in normal tissue, cervical

intraepithelial neoplasia (CIN1, CIN2, and CIN3), and cervical

cancer. We found strong pattern of increased Prx II and III immunostaining

with increasing severity of the lesion. No difference in

staining intensity by grade of lesion was observed for Prx I, and

IV. Therefore, we conclude that Prx II and III are upregulated in

response to the development of cervical cancer.

117


Supplementation of a Selenocystine derivative prevents

the ionizing radiation induced oxidative stress in mice

A. Kunwar and K.I. Priyadarsini

Radiation and Photochemistry Division, Bhabha Atomic Research Centre,

Trombay, Mumbai-400085, India

Radioprotectors are employed to minimize the damaging effects

of ionizing radiation to normal cells during radiotherapy. In

present study we report the in vivo radioprotection effecicay of a

water-soluble synthetic derivative of selenocystine, 3,3'-Diselenodipropionic

acid (DSePA). The radio protective effects of DSePA

was examined in Swiss albino mice, after administration at a dosage

of 2 mg/kg body weight (i.p.), for 5 days prior to whole body

exposure to sub and supra lethal doses of -radiation. DSePA inhibited

radiation induced hepatic lipid peroxidation, protein carbonylation,

loss of hepatic function and deformation in hepatic architecture.

It also protected against the depletion of endogenous

antioxidants viz., glutathione (GSH), GPx, superoxide dismutase

(SOD), and catalase in the hepatic tissue of irradiated mice. Protection

towards GI tract and hematopoietic system was confirmed by

the restoration of radiation induced reduction in villi height, number

of crypt cells and spleen cellularity. The mRNA expression

analysis revealed that DSePA treatment caused augmentation of

GADD45 and inhibition of p21 levels in spleen and the hepatic

tissue. Additionally, it also reversed the radiation induced alterations

in expression of pro-apoptotic BAX and anti-apoptotic Bcl-2

genes, which may be favoring towards survival. In line with these

observations, DSePA improved the 30-day survival of the irradiated

mice by 30%. Based on all these studies it is concluded that

DSePA exhibits potent radioprotective effects against whole body

-radiation and the probable mechanisms of action involves maintenance

of antioxidant enzymes, prophylactic action and inhibition

of apoptosis.

Long-term treatment with schisandrin B ameliorates the impaired

mitochondrial antioxidant status in various tissues of

chronic ethanol-intoxicated rats

Philip Y. Lam, Po Yee Chiu, Hoi Yan Leung, Na Chen,

Pou Kwan Leong, and Kam Ming Ko

Department of Biochemistry, The Hong Kong University of Science &

Technology, Clear Water Bay, Hong Kong SAR, China

Chronic alcohol consumption can lead to multiple organ dysfunction.

In the present study, a rat model of chronic ethanol intoxication

was utilized to investigate the effect of schisandrin B

(Sch B, a dibenzocyclooctadiene compound isolated from Fructus

Schisandrae) on ethanol-induced oxidative damage in the brain,

heart, liver and kidney. Chronic ethanol consumption induced generalized

oxidative tissue damage, as indicated by increases in

plasma sorbitol dehydrogenase (SDH), alanine aminotransferase

(ALT) activities and reactive oxygen metabolites (ROMs) level, as

well as mitochondrial malondialdehyde (mtMDA) production in

various tissues of rats. The ethanol-induced oxidative damage was

associated with impairment in mitochondrial antioxidant status and

activation of heat shock response. Among all tissues examined, the

liver was most susceptible to ethanol-induced oxidative damage, as

evidenced by a significant increase in mtMDA production and decrease

in mitochondrial ATP generation capacity (ATP-GC).

Long-term Sch B treatment was found to enhance mitochondrial

antioxidant and functional status in a tissue non-specific manner.

The ethanol-induced oxidative stress was ameliorated by Sch B

treatment, as indicated by the reversal of altered plasma SDH, ALT

activities and ROMs level, as well as mtMDA production and heat

shock protein 25/70 expression in various tissues of chronic ethanol-intoxicated

rats. The results suggest that long-term Sch B

treatment may increase the resistance of mitochondria to oxidative

stress and thereby ameliorate the impaired mitochondrial antioxidant

status in chronic ethanol-intoxicated rats.

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119


Effect of benzyl isothiocyanates on the toxicity of chemotherapeutic

drugs in cancer cell lines and normal

human lymphocytes

Younghyun Lee 1 ,Yang Jee Kim 1 , Young Joo Choi 1 ,

Hae Dong Woo 1 , Joong Won Lee 1 , Tae Kyung Ha 1 ,Yeonju Choi 1 ,

Sunyeong Lee 1 , and Hai Won Chung 1

1 School of Public Health and Institute of Health and Environmental Sciences,

Seoul National University, Seoul, South Korea

Natural isothiocyanates (ITCs) isolated from cruciferous vegetables

are known to be effective chemopreventive agents. Sulforaphane(SFN)

and Benzyl isothiocyanate(BITC) are naturally

occurring members of the isothiocyanate family. In this study, we

evaluated the effects of ITCs(BITC or SFN) on anticancer drugs

(Cisplatin or 5-Fluorouracil)-induced cytotoxicity in HL-60 cells

and normal human lymphocytes. HL-60 cells were treated with

ITCs (BITC or SFN) for 3 h followed by treatment with chemotherapeutic

drugs(3μM Cisplatin or 10μM 5-Fluorouracil). Pretreatment

of BITC caused a significant decrease of cell viability

and an increase of DNA migration. It also increased apoptotic cell

death compared with either agent alone. However, SFN-mediated

effects were not observed. Cellular exposure to BITC in combination

with anticancer drugs increased reactive oxygen species

(ROS) generation and decreased intracellular glutathione content.

Pretreatment with N-acetyl cysteine effectively inhibited BITC/

anticancer drugs-induced apoptosis. In human lymphocytes, ITCs

did not enhance the cytotoxic effects of anticancer drugs and reduced

anticancer drugs-induced DNA damage. This implies that a

treatment of BITC/anticancer drugs could be useful to increase the

efficacy with avoiding possible side effects following conventional

anticancer drugs. In conclusion, these findings suggest that BITC

may be effective as a therapeutic agent to treat leukemia.

120

A link between mtOGG1-mediated respiratory defect

and mitochondrial ROS

Young-Kyoung Lee 1,3 , June-Hyung Kim 1,3 , You-Mie Kim 1,3 ,

Soo-Han Yoon 2,3 and Gyesoon Yoon 1,3

1 Department of Biochemistry & Molecular Biology and 2 Department of

Neurosurgery, School of Medicine and 3 Department of Molecular Science &

Technology (BK21), The Graduate School, Ajou University, Suwon, 443-721,

Korea. E-mail: ypeace@ajou.ac.kr

Mitochondrial genome is a 16kb-long and double-stranded circular

molecule. It encodes 13 proteins essential for respiratory chain reaction,

but known to be highly prone to oxidative damage because of its

unprotected structure and its environment close to continuous reactive

oxygen species (ROS) production. Diverse mtDNA mutations and deletions

have recently been postulated in carcinogenesis. However,

there is no direct evidence whether and how mtDNA damage contributes

to cancer development. Here, we focused on elucidating the potential

involvement of mtDNA damage in hepatocellular carcinoma

(HCC). Firstly, we assessed mitochondrial function of SNU HCC

cells. Expression levels of respiratory complexes and O 2 consumption

rates were largely decreased whereas ROS production was significantly

increased in most SNU HCC cells compared to Chang cell, an

immortalized human normal hepatocyte. Secondly, we examined deletion

of mtDNA from SNU cells with Southern blotting. Deleted forms

of mtDNA were not detected clearly but there were obviously distinct

digestive patterns by restriction enzyme between Chang and SNU

cells. Surprisingly, when we monitored expression level of mitochondrial

8-oxoguanine DNA glycosylase (mtOGG1), one of the DNA repair

enzymes localized in mitochondria, mtOGG1 expressions were

clearly down-regulated in the SNU cells of which mitochondrial functions

were defective unlike ncOGG1, isotype of mtOGG1 located in

nuclear. Similar results were also found in human HCC tissues. Finally,

we observed that O 2 consumption rate was decreased by

mtOGG1 knockdown using siRNA dose-dependently in Chang cells.

Taken together, our results suggest that mtDNA damage induced by

down-regulated mtOGG1 may contribute to tumor development via

mitochondrial dysfunction and mitochondrial ROS.

121


The antioxidant response element (ARE) has a major role in

the defense against oxidative stress and cancer

Joseph Levy, Michael Danilenko, and Yoav Sharoni

Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University and

Soroka Medical Center, Beer-Sheva, Israel

In recent years, evidence has accumulated indicating that the cancer

preventive action of various dietary ingredients such as carotenoids,

polyphenols and isothiocyanates is, at least in part, due to the

induction of phase II enzymes. Expression of these enzymes is regulated

by the antioxidant response element (ARE) and the transcription

factor Nrf2. Dietary inducers of Nrf2 are diversified in their chemical

structure but are usually electrophiles which react with Keap1 to disrupt

its inhibitory activity on Nrf2. It is interesting that hydrophobic

carotenoids such as lycopene which activate Nrf2 lack any electrophilic

group and thus are unlikely to interact directly with Keap1. Indeed,

we have demonstrated that intact carotenoids do not directly activate

the ARE transcription system and that carotenoid oxidation

products are the active mediators in this stimulation. Furthermore, the

activity of the carotenoid derivatives depends on the relative position

of the methyl group to the terminal aldehyde which determines the reactivity

of the conjugated double bond in reactions such as Michael

addition to SH groups in Keap1.We recently found that Nrf2 is involved

in other anti-cancer effects of phyto-nutrients in hormone dependent

cancers. Various phyto-nutrients inhibit estrogen signaling,

the major risk factor in breast and endometrial cancer. Using overexpression

of Nrf2 and siRNA for this gene, we demonstrated that Nrf2

is involved in the phytonutrient-induced inhibition of the estrogenic

activity. Although the effect of estrogens in breast and endometrial

cancer is harmful, it is beneficial for bone formation. Thus, we investigated

the effect of the phyto-nutrients on estrogenic activity in osteoblasts.

Surprisingly, we found that the same dietary compounds,

which inhibit estrogenic activity in cancer cells, did not inhibit and

even stimulated the expression of estrogen-induced genes in osteoblast

like cells. The results suggest that the Nrf2 transcription factor

is involved in these differential activities of plant-derived phytonutrients

in bone and cancer cells.

PI3K regulation of cellular bioenergetics

Chen Li, Bangyan Stiles, and Enrique Cadenas

Department of Pharmacology and Pharmaceutical Sciences, School of

Pharmacy, University of Southern California, Los Angeles, CA, USA

PI3K regulation of cellular bioenergetics was examined in a

liver-specific PTEN knockout mouse model that exhibits a robust

PI3K/Akt signaling. Bioenergetics (glycolysis and mitochondrial

respiration) of cultured hepatocytes was measured by extracellular

flux analysis and data showed that hepatocytes cultured from

PTEN knockout mice (PTEN -/- ) exhibited higher anaerobic and

aerobic metabolism. Treatment of cultured hepatocytes with the

PI3K inhibitor LY290004 led to a decrease in mitochondrial respiration

suggesting the involvement of the PI3K/Akt pathway in the

mitochondrial energy-transducing process. Mitochondria isolated

from PTEN -/- mouse liver had a higher respiratory control ratio

than those of wild type mice. PTEN -/- hepatocytes showed increased

phosphorylation of Akt (Ser 473 ), GSK3 (Ser 9 ) and

GSK3 (Ser 21 ), increased association of phospho Akt and decreased

association of unphosphorylated GSK3/ isoforms with

mitochondria. Pyruvate dehydrogenase activity was higher in

PTEN -/- hepatocytes. The increased mitochondrial bioenergetics in

PTEN -/- mice may be partially due to PI3K regulation at (a) cytosolic

level, through the phosphorylation of Akt and GSK3/

thereby favoring the translocation of Akt while inhibiting the translocation

of GSK3/ to mitochondria and the subsequent modulation

of mitochondrial protein phosphorylation and activity; and (b)

transcriptional level, through the regulation of NRFs and PGC1

which modulate the transcription of mitochondrial genes. It may be

summarized that PI3K activation enhances mitochondrial bioenergetics

through enhancing substrate availability (glycolysis) and the

regulation of mitochondrial function at the cytosolic and transcriptional

levels.

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123


(-)-Epicatechin reduces L-NAME induced hypertension and

oxidant production in rats

Corina Litterio 1 , Paula D. Prince 1 , Patricia I. Oteiza 2,3 ,

César G. Fraga 1,2 , and Mónica Galleano 1

1 Physical Chemistry-PRALIB, Pharmacy and Biochemistry, University

Buenos Aires-CONICET, Buenos Aires, Argentina; 2 Departments of

Nutrition and 3 Environmental Toxicology, University of California,

Davis, USA

Flavanoids are naturally occurring plant compounds, which

biological effects could explain some of the cardiovascular benefits

linked to the consumption of fruit and vegetables. Dietary intervention

studies in humans and animals indicate that flavanolrich

foods, i.e., wine, tea, and chocolate may exert blood pressure

(BP) lowering effects, and other cardiovascular benefits. However

the biochemical mechanisms mediating those benefits are still under

debate. To advance on the understanding the effects of

flavanols on BP, we used a model of induced hypertension in rats

by treatment with the NO-synthase inhibitor L-NAME. The addition

of (-)-epicatechin (EC) in the diet (0.2-4.0 mg/g diet) significantly

modulated the L-NAME-dependent BP increase in a dosedependent

manner. The decrease in BP was associated with the

presence of EC in plasma, the decrease in markers of oxidative

damage, i.e. malondialdehyde, nitrated proteins, and oxidized glutathione,

and an increase in plasma NO. These results are suggesting

that by lowering cell oxidants EC is maintaining an optimal

NO steady-state level. These conditions finally result in the maintenance

of normal BP values.

This work was partially supported by NIH AT2966; UBACyT (B801 and B802, and

ANPCyT PICT 994. MG and CGF are members of the CIC, CONICET, Argentina

New strategy for high-sensitivity RSNOs assay based on

catalysis of gold nanoparticles

Yang Liu 1 , Hong-ying Jia 1 , Li-bo Du 1 , Xue-ji Zhang 2 ,

Ying-lin Sha 3 , Lu Han 1 , and Qiu Tian 1

1 Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100190,

China.; 2 Department of Chemistry, University of South Florida, 4202 East

Fowler Avenue, CHE-205A, Tampa, Florida 33620-5250 USA; 3 Single Molecule

and Nano-biomedicine Laboratory, Department of Biophysics, Peking

University School of Basic Medical Sciences, Biochemistry Building 204#, Xueyuan

Road 38#, Beijing 100083, China

S-nitrosothiol (RSNOs) by the nitric oxide (NO)-dependent S-

nitrosation of thiol-containing proteins and peptides has multiple

biological functions including platelet deactivation, immunosupression,

neurotransmission, and apoptosis. Most of the functions

are attributed to NO release during the RSNOs decomposition.

Therefore, the quantification of S-nitrosothiols is essential to understanding

the NO-mediated biological events. This study aims to

promote a highly sensitive assay for RSNOs by catalysis of gold

nanoparticles in biological media.

Gold nanoparticles have been shown to quantitively catalyze

NO releasing whenever they come into contact with RSNOs and

the approximate limit of detection of the assay is in the picomole to

fetomole range that rely on the ligand exchange reaction on the

surface of the nanoparticles. The process is ascribed to the formation

of the Au-thiolate on the surface of gold nanoparticles and the

simultaneous cleavage of the S-N bond with release of nitric oxide.

Furthermore, the quantitive detedtion of RSNOs is free from interference

of various endogenous substances, such as NO 2 - , NO 2 - and

GSH, which may co-exist under biological conditions.

Support by NSFC(No. 90813021 & 20875093)

Jia HY, Liu Y, Zhang XJ, et al. Potential Oxidative Stress of Gold Nanoparticles by Induced-NO

Releasing in Serum, J Amer Chem Soc 131(2009)40-41

124

125


NSAIDs and acidic environment induce gastric mucosal

cellular mitochondrial dysfunction


H. Matsui, K. Hiyama, T. Kaneko, Y.N. Nagano,

O. Shimokawa, and A. Hirayama

Graduate School of Comprehensive Human Sciences,

University of Tsukuba, Japan


Backgrounds & Aims: We have previously reported that both

gastric cellular exposure to acidic environment and indomethacin

treatments induced lipid peroxidation and cellular apoptosis (Nagano,

2005). However, possible mechanisms of these phenomena

still remain unknown. In the present study, we elucidate whether

both indomethacin treatment and extracellular acidic environment

cause mitochondrial dysfunction or not. Moreover, we investigate

the effect of rebamipide on these cellular injuries.

Methods; To examine the relations between extracellular pH

and mitochondrial function, cellular oxygen consumptions in various

pH media (1~7.4) were measured with an oxygen electrode.

Mitochondrial membrane potential was measured with a fluorescent

probe JC-1 and a spectrofluorometer. Intercellular pH was examined

with a fluorescent probe BCECF-AM and a

spectrofluorometer. We measured intracellular reactive oxygen

species (ROS) with electron paramagnetic resonance (EPR) using

spin trapping reagents such as CYPMPO. We also treated cells

with both indomethacin and rebamipide in various pH, and

measured mitochondrial function.

Results: Both the indomethacin treatment and the low pH condition

inhibited RGM1 oxygen consumption, and decreased the

mitochondria membrane potential. The intercellular pH decrease

below pH7.0 induced ROS generation. This ROS was decided as

mitochondrial super oxide anion. Moreover, rebamipide inhibited

both acidic environment and indomethacin induced cellular injury.

Conclusion: Extracellular low pH conditions, the same as indomethacin

treatments were likely to induce cellular injuries via

super oxide anion generation under mitochondrial dysfunction.

126

Generation of reactive oxygen species by heating

aqueous solution containing oxygen

Ken-ichiro Matsumoto 1 , Minako Nyui 1 , Masato Kamibayashi 1 ,

Toshihiko Ozawa 1,2 , Ikuo Nakanishi 1 , and Kazunori Anzai 1

1 Radiation Modifier Research Team, Heavy-Ion Radiobiology Research Group,

Research Center for Charged Particle Therapy, National Institute of

Radiological Sciences, Chiba-shi, Chiba 263-8555, Japan, 2 Department of

Analytical Pharmacology, Yokohama College of Pharmacy, Yokohama-shi,

Kanagawa 245-0066, Japan

Interest for hyperthermia clinical cancer treatment has been increased

recently, especially in combination use with existing

treatments, i.e. chemotherapy, radiotherapy and immunotherapy to

increase efficiency of those treatments. Mechanism for hyperthermal

cell killing or sensitization to other stresses/treatments has

been unclear. Relation of reactive oxygen species (ROS) to the effect

of hyperthermia has been widely alleged and/or reported. Nitroxyl

radicals have been conventionally used as chemical redox

probe in vitro and in vivo experiments in fields of electron paramagnetic

resonance (EPR) spectroscopy. In this presentation,

temperature-dependent free radical reactions were investigated using

nitroxyl radicals as redox probes. Gluthathione (GSH)-

dependent EPR signal loss of nitroxyl radicals occurred temperature-dependently.

Details of temperature-dependent reaction of nitroxyl

radicals and GSH were investigated. Heating a solution of a

hydroxylamine showed EPR signal recovery. Heating a spin trapping

agent showed temperature-dependent increase of EPR signal

due to the hydroxyl radical adduct. Most of reactions were suppressed

by bubbling N 2 gas. However, heating carbamoyl-

PROXYL with GSH in the N 2 gas atmosphere showed temporally

enhanced signal decay. Results showed a possibility that heating an

aqueous solution containing oxygen can generate O 2 •- .

127


High-resolution redox imaging of intact cells by rxYFP,

a ratiometric oxidation- sensitive fluorescent protein

Giuseppe Maulucci 1 , Marina Mele 2 , Valentina Labate 2 ,

Marco De Spirito 1 , Tommaso Galeotti 2 , and Giovambattista Pani 2

1

Institute of Physics,LABCEMI Microscopy Core Facility and 2 Institute of

General Pathology, Unit of Redox Signaling, Catholic University Medical

School, Largo F. Vito #1, 00168 Rome, Italy

Plasmid-encoded oxidation sensitive fluorescent probes promise

to pave new avenues for realt time imaging of redox signaling in

live cells. We have recently shown that rxYFP, a redox-sensitive

variant of the Yellow Fluorescent Protein, can be used ratiometrically

to construct high resolution redox maps of live cells. Confocal

analysis of cell fluorescence at two excitation wavelenghts allows in

fact to monitor, voxel by voxel, the distribution of the probe between

its reduced and oxidised states, while normalizing fror probe

concentration and photobleaching. By taking advantage of this

property, we were able to confirm that, in human and murine malignant

cells, the nucleus is significantly more reduced than the cytosol;

additionally, simple deconvolution of redox images constructed

with untargeted rxYFP allowed to identify hypereduced

perinuclear spots corresponding to mitochondria, as further confirmed

by the use of a mitochondrially targeted form of the probe.

In 2D scans, the cell border of adherent cells consistently appears

to be significantly more oxidised than the inner cytosol, while in

3D cell reconstruction oxidation is polarized towards the bottom of

the cell; finally, in a spontaneously migrating cell, the leading front

appears to me more oxidised than the trailing edge. This set of observations

suggests, in keeping with our previous biochemical studies,

that integrin signaling and cytoskeleton rearrangement are associated

with local pro-oxidant activity, with relevant implications

for cell invasion and metastasis. Taken together these examples underscore

the power of rxYFP for the real time, high resolution

analysis of redox signaling in cultured cells.

128

Cellular migration induced by nitric oxide involves the

participation of the GTP-binding proteins Ras and Rac

H.P. Monteiro 1 , R. Eller-Borges 1 , M. Curcio 1 , M.S. Moraes 1 ,

and W.L. Batista 2

1 Department of Biochemistry – UNIFESP – Campus São Paulo, SP – Brazil.

2 Department of Biological Sciences – UNIFESP –

Campus Diadema, SP – Brazil

The small GTP-binding proteins Ras and Rac are molecular

switches exchanging GDP for GTP and converting external signals

in response to a variety of stimuli. Ras and Rac play important

roles in cell proliferation, cell differentiation, and cell migration.

Particularly in the case of Rac, the protein is directly involved in

the reorganization and changes in the cytoskeleton during cell motility.

It is known that nitric oxide (NO) is involved in cell migration,

however only few studies addressed the relationships between

NO and Rac in signaling processes associated with cell migration.

On the other hand, it is well established that NO stimulates the Ras

– ERK1/2 MAP kinases signaling pathway a . In addition, NO also

stimulates the interactions between Ras and the PI3-Kinase signaling

pathway b . In this communication, we describe the participation

of NO in the activation of Rac in rabbit aortic endothelial cells

(RAEC). RAEC cells were stimulated with bradykinin, which

promotes endogenous production of NO. We showed that endogenous

production of NO stimulated Rac and promoted cell migration

over a time period of 24 h. The use of specific inhibitors of

PI3-Kinase and of Ras, resulted in inhibition of NO-stimulated Rac

activation. These results were corroborated by using RAEC cells

permanently transfected with the negative dominant mutant of Ras.

In conclusion, the NO-mediated activation of Rac involves the direct

participation of PI-3 Kinase and Ras.

a FRBM 35: 381-396, 2003.

b JBC 273: 29923-29928, 1998.

Financial Support: We acknowledge the financial support of FAPESP (09/

52730-7) and CNPq

129


Age related oxidase (arNOX) implicated in aging-related

oxidative damage to skin proteins

Dorothy M. Morré 1,2 , D. James Morré 1,2 , Christiaan Meadows 1 ,

Zoe D. Draelos 3 , and Dale Kern 4

1 NOX Technologies, Inc., 1291C Cumberland Avenue, West Lafayette, IN

47906, 2 Purdue University, West Lafayette, IN 47907, 3 2444 N. Main Street,

High Point, NC 27262 and 4 Dale Kern, NuSkin International, 75 W. Center

Street, Provo, UT 84601

Little is known about the sources of reactive oxygen species in

skin that result in protein oxidation and cross-linking of potential

importance to skin aging. Our work has identified arNOX a cell

surface-located hydroquinone oxidase inhibited by coenzyme Q 10

that generates superoxide. arNOX appears at about age 30 and then

increases with increasing age to about age 65. The protein is shed

into body fluids including sera, urine, perspiration and saliva.

arNOX activity may be measured spectrophotometrically using reduction

of ferricytochrome c as the method of quantitation.

arNOX-catalyzed oxidation of protein bound tyrosines was measured

using a fluorescence-labeled tyrosine analog, tyramine, which

when coupled to proteins carrying a tyrosyl radical, rendered the

proteins fluorescent. The assay demonstrated the potential for

arNOX-induced oxidative damage (dityrosine formation) to human

collagen and elastin and to surface proteins of human fibroblasts

and frozen sections from epidermal punch biopsies. Additionally,

autofluoresence indicative of Advanced Glycation Endproducts

correlated with the arNOX activity of epidermal biopsy specimens

consistent with a role for arNOX as a major source of oxidative

damage and cross-linking of skin proteins amenable to topical

(AgeLoc) arNOX-specific inhibitor combinations.

NAC modulates homoaggregation, viability and

proliferation of CD40-activated human B cells

Philippe Nadeau 1 and Sonia Néron 1

1 Héma-Québec, Recherche et Développement, Québec, Canada, G1V 5C3

CD40 ligation by CD154 (CD40 ligand) is essential to B-cell

activation in the germinal center. We have developed a co-culture

model in which proliferation and Ig secretion by purified human

blood B cells can be regulated by the intensity of the CD40-CD154

interaction. In this system, CD154 + fibroblasts and cytokines are

used as surrogates for activated T-cells. Recent studies on tonsillar

B cells, as well as human and mouse B-cell lines, showed that

variations in REDOX potential accompany CD40-mediated B-cell

regulation. Indeed, CD40-induced Reactive Oxygen Species (ROS)

modulate signalling pathways involved, for example, in cell survival,

differentiation and Ig secretion. One objective of our study

was to determine the role of REDOX homeostasis in CD40-

mediated B–cell activation. As a first step, we have tested whether

B-cell homeostasis could be modulated by decreasing REDOX potential.

N-acetylcysteine (NAC), a thiol molecule with antioxidant

properties, was added at 10 and 20 mM to B cells stimulated with a

high ratio of CD154 + cells. NAC treatment induced strong homoaggregation

of B cells, increased cellular viability, while decreasing

proliferation. Besides, analysis of CD45 + CD19 + Ax405 - cells by

flow cytometry indicated that NAC-treated B cells decreased in

size and granularity and exhibited a lower relative fluorescence for

5-(and 6-)chloromethyl-2 ’ ,7 ’ -dichlorodihydrofluorescein diacetate

(CM-H 2 DCF-DA), a ROS detection reagent. Using Fluorescent

Cell Barcoding (FCB), we observed that various signalling pathways

were indeed positively or negatively modulated in NACtreated

B cells. Overall, our results suggest that variations in RE-

DOX potential modulate viability and proliferation of CD40-

activated B cells, likely via activation/inhibition of distinct signalling

pathways. Whether REDOX potential could be altered by the

strength of the CD40 signal, and the precise mechanisms by which

ROS modulate cultured B cells, remain to be further investigated.

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131


Design of pH-sensitive polymeric micelle possessing reduced

forms of TEMPO for imaging of ROS. Anti-oxidative stress

nanoparticles for novel theranostics

Yukio Nagasaki

Graduate School of Pure and Applied Sciences, Graduate School of

Comprehensive Human Sciences, Tsukuba Research Center for Interdisciplinary

Materials Science, Tsukuba Advanced Research Alliance, University of Tsukuba

and Satellite Laboratory of International Center for Materials

Nanoarchitechtonics, Ten-noudai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan

Reactive oxygen species (ROS) are known to play versatile roles

on the occasion of many important events. However, excessive production

of ROS causes significant adverse effect to living body. Such

oxidative stresses must be controlled appropriately. For example, arterial

re-canalizations achieved by thrombolysis and intravascular intervention

are main treatment strategies to restore blood supply in

ischemic stroke and heart attack. However, reperfusion has a dilemma

of ischemia-reperfusion injury caused by ROS, which is produced after

a long ischemic period and can extend a damaged area. Therefore, protection

of organs affected by ROS has been perused not to cause the

larger damaged area than that associated with the arterial occlusion. It

is known that stable nitroxyl radical such as 2,2,6,6-tetramethylpiperidin

1-oxyls (TEMPO) reacts effectively with ROS compounds.

However, these compounds have not been applied clinically because

they were inactivated due to the acute reduction by anti-oxidant systems

such as catalase, glutathione peroxidase in vivo. In order to overcome

poor bioavailability and biocompatibility, we developed a novel

core-shell type nanoparticle containing nitroxyl radicals, amino-

TEMPO, and named radical-containing-nanoparticle (RNP). RNP

formed a micelle possessing amino-TEMPO in the core under the

physiological conditions, and demonstrated high performances of the

prolonged blood circulation time by the compartmentalization of

TEMPO into the micelle and pH-sensitivity to help TEMPO radicals

act as antioxidant due to the collapse of nanoparticle in low pH condition

such as ischemic-tissue in vivo. The preparation, physicochemical

and biological characterization and anti-oxidant properties against

ischemia-reperfusion injury will be summarized in this paper.

The author would like to express his sincere appreciation to Professors H.Matsui, A.Matsumura,

K.Suzuki, Drs. T.Mamiya, Mr.Marushima, (Medical School of University of Tsukuba),

A.Hirayama, (Tsukuba Tech. University), Dr.H.Tsurushima (AIST), Dr.K.Toh, D.Miyamoto and

Mr.T.Yoshitomi (TIMS, University of Tsukuba) for their collaboration.

Intramolecular base-accelerated radical-scavenging reaction

by vitamin E derivatives bearing a pyridine moiety

Ikuo Nakanishi 1 , Haruko Yakumaru 2 , Minako Nyui 1 , Kei Ohkubo 3 ,

Ken-ichiro Matsumoto 1 , Kiyoshi Fukuahra 4 , Haruhiro Okuda 4 ,

Shunichi Fukuzumi 3 , Toshihiko Ozawa 1,5 , Kazunori Anzai 1 , and

Nobuo Ikota 2,6

1 Research Center for Charged Particle Therapy, National Institute of

Radiological Sciences, Chiba 263-8555, Japan, 2 Research Center for Radiation

Emergency Medicine, National Institute of Radiological Sciences, Chiba 263-

8555, Japan, 3 Department of Material and Life Science, Graduate School of

Engineering, Osaka University, Osaka 565-0871, Japan, 4 Division of Organic

Chemistry, National Institute of Health Sciences, Tokyo 158-8501, Japan,

5 Department of Health Pharmacy, Yokohama College of Pharmacy, Yokohama

245-0066, Japan, and 6 School of Pharmacy, Shujitsu University, Okayama 703-

8516, Japan

Fine-tuning of the radical-scavenging activity of antioxidants is

of considerable importance with regard to the development of effective

chemopreventive agents against oxidative stress and associated

diseases. We have reported that the scavenging reaction of

DPPH • (2,2-diphenyl-1-picrylhydrazyl radical) by 2,2,5,7,8-pentamethylchroman-6-ol

(PMC), a vitamin E model, proceeds via a

proton-coupled electron transfer in deaerated methanol (MeOH)

and that pyridines significantly accelerate this reaction by stabilizing

a radical cation intermediate derived from PMC. 1 In this study,

we synthesized three vitamin E derivatives bearing a pyridine moiety

with a linkage of different spacers. All the vitamin E derivatives

thus obtained efficiently scavenged DPPH • in deaerated

MeOH. The second-order rate constant for DPPH • -scavenging by

one of the vitamin E derivatives determined by the stopped-flow

technique is about 3-fold larger than that by PMC. The DPPH • -

scavenging activity is found to be depending on the length of the

spacers.

Nakanishi, I. et al. Org. Biomol. Chem. 2005, 3, 626.

132

133


Beneficial effects of vitamin E on the mitochondrial

dysfunction in hippocampus and frontal cortex of aged rats

Ana Navarro 1 , Manuel J. Bandez 1 , José M. López-Cepero 2 ,

Carmen Gómez 1 , Alejandro D. Boveris 3 , Enrique Cadenas 4 , and

Alberto Boveris 3

1 Department of Biochemistry and Molecular Biology, and 2 Department of Cell

Biology and Histology, Faculty of Medicine, University of Cádiz, 11003 Cádiz,

Spain; 3 School of Pharmacy and Biochemistry, University of Buenos Aires,

C1113AAD Buenos Aires, Argentina; and 4 Department of Pharmacology and

Pharmaceutical Sciences, School of Pharmacy, University of Southern

California, Los Angeles, CA 90089-9121. (anavarro@uca.es)

High doses of vitamin E improved brain mitochondrial function

and neuromuscular and exploratory performances and increased

median life span in aging mice (Navarro et al., Am J

Physiol 289: R1392-R1399, 2005). Age-dependent mitochondrial

dysfunction is more marked in rat hippocampus and frontal cortex

that in whole brain (Navarro et al., Am J Physiol 294: R501-R509,

2008). Such observations prompted us to test vitamin E in the mitochondrial

function of rat hippocampus and frontal cortex upon

aging. Dietary supplementation with vitamin E (2.0 or 5.0 g/kg of

food) from 9 to 12 mo of age was effective in preventing by 93 %

the age-dependent decrease in hippocampus and frontal cortex respiration

from 4 to 12 mo of age. Vitamin E also restored by 95 %

the selective impairment of complexes I and IV activities. The two

effects, inactivation by aging and restoration by vitamin E, were

observed in mtNOS activities, biochemical and functional, in hippocampal

and frontal cortex mitochondria. Vitamin E also prevented

the increase in oxidation products (TBARS and protein carbonyls)

and in H 2 O 2 production in hippocampal and frontal cortex

mitochondria. Hippocampal mitochondrial mass was moderately

decreased, by 19 %, upon aging, an impairment that was restored

by vitamin E.

134

Squalene sensitizes anti-cancer therapy through the inhibition

of DNA damage checkpoint activity by Wip1

Hiroshi Nishida 1 , Naoto Tatewaki 1 , Yuki Nakajima 1 ,

Kayoko Nakayama 1 , Nobuo Ikekawa 2 , and Tetsuya Konishi 1

1 Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan.

2 Niigata Bio Research Center, Niigata, Japan

ATM (Ataxia Telangiectasia Mutated) protein kinase plays a crucial

role in cellular DNA damage responses. The inhibition of ATM

leads to an abolishment of one such signal pathway termed as “checkpoints”.

It is expected that the discovery of checkpoint inhibitor will be

an effective assistance of anti-cancer therapy. Recent study reported

that Wip1, a magnesium dependent protein phosphatase, dephosphorylates

serine 1981 (Ser1981) of ATM during the DNA damage

checkpoint response. On the other hand, Squalene (SQ) has been

thought to assist anti-cancer therapies such as chemo and/or radiation

therapy though the detailed mechanism is unknown. Here, we report

the inhibitory effect of SQ on ATM-dependent checkpoint signaling

pathway. SQ itself did not affect on cell cycle distribution of A549

human lung adenocarcinoma cells. The cell viability was decreased by

SQ treatment after UV (25, 50 and 100 J/m 2 ) or gamma-irradiation

(IR; 2 and 6 Gy) in SQ dose-dependent manner (10, 30, 100 mM). SQ

treatment disrupted G2/M checkpoint in DNA damaged cells analyzed

using phosphor-Histone H3 antibody (Ser10). IR-induced phosphorylations

of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) were

remarkably inhibited by the treatment of SQ in cells. However, In vitro

kinase activities of ATM and AT and Rad3 related (ATR) toward

PHAS-I were not inhibited by SQ. Of importance, SQ increased intracellular

Wip1 protein expression in both of unperturbed and perturbed

cells, and suppressed ATM activation in IR treated cells. The enhanced

expression of Wip1 by SQ treatment lasted for 12 h, and was confirmed

by Wip1-siRNA experiment. Consistent with potential inhibition

of ATM by SQ, xenograft study demonstrated that the tumor

growth of MKN45 gastric cancer cell in vivo was obviously inhibited

by SQ treatment in X-ray exposed or doxorubicine treated mice, significantly.

The expression of Wip1 in tumors were also increased by

oral administration of SQ. Taken together these findings, SQ inhibits

ATM protein kinase activity in cells following DNA damage through

the intracellular induction of Wip1 expression, and was confirmed by

xenograft study with radio/chemo-therapies.

135


Luteolin exerts the anti-gut inflammatory activity

on dextran sulfate sodium induced colitis in mice

Yosuke Nishitani 1 , Yuzo Koda 2 , and Masashi Mizuno 2,3

1 Organization of Advanced Science and Technology, Kobe University, Kobe,

Japan; 2 Faculty of Agriculture, Kobe University, Kobe, Japan; 3 Department of

Agrobioscience, Graduate School of Agricultural Science, Kobe University,

Kobe, Japan

Luteolin is a flavonoid present in significant amounts in vegetables

including celery and carrots, which has anti-inflammatory activities

both in vivo and in vitro. However, the impact of luteolin on

intestinal inflammation has not yet been fully elucidated. In this

study, we evaluated the anti-gut inflammatory activity of luteolin using

in vivo and in vitro gut inflammation models. Colitis was induced

in C57BL/6 mice by the administration of 2% dextran sulfate

sodium (DSS) to drinking water. Luteolin (5, 20, 50 mg/kg) was administered

intragastrically daily, one week prior to commencement

of DSS treatment. Oral administration of 20 or 50 mg/kg of luteolin

significantly ameliorated the shortening of colon length (6.5 ± 0.2

cm and 6.4 ± 0.2 cm, respectively), compared with untreated DSSinduced

colitis mice (5.6 ± 0.2 cm). Similar results were obtained

from histologic analysis of the colon sections in DSS-induce colitis

mice. In addition, the treatment with 20 or 50 mg/kg of luteolin had a

tendency to inhibit the aberrant mRNA expression in inflamed tissue

through notable suppression of TNF-, IL-6, IL-1, and IFN-

mRNA expression. In an in vitro gut inflammation model with the

co-culture system, consisting Caco-2 cells (apical side) and

RAW264.7 cells stimulated by LPS (basolateral side), treatment with

luteolin (100 μM) from apical chamber significantly inhibited TNF-

production from RAW264.7 and IL-8 mRNA expression in Caco-

2. HPLC analysis, post stimulation by LPS, revealed the presence of

luteolin (4 μM) and its glucuronate conjugate (10 μM) in the basolateral

chamber. Furthermore, TNF- production by LPS-stimulated

RAW264.7 was significantly inhibited by the direct treatment of 4

μM luteolin to RAW264.7. In summary, these findings seem to suggest

that oral administration of luteolin exhibits the antiinflammatory

activity in the gut and the agent which exerts the activity

may be luteolin itself and not its metabolite.

136

Zinc and ERK regulation in neurons and in the

developing rat brain

Johnathan R. Nuttall, Yin Htet, and Patricia I. Oteiza

Departments of Nutrition and Environmental Toxicology,

University of California, Davis, Davis, CA 95616, USA

We previously observed that severe and marginal zinc nutrition

during gestation can disrupt brain development and affect associated

cell signals, including the extracellular signal regulated

kinases (ERK). ERK1/2 have central roles in modulating neurogenesis

and neuronal survival, and their inhibition could underlie

the adverse effects of developmental zinc deficiency. We currently

investigated the underlying mechanisms leading to ERK1/2 inhibition

when zinc decreases. Zinc deficiency caused a marked decrease

in ERK1/2 phosphorylation at Thr202 and Tyr204 in proliferating

IMR-32 neuroblastoma cells and differentiated cortical

neurons. Marginal zinc deficiency imposed to rats throughout gestation

led to low ERK1/2 activation in the offspring brain cortex at

postnatal day 2 (P2). Accordingly, a decrease in cellular zinc impaired

the activation of ERK1/2 downstream targets (nuclear

MSK-1). Upstream events in the ERK1/2 pathway, c-Raf (Ser338)

and MEK1/2 (Ser217/Ser221) phosphorylation, were not affected

by zinc deficiency in neuronal cells and P2 rat brain cortex. These

findings suggest that ERK1/2 inactivation in zinc deficiency could

be due to altered activities of downstream phosphatases. PP2A, is

a serine phosphatase regulated by zinc and that plays a key role in

the inactivation of ERK2. While total phosphatase activity was not

affected by zinc deficiency, PP2A activity increased with the time

of exposure to zinc deficient media in IMR-32 cells. In summary,

low zinc availability decreases ERK1/2 phosphorylation in neuronal

cells in culture and in the developing rat brain, which is in

part due to an increased PP2A activation. ERK1/2 inactivation

could explain the adverse effects of zinc deficiency on neurogenesis,

neuronal survival, and ultimately on brain development.

Supported by UC Davis and NIH (HD01743)

137


Rapid diffusion of hydrogen protects the retina:

Administration to the eye of hydrogen-containing saline in

retinal ischemia-reperfusion injury

Hideaki Oharazawa 1 , Tsutomu Igarashi 2 , Takashi Yokota 3 ,

Hiroaki Fujii 1 , Hisaharu Suzuki 2 , Mitsuru Machida 4 ,

Hiroshi Takahashi 2 , Shigeo Ohta 5 , and Ikuroh Ohsawa 4

1 Department of Ophthalmology, Musashikosugi Hospital, Nippon Medical

School, Kanagawa, Japan, 2 Department of Ophthalmology, Nippon Medical

School, Tokyo, Japan, 3 Department of Molecular Biology, 4 The Center of

Molecular Hydrogen Medicine and 5 Department of Biochemistry and Cell Biology,

Institute of Development and Aging Sciences, Nippon Medical School, Kanagawa,

Japan

Introduction: Retinal ischemia-reperfusion (I/R) injury by transient

elevation of intraocular (IOP) is known to induce neuronal damage

through the generation of reactive oxygen species. Previous studies

indicate that molecular hydrogen (H 2 ) is an efficient antioxidant gas

that selectively reduces the hydroxyl radical (•OH) and suppresses

oxidative stress-induced injury in several organs. This study was conducted

to explore the neuroprotective effect of H 2 -loaded eye drops on

retinal I/R injury. Methods: Retinal ischemia was induced in rats by

raising IOP for 60 minutes. H 2 -loaded eye drops were prepared by dissolving

H 2 gas into a saline to saturated level and administered to the

ocular surface continuously during the ischemia and/or reperfusion periods.

One day after I/R injury, apoptotic cells in the retina were quantified

and oxdative stress was evaluated by markers such as 4-

hydroxynonenal and 8-hyroxy-2-deoxyguanosine. Seven days after I/R

injury, retinal damage was quantified by measuring the thickness of

the retina. Result: When H 2 -loaded eye drops were continuously administered,

H 2 concentration in the vitreous body immediately increased

and I/R-induced •OH level decreased. The drops reduced the

number of retinal apoptotic and oxidative stress marker-positive cells,

and prevented retinal thinning with an accompanying activation of

Müller glia, astrocytes and microglia. The drops improved the recovery

of retinal thickness by > 70%. Conclusions: Our results suggest

that H 2 -loaded eye drops will be a highly useful neuroprotective and

anti-oxidative therapeutic treatment for acute retinal I/R injury.

The oxidative potential of hyperbaric oxygen treatment:

A summary of recent experimental work focused on its

exposure time and pressure-related oxidative effects

Sukru Oter 1 , Ahmet Korkmaz 1 , Turgut Topal 1 , Serdar Sadir 1 ,

Mehmet Ozler 1 , Bulent Uysal 1 , Recai Ogur 2 ,

Kemal Simsek 3 , and Hakan Ay 3

Departments of 1 Physiology, 2 Public Health and 3 Undersea & Hyperbaric

Medicine; Gulhane Military Medical Academy; Ankara, Turkey

The risk for oxygen toxicity during hyperbaric oxygen (HBO)

exposure is a known fact. Our previous experimental works have

been focused the pressure- and exposure time-related correlation of

HBO. The persistence of oxidative stress markers following HBO

was also examined. A total of 162 Sprague-Dawley rats were used

for these experiments. The clinically approved maximal pressure/duration

range, namely 3 ATA/2 hours, was chosen as the

highest limits. After HBO exposure, the major target organs of hyperoxic

oxidative stress, i.e., the lung, brain and blood, were taken

for biochemical assays. Malondialdehyde (MDA) as indicator of

lipid peroxidation, and antioxidant enzymes superoxide dismutase

(SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were

the main measured parameters in these studies. In brain tissues, nitrite

plus nitrate values (NO X ), end products of nitric oxide degradation,

were also examined. MDA and SOD levels of blood, brain

and lung tissue as well as lung CAT activity revealed clear pressure-related

rises. Blood, brain and lung tissue MDA and SOD levels

also indicated a relation with HBO exposure time. NO X values

of brain tissue were also found to be increased at maximal exposure

levels. The measured oxidative stress markers usually returned

to their baseline levels within 90 min after HBO exposure.

In conclusion, within its approved limits for therapeutic use, the

safety of HBO was proven by these results. The parallel increase of

antioxidant enzymes and the relative short persistence of oxidation

products support this conclusion.

138

139


Melatonin as an ideal protective agent against

hyperbaric oxygen induced oxidative reactions

Sukru Oter 1 , Bulent Uysal 1 , Mehmet Ozler 1 , Turgut Topal 1 ,

Serdar Sadir 1 , Kemal Simsek 2 , Recai Ogur 3 , and Ahmet Korkmaz 1

Departments of 1 Physiology, 2 Undersea & Hyperbaric Medicine, and 3 Public

Health; Gulhane Military Medical Academy; Ankara, Turkey

Exposure to hyperbaric oxygen (HBO) leads to an increase of

dissolved oxygen in the blood and delivery to the tissues. Therefore

it has been successfully used for the treatment of a variety of

clinical conditions, especially, related to hypoxia. However, the

oxygen toxicity risk of HBO has also been of scientific interest.

Recently, our research team set a series of studies examining the

use of melatonin as a protecting antioxidant against this stressing

condition. In these experiments the effectiveness of exogenously

administered and endogenously secreted melatonin was tested in

acutely exposed and chronically administered HBO treatments. A

total of 247 Sprague-Dawley rats were used for the experimental

sets. Acute one session HBO was administered at 3 ATA for 2 h.

Chronic HBO exposure was set as daily 60 min sessions at 2.5

ATA for 10 days. Exogenous melatonin was injected i.p. prior to

HBO sessions at the doses of 10 mg/kg in the one session and 5

mg/kg in the chronic exposure groups. After HBO exposure blood,

lung and brain tissues were harvested for assays. Oxidative stress

and antioxidant system status were examined. A clear increasing

effect of HBO on almost all oxidative parameters was seen in all

experiments and the antioxidant enzymes presented also higher activities.

All experiments indicated a limiting effect of exogenously

injected as well as endogenously secreted melatonin on HBOinduced

oxidative action. The present experimental results warrant

melatonin as an effective agent against HBO-induced oxidative

stress, but have to be supported by clinical trials in order to be

adopted into clinical conditions.

Beneficial effects of spirulina on non-alcoholic steatohepatitis

model rats by anti-oxidative and anti-inflammatory activities

Wing Pak 1 , Fusako Takayama 1 , Mitsumasa Mankura 1 ,

Toru Egashira 1 , Hiromu Kawasaki 1 , Yasumasa Kodo 2 ,

Manaka Mine 1 , Shigeru Okada 1 , and Akitane Mori 1

1 Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences,

Okayama University, Japan; 2 Spirulina Bio-Lab. Co., Ltd.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease

characterized by necroinflammatory activity, hepatocyte ballooning

with Mallory’s hyaline, and occasional fibrosis. The “two-hit

theory” is widely accepted as the mechanism of NASH pathogenesis.

Recently, Spirulina (SP), a blue-green algae, is drawn more attention,

because of its anti-oxidative, nutritional and medicinal

properties. The aim of this study is to investigate the SP effect on

NASH and to elucidate the mechanism. Methods: Fee radical scavenging

activities of SP against O 2 • , HO • , 1 O 2 and ONOO – were examined

by electron spin resonance method and fluorophotometric

measurement. 8-Hydroxy 2’-deoxyguanosine was quantified by

our ELISA assay. Animal experiment was using NASH model

(PCT/JP 2007/52477) were induced fatty liver in rats by feeding

them a choline deficient high fat diets (CDHF), then treated in addition

with oxidative stress (OS). Animal experiment was performed

with 5 groups; Control (with MF chow diets), CDHF,

NASH, NASH+2SP and NASH+6SP. After experimental period,

blood and liver were collected from rats under anesthesia, to determine

OS related injuries and Western blotting analysis such as

NF-B. Results: SP was shown to have anti-oxidative activities.

The hepatobiliary enzyme leakages and liver fibrosis were exhibited

in NASH rats. The decrease of plasma SOD like activity, and

increases of plasma myeloperoxidase activity, serum iron level,

and the reactive oxygen species from the liver mitochondrial metabolism,

and the NF-B nuclear import were also demonstrated in

NASH rats. The SP administration significantly abated these

changes. Conclusion: The present study demonstrated SP could

prevent the NASH progression through anti-oxidative and antiinflammatory

activities.

140

141


Regulation of mevalonate pathway and Ras signalling by

lycopene in prostatic cancer cells

Paola Palozza, Rossella Simone, Assunta Catalano, and

Maria Colangelo

Institute of General Pathology, Catholic University School of Medicine, 00168

Rome, Italy. E-mail: p.palozza@rm.unicatt.it

Cancer cells have abnormal cholesterol biosynthetic pathways

and farnesylation is a key process in oncogene activation. Tomato

lycopene has been suggested to prevent the risk of prostate cancer,

although the exact molecular mechanism(s) is still unknown. Since

the carotenoid share similar synthetic pathways with cholesterol,

we tested the hypothesis that it may exert its antitumor effects

through changes in mevalonate pathway and in Ras activation. Incubation

of the Ras-activated prostatic carcinoma LNCaP cells,

with a 24-h lycopene treatment (2.5-10 μM) dose-dependently reduced

intracellular total cholesterol by decreasing 3-hydroxy-3-

methylglutaryl-coenzyme A (HMG-CoA) reductase expression and

by inactivating Ras, as evidenced by its translocation from cell

membranes to cytosol. Concomitantly, lycopene reduced the Rasdependent

activation of NF-kB via inhibition of ROS production

and decreased phosphorylation of JNK, ERK1/2 and p38. These

effects were accompanied by an arrest of cell cycle progression

and by apoptosis induction, as evidenced by a decrease in cyclin

D1 and pAKT levels, and by an increase in p21, p27 and p53 levels

and in Bax/Bcl-2 ratio. The addition of mevalonate prevented

the growth-inhibitory effects of lycopene as well as its increase in

Ras cytoplasmatic accumulation and the subsequent changes in

NF-kB. The ability of lycopene in inhibiting HMG-CoA reductase

expression and cell growth and in inactivating Ras was also found

in prostatic PC-3 cancer cells. These findings provide a novel

mechanistic insight into the preventive effects of lycopene in human

prostate cancer.

142

Glutathione disulfide as a cell death signal

Han-A Park, Savita Khanna, Cameron Rink, Surya Gnyawali,

Natalia Kubicki, Sashwati Roy, and Chandan K. Sen

Department of Surgery, Laboratory of Molecular Medicine, Davis Heart &

Lung Research Institute, The Ohio State University Medical Center,

Columbus, OH, 43210

Reduced glutathione (GSH) is a low molecular weight

intracellular thiol in all aerobic cells. Under conditions of oxidative

stress, large amounts of GSH are rapidly oxidized to GSSG. Cell

death is often associated with high GSSG levels. Such results are

interpreted as evidence for oxidative stress without addressing any

functional significance of GSSG in the death process. We hypothesized

that under specific conditions, GSSG functionally participates

in signaling for cell death.

Mechanisms that trigger the oxidation of GSH to GSSG in a

cell such as exposure to ROS or to ROS-generating cytokines also

induce numerous other cellular responses. Thus, it is challenging to

dissect which of those responses actually contributed to the death

process. To address this complication, we raised cellular GSSG

content by microinjection. Control cells were injected with either

GSH or vehicle. GSSG, but not GSH, caused cell death at physiologically

relevant concentrations. GSSG-induced cell death was

protected in the presence of the 12-lipoxygenase(Lox) inhibitors.

Previous work from our laboratory has identified 12-Lox as a key

executioner of neural cell death. Results of this study indicate that

GSSG induces 12-Lox dependent cell death. Furthermore, GSSGdependent

glutathionylation of 12-Lox was identified a critical

player in neural cell death. We tested the hypothesis by using glutaredoxin,

a deglutathionylating enzyme, and noted that glutaredoxin

transfected cells were protected against glutamate challenge.

To test the significance of our findings, GSSG was stereotaxically

injected to the brain in vivo and MRI was performed to quantify

tissue lesion. Strategies directed at improving or arresting of intracellular

GSSG clearance may be effective in minimizing oxidative

stress-related tissue injury or potentiating the killing of tumor cells,

respectively. This work unfolds a new paradigm that is likely to

extend beyond the specific cell type studied and shed light on fundamental

mechanisms underlying cell death.

143


The time-dependent effect of Provinols TM on brain

NO synthase activity in experimental hypertension

O. Pechanova, L. Jendeková, and S. Vranková

Institute of Normal and Pathological Physiology, Slovak Academy of Sciences,

Bratislava, Slovak Republic

Red wine polyphenols have been reported to possess beneficial

properties for preventing cardiovascular diseases but their neuroprotective

effects during chronic L-NAME treatment have not been

elucidated. The aim of this study was to analyze a time course of

Provinols TM effects on brain NO synthase activity and oxidative

damage in L-NAME-induced hypertension. Male Wistar rats, 12

weeks old, were divided into six groups: control groups, groups

treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 40

mg/kg/day) for 4 or 7 weeks and groups receiving Provinols TM (40

mg/kg/day) plus L-NAME for 4 or 7 weeks. At the end of the

treatment, marker of membrane oxidative damage - conjugated dienes

(CD) in the brain and NO synthase activity in the cerebral

cortex, cerebellum and brainstem were determined. L-NAME

treatment for 4 or 7 weeks led to the increase in blood pressure,

elevation of CD concentration and decrease of NO synthase activity

in the brain parts investigated. Provinols TM partially prevented

blood pressure rise and elevation of CD concentration. Comparing

to the L-NAME treated group, Provinols TM increased NO synthase

activity after 4 weeks of treatment. However, the prolonged Provinols

TM treatment for 7 weeks had no effect on NO synthase activity

decreased by L-NAME treatment. In conclusion, Provinols TM partially

prevents L-NAME induced hypertension via the different

mechanisms depending on the duration of treatment. Prevention of

oxidative damage in the brain with modulating effect on NO synthase

activity is suggested.

The study was supported by APVV-0538-07 and VEGA 2/0178/09.

Different mechanisms of melatonin and N-acetylcysteine

effects in adult spontaneously hypertensive rats

O. Pechanova 1 , J. Zicha 2 , L. Paulis 1 , S. Vranková 1 ,

Z. Dobesová 2 , and J. Kunes 2

1 Institute of Normal and Pathological Physiology, Slovak Academy of Sciences,

Bratislava, Slovak Republic, Institute of Physiology, Academy of Sciences of

Czech Republic, Prague, Czech Republic

The imbalance in nitric oxide (NO) and reactive oxygen species

production is often found in both experimental and human hypertension.

The aim of our study was to determine possible effects

of N-acetyl-5-methoxytryptamine (melatonin, 10 mg/kg/day) and

N-acetylcysteine (1.5 g/kg/day) in adult spontaneously hypertensive

rats (SHR) with established hypertension. After a six-weektreatment,

blood pressure was measured and NO synthase (NOS)

activity, concentration of conjugated dienes, protein expression of

endothelial NOS, inducible NOS and nuclear factor-kappaB (NFkappaB)

in the left ventricle were determined. Both treatments improved

the NO pathway by means of enhanced NOS activity and

reduced reactive oxygen species level as indicated by decreased

conjugated diene concentrations and lowered NF-kappaB expression.

N-acetylcysteine (but not melatonin) also increased the endothelial

NOS protein expression. However, only melatonin was able

to reduce blood pressure significantly. Subsequent in vitro study

revealed that both N-acetylcysteine and melatonin lowered the tone

of phenylephrine-precontracted femoral artery via NO-dependent

relaxation. Nevertheless, melatonin-induced relaxation also involved

NO-independent component which was preserved even after

the blockade of soluble guanylate cyclase by oxadiazolo[4,3-

a]quinoxalin-1-one. In conclusion, both N-acetylcysteine and

melatonin were able to improve the NO/reactive oxygen species

balance in adult SHR, but blood pressure was significantly lowered

by melatonin only. This implies that a partial restoration of

NO/reactive oxygen species balance achieved by the antioxidants

such as N-acetylcysteine has no therapeutic effect in adult rats with

established hypertension. The observed antihypertensive effect of

melatonin is thus mediated by additional mechanisms independent

of NO pathway.

The study was supported by APVV-0538-07 and VEGA 2/0178/09.

144

145


Vitamin A and ,-carotene oxidative reactive secondary

sequence chain-growth model explaining antioxidant-related

mortality rate increase

Richard C. Petersen

University of Alabama at Birmingham

The ,-carotene antioxidant analogue for vitamin A was identified

in a smoker study with 29,133 men to form increased risks

for lung cancer in addition to total mortality related to numerous

other varied pathologies such as coronary heart disease, stroke, hypertension

and cardiomyopathy JAMA 290:476-485; 2003. Conjugated

isoprene carotenoids that act as antioxidants have previously

been considered as a source for C=C double-bond addition reactions

by free radicals with the large -system. So, for free-radical

pathogenesis, a chain-growth cardiovascular disease (CVD) polymer

model was tested. 100 gelatin capsules containing a lowviscosity

liquid of vitamin A 8000IU/capsule or ,-carotene

25,000IU vitamin A/capsule, Nature Made, Mission Hills, CA,

were incised and squeezed into separate containment wells 4.0mm

deep and 9.0mm diameters. Each antioxidant was combined with a

Fenton-metal redox couple consisting of cobalt naphthenate and

dibenzoyl peroxide, Sigma Aldrich, St. Louis, MO, to test expected

viscosity increases. When 4% cobalt naphthenate and 3% dibenzoyl

peroxide were added slowly over a 3 day period to both vitamin

A and ,-carotene, viscosity suddenly increased to a point

where both low-viscosity antioxidants began to gel into a sticky

solid for a CVD atherosclerosis model. All liquid flow stopped after

4 days for vitamin A and 9 days for ,-carotene. Over time a

cancer-related model for polymerization cure shrinkage estimated

at approximately 50vol% occurred where both liquid-oil antioxidants

gelled and adhered to the containment substrate surface. Both

polymers also interlaced as ruffled ribbon-like networks glued to

the wells underneath. Further, 0.5mm films exposed to oxygen air

polymerized with shrinkage as harder material for a reperfusion injury

model.

146

Antioxidant properties and protective effects of raw,

boiled and fried garlic on DNA damage in

hypercholesterolemic hamsters

Yara S. Queiroz 1 , Marcelo L. Ribeiro 2 , Demetrius P. Arçari 2 ,

Marcela P. Monteiro 1 , Geni R. Sampaio 1 , and

Elizabeth A.F.S. Torres 1*

1 Department of Nutrition - School of Public Health - University of São Paulo

(USP); 2 Clinical Pharmacology and Gastroenterology Unit - São Francisco

University Medical School, Bragança Paulista, S.P. Av. Dr. Arnaldo 715

São Paulo - S.P. - Brazil - 01246-904

Garlic (Allium sativum L.) is rich in several bioactive compounds

that can act as free radical scavengers. Oxidative stress

plays an important role in the pathogenesis of numerous chronic

age-related free radical-induced diseases. The aim of this study

was to evaluate the antioxidant activity of garlic as well as the ability

to influence DNA in hypercholesterolemic hamsters. Fifty animals

were divided into five diet groups: control, hypercholesterol

(HC), raw garlic/HC, boiled garlic/HC and fried garlic/HC. The

experimental diets were supplemented with 5% lyophilized garlic

for 30 days. The antioxidant capacity was measured using oxygen

radical absorbance capacity (ORAC) and the DNA damage induced

was investigated by the comet assay. ORAC measured in

liver improved by 9%, 17% and 10% for raw garlic/HC, boiled

garlic/HC and fried garlic/HC in relation to control, respectively.

Our results demonstrate that garlic was able to decrease DNA

damage after intervention in liver cells, were significantly lower in

hypercholesterolemic animals treated with boiled garlic (TM =

5.31 ± 0.71) and raw garlic (TM = 7.57 ± 1.14) than those the hypercholesterol

group (TM = 8.69 ± 0.88). The data presented here

show that the forms usually consumed garlic is not genotoxic in

liver. The observed protection may be related to the antioxidant activity

of the garlic bioactive compounds.

147


The Janus face of supplemental oxygen in ischemic stroke:

New insight on therapeutic potential and identifying the

oxygen-sensitive transcriptome

Cameron Rink 1 , Sashwati Roy 1 , Mahmood Khan 2 , Pavan Ananth 1 ,

Periannan Kuppusamy 2 , Chandan K. Sen 1 , and Savita Khanna 1

1 Department of Surgery, 2 Department of Internal Medicine,

The Ohio State University Medical Center, Columbus, OH 43210

To date, the significance of targeting acute ischemic stroke (AIS)-

caused focal hypoxia remains unclear. Despite the support of case reports

and small animal research, three clinical pilot studies that probed

the efficacy of hyperbaric oxygen (HBO) to treat AIS reported insignificant,

or potentially harmful, outcomes. We hypothesize that conflicting

outcomes in literature stem from a limited window of benefit

for supplemental oxygen to treat AIS. The objectives of this study

were two-fold: (1) to define the therapeutic window of opportunity for

supplemental oxygen by employing normobaric (NBO) and HBO at

the onset of ischemia (iNBO, iHBO) or at reperfusion (rNBO, rHBO)

in a rodent model of middle cerebral artery occlusion (MCAO); and

(2) to employ high density transcriptome screening towards uncovering

oxygen-sensitive molecular mechanisms implicated in AIS. The

efficacy for supplemental oxygen to correct AIS-affected tissue pO 2

was evaluated using EPR oxymetry. Stroke lesion volume was quantified

using 4.7T MRI at 48h along with histochemical assessment of

oxidative stress and neurodegeneration. Unbiased query of oxygen responsive

gene networks in stroke-affected tissue was performed using

GeneChip. Verification of gene candidates was achieved by realtime

PCR from laser captured neurons in stroke affected brain tissue.

NBO was sufficient to correct penumbral tissue pO 2 during AIS. iNBO

and iHBO attenuated, while rNBO and rHBO exacerbated, AISassociated

lesion volume, oxidative stress and neurodegeneration. Directed

microarray analysis revealed key oxygen-sensitive gene networks

related to chemokine signaling and neutrophil recruitment. AIS

presents a temporal window of opportunity to minimize brain injury

using supplemental oxygen therapy. Findings provide key information

relevant to the successful design of clinical trials aimed at testing the

effects of supplemental oxygen in stroke affected patients. Supported by

NIH CTSA Pilot Award UL1 RR025755

148

Cyclic adenosine monophosphate (cAMP) mediates the

anti-inflammatory effects of LA and vitamin D

Sonemany Salinthone 1,2 , Vijayshree Yadav 1,2 ,

Dennis N. Bourdette 1,2 , and Daniel W. Carr 1,3

1 Portland Veterans Affairs Medical Center, Portland, OR 97239; 2 Department

of Neurology, Oregon Health & Sciences University, Portland, OR 97239;

3 Department of Endocrinology, Oregon Health & Sciences University, Portland,

OR 97239

Abnormal regulation of the inflammatory response is an important

component of many diseases such as diabetes, Alzheimer’s

disease and multiple sclerosis (MS). Lipoic acid (LA) and vitamin

D are anti-inflammatory agents that are being investigated as alternative/complementary

therapeutics for these conditions. LA is a

sulfur containing fatty acid that has been shown to decrease paralysis,

demyelination and axonal injury in the animal model of MS,

experimental autoimmune encephalomyelitis (EAE). Vitamin D is

a prohormone that has also been shown to be effective in ameliorating

clinical manifestations of EAE. However, the mechanisms

of action of LA and vitamin D are not completely understood. In

this study, we are examining the role of cAMP in mediating the effects

of LA and vitamin D. cAMP is a small molecule second

messenger that acts as a potent immunomodulator and is synthesized

upon activation of G-protein coupled receptors, such as the

histamine, adenosine and beta adrenergic receptors. We found that

LA inhibits IL-2, IL-6 and IL-17 production while enhancing IL-10

synthesis. Using pharmacological inhibitors, we determined that

LA stimulates cAMP production via activation of the histamine

and adenosine, but not the beta adrenergic receptors. In addition,

oral administration of LA to MS patients resulted in increased

cAMP levels. Average cAMP levels in 28 MS subjects were

27.9% higher four hours (13.195 ± 0.779) after ingestion of LA

compared to baseline (9.513 ± 1.271). We also discovered that vitamin

D treatment stimulates cAMP production and inhibits IL-6

secretion in T cell enriched PBMCs. Collectively, the data provide

evidence that LA and vitamin D may be effective as treatment alternatives

in inflammatory diseases by inhibiting the production of

pro-inflammatory mediators via activation of the cAMP signaling

pathway. The information also adds to our understanding of the

bioactivity of LA and vitamin D.

149


The modulation of the redox status of isolated brain

mitochondria by energy linked substrates:

Quantification by high performance liquid chromatography

Harsh Sancheti § , Jerome V. Garcia § , Li-Peng Yap § ,

Derick Han ‡ , and Enrique Cadenas §

§ Department of Pharmacology & Pharmaceutical Sciences, School of

Pharmacy, ‡ Research Center for Liver Diseases, Keck School of Medicine,

University of Southern California, Los Angeles, CA 90089-9121, USA

The balance between oxidized and reduced species, i.e., glutathione

(GSH/GSSG) and pyridine dinucleotides (NADH/NAD,

NADPH/NADP), determine the cellular redox status. Oxidative

stress and altered redox status are widely considered as major

components of aging and age-related diseases. The isolation of mitochondria

from organs is a widely used tool in studying mitochondrial

biology. However, Inherent in the long isolation process,

are alterations in mitochondrial redox status. Previous work from

our laboratory has shown that different isolation methods can alter

the redox status with respect to the glutathione pool. Substrate supplementation

of isolated mitochondria resulted in higher buffering

capacity against H 2 O 2 challenges, in part due to increased GSH

levels. The aim of the present study is to extend our previous work

and monitor changes in the pyridine dinucleotide pool. Upon substrate

supplementation, changes in the pyridine dinucleotide pool

were quantified using HPLC and changes in membrane potential

have been monitored using fluorescent dye Rhodamine 123. Our

data shows that substrate supplementation shifts the mitochondrial

redox status towards reduced state. These data are in agreement

with our previous work and shows changes in redox status of pyridine

dinucleotides when supplemented with mitochondrial energy

substrates.

Fermented garlic, a novel candidate food for the

prevention of diabetic nephropathy

Emiko Sato, Masahiro Kohno, and Yoshimi Niwano

New Industry Creation Hatchery Center (NICHe), Tohoku University, Japan

Our previous study showed that spontaneous fermentation of

garlic under a fixed condition potentiates its fundamental antioxidative

properties. Scavenging activities against O 2 and H 2 O 2 of


80% EtOH extract of the fermented garlic were increased 13-folds

and more than 10-folds respectively, as compared with those of the

control garlic extract. Polyphenol content and potent scavengers

against H 2 O 2 of the extract were also increased in the fermented

garlic. Furthermore, we have shown that the fermented garlic has

an ability to scavenge ·OH, indicating that the fermented garlic

possesses desirable antioxidative properties in terms of scavenging

activities against reactive oxygen species (ROS). Diabetic nephropathy

is one of the three major diabetic complications, and requires

dialysis for the treatment. It is well known that the concentration

of circulating methylglyoxal (MG), a uremic toxin, is

higher in the patients with diabetic nephropathy than in healthy

people. Recently, it has been reported that free radials are generated

via a non-enzymatic reaction between MG and H 2 O 2 in vitro.

Thus we postulate that the free radical species is one of the triggers

of oxidative injury in the kidney, which may in turn lead to diabetic

nephropathy. We have tried to develop a functional food for

the prevention of diabetic nephropathy by applying the fermented

garlic. In this study, we report that the extract of the fermented garlic

have an ability to decrease the free radicals generated via a reaction

between MG and H 2 O 2 . Our results suggest that it is possible

to develop functional food for the prevention of diabetic nephropathy

by applying the extract of fermented garlic, which can decrease

the free radicals suggested as putative triggers of oxidative

injury in the kidney.

150

151


Dietary -tocopherol and plasma -tocopherol are correlated

with the urinary metabolite -CMBHC but not -CEHC in

caucasians and African Americans

Martha G. Sensel 1 , Mary C. Cambou 1 , Weiqing Liu 2 ,

Katie M. Lebold 3 , Maret Traber 3 , and Lenore Arab 1

1 David Geffen School of Medicine and 2 Department of Biomathematics,

University of California, Los Angeles, CA 90095; 3 Linus Pauling Institute,

Oregon State University, Corvallis, OR 97331

Clarity on the relationships between food source and supplemental

vitamin E intake with urinary tocopherol (toc) metabolites are needed

to discern the utility of the latter as biomarkers in human studies. We

examined relationships between dietary intake, plasma (pl.) levels, and

urinary metabolites of -toc in 262 African American (AA) and Caucasian

(CAU) subjects from Los Angeles. Dietary intakes of -toc

were 10.6 (11.2)* and 13.3 (13.7) mg/day for AAs and CAUs, respectively.

Plasma -toc levels were 10. 7 (3.6) and 11.2 (3.2) g/mL for

AAs and CAUs, respectively. Creatinine-adjusted urinary levels of the

major metabolite -CEHC and its precursor, -CMBHC, were

2.59 (3.23) and 0.37 (0.49) mol/g creatinine, respectively in AAs and

3.20 (2.95) and 0.49 (0.71) mol/g creatinine, respectively, in CAUs.

Pearson correlation analysis of log-transformed data showed significant

correlations between dietary -toc and lipid-adjusted pl. -toc (r

= 0.39l; p < 0.0001), dietary -toc and urinary -CMBHC (r = 0.36;

p < 0.0001); and lipid-adjusted pl. -toc and urinary -CMBHC

(r = 0.31; p < 0.0001). Correlations for dietary or pl. -toc with urinary

-CEHC were not significant. In the subset of 154 subjects who did

not consume supplemental vitamin E, correlations were reduced, but

showed the same tendency (dietary vs. lipid-adjusted pl. -toc, r = 0.16

and p = 0.006; dietary -toc vs. urinary -CMBHC, r = 0.28 and

p = 0.0009; lipid-adjusted pl. -toc, vs. urinary -CMBHC: r = 0.20

and p = 0.012). Compared to CAUs, AAs had stronger correlations of

urinary -CMBHC with both dietary -toc (p = 0.008 vs. p = 0.05)

and pl. -toc (p = 0.05 vs. p = 0.17). Although correlations with urinary

-CEHC were not significant in either AAS or the overall group

of non-supplement users, CAUs showed an r = 0.280 and p = 0.025 for

pl. -toc and urinary -CEHC. Further, urinary -CEHC was highly

correlated with urinary -CMBHC (r = 0.528; p < 0.001). The observation

that dietary intake and pl. levels of lipid-adjusted -toc are correlated

with urinary levels of the intermediary -toc metabolite

-CMBCH, but generally not with the terminal metabolite -CECH is

perplexing, but suggests that -CMBCH might serve as a biomarker for

-toc intake. *all descriptive data are expressed as mean (SD)

152

TGF 1 induces mitochondrial ROS through GSK3

inactivation-mediated respiratory defects

Yong-Hak Seo 1,3 , Hyun-Jung Jung 1,3 , Hae-Ok Byun 1,3 ,

You-Mie Kim 1,3 , Soo-Han Yoon 2,3 , and Gyesoon Yoon 1,3

1 Department of Biochemistry & Molecular Biology and 2 Department of

Neurosurgery, School of Medicine and 3 Department of Molecular Science &

Technology (BK21), The Graduate School, Ajou University, Suwon, 443-721,

Korea. E-mail: ypeace@ajou.ac.kr

We previously reported that transforming growth factor 1

(TGF 1) induces senescence through persistent mitochondrial

ROS generation through respiratory defects. In the present study,

we investigated the molecular mechanism of how mitochondrial

respiration is damaged by TGF 1. During the TGF 1-induced senescent

process, no significant changes of respiratory protein expressions

were observed. However, TGF 1 immediately triggered

phosphorylation on negative regulatory sites of both GSK3 and

within 15 min and the phosphorylation levels were continuously

maintained, well corresponding to the intracellular ROS profile.

The GSK3 inactivation was upstream event of the ROS generation,

independent of AKT activation. Interestingly, a conventional

GSK3 inhibitor, SB415286, directly inhibited not only cellular

respiration without alteration of respiratory protein expression in

both Mv1Lu and Chang cells. In addition, the GSK3 inhibition was

enough to induce senescence, accompanying prolonged ROS production.

These results were further confirmed by knockdown of

GSK3 with siRNAs. Our present results suggest that GSK3 inactivation

is involved in the TGF 1-induced respiratory defects,

thereby continuously elevating cellular ROS level and inducing senescence.

153


Investigation of the effects of long-term hyperbaric

oxygenation on the oxidative and antioxidant parameters of

rats’ lung tissue

Kemal Simsek 1 , Hakan Ay 1 , Sukru Oter 2 , Mehmet Ozler 2 ,

Ergun Ucar 3 , Bulent Uysal 2 , Turgut Topal 2 , Ozgur Yesilyurt 4 ,

and Senol Yildiz 1

Departments of 1 Undersea & Hyperbaric Medicine, 2 Physiology, 3 Pulmonary

Medicine and 4 Pharmacology; Gulhane Military Medical Academy; Ankara,

Turkey

Despite to its common clinical use and known benefits, hyperbaric

oxygen (HBO) is also reported to enhance the production of

reactive oxygen species and therefore can cause oxidative stress in

several tissues. Previous studies had shown that HBO induced oxidative

stress is directly proportional to both its exposure pressure

and duration. Nevertheless, these studies were usually performed

with single-session HBO exposures. On the other hand, the clinical

use of HBO commonly depends on a long-term exposure period. In

this study, it was aimed to enlighten the oxidative effect of HBO in

the lung tissue of rats which were exposed to HBO from 5 up to 40

sessions. A total of 60 male Sprague-Dawley rats were divided into

6 study groups exposed to 5, 10, 15, 20, 30 and 40 daily 2.8

ATA/90 min HBO sessions (n = 8 for each). Animals were sacrificed

24 h after the last HBO sessions. An additional control group

was set for obtaining normal data (n = 12). Lung malonyldialdehyde

(MDA) and carbonylated protein (PCO) levels were determined

as measures of oxidative stress, along with the activities of

the antioxidant enzymes superoxide dismutase (SOD) and glutathione

peroxidase. For the first 15 sessions, none of the measured

parameters represented any changes. But in the 20, 30 and 40-

session groups MDA, PCO and SOD were found to be significantly

increased indicating that oxidative stress is established in

rats’ lung tissue: Thus, the standard prophylactic use of antioxidants

during HBO therapy seems to be reasonable, in particular for

patients with longer exposure procedures.

154

Multi-nutrient approach to nitric oxide production

more efficient than L-arginine alone

Tamara Sofi, Joseph Juliano, and Michael Ricciardi

THE Labs, Research & Development Division, 27128 Paseo Espada,

San Juan Capistrano, CA 92675

Nitric Oxide (NO) synthesized from L-arginine by NO synthase

has a large variety of physiological actions in the cardiovascular,

periphery, immune, and nervous system. It is believed to be the

body’s nutrient delivery system and is considered therapeutic

when produced for 30 minutes or more. Via trial and error, various

multi-nutrient applications were tested until increased NO production

crossed the 30 minute threshold using less than 1 gram l-

arginine per dose. In this clinical study 50 human subjects ranging

from 40 to 80 years of age, diagnosed with NO deficiency conditions

to include hypertension and diabetes, were divided into 3

groups receiving either (a) placebo, (b) L-arginine alone, or (c)

Hemoxide, a multi-nutrient formula containing L-arginine and other

nutrient cofactors to include choline bitartrate. Loading dose was 3

capsules per day, 2 times per day, for 3 days. NO levels were

tested via skin surface temperature changes and Siemens’ NO

breathalyzer. Subjects receiving placebo had no spike in NO production

from baseline. Subjects receiving l-arginine alone experienced

an average 8% spike over baseline in NO production within

the first 10 minutes and then gradually declined to flat line at the 20

minute point. Subjects receiving Hemoxide experienced increased

NO levels for 1-4 hours reaching the same initial 10 minute 8% increase

as L-arginine subjects, but then peaked at a 33% average increase

over baseline at the 20 minute point (where L-arginine subjects

flat lined). Levels in the Hemoxide subjects did not drop below

20% increase over baseline until the 65-70 minute point,

gradually declining to 8% over baseline (where l-arginine subjects

peaked) at the 110 minute point. Study results were submitted to

the FDA which made this application a medical food. Since this

study, THE labs has translated the acetylcholine, dopamine, and

serotonin neurotransmitter delivery systems.

155


Novel boron carrier based on core cross-linked micelles

composed of PEG-b-PLA copolymer

with polymerizable boron cluster

Shogo Sumitani 1 , Motoi Oishi 1-3 , and Yukio Nagasaki* 1-5

1 Graduate School of Pure and Applied Sciences, University of Tsukuba,

2 Tsukuba Research Center for Interdisciplinary Materials Science, 3 Center for

Tsukuba Advanced Research Alliance, 4 Graduate School of Comprehensive

Human Sciences, 5 Satellite Laboratory of International Center for Materials

Nanoarchitechtonics

Boron neutron capture therapy has attracted much attention as

the selective cancer therapy using 10 B compounds, which efficiently

generate the a-particles and 7 Li nuclei within ten mm

through the nuclear reaction of 10 B atom with low-energy thermal

neutrons. The success of BNCT is dependent on the delivery systems

to accumulate sufficient quantity of 10 B to tumor tissues.

Herein, we designed and prepared core cross-linked micelles (CL

micelles) composed of poly(ethylene glycol)-block-poly(lactide)

copolymer bearing a methacryloyl group at PLA end using polymerizable

boron cluster. This micelle enables to prolong the blood

circulation time and deliver the 10 B compounds to the tumor tissues

without leakage of 10 B compounds as well as disassociation of the

micelle in the blood stream because of the existence of crosslinked

core through the covalent bonds.

The average diameter of the CL micelle was 85 nm determined

by dynamic light scattering measurements. To clarify biodistribution

of both micelles, 125 I-labeled CL and non cross-linked (NCL)

micelles were injected into BALB/c mice bearing tumor via tail

vein. The CL micelle showed prolonged blood circulation time (t 1/2

= 16.5 h) compared with NCL micelle (t 1/2 = 11.8 h). In addition,

accumulation of CL micelle into tumor tissue was higher than

that of NCL micelles. These results clearly indicate that the stability

of the CL micelle remarkably increased due to the covalently

cross-linked bonds between polymer and boron cluster.

Protective effects of astaxanthin against singlet oxygen-induced

damage in human dermal fibroblasts in vitro

Kumi Tominaga, Nobuko Hongo, Mariko Karato, and

Eiji Yamashita

Life Science Division, Fuji Chemical Industry Co. Ltd., Toyama, Japan

Astaxanthin is widely distributed in nature especially in the

marine organisms such as crustaceans and fish. We recently compared

the singlet oxygen quenching ability of astaxanthin in solvents

with that of 26 common hydrophilic and lipophilic antioxidants,

including vitamins, polyphenols, and certain drugs, under

the same conditions. The result showed that the activity of astaxanthin

was approximately 6,000 times, 800 times, 560 times, and 75

times greater than that of vitamin C, coenzyme Q 10 , catechin, and

alpha-lipoic acid, respectively, which indicates that astaxanthin has

the strongest singlet oxygen-quenching ability among these compounds

1) . Here we studied the protective effects of astaxanthin

against singlet oxygen induced damage in human dermal fibroblasts

in vitro comparing to 7 common antioxidants (-carotene,

lutein, -tocopherol, coenzyme Q 10 , -lipoic acid, vitamin C, and

catechin). Human dermal fibroblasts were treated with 10 μM of

each antioxidant for 24 h. And after singlet oxygen exposure, and

the human dermal fibroblasts were incubated for 24 h and the cell

viability was determined by the MTT assay. Only astaxanthin exhibited

the protective effect in this condition. Even at 3 μM the effect

was identical and an almost 100% protection. Bedsides, a

dose-dependent protective effect was observed for -tocopherol

alone among the tested antioxidants at up to 1,000 μM. A relationship

between the protective effect of astaxanthin and collagen production

ability in the cells and a difference of action mechanisms

of the protective effect between astaxanthin and -tocopherol will

be discussed. The anti-wrinkling effect of astaxanthin, which was

reported in clinical and animal studies, may be attributed to its protective

effect against singlet oxygen-induced damage.

Y. Nishida et al.: Carotenoid Science 11, 16 (2007).

156

157


ESR detection for alkyl-oxy radical and superoxide

radical decay by natural antioxidants

Mitsuko Ukai

Department of Education, Hokkaido University of Education,

Hakodate 040-8567, Japan

Antioxidants play a role as efficient scavengers for radicals.

Oxidation processes are crucial for food storage. Oxidation is the

major causes of chemical spoilage of food. They are rancidity

and/or deterioration of the nutritional quality, color, flavor and texture.

The total antioxidant capacity is an important factor for the

evaluation of the food quality. Basic methods to measure antioxidant

capacity in foods have been reviewed. Three assays should be

recommended for the standardization, oxygen radical absorbance

capacity (ORAC), the Folin - Ciocalteu method and the Trolox (6-

hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalent

antioxidant capacity (TEAC). Methods of analysis of water

soluble and lipid soluble antioxidant capacity in foods were extended

by the modifications of the ORAC procedure. These methods

have been compiled into the USDA food database of antioxidant

capacity of foods. The spin trapping abilities against alkyl-oxy

and superoxide radicals were studied using a new reagent CYP-

MPO. For careful detection of spin adducts, we used borosilicate

ESR flat cells. CYPMPO is a colorless crystalline and very soluble

into aqueous solutions. We examined plant specimens from teas,

vegetables and fruits. CYPMPO successfully trapped alkyl-oxy as

well as superoxide radicals in the plant extracts. Upon illumination

of Hg - Xe arc lamp light onto phosphate buffer solution containing

AAPH and CYPMPO, we generated the alkyl-oxy radicals in

the very pure form. Similarly, superoxide radicals were generated

by HPX, DTPA, CYPMPO and XOD. The spin adducts by CYP-

MPO were sensitive and very stable. The trapping activity for alkyl-oxy

radical was measured by the GSH equivalent, and that for

the superoxide radical by the SOD equivalent. We concluded that

CYPMPO has a high capability for radical trapping and is useful.

158

Complex I syndrome in isolated rabbit heart subjected to

ischemia-reperfusion

1 Laura B. Valdez, 1 Tamara Zaobornyj, 1 Silvina S. Bombicino,

1 Darío E. Iglesias, 2 Martin Donato, 2 Verónica D’Annunzio,

2 Ricardo Gelpi, and 1 Alberto Boveris

1 Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry,

2 Laboratory of Cardiovascular Physiopathology, Department of Pathology,

School of Medicine; University of Buenos Aires, Buenos Aires, Argentina

The effects of ischemia-reperfusion on left ventricle mitochondrial

function were studied in isolated rabbit heart using the Langendorff

technique with 15 min of stabilization, 15 min of ischemia

and 5 and 30 min of reperfusion. Tissue O 2 consumption, mitochondrial

state 3 respiration with malate-glutamate, and complex

I activity were similarly decreased, by 20-25% and 40-45%, after 5

and 30 min reperfusion, respectively. Hydrogen peroxide production

after 30 min of reperfusion was 80% higher than the H 2 O 2

production in control mitochondria. Mitochondrial NO production

decreased 35% in ischemic hearts with a recovery to control values

during reperfusion. The pattern of mtNOS biochemical activity

was in agreement with the changes in mtNOS functional activity in

malate/glutamate-supported O 2 consumption. The mitochondrial

level of protein carbonyls (40%), TBARS (50%) and nitrotyrosine

(3-fold) were markedly increased after the process of ischemiareperfusion.

This experimental model shows an early reduction of

mitochondrial NO production during acute hypoxia, which may release

NO-mediated cytochrome oxidase inhibition. In reoxygenation,

mtNOS activity is up-triggered, increasing mitochondrial NO,

O 2 - and ONOO - productions. During reperfusion, mitochondria develop

to a condition named “complex I syndrome” in which enzymatic

complex I inactivation is associated with protein nitration

and oxidative damage to proteins and phospholipids.

159


Quercetin reduces inflammatory pain by inhibiting

cytokine production and oxidative stress

Waldiceu A. Verri, Jr 1 , Daniel A. Valério 2,3 , Sandra R. Georgetti 4 ,

Danilo A. Magro 2 , Thiago M. Cunha 2 , Fabiana T.M.C. Vicentini 3 ,

Silvio M. Vieira 2 , Maria J.V. Fonseca 3 , Sergio H. Ferreira 2 ,

Fernando Q. Cunha 2 , and Rubia Casagrande 4

1 Departamento de Ciências Patológicas - Centro de Ciências Biológicas,

Universidade Estadual de Londrina; 2 Department of Pharmacology, FMRP,

USP, Ribeirão Preto; 3 Department of Pharmaceutical Sciences, USP, Ribeirão

Preto; 4 Departamento de Ciências Farmacêuticas - Centro de Ciências de

Saúde, Universidade Estadual de Londrina

Quercetin is a flavonoid with antioxidant and antinociceptive

effects. However, the mechanisms involved in its antinociceptive

effect are not fully elucidated. Cytokines and reactive oxygen species

have been implicated in the cascade of events resulting in

inflammatory pain. Therefore, we evaluated the antinociceptive

mechanism of quercetin focusing on the role of cytokines and oxidative

stress. The treatment with quercetin dose- (30-300 mg/Kg)

dependently inhibited the second phase of formalin test, acetic acid

and phenyl-p-benzoquinone overt pain-like behavior, and carrageenin-induced

mechanical hypernociception. Quercetin also inhibited

the hypernociception induced by cytokines (TNF - 43%

and CXCL1 - 38%), but not of inflammatory mediators that directly

sensitize the nociceptor such as PGE 2 and dopamine. Quercetin

did not affect carrageenin- or cytokine (TNF and CXCL1)-

induced leukocyte recruitment, which can be a source of hyperalgesic

mediators. On the other hand, quercetin reduced carrageenininduced

IL-1 production as well as carrageenin-induced decrease

of reduced glutathione (GSH) levels. Concluding, these results

suggest that quercetin exerts its analgesic effect by inhibiting pronociceptive

cytokine production and the oxidative imbalance mediation

of inflammatory pain.

Regulation of cell signals by procyanidins at the

intestinal epithelium

Sandra V. Verstraeten 1 , Matieu Da Silva 2 , Grayson K. Jaeggers 2 ,

Patricia I. Oteiza 2,3 , and César G. Fraga 2,4

1 Department of Biolological Chemistry-IQUIFIB-IIMHNO, School of Pharmacy

and Biochemistry, UBA-CONICET, Argentina; Departments of 1 Nutrition and

3 Environmental Toxicology, University of California, Davis, CA, USA; and

4 Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry,

UBA-CONICET, Argentina

Based on the findings that large procyanidins (LP) protect intestinal

cells from different pro-inflammatory stimuli, we have

now tested the hypothesis that LP interact with intestinal epithelial

cells and through these interactions prevent pro-inflammatory

events initiated at lipid rafts, e.g. calcium mobilization, oxidant

formation, and activation of select cell signal. We used both,

liposomes (as model of membranes with different lipid composition)

and Caco-2 cells (as model of intestinal epithelium), to investigate

the effects of a fraction enriched in LP isolated from cocoa.

LP increased the resistance to Triton-X 100-mediated disruption of

glycolipid-enriched liposomes. In Caco-2 cells, LP inhibited NF-

B activation initiated by different pro-stimulatory compounds at a

different extent. The the highest inhibitory effects were observed

for agents that initiate NF-B -activation at lipid rafts, i.e. tumor

necrosis factor alpha and deoxycholate. Furthermore, LP inhibited

deoxycholate-induced calcium mobilization, oxidant production

and the activation of mitogen-activated kinases (MAPKs). In summary,

LP can interact with synthetic and biological membranes and

protect them from different pro-inflammatory stimuli. The obtained

results suggest that LP has could have selective interactions

with particular areas of the membrane, preventing oxidant and signaling

events initiated at lipid rafts.

Supported by grants from CHNR-State of California Vitamin Price Fixing Consumer Settlement

Fund and Mars Inc., and UBACyT B801.

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161


Labile iron-mediated cerulein-induced oxidative stress in

rat acinar AR42J cells

1 Michal Wozniak, 3 Maciej Sledzinski, 2 Andzelika Borkowska,

2 Alicja Sielicka-Dudzin, 2 Magorzata Halon, and

2 Jedrzej Antosiewicz

1 Department of Medical Chemistry, 2 Bioenergetics and Physiology of Exercise,

3 General and Endocrine Surgery and Transplantation, Medical University of

Gdask, Poland

Reactive oxygen species (ROS) have been implicated in the

pathogenesis of acute pancreatitis (AP). However, the mechanism

of ROS formation in the pancreas remains poorly defined. We observed

that during acute pancreatitis (AP), the iron storage protein

ferritin in the rat pancreas undergoes degradation accompanied by

an increased formation of protein carbonyls. In order to get an insight

into the mechanism of ROS formation, we also performed

experiments on the pancreatic acinar AR42J cells stimulated by cerulein.

In such cells we observed increased labile iron pool (LIP)

that is accompanied by a decrease in the cellular L ferritin level

and an increase in the ROS formation. The changes in ferritin level

were inversely correlated with the ROS formation. Moreover, we

observed that in cells transfected with plasmid encoding inactive

mutant of JNK1 cerulein did not induce ferritin degradation. In

conclusion, our data suggest that LIP significantly participates in

the cerulein induced ROS formation and that both ferritin degradation

and iron dependent ROS formation is controlled by JNK1.

Glucose 6-phospate dehydrogenase deficiency

accelerated Coronavirus 229E infection due to

impaired inflammatory response

Yi-Hsuan Wu # , Shin-Ru Lin # , Mei-Ling Cheng #+ ,

Chuen-Mao Yang * , and Daniel Tsun-Yee Chiu *#+

# Graduate Institute of Medical Biotechnology, Chang Gung University, 259,

Wen-hwa 1 st Rd, Kwei-san, Tao-yuan, Taiwan; + Department of Clinical

Pathology, Chang Gung Memorial Hospital, Kwei-san, Tao-yuan, Taiwan

Glucose 6-phosphate dehydrogenase (G6PD), the key regulatory

enzyme in the pentose phosphate pathway, provides reducing

power to all cells in the form of NADPH to meet the cellular needs

for reductive biosynthesis and maintenance of the cellular redox

status. We have previously demonstrated that cellular susceptibility

toward viral infection is modulated by G6PD status, yet the detailed

mechanism remains elusive. One possibility is that cellular

redox status modulated by G6PD may involve in fine-tuning of

immune response against viral infection. To test this hypothesis,

we used G6PD-knockdown lung epithelial A549 cells as host and

determined the viral production upon coronavirus infection in the

present of TNF- as an infectious model system. Upon 15 ng/ml of

TNF- treatment, G6PD-knockdown A549 cells generated less superoxide

compared to control during earlier time period (


NO stress mediated by neural NO synthase (nNOS),

a potential accelerator of melanoma progression?

Sun Yang and Frank L. Meyskens

Chao Family Comprehensive Cancer Center,

University of California Irvine, Orange, CA 92868

Our laboratory has been extensively involved in the study of abnormal

redox status and redox-sensitive signalings such as APE/Ref-

1, AP-1 and NF-B in the past decade for the experimental therapeutics

of human melanoma. Our goal is to identify key factors underlying

melanoma development. It has been well-documented that UV

radiation exposure especially sunburn at a young age is particularly

linked to melanoma incidence. As an important environmental carcinogen,

UV radiation not only generates ROS, but also produces a

large amount of nitric oxide (NO) in human skin. Utilizing a NOdonor

DETA to mimic NO stress, we demonstrated that melanoma

proliferation and invasion potential were significantly stimulated by

DETA treatment, associated with inductions of many proteins involved

in cell growth (c-Jun, JunD), anti-apoptosis (Bcl-2, APE/Ref-

1) and metastasis signalings (MMP-1, Snail). Notably, long-term exposure

of DETA/NO stress resulted in over-growth of primary normal

human melanocytes with formation of foci in in vitro culture, indicating

gain of additional vertical growth potential. As melanocytes are

originated from neural crest, we proposed that neural NO synthase

(nNOS) might play an important role in generating NO and mediating

NO stress in human melanoma. First, both our in vitro cell culture

(immunoblotting) and in vivo human biopsy (immunohistochemistry)

studies demonstrated marked elevation of nNOS expressions in melanoma.

More interestingly, induction of nNOS was evident with UVB

radiation and bFGF incubation. Knockdown of nNOS with siRNA efficiently

reduced the DETA-induced melanoma proliferation and invasion

potential, with reductions of c-Jun, Bcl-2 and MMP-1. A

novel synthesized nNOS inhibitor JI-11 was utilized to inhibit nNOS

activity; our data showed that co-treatment of JI-11(1μM) significantly

attenuated the alterations induced by UVB radiation and

DETA treatments. Based on our studies, we propose that targeting

nNOS with application of specific synthetic inhibitors to diminish

NO stress represents an innovative and promising strategy for melanoma

prevention.

2-Deoxyglucose diet induces ketosis, enhances

mitochondrial function and reduces Alzheimer’s like pathology

in the triple transgenic Alzheimer’s mouse model

Jia Yao, Ronald Irwin, Shuhua Chen, Eric Hernandez,

and Roberta Díaz Brinton

Department of Pharmacology and Pharmaceutical Sciences,

School of Pharmacy, University of Southern California, Los Angeles, CA 90033

Previously, we have shown that mitochondrial bioenergetic

deficits precede Alzheimer’s disease (AD) pathology in the triple

transgenic AD (3xTg-AD) mouse model(Yao et al., 2009). Both

basic science and clinical studies suggested a change in brain

metabolic profile prior to the onset or diagnosis of AD. In the current

study, we sought to 1) further investigate brain metabolic

change with age and AD progression in the 3xTg-AD mouse

model; 2) to investigate the impact of 2-deoxyglucose (2-DG), a

common compound to induce ketogenesis, on AD pathology. At 3

month female 3xTg-AD mice had higher SCOT expression relative

to non-transgeic (nonTg) female mice, suggesting an early shift to

ketogenic phenotype to compensate the decrease in brain glucose

metabolism in these mice. The expression of SCOT decreased with

age in the 3xTg-AD mice. At 12 months, both SCOT expression

and activity were significantly lower in the 3xTg-AD mice relative

to nonTg. To investigate the impact of 2-DG on AD like pathology,

both nonTg and 3xTg-AD female at 6 month were fed with

either regular diet (AIN-93G) or diet containing 0.04% 2-DG for 3

weeks. Serum ketone levels as well as hippomcapal SCOT levels

were increased with 2-DG in both nonTg and 3xTg-AD mice. In

addition, compared to the control group, 2-DG increased the expression

of enzymes involved in fatty acid metabolism as well as

oxidative phosphorylation. In 3xTg-AD mice, 2-DG diet also reduced

AD like pathology. The reduction in amyloid pathology is

likely due to up-regulation of alpha secretase pathway. All together,

data suggest that in 3xTg-AD mice there is a shift towards

ketogenic phenotype early in AD progression, suggesting the activation

of a compensatory pathway to the decrease in glucose metabolism.

A relative short-term 2-DG treatment induces ketogenesis

in both genotypes, increases fatty acid oxidation and reduces

AD like pathology. The long-term impact of 2-DG remains to be

further investigated.

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165


. NO modulation of glutathonylation of proteins:

Implications for aging and neurodegeneration

Li-Peng Yap, Jerome V. Garcia, Derick Han and Enrique Cadenas

Pharmacology & Pharmaceutical Sciences, School of Pharmacy and Research

Center for Liver Diseases, Keck School of Medicine, University of Southern

California, Los Angeles, CA 90089, USA

The interaction between redox and energy changes establishes

a regulatory mechanism that controls cellular energy levels in response

to redox changes (i.e., increased generation of H 2 O 2 and

. NO) through specific post-translational modifications of cytosolic

and mitochondrial proteins. The role of these protein modifications

in aging and age-related neurodegeneration is established by an

age-dependent increase in the levels of . NO –a consequence of increase

expression of neuronal nitric oxide synthase and inflammation–

leading to nitration, S-nitrosation, and S-glutathionylation of

specific proteins. Acute exposure of primary cortical neurons and

astrocytes to a . NO flux, mirroring neuroinflammation, led to S-

glutathionylation of proteins in a dose- dependent manner due to

oxidation of the cellular redox environment. The significance of a

higher redox buffering capacity was reflected in the extent of intracellular

formation of S-glutathionylated proteins. Increasing concentrations

of GSNO and GSSG formation as a consequence of

. NO exposure correlated with S-glutathionylation of proteins. Glutathonylation

of GAPDH, a key glycolytic enzyme, led to significant

inhibition of its activity.

Glutathionylation of GAPDH and inhibition of its activity were

also observed in a triple transgenic model of Alzheimer’s disease

as a function of age. Metabolic changes observed in Alzheimer’s

disease –decreased pyruvate dehydrogenase activity and mitochondrial

respiration– and redox changes preceded the occurrence

of histopathology and accompanied cognitive deficits and were

compounded by inactivation of cytosolic GAPDH upon S-

glutathionylation.

Regulation of insulin signaling by lipoic acid:

Therapeutic implications for neurodegeneration

Li-Peng Yap, Jerome V. Garcia, Derick Han and Enrique Cadenas

Pharmacology & Pharmaceutical Sciences, School of Pharmacy and Research

Center for Liver Diseases, Keck School of Medicine, University of Southern

California, Los Angeles, CA 90089, USA

Aberrant insulin signaling – entailing decreased Akt (also

known as protein kinase B (PKB) and increased glycogen synthase

kinase (GSK) activities– may account for (a) the early energy

changes due to decreased glucose metabolism and mitochondrial

generation of energy, and (b) the perpetuation of a cycle that leads

to the later neuropathological hallmarks of Alzheimer’s disease:

A plaque and NFT formation. Hence, the modulation of insulin

signaling bears therapeutic potential for the early and late stages of

AD. The use of insulin itself in Alzheimer’s patients has shown increased

cognitive function but with mixed results concerning A

accumulation, as both insulin and A are proteolytically degraded

by the same enzyme (insulin-degrading enzyme). Therefore, compounds

that elicit an “insulin-like” effect may be effective in the

use in AD through the modulation of intracellular insulin signaling

without competition at the IDE level.

Our studies using small animal imaging (microPET) show a

decrease in glucose metabolism in a triple transgenic mouse model

(APP SWE + PS1 M164V + Tau P301L ) that develop both the pathological

and cognitive changes reflected in AD in humans. Decrease in

glucose metabolism in these mice occurs with increasing age and

precedes substantial plaque and tangle formation as well as cognitive

changes. Our studies show that feeding mice lipoic acid induces

insulin-like effects, demonstrated by increase glucose metabolism

as well as modulation of insulin signaling in neurons

through modulation of the phosphorylation status of Akt and

GSK3 in vivo and in vitro.

166

167


Novel EPR imaging of fluctuating oxygenation

associated with a defect of vascular integrity in

transplanted tumors

Hironobu Yasui 1,2 , Shingo Matsumoto 2 ,

Nallathamby Devasahayam 2 , Sankaran Subramanian 2 ,

James B. Mitchell 2 , and Murali C. Krishna 2

1 Laboratory of Radiation Biology, Department of Environmental Veterinary

Sciences, Graduate School of Veterinary Medicine, Hokkaido University,

Sapporo, Japan. 2 Radiation Biology Branch, Center for Cancer Research,

National Cancer Institute, Bethesda, MD 20892, USA

Fluctuation in blood flow is one of tumor phenotype, which

develops heterogeneity of oxygen and subsequently invokes lack

of cancer treatment efficacy. Both to improve therapeutic planning

effectively against solid tumor and to make precise prognosis in

cancer treatment, noninvasive dynamic imaging of spatial and

temporal pO 2 profile density is needed. Here, we report for the first

time to our knowledge that rapid imaging with pulsed electron paramagnetic

resonance imaging (EPRI) capacitated direct monitoring

of oxygen fluctuation within minutes in natural state of murine

squamous cell carcinoma (SCCVII) and human colorectal carcinoma

(HT29) tumors with reliable resolution. Special resonator

applicable for both EPRI and magnetic resonance imaging (MRI)

provided pO 2 maps with anatomical guidance and microvessel

density without positional movement. Oxygen images every 3 min

in two different tumor-bearing mice model disclosed tumor-sizeand

tumor-type-dependent variation of fluctuating oxygenation.

Immunohistochemical analysis for CD31 and SMA revealed that

the fluctuation of oxygenation was correlated with pericyte density

strongly rather than vascular density in tumor. These results suggested

that EPR oxymetric imaging combined with MRI can be a

potent method to detect fluctuating oxygenation in solid tumor

non-invasively.

Signal transduction pathways mediate H 2 O 2 -induced necrosis

in primary cultured hepatocytes

Maria D. Ybanez and Derick Han

Research Center for Liver Diseases, University of Southern California,

2011 Zonal Avenue, LA, CA 90089-9121, USA

In this study, the signaling pathways that regulate necrosis induced

by H 2 O 2 in primary cultured hepatocytes were examined.

H 2 O 2 treated to hepatocytes is consumed within minutes, but hepatocytes

undergo necrosis several hours later. H 2 O 2 treatment induces

a “lag phase” where signaling transduction pathways including

glycogen synthase kinase-3 (GSK-3) and protein kinase C

(PKC) are activated. GSK-3 inhibitor or silencing GSK-3 was

effective in reducing necrosis caused by H 2 O 2 (~ 50- 80%) in primary

cultured hepatocyes. This suggests GSK-3 plays an essential

role in mediating H 2 O 2 -induced necrosis. PKC inhibitor treatment

also protected against H 2 O 2 -induced necrosis. PKC inhibitor

treatment to primary hepatopcytes, however, increased activity of

Akt. GSK-3 is downstream target of Akt, and PKC inhibitor

treatment consequently resulted in increased phosphorylation of

GSK-3 (serine 9, which inactivates GSK-3), likely due through

increase in Akt activity. This suggests PKC is a negative regulator

of Akt and also suggests part of the protective mechanism of PKC

inhibitor against H 2 O 2 -induced necrosis may be mediated through

inactivation of GSK-3 in primary cultured hepatocytes. Taken together

our data demonstrates that signaling pathways involving

GSK-3 and PKC mediate H 2 O 2 -induced necrosis, suggesting

H 2 O 2 induces a “programmed necrosis” in primary cultured hepatocytes.

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169


The mitochondrial energy-redox axis in aging and

caloric restriction:

Potential role of nicotinamide nucleotide transhydrogenase

Fei Yin, Harsh Sancheti, and Enrique Cadenas

Pharmacology and Pharmaceutical Sciences, School of Pharmacy,

University of Southern California, Los Angeles, CA, USA

As cellular powerhouses and established cellular sources of

H 2 O 2 , mitochondria play a central role in the aging progress. The

steady-state level of mitochondrial H 2

O 2

is determined at its approximation

equilibrium by the rate of H 2

O 2

generation (electron

leak during NADH oxidation in the respiratory chain) and of H 2

O 2

removal (NADPH-dependent, GSH and thioredoxin supported

processes in the mitochondrial matrix). Generation of mitochondrial

NADPH is largely dependent on the inner-membrane Nicotinamide

Nucleotide Transhydrogenase (NNT), which catalyzes the

reduction of NADP + to NADPH utlizing the proton gradient as the

driving force and NADH as the electron donor. Thus, NNT represents

a critical link between mitochondrial metabolic function (energy

component) and redox homeostasis (redox component) by

coupling NADPH generation to the TCA cycle and active respiration;

a mitochondrial energy-redox axis is hereby defined. Our results

demonstrate that aging in Fischer 344 rats is accompanied by

(a) impaired energy metabolism; (b) shift of redox state; (c) decreased

NNT activity, and (d) increased H 2

O 2

levels. Furthermore,

these energy and redox changes are attenuated by short-term caloric

restriction in senescent animals but not in young animals.

SiRNA to NNT in PC12 cells elicited changes in both energy and

redox status that validate the role of NNT in the energy-redox axis,

mainly a substantial shift toward anaerobic glycolysis. Data from

the PC12 cell model and an NNT-knockout mouse model

strengthen the importance of the interdependent mitochondrial energy-redox

axis in aging and provides evidence for a regulatory

role of NNT.

Design of pH-sensitive polymeric micelle possessing

reduced forms of TEMPO for imaging of ROS

Toru Yoshitomi 1 , Takashi Mamiya 2 , Hirofumi Matsui 2,3 ,

Aki Hirayama 3,6 , and Yukio Nagasaki 1-5

1

Graduate School of Pure and Applied Sciences 2 Graduate School of

Comprehensive Human Sciences 3 TARA 4 TIMS, University of Tsukuba 5 MANA,

NIMS, Tennoudai 1-1-1, Tsukuba, Ibaraki, Japan

6

Tsukuba University of Technology, Kasuga 4-12-7, Tsukuba, Ibaraki, Japan

Recently, a non-invasive imaging of reactive oxygen species

(ROS) has attracted attentions as a new method of evaluating disease

and response to clinical treatment. For example, inflammation

sites are known to be acidic pH, in which ROS are excessively generated.

A variety of low-molecular-weight electron paramagnetic

resonance (EPR) imaging probes such as 1-hydroxy-2,2,6,6-tetramethylpiperidinyloxy

(TEMPO-H) have been developed for the

detection of ROS. However, such probes are hard to utilize in vivo

due to their preferential renal clearance. To solve this issue, we

have developed pH-sensitive polymeric micelle possessing reduced

forms of TEMPO (RNP(R)) using self-assembling of amphiphilic

block copolymers composed of a hydrophilic poly(ethyleneglycol)

segment and a hydrophobic poly(styrene-TEMPO-H) segment

containing amino groups. Due to a protonation of the amino groups

in the hydrophobic core, RNP(R) disintegrates in response to

acidic pH. When RNP(R) was mixed with horseradish peroxidase

(HRP)/ hydrogen peroxide (H 2 O 2 ) couple as a model of in vivo oxidants,

the EPR spectra of RNP(R) were obtained. The EPR signal

of RNP(R) under acidic condition (pH=5.6) increased much faster

than that under neutral condition (pH=7.4), though the enzymatic

activity of HRP is almost the same under the present experimental

condition. These results suggest that the disintegration of RNP(R)

accelerates the oxidation reaction in acidic region, indicating that

RNP(R) is anticipated as EPR imaging probe for imaging of ROS at

low pH regions.

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171


Anti-senescence effect of natural antioxidants green tea

polyphenols and quinic acid in C. Elegans

L.Z. Zhang 1 , X.Z. Zhao-Wilson 2 , and B.L. Zhao 1

1 State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics,

Chinese Academy of Sciences, Beijing 100101, China. 2 BioMarker

Pharmaceuticals, Inc. 5941 Optical Court, San Jose, CA 95138, USA

Natural antioxidants play important roles in anti-aging process. We

studied the anti-senescence effect of natural antioxidants, green tea

polyphenols and quinic acid in C. Elegans and found they have different

effects and pass through different pathways. We found that Epigallocatechin

gallate (EGCG) extended C. elegans longevity under

stresses. Under heat stress (35°C), EGCG improved the mean longevity

by 13.1% at 0.1mg/ml. Under oxidative stress, EGCG could improve

the mean longevity of C. elegans by 172.9% at 0.1mg/ml. However,

EGCG could not extend the lifespan of C. elegans under normal

culture conditions. Further studies demonstrated that the significant

longevity-extending effects of EGCG on C. elegans could be attributed

to the in vitro and in vivo free radical scavenging effects of EGCG, and

the up-regulative effects of EGCG on stress-resistance-related proteins

including SOD-3 and HSP-16.2 in mutant C. elegans with SOD-

3::GFP and HSP-16.2::GFP expression. Considering that the death rate

of a population is closely related to the mortality caused by external

stress, it could be concluded that the survival-enhancing effects of

EGCG on C. elegans under stresse are very important for anti-aging

research. Quinic acid (QA) from plants can extend the life span of C.

elegans in normal conditions. We found that QA could extend C. elegans

lifespan by 11.4% under normal culturing conditions, 17.8% under

thermal stress, and 29.7% under oxidative stress. Both sir-2.1 and

daf-16 are required for QA to extend worm lifespan, which suggests

that QA causes worms to live longer in a signaling pathway involving

sir-2.1 and daf-16. In the QA-treated worms, the downstream DAF-16-

targeted genes, sod-3 and hsp-16.2, were consequently up-modulated.

However, hsp-16.2 was not indispensable for the lifespan-extending

effect of QA suggesting that HSP-16.2 was only one of the effectors

for QA. Meanwhile, QA exhibited an ability to keep the ROS at a

lower level in worms by scavenging free radicals. The ability of QA to

extend worm lifespan combined with the association of QA with sir-

2.1, daf-16, sod-3, hsp-16.2 and ROS demonstrates that QA possesses

great potential in anti-aging.

Supported by a grant from the National Natural Science Foundation of China (30370361).

Cytosolic ferritin degradation is prerequisite for

mitochondrial ferritin-induced iron mobilization

Yinghui Zhang, Marc Mikhael, Dongxue Xu, Yiye Li,

Shan Soe-Lin, Bo Ning, Yuliang Zhao, Prem Ponka, and

Guangjun Nie*

CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety

National Center for Nanoscience and Technology of China, Beijing 100190

Ferritin sequesters and stores iron, consequently protecting

cells against iron-mediated free radical damage. However, the

mechanism of iron exit from the ferritin cage and its reutilization

are largely unknown. In a previous study we found that mitochondrial

ferritin (MtFt) expression led to a decrease in cytosolic ferritin.

Here we showed that treatment with lysosomal inhibitors

largely blocked ferritin loss in both MtFt-expressing and wild type

cells. Moreover, ferritin in cells treated with lysosomal inhibitors

was found to store more iron than did ferritin in untreated cells.

The prevention of cytosolic ferritin degradation in MtFt-expressing

cells significantly blocked iron mobilization from the protein cage

induced by MtFt expression. These studies also showed that blockage

of ferritin loss by leupeptin resulted in decreased ferritin synthesis

and prolonged ferritin stability, potentially resulting in diminished

iron availability. Lastly, we found that proteasomes were

responsible for ferritin degradation in cells pretreated with ferric

ammonium citrate. Thus, the current studies suggest that ferritin

degradation precedes the release of iron in MtFt-expressing cells;

that MtFt-induced cytosolic ferritin decrease is partially preventable

by lysosomal protease inhibitors; and that both lysosomal and

proteasomal pathways may be involved in ferritin degradation.

172

173


The role of mitochondrial ferritin on hydrogen peroxide

induced SH-SY5Y cell damage

Nan Zhang 1 , Xiang-Lin Duan 1 , Zhen-Hua Shi 1 , Zhen Li 1 ,

Guang-Jun Nie 3 , Bao-Lu Zhao 2 , Yan-Zhong Chang 1*

1 Laboratory of Molecular Iron Metabolism, College of Life Science, Hebei

Normal University, Shijiazhuang 050016, Hebei Province, China

(*chang7676@163.com)

2 State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics,

Academia Sinica, Beijing, 100101, China

3 CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, National

Center for Nanoscience and Technology of China, Beijing, 100190,

China

Mitochondrial ferritin (MtFt) is a newly identified mitochondria

protein encoded by an intronless gene on chromosome 5q23.1

in humans. Previous studies suggested that overexpression of MtFt

caused a redistribution of iron from cytosol to mitochondriathus

high levels of MtFt result in an iron deficient phenotype in cytosol,

it expression also inhibited the in vivo tumor growth due to cytosolic

iron deprivation, and MtFt over-expression in HeLa cells increases

resistance to oxidative stress. In additionthe study of

MtFt may be useful for revealing mitochondria iron metabolism

and finding possible therapeutic applications in neurological disorders

involving increased iron deposition.

In this study, we examined that the role of MtFt on H 2 O 2 induced

cell damage using the MtFt overexpressed SH-SY5Y cells.

Our results showed that overexpression of MtFt restrained the increase

of ROS and the decrease of mitochondrial membrane potential,

maintained the level of anti-apoptotic protein Bcl-2 expression,

inhibited the activation of pro-apoptotic protein caspase3,

thus inhibited the apoptosis of SH-SY5Y cells induced by H 2 O 2 .

Interestingly, when Vector-SY5Y cells were treated with H 2 O 2 for

24 h, the significantly upregulated the levels of ferritin, DMT1

without IRE and downregulated the levels of TfR, DMT1 with IRE

compared to the control groups; while no significant changes were

found in MtFt-SY5Y cells under the same treatment. The mechanism

maybe was that overexpression of MtFt decreased the free

iron level by regulating the iron metabolism related proteins, and

then inhibited the fenton reaction, decreased the production of

ROS, thus inhibited the cell apoptosis induced by H 2 O 2 .

174

Protective effect of green tea polyphenols against

6-OHDA-induced apoptosis through ROS-NO pathway

in Parkinson’s disease models

Baolu Zhao

State key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics,

Academia Sinica, Beijing 100101, China

To investigate the protective effect of green tea polyphenols

against 6-OHDA induced apoptosis in Parkinson’s disease, we developed

a method to simultaneously detect NO and ROS in biological

system using electron spin resonance (ESR) spin trapping

technique. In the models of 6-OHDA induced SH-SY5Y cell and

6-OHDA injected rat neuron, the cell apoptosis was detected by

MTT, flow cytometric analysis using Annexin V and PI and

ELISE methods. ROS and NO free radicals were detected by ESR

technique. The NOS was detected by RT-PCR and Western blot

assays. The protein bound 3-nitro-tyrosine (3-NT) was measured

by competitive ELISE assay. More cell survived and less cell suffered

apoptosis in the culture cells and substantia nigra treated by

green tea polyphenols. ROS and NO free radical generation,

thiobarturic acid reacted substances (TBARS) content, nitrite/nitrate

concentration and protein bound 3-NT were decreased

and antioxidant abilities increased by the treatment of green tea

polyphenols. The protein levels and activity of NOS were decreased

by the treatment of green tea polyphenols. Conclusions:

These results suggest that green tea polyphenols protected cell

apoptosis induced by 6-OHDA through ROS-NO pathway in vitro

and in vivo.

Nie,G..J., Jin,C-F., Zhao,B-L. Distinct effects of tea catechins on 6-

hydroxydopamine-induced apoptosis in PC12 cells. Arch. Biochem. Biophys.

397,84-90,2002

Nie, G.J., Cao, Y-L., Zhao, B-L. Protective effects of green tea polyphenols and

their major component, (-)-epigallocatechin-3-gallate (EGCG), on 6-

hydroxyldopamine-induced apoptosis in PC12 cells. Redox Report, 7,170-

177, 2002

S-H Guo, E Bezard, Baolu Zhao. Protective effect of green tea polyphenols on

the SH-SY5Y cells against 6-OHDA induced apoptosis through ROS-NO

pathway. Free Rad Biol Med 39: 682-695, 2005.

Shuhong Guo, Jingqi Yan, Erwan Bezard, Tangbin Yang, Xianqiang Yang,

Baolu ZhaoProtective effects of green tea polyphenols in the 6-OHDA rat

model of Parkinson’s disease through inhibition of ROS-NO pathwayBiological

Psychiatry 621353-13622007

This work was supported by a grant of National Natural Scientific Foundation

(30170239) and 973 grant (2006CB500700).

175


Effects of aqueous extracts from Vitis coignetiae Pulliat leaves

on non-alcoholic steatohepatitis model rat

Chengzhu Zhao 1 , Fusako Takayama 1 , Toru Egashira 1 ,

Mitsumasa Mankura 1 , Keiji Ueki 2 , Azusa Hasegawa 1 ,

Hiromu Kawasaki 1 , Shigeru Okada 1 , and Akitane Mori 1

1 Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences,

Okayama University. 2 Hiruzen winery Co., Ltd

Background: Vitis coignetiae Pulliat (Yamabudo) is used as

health juice and wine based on the abundant polyphenols and anthocyanins

in its fruit. However, the similar benefits of leaves of

this plant were not been well demonstrated. This study investigated

the therapeutic effects of aqueous extracts from Vitis coignetiae

Pulliat leaves (VCPL) on an animal model of nonalcoholic steatohepatitis

(NASH) at the progressive processes.

Methods: NASH model (PCT/JP 2007/52477) were prepared

by loading oxidative stress following fatty liver formation in rats

by feeding choline deficient high fat diets (CDHF). After the progress

to steatohepatitis, VCPL administration to NASH rats was

performed for 3 weeks. Animal experiment was performed in 5

groups; Control (with MF chow diets), CDHF, NASH, NASH +

100VCPL and NASH+300VCPL. After the experimental period,

blood and liver were collected from anesthetized animals for the

samples to determine the extent of oxidative stress injury and the

overall effects of VCPL , biochemically and histologically.

Results: The electron spin resonance measurements assay

demonstrated the strong anti-oxidative activities of VCPL. In

NASH, hepatobiliary enzyme leakages and liver fibrosis were exhibited.

There were demonstrated OS in liver mitochondria and increases

in nuclear NF-B. VCPL administration after the steatohepatitis,

abated these changes.

Conclusion: The strong anti-oxidation activities of VCPL may

be beneficial in ameliorating NASH. As the OS enhances the NF-

B nuclear import which promotes the induction of inflammatory

proteins.

Anti-oxidation and lifespan-extension activities of

CordyMax in oxidative stress and aging models

Jia-Shi Zhu 1,2 , Yan Zhang 3 , Jieying Yang 3 , Ninzhi Tan 3 ,

and Chunsheng Zhao 3

1 Pharmanex Research Institute, Provo, UT 84601, 2 Pharmanex Beijing Clinical

Pharmacology Center: 2 Xinkang Road, Beijing 100088, China; 3 School of

Pharmacy, Shihezi University, Shihezi City, Xinjiang 832000, China

Cordyceps sinensis is traditionally believed as an anti-aging

herb in China. We have reported the effects of CordyMax (CM), a

mycelia fermentation product of C. sinensis, in glucose-lipidenergy

metabolisms, anti-fatigue and endurance enhancement. In

this study we examined the effect of CM in antioxidant and

lifespan extension in mice. The antioxidant activity was tested in

mice (6 months old) that received 60 days of vehicle or CM (0.5,

1.0, or 1.5 g/kg) and a single dose of 11 Gry 60 Co gamma-radiation

on Day 60. Compared to controls, CM increased plasma total thiolgroups,

GSH and GSH-peroxidase, and liver CAT, SOD and GSHreductase

(p < 0.05). CM reduced liver protein carbonyl-groups

and 8-OHdG (p < 0.05). For examining the lifespan-extension effect

of CM, 250 mice of 12 months of age (both sexes) were received

either vehicle or CM (0.5, 1.0, or 1.5 g/kg) mixed with the

forage. Calorie intake was adjusted to match the levels for controls

twice per week. Compared to controls, the 75% survival time was

extended 94-108 days in the CM dosage groups, the 50% survival

time extended 10-66 days, the 25% survival time extended 29-44

days and the 12.5% survival time extended 7-50 days (86 wks so

far; treatment continues). The Kaplan-Meier Survivor analysis revealed

the extended lifespan and the reduced risks of death by CM

(p < 0.05). In conclusion, CM therapy significantly improves the

body’s antioxidant capacity and extends the lifespan in mice, supporting

the traditional belief on the anti-aging function of CM in

humans.

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177


AUTHOR INDEX

A

Acin-Perez, R............................40

Adams, L.S. ..............................47

Agarwal, A.R............................62

Agbonwaneten, E. ..................105

Aggarwal, B.B. .........................46

Ahmed, S.S. ............................113

Aiman, S. ..................................63

Aimo, L. ....................................64

Albanes, D. ...............................38

Ali, I. .......................................109

Ames, B.N.................................59

Amiranashvili, L..................75,76

Ananth, P.................................148

Anderson, R.F...........................65

Andrei, C.C...............................82

Anghelina, M. ...........................66

Anita, Nor .................................63

Antosiewicz, J....................67,162

Anzai, K. .................... 68,127,133

Arab, L. ...................................152

Arçari, D.P. .............................147

Aronson, W.J. ...........................47

Aruga, T. ..............................87,88

Asghar, M. ................................85

Aung, H.H..........................69,101

Ay, H. ...............................139,154

Azad, A. ....................................70

B

Bairos, V.A. .........................79,80

Bandez, M.J. ...........................134

Bandinelli, S. ............................38

Banerjee, J............................71,72

Banerjee, P. ...............................73

Banerjee, S. ...............................73

Bao, X. ......................................74

Baracat, M.M..................81,82,98

Barbakadze, V. ....................75,76

Baroth, V..........................108,115

Bastiaens, P...............................77

Batista, W.L. ...........................129

Baum, T...................................111

Bendzala, S. ............................114

Bettaieb, A. ...............................77

Beveridge, S............................108

Bhattacharya, S.........................73

Bibus, D. .................................116

Biesalski, H.K...........................41

Biswas, S..............................71,90

Blaner, W.S...............................40

Bombicino, S.S.......................159

Bone, R.A..................................78

Borkowska, A. ...................67,162

Borrás, C. ..................................56

Bourdette, D.N........................149

Boveris, A. .......................134,159

Boveris, A.D. ..........................134

Bratasz, A..................................66

Brinton, R.D............. 105,113,165

Brooks, G.A. .............................93

Budac, S. .................................114

Butt, O.I.....................................66

Byun, H.-O..............................153

C

Cadenas, E. .62,106,107,112,113,

............ 123,134,150,166,167,170

Calcutt, N. .................................91

Cambou, M.C. ........................152

Carr, D.W................................149

Carvalho, R. .........................79,80

Casagrande, R......... 81,82,98,160

Catalano, A. ............................142

Chan, Y.-C. ..........................71,83

Chang, Y.-Z. ...........................174

Chankvetadze, B.......................75

Chen, N. ..................................119

Chen, S. ...................................165

Chen, Z......................................22

Cheng, M.-L............................163

Cherubini, A. ............................38

Chibber, S. ..............................109

Chikvaidze, E.N........................84

Chiu, D.T.-Y...........................163

Chiu, P.Y.................................119

Choe, W. .................................117

Choi, Y. ...................................120

Choi, Y.-J.........................117,120

Chugh, G. ..................................85


Chung, H.W. ...........................120

Cimrova, B..............................114

Clemens, M.G...........................17

Cluett, C. ...................................38

Coelho, A.C. ........................79,80

Colangelo, M. .........................142

Collard, E. .................................86

Compadre, A.J. .......................105

Corsi, A.-M...............................38

Cross, C.E. ..............................101

Cuervo, A.M. ............................57

Cunha, F.Q..............................160

Cunha, T.M.............................160

Curcio, M. ...............................129

D

D’Annunzio, V. ......................159

Da Silva, M.............................161

Danilenko, M. .........................122

Das, D.K....................................25

de Cabo, R.................................43

De Spirito, M. .........................128

Dehpoor, A.R..........................104

Devasahayam, N.....................168

Dobesová, Z............................145

Dohi, K.................................87,88

Donato, M. ..............................159

Dotan, Y....................................89

Draelos, Z.D............................130

Driggs, J. ...................................90

Du, L.-B. .................................125

Du, Y. ........................................20

Duan, X.-L. .............................174

E

Egashira, T. ......................141,176

Ehren, J......................................91

Elkhammas, E...........................99

Eller-Borges, R. ......................129

F

Fan, J. ........................................92

Fan, X........................................93

Fang, H....................................102

Feldman, D................................99

Ferreira, S.H............................160

Ferrucci, L.................................38

Firstenberg, M. .........................99

Fonsecca, M.J.V. ....................160

Font de Mora, J.........................56

Forester, S.C. ............................94

Fraga, C.G........................124,161

Frayling, T.M............................38

Fried, L.P. .................................38

Friedman, A. .............................13

Froozandeh, M........................103

Froyen, E.B...............................95

Fujii, H. ...................................138

Fukuahra, K. ...........................133

Fukuzumi, S............................133

Furber, J.D. ...............................96

G

Galeotti, T. ..............................128

Galleano, M. ...........................124

Gamba, P...................................33

Gambini, J.................................56

Ganesh, K.............................97,99

Garcia, J.V. .........62,150,166,167

Gargiulo, S................................33

Gee, J.......................................115

Gelpi, R. ..................................159

Georgetti, S.R. ........ 81,82,98,160

Ghannadian, N........................103

Gibert, J.C. ................................78

Glotzbach, J. .............................10

Gnyawali, S................ 72,100,143

Gnyawali, U................. 97,99,100

Gogilashvili, L.....................75,76

Gogoladze, T.V. .......................84

Gogvadze, V. ............................26

Gohil, K..............................69,101

Golestani, A. ....................103,104

Gómez, C. ...............................134

Gonçalvez, C........................79,80

Gordillo, G.M. ............. 97,99,102

Grune, T. ...................................58

Guralnik, J.................................38

Gurtner, G.C. ............................10

H

Ha, T.K....................................120

Hagen, T.M...............................28

Haj, F.........................................77

Hajiani, M. .......................103,104

Halon, M. ...........................67,162

Hamilton, R.T. ..105,106,107,113

Hammerling, U. ........................40

Han, D. ..............150,166,167,169

Han, L......................................125

Han, S......................................117

Hargreaves, I.............................45

Hasanzadeh, A........................104

Hasegawa, A...........................176

Haskell, C.F. ....................108,115

Hassan, I..................................109

Heber, D....................................47

Henning, S.M............................47

Herman-Antosiewicz, A. .........67

Hernandez, E...........................165

Hirayama, A.....................126,171

Hiyama, K...............................126

Hodis, H.N. ......................106,112

Holmgren, A. ............................20

Hongo, N.................................157

Hoyos, B. ..................................40

Hristova, M. ..............................24

Hsieh, C.-E..............................110

Htet, Y.....................................137

Huang, J. .................................100

Huang, K. ................................100

Huff, G.K. ...............................111

Hussain, S.-R.A ........................71

Hussien, R.................................93

Hwang-Levine, J..............106,112

I

Ichijo, H. ...................................70

Igarashi, T. ..............................138

Iglesias, D.E............................159

Ikekawa, N. .............................135

Ikota, N....................................133

Irwin, R.W. .............. 105,113,165

J

Jaeggers, G.K..........................161

Jagla, F. ...................................114

Jamora, C. .................................16

Jendeková, L...........................144

Jergelova, M. ..........................114

Jia, H.-Y. .................................125

Jian, J.........................................71

Johnson, E.J. .............................39

Jones, R. ..................................115

Jou, Mei-Jie.............................110

Juliano, J. ................................155

Jung, H.-J. ...............................153

Jung, M....................................117

K

Kagan, V. ..................................42

Kamibayashi, M. ....................127

Kaneko, T................................126

Karato, M. ...............................157

Kasahara, D.I. ...........................24

Kawasaki, H.....................141,176

Kennedy, D.O. .................108,115

Kern, D...............................44,130

Khachatryan, I. .........................84

Khaghani, S.............................103

Khan, M. .................................148

Khanna, S........70-72,83,90,97,99

.................................. 116,143,148

Kim, J.-H.................................121

Kim, K.....................................117

Kim, S. ....................................117

Kim, Y.J. .................................120

Kim, Y.-M........................121,153

Kim-Saijo, M. ...........................23

Kinkhabwala, A........................77

Kirkpatrick, R. ..........................99

Klatte, E. ...................................99

180

181


Ko, K.M. .................................119

Koda, Y. ..................................136

Kodo, Y...................................141

Kohno, M. ...............................151

Konishi, T. ..............................135

Korkmaz, A......................139,140

Kotha, S.R...............................116

Krinsky, N.I. .............................39

Krishna, M.C. .........................168

Kubicki, N...............................143

Kunes, J...................................145

Kunwar, A...............................118

Kuppusamy, P.............. 54,66,148

L

Labate, V.................................128

Lam, P.Y. ................................119

Lame, M....................................69

Langley, P. ................................96

Lebold, K.M............................152

Lee, J.W. .................................120

Lee, P.........................................16

Lee, S..................................16,120

Lee, Y......................................120

Lee, Y.-K. ...............................121

Leonarduzzi, G. ........................33

Leong, P.K. .............................119

Leshno, I.P.M. ..........................89

Leumus, J. ...............................105

Leung, H.Y. ............................119

Levy, J.....................................122

Li, C.........................................123

Li, Y. .......................................173

Li, Z.........................................174

Lichtenberg, D..........................89

Lietz, G......................................37

Lin, S.-R..................................163

Litterio, C................................124

Liu, W. ....................................152

Liu, Y. .....................................125

Lokhandwala, M.F. ..................85

López-Cepero, J.M.................134

López-Grueso, R.......................56

Losordo, D.W. ..........................14

Lu, J...........................................20

Lu, Y. ........................................74

Lüdeman, J.......................108,115

M

Machida, M.............................138

Mackenzie, G.C. .......................64

Maddipati, K.R. ........................97

Maggini, S........................108,115

Magro, D.A.............................160

Maher, P....................................91

Majumder, P. ............................73

Mamiya, T...............................171

Manda, S. ..................................68

Manfredi, G...............................40

Mankura, M. ....................141,176

Maroz, A. ..................................65

Martinez, R.M...........................82

Matsui, H..........................126,171

Matsumoto, A. ..........................68

Matsumoto, K.-I. ....... 68,127,133

Matsumoto, S..........................168

Matsuo, Y..................................22

Matteini, A. ...............................38

Maulik, G. .................................30

Maulik, N. .................................30

Maulucci, G. ...........................128

Mazumder, S.............................73

Meadows, C. ...........................130

Mele, M...................................128

Melzer, D. .................................38

Merlani, M. ..........................75,76

Meyskens, F.L. .......................164

Mihara, Y. .................................87

Mikhael, M..............................173

Miller, M. ..................................99

Miminoshvili, A. ......................84

Mine, Manaka.........................141

Mitchell, J.B............................168

Miyamoto, K........................87,88

Mizuno, M. .............................136

Moldovan, L. ............................66

Moldovan, N.I...........................66

Monleón, D. ..............................56

Montagnier, L. ............................7

Montano, S................................20

Monteiro, H.P. ........................129

Monteiro, M.P.........................147

Moore, J.L.................................99

Moraes, M.S............................129

Moreira, I.C...............................82

Mori, A.............................141,176

Morré, D.M........................44,130

Morré, D.J.. ........................44,130

Mulkijanyan, K....................75,76

Murphy, M.P.............................65

Murray, A..................................38

Mustoe, T.A. .............................15

N

Nadeau, P. ...............................131

Nagano, Y.N. ..........................126

Nagasaki, Y.............. 132,156,171

Nakajima, Y............................135

Nakamachi, T............................87

Nakamura, S. ............................88

Nakanishi, I................ 68,127,133

Nakayama, K. .........................135

Naseem, I. ...............................109

Navarro, A. .............................134

Navas, P. ...................................43

Neel, B. .....................................77

Néron, S. .................................131

Nie, G. ........................ 92,173,174

Ning, B. ...................................173

Nishida, H. ..............................135

Nishitani, Y.............................136

Niwano, Y...............................151

Norberg, E.................................26

Novikova, Z. ...........................767

Nuttall, J.R. .............................137

Nyui, M. ...........................127,133

O

Ogur, R.............................139,140

Oh, I.........................................117

Oharazawa, H. ........................138

Ohkubo, K...............................133

Ohsawa, I. ...............................138

Ohta, S.....................................138

Ohtaki, H...................................87

Oishi, M. .................................156

Okada, S...........................141,176

Okuda, H. ................................133

Omata, Y. ..................................64

Oommen, S. ...........................101

Orlando, L.................................56

Orrenius, S. ...............................26

Otani, H.....................................31

Oteiza, P.I.......64,94,124,137,161

Oter, S. ..................... 139,140,154

Owji, A.A................................103

Ozawa, T. .........................127,133

Ozler, M. .................. 139,140,154

P

Pak, W.....................................141

Pallardó, F.V.............................56

Palozza, P................................142

Pani, G.....................................128

Papadopoulos, K.......................75

Parinandi, N.L....................97,116

Park, A.-A. ..............................143

Pasalar, P..........................103,104

Patel, V......................................99

Paulis, L. .................................145

Payabvash, S...........................104

Pechanova, O. .......... 114,144,145

Peixoto, F. .................................80

Penumathsa, S.V.......................30

Perola, M...................................38

Perry, J.R.B...............................38

Petersen, R.C. .........................146

Pinto, M.L. ...........................79,80

Poli, G. ......................................33

Ponka, P. .................................173

182

183


Prince, P.D. .............................124

Priyadarsani, K.I.....................118

Prolla, T.A.................................49

Q

Qin, J. ........................................39

Queiroz, Y.S. ..........................147

R

Rastegar, R..............................104

Rhee, S.G. .................................21

Ribeiro, M.L. ..........................147

Ricciardi, M. ...........................155

Rice, N. .....................................38

Rink, C. ...............99,116,143,148

Robertson, B. ..........................115

Rodriguez Mañas, L. ................60

Roy, S.........12,70,71,72,83,86,90

.................97,99,102,116,143,148

Russell, R.M. ............................39

Rutledge, J.C.............................69

S

Sadir, S. ............................139,140

Sadoshima, J. ............................32

Saji, H........................................52

Salinthone, S...........................149

Sampaio, G.R..........................147

Samuel, S.M..............................30

Sancheti, H................. 62,150,170

Sanson, J.S. ...............................82

Santos, D. ..................................80

Santos, E. ..................................56

Santos-Ocaña, C. ......................43

Sarkar, A. ..................................99

Sato, E. ....................................151

Satoh, K................................87,88

Schmittgen, T...........................71

Schnitt, R...................................70

Schubert, D. ..............................91

Schultz, C..................................77

Semba, R.D...............................38

Sen, C.K. . 70-72,83,90,97,99,100

...........................102,116,143,148

Sengupta, R...............................20

Sensel, M.G. ...........................152

Seo, Y.-H. ...............................153

Sha, Y.-l. .................................125

Sham, D.....................................24

Shariftabrizi, A. ......................104

Sharoni, Y. ..............................122

Shen, P. .....................................74

Shi, Z.-H..................................174

Shimokawa, O. .......................126

Shinde, S.S................................65

Shioda, S. .............................87,88

Shneker, B.................................99

Sielicka-Dudzin, A. ...........67,162

Siendones, E. ............................43

Silander, K. ...............................38

Simone, R................................142

Simsek, K................. 139,140,154

Singleton, A. .............................38

Sledzinski, M. .........................162

Smith, R.A.J..............................65

Soe-Lin, S. ..............................173

Sofi, T...............................111,155

Son, A........................................23

Sottero, B. .................................33

Spitzer, V. ...............................115

Steinberg, F.M. .........................95

Stiles, B. ..................................123

Subramanian, S.......................168

Sugie, K.....................................23

Sulakvelidze, M........................76

Sumitani, S..............................156

Sun, B........................................99

Sung, B......................................46

Supasai, S..................................64

Surh, Y.-J. .................................48

Suzuki, H.................................138

Swartz, H.M..............................53

T

Takahashi, H...........................138

Takayama, F. ...................141,176

Tan, N......................................177

Tanaka, T. .................................38

Tang, G......................................39

Tatewaki, N.............................135

Thangarajah, H. ........................10

Thirunavukkarasu, M. ..............30

Tian, Q. ...................................125

Tominaga, K. ..........................157

Topal, T.................... 139,140,154

Torres, E.A.F.S.......................147

Traber, M. ...............................152

Tsunawaki, S.............................87

U

Ucar, E. ...................................154

Ueki, K. ...................................176

Ueno, M. ...................................68

Ukai, M. ..................................158

Utsumi, H..................................27

Uysal, B.................... 139,140,154

V

Valdez, L.B.............................159

Valério, D.A............................160

van der Vliet, A. .......................24

Vasu, V.T................................101

Veasey, R. ...............................108

Velazquez, O.C.........................11

Verri, W.A. .................. 81,98,160

Verstraeten, S.V......................161

Vial, N.......................................10

Vicentini, F.T.M.C. ..... 81,98,160

Vicinanza, R. ............................47

Vieira Fonseca, M.J.............81,98

Vieira, S.M..............................160

Viña, J. ......................................55

Virtamo, J..................................38

Von Lintig, J. ............................36

Vranková, S. ....................144,145

W

Walston, J..................................38

Wang, P.....................................47

Waterhouse, A.L.......................94

Watson, A. ..............................108

Wei, J.-Y. ..................................92

Wesley, U..................................24

Wilson, D.W. ............................69

Wong, A....................................47

Woo, H.A..................................21

Woo, H.D................................120

Wozniak, M. ......................67,162

Wu, Y.-H.................................163

X

Xu, D. .................................92,173

Xu, J. .......................................100

Xu, R. ......................................100

Y

Yadav, V. ................................149

Yakumaru, H...........................133

Yamashita, E...........................157

Yang, C.-M. ............................163

Yang, J. ...................................177

Yang, S....................................164

Yannakopouylou, E..................75

Yao, J........................ 105,113,165

Yap, L.-P.................. 150,166,167

Yasui, H. .................................168

Ybanez, M...............................169

Yesilyurt, O.............................154

Yeum, K.-J................................39

Yildiz, S. .................................154

Yin, F................................107,170

Yodoi, J. ....................................22

Yofu, S. .....................................87

Yokota, T. ...............................138

Yoon, G............................121,153

Yoon, K...................................117

Yoon, S.-H. ......................121,153

Yoshihara, E. ............................22

184

185


Yoshitomi, T...........................171

Yu, M. .....................................117

Z

Zaiton, Z....................................63

Zaobornyj, T. ..........................159

Zhan, L. .....................................30

Zhang, L.Z. .............................172

Zhang, N. ................................174

Zhang, X.-j..............................125

Zhang, Y. .........................173,177

Zhao, B.L. ................ 172,174,175

Zhao, C.............................176,177

Zhao, Y...............................92,173

Zhao-Wilson, X.Z...................172

Zhivotovsky, B. ........................26

Zhu, J.-S. .................................177

Zicha, J. ...................................145

Zimermann, V.V.M..................82

zur Hausen, H. ............................6

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