Vol 9 No1 - Journal of Cell and Molecular Biology - Haliç Üniversitesi

jcmb.halic.edu.tr

Vol 9 No1 - Journal of Cell and Molecular Biology - Haliç Üniversitesi

Journal of Cell and

•Notch in apoptosis

Molecular Biology

Volume 9 · No 1 · June 2011

http://jcmb.halic.edu.tr

Xenobiotic genes and susceptibility to bladder cancer

MMP1 and MMP2 polymorphisms in TMJ disorder

Silencing suppressors of PVM

Bio-database compression using enhanced suffix array


Journal of Cell and

Molecular Biology

Volume 9 · Number 1

June 2011

İstanbul-TURKEY


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Adile ÇEVİKBAŞ, İstanbul, Turkey

Beyazıt ÇIRAKOĞLU, İstanbul, Turkey

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Aglika EDREVA, Sofia, Bulgaria

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Journal of Cell and

Molecular Biology

Published by

Haliç University

Faculty of Arts and Sciences

Editor

Nagehan ERSOY TUNALI

Editorial Board

M.Baki YOKEŞ

M.Burcu IRMAK YAZICIOĞLU

Kürşat ÖZDİLLİ

Aslı BAŞAR

Editorial Assistance

Ozan TİRYAKİOĞLU

Özlem KURNAZ

Nermin GÖZÜKIRMIZI, İstanbul, Turkey

Ferruh ÖZCAN, İstanbul, Turkey

Füsun GÜMÜŞEL, İstanbul, Turkey

Candan JOHANSEN, İstanbul, Turkey

Asım KADIOĞLU, Trabzon, Turkey

Maria V. KALEVITCH, Pennsylvania, USA

Valentine KEFELİ, Pennsylvania, USA

Meral KENCE, Ankara, Turkey

Uğur ÖZBEK, İstanbul, Turkey

Sevtap SAVAŞ, Toronto, Canada

Müge TÜRET SAYAR, İstanbul, Turkey

İsmail TÜRKAN, İzmir, Turkey

Mehmet TOPAKTAŞ, Adana, Turkey

Meral ÜNAL, İstanbul, Turkey

Selma YILMAZER, İstanbul, Turkey

Ziya ZİYLAN, İstanbul, Turkey


Journal of Cell and Molecular Biology

CONTENTS Volume 9 · Number 1 · June 2011

Review Articles

Decision making mechanism influences the regulatory function of Notch in

Apoptosis

1

A.NAIR

Polymorphisms in the xenobiotic genes and susceptibility to bladder cancer

5

N.ERSOY TUNALI and N.O.TİRYAKİOĞLU

Research Articles

Identification of silencing suppressors of potato virus M

R.KRYLDAKOV, R.AKBERGENOV, T.HOHN and B.ISKAKOV

Identification of a novel dehydration responsive transcript from tossa jute

(Corcohrus olitorius L.)

S.SHARMIN, M.M.MOOSA, Md.S.ISLAM, I.KABIR, A.AKTER and H.KHAN

Protective effects of curcumin on cadmium chloride induced colon toxicity in

Swiss albino mice

P.SINGH, P.MOGRA, V.SANKHLA and K.DEORA

In vitro regeneration of Cleome viscosa – an important medicinal herb

J.ANBURAJ, C.R.SINGH, S.SUNDARRAJ and S.KANNAN

Bio-database compression using enhanced suffix array for pairwise sequence

alignment

A.KUNTHAVAI and S.VASANTHA RATHNA

Cloning and expression of Lentinula edodes cellobiohydrolase gene in E. coli

and characterization of the recombinant enzyme

S.TAIPAKOVA, B.SMAILOV, G.STANBEKOVA and A.BISSENBAEV

Investigation of the MMP1 and MMP3 promoter polymorphisms in

temporomandibular joint disorder

N.TASKIN, K.ULUCAN, G.DEGIN, A.AKCAY, B.KARATAS and T.AKCAY

Lead induced alterations in blood cell counts and hemoglobin during gestation

and lactation in Swiss albino mice

I. BARBER, R. SHARMA, S. MOGRA, K. PANWAR and U. GARU

The effects of aqueous and ethanolic leaf extracts of Vernonia amygdalina on

some vital organs in adult Wistar rats

S.NWANGWU, D.A.OFUSORI, S.JOSIAH, O.F.AMEGOR, H.NJOYA and

A.O.AYOKA

Short Communication

Investigation of the EGFR gene variations in bladder cancer patients using

INFINITI TM Analyzer

N.O.TİRYAKİOĞLU, Ö.KURNAZ, Ö.O.ÇAKIR and N.ERSOY TUNALI

Instructions for Authors

15

21

31

37

45

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Journal of Cell and Molecular Biology 9(1): 1-4, 2011 Review Article 1

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Decision making mechanism influences the regulatory function of

Notch in apoptosis

Ajay NAIR*

Laboratory for Evolution and Development, Department of Biochemistry, University of Otago, Dunedin

9054, New Zealand

(* author for correspondence; rudraaj14@gmail.com)

Received: 24 January 2011; Accepted: 28 February 2011

Abstract

The anti-apoptotic role of Notch and its interactors has been cited by many in the recent past. This intriguing

phenomenon however involves a non-canonical pathway that is independent of the usual nuclear-

localization of the Notch intracellular domain (NICD) which is formed following a series of cleavage events

set off by the ligand dependent activation of the Notch receptor. This finding has in turn led to the general

postulation that there is perhaps a membrane-tethered or cytoplasm localized form of NICD which is

responsible for its non-canonical pathway. However, this notion has been a subject of scrutiny from the

moment of its origin. This review will make an attempt to compile most of these current ambiguities and will

furthermore try to substantiate, a likely evolutionary significance of the regulatory function of Notch on

apoptosis, by using the theory of Natural Selection to intervene.

Keywords: Notch, nuclear localization, Notch intracellular domain, apoptosis, natural selection.

Karar verme mekanizması Notch'un apoptozdaki düzenleyici rolünü etkiler

Özet

Notch ve etkileşimde bulunduğu proteinlerin anti-apoptotik rollerinden yakın geçmişte fazlaca

bahsedilmiştir. Ancak bu ilgi çekici fenomen, Notch reseptörünün ligand bağımlı aktivasyonuyla başlayan

kesilme olayları sonucunda gerçekleşen Notch intrasellüler bölgesinin nükleer lokalizasyonundan bağımsız

kanonik olmayan bir yolakla ilişkilidir. Bu bulgu kanonik olmayan bu yolaktan sorumlu NCID'nin

membrana bağlı veya sitoplazmik bir formu olabileceği varsayımına yol açmıştır. Fakat bu görüş baştan

itibaren detaylı incelemelere maruz kalmıştır. Bu derleme, bu konudaki belirsizlikleri toplamaya ve dahası

doğal seleksiyon teorisi aracılığıyla Notch proteininin apoptozda düzenleyici işlevinin muhtemel evrimsel

önemini göstermeye çalışacaktır.

Anahtar Sözcükler: Notch, nükleer lokalizasyon, Notch intraselüler bölgesi, apoptoz, doğal seleksiyon.

Introduction

The Notch protein is a transmembrane receptor

possessing distinct extracellular and intracellular

domains. Specific ligand proteins (single pass

transmembrane proteins belonging to the

Delta/Serrate/ LAG-2 family of proteins) bind to

the extracellular domain which in turn triggers

successive proteolytic cleavage events starting with

that of the extracellular domain, catalyzed by a

metalloprotease, TACE (Tumor Necrosis Factor

Alpha Converting Enzyme). This is then followed

by the cleavage of the remaining notch protein

catalyzed by γ-secretase (Perumalsamy et al.,

2009). Once cleaved, it is referred as the Notch

intracellular domain and migrates to the nucleus to

influence gene expression. This is the conventional

Notch signaling pathway. Apart from the canonical

pathway of Notch signaling there also an


2 Ajar NAIR

alternative pathway which culminates in the release

of anti-apoptotic factors and the inactivation of proapoptotic

proteins, especially in T cells (Sade et al.,

2004). However, this unorthodox pathway avoids

the usual nuclear localization. Recent research on

the same has engendered the possibility of a

membrane-tethered or cytoplasm- localized form,

something that has been a subject of intense

research since then. Now the question regarding the

membrane or cytoplasmic localization of the Notch

intracellular domain during its anti-apoptotic non

canonical signal transduction is certainly puzzling.

But what is utmost intriguing is that the structural

composition of NICD doesn’t completely and

substantially support a membrane tethered or

cytosolic form separately. This assertion can be

made more explicit with the following findings

from various works done in this regard:

1. The existence of a nuclear localization sequence,

intact in the NICD.

2. Notch intracellular domain construct with a

partially truncated PEST (P (Proline), E (Glutamic

acid), S (Serine), T (Threonine)) domain is

abbreviated as NIC (Figure 1); whereas the active,

full-length Notch intracellular domain is

abbreviated as NICD. While investigating the

regulatory function of Notch-1 on apoptosis, one

anti-apoptotic protein in particular is significantly

upregulated in NICD-expressing Jurkat cells: the

XIAP (X-linked inhibitor of apoptosis). A nearly

three-fold increase of XIAP protein was detected in

NICD-expressing T cells. Over expression of the

full-length Notch intracellular domain also resulted

in elevated levels of XIAP (Liu WH et al., 2007).

3. Notch increases XIAP stability through

downregulation of proteasome-mediated protein

degradation and upregulation of XIAP by Notch

activation is not Akt-dependent (Liu et al., 2007;

Dan et al., 2004).

4. NICD is composed of a RAM domain at the N

terminal, then followed by six repeats of Ankyrin

(ANK) followed by a NLS. This is ensued by a

Transactivation domain (TAD) and the NICD

structure ends at the C terminal, by a PEST domain

(Figure 1). Deletions in the RAM or ANK repeat

domains, do not affect the binding of NICD to

XIAP. Truncation of the TAD from NICD on the

other hand abolishes its association with His-XIAP,

indicating that TAD is the XIAP binding region on

NICD. NICD directly binds XIAP, where the Cterminal

domain on NIC and the RING-finger

domain of XIAP are critical for such interaction

(Liu et al., 2007).

5. NICD interacts with XIAP in the cytoplasm;

which was confirmed by the finding that coexpression

of XIAP decreases NIC-directed HES5

(Hairy and enhancer of split5) trans-activation (Liu

et al., 2007).

6.TAD dictates the transcription activity of NICD.

Partial deletion of TAD domain retains most of the

NICD transcription activity, shown by activation of

the HES5 promoter, yet completely eliminating the

capacity of NICD to bind XIAP (Figure 2) (Liu et

al. 2007). If there is an independent membrane

tethered form, then the canonical pathway must not

get influenced by any means.

Figure 1. Structural makeup of NICD. RAM

domain at the N terminal is followed by six ANK

repeats, a nuclear localization sequence, a TAD

(Trans-activation domain) and a PEST domain at

the C terminal. Truncation of the TAD affects

NICD’s association with XIAP; altering NICD’s

anti-apoptotic function.


The aforesaid, thus, strongly suggests that there

is more to be added to complete the puzzle. From

the above points it seems that there exists a

decision making mechanism, within the cytoplasm

which decides the fate of the NICD, which either

complies with its usual nuclear retention or NCID

gets stabilized in the cytoplasm. And certainly this

point out to the possibility of an alternate

modification of the NICD, other than those

mentioned above, that perhaps answers for such a

diversity seen in Notch, in terms of its action.

Figure 2. Activation of transcription, via NICD’s

usual nuclear retention in a cell. A) activated notch

receptor. B) cleaved NICD with intact TAD. C)

XIAP. D) NICD with truncated or deleted TAD;

NICD fails to bind XIAP. E) HES5; NICD

localizes to nucleus and associates with HES-5 to

turn on transcription.

An evolutionary rationale

This perplexing behavioral change exhibited by

Notch receptors and its signaling cascade may

perhaps be explained in an alternate fashion, by

observing it from an angle that concentrates on

reasoning out the advantage of a T cell that may

hold when urged to respond to an environmental

stimulus like neglect or etoposide or DNA damage

induced death signals. It’s recognized that energy is

consumed for two central processes in any

biological cell; one is for reproduction and the other

for cell-maintenance. Although indirectly, the notch

canonical pathway is responsible for meeting the

requirements pertaining to development,

Processing of Notch rescues cells from apoptosis 3

reproduction etc. But a situation like neglect or

other antagonist stimulus poses an unfavorable

condition. Hence it is certainly in a cell’s interest to

switch to a mode which would momentarily

conserve much of the cells free energy for cell

maintenance by promoting cell survival signaling

pathways rather than promoting the customary

transcriptional activities (developmental and

metabolic); for it is utterly uneconomical to spend

the same energy on reproducing the cells in an

attempt to revitalize the cell population. Therefore,

notch signaling in entirety is bound to have a

decision making mechanism from the time of its

activation. But does notch directly respond to the

demand or the environmental stimulus. Perhaps

there is more than a straight intrusion from Notch.

Notch interactors, other than the canonical and noncanonical,

are mostly playing a pivotal role in

answering this question which still stands

challengeable inspite of the various breakthroughs

made in the context of Notch and its regulatory

function on apoptosis. The question that continues

to seek an answer is, why does a mechanism like

the Notch signaling pathway, which is one of the

highly conserved cell signaling systems present in

most multicellular organisms in terms of involving

gene regulation mechanisms that control multiple

cell differentiation processes, incorporate a

different role such as anti apoptosis? Because notch

holds the potential to influence cell fate decisions,

it is imperative that there be a decision making

mechanism that can modulate this omnipotent

transmembrane protein and its actions, depending

upon the need of the hour from a toll of billion

deaths (Neglect or DNA damage induced).

Probably cells have earned and evolved towards

this non conventional Notch pathway, that rescues

cells from self flattening. This postulation finds

support from a concept called- The Selfish Gene

Theory or Gene Selection Theory, according to

which “adaptations are the phenotypic effects of

genes to maximize their own representation and

continued survival in the future generations”.

Genes build vehicles to meet their mutual interests

of jumping into the next generation of vehicles

(Dawkins, 2006). Cells are the survival machines of

genes. So it is important that there exist a sequential

set of events which end up modulating the NICD in

response to an apoptotic stimulus; this change will

ensure cell survival and as a consequence, promote

the perpetuation of genes.

Neglect like situation, (known to activate antiapoptotic

factors via Notch, in T cells (Sade et al.,


4 Ajar NAIR

2004)) is extremely stressful; leading to a shift in

the cell’s energy. The cell prepares itself for

apoptosis; apoptosis promoting factors are

activated. Such an event forces mitochondria

towards an immediate fragmentation which marks

the onset of apoptosis; fragmentation induces

release of intrinsic pro-apoptotic factors (for e.g.

cytochrome-c) (Suen et al., 2008). Perhaps an

apoptotic distress signal, such as the one mentioned

above, prevents the usual processing of NICD,

which causes the partial or complete deletion of its

trans-activation domain; in so doing prevent its

nuclear localization and its ability to stimulate

transcription by associating with transcription

factors (for e.g. HES5). Possibly, a decision making

mechanism in the form of an additional physical

modification of NICD, in the cytoplasm, changes

its typical course, so as to meet the need, which is

to respond to the induced stress. Besides activating,

anti-apoptotic factors like XIAP, this tailored NICD

may well be acting upstream to the factors which

promote mitochondrial connectivity (for e.g.

Mitofusin (Mfn) 1&2), causing a delay in bax

activation, cytochrome-c release and eventually halt

cell demolition (Figure 3).

Figure 3. Proposed alteration in notch activity

during cell neglect. A) Notch receptor. B) Induced

stress leads to, a shift in the energy metabolism,

and the release pro-apoptotic factors (shown as

blue circles); this results in mitochondrial

fragmentation. C) This might act as an alarm

upstream notch to stop its usual course to the

nucleus; HES5 (shown in blue inside the nucleus)

remains unbound to trigger transcription. D)

Cleaved NICD binds to XIAP via its intact TAD.

E) This interaction might activate other antiapoptotic

factors and also release factors (for e.g.

Mfn) to restore mitochondrial connectivity.

Conclusion

For each and every phenomenon or process that

exists, there must in every case be a reason for its

occurrence in terms of the benefit it provides to a

cell. This leads to a specific adaptation with respect

to the cell’s requirement at a particular instance

(such as the long term survival of Memory T cells

due to the regulatory function of Notch-1 on

apoptosis). Through rigorous selective pressures on

a cell, several individual molecular processes are

scrutinized. The one that makes a cell evermore

specialized with respect to the immediate demand,

is selected by natural selection. It is this long

evolutionary history that, in all probabilities,

endowed notch with this non-canonical

mechanism. Hence, notions that speculate a

discrete membrane localized form for Notch, do

not seem to hold up.

References

Liu WH, Hsiao HW, Tsou WI and Lai MZ. Notch

inhibits apoptosis by direct interference with

XIAP ubiquitination and degradation. The

EMBO Journal. 26: 1660–1669, 2007.

Perumalsamy LR, Nagala M, Banerjee P and Sarin

A. A hierarchical cascade activated by noncanonical

Notch signaling and the mTOR–

Rictor complex regulates neglect-induced death

in mammalian cells. Cell Death and

Differentiation. 16: 879-889, 2009.

Sade H, Krishna S and Sarin A. The Anti-apoptotic

Effect of Notch-1 Requires p56lck-dependent

Akt/PKB-mediated Signaling in T Cells, The

Journal of Biological Chemistry. 279: 2937–

2944, 2004.

Suen DF, Norris KL and Youle RJ. Mitochondrial

dynamics and apoptosis. Genes & Development.

22: 1577-1590, 2008.


Journal of Cell and Molecular Biology 9(1): 5-13, 2011 Review Article 5

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Polymorphisms in the xenobiotic genes and susceptibility to

bladder cancer

Nagehan ERSOY TUNALI* and Necip Ozan TİRYAKİOĞLU

Haliç University, Department of Molecular Biology and Genetics, Istanbul, TURKEY

(* author for correspondence; nagehanersoy@halic.edu.tr)

Received: 13 May 2011; Accepted: 20 June 2011

Summary

It is thought that the interaction between genetic and environmental factors accounts for the different levels

of susceptibilities to the development of bladder cancer. The well known etiological risk factors of bladder

cancer are occupational exposure to certain carcinogens and cigarette smoking. Therefore, polymorphisms in

the genes coding for the xenobiotic-metabolizing enzymes may cause individual variations in metabolizing

the exposed carcinogens. Here we aimed to review the metabolising genes and respective polymorphisms

and to demonstrate the association between xenobiotic-metabolising gene polymorphisms and bladder

cancer. Among metabolizing enzymes, we will introduce N-acetyltransferases (NAT), glutathione-Stransferases

(GST), sulphotransferases (SULT), NAD(P)H dehydrogenase quinone 1 (NQO1),

methylenetetrahydrofolate reductase (MTFR), cytochrome P-450 (CYP) and aldo-Ketoreductases (AKR)

with special emphasis on the last two enzyme group.

Keywords: Bladder cancer, xenobiotic metabolism, metabolising genes, genetic susceptibility, genetic

polymorphism

Ksenobiyotik genlerdeki polimorfizmler ve mesane kanserine yatkınlık

Özet

Genetik ve çevresel faktörler arasındaki etkileşimin mesane kanseri oluşumu için farklı seviyelerde

yatkınlık oluşturduğu düşünülmektedir. En iyi bilinen etiyolojik risk faktörleri, meslek sebebi ile

maruz kalınan kanserojenler ve sigara içimidir. Bu nedenle, ksenobiyotik-metabolize edici

enzimlerdeki polimorfizmler maruz kalınan karsinojelerin metabolize edilmesinde kişisel

varyasyonlara sebep olabilir. Biz burada metabolize edici genleri ve ilgili polimorfizmleri derlemeyi

ve ksenobiyotik-metabolize edici gen polimorifzmleri ile mesane kanseri arasındaki bağlantıyı

göstermeyi amaçladık. Metabolize edici genlerden N-asetiltransferazları (NAT), glutatyon-Stransferazları

(GST), sulfotransfeazları, NAD(P)H dehidrogenaz kinon 1’i (NQO1),

metilentetrahidrofolat redüktazı, sitokrom P-450’yi (CYP) ve aldo-ketoredüktazları (AKR), son iki

enzim grubuna özel vurgu yaparak tanıtacağız.

Anahtar kelimeler: Mesane kanseri, ksenobiyotik mekanizma, metabolize edici genler, genetik

yatkınlık, genetik polimorfizm

Introduction

Cancer is regarded as a multifactorial disorder

affected both by genetic and environmental factors,

which culminate in dysregulated cell cycle.

Although environmental risk factors are considered

as the most important among others,

epidemiological studies have shown that a certain

part of the population is in fact more prone to

develop cancers despite being exposed to the same

type and amount of environmental risks. This


6 Nagehan ERSOY TUNALI and Necip Ozan TİRYAKİOĞLU

finding has led the molecular epidemiology studies

to focus on predisposition to cancer via genetic

factors. It has been speculated that either the

polymophisms in the DNA repair genes or those in

carcinogen metabolizing genes should primarily be

questioned as possible candidates of genetic

modifiers. The polymorphisms in the DNA repair

genes may alter the function of the related enzyme

and thereby affect the repair capacity of the

individual. This kind of alteration in the DNA

repair mechanism may get the individual more

sensitive to carcinogens and more susceptible to

cancer formation (Franekova et al., 2008).

Likewise, polymorphisms in the genes coding for

the xenobiotic-metabolizing enzymes may cause

individual variations in metabolizing the exposed

carcinogens (Longuemaux et al., 1999). As a result,

individuals with a certain genetic background may

become more susceptible to develop cancer.

Xenobiotic metabolism

Xenobiotic metabolism (from the Greek xenos

"stranger" and biotic "related to living beings") is

composed of several metabolic pathways that

modify the chemical structure of xenobiotics, e.g.

drugs, carcinogens, poisons. Upon their intake by

an organism, xenobiotics undergo several metabolic

Metabolising genes and susceptibility to bladder

Cancer

Polymorphisms in the xenobiotic-metabolising

genes are pivotal to the above-mentioned

alterations and therefore have been intensively

investigated in many cancer types. Foremost of

Figure 1. Activation pathways of carcinogens

conversions to facilitate their solubility in order to

expedite their removal from the organism. These

so-called biotransformation processes consists of

two consecutive steps; an initiation phase, where

carcinogens are modified to accept reactive groups

(Phase I) via the action of cytochrome P450

oxidases; and a second phase where they are

converted into more easily extractable molecules by

conjugation reactions (Phase II) via transferase

enzymes. The xenobiotic metabolism can be

regarded as a detoxification mechanism consisting

of two phases, however, conversion reactions

catalyzed by xenobiotic-metabolising enzymes can

also activate potential carcinogens (Figure 1.).The

reason for this is the vastly diverse chemical

structures of carcinogens which can not be

compensated by the limited number of conversion

reactions catalyzed by xenobiotic enzymes. The

capability of the xenobiotic metabolism to either

activate or detoxify carcinogens constitute a fragile

balance between activation and detoxification of

carcinogens, which defines the amount of DNA

damage accumulated in the cells. Alterations

affecting enzyme activity shifts this balance

towards either detoxification or activation of

carcinogens.

these cancers is the bladder cancer, for which the

well-known etiological risk factors are occupational

exposure to certain carcinogens and cigarette

smoking. In fact, smoking is proved to be the

primary cause of approximately 70% of the bladder

cancer cases (Parkin 2008). It is thought that the

interaction between genetic and environmental


factors accounts for the different levels of

susceptibilities to the development of bladder

cancer. Bladder cancer incidence varies highly

between distinct regions worldwide, with

developed countries having the highest incidence

(Silverman et al., 1999; Parkin et al., 2002). Higher

bladder cancer incidence in developed countries

despite increased awareness against risk factors

indicates the existence of genetic factors affecting

bladder cancer susceptibility. Thereby,

polymorphisms with possible association to bladder

cancer have been widely investigated. Many

metabolizing genes and their polymorphisms have

been identified as responsible for individual

susceptibility to cause BC.

This review is written as an attempt to

demonstrate the association between xenobioticmetabolising

gene polymorphisms and bladder

cancer. Among metabolizing enzymes, we will

introduce N-acetyltransferases (NAT), Glutathione-

S-transferases (GST), Sulphotransferases (SULT),

NAD(P)H dehidrogenase Quinone 1 (NQO1),

Methylenetetrahydrofolate reductase (MTFR),

Cytochrome P-450 (CYP) and Aldo-Ketoreductases

(AKR) with special emphasis on the last two

enzyme group and related polymorphisms.

N-acetyltransferases

In humans, genetic locus for the two isoenzymes of

NAT, NAT1 and NAT2, resides at chromosome

8p22. NAT1 and NAT2 are polymorphic and both

catalyze N- and O-acetylation of aromatic and

heterocyclic amines (Franekova et al., 2008).

NAT2, one of the Phase II enzymes, play roles in

the bio-conversion of heterocyclic amines to

electrophilic nitrenium ions. NAT2 catalyzes the

activation and deactivation of various aromatic

amines, heterocyclic amines and hydrazin drugs.

Polymorphisms in the NAT2 gene have been

strongly associated with bladder cancer. Based on

the 13 SNPs, 30 different NAT2 alleles have been

defined

(http://louisville.edu/medschool/pharmacology/NA

T.html). Depending on the effects of the

polymorphisms, NAT2 alleles have been

categorised in two groups as slow and fast

acetylators. Individuals who lack both of the two

functional NAT2 alleles have been designated as

slow acetylators. A meta-analysis with combined

results from 31 different studies has shown that

NAT2 slow acetylation alleles increase the risk of

Genetic susceptibility to bladder cancer 7

bladder cancer (OR=1,4; %95CI=1,2-1,7) (Garcia-

Closas et al., 2005).

NAT1 enzyme transfers one acetyl group from

acetyl-coA to arylamine and hydrazin substrates.

Many NAT1 variants have been defined to date

(http://louisville.edu/medschool/pharmacology/NA

T.html), however studies have revealed conflicting

results (Hung et al., 2004) and their functional

significance could not be fully identified yet.

Since aromatic amines are present in cigarette

smoke are the major risk factors for bladder cancer

and are metabolized by NAT enzymes, it is for sure

that the polymorphisms of the NAT genes have

important roles in predisposing the individuals to

bladder cancer (Franekova et al., 2008).

Glutathione-S-transferases

Cytosolic GSTs belong to a superfamily of Phase II

enzymes, which contain sub-families and

polymorphic isoenzymes. The genetic

polymorphisms of these enzymes affect the

individual susceptibility to cancer, cardiovascular

and respiratory diseases. GSTs facillitate the

detoxification of electrophilic compounds by

gluthathione conjugation. They interact with a wide

variety of substrates including polycyclic aromatic

hydrocarbon (PAH) epoxides and oxidative stress

by-products. GSTM1 detoxifies carcinogenic PAHlike

benzopyrene. A common deletion

polymorphism in GSTM1 gene causes the loss of

enzyme activity and has been shown to be strongly

associated with increased risk for bladder cancer

(Jiang et al., 2011, Salinas-Sánchez et al., 2010)

According to a meta-analysis conducted to show

the overall effect of this polymorphism, an odds

ratio (OR) of 1,5 has been calculated (%95CI=1,3-

1,6) (Garcia-Closas et al., 2005). GSTT1 detoxifies

smaller reactive hydrocarbons, like ethylene oxide.

GSTT1 deletions, which have high frequency in

Caucasians, have been associated with increased

risk for bladder cancer (Abdel- Rahman et al.,1998;

Srivastava et al., 2004). GSTP1 is involved in the

conjugation and detoxification of many

xenobiotics. The results of the studies investigating

GSTT1 and GSTP1 gene polymorphisms are

generally conflicting (Brockmoller et al., 1996;

Lee et al., 2002.).

In summary, polymorphisms of GSTM1,

GSTT1 and GSTP1 constitute risk factors for many

cancer types. Especially the null alleles of GSTM1


8 Nagehan ERSOY TUNALI and Necip Ozan TİRYAKİĞLU

and GSTT1 are risk factors for bladder cancer in

chain smokers.

Sulphotransferases

SULTs are encoded by a supergene family and

catalyze the sulphonation of many xenobiotic

compounds, including drugs and carcinogens.

SULT enzymes play role as one of the main

detoxification systems in adults and in developing

human fetuses. This gene family is not very

polymorphic, except SULT1A1. SULT1A1

Arg213His polymorphism, which causes a decrease

in enzyme activity and thermal stability, has been

suggested as a protective factor for bladder cancer

(Zheng et al., 2003).

NAD(P)H dehidrogenase, Quinone 1

NQO1 enzyme protects the cell against

electrophilic and oxidizing metabolites of

xenobiotic and endogenous quinone compounds.

The gene coding for the NQO1 enzyme harbors

several polymorphisms affecting enzyme activity.

A187C transition results in the replacement of

proline with serine and decreases enzyme activity.

Accordingly, Ser187 allele has been shown to

increase bladder cancer risk (Franekova et al.,

2008).

Methylenetetrahydrofolate reductases

MTHFR converts 5,10-methylenetetrahydrofolate

(a methyl donor in deoxythymidine monophosphate

synthesis) to 5-methyltetrahydrofolate. Folate

deficiency is related to breakage of DNA strands

and binding of urasil to DNA. If an MTHFR variant

causes a decrease in the folate levels by decreasing

the enzyme activity; one can expect a tendency to

DNA strand breakage and cancer formation. On the

other hand, variant MTHFR activity alters the

amount of methyl donors and thereby involves in

bladder cancer formation by changing the status of

promotor methylation. MTHFR 677C>T and

1298A>C polymorphisms are proved to decrease

enzyme activity. However, the relationship between

the mentioned polymorphisms, enzyme activity and

susceptibility to bladder cancer should be more

extensively studied to seek for confirming results

(Karagas et al., 2005).

Cytochrome P-450 genes

CYP enzymes catalyze Phase I reactions and

belong to the microsomal enzyme super family.

CYP enzyme system consists of over 20 highly

polymorphic CYP enzymes

(http://www.cypalleles.ki.se/). Human CYP1A1

enzyme is expressed in epithelium and plays a key

role in the activation of many procarcinogens

including PAHs and aromatic amines arising from

tobaccco-related products. Ile-Val (m2) mutation

located in heme-binding domain and MspI (m1)

mutation has been shown to increase enzyme

activity. White blood cells of smokers harboring a

Ile-Val mutation have been shown to contain more

PAH-DNA adducts in comparison to non-smokers.

Expression of CYP1A1 has been shown to elevate

in bladder cancer patients in correlation with

increased tumour stage (Bartsch et al., 2000). m1

and m2 mutations, on the other hand, are directly

associated with increased risk for lung cancer

(Aynacioglu et al., 1998). CYP1A1 mutations

exhibit variations among different ethnic groups. In

contrast to Causians, prevalances of m1 and m2 are

high among Asian populations. M3 mutation was

identified only in Africans. In molecular and

epidemiologic studies, CYP1A1*2B allele,

harboring m1 and m2 mutations, was found to be

correlated with increased risk for lung cancer.

Figure 2 shows the localisation of these four

important mutations (m1, m2, m3, m4) on CYP1A1

gene. Human CYP1B1 gene is located at

chromosome 2p21 and contains three exons in a

10kb region. It codes for the 543 amino acid long

enzyme (Figure 3). It was found to be

overexpressed in many cancer types including,

colon, lung, skin, brain cancers in contrast to its

lower expression in normal somatic tissues (Thier

et al., 2002). CYP1B1 activates PAHs, aromatic

and heterocyclic amines. It also catalyzes the

conversion of benzo[a]pyrene to carcinogenic

metabolite diol epoxide-2, which indicates its

significant role in the activation of tobacco

carcinogens. Studies conducted so far indicate that

CYP1B1 polymorphisms, especially codon 432

polymorphism, change enzyme activity and

contribute to carcinogenesis. CYP1B1 codon 432

polymorphism increases risk for smokers for head

and neck cancers (Thier et al., 2002).

CYP2D6 gene codes for debrisoquine

hydroxylase. Its subtrates include aromatic amines

and tobacco nitrosamines. The existence of

homozygous recessive mutations in this gene

results in the inability of metabolising certain

compounds. Individuals with these mutations


compromise nearly 5-10% of the Caucasian

population which are categorised as poor

metabolizers (PM) because of their inability to

metabolise certain compunds. The PM phenotype is

Genetic susceptibility to bladder cancer 9

associated with increased susceptibility to cancer,

indicating a possible role for this enzyme in the

conversion of procarcinogens to active carcinogens.

Figure 2. m1, m2, m3 and m4 mutations in the CYP1A1 gene

Most common mutations responsible for the PM

phenotype have been identified. G/A transition

located on exon3-intron4 conjunction creates an

early stop codon resulting in defective mRNA. This

CYP2E1 metabolizes many procarcinogens, e.g.

N´-nitrosonornicotine, 4-methylnitrosamino-1,3pyridyl-1-butanone

and other volatile nitrosamines,

which are inhaled via cigarette smoking.

Inter-individual expression variants of human

CYP2E1 gene have been identified. The most

frequently studied RFLPs on the CYP2E1 gene are

those residing at the 5’ end of the gene which are

proved to alter enzyme activity. Among them,

PstI/RsaI (mutant allele: CYP2E1*5B) and DraI

(mutant allele: CYP2E1*6) polymorphisms. These

two polymorphisms together constitute the

CYP2E1*5A allele. In addition to this, RsaI (G-

1259C) and PstI (C-1091T) alleles are in complete

linkage disequilibrium with eachother. CYP2E1*6

allele variant is present in nearly 10% of the

European population. Recently, Haufroid et al.

(2002) have shown that people with at least one

CYP2E1*6 allele have lower chlorzoxazone than

homozygous wild type individuals. There are ethnic

variations in CYP2E1*5A allele frequencies among

Figure 3. Structure of the CYP1B1 gene (Thier et al., 2002)

mutation accounts for 80% of the PM phenotype.

While the deletion of the whole CYP2D6 gene

accounts for the 15% of PM phenotypes (Sobti et

al., 2005; Febbo et al., 1998).

Asians and Europeans. Europeans are 5%

heterozygous; Asians are 37% heterozygous and

6% homozygous for this allele. Because of the

certain inter-ethnic variations of the CYP2E1*5A

allele, genetic polymophisms, enzyme expression

and alterations in the chemical metabolism are still

not clear and the studies are conflicting each other

(Bolt et al., 2003).

Aldo-Ketoreductases

AKRs catalyze the conversion of carbonyl groups

to alcohol derivatives. Their most common

endogenic substrates are lipid aldehydes, steroids

and prostoglandines. They also activate prodrugs

and polycyclic aromatic hydrocarbons (PAHs). In

addition, they have been shown to induce resistance

against chemotherapeutic agents like cysplatine and

doxorubicin. The essential function of AKRs is to

convert aldehydes to primary and secondary

alcohols by reduction (Flynn and Green 1993;

Penning et al., 2004). This conversion enables the


10 Nagehan ERSOY TUNALI and Necip Ozan TİRYAKİOĞLU

conjugation reactions and therefore aldoketoreductases

are considered as Phase I enzymes.

Human AKRs interact with various substrates

including drugs, carcinogens and reactive

aldehydes. Their important role in the metabolism

of these substrates signifies them as key enzymes in

designating the carcinogenicity of these substrates.

There are 15 AKR families discovered so far.

Human AKRs are categorised into 3 families as

AKR1, AKR6 and AKR7. 8 of the 13 discovered

human AKRs belong to the AKR1 family

(AKR1A1, AKR1B1, AKR1B10, AKR1C1,

AKR1C2, AKR1C3, AKR1C4 and AKR1D1).

Most of the AKR enzymes are 34-37 kDa

monomeric proteins with a catalytic tetrade region,

conserved among family members. Their substrate

binding domains, on the other hand, show great

variety, representing the diversity of their substrates

(Jin and Penning, 2007).

AKRs and activation of PAHs

There are three different pathways of PAH

activation. These are P-450 peroxidase induced

formation of radical cations, P-450 induced

formation of diol-epoxides and AKR induced

formation of reactive o-quinones. The latter are

highly reactive molecules which can bind to most

macrobiomolecules including DNA, RNA and

proteins preemminently (Jin and Penning, 2007).

The formation of o-quinones generates reactive

oxygen species as by-products, which activate RAS

targets by inducing the expression of antioxidant

response element (ARE) containing genes. Since

RAS pathway is responsible for transmitting the

extracellular growth signals to nucleus, o-quinones

produced by AKRs are considered to effect both the

induction and progress of carcinogenesis (Jin and

Penning, 2007).

Function and tissue specific expression of Aldo-

Ketoreductases

Human AKR1C family members; AKR1C1,

AKR1C2, AKR1C3 and AKR1C4; show great

homology to each other. They all contain 9 exons

and are located on chromosome 10. The expression

of AKR1C genes are regulated by AREs located in

their promoters.

AKR1C1 and AKR1C2 have been shown to be

expressed 50-fold more in lung tumors (Hsu et al.

2001). To confirm these results Palackal and

colleagues have measured the expression levels of

AKR1C1, AKR1C2 and AKR1C3 isoforms in lung

adenocarcinoma cells and obtained similar results

(Palackal et al., 2002). Furthermore a study

conducted by Dozmorov et al. have shown that

increased AKR1C3 expression induce angiogenesis

and increase cell survival (Dozmorov et al., 2010)

.AKR polymorphisms

Even though the structural difference between

AKR1C1 and AKR1C2 arises only from 7 different

aminoacids, they have vastly different functions.

Nonhomologous aminoacids are T38V, R47H,

L54V, C87S, V151M, R170H and Q1721 which

are highly polymorphic. Common polymorphisms

in the AKR1C1 gene are T38A, T38I and R47H,

whilst H47R, S87C, L172Q and V38A are common

polymorphisms in AKR1C2. There have been 17

polymorphisms identified in the coding regions of

AKR1C3. Nine of these polymorphisms results in

amino acid changes while the other eight are

synonymous (Table 1.).

One of the nonsynonymous polymorphisms, the

G106T polymorphism, has been shown to produce

truncated protein; and another nonsynonymous

polymorphism, A230G, has been associated with

decreased cholesterol levels in the blood

(Jakobsson et al., 2007). AKR1C4*5 (L311V)

polymorphism is located on the C-terminal and has

been shown to affect substrate binding specifity

(Kume et al., 1999).

There are a few studies conducted to investigate

the association between the AKR polymorphisms

and cancer. AKR polymorphisms have been

investigated in Non-Hodgkin lymphoma and

AKR1A1 rs2088102 polymorphism has been

shown to be associated with increased risk (Lan et

al., 2004). Another study, where the association

between prostate cancer and AKR1C3

polymorphism have been investigated has shown an

increased risk for rs7741 polymorphism

(Cunningham et al., 2007).

Studies concerning rs12529 polymorphism gave

conflicting results. rs12529 has been shown to be

associated with increased lung cancer (OR=1,84,

%95CI= 0,98-3,45), while it has been shown to

confer protection for bladder cancer (Lan et al.,

2007; Figuerova et al., 2008; Tiryakioğlu, 2011).

Figuerova et al.(2008) investigated 8

polymorphisms located in two regions of AKR1C3

gene, which are likely to affect bladder cancer risk

(Table2).


Genetic susceptibility to bladder cancer 11

Table 1. Exonic polymorphisms of AKR1C3 gene (http://www.med.upenn.edu/akr/polymorphisms.shtml)

Exon Polymorphism Function Nucleotide change Aminoacid change Allele

3 rs7741 synonymous G90A P30 AKR1C3*1A

4 rs12387 synonymous G312A K104 AKR1C3*1B

6 rs1131124 synonymous G495A V165 AKR1C3*1C

3 rs1804061 synonymous A117G K39 AKR1C3*1D

6 rs1937839 synonymous G459A K153 AKR1C3*1E

8 rs7342072 synonymous C705T L235 AKR1C3*1F

6 rs17849485 synonymous C546T L182 AKR1C3*1G

3 rs28943574 synonymous A231G E77 AKR1C3*1H

1 rs12529 nonsynonymous C15G Q5H AKR1C3*2

6 rs1131132 nonsynonymous C525G M175I AKR1C3*3

3 rs1804062 nonsynonymous G106T E36term AKR1C3*4

9 rs1804059 nonsynonymous G879T M293I AKR1C3*5

4 rs4987102 nonsynonymous G316A A106T AKR1C3*6

3 rs11551177 nonsynonymous A230G E77G AKR1C3*7

9 rs11551178 nonsynonymous G883T A295S AKR1C3*8

5 rs28943579 nonsynonymous G434A C145Y AKR1C3*9

5 rs28943580 nonsynonymous C772T R258C AKR1C3*10

In this study, rs3763676, rs12775701 and

rs1937843 polymorphisms were found to be

associated with increased bladder cancer risk;

rs1937845, rs12529, rs4881400 and rs4641368

polymorphisms were found to confer protection

against bladder cancer. Recently, rs12529

polymorphism has been shown to be associated

with decreased bladder cancer risk (Tiryakioğlu,

2011). According to this retrospective study,

Conclusion

homozygous GG variant genotype protects against

bladder cancer (OR=0,255; %95CI=0,101-0,644),

which supports the findings of Figuerova et

al.(2008), where OR=0,78; %95CI= 0,52-1,18.

Upon adjustment of these figures according to

important risk factors in bladder cancer, namely,

sex and cigarette smoking status, Tiryakioğlu

(2001) have shown that rs12529 GG genotype still

conserves its protective effect (OR=0,243).

Table 2. AKR1C3 polymorphisms associated with bladder cancer

Polymorphism Genotype Odds Ratio

rs12529 GG OR= 0,78; %95CI= 0,52-1,18

rs1937845 GG OR=0,74; %95CI= 0,57-0,96

rs3763676 AG OR=1,28; %95CI= 1,05-1,55

rs4881400 GG OR=0,53; %95CI= 0,33-0,85

rs461368 TT OR=0,42; %95CI= 0,20-0,91

rs127755701 GG OR=1,41; %95CI= 1,05-1,90

The genetic variations in the human genome is truly

a powerful source for the investigation of the

complex disesases where genetic and

environmental factors interact to characterize the

final outcome. Over the past ten years, researchers

agreed on that rare genetic defects imputed to

certain populations are not usually responsible for

ever increasing cancer cases; rather polymorphic


12 Nagehan ERSOY TUNALI and Necip Ozan TİRYAKİOĞLU

variations in the DNA sequences confer individual

susceptibilities. This notion has been accepted for

many cancer promoting mechanisms, including

bladder cancer (Kim and Quan, 2005). Bladder

cancer still conserves its importance regarding

genetic and epidemiologic studies, since

environmental factors, mainly cigarette smoking

and occupational exposure to certain carcinogens

play major roles in the formation of this cancer

type. There are reasons for the high importance of

this cancer type in terms of molecular research. In

the first place, genetic polymorphisms in the

metabolizing genes may alter the functions of the

related enzymes, which will result in abnormalities

in the xenobiotic functions of the cell. As an

outcome, the environmental carcinogens may not

be detoxified properly and hence cause bladder

cancer. Identification of the polymorphisms in the

xenobiotic genes and their functional relevance to

bladder cancer formation will certainly highlight

the importance of inter-individual genetic

variations and the phenomenon of genetic

susceptibility, which can be adapted easily to other

genetic conditions. Looking at the same view from

another direction we can still come up with the

importance of genetic polymorphisms in defining

disease risks. Since bladder cancer is caused mainly

by environmental factors, people living at the same

standards in terms of cigarette smoking, diet and

other environmental exposures can be investigated

for the frequency of occurrence of bladder cancer.

Keeping the environmental factors constant, the

only remaining factor will be the genetic variations.

Comparing the frequencies of the genetic

polymorphisms among bladder cancer cases and

healthy controls exposed to the same environments,

genetic polymorphims responsible for the bladder

cancer formation can be identified. There are for

sure hundreds of genetic loci to investigate,

however, it will be wise to begin with the genes

coding for the xenobiotic-metabolizing enzymes.

On the other hand, the above-mentioned studies

should be performed for each ethnic and

geographic population in order to delineate

genotypic distributions and to define genetic

susceptibility loci. Using the accumulated data,

researchers can establish diagnostic and even presymptomatic

criteria on the basis of genetic

polymorphisms. Until now, the prognostic and

diagnostic value of the current accumulated data

prove to be problematic to utilize. The main reason

for this is the conflicting results in addition to

overlooked environmental and demographic

factors. Another reason is the small scope of the

most studies, focusing on a few genes instead of

taking the whole metabolic systems into account.

Also, sample sizes of the studies are one of the

major problems; wider retrospective research

studies including hundreds of samples should be

constructed. With the ever reducing time and

money costs of whole genome association studies,

it becomes rapidly easier to perform wide-range

studies, paving the way for more rewarding

research.

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Thier R, Brüning T, Roos PH and Bolt HM.

Cytochrome P450 1B1, a new keystone in

gene-environment interactions related to

human head and neck cancer? Toxicol. 76:

249-256, 2002.

Tiryakioğlu O. Investigation of aldoketoreductase

1C3 rs12529 polymorphism in

bladder cancer patients. MSc Thesis. Haliç

University, 2011.

Zheng L, Wang Y, Schabath MB, Grossman

H.B and Wu X. Sulfotransferase 1A1

(SULT1A1) polymorphism and bladder

cancer risk: A case-control study. Cancer

Letters. 202:61-69, 2003.


Journal of Cell and Molecular Biology 9(1): 15-20, 2011 Research Article 15

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Identification of silencing suppressors of potato virus M

Ruslan KRYLDAKOV 1* , Rashid AKBERGENOV 2 , Thomas HOHN 2 and Bulat

ISKAKOV 1

1

Laboratory of Proteins and Nucleic Acids Research, M.A. Ajtkhozhin Institute of Molecular Biology and

Biochemistry, Almaty, Kazakhstan

2

Laboratory of Molecular Plant Virology, Botanical Institute, University of Basel, Basel, Switzerland

(* author for correspondence; kryldakov@yahoo.com)

Received: 10 May 2010; Accepted: 20 January 2011

Abstract

Gene silencing is an important regulatory and defense mechanism in plants. Plant viruses evolved some

proteins that suppress cellular RNA interference mechanism. In this work identification of silencing

suppressors of potato virus M (PVM) is reported. The cDNA sequences of different PVM open reading

frames were cloned into the Gateway-compatible binary T-DNA destination vector pMDC32, harboring a

double 35S promoter. Each of these constructs was electroporated into Agrobacterium tumefaciens C58C1

which then were agroinfiltrated into Nicotiana benthamiana 16C line. It was shown that viral coat protein

(34K) as well as some other PVM proteins (12K, 11K) possessed suppressor properties of cellular silencing

activity.

Keywords: Gene silencing, suppressors of silencing, potato virus M, agroinfiltration, Nicotiana benthamiana

16C line

Patates virüsü M’nin susturucu baskılayıcılarının tayini

Özet

Gen susturma bitkilerde önemli bir düzenleme ve savunma mekanizmasıdır. Bitki virüsleri hücresel RNA

müdehalesi mekanizmalarını baskılayan bazı proteinler evrimleştirmiştir. Bu çalışmada patates virüsü M’nin

(PVM) susturucu baskılayıcılarının belirlenmesi rapor edilmiştir. Farklı PVM açık okuma çerçevesi olan

cDNA dizileri çift 35S promotor içeren ikili T-DNA hedef vektörü pMDC32’ye klonlandı. Bu düzeneklerin

herbiri elektroporasyon ile Agrobacterium tumefaciens C58C1’e verilip daha sonra agroinfiltrasyon ile

Nicotiana benthamiana 16C hattına süzdürüldü. Viral kılıf proteininin (34K) yanı sıra bazı diğer PVM

proteinlerinin (12K, 11K) hücresel susturma aktivitesinin baskılayıcı özelliklerine sahip oldukları gösterildi.

Anahtar Sözcükler: Gen susturma, susturmanın baskılayıcıları, patates virüsü M, agroinfiltrasyon, Nicotiana

benthamiana 16C hattı

Introduction

Recently a new defense system was detected that

does function in plants, namely post-transcriptional

gene silencing (PTGS) or RNA interference

(RNAi). In response to virus infection, this

“adaptive immune-system” is induced based on the

recognition of double-stranded (ds) RNA or

excessive quantities of RNA (Jorgensen, 2003).

RNAi is an evolutionary conserved mechanism in

many, if not all, eukaryotes, to target and degrade

aberrant endogenous or exogenous RNA molecules

(Sontheimer, 2005; Voinnet, 2005).

Due to this silencing defense system, plant

viruses are thought to need a counter defense

mechanism to successfully infect a plant.

Depending on the virus, such PTGS suppression

can be a secondary function of almost any type of

viral protein. In plants, RNA silencing has been

demonstrated to be one of the most important


16

Ruslan KRYLDAKOV et al.

antiviral mechanisms (Li and Ding, 2001; Ding et

al., 2004). As a response to this highly efficient

antiviral RNA silencing pathway, plant viruses

have evolved specific suppressor proteins. Many of

these proteins were previously described as

virulence factors or pathogenicity determinants. It

has been reported that many plant viruses encode

suppressor proteins to combat against RNA

silencing (Voinnet, Pinto and Baulcombe, 1999; Li

and Ding, 2001). Identification of silencing

suppressors of different viruses is a crucial step in

direction of development of resistance against viral

infection (Ding and Voinnet, 2007).

Silencing suppressors can interfere with several

steps of the silencing pathway, i.e. dsRNA cleavage

to produce siRNAs, destabilization of siRNAs,

binding of siRNAs and siRNA usage as part of

RISC, which is required for target RNA

elimination. Silencing suppressors are usually

multifunctional viral proteins and in different virus

families different viral proteins gain suppressor

function as an additional property. Interference

with suppressor function would inhibit the viral

counter-defense system.

The research and identification of silencing

suppressors of PVM were carried out. Potato is one

of the most important crops, and PVM, together

with PVY, is responsible for 80% of all viruscaused

losses of potato yields, especially in

Kazakhstan (Sozinova et al., 2007). Thus the

identification of PVM’s silencing suppressors

points to the role of gene silencing as a natural

antiviral defense system in plants.

Materials and methods

RNA isolation

A PVM isolate was maintained in Nicotiana

tabacum plants. Leaves of N. tabacum were

harvested and frozen in liquid nitrogen, ground to a

fine powder and mixed with Tri-reagent (“Sigma”),

extracted once with 1/5 volume of chloroform.

RNA was precipitated with the addition of an equal

volume of isopropanol and incubated at +4°C for

30 min. RNA was washed with 70% ethanol and

dissolved in water.

Construction of PVM vectors

The last 5 of 6 protein sequences of PVM were

obtained with using SuperScript II Reverse

Transcriptase according to manufacture protocol.

PVM cDNA fragments encoding the 25kDa,

12kDa, 7 kDa, coat protein (CP) and 11 kDa were

PCR-amplified from cDNA and individually cloned

using Gateway cloning kit (Invitrogen). First, the

PCR products were cloned into pDONR207 vector

using BP clonase, then they were re-cloned into

pMDC32 destination vector harbouring double 35S

promoter.

Plant material and A. tumefaciens infiltration

GFP transgenic 16C line of Nicotiana benthamiana

plants were grown as described previously (Marano

and Baulcombe, 1998; Voinnet et al. 1998).

Recombinant Agrobacterium tumefaciens strain

C58C1, carrying different constructs, were grown

at 28°C in 2 ml of LB medium with 2,5 μg/ml

tetracycline and 50 μg/ml kanamycin for two days,

then transferred to 18 ml of fresh LB medium with

appropriate antibiotics and were grown overnight at

28°C. The bacteria were centrifuged at 4000 g for

10 min. The pellet was resuspended in

agroinfiltration solution (10mM MgCl2, 10 mM

MES and 100 μM acetosyringone) up to desired

optical density (OD600). The bacteria solution was

left at room temperature for overnight before

infiltration. Infiltration carried on underside leaves

of 5-7-week-old N. benthamiana plants. For coinfiltration,

equal volumes of indicated A.

tumefaciens cultures (OD600=1) were mixed before

infiltration. After 5-10 days post infiltration, plants

were observed for silencing effect.

GFP Imaging

Visual detection of GFP fluorescence in whole

plant was performed using a 100W hand-held longwave

ultraviolet lamp. Plants were photographed

with a yellow filter.

Results

Potato virus M belongs to the genus Carlavirus of

the Flexiviridae family. The PVM genome is a

single positive-strand RNA of 8534 nucleotides.

The sequence contains six open reading frames

(ORFs) (Figure 1), and non-coding regions

consisting of 75 nucleotides at the 5 ’ -end and 70

nucleotides followed by a poly(A) tail at the 3 ’ -end

(Zavriev et al., 1991).


Identification of silencing suppressors of PVM 17

Figure 1. Scheme of PVM genome. Genes are shown as boxes and molecular masses of encoded proteins are

indicated in kDa. (PRO: gene of polyprotein encoding for several peptides; CP: coat protein gene)

The ORF beginning at the first initiation codon at nucleotide 74 encodes a polypeptide of 223K, virus

RNA replicase (Table 1). The next coding block consists of three ORFs encoding polypeptides of 25K, 12K

and 7K, which is responsible for viral cell-to-cell movement. The third block consists of two ORFs encoding

polypeptides of 34K (coat protein) and 11K. The 11K polypeptide contains a pattern resembling the

consensus for a metal-binding nucleic acid-binding zinc-finger motif.

Table 1. Proteins of PVM and their sizes

Symbol of protein Protein Start

End

Length

(nt)

(nt)

(bp)

ORF 1 polyprotein 74 5980 1968

ORF 2 25kDa protein 6019 6708 229

ORF 3 12kDa protein 6686 7015 109

ORF 4 7kDa protein 7012 7203 63

ORF 5 coat protein 7225 8139 304

ORF 6 11kDa protein 8136 8462 108

According to articles for last years (Chiba et al.,

2006), the protein 11K is most putative silencing

suppressor. The aim of our investigation was the

check of suppressor activity of all PVM proteins

except first one, largest polyprotein due to its size.

The primers for cloning were designed using

the GeneRunner programme version 3.0

(ORF2sense 5 ’ -

gggacaagtttgtacaaaaaagcaggctatggatgtgattgtagatttg-

3 ’ and ORF2antisense 5 ’ -

ggggaccactttgtacaagaaagctgggtctcaggcggcggtgtaagt

gg-3 ’ for ORF2; ORF3sense 5 ’ -

ggggacaagtttgtacaaaaaagcaggctatgccacttacaccgccgc

c-3 ’ and ORF3antisense 5 ’ -

ggggaccactttgtacaagaaagctgggtctcatgtgtgtgagcccgca

c-3 ’ for ORF3; ORF4sense 5 ’ -

ggggacaagtttgtacaaaaaagcaggctatgatagtgtatgtacttgta

g-3 ’ and ORF4antisense 5 ’ -

ggggaccactttgtacaagaaagctgggtcttatgacctaaaggaacca

cac-3 ’ for ORF4; ORF5sense 5 ’ -

ggggacaagtttgtacaaaaaagcaggctatgggagattcaacgaaga

aag-3 ’ and ORF5antisense 5 ’ -

ggggaccactttgtacaagaaagctgggtctcattttctattagactttac

at-3 ’ for ORF5; ORF6sense 5 ’ -

ggggacaagtttgtacaaaaaagcaggctatgaaggacgtaaccaag

gtggctt-3 ’ and ORF6antisense 5 ’ -

ggggaccactttgtacaagaaagctgggtcctactctcgcttgttgatga

c-3 ’ for ORF6). The Gateway-kit (Invitrogen) was

used for cloning. The DNA of the different PVM

ORFs were obtained by RT-PCR, and cloned into

the Gateway-compatible binary T-DNA destination

vector pMDC32. Each of these constructs were

electroporated into Agrobacterium tumefaciens

C58C1 strain according to the manufacturer’s

instructions. All constructs described above were

verified by nucleotide sequencing.

The Nicotiana benthamiana plant, constitutively

expressing GFP transgene (line 16C; a gift from

David Baulcombe), and the Agrobacterium

infiltration operation have been described

previously (Hamilton et al., 2002). The N.

benthamiana line 16C were cultured in growth

chambers at 22 to 24°C before and after infiltration.

For coinfiltration, equal volumes of individual

Agrobacterium cultures (OD600= 1) were mixed


18

Ruslan KRYLDAKOV et al.

prior to infiltration. HC-Pro of potato virus Y and

P19 of tombusviruses were used as a positive

control. GFP fluorescence was observed under

long-wavelength UV light and photographed using

a digital camera with a yellow filter.

GFP expression reached the highest level in all

leaves infiltrated with GFP, as well as GFP plus

other genes at 5 to 7 days postinfiltration (dpi), as

shown by the enhanced green fluorescence in the

infiltrated patches. The green fluorescence intensity

remained strong in the patches coinfiltrated with

ORF 3 and ORF 5 during the 7-9 dpi. Coinfiltration

with ORF 6 also resulted in GFP fluorescence over

a similar period of observations, but not as strong

and clear as above described samples (Figure 2).

On the other hand, GFP was silenced upon

inoculation with OFR 2 and ORF 4.

Figure 2. Effects of different ORFs of PVM on

gene silencing in GFP transformants of Nicotiana

benthamiana. Infiltrated leaves are shown.

Silencing of GFP is manifested in red fluorescent.

Green spots indicate suppression of silencing.

Photographs were taken of UV-illuminated plants.

1 and 2: HC-Pro and P19, respectively, are shown

as control; 3: ORF2, 25K protein; 4: ORF3, 12K

protein; 5: ORF4, 7K protein; 6: ORF5, coat

protein; 7: ORF6, 11K protein

According to these data the most promising

candidates as silencing suppressors are ORF 3 (12K

protein; Table 1), ORF 5 (coat protein; Table 1)

and, in lesser extent, ORF 6 (11K protein; Table 1),

while ORF 2 and ORF 4 revealed no suppressor

activity. We have repeated this experiment three

times which yielded the same results.

Discussion

Most of the viruses have some sort of silencing

suppressor system as an evolutionary counterresponse

to the antiviral activity of the plant gene

silencing defence. Gene silencing suppressors can

inhibit silencing of transgenes or endogenous

genes. Test for silencing suppression was based on

a previously described experimental system

(Brigneti et al., 1998). When leaves of N.

benthamiana line 16C plants were agroinfiltrated

with an A. tumefaciens C58C1 strain carrying the

construct 35S GFP, the strong green fluorescent

signal disappeared under UV light due to GFP

silencing. However, if 35S-ds GFP and any strong

silencing suppressor are co-agroinfiltrated, the

fluorescent signal does not disappear and the area

does not look dark under UV light. This is due to

the inhibition of gene silencing caused by that

suppressor.

Thus, the result of experiments shows that

ORF3 (12K protein; part of triple block, cell-to-cell

movement), ORF5 (coat protein) and ORF6 (11K

protein) of potato virus M has a suppressor activity.

It corresponds to the results of recent articles about

suppression activity of virus proteins responsible

for long-distance movement (Voinnet, 2001), viral

coat proteins (Roth, Pruss and Vance, 2004) and

10K-16K proteins with a conserved amino acid

sequence motif encoded by diverse filamentous

viruses (Chiba et al., 2006).

The 12K protein encoded by the second ORF of

PVM triple gene block (TGB) which involved in

the cell-to-cell and long-distance movement of

virus. This protein possesses conserved

hydrophobic domain and plays significant role in

TGB functions (Seppänen et al., 1997). TGBcontaining

viruses have evolved special

mechanisms to suppress RNA silencing (Voinnet et

al., 2000; Morozov and Solovyev, 2003).

The potex-like Carlavirus PVM requires the

coat protein for virus cell-to-cell movement

(Morozov and Solovyev, 2003; Verchot-Lubicz,

2005). In RNA silencing assays, plant viral

suppressors differ by their ability to suppress

intracellular and/or intercellular silencing (Li and

Ding, 2001; Roth, Pruss and Vance, 2004).

Spreading of silencing in plants occurs through

plasmodesmata and results from a cell-to-cell

movement of a short-range silencing signal, most

probably 21-nt short interfering RNAs (siRNAs),

that are produced by one of the plant Dicer

enzymes (Kalantidis et al., 2008).

11K protein had been shown to be a nonspecific

nucleic acid-binding protein (Gramstat,

Courtpozanis and Rohde, 1990), similar to p10

which is silencing suppressor of Grapevine virus A

(Zhou et al., 2006). Interestingly, 11K is supposed

to be translated from the CP subgenomic RNA by a

(-1) frameshift within the A-rich region


AAUAGAAAAUGA preceding the 12K AUG

translational start codon (underlined) and

representing the 3’-terminus of the CP coding

region (UGA stop codon in bold letters) (Gramstat,

Courtpozanis and Rohde, 1990).

This article describes the identification of viral

suppressors of RNA silencing encoded by a natural

pathogen. An exact role for any of the identified

PVM suppressors in host infection remains to be

established.

The potential of using viral suppressors to help

understand the mechanism of RNA silencing in

plants is largely unexplored, and these studies

promise to be a fertile area of research. Viral

suppressors of silencing also provide unique tools

to understand the mechanism of RNA silencing as

well as interaction between virus and their hosts.

Moreover, it became an extremely useful technique

for molecular biology and very powerful

biotechnological tool.

Acknowledgement

This work has been supported by funds from

INTAS, Young Scientist Fellowship Nr. 06-

1000014-6200.

References

Brigneti C, Voinnet O, Li WX, Ji LH, Ding SW

and Baulcombe DC. Viral pathogenicity

determinants are suppressors of transgene

silencing in Nicotiana benthamiana. The

EMBO Journal. 17: 6739-6746, 1998.

Chiba M, Reed JC, Prokhnevsky AI, Chapman EJ,

Mawassi M, Koonin EV, Carrington JC and

Dolja VV. Diverse suppressors of RNA

silencing enhance agroinfection by a viral

replicon. Virology. 346: 7-14, 2006.

Ding SW, Li H, Lu R, Li F and Li WX. RNA

silencing: a conserved antiviral immunity of

plants and animals. Virus Research. 102: 109-

115, 2004.

Ding SW and Voinnet O. Antiviral Immunity

Directed by Small RNAs. Cell. 130: 413-426,

2007.

Gramstat A, Courtpozanis A and Rohde W. The 12

kDa protein of potato virus M displays

properties of a nucleic acid-binding regulatory

protein. FEBS Letters. 276: 34-38, 1990.

Identification of silencing suppressors of PVM 19

Hamilton A, Voinnet O, Chappell L and Baulcombe

D. Two classes of short interfering RNA in

RNA silencing. The EMBO Journal. 21: 4671-

4679, 2002.

Jorgensen RA. RNA silencing in plants:

mechanisms, methods, and applications. Plant

& Animal Genomes XI Conference, USA.

2003.

Kalantidis K, Schumacher HT, Alexiadis T and

Helm JM. RNA silencing movement in plants.

Biology of the Cell. 100: 13-26, 2008.

Li WX and Ding SW. Viral suppressors of RNA

silencing. Current Opinion in Biotechnology.

12: 150-154, 2001.

Marano MR and Baulcombe D. Pathogen-derived

resistance targeted against the negative-strand

RNA of tobacco mosaic virus: RNA strandspecific

gene silencing? Plant Journal. 13:

537-546, 1998.

Morozov SYu and Solovyev AG. Triple gene block:

modular design of a multifunctional machine

for plant virus movement. Journal of General

Virology. 84: 1351-1366, 2003.

Roth BM, Pruss GJ and Vance VB. Plant viral

suppressors of RNA silencing. Virus Research.

102: 97-108, 2004.

Seppдnen P, Puska R, Honkanen J, Tyulkina LG,

Fedorkin O, Morozov SYu and Atabekov JG.

Movement protein-derived resistance to triple

gene block-containing plant viruses. Journal of

General Virology. 78: 1241-1246, 1997.

Sontheimer EJ. Assembly and function of RNA

silencing complexes. Nature Reviews. 6: 127-

138, 2005.

Sozinova L, Abay A, Ramankulov E, Nachmias A

and Loebenstein G. Reducing Virus Incidence

in Potato Seed Tubers by Rapid Propagation: A

Pilot Scheme in Kazakhstan, The 28 th Congress

of the Israeli Phytopathological Society, The

Volcani Center, Israel. 2007.

Verchot-Lubicz J. A New Cell-to-Cell Transport

Model for Potexviruses. Molecular Plant-

Microbe Interactions Journal. 18: 283-290,

2005.

Voinnet O, Vain P, Angell S and Baulcombe DC.

Systemic spread of sequence-specific transgene

RNA degradation is initiated by localized


20

Ruslan KRYLDAKOV et al.

introduction of ectopic promoterless DNA.

Cell. 95: 177-187, 1998.

Voinnet O, Pinto YM and Baulcombe DC.

Suppression of gene silencing: A general

strategy used by diverse DNA and RNA

viruses of plants. Proceedings of the National

Academy of Sciences. 86: 14147-14152, 1999.

Voinnet O, Lederer C and Baulcombe DC. A viral

movement protein prevents spread of the gene

silencing signal in Nicotiana benthamiana.

Cell. 103: 157-167, 2000.

Voinnet O. RNA silencing as a plant immune

system against viruses. Trends in Genetics. 17:

449-459, 2001.

Voinnet O. Induction and suppression of RNA

silencing: insights from viral infections. Nature

Reviews Genetics. 6: 206-220, 2005.

Zavriev SK, Kanyuka KV and Levay KE. The

genome organization of potato virus M RNA.

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Journal of Cell and Molecular Biology 9(1): 21-29, 2011 Research Article 21

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Identification of a novel dehydration responsive transcript from

tossa jute (Corcohrus olitorius L.)

Sazia SHARMIN 1,δ , Mahdi Muhammad MOOSA 1,δ , Md. Shahidul ISLAM 1,2 , Inamul

KABIR 3 , Arzuba AKTER 1 1, *

and Haseena KHAN

1

University of Dhaka, Department of Biochemistry & Molecular Biology, Dhaka 1000, Bangladesh

2

Bangladesh Jute Research Institute, Dhaka 1207, Bangladesh

3

University of Dhaka, Department of Genetic Engineering & Biotechnology, Dhaka 1000, Bangladesh

δ

Contributed equally to the study.

(* author for correspondence; haseena@univdhaka.edu)

Received: 18 January 2011; Accepted: 23 April 2011

Abstract

Water deficit stress is one of the major environmental stress conditions faced by sessile land plants.

Acquisition of resistance to this stress enabled plants to settle into terrestrial habitats. Dehydration stress is a

specific form of water deficit stress. A number of functionally characterized genes are involved in

dehydration response pathway. Here we report a novel dehydration responsive transcript from tossa jute

(Corcohrus olitorius L.) which shows reduced expression under dehydration stress. Study of tissue specific

expression revealed that the transcript is expressed in root, shoot and leaf under normal conditions. Search for

known homologues of the transcript failed to identify functionally characterized counterparts in any other

plant species.

Keywords: Drought, stress tolerance, water deficit stress, dehydration responsive transcript, drp.

Tossa jute‘den (Corcohrus olitorius L.) yeni bir dehidrasyon cevap transkripti

tanımlanması

Özet

Su eksikliği stresi sapsız kara bitkilerince karşılaşılan en büyük çevresel stress koşullarından biridir. Bu

strese dirençlilik kazanmak bitkilerin karasal yetişme ortamına yerleşmesini sağlamıştır. Dehidrasyon stresi

su eksikliği stresinin özgün bir formudur. Birçok fonksiyonel olarak karakterize edilmiş gen dehidrasyon

tepki yolağıyla ilişkilidir. Bu makalede hint kenevirinden (Corcohrus olitorius L.) dehidrasyon stresinde

azalmış ifade gösteren yeni bir dehidrasyona cevap transkripti rapor etmekteyiz. Dokuya özgün ifade

çalışması transkriptin kök, budak ve yaprakta ifade edildiğini ortaya koymuştur. Transkriptin bilinen

homologlarının araştırılması sonucunda diğer başka bitkilerde fonksiyonel olarak karakterize edilmiş bir eşi

bulunamamıştır.

Anahtar Sözcükler: Kuraklık, stres toleransı, su eksikliği stresi, dehidrasyon cevap transkripti, drp

Introduction

Unlike their mobile animal counterparts, sessile

plants have to adjust their gene expression pattern

as well as metabolite profile to cope with altered

local environmental conditions (Kulheim et al.

2002). A number of genes are involved in these

pathways which can either be up-regulated (Hannah

et al. 2005; Holappa and Walker-Simmons, 1995;

Yan et al. 2005.) or down-regulated (Hannah et al.


22 Sazia SHARMIN et al.

2005; Yan et al. 2005) based on their mode of

function. Identification of these genes may lead to

the development of commercial crops, which are

better suited to grow in a particular unfavourable

condition. Due to cross-species transferability of

gene functions, this improvement can be achieved

in non-source plant species as well.

Jute (Corchorus olitorius L.) is the most

important bast fibre producing plant (Kundu, 1956).

It is the major fibre producing plant of Bangladesh

(Hossain et al. 2003) and one of the major fibre

crops of the Indian subcontinent (Basu et al. 2004).

The production quality and yield of this

economically important crop is affected by several

biotic e.g. fungi, pest, insect, nematode, virus, mite

(BBS, 2004; Ghosh, 1983; Keka et al. 2008) and

abiotic factors e.g. salinity, submergence and low

temperature (Hossain et al. 2003; Samira et al.

2010). Thus, development of jute varieties with

increased resistance to both biotic and abiotic stress

conditions have considerable economic importance.

In spite of its enormous commercial value, only

a few genes or part of genes from jute have been

identified or sequenced (Ahmed et al. 2009; Alam

et al. 2010; Islam et al. 2005; Mahmood et al.

2010; Samira et al. 2010; Wazni et al. 2007). Here

we report a novel transcript from Corchorus

olitorus var. 9897 responsive to dehydration stress.

Search for known homologues failed to identify

functionally characterized counterparts in any other

plant species.

Materials and methods

Stress treatment of plant seedlings

Plant seeds were incubated at room temperature in

the absence of light for two days in petri dishes in

the presence of water only. Germinated plant

seedlings were subjected to different stress

conditions such as low temperature, dehydration,

fungus and abscisic acid (ABA) beginning third

day of their germination according to methods

described by Alam and co-workers (Alam et al.

2010). Fungus treatment was done by spraying

suspension of Macrophomina phaseolina on the

plate with germinated seedlings (Alam et al. 2010;

Mahmood et al. 2010). For dehydration treatment,

the seedlings were treated with 50 mM manitol to

create an environment similar to water deficit

condition. The concentration of manitol was

increased to 100 mM on day 5 (Alam et al. 2010).

Salt stress was applied by adding 50 mM NaCl to

the plate on day 3. The concentration was increased

by 50 mM per day, reaching to 150 mM NaCl on

day 5 (Alam et al. 2010). Low temperature

treatment was carried out by growing the seedlings

at 14 O C in an incubator from day 3 (Alam et al.

2010).

Isolation of DNA and RNA

Genomic DNA from different Corchorus species

was isolated using the CTAB method (Murray and

Thompson, 1980). RNA was isolated using

TRIZOL reagent (Invitrogen) according to the

provided manual. For tissue specific RNA isolation,

leaf, stem and root was cut into pieces, ground in

liquid N2. RNA was isolated from ground tissue

using TRIZOL reagent (Invitrogen) as per

manufacturer’s instruction

Identification of the dehydration responsive

transcript

The transcript was identified as an additional band

while carrying out RT (Reverse Transcriptase)-

PCR of a cold stress responsive gene, LDLP (Low

density lipoprotein like protein) from C. olitorius

using the primer RevP (unpublished result). RT-

PCR was carried out using RNA isolated from C.

olitorius grown under normal condition. This

additional band was extracted from agarose gel

using QIAGEN MinElute Gel Extraction Kit and

sequenced (1 st Base PTE Singapore) using ForP

and RevP as primers. Gene specific primers

(GSPF1, GSPR1 and GSPR2) for the transcript was

designed using Primer3Plus (Untergasser et al.

2007). Sequences of primers are given in Table 1.

These primers were used to sequence partial CDS

(coding sequence) of drp from C. capsularis, C.

tridens, C. aestuans, C. pseudo-olitorius, C.

trilocularis.

Table 1. Primers used in the study

Primer Sequence

ForP 5’- ATACTTATGGGAGAATGAAAG -3’

RevP 5’- TACAGGTAAAATGTCACCAA -3’

GSP-R1 5’- GAGGCTTTTCTTGGAAC -3’

GSP-R2 5’- GTCCCGATCCCTTGAATTAC -3’

GSP-F1 5’- CTTAATCGGAGCAGGGTGAG -3’


Semiquantitative RT-PCR

Total RNA was isolated from jute seedlings and

quantified using a nanodrop (ND-1000). Poly (A)

RNA was isolated from total RNA using mRNA

extraction kit (SIGMA, MRN 10KT, 046k6879)

and quantified. First strand cDNA was synthesized

from 50 ng of mRNA using gene specific primer

GSP-R2. PCR amplification of the cDNA was

carried out for 25 cycles using primers GSP-F1 and

GSP-R2 (95°C for 40 s, 58°C for 40 s, and 72°C

for 50 s). The housekeeping gene β-actin was used

as the internal standard for reverse transcriptase

polymerase chain reaction (Alam et al. 2010).

5′ RACE

277 bp sequence at the 5′ end of the transcript was

determined by 5′- Rapid Amplification of cDNA

Ends (RACE) protocol. The first strand cDNA was

prepared from mRNA using the primer GSPR1.

Purification of first strand cDNA and TdT tailing

was carried out as recommended by the

manufacturer (5′ RACE system, Invitrogen

Corporation, Carlsbad, CA). First round PCR of

target cDNA was carried out using the primer

GSPR2 for 35 cycles (95°C for 1 min, 55°C for 1

min, and 72°C for 2 min).

Southern blot

15 μg DNA was digested with EcoRI and SacI and

size-fractionated on 1% agarose gel and then

transferred onto a positively charged nylon

membrane (Amersham Hybond-N). Probe was

synthesized from RT-PCR product using primer

GSP-F1 and GSPR2 and Dig nucleic acid labeling

and detection kit (Roche, IN). Manufacturer’s

guideline for highly stringent hybridization and

detection conditions was followed (probe 10

ng/mL, stringency washes with 0.5X SSC, 0.1%

SDS).

Northern blot

10 μg RNA was separated by electrophoreses in 1X

MOPS and transferred to a positively charged

nylon membrane (Amersham Hybond-N). Probe

was synthesized from RT-PCR product by using

primer GSPF and GSPR2 and Dig nucleic acid

labelling and detection kit (Roche, Cat No.

11175033910). Manufacturer’s guideline for highly

stringent hybridization and detection conditions

Novel dehydration responsive transcript from jute 23

was followed (probe 10 ng/mL, stringency washes

with 0.1X SSC, 0.1% SDS).

Sequence assembly and analysis

Sequences from gel extracted band and 5′ RACE

was assembled using CAP3 (Huang and Madan,

1999) from PBIL web-server (Perriere et al. 2003).

Coding sequences were identified using ESTScan2

(Iseli et al. 1999). Phylogenetic tree was

constructed using MEGA4 by UPGMA method

with default parameters (Tamura et al. 2007).

Multiple sequence alignment was done using

Clustalx (Larkin et al. 2007). Protein secondary

structure was predicted from NPS@ webserver

(Combet et al. 2000).

Results

Identification of the transcript

The dehydration responsive transcript was

identified by sequencing of additional band

obtained from RT-PCR of LDLP gene. The 396 bp

sequenced transcript failed to show significant

similarity to any of the so far functionally

characterized proteins in blast search at NCBI

database. An additional 277 bp sequence at the 5′

end was determined by 5′ RACE. Partial CDS of

drp from five other Corchorus spp (C. capsularis,

C. tridens, C. aestuans, C. pseudo-olitorius, C.

trilocularis) were obtained using gene specific

primer (GSP) pairs. Sequence information was

deposited to DDBJ (Accession numbers

AB571589-AB571594).

Copy number determination

To determine the copy number of the gene within

the C. olitorius genome, isolated genomic DNA

was digested EcoRI and SacI. Southern blot with

both enzyme digests showed four distinct bands

(Figure 1). Thus, it is likely that C. olitorius

genome has four copies of drp.

Expression study: Tissue and stress specific

expression

Reverse transcriptase PCR (RT-PCR) from RNA

isolated from leaf, stem and root suggested that drp

was expressed in all three tissues (Figure 2).

Semiquantitative RT-PCR showed reduced

expression under dehydration stressed condition


24 Sazia SHARMIN et al.

(Figure 3). Similar expression pattern was observed

in Northern blot of C. olitorius transcript with a

cDNA probe specific for the sequence (Figure 4).

The transcript also showed somewhat reduced

Figure 1. Southern blot of drp after EcoRI (a) and

SacI (b) digestion. Arrows indicate bands to which

probe was hybridized.

Figure 3. Semiquantitative RT-PCR of drp (top)

and actin control (bottom) under different stress

conditions (N, L, S, D, F and A stands for Normal,

Experimental, Low temperature, Salt, Dehydration,

Fungal and ABA treatment, respectively).

Comparative analysis of drp from different

Corchorus spp.

Translated protein sequence alignment showed a

highly conserved segment of 32 amino acid

expression in low temperature and fungus treated

plants (Figures 3 and 4).

Figure 2. RT-PCR of RNA isolated from stem, root

and leaf (S, R and L stands for stem, root and leaf,

respectively).

Figure 4. Northern blot of drp under different

stress conditions (N, L, S, D, F, A stands for

Normal, Experimental, Low temperature, Salt,

Dehydration, Fungal and ABA treatment,

respectively). rRNA is shown below as a control

for equal loading.

residues (Figure 5a). Secondary structure prediction

suggested that the region is mainly composed of

Random Coils (Figure 5b and Table 2). A

phylogenetic tree (Figure 6) of drp nucleotide

sequence from different Corchorus species was

constructed using MEGA4 (Tamura et al. 2007).


A

Corchorus_olitorius SAPKARQRCVADGKQVNIPAPSYDAMGGRIAE

Corchorus_capsularis SAPKARQRCVADGKQVNIPAPSYDAMGGRIAE

Corchorus_tridens SAPKARQRCVADGKQVNIPAPSYDAMGGRIAE

Corchorus_pseudo-olitorius SAPKARQRCVADGKQVNIPAPSYDAMGGRIAE

Corchorus_trilocularis SAPKARQRCVADGKLVNIPAPSYSAMGGRIAE

Corchorus_aestuans SAPKARQRCVADGKQVNIPAPSYNAMGGRIAE

************** ********.********

B

10 20 30

| | |

UNK_136050 SAPKARQRCVADGKQVNIPAPSYDAMGGRIAE

DPM ctchhhhhhehtcccecccccccchhcchhcc

DSC ccccceeeeecccccccccccccccccccccc

GOR1 hhhhhheeeeettteeeeeeceeeehhhhhhh

GOR3 cccchhheehhccceecccccccccccceehh

HNNC cccccccccccccccccccccccccccccccc

MLRC cccccceeeecccccecccccccccccccccc

PHD ccccccchhhhccccccccccccccccccccc

Predator ccchhhhhhhcccccccccccccccccccccc

SOPM ccccccheeecttccccccccccchhtchhhh

Sec.Cons. ccccc?heeecccccccccccccccccccccc

Sequence length : 32

Novel dehydration responsive transcript from jute 25

Figure 5. A) Highly conserved segment of the translated protein sequence of drp gene from different

Corchorus spp; B) Predicted secondary structure of the conserved segment. The last line shows consensus

secondary structure from different methods of prediction.

Table 2. Summary of predicted secondary structures from different prediction tools. The last row is for

consensus secondary structure.

Software Random coil (c) Alpha helix (h) Extended strand (e)

DPM (Deléage and Roux, 1987) 53.12 % 34.38% 6.25%

DSC (King and Sternberg, 1996) 84.38% 0.00% 15.62%

GOR1 (Garnier et al. 1978) 3.12% 40.62% 46.88%

GOR3 (Gibrat et al. 1987) 59.38% 21.88% 18.75%

HNNC (Guermeur, 1997) 100.00% 0.00% 0.00%

MLRC (Guermeur et al. 1999) 84.38% 0.00% 15.62%

PHD (Rost and Sander, 1993) 87.50% 12.50% 0.00%

Predator (Frishman and Argos, 1996) 78.12% 21.88% 0.00%

SOPM (Geourjon and Deléage, 1994) 59.38% 21.88% 9.38%

Sec.Cons. (Consensus) 84.38% 3.12% 9.38%


26 Sazia SHARMIN et al.

Figure 6. Evolutionary relationship of 6 Corchorus species, poplar and rice. The percentage of replicate trees

in which the associated taxa clustered together in the bootstrap test (10000 replicates) is shown next to the

branches. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1).

Discussion

Acquisition of dehydration tolerance is one of the

traits that enabled land-plants to settle into

terrestrial habitats (Richardt et al. 2007).

Dehydration stress is a specific form of water

deficit stress that results in alteration of plants at

the cellular, morphological and developmental

levels (Bray, 1993). At the molecular level, the

pathway can be classified into two broad categories

viz. ABA dependent and ABA independent

pathway (Ramanjulu and Bartels, 2002). Individual

pathways are discussed in detail elsewhere

(Agarwal and Jha, 2010; Ingram and Bartels, 1996;

Nakashima et al. 2009; Umezawa et al. 2006;

Yamaguchi-Shinozaki and Shinozaki, 2006).

The newly identified transcript, namely drp,

was found to be expressed in all three tissues tested

(root, stem and leaf) in normal condition (Figure 2).

Analysis of the expression pattern after stress

treatments suggests decreased expression in

dehydration stress, but increased expression in

ABA stress condition (Figures 3 and 4).

Comparative analyses of water deficit stressed

transcriptome of a number of plants identified plant

genes showing altered expression in either

dehydration stress alone or in combination with any

other water deficit stress or ABA treatment (Bray,

2004; Rabbani et al. 2003; Seki et al. 2002; Talame

et al. 2007). Nevertheless, none of the studies

identified single transcript showing opposite

expression patterns in water deficit stress and ABA

treatment as shown by drp. It is to be noted that the

transcript also shows somewhat reduced expression

in low temperature treated plants. Previous studies

on dehydration stress responsive genes also found

similar correlation with low temperature response

pathways (Shinozaki and Yamaguchi-Shinozaki,

2000).

Comparative analysis of different experimental

methods of water deficit stress treatment revealed

that three transcripts (At2g06850, At1g72610 and

At5g20630) are down-regulated in Arabidopsis

irrespective of the method (filter paper, mannitol or

soil water deficit) applied (Bray, 2004).

Interestingly all the three genes are either known or

hypothesized to function in plant cell wall

modification pathways that control growth (Bray,

2004). Both semi-quantitative RT (reverse

transcriptase) -PCR and northern blot suggests

reduced expression of drp in fungus treated plants.

Study by Schenk and co-workers (Schenk et al.

2000) revealed that a significant portion of fungal

inoculation responsive genes in Arabidopsis are

associated with cell wall synthesis and

modification. Thus, drp might also play a role in

defence pathways against fungal infection in jute

by playing a role in cell wall synthesis and/or

modification.

Southern blot of the transcript suggests that C.

olitorius genome has four copies of drp gene

(Figure 1). Thus, it is also possible that different

copies of the drp gene are under different

regulatory mechanisms and the observed

expression patterns are of different drp genes which


are indistinguishable in the region selected for

analyses.

The newly identified transcript failed to show

significant similarity to any of the so far

functionally characterized proteins. It is to be noted

that plant specific nucleotide blast in NCBI

database shows significant similarity to a

hypothetical Poplus trichocarpa protein

(XM_002337327.1) and other plant transcripts.

Paralogues of the gene were found to be present in

all tested Corchorus species. Phylogenetic analysis

of drp gene and its homologues suggests that C.

capsularis is the closest neighbour of C. olitorius in

terms of drp. It is to be noted that all considered

dicots grouped into same clade in the tree (Figure

6).

Multiple sequence alignment of protein

sequences from different Corchorus spp. identified

a highly conserved segment of 32 amino acid

residues. Secondary structure prediction at NPS@

webserver (Combet et al. 2000) suggested these

amino acids are most likely to form random coils

(Figure 5b and Table 2). Random coils or more

specifically coils add flexibility to the protein

structure (Buxbaum, 2007). Our literature survey

revealed an interesting relationship between

random coils and their possible role in dehydration

response. It has been long known that different

organisms release osmolytes when subjected to

water stresses (Yancey et al. 1982). Study by Qu

and co-workers (1998) identified that osmolytes

cause contraction of random coils leading to

decrease of conformational entropy and subsequent

stabilization of protein folding.

The present study has identified a novel

dehydration responsive transcript from different

jute (Corchorus) species. Further studies are

required to elucidate the detailed mechanism of

action of this transcript.

Acknowledgement

This research was supported by the United States

Department of Agriculture (USDA). We are

thankful to Bangladesh Jute Research Institute

(BJRI) for providing seeds of different jute species.

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Journal of Cell and Molecular Biology 9(1): 31-36, 2011 Research Article 31

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Protective effects of curcumin on cadmium chloride induced colon

toxicity in Swiss albino mice

Preeti SINGH * , Priya MOGRA, Vandana SANKHLA and Kanchan DEORA

Department of Zoology, College of Science, M.L.S. University, Udaipur-Rajasthan, 313001 India.

(* author for correspondence; priti1960@yahoo.co.in )

Received: 26 October 2010; Accepted: 29 April 2011

Abstract

Cadmium, an omnipresent, hazardous, heavy metal, is recognized to produce severe toxic effects in humans.

The Agency for Toxic Substances and Disease Registry (ATSDR) in Atlanta, Georgia has listed cadmium as

number 7 in its top 20 list of hazardous substances. In contrast to the toxic activity of cadmium chloride,

curcumin derived from Curcuma longa (Turmeric) is attributed with numerous beneficial properties and is

supposed to possess multifaceted healing properties. The purpose of the present study was to elucidate the

protective efficacy of curcumin against cadmium chloride induced colon toxicity in Swiss albino mice. Four

batches of Swiss-albino mice, each batch comprising six mice, were treated with cadmium chloride and

curcumin. Comparative observations of colon in cadmium-treated groups clearly showed induction of

histopathological damage as evidenced by emptied goblet cells, lacerated and suppressed mucosa, destruction

of surface epithelium, marked decline in mucin production and hemorrhagic colon, which were not seen in

control and curcumin treated group. In animals pretreated with curcumin and subsequently treated with

cadmium chloride, the histopathology of colon clearly delineates the protection accorded by curcumin, as the

induced damage by cadmium chloride was negligible and the number of empty goblet cells, hemorrhagic

areas, suppression of mucosa was significantly reduced.

Keywords: Curcumin, cadmium chloride, colon, goblet cells, mucosa

Swiss albino farelerde zerdeçalın kadmiyum klorür ile indüklenmiş kolon

toksisitesine koruyucu etkileri

Özet

Her yerde bulunan, tehlikeli bir ağır metal olan kadmiyumun insanda şiddetli toksik etkiler yarattığı fark

edilmiştir. Atlanta, Georgia’daki Toksik Maddeler ve Hastalık Kayıt Acentası (ATSDR) kadmiyumu ilk 20

tehlikeli maddeler listesinde 7. sırada listelemiştir. Kadmiyum kloridin toksik aktivitesine karşın, Curcuma

longa’dan (Turmeric) elde edilen zerdeçala çok sayıda yararlı özellikler atfedilmiş ve iyileştirici özelliklere

sahip olduğu tahmin edilmiştir. Bu çalışmanın amacı Swiss albino farelerde zerdeçalın kadmiyum klorid ile

indüklenmiş kolon toksisitesine karşı koruyucu etkisini açıklamaktır. Her birinde altı fare bulunan dört grup

Swiss albino fare kadmiyum klorid ve zerdeçal ile muamele edilmiştir. Kadmiyum ile muamele edilmiş

grupta kolonunun karşılaştırmalı gözlemi açıkça boşalmış goblet hücreleri, yırtılmış ve bastırılmış mukoza,

yüzey epitelin yıkımı, müsin üretimindeki belirgin düşüş, kolon kanaması gibi durumlarla kanıtlanan

histopatolojik hasar indüklenmesi göstermiştir. Öncelikle zerdeçal ve daha sonra kadmiyum klorid ile

muamele edilmiş hayvanların kolon histopatolojisi, kadmiyum klorid ile indüklenen hasarın gözardı edilebilir

olması ve boş goblet hücreleri ve kanama alanlarının sayısı ile mukoza baskılanmasının önemli derecede

azalması ile zerdeçalın koruyucu rolünü anlaşılır bir biçimde açıklar.

Anahtar Sözcükler: Curcumin, kadmiyum klorür, kolon, goblet hücreleri, mukoza


32 Preeti SINGH et al.

Introduction

Cadmium, an omnipresent, hazardous, heavy metal

is recognized to produce severe toxic effects in

humans (Lee et al., 1980; Jarup et al., 1998a;

ATSDR, 1999 and Singh et al., 2007). The Agency

for Toxic Substances and Disease Registry

(ATSDR, 1989) in Atlanta, Georgia has listed

cadmium as number 7 in its top 20 list of hazardous

substances. Cadmium will invariably be present in

our society, either in useful products in the form of

nickel-cadmium batteries, dyes, plastics,

electrochemistry, paint pigments or in controlled

wastes as a major source of pollution, in water and

as constituent of food material (Friberg et al., 1975;

Goyer et al., 1984; Friberg et al., 1985a; Jarup et

al., 1998 and Ikeda et al., 2000). In humans it has

been found to produce wide range of biochemical

and physiological dysfunctions as manifested in the

forms of various diseases viz. Itai-itai, kidney

malfunction, inflammation, Parkinsons disorder,

liver malfunction etc. (Friberg et al., 1974; Nogawa

et al.,1977; RAIS ,1991; Jarup et al., 1998; and

Ikeda et al.,2000).Various modes and

methodologies are being devised to combat

cadmium-induced toxicity with a focus on herbal

formulations. The use of herbal medicine increases

every day and still finds a wide use worldwide.

Traditional herbs have more acceptance than

prescription drugs in many cultures. One of such

drug is curcumin derived from Curcuma longa the

dried powered form of which is known as Turmeric

and is a mandatory condiment of every Indian

kitchen. Turmeric extract containing the ingredient

curcumin is marked widely in the western world as

a dietary supplement for the treatment and

prevention of a variety of disorder, including

inflammation, arthritis and other proposed uses of

curcumin is in Alzheimer's Disease, cancer

prevention, cataract prevention, chronic, high

cholesterol, liver protection, menstrual pain,

multiple sclerosis, osteoarthritis, rheumatoid

arthritis etc. (Deodhar et al., 1980; Chen et al.,

1996; Eigner and Scholz, 1999). Curcumin is also

reported to have antibacterial, antiamoebic,

antifungal, antiviral and anti HIV activities

(Ammon, 1992; Azuine, 1992; Ruby, 1995 and

Mortellini et al., 2000). In some experiments it has

been shown to be 300 times more potent than

vitamin E. However no concrete evidence in

scientific field is available pertaining to the colonic

protective effects of curcumin on cadmium chloride

induced toxicity. Hence, the aim of the present

research was to fulfill this lacuna and to determine

the efficacy of curcumin in rendering protection

against cadmium chloride induced colonic toxicity

at the cellular level.

Materials and methods

In the present experiment the following chemical

reagents were used. Cadmium chloride: CdCl2.

H2O was procured from Glaxo company (India),

with following features: Molecular Weight =

201.32, Minimum assay (ex Cl) 95%. Curcumin

Crystalline: C21H20O6 was procured from Loba

Chem. Pvt. Ltd., with following features: Molecular

Weight = 368.39 Minimum assay (acidimetric)

99%, Melting point= 170- 180°C.

The present study was conducted after taking

prior approval by Indian Animal Ethical Committee

(No .CS\Res\07\759), on twenty four adult male

Swiss albino mice 32-50 days old and weighing

around to 30-40g were maintained in plastic cages

under controlled lighting conditions (12:12 light:

dark cycle) relative humidity (50 ± 5%) and

temperature (37 ± 2°C), fed with mice feed and

were given ad libitum access to water. A group of 6

mice per experiment were taken and treated with

CdCl2 and curcumin. The doses were prepared

daily in distilled water and were administered by

gastric gavage method.

The dose protocol was as follows: Group-I

Control group (a) were treated with only vehicle

(water) for a day; Group-II Control groups (b) were

treated with only curcumin 10mg/animal/day for 16

days; Group-III Experimental batch were

administered CdCl2 50mg/kg/animal for a single

day; Group-IV Experimental batch with were pretreated

with curcumin 10mg/animal/day for 15 days

and on the 16 th day a single oral dose of CdCl2

50mg/kg/day/animal was administered. After

administration of the last dose, the control and the

experimental animal were sacrificed. Their colon

was excised and subsequently fixed in Bouins

solutions. After fixation, wax block and paraffin

sections of the colons were prepared, processed and

differentially stained in hematoxyline and eosin for

general histopathological assessment. The sections

were stained in bromophenol blue for assessment of

protein profile, and in Schiff’s for the study of

glycoproteins. They were then observed under a

light microscope and the gross histopathological

profile was assessed.


Results

The colonic histopathological profile of control

group mice revealed no aberrations the colonic

mucosa was distinct with no lacerations and the

goblet cells present were filled. Mucin production

was distinctly evident (Figure 1a and 1b).

Similarly, no significant variations were observed

in curcumin treated mice, which showed a similar

structural profile as that of control group mice

(Figure 1c and 1d). The histopathology of colon in

animals treated with CdCl2 was blunt and

suppressed, clearly delineating histopathological

Protective effects of curcumin on colon toxicity 33

damage. Emptied goblet cells, lacerated mucosa,

destruction of surface epithelium, marked decline

in mucin production and hemorrhagic areas in

colon were also visible (Figure 1g and 1h). In

animals pretreated with curcumin and subsequently

treated with CdCl2 the histopathology of colon

clearly delineates the protection accorded by

curcumin as the induced damage by CdCl2 was

negligible and the number of empty goblet cells,

hemorrhagic areas, suppression of mucosa was

significantly reduced (Figure 1e and 1f).

Figure 1. (a) Photomicrograph showing the colonic surface of control group. Prominent mucosa and filled

goblet cells is distinctly visible. (10X magnification), (b) Photomicrograph showing the colonic surface of

control group. Filled goblet cells are clearly visible. (40X magnification), (c) Photomicrograph of colon of

cur cumin treated group. Mucosa is similar to that of control group. (10X magnification), (d)

Photomicrograph of colon of cur cumin treated group showing distinct goblet cells (40X magnification), (e)

Photomicrograph of colon of group which was pretreated with cur cumin and subsequently treated with

cadmium. Suppression of colonic mucosal surface is less as compared to mucosa of only cadmium chloride

treated group. (10X magnification), (f) Photomicrograph of colon of group which was pretreated with cur

cumin and subsequently treated with cadmium. Filled and empty goblet cells are clearly visible. (40X

magnification), (g) Photomicrograph of colon of cadmium treated experimental group. Emptied goblet cells,

suppressed mucosa, and destruction of surface epithelium are evident. (10X magnification), (h)

Photomicrograph of colon of cadmium treated experimental group. Empty goblet cells are visible. (40X

magnification).


34 Preeti SINGH et al.

Discussion

Cadmium is an omnipresent heavy metal which

enters the biological systems from natural sources,

such as volcanic emissions, weathering of rocks,

mining processes as well as from industrial

applications, agricultural practices and human

usages. Cadmium emissions into the environment

are normally continuous between the three main

environmental compartments air, water and soil.

The majority of cadmium exposure arises from

ingestion of food substances due to uptake of

cadmium by plants from fertilizers, sewage sludge,

manure and atmospheric deposition (Lee and

White, 1980; Anderson et al 1988; Hotz et al.,

1989; Lauwerys et al., 1991; Iwata et al., 1992;

Bernard et al 1992, Ikeda et al., 2000). Human

uptake of cadmium is mainly through cigarette

smoking and food intake. In vegetarian diet,

mushrooms, cocoa powder, potatoes, fruits, wheat,

grains, bran, sugerbeet fiber, carrot, dried seaweeds

etc. are the source of cadmium intake. Similarly, in

non-vegetarian diets shellfish, mussel, meat and

fish are rich in cadmium. These cadmium rich

foods can greatly increase cadmium concentration

in the human body (Friberg et al., 1985b; Vahter et

al., 1996 and WHO, 2000). In humans cadmium

has a long half life, which is reported to be of 10-30

years in kidney, and 4.7-9.7 years in liver (Chen

and Wang, 1990). On average, 5% of the total

orally ingested cadmium is absorbed in the

intestines, but individual values range from less

than 1% to more than 20%.This reflects the fact

that humans do not have effective pathways for

cadmium elimination, hence cadmium shows the

phenomenon of bioaccumulation. Once absorbed

by an organism, it remains resident for many years.

Where cadmium toxicity is concerned, colon is of

prime importance, for of the total cadmium that

enters the gastrointestinal tract 90-95% is excreted

out and hence colonic cells are exposed to cadmium

in fecal matter as well as to cadmium present in the

circulation. Hence in the present experiment we

aimed to assess the toxic effects of cadmium

chloride as well as to monitor the protective effects

of curcumin in colon of Swiss albino mice on the

basis of histopathological observations. The

integrity of colon depends upon the balance

between the hostile factors, one of them being

cadmium, which damages the mucosa ,goblet cells

etc. and the protective factors such as certain

internal secretions like mucin and certain external

agents like curcumin which render protection

against cadmium toxicity. Pertaining to cadmium

toxicity our results are similar to the results of

various studies where cadmium has been reported

to cause variations in histoarctecture of colon.

These reports revealed that oral exposure to

cadmium caused severe necrosis, hemorrhage and

ulcers in the colonic epithelium as well induced

decreased body weight and muscle atrophy (Barret

et al., 1947; Stawe et al., 1972; Richardson et al.,

1974 and ATSDR, 1999). These reports give

validity to our experiment, where we have also

discerned similar cadmium induced aberrations in

colon. In contrast to the injurious activity of

cadmium, curcumin serves as protective agent

where it has been shown to decrease congestion and

inflammation in stagnant mucus membranes and no

negative side effects have been associated with

curcumin supplementation (Mukhopadhyay et

al.,1982; Rao et al., 1982; Dikshit et al., 1995;

Shukla et al., 2002 and Egan et al., 2004).

Curcumin has also been reported to have proposed

uses in ulcerative colitis (Deodhar et al., 1980 and

Chen et al., 1996). Curcumin has been shown to

protectively coat esophagus, stomach, intestinal

mucus membrane and reduce acid secretion.

However no information is available pertaining to

protective effect of curcumin in relation to

cadmium induced toxicity. Hence, this study clearly

delineates the protective effect of curcumin against

CdCl2 induced colonic toxicity.

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Journal of Cell and Molecular Biology 9(1): 37-44, 2011 Research Article 37

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

In vitro regeneration of Cleome viscosa – an important medicinal

herb

Jeyaraj ANBURAJ *,1 , Chinnappan Ravinder SINGH 1 , Shenbagamoorthy SUNDARRAJ 2

Soundarapandian KANNAN 2

1

Ayya Nadar Janaki Ammal College, Department of Biotechnology, Sivakasi, India.

2

Bharathiar University, Proteomics and Molecular Cell Physiology Lab, Department of Zoology,

Coimbatore, India.

(* author for correspondence; geneanbu@yahoo.co.in)

Received: 30 August 2010; Accepted: 26 May 2011

Abstract

Cleome viscosa is an important medicinal herb. Mass collection of the plant from natural habitats has led to

its depletion. We have established a protocol for its mass propagation through in vitro organogenesis using

leaf explants. For callus induction it was found that Indole-3acetic acid (2mg/l) has a significant effect on the

callus induction, callus index and callus physical appearance. The moderate response was observed with

Indole-3butyric acid (2mg/l), and a low response was observed with 2-Napthalene acetic acid (2mg/l). 2mg/l

concentration of Benzyl aminopurine significantly influenced shoot proliferation. Optimum response was

observed with Benzyl aminopurine + Kinetin (5+2.5mg/l), and low response was observed with Kinetin

(1mg/l). About 91% of rooting response was observed with NAA (0.1mg/l). Moderate response was observed

from Indole-3butyric acid (0.1mg/l) and low response observed from IAA (0.1mg/l). After two weeks of

acclimatization 90% of survival rate have been observed. The present study describes a well-documented and

reliable protocol for Cleome viscosa from leaf explants with a high rate of multiplication. This protocol could

be used as a basic tool for commercial cultivation of this medicinally important plant Cleome viscosa.

Keywords: Cleome viscosa, organogenesis, medicinal plants, in vitro regeneration, auxins

Önemli bir tıbbi bitki olan Cleome viscosa’nın in vitro rejenerasyonu

Özet

Cleome viscosa önemli bir tıbbi bitkidir. Bitkinin doğal ortamından toplu halde toplanması tükenmesine

sebep olmuştur. Yaprak eksplantları kullanarak in vitro organogenez ile toplu üretimini sağlayacak bir

protokol oluşturduk. İndol- 3-asetik asitin (2mg/l) kallus oluşumunda, kallus indeksinde ve kallus fiziksel

görünümünde önemli derecede etkili olduğu belirlenmiştir. İndol-3-butirik asitin (2mg/l) orta derecede, 2naptalen

asetik asitin de (2mg/ml) düşük derecede etki gösterdiği belirlenmiştir. 2mg/ml konsantrasyonda

benzil aminopürin ise filiz proliferasyonunu önemli derecede arttımıştır. Benzil aminopürin + Kinetin (5+2

mg/l) ile optimum cevap, Kinetin (1mg/ml) ile de düşük dereceli cevap gözlemlenmiştir. İndol-3-bütirik

asitin (0,1mg/l) orta derecede, İAA' nın ise düşük derecede cevaba sebep olduğu gözlemlenmiştir.

İklimlendirmenin iki hafta sonrasında %90 hayatta kalma oranı gözlemlenmiştir. Bu çalışmada Cleome

viscosa yaprak eksplantlarından yüksek oranda çoğaltım sağlayan güvenilir ve iyi belgelenmiş bir protokol

tarif edilmektedir. Bu protokol tıbbi önemi olan bir bitki olan Cleome viscosa'nın ticari amaçlı kültürünün

yapılabilmesi için temel bir araç olarak kullanılabilir.

Anahtar Sözcükler: Cleome viscosa ,organogenez, tıbbi bitkiler, in vitro rejenerasyon, oksinler.


38 Jeyaraj ANBURAJ et al.

Introduction

Cleome viscosa Linn. (Capparidaceae) is

commonly known as Tickweed, wild mustard or

Spider plant (English), Hurhur (Hindi) and

Hurhuria (Bengali). It occurs in woodland and

grassland, and is a weed of fallow land, fields,

roadsides and wasteland, often occurring on sandy

soils, but sometimes on calcareous and rocky soils.

It is a widely distributed herb with yellow flowers

and long slender pods containing seeds. The whole

plant is sticky in nature and has a strong odour

resembling asafoetida. It is found in both seasonal

dry and humid conditions, from mean sea level up

to an altitude of 1000 m. The seeds have no

dormancy and germinate readily after shedding.

Plants start flowering 3–4 weeks after germination

and the lifecycle is about 3 months. Cleome viscosa

Linn (Capparidaceae), also called “Dog mustard”,

is a herb that grow up to 1m height in India

(Parimala et al., 2004). Cleome is a large genus

included in the Capparaceae family, which

comprises 427 species occurring in tropical and

subtropical regions of the world (Brummit, 1992).

In Asia and Africa the leaves and seeds are used

as a rubefacient and vesicant and to treat infections,

fever, rheumatism and headache. The whole herb is

used in the treatment of ringworm, flatulence, colic,

dyspepsia, constipation, cough, bronchitis, cardiac

disorders (Kirtikar and Basu, 1975; Saxena et al.,

2000). Leaves are used as the external application

to inflammation of the middle ear and applied on

wounds and ulcers (Chopra et al., 1986),

hepatoprotective activity (Gupta and Dixit, 2009).

Traditionally, this plant is used in various disorders

such as diarrhoea, fever, inflammation, liver

diseases, bronchitis, skin diseases, and malarial

fever. Its juice is useful in piles, lumbago and

earache (Rukmini, 1978). The analgesic, antipyretic

and anti-diarrhoeal activities of the extract have

been reported by researchers. A decoction is used

as an expectorant and digestive stimulant and the

vapour from a steaming decoction of the whole

plant is inhaled to treat headache (CSIR, 1950). The

seeds and its oil have anti-helminthic properties but

they are ineffective in treating roundworm

infections (Saxena et al., 2000). They also have

anti-convulsant effect in Swiss albino mice (Mishra

et al., 2010). The root is a remedy for scurvy and

rheumatism (Rukmini, 1978). An aqueous seed

extract displayed significant analgesic activity in

mice and local anaesthetic activity in guinea pigs

(Singh and West, 1991; Parimaladevi et al., 2003).

In tests with rats, the anti-diarrhoeal (Devi et al.,

2002) and anti-pyretic (Devi et al., 2003) activities

of the extracts have been confirmed. Leaves and

young shoots are cooked as a vegetable

(Manandhar, 2002). It has a sharp mustard like

flavor (Facciola, 1990). The pungent seed can be

pickled or used as a mustard substitute in curries

(Manandhar, 2002; Facciola, 1990). The juice of

the plant is used as a condiment (Facciola, 1990).

Oil obtained from the seeds is used for cooking

(Manandhar, 2002). All parts of the plant are used

in liver diseases, chronic painful joints and mental

disorders (Chatterjee and Pakrashi, 1991).

The induction of callus growth and subsequent

differentiation and organogenesis is accomplished

by the differential application of growth regulators

and the control conditions in the culture medium.

With the stimulus of endogenous growth substances

or by addition of exogenous growth regulators to

the nutrient medium cell division, cell growth and

tissue differentiation are induced (Tripathi and

Tripathi 2003). Organogenesis permits the

manipulation of large numbers of high quality plant

material within a short period of time. The vast

usage of the Cleome viscosa in medicinal purpose,

based on the above reason, it is increasing

commercial demand of Cleome viscosa. The

present study was undertaken to develop a more

efficient protocol for rapid in vitro multiplication of

Cleome viscosa using leaf explants as an initial

plant material.

Materials and methods

The plants were collected from in and around

Virudhunagar district, Tamil Nadu, India (Figure

1). Small young leaves were collected from healthy

plants of Cleome viscosa L defoliated and sectioned

into many segments. They were washed under

continuous flashing of running tap water for 30

minutes and then with distilled water three times.

Leaves were treated with 0.1% mercury chloride

for 1.5 minutes and washed with sterilized double

distilled water for 3 times. The small washed

explants were treated with 3% hydrogen peroxide

for 1.5 minutes and then washed with sterilized

double distilled water for 3 times. At final the

explants were treated with 70% ethanol for 0.5

minutes and then washed with sterilized double

distilled water for 3 times in laminar air flow

cabinet.


Figure 1. Natural habitat of Cleome

(Linn.).

Culture media and growth condition

viscosa

The medium comprised of macro and

micro

elements according to Murashige and Skoog (1962)

with myoinositol (100mg/l), thamine

HCl

(0.1mg/l), pyridoxine (0.5mg/l), nicotinic acid

(0.5mg/l) and sucrose (30g/l), solidified with 0.8%

agar. The plant growth regulators used were

Kinetin (Kn), Indole-3butyric acid (IBA), Indole-

3acetic acid (IAA), 2-Napthalene acetic acid

(NAA) and Benzyl aminopurine (BAP). All

experiments were carried out in culture tubes

(25×150mm) containing 10ml of culture medium.

The pH of the medium was adjusted to 5.8 prior to

autoclaving at 121ºC for 20 minutes. Cultures were

incubated under 16 hours/8 hours light/dark cycles.

Callus induction and shoot regeneration

The small segments of leaf explants were cultured

on MS medium fortified with different

concentration of auxins such as, IAA, NAA and

IBA ranging from 0.5 – 4.0mg/l for callus

initiation. Explants were found to give profuse

callusing and when callusing was observed in entire

explant, the callus was transferred to MS medium

supplemented with various concentrations of BAP

(0.5-4.0mg/l), Kinetin (0.5-4.0mg/l) and BAP (1.0-

5.0mg/l) in combinations with Kinetin (0.5-

2.5mg/l). All the cultures were maintained at 25 ± 2

ºC for a photoperiod of 16h light per day. A total of

40 explants was used for each experiment and was

repeated 5 times. Data on percent of response,

number of shoots and shoot length formation per

explant was recorded after 30 days of culture.

Callus Growth Measurement

In order to minimize the differences in growth that

may have been the result of variation in inoculum

Regeneration of Cleome viscosa 39

size, a growth index was used to evaluate the callus

growth after 30 days of culture. Dry weight of calli

was evaluated by drying fresh calli in an air forced

oven at 60ºC for 36 hrs. The growth index was

determined according to the formula:

Regeneration of roots and development of complete

plantlets

For rooting individual microshoots (1.5-2.5mm

length) were isolated from callus clumps and

cultured on MS medium containing 0.1-1.0mg/l of

different auxins such as, IBA, IAA and NAA to

induce of root formation. A total of 40 plantlets

were taken for each experiment and were repeated

5 times. Percent of rooting, number of root

formations and root length was recorded 40 days

after root initiation. All the rooted plants were

carefully removed from culture tubes and washed

in tap water to remove agar gels and transferred to

plastic pots containing soil mixture (sterile soil,

vermicompost and sand) in the ratio of 1:2:1. In

vitro regenerated plants covered with polythene

bags to maintain high humidity. These were

acclimatized at 25 ± 3ºC less than 16/8 hours

photoperiod and watered regularly. After 3 – 4

weeks, the polythene bags were removed and

established plantlets were transplanted to earthen

pots in a greenhouse.

Statistical analysis

All results reported are the means of five replicate.

The result are expressed as mean values ± standard

deviation (n=5). Data were statistically analyzed at

P≤0.05 levels using one way analysis of variance

(ANOVA). Mean comparisons were carried out by

Duncan's multiple ran test was used as a post hoc

analysis (SPSS package, Version 17).

Results

The fresh smaller size of explants provide less

chance of contamination, as well as, longer leaves

showed, total loss of morphogenic potential. 3%

hydrogen peroxide, 0.1% mercuric chloride and

70% ethanol was used to sterilize the leaves.


40 Jeyaraj ANBURAJ et al.

Surface of the leaves were sterilized with 0.1%

mercuric chloride for 1.5minutes, 3% hydrogen

peroxide for 1.5minutes and 70% ethanol for

0.5minutes reported the maximum growth without

contamination.

Interactive effects of culture medium and

explant type were statistically significant (P≤0.05)

for calli fresh weight, and calli growth index (Table

1). Calli were initiated from leaf explants of

Cleome viscosa (Figure 2A&B). After two weeks

of culture on MS medium fortified with different

concentrations of auxins IBA (0.5-4 mg/l), NAA

(0.5-4 mg/l) and IAA (0.5-4 mg/l). The highest

mean for calli growth index, and calli fresh weight

of leaf explant belonged to the MS culture medium,

supplemented with 2mg/L IAA. Leaf explant was

superior to the nodal explants regarding the growth

index, and calli fresh weight. About 81% callusing

response with maximum of 178±0.71mg (P≤0.05)

fresh mass and calli growth index 2.3±0.07

(P≤0.05) was observed. The optimum concentration

of IBA (2mg/l) showed 70% callusing response

with 122±0.54mg fresh mass was observed in leaf

explants. Here IBA (2mg/l) was more effective

callusing response and callus growth index

compare with IBA (1mg/l) (P=0.526). Low

callusing response (55%) with 107±0.57mg fresh

mass was observed in leaf explants cultured on

NAA (2mg/l) fortified medium (Figure 2C, D & E).

The callus cultured on MS medium with 3.0%

sucrose and 0.8% agar medium supplemented with

hormones such as, BAP, KN and BAP+KN

produced microshoots. High frequency (95%)

multiple shoot induction with 7.6±0.54 (P≤0.05)

number of shoots and an average length of

4.10±0.18cm shoots in MS medium supplemented

with 2.0mg/l BAP. On the other hand, the callus

cultured on cytokinin combination BAP and KN

(5+2.5mg/l) containing medium promoted the

microshoots. About 85% moderate shooting

response with 7.2±0.83 number of shoots and an

average length of 3.62±0.15cm shoots from leaf

callus. Low frequency (69%) multiple shoot

induction with 5.6±0.55 (P=0.149) number of

shoots and an average length of 3.15±0.28cm

shoots in MS medium supplemented with 1.0mg/l

Kn (Table 2, Figure 2F, G & H).

The healthy shoots with two to three leaves

were isolated from shoot clumps and cultured on

root induction medium to induce adventitious

rooting. MS medium fortified with different

concentration of auxins IBA (0.1-1.0mg/l), NAA

(0.1-1.0mg/l) and IAA (0.1-1.0mg/l). The

individual plantlets cultured on MS medium

supplemented with NAA (0.1mg/l) fortified

medium induced high frequency (91%) rooting

with 3.4±0.89 (P≤0.05) number of microroots and

an average length of 3.00±0.25cm roots. There is

no significant compared with concentration of

0.2mg/l NAA (P=0.6). When cultured on the MS

medium containing other hormones such as IBA

(0.1mg/l) and IAA (0.1mg/l) both explants showed

low response than 0.1mg/l of NAA (Table 3, Figure

2 I). The healthy rooted plants were carefully

removed from the culture tubes and washed in

running tap water to remove agar gels and

transferred to plastic pots containing soil mixture.

Finally, the regenerated plants were successfully

transferred to earthen pot containing soils. Almost

all (90%) the in vitro regenerated plants

successfully survived in green house condition

(Figure 2J, K, L & M).

Figure 2. A) Callus initiation in Cleome viscosa

from leaf explants in MS media containing 2mg/l

IBA. B) Callus initiation in Cleome viscosa from

leaf explants in MS media containing 2mg/l NAA.

C) Callus induction in Cleome viscosa from leaf

explants in MS media containing 2mg/l IAA. D)

Kinetin MS media. H) Shoot regeneration from

callus on BAP + KN (4+2mg/l) MS media. I)

Rooting generation on 0.5mg/l IAA & NAA. J) In

vitro regeneration of complete plantlets of Cleome

viscosa from leaf explants. K) Plants growing in

Callus induction in Cleome viscosa from leaf

explants in MS media containing 2mg/l NAA. E)

Callus induction in Cleome viscosa from leaf

explants in MS media containing 2mg/l IBA. F)

Shoot regeneration from callus on 2mg/l BAP MS

media. G) Shoot regeneration from callus on 2mg/l

earthen pots containing soil under green house

condition.


Regeneration of Cleome viscosa 41

Table 1. Effects of different concentration of auxins on callus growth index from leaf of Cleome viscosa after

30days.

Growth regulators Callusing Callus growth Callus growth

Callus

(mg/l)

IAA

response % (mg fresh mass) index (Mean ±SE) morphology

0.5 60 129±0.83a 1.2±0.09ac Green nodular calli

1 65 140±0.89a 1.4±0.09b Green nodular calli

2 81 178±0.71a 2.3±0.07abc Green nodular calli

3 80 156±0.55a 1.8±0.07ab Green nodular calli

4

IBA

75 147±0.45a 1.6±0.09c Green nodular calli

0.5 65 114±0.84ab 1.3±0.05a White green compact calli

1 60 109±0.71b 1.2±0.03b White green compact calli

2 70 122±0.54a 1.6±0.04c White green compact calli

3 60 110±0.57ab 1.2±0.04abc White green compact calli

4

NAA

60 80±0.99ab 1.0±0.03abc White green compact calli

0.5 45 100±0.45a 1.4±0.02a White yellow friable calli

1 50 103±0.83a 1.8±0.11ab White yellow friable calli

2 55 107±0.57a 2.1±0.12ab White yellow friable calli

3 40 84±0.55a 1.4±0.05b White yellow friable calli

4 15 39±0.89a 1.0±0.06ab White yellow friable calli

Values followed by the same letter within each column are not significantly different.

Table 2. Effects of different concentration of cytokinins and cytokinins combinations on multiple shoot

induction from leaf of Cleome viscosa.

Growth regulators Callusing

Number of

Shoot length

(mg/l)

Bap

response % total shoots)

(cm)

0.5 60

5.4±0.54ac

3.50±0.24a

1 80

6.2±0.83bd

3.60±0.16b

2 95

7.6±0.54ac

4.10±0.18abc

3 55

2.6±0.89ab

3.20±0.25c

4 50

2.2±0.45cd

3.10±0.18b

Kn

Bap+Kn

0.5

1

2

3

4

60

69

54

45

40

5.2±0.45a

5.6±0.55b

5.1±0.84c

4.8±0.95d

4.2±0.84e

2.85±0.21a

3.15±0.28b

3.08±0.15c

3.20±0.25d

3.00±0.13e

0.5

1

2

3

4

60

45

75

80

85

5.4±0.89a

4.2±0.84bc

6.0±0.71c

4.4±0.89d

7.2±0.83abd

2.95±0.31a

2.63±0.18b

3.25±0.15bc

2.80±0.14c

3.62±0.15abc

Values followed by the same letter within each column are not significantly different.


42 Jeyaraj ANBURAJ et al.

Table 3. Effects of different concentration of auxins on efficient rooting from shooted callus of Cleome

viscosa.

Growth regulators

(mg/l)

NAA

0.1

0.2

0.5

1

IBA

IAA

0.1

0.2

0.5

1

0.1

0.2

0.5

1

Callusing

response %

91

80

55

40

85

70

40

35

65

40

35

25

Number of

total shoots)

3.4±0.89ac

3.2±0.45bd

2.6±0.89ab

2.4±0.54cd

3.2±0.83a

3.0±0.71b

2.2±0.84ab

1.6±0.54ab

2.8±0.84ab

1.6±0.89a

1.4±0.55b

1.2±0.44a

Values followed by the same letter within each column are not significantly different

Discussion

The fresh smaller size of explants provide less

chance of contamination, longer leaves showed a

total loss of morphogenic potential as well. A

similar report has been published in Rauvolfia

serpentine by Singh et al. (2009) who reported that

shoot regeneration from small leaf explants of

Rauvolfia serpentine L. The surface sterilization of

explants is very essential for establishment, as well

as for optimum induction of callus and plantlet

regeneration in vitro condition (Yeoman and

Macleod, 1977). In the present study 3% hydrogen

peroxide, 0.1% mercuric chloride and 70% ethanol

was used to sterilize the leaves, resulted the

maximum growth without contamination.

Similarly, Sairkar et al. (2009) reported that the

cultured explants showed more than 80%

contamination free cultures when treated with 0.1%

HgCl2 for surface sterilization.

The MS medium was the most effective for

callusing of leaf explants. The explants cultured on

MS medium supplemented with different auxins of

IBA, NAA and IAA showed varied response for

callusing. Among three types of auxins, IBA and

IAA were previously found to be very effective

(Singh et al., 2009). However, in the present

Shoot length

(cm)

3.00±0.25abc

2.50±0.28a

2.45±0.23b

2.30±0.23c

2.10±0.23ac

1.85±0.41b

1.80±0.19a

1.53±0.18bc

2.00±0.18ac

1.85±0.12b

1.65±0.16a

1.50±0.20bc

investigation calli were initiated from leaf explants

of Cleome after two weeks of culture on MS

medium fortified with different auxins IBA (0.5-4

mg/l), NAA (0.5-4 mg/l) and IAA (0.5-4 mg/l).

About 81% callusing response with maximum of

178mg fresh mass was observed in leaf explants

cultured on MS medium supplemented with IAA

(2mg/l).

In this present research that the callus cultured

on MS medium, hormones such as BAP, KN and

BAP+KN produced microshoots. High frequency

(95%) multiple shoot induction with 7.6±0.54

number of shoots and an average length of

4.10±0.18 shoots in MS medium supplemented

with 2.0mg/l BAP, was observed. Similarly, the

two cytokinins used BAP induced significantly

higher percentage of shoot initiation and mean

number of shoot, whereas higher mean shoot length

was obtained in the shoots obtained on media

supplement with KN (Gokhale and Bansal 2009).

The healthy shoots were isolated from shoot

clumps and cultured on root induction medium to

induce adventitious rooting. MS medium was

fortified with different concentrations of auxins

IBA (0.1-1.0mg/l), NAA (0.1-1.0mg/l) and IAA


(0.1-1.0mg/l). The individual plantlets cultured on

MS medium supplemented with NAA (0.1mg/l)

fortified medium induced high frequency (91%)

rooting with 3.4±0.89 number of microroots and an

average length of 3.00±0.25 roots. When cultured

on the MS medium containing other hormones such

as IBA (0.1mg/l) and IAA (0.1mg/l), both explants

showed lower response than that of 0.1mg/l of

NAA. A similar report has been published by Amin

et al, (2003), who reported the root formation was

induced in in vitro regenerated shoots by culturing

them on half strength of MS medium with 0.1-

1.0mg/l either of NAA, IBA and IAA. Among three

types of auxins, NAA was found to be most

effective at different concentrations tested for root

production. The findings are in agreement with

those observed in other plant species. Although

Gokhale and Bansal (2009) reported that the three

auxins (IAA, IBA, NAA) induced roots in in vitro

raised shoots of O. indicum, yet NAA responded

best for all parameters of rooting. For shoots with

highest percentage of root induction, maximum

number of elongated roots was developed on MS

medium containing NAA. Long, branched healthy

roots were produced in this rooting media.

The in vitro derived plants acclimated better

under ex vitro conditions when they were

transferred on specially made plastic trays

containing coco-peat as potting mix and moistened

uniformly at periodic intervals taking special care

not to damage the roots. The rest of the procedure,

followed from this stage up to their establishment

in soil was as usual (Amin et al., 2003). In the

present investigation the healthy rooted plants were

carefully removed from the culture tubes and

washed in running tap water to remove agar gels

and transferred to plastic pots containing soil

mixture. Finally, the regenerated plants were

successfully transferred to earthen pot containing

soils. Almost all (90%) the in vitro regenerated

plants successfully survived at green house

conditions. Similarly, the success of transplantation

was 85% when plantlets were sufficiently healthy

with new growth.They were subsequently

transferred to larger pots and gradually acclimated

to outdoor conditions. The protocol reported here is

reproducible, it has a potential for being utilized to

conserve the germplasm and allowing at the same

time, a large scale micropropagation of this

important medicinal plants (Jeyakumar and

Jayabalan, 2002).

In conclusion, a significant progress has been

made in the in vitro regeneration system of this

Regeneration of Cleome viscosa 43

medicinally important Cleome viscosa. Using this

technique, it is possible to produce healthy and

disease free clones which could be released to their

natural habitat in large scale. It may also be useful

for gene manipulation for this important medicinal

species.

Acknowledgements

I am extremely grateful to Dr. M. Vimala Devi, the

head at Department of Biotechnology, Ayya Nadar

Janaki Ammal College, Sivakasi, India for his

stimulating guidance, invaluable suggestions and

critical and persistent encouragement. I wish to

thank Mr. Ga. Bakavathiappan and Mr. M. Pavaraj,

for helping me to carry out my project work in

Ayya Nadar Janaki Ammal College, Sivakasi,

India.

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Journal of Cell and Molecular Biology 9(1): 45-52, 2011 Research Article 45

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Bio-database compression using enhanced suffix array for pairwise

sequence alignment

Arumugam KUNTHAVAI *1 and Somasundaram VASANTHA RATHNA 2

1 Department of CSE/IT, Coimbatore Institute of Technology, Coimbatore, Tamilnadu, India

2 Department of EEE, Coimbatore Institute of Technology, Coimbatore, Tamilnadu, India

(* author for correspondence; kunthavai@cit.edu.in)

Received: 19 February 2011; Accepted: 10 June 2011

Abstract

Sequence alignment is a bioinformatics application that determines the degree of similarity between

nucleotide or amino acid sequences which is assumed to have same ancestral relationships. This sequence

alignment method reads query sequence from the user and makes an alignment against large and genomic

sequence data sets and locate targets that are similar to an input query sequence. Traditional accurate

algorithm, such as Smith-Waterman and FASTA are computationally very expensive, which limits their use

in practice. The current set of popular search tools, such as BLAST and WU-BLAST, employ heuristics to

improve the speed of such searches. However, such heuristics can sometimes miss targets, in which many

cases are undesirable. This paper provides BioDB Compression Tool using enhanced suffix array, to perform

accurate and faster biological sequence analysis as an improvement on the computation time of existing tools

in this area using data base compression. The main idea is to pick matched patterns of the query sequence and

identify sequences in the database which share a large number of these matched patterns, thereby

compressing the size of the database to very few sequences. Experiment results are cross validated using data

mining technique. This shows that a new BioDB Compression developed effectively compresses the database

and obtains very similar results compared to that traditional algorithm in approximately half the time taken

by them.

Keywords: Sequence alignment, enhanced suffix array, compression, local alignment, data mining

Çiftli dizi sıralaması için kuvvetlendirilmiş sonek dizilişi kullanarak biyo-veritabanı

sıkıştırması

Özet

Dizi sıralaması aynı atasal ilişkiye sahip olduğu tahmin edilen nükleotid ve amino asit dizisi arasında

benzerlik derecesini tayin eden bir biyoenformatik uygulamadır. Dizi sıralama metodu kullanıcıdan sorgu

dizisini okur, büyük ve genomik dizi veri setlerine karşı sıralama yapar ve giriş sorgu dizisine benzer olan

hedefleri yerleştirir. Smith-Waterman ve FASTA gibi geleneksel güvenilir algoritmaların çok pahalı olması

pratik olarak kullanımlarını kısıtlar. BLAST ve WU-BLAST gibi güncel popüler araştırma araç setleri bu tür

araştırmaların hızını geliştirmek için buluşsal yöntemler kullanırlar. Ancak, bu tür buluşsal yöntemlerin çoğu

istenmeyen durumlarda bazen hedefleri kaçırabilir. Bu yayın bu alanda var olan araçların hesaplama

zamanını geliştirmek amaçlı kesin ve daha hızlı biyolojik dizi analizi yapmak üzere kuvvetlendirilmiş sonek

dizilişi kullanarak BioDB Sıkıştırma Aracını sunar. Ana fikir sorgu dizisinin eşleşen paternlerini seçmek ve

veri tabanında büyük sayıda eşleşen paterni paylaşan dizileri tayin ederek veri tabanı büyüklüğünü çok az

sayıda diziye sıkıştırmaktır. Deneysel sonuçlar veri madenciliği tekniği kullanılarak çapraz onaylanmıştır. Bu

da yeni geliştirilen BioDB Sıkıştırmanın veritabanını efektif olarak sıkıştırdığını ve klasik algoritmalarla

karşılaştırıldığında yarı zamanda çok benzer sonuçlar elde edildiğini gösterir.

Anahtar Sözcükler: Dizi sıralaması, kuvvetlendirilmiş sonek dizilişi, sıkıştırma, veri madenciliği


46 Arumugam KUNTHAVAI and Somasundaram VASANTHA RATHNA

Introduction

Bioinformatics is the field of analyzing the

biological information using computers and

statistical techniques; the science of developing and

utilizing computer databases and algorithms to

accelerate and enhance biological research. The

primary goal of bioinformatics is to increase the

understanding of biological processes. Common

activities of bioinformatics include mapping and

analyzing DNA and protein sequences, aligning

different DNA and protein sequences to compare

them and creating and viewing 3-D models of

protein structures. It has been successfully applied to

predict the function, structure and evolution of

biological sequences. It can reveal biological

relationship among organisms, for example, finding

evolutionary information, determining causes and

cures of diseases.

Sequence comparison is a basic operation of the

sequencing problem. Sequences can be aligned

across their entire length (global alignment) or only

in certain regions (local alignment). Local sequence

alignment plays a major role in the analysis of DNA

and protein sequences (GusFeild, 1997). This paper

describes the pair wise local alignment, which is the

basic step for many other applications like detecting

homology, finding protein structure and function,

deciphering evolutionary relationships, etc. Smith

Waterman developed a dynamic programming

approach to sequence alignment problem that is

widely used (Smith and Waterman, 1999). BLAST

(Altschul et al., 1990), FASTA (Pearson, 2000), WU-

BLAST (blast.wustl.edu) and (Matthew et al., 2003)

is two commonly used programs for similarity

searching on biological sequences. The methods

widely used are based on heuristic paradigms and

have relatively a fast execution time, they do not

produce optimal alignments sought by entirely

sequenced. This reality presents the need for

comparing long DNA or protein sequences, which is

a challenging task due to its high demands for

computational requirements (power and memory).

One important feature of BLAST is its ability to

compare a query with a database of sequences.

Considering the rapid growth of database sizes, this

problem demands ever-growing computation

resources, and remains as a computational challenge.

Divya in Sequence Comparison Tool - SCT (Divya

et al., 2006) developed sequence similarity in a

database instead of pair wise sequence alignment.

This tool preprocesses the database to create a

special generalized suffix tree from the sequences in

the database. The suffix tree (Divya et al., 2006) is

one of the most important data structures in string

processing and comparative genomic. The space

consumption of the suffix tree is a bottleneck in

large-scale applications such as genome analysis. To

overcome this bottleneck, in this paper suffix tree is

replaced with suffix arrays enhanced with the Lcptable.

Every algorithm that uses a suffix tree as data

structure can systematically be replaced with an

algorithm that uses an enhanced suffix array

(Abouelhoda et al., 2004) and solves the same

problem in the same time complexity but with

improved space complexity. The generic name

enhanced suffix array (Manber and Myers, 1993)

stands for data structures consisting of the basic

suffix array enhanced with additional tables.

Generalized suffix array (GSArray) is formed for

the query sequence and longest common

subsequence (LCS) is obtained and is compared

against the database; sequences, which consist of

LCS, are identified from the database. Sequences are

ranked with respect to the number of significant

patterns they share with the query sequence. Finally

database is compressed by selecting only a given

number of sequences with topmost ranks.

Materials and methods

Let S be a string of length ‘n’ over an ordered

alphabet ∑. It is assumed that the size of the alphabet

is a constant, and i.e. n < 232.The latter implies that

an integer in the range [0: n] can be stored in 4 bytes

and also assumed that the special symbol $ is an

element of ∑ (which is larger than all other

elements) but does not occur in S. S[i] denotes the

character at position i in S, for 0 ≤ i < n. For i ≤ j, S

[i...j] denotes the substring of S starting with the

character at position i and ending with the character

at position j. The substring S [i...j] is also denoted by

the pair of positions (i, j).

Suffix Tree: A suffix tree for the string S is a

rooted directed tree with exactly n+1 leaves

numbered 0 to n. Each internal node, other than the

root, has at least two children and each edge is

labeled with a nonempty sub string of S$. No two

edges out of a node can have edge-labels beginning

with the same character. The key feature of the suffix

tree is that for any leaf i, the concatenation of the

edge-labels on the path from the root to leaf i exactly

spells out the string Si, where Si = S [i...n – 1]


denotes the ith nonempty suffix of the string S$, 0 ≤ i

< n. The space and time complexity of this suffix

tree is O (n). Consider the suffix tree as an example

shown in Figure 1 for the string S = acaaacatat.

Using the suffix tree of S1 # S2, MUMs(Maximum

Unique Match) (Delcher et al., 1999) can be

computed in O (n) time and space, where n = [S1 #

S2] and # is a symbol neither occurring in S1 nor in

S2.

However, the space consumption of the suffix tree

is a major problem when comparing large genomes.

Figure 1. The suffix tree for S = acaaacatat.

Suffix array

Suffix array is designed for efficient searching of a

large text. It requires only 4n bytes (4 bytes per

input character) in its basic form. Searching a text

can be performed by binary search using the suffix

array. Suffix trees can be constructed in O (n) time

in the worst case, versus O (nlogn) time for suffix

arrays. Suffix arrays will prove to be better than

suffix trees for many applications. The suffix array

(denoted by suftab) of the string S is an array of

integers in the range 0 to n, specifying the

lexicographic ordering of the n + 1 suffixes of the

string S$. That is, S (suftab [0]), S (suftab [1] … S

(suftab[n]) is the sequence of suffixes of S$ in

ascending lexicographic order as shown in Table 1.

Suffix array generation

Creating suffix array requires time O (nlogn) and

searching for a pattern in it requires time O

(nlogm), where n is the length of the pattern and m

is the length of the string. In (Abouelhoda et al.,

2002), the authors provides a systematic way of

replacing string processing algorithm based on a

bottom-up traversal of a suffix tree by a

corresponding algorithm that is based on an

enhanced suffix array. The advantages of this

approach are better search, more space efficient

Bio-database compression using enhanced suffix array 47

than suffix tree since the implementation of the

enhanced suffix array requires only 5n bytes and

the running times of the algorithms are much better

than those based on the suffix tree and the

algorithm is easier to implement on enhanced suffix

arrays than on suffix trees.

Table 1. The Lcp-interval table of S = acaaacatat$

s.no Suftab Lcptab S(suftab[i])

0 2 0 aaacatat$

1 3 2 aacatat$

2 0 1 acaaacatat$

3 4 3 acatat$

4 6 1 atat$

5 8 2 at$

6 1 0 caaacatat$

7 5 2 catat$

8 7 0 tat$

9 9 1 t$

10 10 0 $

A pair of substrings R = ((i1, j1); (i2, j2)) is a

repeated pair if and only if = ((i1, j1) ≠ (i2, j2)) and

S [(i1… j1)] = S [(i2 …j2)]. The length of R is j1 -

i1+1. A repeated ((i1, j1); (i2, j2)) is called left

maximal if S[i1 ─ 1] ≠ S[i2 ─ 1] and right maximal

if S[j1 ─ 1] ≠ S[j2 ─ 1]. A repeated pair is called

maximal if it is left and right maximal. A substring

ω of S is a (maximal) repeat if there is a (maximal)

repeated pair ((i1, j1); (i2, j2)) such that ω = S

[i1...j1]. A super maximal repeat is a maximal

repeat that never occurs as a substring of any other

maximal

repeat.

The suffix array suftab is an array of integers in

the range 0 to n, specifying the lexicographic

ordering of the n+1 suffixes of the string S$. That

is, Ssuftab [0]; Ssuftab [1] … Ssuftab[n] is the sequence

of suffixes of S$ in ascending lexicographic order,

where Si = S [i ... n -1 $ denotes the ith nonempty

suffix of the string S$ , 0 ≤ i ≤ n. The suffix array

requires

4n bytes.

The Lcp-interval table Lcptab is an array of

integers in the range 0 to n and Lcptab [0] = 0

shown table 1. Lcptab[i] is the length of the longest

common prefix of Ssuftab [i−1] and Ssuftab[i], for 1 ≤

i ≤ n. Since Ssuftab[n] = $, it is always have

Lcptab[n] = 0. The Lcp-table can be computed as a

by-product during the construction of the suffix


48 Arumugam KUNTHAVAI and Somasundaram VASANTHA RATHNA

array. The lcp-interval tree of S = acaaacatat$ is

shown in Figure 2.

Figure 2. The Lcp-interval tree of S = acaaacatat$

An interval [i...j], where 0 ≤ i ≤ j ─ n, in an Lcparray

is called an Lcp-interval

of Lcp-value ℓ

(denoted by ℓ-[i...j]) if

Lcptab[i] < ℓ

Lcptab[k] ≤ ℓ for all k with i+1 ≤ k ≤ j

Lcptab[k] = ℓ for at least one k with

i+1 ≤ k ≤ j

Lcptab [j + 1] < ℓ

Every index k, i+1 ≤ k ≤ j, with Lcptab[k] = Ssuftab

is called ℓ index. The set of all ℓ indices of an ℓ

interval [i...j] will be denoted by ℓ Indices (i...j). If

[i...j] is an ℓ-interval such that ω =

S[suftab[i]..Suftab[i]+ ℓ -1] is the longest common

prefix of the suffixes Ssuftab[i]; Ssuftab[i+1]; … ;

Suftab[j], then [i...j] is also called ω-interval. Based

on the analogy between the suffix array and the

suffix tree, it is desirable to enhance the suffix array

with additional information to determine, for any ℓ-

interval [i..j], all its child intervals in constant time

using enhancing the suffix array with two tables.

Enhanced Suffix array

The new data structure consists of the suffix array,

the Lcp-interval table, and an additional

table: the

child-table cldtab shown in Table 2.

The child-table is a table of size n+1 indexed

from 0 to n and each entry contains three values:

up, down, and nextℓIndex. Each of these three

values requires 4 bytes in the worst case. The

values of each cldtab-entry are defined as follows

(it is assumed that min Φ = max Φ = 1):

1. cldtab[i].up =

Min {q Є [0..i - 1] | Lcptab[q] > Lcptab[i]

and for all k Є [q + 1..i - 1] :

Lcptab[k] ≥ Lcptab[q]}

2. cldtab[i].down

=

Max {q Є [i + 1.. n] | Lcptab[q] > Lcptab[i]

and for all k Є [i + 1..q - 1] : Lcptab[k] ≥

Lcptab[q]}

3. cldtab[i].next ℓ Index =

Min {q Є [i + 1.. n] | Lcptab[q] = Lcptab[ i]

and for all k Є [i + 1..q - 1] : Lcptab[k] >

Lcptab[i]}

The child-table stores the parent-child

relationship of Lcp-intervals. For an ℓ -interval

[i...j] whose ℓ -indices are i1 < i2


with a highly conserved region. It is calculated

based on the length of the pattern and frequency

i.e., number of occurrences in the database. The

given pattern p is classified as significant if it

satisfies the following constraints:

The length of p ≥ a given length-threshold: a

significant pattern must be sufficiently long

to carry important biological information.

The score of p ≥ a given score-threshold: a

significant pattern

must have a sufficiently

high score.

GSArray is constructed for the entire sequences

in the database. While constructing GSArray, at

each node i, the length and the

frequency ) (i.e. the number of occurrences of

pi in the database) is stored for the corresponding

pattern pi this frequency is incremented for every

new node. Then the score function

is

calculated using the equation (1)

(1)

Where = Function value of the pattern,

= frequency of the node,

= length of the label of prefix,

i = node number,

|DB| = Database size.

The score function is used as a measure

to determine whether a particular pattern is

significant to be included in the post-processing for

evaluation of sequences, to determine the closest

set of sequences. Then the query sequence Q and

number of sequences to be selected from the

database is read from the user, temporarily added

onto GSArray. This enables to determine which

suffixes of the query are shared by the sequence in

the database. The query sequence is only

temporarily added to the tree so that GSAlign is not

affected for future sequence searches. Initially all

the nodes in the GSArray are 0. When the query

sequence Q is added as a suffix, the nodes visited is

set to 1. This expedites the search for common

patterns within the GSArray because only those

paths in the tree for patterns that contain substrings

of the query sequence are examined. In depth-first

manner, starting at the root all the nodes are visited

to check the value 1. If the current node has no

child whose value is 1, then the search backtrack to

its parent node. During this traversal all the

significant patterns are collected. The sequence

may have other common patterns that are not

significant. An optimal alignment between these

two sequences in an ideal case contains all

significant patterns. After this process the query

Bio-database compression using enhanced suffix array 49

sequence from GSArray is deleted. Top ten

significant patterns are selected and stored. The

sequence that contains significant patterns is

extracted and stored. Reverse check is made to

obtain the accuracy of the results; it computes how

many chosen patterns are being shared by each of

the sequences extracted already. Higher the number

(weight), greater will be the similarity of the

corresponding sequence to the query. Based on this

weight

the sequences are ranked.

A top n sequences are transferred to new

database. Algorithm for

BioDB Compression Tool

is shown in Figure 3.

//Input: Set of DNA and Protein Sequences

// Output: Compressed set of DNA and Protein sequences

S1: Read DNA or Protein database;

S2: Construct GSArray for the input sequences, Set node visit

=0;

S3: While constructing the suffix array, store the information of

label length, frequency at nodes and sequences traversing

through the branches;

S4: Use the equation (1) to calculate the degree of similarity of

patterns in the form of prefixes;

S5: Read query sequence Q and the number of sequences n to

select from the user;

S6: Temporarily add the suffixes of query to the generalized

GSArray;

S7: While adding the suffixes highlight nodes of the paths which

are traversed by the query sequence;

S8: Post process the GSArray to extract patterns shared by the

query Sequence, which lies above a defined threshold on

the

function-value;

S9: Pick the top ten of these patterns and store;

S8: Do a reverse check to compute the weight of each sequence

in the subset;

S9: Rank the sequences according to these weights;

S11: Pick top n sequences from the subset and write to a new

database;

Figure 3. Algorithm for BioDB Compression Tool

Cross validation

The real world DNA (GSS, EST) and Protein

sequence (Protein C, S) databases from Oryza

sativa group is extracted from NCBI website and

enhanced suffix array is formed for all the

sequences in the database. Based on the user given

query sequence, the significant patterns are

generated. The sequences in the DNA or Protein

database are given weights according to the number

of patterns they contain. The compressed database

is formed by selecting top ‘n’ sequences with

highest

ranks and written into a new database.

The latest data mining technique, 7-fold cross

validation is used to validate the results obtained

from BioDB Compression Tool and WU-BLAST.


50 Arumugam KUNTHAVAI and Somasundaram VASANTHA RATHNA

The main idea of 7-fold cross validation approach

is ‘train on 6 folds, test on 1 fold. The data set is

divided into 7 parts. Among the 7 parts 6/7 of the

data are used for training data set and the remaining

1/7 is used for testing data set. BioDB Compression

Tool is applied on training data set and then on

WU_BLAST for single user given query sequence.

For the same query sequence WU-BLAST alone is

applied on the testing data set. The average of this

seven runs is computed for analysis. In this paper

such seven queries are taken and analyzed. 49

sequences from Oryza sativa GSS gi: 288881557 to

gi: 288881606 are taken as a database set, 42

sequences are used for training data set and 7

sequences are used for test data set. Single query

sequence is applied first on BioDB Compression

Tool, data base is compressed. WU-BLAST is

performed on the new data base and the same query

sequence. Then for same sequence WU-BLAST

alone is applied. The sequences in the training and

test data set are interchanged and the above steps

are repeated until every fold is used for training.

Average of the result from 7 runs are calculated and

stored. This is experimented for 7 different queries.

The objective is to test whether the results are

consistent for all the queries on a particular

database in terms of computational time.

Results

Compressed database from BioDB Compression

Tool is cross validated using 7-fold cross validation

approach. The cross validation results of seven

different queries for DNA database set GSS gi:

288881557 to gi: 288881606 are shown in Figure 4

and EST gi: 288886142 to gi: 288886191 are

shown in Figure 5 and the results of seven different

queries Protein C &S are shown in Figures 6 and 7.

Figure 4.

Results of cross validation for GSS DNA

dataset

Figure 5. Results of cross validation for EST DNA

dataset

The idea is to test whether the results are

consistent for all queries on a particular database in

terms of computation time. From the figures 4-6 it

is inferred that the developed BioDB Compression

Tool performs much better than WU-BLAST.

Computational complexity of using developed

BioDB Compression Tool is reduced compared to

WU-BLAST because; compression takes place

along sequence alignment. The Enhanced suffix

array algorithm used in BioDB Compression Tool

requires 5n bytes character where SCT requires 20n

bytes which uses suffix tree.

Figure 6. Results of cross validation for Protein C

Hence it is proved that space complexity is

approximately five times less than SCT.

Implementation results show that the running time

of developed algorithm BioDB Compression Tool

using enhanced suffix array is much better than

SCT which uses suffix tree.


Figure 7. Results of cross validation for Protein S

Discussion

In this paper the BioDB Compression Tool using

enhanced suffix array has been developed. BioDB

Compression Tool pre-processes the database to

create a generalized enhanced suffix array and

extended by adding frequency and length

information for the patterns. BioDB Compression

Tool distinguishes patterns by computing

significance-scores. A pattern is regarded as

significant if it is long enough, and it appears

frequently enough in the database. The scoring

function takes into account a pattern's length and

frequency, the given threshold values, and

determines if a pattern is significant. Using these,

for a given query sequence BioDB Compression

Tool compresses the database to only a few

sequences that share the most significant patterns

with the query. This compression in database size

speeds-up the local alignment of the query

sequence against the database. Experimental results

have shown that BioDB Compression Tool

provides a speed-up over WU-BLAST. It is able to

reduce the time of a database search to nearly five

times originally taken by WU-BLAST. Results

from WU-BLAST have shown that this method is

experimentally effective, as the results obtained by

BioDB Compression Tool produces accurate

alignment. Combined with the extended suffix

array, BioDB Compression Tool has the advantage

of using WU-BLAST to do the local sequence

alignment. Latest Data Mining technique, 7-fold

cross validation is applied to attain a greater

accuracy in the results and the results are

satisfactory. The Enhanced suffix array algorithm

used in BioDB Compression Tool requires 5n bytes

/character where SCT requires 20n bytes which

uses suffix tree. So, space complexity is

approximately five times more than SCT.

Experimental results show that the running time of

Bio-database compression using enhanced suffix array 51

BioDB Compression Tool is much better than SCT

which uses suffix tree.

In this paper, a small domain of sequences have

been selected from the DNA and Protein database

and experimentally proved that, enhanced suffix

array reduces space complexity by five times.

BioDB Compression Tool can also be applied to

Global Sequence Alignment and Multiple Sequence

Alignment. This work can also be applied on

databases of protein sequences for alignment.

References

Abouelhoda MI, Kurtz S and Ohlebusch E. The

enhanced suffix array and its applications to

genome analysis. Proc. Workshop on

Algorithms in Bioinformatics. Springer- Verlag,

Berlin. 2452: 449- 463, 2002.

Abouelhoda MI, Stefan Kurtz and Enno Ohlebusch.

Replacing suffix trees with enhanced suffix

arrays. Journal of Discrete Algorithms. 2:53-86,

2004.

Altschul SF, Gish W, Miller W, Myers E and

Lipmann D. Basic Local Alignment Search

Tool. Journal of Molecular Biology. 215:403 -

410, 1990.

Delcher AL, Kasif S, Fleischmann RD, Peterson J,

White O and Salzberg SL. Alignment of Whole

Genomes. Nucleic Acids Res. 27:2369-2376,

1999.

Divya RS, Abdullah NA and Xindong Wu. Using

an extended suffix Treeto speed-up sequence

alignment. IADIS International Conference

Applied Computing. 655-660, 2006.

Gus Field D. Algorithms on Strings, Trees and

Sequences. Cambridge University Press. 1997.

Basic Local Sequence Alignment Tool. Retrieved

May 4, 2009, from http://blast.wustl.edu/

Manber U and Myers EW. Suffix Arrays: A New

Method for On-Line String Searches. SIAM

Journal on Computing. 22(5):935-948, 1993.

Matthew Bellgard, Thomas Gamble, Mark

Reynolds, Adam Hunter, Ed Trifonov, and Ross

Taplin. Gap apping: a paradigm for aligning two

sequences. Applied Bioinformatics. 2(3 Suppl):

31-35, 2003.

Pearson WR. Flexible sequence similarity

searching with the FASTA3 program package.

Methods in Molecular Biology.132: 185-219,


52 Arumugam KUNTHAVAI and Somasundaram VASANTHA RATHNA

2000.

Smith TF and Waterman MS. Identification of

common molecular subsequences. Journal of

Molecular Biology. 147:195-197, 1981


Journal of Cell and Molecular Biology 9(1): 53-61, 2011 Research Article 53

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Cloning and expression of Lentinula edodes cellobiohydrolase gene

in E. coli and characterization of the recombinant enzyme

Sabira TAIPAKOVA 1 , Bauyrzhan SMAILOV 1 , Gulshan STANBEKOVA 2 and

Amangeldy BISSENBAEV 1, *

1

Department of Genetics and Molecular Biology, Faculty of Biology, al-Farabi Kazakh National University,

050038, Almaty,al-Farabi 71, Kazakhstan

2

Institute of Molecular Biology and Biochemistry, Dosmukhamedov 86, 480012, Almaty, Kazakhstan

(* author for correspondence; Amangeldy.Bisenbaev@kaznu.kz)

Received: 11 March 2011; Accepted: 10 June 2011

Abstract

A gene encoding cellobiohydrolase CEL7A was successfully isolated from the L. edodes mushroom strain

N127 using RT-PCR. The deduced amino acid sequence encoded by cel7A showed high homology with the

sequence of glycoside hydrolase family 7. To confirm the gene sequence encoding the CEL7A the cloned

gene was expressed in E. coli. For the first time the cel7A gene from the L. edodes was expressed in E. coli

and characterized. The recombinant CEL7A has the ability to hydrolyze Avicel, Filter paper, p-Nitrophenyl

β-D-lactopyranoside (pNP-Lac) and p-Nitrophenyl β-D-cellobioside (pNP-Cel). The activity of the cloned

enzyme towards carboxymethylcellulose (CMC) is much lower. It showed an optimal working condition at

50 0 C and pH 7.

Keywords: Cellulose, cellobiohydrolase, Lentinula edodes, gene expression, enzyme activity

Lentinula edodes selobiyohidrolaz geninin E. coli’de klonlanması, ekspresyonu ve

recombinant enzimin karakterizasyonu

Özet

L. edodes mantarı N127 suşundan selobiyohidrolaz CEL7A’yı kodlayan bir gen RT-PCR kullanarak

başarıyla izole edilmişti. Cel7A tarafından kodlanan tahmini aminoasit dizisi gilikosid hidrolaz ailesi 7 dizisi

ile yüksek homoloji göstermiştir. CEL7A’yı kodlayan gen dizisini doğrulamak için klonlanan gen E. coli’de

eksprese edilmiştir. L. edodes’den izole edilen cel7A ilk kez E. coli’de ifade edilip karakterize edilmiştir.

Rekombinant CEL7A, Avisel’i, Filtre kağıdını, p-Nitrofenil β-D-laktopiranosidi (pNP-Lac) ve p-Nitrofenil β-

D-selobiosidi (pNP-Cel) hidrolize etme yeteneğine sahiptir. Klonlanan enzimin karboksimetilselüloza (CMC)

aktivitesi daha azdır. 50 0 C ve pH 7’nin optimal çalışma şartları olduğu gösterilmiştir.

Anahtar Sözcükler: Selüloz, selobiyohidrolaz, Lentinula edodes, gen ifadesi, enzim aktivitesi

Introduction

Cellulose, consisting of glucose units linked

together by β-1,4-glycosidic bonds, is the most

abundant carbohydrate in the biosphere. An

estimated rate of cellulose synthesis is

approximately 4 x 10 7 tons per year. For a longrange

solution for problems of energy, chemicals,

and food, cellulose is the most promising renewable

carbon source that is available in large quantities

(Murai et al., 1998).

Full hydrolysis of cellulose requires synergistic

action of three major types of enzymatic activity:

1,4-β-D-endoglucanases (EC 3.1.2.4), 1,4-β-Dcellobiohydrolases

(CBH; EC 3.1.2.91) and βglucosidases

(EC 3.2.1.21). CBHs hydrolyze


54 Sabira TAIPAKOVA et al.

crystalline cellulose by initiating their action from

the ends of the cellulose chains and producing

primarily cellobiose. Endoglucanases catalyze

hydrolysis internally in the cellulose chain and

attack the amorphous regions in cellulose,

providing new chain ends for CBHs. β-

glucosidases hydrolyze cellobiose to glucose. These

enzymes are collectively known as cellulases and

act in a synergistic manner to facilitate complete

cleavage of the cellulose β -1,4-glycosidic bonds

(Teeri, 1997)

CBHs are key components in the multi-enzyme

cellulose complexes. Most of them belong to

glycosyl hydrolase families 6 or 7. Cellulolytic

fungus generally produces two different CBHs,

CBHI and CBHII. These two types of enzymes

which are classified based on sequence identity and

can achieve complete, although slow, solubilization

of cellulose crystals even without help of

endoglucanases (Teeri, 1997).

Lentinula edodes, commonly referred to as the

Shiitake mushroom, is the most popular and

economically important edible mushroom in the

world because of its taste, nutritional and medicinal

properties (Mizuno, 1995). L. edodes has

traditionally been grown on freshly cut logs

(Leatham, 1985). It is a white rot fungus that

decays for nutrients all the major polymers

(cellulose, hemicelluloses, and lignin) found in

wood lignocelluloses (Lee et al., 2001). Thus, L.

edodes produces a wide variety of enzymes that

may have high activities against cellulosic biomass.

Several papers reported that L. edodes has a

strong ability to produce thermostable cellulases,

including CBH (Leatham, 1985; Lee et al., 2001;

Pereira Júnior et al., 2003). L. edodes produces at

least two forms of cellobiohydrolase (CEL7A and

CEL6B). Genes encoding each of these enzymes

were cloned from L. edodes grown on a wood

substrate using a PCR based strategy with

degenerate primers directed at the cellulose-binding

domain (Lee et al., 2001). On the basis of

nucleotide sequence analysis it was shown that

cel7A encodes a 516-amino acid (aa) protein that

belongs to glycosyl hydrolase family 7 and has

sequence similarities to CBHI genes from other

fungi. Whereas cel6B gene encodes a 444 aa

protein that belongs to glycosyl hydrolase family 6

and has sequence similarities to CBHII genes from

other fungi. However, both fungal CBHs genes

have not been previously expressed and

characterized.

In this paper we report the cloning of a L.

edodes cel7A gene and its successful expression in

a heterologous host, E. coli.

Materials and methods

Materials

Restriction enzymes, T4 DNA ligase and Taq DNA

polymerase were purchased from Fermentas Life

Sciences (St. Leon-Rot, Germany). Plasmid DNA

was isolated using High Pure Plasmid Isolation Kit

and DNA fragments were purified using Agarose

Gel DNA Extraction Kit (Roche Diagnostics

GmbH. Mannheim, Germany). All other chemicals

and reagents were analytical grade and were

supplied by Sigma-Aldrich Corp (St. Louis, MO

USA). The expression vector pET11d was from

Invitrogen, USA.

Strains and culture conditions

L. edodes strain N127 was obtained from collection

of Kazakh National Agrarian University (Almaty,

Kazakhstan). L. edodes strain N127 was cultured at

room temperature on poplar sawdust blocks. From

sawdust blocks the mycelium was harvested with a

scalpel. The mycelium samples were pooled and

frozen in liquid nitrogen and stored at -80 0 C until

further processing. Escherichia coli DH5a were

used as a host for plasmid propagation. E.coli

Rosetta (DE3) (Invitrogen, USA) was used as a

host for expression of the recombinant CEL7A.

Total RNA extraction and RT-PCR

Total RNA extraction of L.edodes and RT-PCR

was performed as described (Sambrook et al.,

1989). Total RNA isolated as described above was

used as a template for RT reactions. Using the

sequence of the L. edodes cel7A cDNA available in

the GenBank database (GenBank accession number

AF411250), the forward primer, сel7A Dir: 5’-

GATCACCATGGTCCGAACAGCAGCTCTCCT

CT-and the reverse primer, сel7А Rev: 5’-

CTAGGATCCCTACAAACATTGACTGTAGTA

AGG-3’ were designed and used for RT-PCR. The

underlined bases in primers are the sites of

restriction enzymes NcoI and BamHI, respectively.


Construction of expression vector and protein

expression

The PCR product and pET11d vector were doubledigested

by NcoI and BamHI at 37 0 C according to

the protocol. The digested products were ligated by

T4 ligase at 4 0 C for overnight. The recombinant

vector pET-11d/cel7A was transformed into E.coli

Rosetta (DE3) competent cells and the

transformants were selected on LB plates with

ampicillin. The different resistant transformants

were picked and inoculated into 20 mL LB medium

with 200 μg/ml ampicillin and grown at 37°C in a

shaking incubator (150 rpm) for 12 to 16 h. Then,

the incubation was continued in 1L LB medium.

When the culture grown at 30°C reached an

OD600=0.4–0.6, isopropyl-D-thiogalactopyranoside

(IPTG) was added to a final concentration of 0.2

mM to induce expression. After incubation cells

were harvested by centrifugation at 6000 rpm for 5

min at 4°C. Cells were resuspended in storage

buffer containing 20 mM HEPES-KOH pH 7.6, 40

mM NaCl. The cells were lysed by successive

cycles of freezing (liquid nitrogen) and thawing by

sonication for 20 s at half power (14 mA) until a

clear cell lysate was obtained. This mix was used as

crude protein extracts or alternatively the sonicated

mixture was centrifuged at 14 000 g for 10 min to

remove cell debris, and this cleared supernatant

fluid was used as crude proteins extracts. The crude

protein extracts and cellular fractions regarded as

crude enzyme extracts were used for identifying

CEL7A expression by SDS-PAGE, Western

blotting and enzyme analysis.

Preparation of anti-CEL7A antibodies and Western

blotting

The enzyme sample (preincubated with Complete

EDTA-free Protease Inhibitor Cocktail) was

electrophoresed on a 10% (w/v) polyacrylamide gel

contaning 0.1% (w/v) SDS. The gel was stained

with Coomassie Brillant Blue R-250 and the major

protein band was excised from the gel and used as

antigen. The gel piece was homogenized with

liquid nitrogen and emulsified in Freund’s complete

adjuvant (Sigma) and injected subcutaneously into

a rabbit. The same amount of booster injections

was given every two weeks using incomplete

Freund’s adjuvant. The rabbit was bled before the

first injection (preimmune serum) and then one

week after the last booster to obtain immune serum

(anti-CEL7A antibodies). Western blots were

Expression of L. edodes cel7A gene in E.coli 55

performed by standard protocols using 1:400 anti-

CEL7A polyclonal antibody diluted in blocking

buffer and 1:10000 horseradish peroxidaseconjugated

secondary antibody.

Mass spectrometry

The target bands on SDS-PAGE gels were excised

and subjected to in-gel digestion with trypsin

followed by peptide mass fingerprinting by matrix

assisted laser desorption ionization time-of-flight

mass spectrometry (MALDI-TOF) using a

MALDI-TOF-TOF spectrometer, Ultraflex II

(Bruker). Spectra from all experiments were

converted to DTA files and merged to facilitate

database searching using the Mascot search

algorithm v2.1 (Matrix Science, Boston, MA)

against the non-redundant protein sequences of

GenBank (National Center for Biotechnology

Information).

Protein and enzyme assays

The protein content was determined by Bradford

method (Bradford, 1976) Enzyme activity was

assayed with Avicel, filter paper,

carboxymethylcellulose (CMC) as a substrate by

measuring the amount of reducing sugar according

to the Nelson-Somogyi method (Somogyi, 1952;

Nelson, 1944). The reaction mixture consisted of

50 mM sodium phosphate buffer (pH 7.0), 1%

(w/v) of CMC or Avicel or 50 mg of filter paper

(1x 6 cm) and an appropriate amount of enzyme.

The generated reducing sugar was quantified at 540

nm and using D-glucose as a standard for

spectrophotometric estimation. One unit of

cellobiohydrolase activity was defined as the

amount of enzyme that releases 1 μM of reducing

sugar per minute per mg of total protein under the

assay conditions.

When p-Nitrophenyl β-D-lactopyranoside

(pNP-Lac) and p-Nitrophenyl β-D-cellobioside

(pNP-Cel) were used as a substrate, assays were

performed in 500 μl reaction volume in 50 mM

sodium phosphate buffer (pH 7.0) containing 8 mM

of pNP-Cel or pNP-Lac with appropriate amounts

of enzyme. The reaction was terminated by the

addition of 2 ml of 1 M Na2CO3 after 1 h

incubation. One unit of enzyme activity was

defined as the amount of the enzyme that produced

the equivalence of 1 μM pNP at the optimal

conditions in 1 min per 1 mg of total protein.


56 Sabira TAIPAKOVA et al.

Results

To isolate a gene coding CEL7A, we designed two

oligonucleotide primers based on the previously

published sequence (Lee et al., 2001). First, PCR

was done using genomic DNA of L. edodes N127

as a template. PCR product showed a clear band

about 2000 bp (Figure 1B).

In the following experiments we amplified

cel7A cDNA by reverse transcriptase (RT) PCR

starting from total RNA. High quality RNA was

isolated from mycelium of L. edodes grown on the

poplar sawdust using the CTAB-based extraction

method. RNA examined by electrophoresis on

0.8% agarose gel showed two bands corresponding

to 18S and 25S rRNA (Figure 1A), that little or no

RNA degradation occurred during the isolation.

The A260/A280 and A260/A230 absorbance ratios

were 1.9 and 2.0, respectively. This indicates low

contamination by protein substances and secondary

metabolites in the isolated RNA samples. In

general, the RNA obtained was of high quality and

integrity. Total RNA prepared as above was

subjected to RT-PCR with the same primers. A

single DNA band about 1551 bp in size was

amplified (Figure 1B). The 1551 bp DNA fragment

was ligated into pET11d vector and propagated in

E.coli. The clones were sequenced from both

directions.

Figure 1. A) Extraction of total RNA from L. edodes N127, B) PCR amplification of cel7A gene. M: marker

(bp), Lane1: Genomic DNA as template, Lane2: total RNA as template.

Determination of the nucleotide sequence

revealed complete coincidence with the nucleotide

sequence of the cel7A gene L. edodes strain

Stamets CS-2, that was published earlier (Lee et al.,

2001).

The blastp results in Table 1 showed that the

deduced amino acid sequence of 516 amino acids is

identical with L. edodes cellobiohydrolase I amino

acid sequence and showed high homology to other

fungal CBHI enzymes belonging to glycosyl

hydrolases family 7: 99% identity to cellulase of

Irpex lacteus, 96% to cellobiohydrolase I of

Schizophyllum commune and 95% to

cellobiohydrolase I–II of Volvariella volvacea.

Results in this study show that amino acid

sequence deduced from the nucleotide sequence of

cel7A is homologous to other CBHs. However,

until now the cel7A gene of L. edodes

cellobiohydrolase has not been expressed in

bacterial and yeast systems. Therefore, it was

needed to be determined if it really codes a CBH.

In order to confirm that the cel7A gene encodes

a cellobiohydrolase we used E. coli Rosetta (DE3)

expression system. The synthesized by RT-PCR

1551bp fragment was digested with restriction

endonucleases NcoI and BamHI and cloned in the

pET11d plasmid with retention of the reading

frame. The inserted fragment was tested for the

absence of mutations by sequencing. The resulting


plasmid pET11d/cel7A was then used to transform

E. coli Rosetta (DE3).

Table 1. The significant matches of blastp analysis for cDNA of L.edodes N127

Expression of L. edodes cel7A gene in E.coli 57

Genbank accession Organism and gene Score E value Identity

AAK95563.1

Lentinula edodes cellulase CEL7A

mRNA

1043 1043 100%

BAA76365.1 Irpex lacteus cellulase mRNA 639 639 99%

AAX55505.1

Schizophyllum commune H4-8

glycoside hydrolase family 7 protein

586 625 96%

AAT64007.1

Volvariella volvacea

cellobiohydrolase I-II mRNA

The transformation of competent E.coli cells

with the ligation products of the pET11d vector and

the L. edodes cel7A gene amplicon yielded over 47

colonies, among which 4 individual clones were

selected. All the selected colonies were assayed by

PCR and restriction analysis for the presence of the

recombinant plasmids carrying the cel7A gene.

Plasmid DNAs were isolated and purified

according to the protocol of High Pure Plasmid

Isolation Kit. Plasmid DNAs were digested by NcoI

and BamHI, followed by agarose gel

electrophoresis. From Figure 2 we can see that

plasmid DNAs was cut into two fragments, one

Figure 2. Verification of the recombinant plasmid

pET11d/cel7A digested by Nco I and BamHI M -

Marker (bp); 1-4 Clones

647 681 95%

about 1551 bp corresponding to cel7A gene, and the

other about 5674 bp corresponding to the vector.

We amplified the cel7A gene by PCR, using

transformant plasmid DNA as a template and gene

specific primers. The fragment detected by agarose

gel electrophoresis corresponded in length to the

cloned cel7A gene (Figure 3). Detection of the PCR

product corresponding in length to the cloned cel7A

gene sequence indicated that the analyzed colonies

contained plasmids carrying the relevant gene. As a

result of screening, we identified four colonies

carrying the recombinant plasmids.

Figure 3. Verification of the recombinant plasmid

pET11d/cel7A M - Marker (bp); 1- 4 Clones


58 Sabira TAIPAKOVA et al.

Synthesis of CEL7A protein was assessed in

recombinant E. coli. Cell lysate of an E. coli

expressing the cel7A gene was analyzed by SDS-

PAGE and Western blotting using polyclonal anti-

CEL7A antibodies.

After induction with IPTG for 4-12 hours the

cells were lysed and the protein samples were

prepared for SDS-PAGE by boiling in 2x sample

buffer. 53,5kDa and 49 kDa protein bands from

crude extracts of cells harboring the plasmid

pET11b/cel7A were shown on SDS-PAGE (Figure

4A), whereas no similar protein bands from crude

extracts of cells harboring the plasmid pET11d

were detected (Figure 4A). In Figure 4B, the SDS-

PAGE gel was transferred to a PVDF membrane

and probed with anti-CEL7A polyclonal antibody.

Western blot analysis revealed a major protein

band of 53,5-kDa specific to CEL7A in the crude

extracts of the cells harboring the plasmid

pET11d/cel7A, but not pET11d (Figure 4B). To

validate the identity of these proteins to CEL7A,

the polypeptides were excised from the gel,

subjected to trypsin digestion and MALDI-TOF-

MS analysis. MASCOT search results revealed a

top score of 659 for CBHI, where probability based

mowse score > 44 are significant and indicate

identity or extensive homology (p


Expression of L. edodes cel7A gene in E.coli 59

Figure 5. Effects of pH and temperature on the activities of recombinant CEL7A. A) pH profile was

determined by incubating the enzyme at 50 0 C for 1 h at varying pHs (sodium acetate buffer pH 4-6, sodium

phosphate buffer pH 6-7 and glycine buffer pH 9). B) Temperature profile was determined by incubating the

enzyme in 0.05M sodium phosphate buffer pH 7 for 1 h at different temperatures.

Crude extract of an E. coli expressing the cel7A

gene was examined for its ability to hydrolyze

various cellulosic substrates at pH 7 and 50 0 C

(Table 2). It could hydrolyze Avicel, Filter paper,

pNP-Lac and pNP-Cell, but activity towards

carboxymethylcellulose (CMC) was much lower

compared to Avicel and Filter paper. These results

strongly indicate that CEL7A has cellobiohydrolase

activity.

Table 2. Activities of the recombinant enzyme toward different cellulosic substrates at pH 7.0 and 50 0 C

Substrate

Activity

(U/mg total protein)

Avicel 21.4 ± 1.4

Filter paper 23.2 ± 0.3

CMC 2.9 ± 0.5

pNP-Lac 4.1 ± 0.08

pNP-Cell 3.8 ± 0.1


60 Sabira TAIPAKOVA et al.

Discussion

CBH enzymes are key components in fungal

cellulase systems, and their functional activity is

critical for consolidated bioprocessing for

bioethanol production. For example, CBHs make

up to 80% of the total mass for the T. reesei system,

and CBH plays a particularly important role,

making up 60% of the total mass (van Zyl et al.,

2007). A number of cellulase genes from bacteria

and fungus have been cloned and expressed in yeast

or E. coli (Qiao et al., 2004; Hong et al., 2001,

Hong et al., 2003). However fungal and bacterial

endoglucanase (EG) production in recombinant

strains was more successful than CBH production

(van Zyl et al., 2007). This is not surprising

considering that EG enzymes usually have specific

activities of 2 to 3 orders higher in magnitude on

synthetic and amorphous cellulose substrates, such

as carboxymethylcellulose (CMC), in comparison

to CBHs. Thus it is easier to measure the presence

of even small amounts of heterologous EG

compared to CBHs.

Expression of fungal cellulases in E. coli as

non-glycosylated forms has been attempted with

limited success. Only a few cellulose genes, such as

cbhI and egl3 of the well-studied fungus T. reesei,

were expressed in E. coli, but the productivity of

cellulases was very low (Ekino et al., 1999).

In this study, we have successfully expressed the

cel7A gene of L. edodes in E. coli under the control

of the T7 promoter. SDS–PAGE and Western

blotting analysis showed that CEL7A constitutes a

major protein produced in E. coli with a molecular

weight of 53,5 kDa, which agrees with the

predicted size from the deduced amino acid

sequence. Amino acid sequencing of the putative

recombinant protein by MALDI-TOF and its

analysis using NCBI BLAST indicated that the

enzyme contained putative conserved domains of

glycosyl hydrolase family 7.

As shown in Figure 4A plasmid vector

pET11d/cel7A expresses two additional bands

which in SDS-PAGE migrate as 53 kDa and 49

kDa proteins. Since the difference in molecular

weight between these two proteins is approximately

5 kDa we may suggest that the CEL7A protein was

partially degraded possibly during cell lysis. The

cel7A enzyme contains 430 aa catalytic domain and

57 aa C-terminal cellulose-binding domain (CBD)

and serine/threonine-rich linker sequence (Lee et

al., 2001). We may speculate that the proteolytic

degradation of 57 aa C-terminal CBD and linker

sequence results in truncated 49 kDa protein.

Indeed, western blot analysis confirms the presence

of low molecular weight CEL7A proteins which

migrate below the full-length recombinant protein

in SDS-PAGE suggesting proteolytic degradation

of CEL7A during expression and/or cell lysis.

Furthermore we hypothesize that the linker region

of E. coli-expressed CEL7A might be more

susceptible to proteolysis as compared to

glycosylated form of this protein expressed in yeast

cells (unpublished observation). It seems likely that

glycosylated linker region in native CEL7A of L.

edodes protects the enzyme from proteolytic

degradation nevertheless further investigations are

required.

The substrate specificities indicated that CEL7A

contains cellobiohydrolase activity since it could

hydrolyze crystalline cellulose (Avicel, Filter

paper). Activity of cloned enzyme toward CMC

was much lower than to Avicel and filter paper.

These data are consistent with the established

results that fungal CBH proteins have very limited

action on substituted cellulose such as CMC or

hydroxyethylcellulose (Basiria and Mishra, 1989;

Kanokratana et al., 2002). Since most of the fungal

cellulases are glycoproteins, the modification of the

native protein by glycosylation has been reported to

play an important role in synthesis, secretion, and

stability of extracellular cellulases (Basiria and

Mishra, 1989). Here we have demonstrated that

CEL7A synthesized in E. coli without

glycosylation has an enzymatical activity. This

indicates that glycosylation is not necessary for

enzymatic activity of CEL7A.

In summary, a gene encoding CEL7A was

successfully isolated from L. edodes strain N127

using RT-PCR technique. The determined

nucleotide sequence is completely corresponded to

the nucleotide sequence of the cel7A gene of L.

edodes strain Stamets CS-2 which was described

previously (Lee et al., 2001). The enzyme was

successfully expressed in E. coli in an active form.

Enzymatic properties of CEL7A were also

determined. The optimal temperature for enzyme

activity was 50 0 C and the optimal pH was 7.0.

Acknowledgement

This work was supported by grant from National

Centre for Biotechnology of the Republic of

Kazakhstan.


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Journal of Cell and Molecular Biology 9(1): 63-68, 2011 Research Article 63

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Investigation of the MMP1 and MMP3 promoter polymorphisms in

temporomandibular joint disorder

Necati TASKIN 1 , Korkut ULUCAN 2,* , Guhan DEGIN 3 , Arzu AKCAY 4 , Berfin

KARATAS 3 and Teoman AKCAY 5

1

Istanbul Education and Research Hospital, Department of Pediatrics, Istanbul, Turkey.

2

Marmara University, Faculty of Dentistry, Department of Medical Biology and Genetics, Istanbul, Turkey.

3

Marmara University, Faculty of Dentistry, Department Of Oral And Maxillofacial Surgery, Istanbul,

Turkey.

4

Bakirkoy Maternity and Child Education and Research Hospital, Pediatric Hematology, Istanbul, Turkey.

5

Sisli Etfal, Education and Research Hospital, Istanbul, Turkey.

(* author for correspondence; korkutulucan@hotmail.com)

Received: 24 May 2011; Accepted: 16 June 2011

Abstract

Matrix metalloproteinases 1 and 3 (MMP1 and MMP3) are metal dependent endopeptidases responsible for

hydrolyzing extracellular matrix molecules, and also have important roles during the matrix destruction in

temporomandibular joint (TMJ) degeneration. In the present study, we aimed to investigate the relation

between characterized promotor polymorphisms of MMP1 and MMP3 genes and TMJ in a Turkish

population. 35 TMJ patients and 50 healthy controls were recruited to the study. MMP1 and MMP3 promotor

polymorphisms were examined by PCR-RFLP methodology. For MMP1 polymorphism; 8, 14 and 13 of 35

patients were 1G/1G, 1G/2G and 2G/2G, respectively. For the same polymorphism; 11, 28, 11 of 50 controls

were 1G/1G, 1G/2G and 2G/2G, respectively. 6, 14, 15 of 35 patients were 5A/5A, 5A/6A and 6A/6A,

respectively, for MMP3 genotype. Of the 50 controls, 8, 23, 19 were 5A/5A, 5A/6A and 6A/6A, respectively.

Comparison of the polymorphism between healthy subjects and patients yielded no statistically significant

difference (p0,05). The examined polymorphisms didn’t have major effects on the TMJ formation. With the

increasing number of the both groups and classification of the patients with their pain level and disease

severity will give us more informative data about the polymorphism of the genes and TMJ formation.

Keywords: Temporomandibular joint disorder, matrix metalloproteinase, MMP1 promotor polymorphism,

MMP3 promotor polymorphism, orofacial pain.

Temporomandibular bağlantı bozukluklarında MMP1 ve MMP3 promotor polimorfizmlerinin

incelenmesi

Özet

Matriks metaloproteinaz 1 ve 3 (MMP1 ve MMP3) ektraselüler matriks moleküllerini parçalayan metal

iyonlarına bağımlı endopeptidazlardır ve temporomandibular bağlantı bozukluklarında (TBB) hücrelerarası

matriks yıkımında önemli rolleri bulunmaktadır. Bu çalışmada 35 TBB hastasında, hastalığın oluşumunda rol

oynayan MMP1 ve MMP3 genlerindeki promotor polimorfizmlerinin ilişkisini saptamayı amaçladık. Bu

nedenle 35 hasta ve 50 sağlıklı kontrolü çalışmaya dahil ettik. Polimorfizm analizleri standart PCR-RFLP

metodu ile gerçekleştirildi. MMP1 polimorfizm sonuçlarına göre 35 hastanın 8’i 1G/1G, 14’ü 1G/2G ve 13’ü

ise 2G/2G, 50 kontrolün ise 11’i 1G/1G ve 2G/2G, 28’i ise 1G/2G genotiplerinde bulunmuştur. MMP3

polimorfizm sonuçları ise hasta grubunda 6’sı 5A/5A, 14’ü 5A/6A ve 15’I 6A/6A, kontrol grubunun ise 8’i

5A/5A, 23’ü 5A/6A ve 19’u ise 6A/6A genotipinde bulunmuştur. Kontrol grubu ve hasta gruplarının

istatistiksel olarak karşılaştırıldıklarında anlamlı bir fark bulunamamıştur. Bu sonuçlara dayanarak analiz

edilen polimorfizmler ve TBB arasında çalışılan populasyonda bir ilişki saptanamamıştır. Hasta ve kontrol

sayısının artırılması ve TBB’li hastaların ağrı ve hastalığın ilerleme durumlarına göre sınıflandırmalarının

yapılması ile daha anlamlı sonuçların alınacağına inanmaktayız.


64 Necati TASKIN et al.

Anahtar Sözcükler: Temporomandibular bağlantı bozuklukları, matriks metaloproteinaz, MMP1 promotor

polimorfizmi, MMP3 promotor polimorfizmi, orofasyal ağrı

Introduction

Temporomandibular joint disorders (TMJD) are the

orofacial problems involving masticatory

musculature, the temporomandibular joint (TMJ),

and related structures. This disorder frequently

causes chronic pain in the orofacial region

(Dworkin et al., 2002). Degeneration of articular

cartilage, capsule, ligaments and synovial

membrane are the major clinical outcomes of TMJ.

These degenerative changes in the tissues are slow

at the beginning but with the progression of the

disease, irreversible changes occur in the joint

(Stegenga, 2001).

The main characteristic change in TMJ is the

alteration of the equilibrium between synthesis and

degradation of extracellular matrix (ECM)

collagens, especially by proteolysis (Planello et al.,

2011). Accumulation of the hydrolyzed proteins,

catabolites formed by proteolytic activities,

inflammatory modulators like cytokines and nitric

oxide, matrix metalloproteinases (MMPs) found in

the synovial fluid are the reason of inflammatory

process of TMJ (Dijkgraaf et al,1995; Srinivas et

al., 2001; Kubota et al., 1998). On the other hand,

MMPs are considered to be the key proteolytic

enzymes for the destruction of ECM in TMJ

(Wadhwa and Kapila, 2008).

MMPs are metal dependent endopeptidases that

are capable of cleaving most, if not all of the

constituents of the extracellular matrix including

collagen, fibronectin, and proteoglycans (Astolfi et

al., 2006; Plenello et al., 2011). They are secreted

as pro-enzymes, inactive state of the enzyme, and

activated after the cleavage of N- terminal domain

(Visse and Negasse, 2003) by other proteinases.

Also several promotor polymorphisms were

reported to regulate transcription activity and these

have been associated with degenerative diseases

like periodontitis and arthritis (Barlas et al., 2009;

Astolfi et al., 2006). These promotor variations can

influence the basal and inducible levels of MMP

expression. An insertion/ deletion variation of

MMP1 promotor at position -1607 creates two

alleles, 1G having one guanin and 2G, having two

guanines at this position (Rutter et al., 1998).

MMP3 also has a functional variation at -1612

which give rise to 5A and 6A alleles, according to

repeat number (de Maat, 1999). MMP3 degrades a

wide range of extracellular matrix proteins and

should be regarded as a potential interest in DNA

polymorphism- TMJ studies.

Of the big family of MMPs, MMP1 and MMP3

are synthesized and exocytosed in fibroblasts and

chondrocytes, increasing the possibility of their

probable role in TMJ. In this study, we aimed to

identify the effect of 1G/2G MMP1 and 5A/6A

MMP3 polymorphisms in TMJ formation.

Materials and methods

Study Group

35 TMJ patients and 50 healthy subjects with no

history of any genetically based disease were

enrolled to the study. All the participants were

informed in detail about the study. The participants

were unrelated Turkish people from the eastern

region of Turkey. The TMJ degeneration group

included 30 individuals of both genders and

undergone MRI scans in Marmara University,

Faculty of Dentistry, Department of Oral and

Maxillofacial Surgery. The individuals who have

shown at least one sign of degenerative process in

any of the mandibular condyles were included to

the TMJ group, according to their MRI scans.

Amplification and Genotyping of SNPs

Genomic DNA isolated from peripheral blood by

using a commercial kit (Roche, Germany) by

following the instruction guide. For MMP1

analysis, PCR reactions were carried out in a

volume of 50 µl containing 50–100 ng DNA

template in 10 mM Tris–HCl (pH 8.0), 50 mM

KCl, 1.5 mM MgCl2, 1 mM each of dNTPs, 1.0 U

Taq DNA polymerase (Fermentas, Germany) and

1.0 mM of each primer previously described

(Rutter 98). The conditions of PCR amplification

were as follows: a denaturation step at 95˚C for 5

min followed by 38 cycles at 95˚C for 1 min, 55˚C

for 1 min, 72˚C for 1 min, a final extension at 72˚C

for 10 min and stop at 4˚C. For MMP3 analysis,

PCR reactions were carried out in a volume of 50


µl containing 50–100 ng DNA template in 10 mM

Tris–HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 1

mM each of dNTPs, 1.0 U Taq DNA polymerase

(Fermentas, Germany) and 1.0 mM of each primer

previously described (Ye et al., 1996) The

conditions of PCR amplification were as follows:

denaturation step at 94˚C for 5 min followed by 35

cycles at 94˚C for 1 min, 65˚C for 45 s, 72˚C for 1

min, a final extension at 72˚C for 7 min and stop at

4˚C.

In order to complete genotype analyses, amplicons

MMP1 and MMP3 promotor polymorphisms in TMJ disorder 65

of MMP1 and MMP3 were digested by XmnI (New

England Biolabs) and Tth111I (New England

Biolabs) restriction enzymes, respectively, as

previously described (Planello et al., 2011). 1G

allele of MMP1 was digested to 89 and 29 bp

whereas 2G allele was not digested and remained in

its original amplicon length, 118 bp. 5A allele of

MMP3 yielded 97 and 33 bp products by digestion,

6A allele was not digested and maintained the

original length of 130 bp. All the visualization

processes were carried out by 12% polyacrylamide

gel stained with etidium bromide (Figure 1)

Figure 1. RFLP analyses of MMP1 and MMP3 genes, M: Molecular marker (Fermentas, Germany), 2G/2G:

Homozygous genotype of 2G allele for MMP1, 1G/2G: Heterozygous genotype for MMP1, 1G/1G:

Homozygous of 1G allele for MMP1, 5A/5A: Homozygous genotype of 5A allele for MMP3, 5A/6A:

Heterozygous genotype for MMP3, 6A/6A: Homozygous genotype of 6A allele for MMP3.

Statistical analysis

Statistical analysis for identifying the differences

between TMJ and control groups was performed

with Chi-square test by using SPSS 18.0. p0,05

was accepted as statistically significant whereas

values higher than 0,05 was not.

Results

For the MMP1 genotypes, 8 of the 35 patients and

11 of the 50 controls were 1G/1G, 14 patients and

28 were 1G/2G and 13 patients and 11 controls

were 2G/2G, respectively. Genotypes of MMP1

polymorphisms and p- values were summarized in

Table 1. When we compare two groups for MMP1

genotypes, we could not find any statistically

significant association, all of the p- values are

0,5%.

For the MMP3 genotypes, of the 50 controls, 8

were 5A/5A, 23 were 5A/6A and 19 were 6A/6A,

respectively. 6 of the 35 patients were 5A/5A, 14

were 5A/6A and 15 were 6A/6A. Genotypes of

MMP1 polymorphisms were summarized in Table

2. Like in MMP1 genotypes, we could not find any

statistically significant association in patient and

control group, the p- values are 0,5%.


66 Necati TASKIN et al.

Discussion

Table 1. Genotype distribution and OR of MMP1 polymorphism in control and TMJ groups

Genotypes

Control Group

(n=50)

TMJ Group

(n=35)

p- value

OR (95% CI)

Control/TMJ Group

1G/1G 11 8 0,56 0,952 (0,338-2,678)

1G/2G 28 14 0,14 1,909 (0,794-4,589)

2G/2G 11 13 0,1 0,541 (0,206-1,422)

Table 2. Genotype distribution and OR of MMP3 polymorphism in control and TMJ groups

Genotypes

Control Group

(n=50)

TMJ Group

(n=35)

p- value

OR (95% CI)

Control/TMJ Group

5A/5A 8 6 0,889 0,921 (0,289-2,935)

5A/6A 23 14 0,659 1,278 (0,532-3,067)

6A/6A 19 15 0,653 0,817 (0,339-1,970)

MMPs are secreted by pro- enzymes and activated

after the removal of their N- terminal domain

(Visse and Negase, 2003). For MMP1 and MMP3

enzymes, there is an additional activation process,

transcriptional regulation. Promoter 1G/2G

polymorphism at the -1607 position of MMP1 gene

and 5A/6A promoter polymorphism of MMP3 were

reported to influence gene expression levels of

related MMPs, and associate these SNPs with

degenerative diseases (Ye et al., 1996; Barlas et al.,

2009; Astolfi et al., 2006).

Rutter et al. reported that 2G allele of

MMP1gene increases the gene expression and

mRNA levels of MMP1 gene (Rutter et al., 1998).

This allele creates a binding site for Ets

transcription factor family, which promotes 37-

fold in vitro increase of transcription activity. Also

another study, in transgenic mice, showed the in

vivo effect of 2G allele on transcription activity

(Coon et al., 2009).

We could not observe a significant difference in

1G/2G SNP of MMP1 gene between TMJ patients

and control group. Our findings are in accordance

with the study reporting no association between

MMP1 -1607 polymorphism and rheumatoid

arthritis progression, degenerative disease including

systemic joints. Although there are some reports

indicating the molecular difference of systemic

joints and TMJ, the exact mechanism of

degenerative effect, in which MMP1 is in charge, is

still unclear (Wadhwa and Capila, 2008). There are

also other studies that our findings are not in

agreement. These studies reported the association

of 2G allele and certain types of cancer (Peng et al.,

2010), periodontitis (de Souza et al., 2003) and

TMJ (Planello et al., 2011). The latter associated

the 2G allele and TMJ degeneration and showed

that 2G/2G individuals have a 2,47 time higher

probability of developing TMJ when compared to

1G/1G and 1G/2G individuals. At the same study,

authors couldn’t find any allelic association,

claiming only 2G/2G individuals are in risk of

developing TMJ, individuals without 2G allele are

protected from the disease. Small sample size,

heterogenetic nature of genetic diseases and

probable multifactorial background of TMJ may be

the reasons of our results that are not in agreement

with previous mentioned studies.

Like MMP1, MMP3 has an SNP at position -

1612 which shows variation in enzyme activity. 5A

allele in this position of the gene has a greater

activity to 6A allele, in vitro (Ye at al., 1996).

Allele 5A is associated to increase the risk of acute

myocardial infarction (Terashima et al., 1999) and

in another study, 5A allele has been suspected to

atherosclerosis (Ye, 2000). MMP3 has an

accumulative activity during the initial phase of

TMJ, degrades various types of matrix proteins and

activates other MMPs like MMP1, MMP8 and

MMP13, leading to an increase in tissue

degradation (Planello et al., 2011; Fujita et al.,

2009). We couldn’t find any association with the

polymorphism and TMJ, like Planello et al (2011).

The exact activity of MMP3 in tissue degradation is

not clear and may be the SNP examined has a

limited influence on the enzyme activity.


There are number of factors which affect

unexpected results on polymorphism- disease

association studies. Ethnicities of the races,

different allele frequencies between populations,

classifications of patient groups are only some of

them. Also the metabolism or the activity processes

of the examined molecules are important. In our

study, MMP1 and MMP3, they are regulated not

only by transcription, but also activation of the pro-

enzyme and inhibitions of enzymes by their tissue

inhibitors (TIMPs) (Astolfi et al., 2006). There are

other factors that influence secretion, cell surface

localization and clearance of the degraded MMPs.

These regulation factors may help us to explain

why our results didn’t support the information on

the literature. Astolfi et al. (2006) also supported

the idea that increase in mRNA levels may not

necessarily lead to increased levels of MMPS.

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MMP1 and MMP3 promotor polymorphisms in TMJ disorder 67

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Journal of Cell and Molecular Biology 9(1):69-74, 2011 Research Article 69

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Lead induced alterations in blood cell counts and hemoglobin

during gestation and lactation in Swiss albino mice

Isha BARBER, Ragini SHARMA*, Sheetal MOGRA, Khushbu PANWAR and Umesh

GARU

Environmental and Developmental Toxicology Research Lab, Department of Zoology, M. L. S. University,

Udaipur- 313001 Rajasthan (INDIA)

(* author for correspondence; taurasragini@yahoo.com)

Received: 31 March 2011; Accepted: 20 June 2011

Abstract

Although a number of studies in animal models have shown changes in hemoglobin content and blood cell

counts after lead administration during gestation and lactation, lead induced hematological changes are not

well established. In the present study, hemoglobin content and blood cells of normal and lead exposed

pregnant and lactating Swiss mice were compared in which selected pregnant females were treated with lead

acetate by gavages (266.66, 533.33, 1066.66 mg/kg BW) during gestation and lactation. Animals were

exposed to heavy metal lead orally from day 10 of gestation to 3 rd weeks of lactation. Hemoglobin content

and blood cell counts were examined on 17 th day of gestation and on 1 st , 7 th , 14 th and 21 st day after birth. The

results indicated that in lead intoxicated pregnant females, hemoglobin content and red blood cell (RBC)

counts decreased and white blood cell (WBC) counts increased. From the results of above study it can be

concluded that high levels of lead exposure during gestation and lactation can severely damage heme

synthesis and alter the number of RBC and WBC.

Keywords: Lead acetate, Swiss albino mice, hemoglobin, RBC, WBC.

Swiss albino farelerde gebelik ve süt verme süresince kurşun ile tetiklenen

hemoglobin ve kan hücreleri sayılarındaki değişimler

Özet

Pek çok hayvan modeli çalışmalarının gebelik ve süt verme boyunca kurşun yüklemesi sonrasında kan

hücrelerinin sayıları ve hemoglobin miktarlarındaki değişiklikleri göstermesine rağmen, kurşun ile tetiklenen

hematolojik değişiklikler iyi belirlenememiştir. Bu çalışmada normal ve kurşuna maruz kalan hamile ve süt

veren Swiss farelerin hemoglobin miktarı ve kan hücreleri gebelik ve emzirme sırasında gavajla kurşun asetat

(266.66, 533.33, 1066.66 mg/kg BW) uygulanan seçilmiş hamile dişilerinkiyle karşılaştırılmıştır. Hayvanlar

gebeliğin 1. gününden süt vermenin 3. haftasına kadar oral olarak ağır metal kurşuna maruz bırakılmıştır.

Hemoglobin miktarı ve kan hücresi sayıları gebeliğin 17. günü ve doğumdan sonraki 1., 7., 14., ve 21.

günlerde incelenmiştir. Sonuçlar, kurşun ile zehirlenmiş gebe dişilerde hemoglobin miktarının ve kırmızı kan

hücresi (RBC) sayılarının azaldığını ve beyaz kan hücrelerinin (WBC) arttığını göstermiştir. Yukarıda

belirtilen sonuçlardan, gebelik ve süt verme sırasında yüksek miktarda kurşuna maruz kalmanın hem grubu

sentezine ciddi zarar verebileceği ve RBC ve WBC sayılarını değiştireceği sonucuna varılabilir.

Anahtar Sözcükler: Kurşun asetat, Swiss albino fare, hemoglobin, RBC, WBC.


70 Isha BARBER et al.

Introduction

In the modern society, thousands of hazardous

chemicals and heavy metals are being produced and

used in a wide variety of work places all over the

world. Heavy metals are trace metals that are at

least five times denser than water and are taken into

body via inhalation, ingestion and skin absorption.

It should be noted that most of the pathological

conditions in body arise as a result of the exposure

to these injurious substances.

Lead and other heavy metals create reactive

radicals which damage cell structure including

DNA and cell membrane (Flora et al., 2008). Lead

poisoning can cause a variety of symptoms and

signs which vary depending on the individual and

the duration of lead exposure (Karri et al., 2008;

Kosnett, 2005). Gestational lead exposure has many

adverse effects on development; a few of them may

be most pronounced during the first trimester

(Mogra et al., 2009)

Many investigators studied the lower as well as

higher exposure levels to lead. According to

Escribano et al., (1997) 140 mg/ kg BW dose was

an approximate environmental daily-exposure

level. This dose was used as the minimum dose.

Gurber et al. (1997) studied the higher

concentration of lead that was 1000 mg/kg BW

exposure in industrial areas.

The amount of lead in blood and tissues, as well

as the time course of exposure, determines the level

of toxicity (Pearson and Schonfeld, 2003). Blood

often shows pathological changes before the

external signs of poisoning become apparent.

The absorbed lead enters the blood stream

where over 90 percent of it is bound to the red cells

with a biological half life of 25-28 days (Azar et

al., 1975). Toxicological effects of lead have their

origin in perturbation in cell function of various

organ systems. The major biochemical effect of

lead is its interference with heme synthesis which

leads to hematological damage (Awad and William,

1997). Despite several published accounts on

pathophysiological alterations of lead toxicity and

the cure of lead poisoning by sequestering agents

(Royce and Rosenberg, 1993), the approaches are

limited in scope. Therefore the present

investigation was focused to evaluate the changes

in the number of blood cells and Hb content during

pregnancy and lactation followed by comparison of

this data with the high levels of lead intoxication

during gestation and lactation.

Materials and methods

Random breed Swiss albino mice were used for the

present study. Sexually mature male and females

weighing 28-30 gm were put in breeding cages in

the ratio of 1:4 and provided standard diet and

water ad libitum. The cages were checked every

day in the morning and females showing vaginal

plug were isolated. The selected pregnant females

were divided into 4 groups and exposed orally by

lead acetate (266.66, 533.33 and 1066.66 mg/kg

BW) with the help of canula. All the experimental

work was approved by the institutional animal

ethics committee (No.CS/Res/07/759).

(1) Group 1- Control (distilled water only)

(2) Group 2- Exposure of lead acetate (266.66

mg/kg BW) from 10 th day of gestation up to 21 st

day of lactation.

(3) Group 3- Exposure of lead acetate (533.33

mg/kg BW) from 10 th day of gestation up to 21 st

day of lactation.

(4) Group 4- Exposure of lead acetate (1066.66

mg/kg BW) from 10 th day of gestation up to 21 st

day of lactation.

During the respective tenure of experiment,

hemoglobin, RBC and WBC counts of female

Swiss mice were recorded on 17 th day of gestation

and on 1 st , 7 th , 14 th and 21 st days of lactation. Blood

samples for hemoglobin and blood cell counts were

obtained from the tail of each mouse. The tip of the

tail was cleaned with spirit before being cut with a

sharp blade and was not squeezed to avoid dilution

of blood by tissue fluid. The first few drops of

blood were discarded and the blood was diluted, for

cell counting with the help of haemocytometer. The

hemoglobin was estimated by hemoglobinometer.

Number of (RBCs) and (WBCs) were estimated

with haemocytometer adopting the method

described by Dacie and Lewis (1975). The

statistical analysis was performed by using analysis

of variance (ANOVA) for the comparison of data

between different experimental groups.

Results

Introducing lead acetate to female Swiss mice

during gestation and lactation period produced a

significant decrease in the hemoglobin content and

number of RBC. Exposure during the gestation

period produced a significant decrease in WBC

counts at the time of birth whereas number of cells

increase during lactation period.


Data regarding changes in hemoglobin, RBC

and WBC counts among different experimental

groups and the results obtained for hemoglobin,

RBC and WBC are summarized in Tables 1-3. It is

evident from the tables that control animals have

12.5 g/dL Hb, 9.21×10 6 RBC/mm 3 and 8.22×10 3

WBC/mm 3 in their blood on 17 th day of gestation.

As compared to controls, hemoglobin content, RBC

and WBC counts of lead- treated female mice were

significantly decreased at the time of birth, after

introduction of 266.66, 533.33 and 1066.66 mg/kg

BW of lead acetate.

Lead induced hematological changes 71

The results collected for the hemoglobin is

summarized in Table 1.

It is evident that control animals have 11.5,

12.0, 13.1 and 14.0 g/dL Hb in their blood on 1 st ,

7 th , 14 th and 21 st day of lactation, respectively.

Introduction of 266.66, 533.33 and 1066.66mg/kg

BW doses of lead acetate produced a significant

decrease (p


72 Isha BARBER et al.

Table 3. WBC count (×10 3 cells/mm 3 ) in pregnant and lactating female mice treated with lead acetate.

Treatment group

Prenatal

Days

Postnatal

17 1 7 14 21

Control 8.22±3.92 8.07±3.30 8.33±4.30 8.56±3.56 8.86±3.47

Lead acetate (266.66 mg/kg BW) 8.62±5.00* 8.48±1.79* 8.70±5.77 8.71±7.50 9.18±1.25

Lead acetate (533.33 mg/kg BW) 8.98±9.46* 8.62±2.95* 9.10±1.47* 9.20±2.04* 9.31±2.09*

Lead acetate (1066.66 mg/kg BW) 9.26±1.70* 9.00±1.73* 9.17±8.66* 9.35±1.68* 9.46±1.43*

Values were expressed as means ± S.D., values are significantly different (p


elated to major structural and functional changes

of the intimate relation between fetus and mother.

These structural changes are related to implantation

and placentation; and the functional changes to

increasing hypoxia in the crowded condition of the

gravid uterus (Robert and Rosenberg, 1993).

In conclusion, the present study indicates that

lead adversely affects hemoglobin and blood cell

counts. Dose dependent significant decrease in

hemoglobin and RBC counts were observed after

administration of 266.66, 533.33 and 1066.66

mg/kg/body weight doses of lead acetate. However,

WBC counts showed increase in their number.

The results observed in this paper can be used

as a background information for the evaluation of

reproductive toxicity induced by lead during

gestation and lactation in Swiss mice. Moreover,

the mice seem to be a useful animal model for

investigating the mechanisms of alterations in the

blood cells and hemoglobin content which is

reported to occur during pregnancy and lactation in

mice.

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Journal of Cell and Molecular Biology 9(1): 75-82, 2011 Research Article 75

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

The effects of aqueous and ethanolic leaf extracts of Vernonia

amygdalina on some vital organs in adult Wistar rats

Spencer NWANGWU 1 , David Adesanya OFUSORI 2,* , Sunday JOSIAH 1 , Osasere Frank

AMEGOR 3 , Helen NJOYA 1 and Abiodun Oladele AYOKA 4

1 Department of Biochemistry, School of Basic Medical Sciences, Igbinedion University, Okada, Edo-State,

Nigeria

2 Department of Anatomy and Cell Biology, Faculty of Basic Medical Sciences, Obafemi Awolowo University,

Ile-Ife, Osun-State, Nigeria

3 Department of Medical Laboratory Science, College of Health Sciences, Igbinedion University, Okada, Edo

State, Nigeria

4 Department of Physiological Sciences, Faculty of Basic Medical Sciences, Obafemi Awolowo University,

Ile-Ife, Osun-State, Nigeria

(* author for correspondence; davidofus234@yahoo.com

Received: 15 February 2011; Accepted: 20 June 2011

Abstract

The aim of this study was to comparatively determine the effects of aqueous and ethanolic extracts of

Vernonia amygdalina (Asteraceae) leaves, adopting histological procedures on the liver, stomach and kidney

in adult wistar rats.There were four groups of animals: A, B, C and D. Groups A, B and C served as the

treated animals while group D served as the control animals. Each of the groups was further subdivided into

two i.e A1 & A2; B1 & B2; C1 & C2 and D1 & D2. Animals in groups A1, B1 and C1 were administered

orally with aqueous extract of V. amygdalina 100, 200 and 300 mg/kg respectively while animals in groups

A2, B2 and C2 were administered orally with ethanolic extract of V. amygdalina 100, 200 and 300 mg/kg

respectively. The control groups, D1 and D2, received equal volume of normal saline. There were no

significant derangement in the cytoarchitecture of the stomach, liver and kidney. Rather, the cytoarchitecture

of the animals treated with both the ethanolic and aqueous extracts (300mg/kg) of V. amygdalina were better

organized when compared with the control. It was therefore concluded that both ethanolic and aqueous leaf

extracts of V. amygdalina are non-toxic and may possess cytoprotective potential.

Keywords: Vernonia amygdalina, histological procedures, liver, stomach, kidney

Vernonia amygdalina yaprağının sulu ve etanolik özütlerinin erişkin Wistar

sıçanlarının bazı hayati organlarına etkileri

Özet

Çalışmanın amacı, erişkin Wistar sıçanlarda Vernonia amygdalina (Asteraceae) yapraklarının sulu ve

etanolik özütlerinin karaciğer, mide ve böbrek üzerindeki etkilerini histolojik yöntemlerle karşılaştırmalı

olarak belirlemektir. Çalışmada dört grup hayvan bulunmaktadır: A, B, C ve D. D grubu kontrol hayvanları

olarak belirlenirken; A, B ve C grupları muamele edilmiş hayvanları göstermektedir. Her bir grup ayrıca iki

alt gruba bölünmüştür; A1 & A2; B1 & B2; C1 & C2, D1 & D2. A1, B1 ve C1 gruplarındaki hayvanlara

sırasıyla 100, 200 ve 300 mg/kg V.amygdalina sulu özütü oral olarak uygulanırken A2, B2 ve C2’ye ise

sırasıyla 100, 200 ve 300 mg/kg V.amygdalina etanolik özütü oral olarak uygulanmıştır. Kontrol grupları

olan D1 ve D2 eşit hacimde normal tuz almışlardır. Mide, karaciğer ve böbreğin hücresel yapısında belirgin

bir bozulma bulunmamıştır. Bilakis hem sulu, hem de etanolik V. amygdalina (300mg/kg) özütü uygulanan


76 Spencer NWANGWU et al.

hayvanların hücresel yapıları kontrollerle karşılaştırıldığında daha iyi organizedir. Bu nedenle sulu ve

etanolik V. amygdalina yaprak özütlerinin her ikisinin de toksik olmadığı ve hücre koruyucu potansiyele

sahip olabileceği sonucuna varılmıştır.

Anahtar Sözcükler: Vernonia amygdalina, histolojik süreçler, karaciğer, mide, böbrek.

Introduction

Vernonia amygdalina (Del. Asteraceae)

commonly known as “bitter leaf” is a valuable

shrub that is widespread in East and West Africa.

It is 2-5 m tall with petiolate green leaves of about

6 mm diameter (Ojiako and Nwanjo, 2006). In

Nigeria the stem is used as chew-sticks, while the

leaves are being used as a popular vegetable for

soups particularly among the Igbos of Southern

Nigeria, Africa (Ojiako and Nwanjo, 2006). Its

medicinal values for fever, laxative, pile

(haemorrhoids) and gastro-intestinal troubles have

been investigated and reported by the following

authors: (Oliver, 1960; Ainslie, 1973; Kupcham,

1971; Akah and Okafor, 1992; Abosi and

Raseroka, 2003; Huffman, 2003; Izevbigie et al.,

2004). In fact all parts of the plant have been

known to be pharmacologically useful. Oral

administration of the aqueous leaf extract of the

plant was found to relieve pain (Tekobo et al.,

2002) and to lower body temperature (Tekobo et

al., 2002). Atangwho et al. (2010) reported

instantaneous reduction of blood glucose and a

variation in blood glucose similar to that of

insulin-treated rats in animals administered with

ethanol extract of V. amygdalina.

Nutritionally, V. amygdalina is used mainly in

soup making in the tropics and also as an

appetizer and febrifuge (Ijeh et al., 1996; Iwu,

1996, Ojiako and Nwanjo, 2006) and has proven

to be a successful supplement in weaning foods

(Ojiako and Nwanjo, 2006). In Nigeria, as in

other tropical countries of Africa where the daily

diet is dominated by starchy staple foods,

vegetables are the cheapest and most readily

available sources of important proteins, vitamins,

minerals and essential amino acids (Okafor, 1983,

Ojiako and Nwanjo, 2006). The importance of V.

amygdalina in animal nutrition in Nigeria has also

been well documented (Onwuka et al., 1989;

Aregheore et al., 1998; Ojiako and Nwanjo 2006).

Despite these beneficial uses of the plant, there

has been conflicting reports on its exact

toxicological potentials on some visceral organs.

For instance Aregheore et al., (1998) reported the

presence of toxic phytochemicals. There are also

reports of actual hepatotoxicity in mice (Igile et

al., 1995), also, there was a report on

hepatoprotective effects in rats (Babalola et al.,

2001). Ojiako and Nwanjo (2006) reported that V.

amygdalina leaves may be toxic (just like several

other vegetables) if consumed in very large

quantities but the potential danger is not higher

than has been observed for other common

vegetables that are routinely consumed in Africa

in even larger quantities.

In view of these conflicting reports, we

therefore set to comparatively determine the

effects of aqueous and ethanolic leaf extracts of V.

amygdalina, adopting histological procedures on

the stomach, liver and kidney in adult wistar rats.

Materials and methods

Experimental Site

This research was conducted in the Department of

Biochemistry, School of Basic Medical Sciences,

Igbinedion University, Nigeria.

Experimental Animals

A total of forty healthy wistar rats obtained from a

private farm in Benin City were used for the

experiment. These animals were acclimatized for

two weeks before the commencement of the

study. The animals were treated in accordance

with the “Guide for the Care and Use of

Laboratory Animals” prepared by the National

Academy of Sciences and published by the

National Institutes of Health (NIH, 1985) .

Experimental Design

There were four groups of animals: A, B, C and

D. Groups A, B and C served as the treated

animals while group D served as the control

animals. Each of the groups (n=10) was further

subdivided into two i.e A: A1 & A2; B: B1 & B2;


C: C1 & C2 and D: D1 & D2. Animals in groups

A1, B1 and C1 were administered orally with

aqueous extract of V. amygdalina 100, 200 and

300 mg/kg respectively while animals in groups

A2, B2 and C2 were administered orally with

ethanolic extract of V. amygdalina 100, 200 and

300 mg/kg respectively. The control groups D1

and D2 received equal volume of normal saline.

Collection of plant materials

Fresh but matured leaves of Vernonia amygdalina

was procured from a local market in Okada, Edostate.

They were authenticated in the Botany

Department, Igbinedion University, Nigeria. The

leaves were rinsed severally with clean tap water

to remove dust particles and debris and thereafter

allowed to completely drain.

Preparation of plant extracts

Plant materials were separately chopped into bits

with a knife on a chopping board. The leaves

were then air dried and one kilogram (1kg) V.

amygdalina was reduced to powder with an

electric blender. The powder was divided into two

portions. One of the portions was percolated with

80% ethanol while the other was percolated with

distilled water. The mixtures were allowed for 48

h in the refrigerator at 4 0 C for thorough extraction

of the plants active components. These were then

filtered with cheesecloth and later with Whatman

No. 1 filter paper to obtain a homogenous filtrate.

These filtrates were then concentrated in vacuo at

low temperature (37- 40 0 C) to about one tenth the

original volume using a rotary evaporator. The

concentrates were allowed open in a water bath

(40 0 C) for complete dryness for both ethanol and

aqueous extracts of V. amygdalina. The extracts

(26.7% yield) were then refrigerated at 2- 8 0 C

until use.

Histological Procedure

Histological study was carried out using the

method of Carleton (1967). These procedures

Effects of Vernonia amygdalina extracts on vital organs 77

involved dehydration of the liver, kidney and

spleen tissues with graded ethanol concentrations

(50%, 70%, 90% and 100%, respectively),

clearing in xylene, followed by infiltration in

paraffin wax for 2 h at 56 o C and embedding in

paraffin wax for 48 h. Sections (5 μm thick) were

then obtained, using a rotary microtome,

subjected to haematoxylin and eosin (H & E)

staining procedure and examined under a light

microscope. Permanent photomicrographs of the

observations were taken, using an Olympus

Research Microscope (model BX51).

Results

There were no significant derangement in the

cytoarchitecture of the stomach, liver and kidney.

Rather, the cytoarchitecture of the animals treated

with both the ethanolic and aqueous extract

(300mg/kg) of V. amygdalina were better

organized when compared with the control

(Figures 1-3). In the stomachs of treated animals,

there were no disruptions of surface epithelium

and no presence of submucosal edema as well as

leucocytes infiltration as compared with control

groups (Figure 1). There were no hypertrophy of

the liver and no necrosis of the hepatocytes in the

treated animals as compared with control groups

(Figure 2). Also, the kidneys of animals in the

treated groups presented well preserved renal

corpuscles and Bowman’s spaces as compared

with control groups (Figure 3). Animals in both

the treated and control groups showed no physical

changes in their appearances. The present study

demonstrated that oral administration of both

ethanolic and aqueous extracts (300 mg kg -1 )

preserved the hepatic, renal and gastric integrity

as compared to animals administered with lesser

dose (200 and 100 mg kg -1 ) and only distilled

water (Figures 1-3). There were no significant

differences between the cytoprotective abilities of

the animals treated with ethanolic or aqueous

extracts compared to the animals treated with

control.


78 Spencer NWANGWU et al.

A1 A2

C1

B1 B2

Figure 1. Histological section of gastric mucosa treated with A: aqueous extract V. amygdalina (A1-100, B1-

200, C1-300mg/kg); B: ethanolic extract of V. amygdalina (A2-100, B2-200, C2-300mg/kg). Note that there

were no disruptions of surface epithelium. No presence of submucosal edema and leucocytes infiltration as

compared with control groups D1 and D2 (H and E stain, 100x).

C2

D1 D2


Effects of Vernonia amygdalina extracts on vital organs 79

Figure 2. Histological section of hepatic parenchyma treated with A: aqueous extract V. amygdalina (A1-

100, B1-200, C1-300mg/kg); B: ethanolic extract of V. amygdalina (A2-100, B2-200, C2-300mg/kg). Note

that there were no hypertrophy of the liver and no necrosis of the hepatocytes as compared with control

groups D1 and D2 (H and E stain, 100x).


80 Spencer NWANGWU et al.

Figure 3. Histological section of renal parenchyma treated with A: aqueous extract V. amygdalina (A1-100,

B1-200, C1-300mg/kg); B: ethanolic extract of V. amygdalina (A2-100, B2-200, C2-300mg/kg). Note that

the renal corpuscles and Bowman’s spaces were well preserved. Also, there were no evidence of cellular

necrosis as compared with control groups D1 and D2 (H and E stain, 100x).

Discussion

The histopathological changes observed in the

liver, kidney, stomach of aqueous and ethanolic

extracts of V. amygdalina on rats showed no

evidence of lesions in the treated animals when

compared with control. Our histological findings

further revealed that the cyto-architecture of the

liver parenchyma was better organized in the

groups treated with 300mg/kg of both ethanolic and

aqueous extracts of V. amygdalina. The functions

of the liver which includes detoxification,

metabolic activities, synthesis of plasma protein

and destruction of spent red blood cells among

other functions will be better enhanced based on


histological evidences from our study. This shows

that administration of ethanolic and aqueous

extracts of V. amygdalina provides a protective role

for the liver which is the first organ susceptible to

any injurious substances in case of toxicity. This is

in line with previous work (Ofusori et al. 2008)

who investigated the effect of Croton zambesicus

(euphorbiaceae) on the liver. The renal parenchyma

showed no evidence of vacuolations or distortion of

any kind rather, renal corpuscles and Bowman’s

spaces were well preserved as compared with

control groups in all the experimental groups. It

was evident on the photomicrograph that the

tubules constitute the bulk of the renal parenchyma

with different shapes, diameters and staining

intensities. This points to the fact that V.

amygdalina may have a vital role to play in

osmoregulation and excretion. Also, the gastric

mucosa of the treated groups were better organized

when compared with the control (Figure 1). There

was no evidence of ulceration in the surface

epithelium, no presence of submucosal edema and

leucocytes infiltration. Studies have shown that

anti-oxidants significantly strengthen the gastric

walls and protect tissue from oxidative damage

(Marins, 1996).

All these evidences revealed that V. amygdalina

is a very important plant that can be exploited for

cyto-protective purposes. The cyto-protective

mechanism may be by moping up free radical there

by protecting the cells of the organs from any type

of assaults. Several other studies have shown that

the aqueous and ethanolic leaf extracts of V.

amygdalina is non-hazardous, even when taken for

some time. The potency of these extracts may have

been amplified on account of increased

concentration or build up of specific

phytochemicals, although the chemistry is yet

unknown. Several findings showed that V.

amygdalina has strong antioxidant activity

corresponding to mitigation of the generation of

hydroxyl radicals. Yeh et al., (2003) and Battell et

al., (1999) postulated that this antioxidant activity

may provide possible rationale for the observed

therapeutic effects of V. amygdalina.

Based on the results obtained, it can be

concluded that ethanolic and aqueous leaf extracts

of V. amygdalina are non toxic and may possess

cytoprotective potential.

Effects of Vernonia amygdalina extracts on vital organs 81

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activity of Vernonia amygdalina. Br. J.

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Akah PA and Okafor CI. Hypoglycaemic effect of

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Battell ML, Rodrigues B, Yuen VG and McNeill

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the Nutr. Society. 62(2):371-81, 2003.

Igile GO, Oleszek W, Jurzysta M, Burda S, Fafunso

M and Fasanmade AA. Nutritional assesment of

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Ijeh I, Nwugo VO and Obidoa O. Comparative

studies on the nutritive Phyto-chemical and

antimicrobial properties of two varieties of

Vernomia amygdalina. Plant Pros. Res. Comm.

1: 71-75, 1996.

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natural inhibitor of extracellular signalregulated

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growth. Experimental Biol. Med. (Maywood)

229(2): 163-169, 2004.

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Carley DG. Dioscoretine: The hypoglycaemic


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principles of Dioscorea dumentorium. Plant

Med. 56: 119-126, 1996.

Kupcham SM. Drugs from Natural products. Plant

source in drugs discovery, science and

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Dermatol. Surg., 22: 156-160, 1996.

Ofusori DA, Adelakun AE and Ayoka AO.

Histological Investigation of the Effects of

Croton zambesicus on the Liver of Swiss albino

mice. The Int. J. Gastroenterology. (7)1: 2008.

Ojiako OA and Nwanjo H. Is Vernonia amygdalina

hepatotoxic or hepatoprotective? Response from

biochemical and toxicity studies in rats. Afr J.

Biotech. 5 (18): 1648-1651, 2006.

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Journal of Cell and Molecular Biology 9(1): 83-86, 2011 Short communication 83

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Investigation of the EGFR gene variations in bladder cancer

patients using INFINITI TM Analyzer

Necip Ozan TİRYAKİOĞLU 1 , Özlem KURNAZ 1 , Ömer Onur ÇAKIR 2 , Nagehan ERSOY

TUNALI 1,*

1

Haliç University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, Istanbul,

Turkey

2

T.R. Ministry of Health, Bağcılar Training and Research Hospital, Istanbul, Turkey

(* author for correspondence; nagehanersoy@halic.edu.tr)

Received: 26 May 2011; Accepted: 22 June 2011

Abstract

Dysregulation of epidermal growth factor receptor (EGFR), which leads to increased proliferation, is a

common event in most cancers. Since its recognition as a keyplayer in carcinogenesis, several anti-EGFR

agents have been developed as chemotherapeutic agents. Studies investigating the effect of EGFR mutations

on the activity of anti-EGFR agents resulted in the discovery of a number of cancer tissue-specific mutations

which either increase or decrease sensitivity to tyrosine kinase inhibitors (TKI). Determination of these

mutations is essential to prognostic processes, but is limited to tissue availability. Being able to determine

tumour specific EGFR mutations by using blood sample as the genotyping material will eliminate the need

for invasive techniques. This study is the first step of a wider research with the final goal of matching

mutations in tumour tissues with that of normal somatic cells from the same individuals. All the known exon

18-21 mutations were screened in 48 bladder cancer patients with INFINITI TM analyzer using leukocytes as

the DNA source. However, none of the investigated mutations have been detected in the genomic DNA

samples of bladder cancer patients.

Keywords: INFINITI TM , epidermal growth factor receptor, tyrosine kinase inhibitor, drug sensitivity, bladder

cancer.

Mesane kanseri hastalarında EGFR geni varyantlarının INFINITI TM ile incelenmesi

Özet

Artmış çoğalmaya sebep olan epidermal büyüme faktörü reseptörünün (EGFR) düzenlenmesindeki

bozukluklara çoğu kanser türünde rastlanır. Karsinogenezde oynadığı kilit rol farkedildiğinden beri anti-

EGFR etkili bir çok kemoterapötik ajan geliştirilmiştir. EGFR mutasyonlarının anti-EGFR ajanlarının

aktivitesine etkisinin incelendiği araştırmalarda tirozin kinaz inhibitörüne hassasiyetini artıran veya azaltan

bir çok kanser dokusuna özgün mutasyon belirlenmiştir. Bu mutasyonların belirlenmesi tedavi aşamasının

temelini oluşturur, fakat mutasyonların belirlenmesi kanser dokusuna erişebilirlik ile sınırlıdır. Tümör

spesifik EGFR mutasyonlarının genotipleme aracı olarak kan örnekleri kullanılarak belirlenebilmesi invaziv

tekniklere olan gerekliliği ortadan kaldıracaktır. Bu çalışma nihai olarak olarak tümör dokusu

mutasyonlarının normal somatik hücre mutasyonları ile eşlenmesinin amaçlandığı daha geniş bir araştırmanın

ilk aşaması olarak gerçekleştirilmiştir. Bilinen tüm exon 18-21 mutasyonları 48 mesane kanseri hastasında

INFINITI TM cihazı ile DNA kaynağı olarak lökositler kullanılarak taranmıştır. Ancak, araştırılan hiçbir

mutasyon mesane kanseri hastalarının genomik DNA’larında belirlenememiştir.

Anahtar Sözcükler: INFINITI TM , epidermal büyüme faktörü reseptörü, tirozin kinaz inhibitörü, ilaç

hassasiyeti, mesane kanseri.


84 Necip Ozan TİRYAKİOĞLU et al.

Introduction

Epidermal growth factor receptor (EGFR) is a cell

surface receptor that activates signalling cascades

which mainly induce proliferation and cell survival.

Human genome codes for four members of the

EGFR family, EGFR/ErbB1, HER2/ErbB2/neu-2,

HER3/ErbB3 and HER4/ErbB4. All the family

members consists of a ligand-binding, a

transmembrane and a cytoplasmic domain with

tyrosine kinase activity (Gschwind et al., 2004).

EGFR members reside on the cell membrane as

monomers which dimerize upon phosphorylation

by ligand binding. Eight different molecules have

been identified as EGFR/ErbB1 ligands. All the

EGFR ligands, except Crypto, are synthesized as

precursor molecules and become functional only

after proteolytic clevage. In addition, EGFR family

members have been shown to be activated

indirectly by chemokines, cell adhesion molecules

and G-protein coupled receptors (Yarden and

Sliwkowski, 2001). The dimerisation of EGFR

members can occur as homodimerisation or as

heterodimerisation with another member of the

family. Following dimerisation,

autophosphorylation in homodimers and

transphosphorylation in heterodimers occurs.

During this process five tyrosine residues, Y992,

Y1045, Y1068,Y1148 and Y1173, located on Cterminal

are phosphorylated. Consequently, EGFRs

activate a multitude of transducer and adapter

proteins including HP-2, GRB2, PI3K and Akt.

Activation of these proteins induce a variety of

cytoplasmic pathways, mainly MAPK and JNK,

which trigger cellular events like DNA synthesis

and proliferation (Oda et al., 2005).

Dysregulation of EGFRs are mainly caused by

overexpression of the receptor itself, their ligands

or both. In addition to overexpression, mutations in

the tyrosine kinase domain causing consistent

activation of the receptor account for another major

mechanism affecting EGFR signalisation. Such

mutations have been identified in lung, stomach

and breast cancer tissues, but not in normal tissues.

Dysregulation of EGFR can also be the result of

HER2/ErB2 heterodimerisation. HER2 does not

have ligand binding activity, instead it increases

the ligand binding affinity of the protein it

dimerises with. Thus, EGFR/HER2 dimerisation

leads to a more active receptor and increased

activity of the EGFR pathways (Herbst, 2004).

Upon discovery of disturbed EGFR functions in

multiple cancers, abundant number of studies have

been conducted in order to develop drugs targeting

EGFR. Inhibitory molecules targeting EGFR can be

classified in three main categories; monoclonal

antibodies, chemical inhibitors and antisense

oligonucleotides.

Monoclonal antibodies exert their inhibitory

effect by binding to the ligand binding domain and

disrupt activation upon ligand binding. Monoclonal

antibodies against EGFR have been showed to

inhibit cell growth and induce apoptosis. However,

since they exert their effect only on ligand binding

domain, EGFRs with tyrosine domain mutations

are prone to be irresponsive to the inhibitory effects

of monoclonal antibodies. In contrast to

monoclonal antibodies, chemical inhibitors target

tyrosine kinase domain and inhibit EGFR by

blocking ATP from interacting with the tyrosine

kinase domain. Antisense nucleotides, on the other

hand, prevent translation of the EGFRs and their

ligands and carry out their inhibitory effect at a

much essential level.

90% of the somatic EGFR mutations are located

in the tyrosine kinase domain, namely exons 18-21

(Table 1). Since tumor cells depend on the

activating mutations on tyrosine kinase domain of

EGFR for proliferative signals, it has been shown

that tumor cells harboring such mutations are more

sensitive to thymidine kinase inhibitors (TKI).

Thus, determination of the EGFR mutations has

both prognostic value as an indicator of drug

response and diagnostic value as a biomarker

(Lynch et al. , 2004).

Since the determination of EGFR mutations

requires tumour samples and collecting a tumour

sample is only possible through invasive

techniques, EGFR mutation surveys are limited to

tissue availability. Being able to scan for the EGFR

mutations by using whole blood as an alternative

DNA source may provide information about the

genotype of the original tumour tissue without the

need for invasive techniques. Therefore, the study

presented here is initiated as the first step of a wider

research with the final goal of matching mutations

in tumour tissues with that of normal somatic cells

from the same individuals.

Table 1. Common EGFR Mutations

Mutations Frequency Effect

G719A/C/S 3% Increased sensitivity

Ex 19 Deletion 48% Increased sensitivity

Ex20 Deletion 4% Decreased sensitivity

T790M 5% Decreased sensitivity

L858R 43% Increased sensitivity

L861Q 2% Increased sensitivity


EGFR variations in bladder cancer 85

Materials and methods

Ethical Committee for Clinical Research. DNA was

As the first step, genomic DNA extracted from the

leukocytes of 48 bladder cancer patients have been

surveyed for the total of 50 mutations identified to

date in exons 18-21 of the EGFR gene. The

selected study population consisted of 40 males and

8 females with bladder cancer. Tumour tissue and

blood samples were obtained from each induvidial

and categorised according to their tumour stage and

grade. Tumour samples were collected in tubes

containing RNase Later (Invitrogen) solution, while

blood samples were collected in tubes containing

K2EDTA. Ethical approval for his study was

obtained from Istanbul University Medical Faculty,

extracted from blood samples by using QiaAmp

DNA Blood Mini kit (Qiagen) as described in the

package insert.

Extracted DNA samples were surveyed for a

total of 50 mutations in exons 18-21 by

INFINITI Analyzer (Table 2). INFINITI

Analyzer has different protocols for different

assays, but the five essential steps are common to

all; 1) multiplex PCR amplification of DNA, 2)

fluorescent label incorporation using analyte

specific primer extension (ASPE), 3) hybridization

of the ASPE primers to a microarray followed by

washing, 4) scanning of the microarray and 5)

signal detection and analysis.

Table 2. Scanned EGFR Mutations

Exon 18 Exon 19 Exon 20 Exon 21

Glu709 Lys Lys739_Ile744dup Glu762insEAFQ Asn826Ser

Glu709 Gln Lys745_Glu746del Glu762ins His835Leu

Glu709 Ala Glu746_Ala750del Val769Met Leu858Arg

Glu709 Gly Glu746_Thr751delins Val769Leu Leu858Met

Glu709 Val Glu746_Thr751delins Asp770ins Leu858Arg

Gly719 Ser Glu746_Ala750delins Ala767_Val769dup Leu861Gln

Gly719 Arg Glu746_Ala750del Asp770fs Leu861Arg

Gly719 Cys Glu746 Lys Pro772Arg

Gly719 Ala Glu746_Ala750delins Ser768_Asp770dup

Ser720 Phe Glu746_Pro753delins His773Arg

Glu746_Ser752del His773Leu

Glu746_Ser752delins Asn771_His773dup

Glu746_Thr751delins Val774Met

Glu746_Thr751delins Arg776Cys

Glu746_Ser752delins Gly779Phe

Glu746_Thr751delins Thr790Met

Glu746_Ser752delins

Leu747_Glu749del

Leu747_ Thr751delins

Leu747_Ser752del

Leu747_Ser752delins

Leu747_Pro753delins

Glu746_Glu749del

Leu747_Glu749del

Glu746_Thr751del

Leu747_ Thr751del

Leu747_ Thr751delins

Leu747_ Lys754delins

Leu747_Pro753delins

Leu747_Ala750del

Leu747_ Thr751del

Arg748_Thr751delins

Glu749_Thr751del

Thr751_Ile759delins

Ser752_Ile759del


86 Ozan TİRYAKİOĞLU et al.

Results

INFINITI Analyzer detects EGFR mutations

according to the ratio of the specific fluorescence

signal emitted from the analyte, represented in

Relative Fluorescence Unit (RFU), to the

fluorescence signal of the non-specific

oligonucleotide background control spots. A ratio

of 2 and higher indicates the existence of the

particular EGFR mutation and a ratio lower than 2

indicates the absence of the particular EGFR

mutation.

According to specific RFUs for each mutation,

none of the 48 DNA samples extracted from blood

carry inherited EGFR mutations within the tyrosine

kinase domain (exons 18-21) of EGFR.

Discussion

The absence of any mutations in the surveyed

specimens might be attributed to the rarity of EGFR

mutations in bladder cancer cases as shown by

other studies before (Blehm et al., 2006; Villares et

al., 2007). However, it might also arise from the

fact that using whole blood as a source of

information for tumour genotype is an inaccurate

method. The underlying reason can only be

identified upon the completion of the second step of

the study, which will provide us the genotype data

obtained directly from tumour tissues and thus

enable to perform a cross-comparison between

tumour tissue and leukocyte genotype.

Acknowledgements

This study was supported by Haliç University and

CDK Tıbbi Malzeme Tic. Ltd. Şti. (Istanbul,

TURKEY).

References

Gschwind A, Fischer OM and Ullrich A. The

discovery of receptor tyrosine kinases: targets

for cancer therapy. Nat Rev Cancer. 4 . 361–

370, 2004.

Blehm KN, Spiess PE, Bondaruk JE, Dujka ME,

Villares GJ, Zhao YJ, Bogler O,Aldape KD,

Grossman HB, Adam L, McConkey DJ,

Czerniak BA, Dinney CP and Bar-Eli M.

Mutations within the kinase domain and

truncations of the epidermal growth factor

receptor are rare events in bladder cancer:

implications for therapy. Clin Cancer Res.

12(15):4671-7, 2006.

Herbst RS. Review of epidermal growth factor

receptor biology. International Journal of

Radiation Oncology .59(2): S21-S26, 2004.

Oda K, Matsuoka Y, Funahashi A and Kitano H.

Comprehensive pathway map of epidermal

growth factor receptor signaling. Mol Syst Biol.

1: 20050010, Epub 2005, May25.

Lynch TJ, Bell DW, Sordella R, Gurubhagavatula

S, Okimoto RA, Brannigan BW,Harris PL,

Haserlat SM, Supko JG, Haluska FG, Louis

DN, Christiani DC, Settleman J and Haber DA.

Activating mutations in the epidermal growth

factor receptor underlying responsiveness of

non-small-cell lung cancer to gefitinib. N Engl J

Med. 350(21):2129-39, 2004.

Villares GJ, Zigler M, Blehm K, Bogdan C,

McConkey D, Colin D, Bar-Eli M. Targeting

EGFR in bladder cancer.. EGFR in bladder

cancer. World J Urol. 25(6):573-9. 2007

Yarden Y and Sliwkowski MX. Untangling the

ErbB signalling network. Nat Rev Mol Cell

Biol. 2(2):127-37,2001.


Journal of Cell and Molecular Biology - INSTRUCTIONS for AUTHORS

General

Journal of Cell and Molecular Biology

(JCellMolBiol) is an international journal which

covers original works in the field of cell biology,

molecular biology, genetics, microbiology,

neurobiology, bioinforma-tics and related topics.

The official language of the journal is English,

however manuscripts in Turkish are accepted as

well.

Conditions for publication

This journal publishes research articles, review

articles, short communications, book/software

reviews, case reports and letters to the editor.

Research articles: Only original contributions will

be accepted which have not been published

previously. Manuscripts should not exceed 15

papers of printed text, including tables, figures and

references

Review articles: Reviews of recent developments in

a research field and ideas will be accepted.

Manuscripts should not exceed 15 papers of printed

text. Illustrations are encouraged.

Short communications: These include small-scale

investigations or innovative methods, techniques,

clinical trials and epidemiological studies. It should

not exceed 3 pages.

Letters to editor: These include opinions, news and

suggestions. Letters should not exceed 2 papers of

printed text.

Case Reports: These include individual

observations based on small numbers of subjects.

This type of research cannot indicate causality but

may indicate areas for further research.

REVISED

June 30 th , 2011

Manuscripts should be submitted by e-mail to:

Journal of Cell and Molecular Biology

Haliç Üniversitesi

Fen Edebiyat Fakültesi

Moleküler Biyoloji ve Genetik Bölümü

Sıracevizler Cad. No:29

Bomonti-Şişli 34394, İstanbul-TÜRKİYE

Tel: +90 212 343 08 87, Fax: +90 212 231 06 31

E-Mail: jcmb@halic.edu.tr

87

Book/software reviews: Short but concise

description of the book/software, not exceeding a

page. Book/software reviews are not peer reviewed.

Presentation

Papers should be typed clearly, double-spaced with

3 cm wide margins.

Manuscripts should be prepared using Word

Processor.

Cover Letter: You may briefly explain your work

and its contribution to present knowledge.

Title Page: The first page of your manuscript

should be a title page containing the type of paper;

the title; all authors' full names, and affiliations;

and the corresponding author's contact address

(including phone and fax numbers) and e-mail

address. The title should be as short as possible, but

should give adequate information regarding the

contents. Authors should also state a running title

of no more than 50 characters including spaces.

All pages must be numbered.

Full Paper

The full paper should be divided into the following

parts in the order indicated:


88

Abstract: A brief, informative abstract, not

exceeding 200 words, should be provided in

English and in Turkish. For authors who are not

native Turkish speakers, JCellMolBiol can provide

the Turkish abstract.

Keywords: Immediately following the abstract,

authors should provide 5 keywords or phrases that

reflect the content of the article.

Introduction should include theory, hypotheses,

prior work

Material and methods may include subheadings

Results: If the study consists of different parts,

subheadings in this section should be consistent

with subheadings in the methods.

Discussion

Acknowledgements should precede the list of

references

References: Papers cited in the manuscript should

be listed in alphabetical order according to the first

author's surname.

Tables and Figures

Tables and figures should be embedded within

the text in their appropriate positions.

Each table should be accompanied by a short

instructive title line plus an explanatory caption at

the top. Indicate footnotes within tables by

superscript letters and type footnotes below the

table.

Electronically submitted figures are preferred

in *.jpg or *.tiff (min. 300 dpi) formats. Each figure

should be supplied with a short instructive title line.

Do not give magnification on scales in the figure

titles; instead draw bar scales directly on the

figures.

All the tables and figures must be referred to

within the text.

Units, Abbreviations and Scientific Names

Only SI units should be used. Current

abbreviations can be used without explanation,

others must be explained.

All acronyms/abbreviations must be explained

in parenthesis after their first occurrence. If many

REVISED

June 30 th , 2011

unfamiliar acronyms/abbreviations are used, please

compile them in an "Abbreviations" section at the

end of the paper.

Latin expressions should be underlined or typed

in italics.

Referencing

In the text, citations with two authors should

take the form: Smith and Robinson,1990. If several

papers are cited by the same author in the same

year, they should be lettered in sequence (1990a),

(1990b), etc. When papers are by more then two

authors they should be cited as Smith et al.,1990.

In the list, references must be placed in

alphabetical order. The following models for the

reference list cover all situations. The punctuation

given must be exactly followed.

Redford IR. Evidence for a general relationship

between the induced level of DNA double

strand breakage and cell killing after Xirradiation

of mammalian cells. Int J Radiat

Biol. 49: 611- 620, 1986.

Tccioli CE, Cottlieb TM, Blund T. Product of the

XRCCS gene and its role in DNA repair and

V(D)J recombination. Science. 265: 1442-1445,

1994

Ohlrogge JB. Biochemistry of plant acyl carrier

proteins. The Biochemistry of Plants: A

Comprehensive Treatise. Stumpf PK and Conn

EE (Ed). Academic Press, New York. 137-157,

1987.

Brown LA. How to cope with your supervisor. PhD

Thesis. University of New Orleans, 2005.

Web document with no author: Leafy seadragons

and weedy seadragons 2001. retrieved

November 13, 2002, from http:// www.

windspeed.net.au/jenny/seadragons/

Web document with author: Dawson J, Smith L,

Deubert K. Referencing, not plagiarism.

Retrieved October 31, 2002 from http:

//studytrekk.lis.curtin.edu.au/

Only papers published or in press should be

cited in the literature list. Unpublished results,

including submitted manuscripts and those in

preparation, should be indicated as unpublished

data in the text.


Submission Policies and Authorship

Upon submission of a manuscript, it is accepted

that all co-authors have approved the contents of

the manuscript and its submission by the

corresponding author, and that the corresponding

author is authorized to represent all co-authors in

pre-publication discussions with JCellMolBiol.

The corresponding author is responsible for

ensuring that all the contributors to the relevant

work are listed as authors and that all authors have

aggreed to the manuscript’s content and its

submission to the JCellMolBiol. In case the Journal

happens to be aware of an authorship dispute,

authorship must be approved in writing by all of the

parties.

Cost

There are no submission fees or page charges.

Criteria for the Selection of Manuscripts

Manuscripts should meet the following criteria: the

study conducted is material is original and ethical,

the writing is clear; the study methods are

appropriate, the data are valid, the conclusions are

reasonable and supported by the data; the

information is important; and the topic is

interesting to our readership.

Editorial Processes

Researchers may request informal feedback from

the editors in a particular manuscript. The

presubmission process aids in the submission

decision for authors

When JCellMolBiol receives a manuscript, the

Editor and Associate Editor will first decide

whether the manuscript meets the formal criteria

specified with “Guidelines for Authors” and

whether it fits within the scope of the Journal. In

case of doubt on the basis of initial review, the

editor will consult other members of the Editorial

Board.

Manuscripts that are found suitable for peer

review will be assigned to two expert reviewers.

Reviewers may either be Editorial Board members

or external experts selected by the Editorial Board.

The corresponding author is notified by e-mail

when the editor decides to send a paper for review.

The reviewers will have up to three weeks to

review the submitted article. After peer review, the

REVISED

June 30 th , 2011

89

editor will contact the author. If the author is

required to submit a revised version, the revised

version has to be submitted by the author within

two weeks. Otherwise, the manuscript will be

removed from the manuscript submission queue

and will be considered rejected.

In cases where the referees have requested welldefined

changes to the manuscript, editors may

request a revised manuscript that addresses to

referees’ concerns. The revised version is sent back

to the original referees for re-review. In cases

where the referees’ concerns are more wideranging,

editors may reject the manuscript. The

revised manuscript should be accompanied by a

cover letter that includes a point-by-point response

to referees’ comments and an explanation of how

the manuscript has been changed.

As a matter of policy, we do not suppress

referees’ reports, any comments directed to authors

are transmitted regardless of what we may think of

the content. On rare occasions, we may edit a report

to remove offensive language or comments to

reveal confidentiality.

The final decision to accept or reject a

manuscript will be made by the Editor. If it

becomes apparent that there are serious problems

with the scientific content or with violations of our

publishing policies, the Editor also reserves the

right to reject a paper even after it has been

accepted

After acceptance, the Editor may make further

changes to the text and figures so that the

manuscript is readable and clear. Page proofs will

be sent to the corresponding author via email for

checking before publication. Corresponding authors

are sent proofs and are welcome to discuss

proposed changes with the Editor, but

JCellMolBiol reserves the right to make the final

decision about the style. Corrected proofs should be

sent back to the Editor within three days of receipt,

otherwise the Editor reserves the rights to correct

the proofs himself and to send the material for

publication.

Appeals

Authors have the right to ask the Editor to

reconsider a rejection decision, which is considered

an appeal. Decisions are reversed only if the Editor

is convinced that the original decision was a serious

mistake. If an appeal merits further consideration,

the Editor may send the author’s response or the

revised paper to one or more referees, or Editor


90

may ask one referee to comment on the concerns

raised by another referee.

Advance Online Publication

JCellMolBiol provides Advance Online Publication

of articles, which benefit authors with an earlier

publication date and allows the readers’ access to

accepted papers several weeks before they appear

in print

Ethical Issues

For manuscripts reporting experiments on live

vertebrates or higher invertebrates, authors must

declare that the study was approved by the

institutional ethics committee. Papers describing

investigations on human subjects must include a

statement that informed consent was obtained from

all subjects.

Plagiarism

If portions of the manuscript have already been

published by the author on other journals or

websites, JCellMolBiol Editorial Board needs to

know which portions of the manuscript have been

previously published and where. The author should

include a note in the cover letter indicating which

portions have been published elsewhere.

In case of any suspicion on scientific misconduct or

dishonesty in research, JCellMolBiol reserves the

right to forward any submitted manuscript to an

appropriate authori-ty for investigation.

Copyright Notice

It is the responsibility of the submitting author to

ensure that the authorship of the paper reflects the

contributions of the authors to the work described,

and that all listed authors have agreed to the

submission of the manuscript in its current form.

Conditions of publication in JCellMolBiol are

that the paper has not already been published

elsewhere; that it is not currently being considered

for publication else-where; all persons designated

as authors should qualify for authorship, and all

those who qualify should be listed. If accepted,

Haliç University and JCellMolBiol have the

exclusive license to publish.

JCellMolBiol is freely available to individuals

and institutions. Copies of this Journal and articles

REVISED

June 30 th , 2011

in this journal may be distributed for research or for

educational purposes free of charge. However,

commercial use of articles contained herein is

prohibited without the written consent of the editor.

Publication Agreement

The corresponing author is required to assign the

Publication Agreement Form in order to publish the

submitted manuscript in JCellMolBiol.


Journal of Cell and

Volume 9 · No 1 · June 2011

Review Articles

Molecular Biology

Decision making mechanism influences the regulatory function of Notch in Apoptosis

A.NAIR

Polymorphisms in the xenobiotic genes and susceptibility to bladder cancer

N. ERSOY TUNALI and N. O. TİRYAKİOĞLU

Research Articles

Identification of silencing suppressors of potato virus M

R. KRYLDAKOV, R. AKBERGENOV, T. HOHN and B. ISKAKOV

Identification of a novel dehydration responsive transcript from tossa jute (Corcohrus olitorius L.)

S. SHARMIN, M. M. MOOSA, Md. S. ISLAM, I. KABIR, A. AKTER and H. KHAN

Protective effects of curcumin on cadmium chloride induced colon toxicity in Swiss albino mice

P. SINGH, P. MOGRA, V. SANKHLA and K. DEORA

In vitro regeneration of Cleome viscosa – an important medicinal herb

J.ANBURAJ, C. R. SINGH, S. SUNDARRAJ and S. KANNAN

Bio-database compression using enhanced suffix array for pairwise sequence alignment

A.KUNTHAVAI and S.VASANTHA RATHNA

Cloning and expression of Lentinula edodes cellobiohydrolase gene in E. coli and characterization of

the recombinant enzyme

S. TAIPAKOVA, B. SMAILOV, G. STANBEKOVA and A. BISSENBAEV

Investigation of the MMP1 and MMP3 promoter polymorphisms in temporomandibular joint

disorder

N. TASKIN, K. ULUCAN, G. DEGIN, A. AKCAY, B. KARATAS and T. AKCAY

Lead induced alterations in blood cell counts and hemoglobin during gestation and lactation in Swiss

albino mice

I. BARBER, R. SHARMA, S. MOGRA, K. PANWAR and U. GARU

The effects of aqueous and ethanolic leaf extracts of Vernonia amygdalina on some vital organs in

adult Wistar rats

S. NWANGWU, D. A. OFUSORI, S. JOSIAH, O. F. AMEGOR, H. NJOYA and A. O. OYAKA

Short Communication

Investigation of the EGFR gene variations in bladder cancer patients using INFINITI TM Analyzer

N. O. TİRYAKİOĞLU, Ö. KURNAZ, Ö. O. ÇAKIR and N. ERSOY TUNALI

Instructions for Authors

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