Full Journal - Journal of Cell and Molecular Biology - Haliç Üniversitesi

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Full Journal - Journal of Cell and Molecular Biology - Haliç Üniversitesi

‹STANBUL

1998

VOLUME 3 • NO. 1 • 2004 • ISSN 1303-3646

HAL‹Ç UNIVERSITY

FACULTY OF ARTS AND SCIENCES

Journal of Cell and

Molecular Biology


Haliç University

Faculty of Arts and Sciences

Journal of Cell and Molecular Biology

Founder

Prof. Dr. Gündüz GED‹KO⁄LU

President of Board of Trustee

Rights held by

Prof. Dr. Ferruh KORKUT

Rector

Correspondence Address:

The Editorial Office

Journal of Cell and Molecular Biology

Haliç Üniversitesi, Fen-Edebiyat Fakültesi,

Ahmet Vefik Pafla Cad., No: 1, 34280, F›nd›kzade,

‹stanbul-Turkey

Phone: 90 212 530 50 24

Fax: 90 212 530 35 35

E-mail: jcmb@halic.edu.tr

Journal of Cell and Molecular Biology is

indexed in EBSCO database.

Summaries of all articles in this journal are

available free of charge from www.halic.edu.tr

ISSN 1303-3646

Printed at Yaflar Printing House

Igor ALEXANDROV, Dubna, Russia

Çetin ALGÜNEfi, Edirne, Turkey

Aglaia ATHANASSIADOU, Patras, Greece

fiehnaz BOLKENT, ‹stanbul, Turkey

Nihat BOZCUK, Ankara, Turkey

‹smail ÇAKMAK, ‹stanbul, Turkey

Adile ÇEV‹KBAfi, ‹stanbul, Turkey

Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey

Ayfl›n ÇOTUK, ‹stanbul, Turkey

Fevzi DALDAL, Pennsylvania, USA

Zihni DEM‹RBA⁄, Trabzon, Turkey

Mustafa DJAMGOZ, London, UK

Aglika EDREVA, Sofia, Bulgaria

Ünal EGEL‹, Bursa, Turkey

Advisory Board

Journal of Cell and

Molecular Biology

Published by Haliç University

Faculty of Arts and Sciences

Editor

Atilla ÖZALPAN

Associate Editor

Narç›n PALAVAN ÜNSAL

Editorial Board

Çimen ATAK

Atok OLGUN

P›nar ÖZKAN

Nihal BÜYÜKUSLU

Nefle AKIfi

Kürflat ÖZD‹LL‹

Damla BÜYÜKTUNÇER

Özge EM‹RO⁄LU

Mehmet Ali TÜFEKÇ‹

Merve ALO⁄LU

Asl› BAfiAR

Secretary

Burçin SARGIN

Aynur GÜREL, ‹zmir, Turkey

Candan JOHANSEN, ‹stanbul, Turkey

As›m KADIO⁄LU, Trabzon, Turkey

Valentine KEFEL‹, Pennsylvania, USA

Michael P. MADAIO, Pennsylvania, USA

Göksel OLGUN, Edirne, Turkey

U¤ur ÖZBEK, ‹stanbul, Turkey

Zekiye SULUDERE, Ankara, Turkey

‹smail TÜRKAN, ‹zmir, Turkey

Mehmet TOPAKTAfi, Adana, Turkey

Meral ÜNAL, ‹stanbul, Turkey

Mustafa YAT‹N, Boston, USA

Ziya Z‹YLAN, ‹stanbul, Turkey


Journal of Cell and

Molecular Biology

Volume 3/2004

Haliç University

Faculty of Arts and Sciences

‹stanbul-TURKEY


Haliç University

Faculty of Arts and Sciences

Journal of Cell and Molecular Biology

Founder

Prof. Dr. Gündüz GED‹KO⁄LU

President of Board of Trustee

Rights held by

Prof. Dr. Ferruh KORKUT

Rector

Correspondence Address:

The Editorial Office

Journal of Cell and Molecular Biology

Haliç Üniversitesi, Fen-Edebiyat Fakültesi,

Ahmet Vefik Pafla Cad., No: 1, 34280, F›nd›kzade,

‹stanbul-Turkey

Phone: 90 212 530 50 24

Fax: 90 212 530 35 35

E-mail: jcmb@halic.edu.tr

Journal of Cell and Molecular Biology is

indexed in EBSCO database.

Summaries of all articles in this journal are

available free of charge from www.halic.edu.tr

ISSN 1303-3646

Printed at Yaflar Printing House

Igor ALEXANDROV, Dubna, Russia

Çetin ALGÜNEfi, Edirne, Turkey

Aglaia ATHANASSIADOU, Patras, Greece

fiehnaz BOLKENT, ‹stanbul, Turkey

Nihat BOZCUK, Ankara, Turkey

‹smail ÇAKMAK, ‹stanbul, Turkey

Adile ÇEV‹KBAfi, ‹stanbul, Turkey

Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey

Ayfl›n ÇOTUK, ‹stanbul, Turkey

Fevzi DALDAL, Pennsylvania, USA

Zihni DEM‹RBA⁄, Trabzon, Turkey

Mustafa DJAMGOZ, London, UK

Aglika EDREVA, Sofia, Bulgaria

Ünal EGEL‹, Bursa, Turkey

Advisory Board

Journal of Cell and

Molecular Biology

Published by Haliç University

Faculty of Arts and Sciences

Editor

Atilla ÖZALPAN

Associate Editor

Narç›n PALAVAN ÜNSAL

Editorial Board

Çimen ATAK

Atok OLGUN

P›nar ÖZKAN

Nihal BÜYÜKUSLU

Nefle AKIfi

Kürflat ÖZD‹LL‹

Damla BÜYÜKTUNÇER

Özge EM‹RO⁄LU

Mehmet Ali TÜFEKÇ‹

Merve ALO⁄LU

Asl› BAfiAR

Secretary

Burçin SARGIN

Aynur GÜREL, ‹zmir, Turkey

Candan JOHANSEN, ‹stanbul, Turkey

As›m KADIO⁄LU, Trabzon, Turkey

Valentine KEFEL‹, Pennsylvania, USA

Michael P. MADAIO, Pennsylvania, USA

Göksel OLGUN, Edirne, Turkey

U¤ur ÖZBEK, ‹stanbul, Turkey

Zekiye SULUDERE, Ankara, Turkey

‹smail TÜRKAN, ‹zmir, Turkey

Mehmet TOPAKTAfi, Adana, Turkey

Meral ÜNAL, ‹stanbul, Turkey

Mustafa YAT‹N, Boston, USA

Ziya Z‹YLAN, ‹stanbul, Turkey


Journal of Cell and Molecular Biology

CONTENTS Volume 3, No.1, 2004

Dedication

Review articles

Gene therapy for the haemoglobinopathies

Hemaglobinopati için gen terapisi

A. Athanassiadou 1-7

Systemic acquired resistance: Characterization of genes associated with plant

defence response

Sonradan sistemik olarak elde edilmifl direnç: Bitki savunma cevab› ile ilgili

genlerin karakterizasyonu

‹. Tiryaki, H. Tunaz 9-14

Research papers

Biological investigations into AAnnttiiddeessmmaa mmaaddaaggaassccaarriieennssee Lam. (Euphorbiaceae),

FFaauujjaassiiooppssiiss fflleexxuuoossaa (Lam.) C. Jeffrey (Asteraceae), TTooddddaalliiaa aassiiaattiiccaa (L.) Lam.

and VVeepprriiss llaanncceeoollaattaa (Lam.) G. Don (Rutaceae)

Antidesma madagascariense Lam. (Euphorbiaceae), Faujasiopsis flexuosa (Lam.)

C. Jeffrey (Asteraceae), Toddalia asiatica (L.) Lam. and Vepris lanceolata (Lam.)

G. Don (Rutaceae) da biyolojik araflt›rmalar

F. B. Narod, A. Gurib-Fakim, A. H. Subratty 15-21

The role of mmeettaa-topolin in senescence of wheat leaf segments

Meta-topolinin bu¤day yaprak segmentlerinin senesensindeki rolü

N. Palavan-Ünsal, S. Ça¤, E. Çetin 23-31

Characterization of BBaacciilllluuss species by numerical analysis of their SDS-PAGE

p rotein profiles

Bacillus Türlerinin SDS-PAGE protein profillerinin nümerik analiziyle karakterizasyonu

‹. Berber 33-37

A kinetic model for iinn--vviittrroo intestinal uptake of LL-tyrosine and D (+)-glucose across

rat everted gut sacs in the presence of MMoommoorrddiiccaa cchhaarraannttiiaa, a medicinal plant used

in traditional medicine against diabetes mellitus

Diabetes mellitus’a karfl› kullan›lan geleneksel t›bbi bitki Momordica charantia varl›¤›nda ters

yüz edilmifl s›çan ba¤›rsa¤›ndan L-tirozin ve D(+)-glukoz geçifli için in vitro kinetik model

M. F. Mahomoodally, A.Gurib-Fakim, A. H. Subratty 39-44

P rostatic intraepithelial neoplasm: Retrospective results of clinical,

histopathological approaches

Prostat intraepitel neoplasm: Klinik ve histopatolojik retrospektif sonuçlar

O. N. Akbulut, S. Güney, ‹. Duman, ‹. Çömez, S. Arisan, E. Ergenekon 45-50

Bladder tumor antigen sensitivity in bladder cancer patients

Mesane kanseri hastalar›nda mesane tumör antijen hassasiyeti

S. Güney, S. Arisan, ‹. Duman, T. Çaflkurlu, C. Sönmez, E. Ergenekon 51-55

Book Reviews 57

Instructions to authors 58-59


This issue is dedicated to

P rof. Dr. M. At›f fiengün

for his invaluable contribution to biology


M. At›f fiengün, Curriculum Vitae

M. At›f fiENGÜN was born on the 29 th of August, 1915 in Çal/ Denizli. His father, a judge, Mr. Ali R›za

and his mother Ms. Saliha Feride had moved to Izmir right after it was saved, together with the family

members. Hence Mr. fiengün had completed his primary, secondary and high school education in Izmir.

Having accomplished the exam the Ministry of Education organized to send high school graduates abroad,

he went to Germany in 1932 for further study. He acquired his title as a “Dr.”, having graduated the Faculty of

Natural Science, University of Berlin, in June 1939. As soon as he came back to Turkey, fiengün started his

teaching career in Istanbul Vefa High School as an intern Biology teacher.

fiengün started his position as an assistant in Istanbul University, Faculty of Science, Zoology Institute in

1943 after he had completed his military service. He worked in the Institute of Education, Ankara Gazi

University in 1944. He proceeded in his academic career in Istanbul University, Institute of Zoolgy

respectively, as a research assistant, associate professor, and finally as the head of the department. As the Dean

of Istanbul University, Faculty of Science, he started many innovations, between 1960 –1962. For instance, he

initiated the construction of the Biology Building which was then called “New Zoology Building” in 1960.

He was in charge of Faculty of Science first as the Head of Radiobiology (which he founded in 1969) as

well as the Director of Institute of Hydrobiology, Istanbul University. With the approval of the New Act

concerning the universities in 1981, he was honoured as the head of General Biology subdepartment. Having

served the academic circle for years efficiently, fiengün announced his retirement in January 1984.

As a participant of many international gatherings as well as seminars, Prof. Dr. At›f fiengün had acquired

the chance to develop his vision and wisdom in international academic arena. Besides, he had both encouraged,

organized and actualized such academic meetings in Turkey. Among these organizations, one can recall the two

NATO courses held in Istanbul in 1964 and 1982 owing to his individual efforts.

Prof. Dr. At›f fiengün had contributed to the founding and development of Çekmece, Nuclear Research and

Training Center as well as TUBITAK.

Prof. Dr. At›f fiengün who deceased in 06.10.2002 played a crucial role in the foundation of biology as a

field of science besides his great efforts to introduce many young biologists to the academic circle. Owing to

his notable contributions, he was honoured by TUBITAK with “science award”. His area of research involved

“The Influence of Genes on Vitality”, “The Variation of Giant Chromozomes According to Tissue” and

“Anticancerogens”, briefly. He was the author of 65 articles in other languages, 63 articles in Turkish, 17 books

as well as the director of 25 Ph.D. thesis.

We feel proud to dedicate this issue to him and thank all contributors on his behalf.


Journal of Cell and Molecular Biology 3: 1-7, 2004.

Haliç University, Printed in Turkey.

Gene therapy for the haemoglobinopathies

Aglaia Athanassiadou

Dept of Biology, Faculty of Medicine, University of Patras, Greece

Received 4 August 2003; Accepted 4 October 2003

Abstract

The haemoglobinopathies have been among the first diseases to be considered for the development of gene therapy

protocols. Initial attempts involved the transfer of the β-globin gene into established cell lines and later mouse

models of the disease, by means of viral vectors. These attempts were not successful, due to various disadvantages

of the vector system used, as well as to the complex regulation of β-globin gene expression. More promising are

current attempts that make use of vectors, viral or episomic, that can accommodate large insert DNA, can support

long term expression of the transgenes and can transduce human haemopoetic stem cells.

KKeeyy wwoorrddss:: Haemoglobinopathie, β-globin, gene therapy

Hemoglobinopati için gen terapisi

Özet

Hemoglobinopatiler gen terapi protokollerinin gelifltirildi¤i ilk hastal›klardand›r. ‹lk denemeler β-globin geninin

bilinen hücre hatlar› ve daha sonras›nda fare modellerde viral vektörlerle transfer edilmesi üzerine gerçekleflti. Bu

denemeler baflar›ya ulaflamad›. Çünkü viral vektörler kullan›lan sistemlerde dezavantajlara sahipti. Ayn› zamanda

β-globin geni oldukça kompleks bir ekspresyona sahip oldu¤u için baflar›l› olunamad›. Bugün daha fazla umut

vadeden vektörler, viral veya episomik olup, daha büyük DNA parçalar›n›n aktar›lmas› ve transgenlerin uzun

dönemli ekspresyonlar›n›n desteklenerek insan kök hücrelerinin uyar›lmas›n› sa¤l›yorlar.

AAnnaahhttaarr ssöözzccüükklleerr:: Hemoglobinopati, β-globin, gen terapi

Introduction

The Haemoglobinopathies constitute a very common

and heterogeneous group of genetically inherited

disorders. They are caused by either an imbalance in the

α:ß polypeptide ratio of haemoglobin, in which case the

resulting disease is thalassaemia, or abnormalities in the

function of ß-globin chain, as in the case of sickle cell

disease. (Stamatoyannopoulos et al., 1987, Weatherall

and Clegg, 1981). The heamoglibinopathies were

among the first diseases to be considered for gene

therapy being monogenetic disorders, whose

pathophysiology correlated very well with the deficient

genetic background, meaning that a simple correction

of the gene responsible through somatic gene therapy

could reverse the disease phenotype.

In terms of the identity of globin gene transfer, two

a re the main strategies used so far:

• One aims at the transfer of the normal ß-globin

gene, to replace the function of the mutated

endogenous ß-globin genes

• The second one aims at the transfer of the

normal γ-globin gene, or of a molecule that

induces globin gene expression, e.g.

erythropoetin, in an attempt to cause an

increase of the endogenously produced Fetal

1


2 Aglaia Athanassiadou

Haemoglobin (α2-γ2) to a therapeutic level, as

it may replace the missing adult Haemoglobin A.

However, most of the procedures employed so far,

concentrate on the first case.

The development of a gene therapy strategy for the

haemoglobinopaties has been a long process with no

conclusive, concrete results yet, in spite the variety of

approaches and means employed. This review focuses

on the basic aspects of this process with emphasis on

problems that arose in the various phases of the

respective research.

A. The complex regulation of ß-globin gene

expression

The globin genes structure and expression has been the

object of extensive studies worldwide and, in

particular, the molecular mechanisms underlying the

regulation of gene expression from the human ß-globin

locus are, to a great extent, well understood. The genes

of the human ß-globin locus are expressed in various

tissue-locations and at various times, being switched

on and off during development, so they are subjected

to a fine, precise and complex regulation.

The primary level of regulation of the genes within

the ß-globin locus is activation by the Locus Control

Region (ßLCR). The ßLCR consists of five,

developmentally stable, erythroid-specific DNase I

hypersensitive sites (HS1,2,3,4,5) distributed over

16kb of DNA located 5’ of the entire ß-globin gene

cluster. The role of the ßLCR is to determine the tissue

specificity (erythroid) and the developmental stage at

which every one of the five genes located in the ßglobin

gene cluster (namely the embryonic ε gene, the

fetal G γ and A γ genes and the adult δ, and ß genes) are

expressed. This function of the ßLCR was elucidated

by gene transfer experiments. Crucial experiments

were carried out, in which the ß-globin gene was

transferred –by stable transfection- into tissue culture

cells and transgenic animals. In such experiments the

foreign DNA that enters the cells integrates into the

genome of the host cells, (intergrated transgene).

Considering that a very big portion of the genome is in

heterochromatic state, the foreign DNA often is

incapable for expression, after integration into the

target cell’s genome. This is also true for the initial

experiments using ß-globin gene and the respective

promoter only. However, the inclusion of ßLCR along

with the ß-globin gene in the transferred construct was

found to confer on it physiological level of erythroidspecific

gene expression that is independent of the siteof-integration.

Furthermore, this level of expression is

directly proportional to the copy number “ßLCR, ßglobin

gene” system that entered into the cells

(Grosveld et al., 1998).

The total ßLCR is relatively long to be included in

the usual vector systems and, therefore, investigations

were carried out to test whether one or two of its

elements would be enough to drive transgene

expression, in a sufficient level. The use of the ßLCR

HS2 site alone, which possesses a classical enhancer

activity, can act as an LCR only at multiple copy

number (Ellis et al., 1993) and HS3 functions as single

transgene copies but give significant variable levels of

expression that are bellow a therapeutic value (Ellis et

al., 1996). It is therefore clear from these and

numerous other studies (see Antoniou and Grosveld,

1999; May et al., 2000; May et al., 2002), that a

combination of ßLCR elements is required to

preserve full physiological gene function and, from a

gene therapy perspective, therapeutic levels of

expression of the linked gene. It is desirable that the

whole ßLCR is included in the therapeutic construct

(Chow et al., 2002) but this is not always possible.

B. The gene delivery systems

I. Viral vectors

Considering gene delivery, viruses were the first

vectors to be employed: their biology was already

known, they are capable of targeting a type of cell

(tissue targeting) and they can be produced in high

quantities. They had, of course, to be engineered so as

not to be infective, after entrance to the target cell.

A high number of systems based on known

genomes of viruses, carrying different transcription

units have already been tested in cell cultures and

animal model systems. Retroviral (Leboulch et al.,

1994; Sadelain et al., 1995) and adeno-associated type

2 (Miller et al., 1994; Einerhand et al., 1995) vectors

containing multiple ßLCR sites have been tested, but

have been proved inadequate. The major problems

encountered with these vectors are of three types:

1. Viral vectors can accept only a relatively small

size of insert DNA, usually less than 8Kb and

they suffer the problem in that they can only

accommodate a limited size of genes to be


transferred. This poses limitations to the

inclusion of transcriptional units with fulllength

regulatory elements.

2. As far as transgene expression is concerned,

despite the inclusion of elements from the

ßLCR, the levels of expression were low and

decayed over time (Derek, 2000). This is due

to the heterochromatin effect on the intergrated

transgene. To overcome this problem, element

considered to protect euchromatin areas from

heterochromatin invasion were used, like the

chicken HS4 chromatin insulator (Yannaki et

al., 2002)

3. These vectors usually do not transduce

Heamopoetic Stem Cells, as the latter do not

replicate.

The use of lleennttiivviirraall vveeccttoorrss as gene delivery

systems complement the successful features of

previously developed retroviral vectors in that they

have the ability to infect non-dividing cells, integrate

efficiently, have a larger carrying capacity for the

therapeutic transcriptional units and transfer genes free

of viral sequences (Vigna et al., 2000; Follenzi et al.,

2000). Lentiviral vectors containing human ß-globin

transcription units have been the most successful to

date, when tested in mouse models of ß-thalassaemia,

but still function at levels that would be below a

therapeutic value in patients with thalassaemia (May et

al., 2002).

However, all the above vector systems involve

random integration and so, insertional mutagenesis is

of concern. Recently, successful gene therapy of

children with X-linked severe combined immune

deficiency was overshadowed by the development of

leukemia in two patients (Kohn et al., 2003). The

mechanism by which these two patients developed

leukemia is not clear. It is known however that the

transgene was integrated into or near the oncogene

LMO (chromosome 11) in both of these two unrelated

cases and this indicates that LMO2 transactivation is

likely to be involved in the development of leukemia.

The whole event reveals that the random integration of

the transgene into the host genome may be linked to

insertional mutagenesis, leading to malignant

transformation.

II. Episomal vectors

The use of Replicating Episomal Vectors (REVs)

(Sclimenti et al., 1998) offers an attractive alternative

Gene therapy 3

to integrating retroviral and adeno-associated viral

(AAV) vectors for producing long-term gene

expression within a gene therapy context. REVs, being

episomal, do not integrate into the host genome. Their

main advantages are that:

1. They are not connected with potential hazards

deriving from insertional mutagenesis that is an

inherent problem with integrating viral vectors.

2. They do not pose the same size limitations on

the therapeutic transcription unit as do viral

vectors, with inserts in excess of 300kb being a

possibility (Sun et al., 1994).

3. They can be introduced into the target cells

using non-viral delivery systems that can be

produced more cheaply at scale than with viral

vectors (Satoh et al., 1997; Saeki et al., 1998;

Tsukamoto et al., 1999).

The development of REVs is currently gaining

momentum and REVs based on viral origins of

replication such as those from EBV (Yates et al.,

1985), human papovavirus BK (De Benedetti et al.,

1991; Sabbioni et al., 1995), BPV-1 (Ohe et al., 1995)

and SV40 (Cooper et al., 1997) hold the greatest

promise. Combinations of viral and mammalian

chromosomal origins of replication have also been

found to improve nuclear retention of the episomes

(Krysan et al., 1989; Wohlgemuth et al., 1996).

Episomal vectors for the transfer of human ßglobin

gene appeared only last year in the literature

and they are still in the phase of evaluation of their

potential, after transfer in established cell lines.

The use of self-replicating episomal vectors

(REVs) based on viral origins of replication such as

from EBV (Yates et al., 1985) and SV40 (Cooper et al.,

1997), have offered several potential advantages to the

transfer of ß-globin gene transcription units based on

work in tissue culture cell lines. These benefits are:

(i) the possibility of constructing large size

therapeutic transcriptional units that can

contain the total LCR and other controlling

elements of ß-globin gene regulation

(ii) the possibility of long term expression of ßglobin

gene under the control of the total LCR.

Tissue-specific gene expression of the human ßglobin

from within REVs has been assessed in two

recent works. In the first study (Chow et al., 2002)

with REVs based on the oriP/EBNA1 system of EBV,

we assessed the expression of the human ß-globin gene

either alone or in combination with core elements of

the ßLCR within plasmid REVs and show that high


4 Aglaia Athanassiadou

Figure 1: Construct ß-cos203. This cosmid vector is based on the ori P/EBNA I system of EBV and contains the hygromycin

resistance gene (Hygro) for selection in mammalian cells and the ampicillin resistance gene (Amp) for selection in prokaryotic

cells. The insert contains the “ß-LCR minilocus” that is: the genomic ß-globin gene along with the complete LCR, which

normally resides 50Kb 5’ the ß-globin gene and the 3’ DNase hypersensitive enhancer, which normally resides 20Kb 3’ the

ß-globin gene.

levels of erythroid-specific expression can, in cases, be

conferred by this element. In addition, a 48 kb ßLCR

minilocus cosmid REV, (Figure 1) containing the total

ßLCR and the 3’ DNase hypersensitive enhancer of the

ß-globin gene, is efficiently expressed and

p ropagated in an episomal state in K562 and

importantly, in MEL cells (Table 1). Each of these

transfected cell lines contains on average 1 to 2 copies

of replicating transgenes, as estimated by Southern

blot analysis, after double digestion with EcoRI and

the DpnI. The latter digests only DNA methylated by

adenosine methyltransferase, and therefore DNA

grown in procaryoyic cells, thus eliminating the nonreplicating

transferred cosmids. Most interestingly, in

the K562 cells tested, the cosmid retains high levels of

ß-globin gene expression for three months, in the

presence as well as in the absence of selection pressure

(hygromycin).

In the second work (Black and Vos, 2002), based

on the same REV system but containing a 185kb

fragment, encompassing the whole of the ß-globin

locus and more, the ß-globin transgene is efficiently

expressed in cultures of murine myoblasts, again for

three months, with and without selection.

These data demonstrate the potential utility of this

system for gene therapy applications

C. The target cells

These are usually immopralised cell lines, in which

the potential for regulated expression of the transgene

is estimated, e.g.:

1. K562, a human, chronic myelogenous leukemia

line, trisomic for chromosome 11, displaying

erythroid properties. It constitutively expresses


the embryonic ε- and foetal γ-globin, but not

the adult ß-globin gene, thus facilitating

detection of ß-globin transgene expression.

2. MEL cells, an adult-stage murine

erythroleukemia cell line, inducible to terminal

erythropoiesis.

When experiments in such cell lines are promising,

it is appropriate to proceed with transfer into mice

models of the disease, to see if correction of disease

phenotype is possible.

The final goal for the gene therapy of the

Haemoglobinopathies is ß-globin gene transfer into

Human Haemopoetic Stem Cells (HSC).

Hematopoietic stem cells as target cells for gene

transfer, have been central in the development of gene

therapy strategies for the treatment of genetic disorders

that affect the function of the various blood cell types.

They can be in culture, thereby allowing eexx vviivvoo gene

transfer, as opposed to the iinn vviivvoo gene transfer into

organs or tumours. Additionally they are unique for

their ability to produce all blood cell types.

Eventually the transduced HSC will be transferred

into the patient bone marrow from whom they derive

and their effect on reversing the disease phenotype will

be estimated.

D. Concluding remarks

Gene therapy 5

Table 1: Data on ß-cos203 transfections: Two transfected cell lines in K562 cells (# 1 and # 2) and two transfected cell lines

in MEL cells ( # 3 and # 4) are shown. Copy number of episomes per cell refer to average per cell line, as estimated by Southern

blot analysis of cosmid DNA isolated along with genomic DNA, normalized against endogenous two copies /cell genes.

Replicating copies of episomes were estimated after digestion with DpnI restriction enzyme (see text). Expression is estimated

by S1 nuclease protection assay on total RNA from transfected lines and normalized against the expression of endogenous genes

as internal controls.

A. K562 cell lines:

Cell line No # 1 # 2

COPY number 4 2

of episomes / cell

COPY number of 1 2

replicating episomes / cell

EXPRESSION 25% 30%

per replicating transgene

+Hygro-cultures

EXPRESSION 27% 29%

per replicating transgene

-Hygro-cultures

B. MEL cell lines:

Cell line No # 3 # 4

COPY number 10 2

of episomes / cell

COPY number of 1 1

replicating episomes / cell

EXPRESSION 20% 38%

per replicating transgene

+Hygro-cultures

The gene therapy for the Haemoglobinopathies is not

here yet. The ß-globin gene is a textbook paradigm for

the molecular genetic analysis of human genes and its

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It seems that unknown so far elements that reside in

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vectors that can take in large enough pieces of insert

DNA to include the total ßLCR and other control

elements came along with the episomal vectors.

The demand is still on a vector system that will


6 Aglaia Athanassiadou

combine all the desired properties in one. It is believed

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Gene therapy 7


Journal of Cell and Molecular Biology 3: 9-14, 2004.

Haliç University, Printed in Turkey.

Systemic acquired resistance: Characterization of genes associated with

plant defence response

‹skender Tiryaki 1 * and Hasan Tunaz 2

1Department of Agronomy, Kahramanmarafl Sütçü ‹mam University Kahramanmarafl 46060, Turkey;

2Department of Plant Protection, Kahramanmarafl Sütçü ‹mam University Kahramanmarafl 46060,

Turkey (*author for correspondence)

Received 30 September 2003; Accepted 1 December 2003

Abstract

Plants show their defence mechanism against pathogens and insects by induction of both localized and systemic

responses. Of these, systemic acquired resistance (SAR) provides a protection on the uninfected tissues by activating

different signaling pathways leading to induction of several pathogen related (PR) genes upon contact with invaders.

Details of the molecular mechanisms of SAR, including pathogenesis-related proteins and their expression, and the

signals including salicylic acid and jasmonates are still unclear. However, characterization of mutants involved in

this response pathway helps to dissect this complex mechanism of SAR. In this paper, it will be discussed genes

associated with SAR and the identification of mutants involved in this response pathway.

KKeeyy wwoorrddss:: Resistance, mutants, salicylic acid, jasmonates, gene

Sonradan sistemik olarak elde edilmifl direnç: Bitki savunma cevab› ile ilgili genlerin

karakterizasyonu

Özet

Bitkiler patojen ve böcek sald›r›lar›lar›na karfl› hem lokalize hem de sistemik olan savunma mekanizmalar›n›

harekete geçirmek suretiyle tepki gösterirler. Bunlardan bu sald›r›lara karfl› koymak amac›yla bitkide bir bütün olarak

gelifltirilen “Sistemik kazan›lm›fl direnç” (SAR), infekte olmayan dokularda patojenlerle ilgili birçok genin

uyar›lmas›na öncülük eden farkl› sinyal ak›fl a¤lar›n› harekete geçirmek suretiyle bir koruma sa¤lar. SAR’›n

moleküler mekanizmas›n› oluflturan patojeniteyle ilgili proteinler ile bunlar›n ekspresyonlar› ve bu mekanizmada

görev alan salisilik asit ve jasmonat gibi sinyal moleküllerinin detaylar› tam anlam›yla bilinememektedir. Ancak

tepkiye ba¤l› sinyal ak›fl a¤lar›nda görev alan mutant bitkilerin karakterizasyonu, kompleks bir yap›ya sahip olan

SAR’›n moleküler mekanizmas›n› küçük bileflenlerine ay›rarak daha kolay anlafl›labilmesine yard›mc› olmaktad›r.

Bu makalede SAR ile ilgili genler ile bu tepki ak›fl›nda görev alan mutantlar›n belirlenmesi tart›fl›lacakt›r.

AAnnaahhttaarr ssöözzccüükklleerr:: Direnç, mutant, salisilik asit, jasmonat, gen

9


10 ‹skender Tiryaki and Hasan Tunaz

Introduction

Plant diseases and insects are major constraints to

plant growth and development. The primary control

tactic for managing pathogens has relied solely on

chemical control. In recent years, increased awareness

of the potential adverse effects that pesticides can have

on the environment has underscored the need to

develop alternative, nonchemical methods of

controlling diseases. The development of plants with

enhanced resistance to pathogens is one alternative

method for controlling pathogens; however, the

incorporation of disease resistance into plants has been

restricted by limited genetic variability and the

potential for pathogens to evolve resistant biotypes.

Molecular techniques, such as recombinant DNA and

gene transformation techniques provide useful tools

for augment traditional breeding efforts.

One area of research related to diseases resistance

that scientists are focusing on is the plant’s defence

mechanisms. Plants successfully resist invasion from

an array of pathogens by inducing plant responses. A

hypersensitive response is one example of a defence

response utilized by plant to combat pathogens. The

hypersensitive response causes necrosis of plant cells

around the cite of infection which serves to limit the

spread of the infection throughout the plant. Besides

localization of infection, hypersensitive response

triggers a phenomenon known as systemic acquired

resistance (SAR). SAR has recently generated

considerable attention; however, details of the signal

transduction pathway leading to SAR are still unclear.

This paper will strictly discuss SAR related genes and

the identification of mutant involved in this response

pathway.

Plant defence pathways

Plants response in a variety of ways to pathogen attack

that refers not only microbial pathogens but also

nematodes, insects, or herbivores, as well as treatment

with certain chemicals, or other types of stresses

(Sticher et al., 1997). Plant defence responses to these

attacks have been categorized into three primary

pathways: the gene for gene resistance pathway,

pathways that serve to limit virulent pathogen from

spreading throughout the plant, and the SAR pathway

(Glazebrook et al., 1997).

In the gene for gene resistance pathway, the host is

resistant when the pathogen has a particular avirulence

gene that corresponds to a specific resistance (R) gene

in the host. Plants containing a specific R gene are able

to recognize pathogens that carry a corresponding

aviru-lence (avr) gene that leads to localized cell death

called the hyper-sensitive response (HR) and limits the

spread of the pathogen (Glazebrook et al., 1997;

Rairdan and Delaney, 2002).

The gene for the gene pathway not only stimulates

the localized hypersensitive response in plant, but also

SAR for future pathogen attacks. In the SAR pathway,

infection by a pathogen that causes host cell that

triggers a signal to be emitted throughout the plant, this

signal triggers the activation of defence genes in

uninfected tissues, and results in the plant showing

enhanced resistance to subsequent infection by an

array of pathogens (Glazebrook et al., 1997).

Systemic acquired resistance (SAR)

Studies to discern the factors responsible for

stimulating and controlling SAR proceeded the

discovery that salicylic acid produces resistance in

plants to an array of pathogens (Gaffney et al., 1993;

Delaney et al., 1994). This finding suggested that

salicylic acid is an important component for the

induction of SAR. Salicylic acid is widely distributed

in both monocot and dicot plants (Raskin et al., 1990).

Evidence for the support of salicylic acid to play an

important role in SAR come from experiments using

plants transformed with bacterial nahG gene (Gaffney

et al., 1993). This gene encodes salicylate hydroxylase

which is an enzyme that converts salicylic acid to an

inactive compound called catechol. These experiments

showed that plants expressing salicylate hydroxylase

were unable to accumulate salicylic acid following

pathogen attack and as a result these plants lacked the

ability to activate SAR genes or to develop resistance

(Gaffney et al., 1993). Several researches have also

demonstrated that the application of salicylic acid or

salicylic analogs: 2,6 dichloroisonicotinic acid and

benzo-(1,2,3)-thiadiazole-7-carbothioic acid have the

ability to stimulate SAR (White, 1979; Metraux et al.,

1991; Gaffney et al., 1993; Gorlach et al., 1996);

whereas, the elimination of salicylic acid from the

plant leads to plants that are incapable of establishing

a SAR response (Gaffney et al., 1993; Delaney et al.,

1994). These studies further support the notion that

salicylic acid plays a role in the SAR pathway. It is still


unclear if salicylic acid serves as systemic signal for

plant defence pathway, nonetheless, it is evident that if

salicylic acid cannot build-up in the plant, SAR gene

expression and disease resistance cannot be induced.

Pathogenesis-related proteins (PRs) have also been

shown to be associated with the initiation of SAR. PRs

accumulate after pathogen attack or related situations

(van Loon et al., 1994). Research has identified the

expression of PR-1 (Pathogen-Related -1), PR-5, and

BGL2 (1, 3-ß-glucanase) to be correlated with the

SAR response (Uknes et al., 1992). Because PR genes

are associated with the onset of SAR, they can be

utilized as target genes to study defence pathways.

White (1979), Ward et al. (1991), and Uknes et al.

(1992) demonstrated that an increase in salicylic acid

levels stimulates the accumulation of PR proteins.

These experiments gave the basis for the proposed

hypothesis that the accumulation of salicylic acid

triggers the expression of PR proteins that serve to

limit infection of the host (Ward et al., 1991). The PR

genes: PR-1, BGL2, and PR-5 have been identified as

genes regulated by salicylic acid (Uknes et al., 1992).

Ward et al. (1991) demonstrated that tobacco salicylic

acid stimulates the production of the same PR proteins

that infection by the tobacco mosaic virus does.

The isolation of plant mutants with defects in

specific defence responses, such as SAR has allowed

the roles these responses play in fighting pathogens to

be investigated by studying the effects of their absence

on plant pathogen interactions. Method employed to

identify and characterize genes associated with SAR

include genetic screens of mutants that display altered

SAR responses and studying the activity of the BLG2-

GUS reported gene (Delaney, 1997; Glazebrook et al.,

1997). Using these techniques, several Arabidopsis

mutants have been isolated and studied to investigate

the regulatory effect they have on SAR signaling

(Delaney, 1997; Maleck et al., 2002; Rairdan and

Delaney, 2002).

Arabidopsis thaliana has been the primary plant

species, along with tobacco and tomato, for genetic

analysis because it possess several characteristics that

make it desirable for identifying and cloning genes of

interest, including a small genome size, rapid

generation time, its map and genome sequence are

available, and mutation and transgenic techniques

have been developed and optimized for this species

(Baker et al., 1997).

Several mutants have been isolated that display

altered SAR responses. This review will strictly focus

Genes in plant defence response 11

on identification of these mutants (cpr, cim, lsd, acd2,

nim1, and npr1) and a brief discussion of their role in

the SAR defence response. Mutants that have been

found to be associated with SAR can be divided into

two categories: mutants that are constitutive

expressors of PR genes and SAR, and mutants that fail

to express PR and develop SAR (Delaney, 1997).

The identification of mutants that display

constitutive expression of SAR has been achieved by

analyzing RNA from mutagenized seedlings using

northern blot analysis. SAR genes were used as probes

for the hybridization experiments. Three classes of

mutants identified using this technique were the cim

(constitutively immune) mutants, the lsd (lesion

stimulating disease) mutants, and the acd2

(accelerated cell death) mutant (Lawton et al., 1993;

Dietrich et al., 1994; Greenberg et al., 1994). These

mutants all have constitutive expression of SAR,

however, the lsd and acd2 mutants are different from

the cim mutant because they develop lesions as a result

of spontaneous cell death. The results of this research

have demonstrated that the cim, lsd, and acd2 mutants

are associated with increased levels of salicylic acid,

expression of defence genes (PR-1, PR-5, and BGL2),

and resistance to virulent pathogens.

A second method developed for the identification

of mutants that exhibit constitutive expression of SAR

is the use of transgenic plants possessing the ß-1, 3glucanase

(PR-2) promoter (BGL2) attached to a

reporter gene that encodes GUS. The idea behind this

method is to identify mutants with increased

expression of PR-2. Using this technique, the cpr1 and

cpr6 mutants, a fourth class of mutants having

increased expression of defence related proteins (PR-

1, PR-5, and BGL2) and enhanced diseases resistance,

were identified (Bowling et al., 1994; Clarke et al.,

1998) that the cpr6 mutant also expresses the defensin

proteins PDF1.2 and Thi2.1. Defensins are small

antifungal proteins found in the animal and plant

kingdom (Broekaert et al., 1997). In Arabidopsis,

systemic expression of the defensin gene PDF1.2 is

induced during infection by the fungus Alternaria

brassicicola (Penninckx et al., 1996). The PDF1.2

gene is up-regulated by jasmonic acid (JA) but not by

salicylic acid (SA) and, importantly, after infection

with A. brassicicola, JA accumulates in both infected

and non-infected leaves (Kachroo et al., 2001; Shah et

al., 2001; Yoshioka et al., 2001).

Genetic studies have determined the cpr1 mutation

to be recessive which suggests that CPR1 gene may


12 ‹skender Tiryaki and Hasan Tunaz

serve to suppress salicylic acid build-up in the plant

(Bowling et al., 1994). The cpr6 and cim mutations, on

the other hand, are dominant and therefore these

mutants may be responsible for constitutive expression

of factors that are positive regulators of salicylic acid

accumulation. It is believed that these mutations act

upstream of salicylic acid in the SAR pathway because

depletion of salicylic acid prevents SAR gene

expression (Glazebrook et al., 1997; Clarke et al.,

1998).

In addition to mutants that have constitutive

expression of SAR, several mutants have been

identified that fail to develop SAR. Screening BGL2-

GUS transgenic plant treated with salicylic acid or 2,6dichloroisonicotinic

acid and identifying mutants that

fail to exhibit increased expression of GUS has led to

identification of the npr mutant (Cao et al., 1994). This

mutant fails to exhibit expression of PR proteins and

induction of SAR genes despite treatment with

salicylic acid or 2,6-dichloroisonicotinic acid (Cao et

al., 1994).

Screening plants treated with chemicals that induce

SAR for resistance to certain pathogens has been

another technique for isolating mutants that fail to

exhibit SAR. A nim1 mutant has been isolated that

does not express PR proteins and fails to develop

resistance to Pseudomonas parasitica after treatment

with 2,6-dichloroisonicotinic acid must act through

NIM induction of SAR pathway since treatment of the

nim1 mutant with 2,6-dichloroisonicotinic acid does

not activate SAR (Delaney et al., 1995).

Experiments conducted by Delaney (1997) and

Shah et al. (1997) showed that the nim1 and npr1

mutants are allelic. Because both of these mutants fail

to induce expression of SAR despite treatment with

salicylic acid and 2,6-dichloroisonicotinic acid, it is

proposed that NPR1 and NIM1 genes act in the SAR

signal transduction pathway; however, at a location

downstream from salicylic acid (Delaney et al., 1995).

A tomato mutant, def1, deficient in jasmonate

biosynthesis, fails to accumulate PinII in response to

wounding and exhibits susceptibility to feeding by

tobacco hornworm, Manduca sexta (Howe et al.,

1996). However, methyl jasmonate when applied to

the surface of tomato leaves, induces the synthesis of

defensive proteinase inhibitor proteins in the treated

plants and in nearby plants incubated in the same

chamber with MeJA sprayed plants (Farmer and Ryan,

1990). It was also shown that when tomato and 5 g of

leafy branches of Artemisia tridentata, a plant shown

to possess methyl jasmonate in leave surface structure,

were incubated in the same chamber with no physical

contact, proteinase inhibitor accumulation is induced

in tomato leaves (Farmer and Ryan, 1990).

Clearly, further studies are needed to assess the

relationship among these components discussed to

determine their exact regulatory effect and/or role in

the SAR response. These types of studies will

ultimately help us to understand the SAR signal

transduction pathway. For instance, Clarke et al.

(1998) have investigated the cpr6 mutant and as a

result of their work they have proposed that CPR6 may

express PR genes by the two distinct mechanisms: (1)

direct interaction by which CPR6 expresses PR genes

will provide useful insight into the plant’s defence

response to pathogen attack. In addition, microarray

analysis of gene expression related to SAR started to

give a broader perspective on this manner (Glazebrook

et al., 2003).

Conclusion

Genetic analysis of mutants has been demonstrated to

be a useful and powerful tool for investigating defence

pathways. The understanding of the mechanisms

associated with SAR pathway has advanced

significantly since the initial discovery of this

pathway. However, much work remains to be done in

elucidating the signal transduction pathway

responsible for the induction of this defence response

and components that regulate this response.

The study of SAR has provided valuable insight

into plant disease resistance and will undoubtedly aid

in the development of novel strategies for achieving

disease control. Delaney (1997) proposed three

potential ways the information generated from studies

on the SAR pathway can be used for developing novel

disease suppression methods: (1) the development of

agrochemicals that elicit the SAR; (2) the development

of plants with enhanced disease resistance, i.e. the

isolation and incorporation of traits with increased

inducible defence systems or constitutive expression

of SAR; and (3) the isolation and incorporation of

genes that regulate the SAR pathway in an attempt to

manipulate plants to express SAR at optimal times and

conditions.

It is evident after preparing this review that

significant progress has been made in identifying the

SAR signal transduction pathway and components that


serve to regulate this defence response. Nonetheless,

additional research is needed in order to fully

understand how this pathway functions and the

potential for manipulation of this defence for the

development of novel disease control strategies.

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10: 69-78, 1997.


14 ‹skender Tiryaki and Hasan Tunaz

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Plant J. 26: 447-459, 2001.


Journal of Cell and Molecular Biology 3: 15-21, 2004.

Haliç University, Printed in Turkey.

Biological investigations into AAnnttiiddeessmmaa mmaaddaaggaassccaarriieennssee Lam.

(Euphorbiaceae), FFaauujjaassiiooppssiiss fflleexxuuoossaa (Lam.) C. Jeffrey (Asteraceae),

TTooddddaalliiaa aassiiaattiiccaa (L.) Lam. and VVeepprriiss llaanncceeoollaattaa (Lam.) G. Don

(Rutaceae)

Fawzia Bibi Narod 1 , Ameenah Gurib-Fakim 1 * and Anwar Hussein Subratty 2

1 Department of Chemistry, 2 Department of Health & Medical Sciences; Faculty of Science, University of

Mauritius, Reduit, Mauritius (*author for correspondence)

Received 1 September 2003; Accepted 30 September 2003

Abstract

Extracts obtained from four endemic medicinal plants of Mauritius, namely, Antidesma madagascariense Lam.

(Euphorbiaceae), Faujasiopsis flexuosa (Lam.) C. Jeffrey (Asteraceae), Toddalia asiatica (L.) Lam. and Vepris

lanceolata (Lam.) G. Don (Rutaceae), were screened for antimicrobial activity. Our results showed that twenty out

of twenty-eight tested extracts possessed antibacterial activity against at least one of the four test bacteria, while 17

extracts showed antifungal activity against at least one of the two test fungi. Furthermore, eight extracts obtained

from these plants were found to elicit contractile responses in isolated rat smooth muscles in-vitro. Attempts were

also made towards the validation of three herbal teas prescribed by traditional healers.

KKeeyy wwoorrddss:: Medicinal plants, antibacterial activity, antifungal activity, smooth muscles, Mauritius

AAnnttiiddeessmmaa mmaaddaaggaassccaarriieennssee Lam. (Euphorbiaceae), FFaauujjaassiiooppssiiss fflleexxuuoossaa (Lam.) C.

Jeffrey (Asteraceae), TTooddddaalliiaa aassiiaattiiccaa (L.) Lam. and VVeepprriiss llaanncceeoollaattaa (Lam.) G. Don

(Rutaceae) da biyolojik araflt›rmalar

Özet

Mauritus’un endemik t›bbi bitkileri olan Antidesma madagascariense Lam. (Euphorbiaceae), Faujasiopsis flexuosa

(Lam.) C. Jeffrey (Asteraceae), Toddalia asiatica (L.) Lam. and Vepris lanceolata (Lam.) G. Don (Rutaceae) dan elde

edilen ekstreler antimikrobiyal aktivitelerini belirlemek üzere tarand›. Sonuçlara göre denenen 28 ekstreden 20 sinin

denenen dört bakteriden en az birine karfl› antibakteriyel aktivite gösterdi¤i, di¤er taraftan 17 ekstrenin denenen iki

mantardan en az birine karfl› antifungal aktivite gösterdi¤i saptand›. Ayr›ca bu bitkilerden elde edilen sekiz ekstrenin,

in vitro da izole edilen s›çan düz kaslar›nda kas›lmaya sebep oldu¤u belirlendi. Ayr›ca geleneksel yöntemlerle tedavi

yapanlar›n reçetelerine yazmalar› için üç bitki çay›n›n yürürlü¤e girmesi girifliminde bulunuldu.

AAnnaahhttaarr ssöözzccüükklleerr:: T›bbi bitki, antibakterial aktivite, antifungal aktivite, düz kaslar, Mauritus

15


16 Fawzia Bibi Narod et al.

Introduction

It is known that phytochemicals with biological

properties will continue to offer potential sources of

new antibiotics, anticancer and cardiovascular drugs

amongst other pharmaceutical agents. Following this

biorational approach and as part of a study on the flora

of Mauritius (a tropical island located in the South

West of Indian Ocean), 4 endemic medicinal plants,

namely

1. Antidesma madagascariense Lam.

(Euphorbiaceae),

2. Faujasiopsis flexuosa (Lam.) C. Jeffrey

(Asteraceae),

3. Toddalia asiatica (L.) Lam. (Rutaceae) and

4. Vepris lanceolata (Lam.) G. Don (Rutaceae)

were tested for potential antibacterial and antifungal

properties as well as their possible effects on isolated

rat ileal and aortal smooth muscles in vitro.

Both Antidesma madagascariense Lam. and

Faujasiopsis flexuosa (Lam.) C. Jeffrey have been

traditionally used in the treatment of dysentery and

diabetes in the local pharmacopoeia of Mauritius

(Gurib-Fakim et al., 1995, 1996). A. madagascariense

Lam. is also reported to possess diuretic, astringent

and febrifuge properties (Gurib-Fakim et al., 1996) .

On the other hand, Toddalia asiatica (L.) Lam. and

Vepris lanceolata (Lam.) G. Don are reported to be

effective against asthma and related respiratory

ailments (Gurib-Fakim et al., 1997). Furthermore, T.

asiatica (L.) Lam. is also used in the local traditional

medicine to relieve fever (Gurib-Fakim et al., 1997).

In the present study, we report the antimicrobial

properties of extracts obtained from A.

madagascariense Lam., F. flexuosa (Lam.) C. Jeffrey,

T. asiatica (L.) Lam. and V. lanceolata (Lam.) G. Don

as well as the effects of the plant extracts on rat ileal

and aortal smooth muscles in vitro. An attempt has also

been made to validate the ethnobotanical uses of three

different herbal teas containing A. madagascariense

Lam., T. asiatica (L.) Lam. and V. lanceolata (Lam.)

G. Don respectively.

Materials and methods

Plant materials

Plant materials were collected from Macchabe and

Petrin forests and were identified by the Curator of the

National Herbarium, Mauritius Sugar Industry

Research Institute. Voucher specimens were deposited

at the herbarium collection of the Department of

Chemistry, Faculty of Science, University of

Mauritius.

Extraction and fractionation

Dried powdered plant material was extracted to

exhaustion with methanol in a Soxhlet apparatus and

the solvent was then distilled off under reduced

pressure to afford crude plant extracts. Fractionation

was performed by subjecting dried powdered plant

material to Soxhlet extraction using a gradient elution.

Each plant fraction was then evaporated to dryness

under reduced pressure.

Preparation of plant extracts for antimicrobial tests

Plant extracts were dissolved in a mixture of methanol

and sterilized distilled water (1:1 v/v) before mixing

with the agar; non-polar plant fractions were

homogenized in a mixture of chloroform, water and

Tween 80 (1.5: 0.25: 0.25 v/v). Plant extract was

initially prepared at a concentration of 32 mg/ml;

extracts found to inhibit microbial growth at 32 mg/ml

were further investigated to determine their minimal

inhibitory concentration (MIC) values (Mitscher et al.,

1972). The plant extracts were tested against three

Gram-negative bacteria (Escherichia coli,

Pseudomonas aeruginosa, Salmonella typhimurium),

the Gram-positive bacterium Staphylococcus aureus

and the fungi Candida albicans and Aspergillus niger.

The test bacteria and Candida albicans are clinical

isolates obtained from the local hospital. Aspergillus

niger was obtained from the Department of Health and

Medical Sciences of the Faculty of Science, University

of Mauritius.

Antimicrobial testing

Antimicrobial screening was done by the agar dilution

method (Mitscher et al., 1972). Test fungi were

incubated for 48 h (Candida albicans at 37°C and

Aspergillus niger at 27°C) while the test bacteria were

incubated at 37°C for 24 h. The positive controls were

the antibacterial drugs Ciproglen (ciprofloxacin)

(Glenmark Pharmaceuticals Ltd, India) and Amoxil

(amoxycillin) (SmithKline Beecham Pharmaceuticals,

England) and the antifungal drug Nizoral


(ketoconazole) (Janssen, U.K.). Agar plates containing

only solvent and agar were used as negative controls.

MIC values for amoxycillin and ciprofloxacin are

given in Table 1. The MIC value for ketoconazole was

found to be 5.96 x 10 -6 mg/ml against C. albicans but

its MIC value was not determined against A. niger.

Antimicrobial testing of herbal teas

In order to test plants prepared in the way people use

herbal remedies, 3 herbal teas made out of A.

madagascariense Lam., T. asiatica (L.) Lam. and V.

lanceolata (Lam.) G. Don were investigated for their

antimicrobial activity. Herbal teas were made in water

according to the recipes prescribed by professional

herbalists (Gurib-Fakim et al., 1996,1997). Recipes for

the herbal teas that have been tested are:

(a) A. madagascariense Lam. herbal tea

10 leaves of A. madagascariense Lam.; a handful of

roots of Collier cipaye (Coix lacryma-jobi, Poaceae);

15 leaves of fandamane (Aphloia theiformis,

Flacourtiaceae); 15 cm of the bark of Bois de ronde

(Erythroxylum laurifolium Lam., Erythroxylaceae);

0.5 cm of the root of Manglier (Rhizophora

mucronata, Rhizophoraceae); 3 whole plants of La

Villebague (Bidens pilosa, Asteraceae) and 10 leaves

and liana of Bétel sauvage (Piper species, Piperaceae).

The mixture was boiled in water (1.5 litres) until

reduced to 1 litre. This infusion taken orally is reported

to be effective in cases of kidney ailments and

dysentery (Gurib-Fakim et al., 1996).

(b) T. asiatica (L.) Lam. herbal tea

An infusion of leaves of T. asiatica (L.) Lam. together

with leaves of citronelle (Cymbopogon citratus,

Poaceae) and a piece of ginger (Zingiber officinale,

Zingiberaceae) was prepared by boiling in water. Such

an infusion is reported to be effective in alleviating

fever (Gurib-Fakim et al., 1997).

(c) V. lanceolata (Lam.) G. Don herbal tea

Leaves and stems of V. lanceolata (Lam.) G. Don were

boiled together in water to produce an infusion, which

is reported to be effective against asthma, bronchitis

and related respiratory ailments (Gurib-Fakim et al.,

1997).

In-vitro testing of plant extracts on isolated rat ileal

and aortal smooth muscles

Ethical clearance for conducting animal

experiments was obtained from the Faculty of Science

Investigations in medicinal plants 17

Research Committee of the University of Mauritius.

Sprague-Dawley rats weighing between 50-100 g were

killed by a severe blow to the head. A stretch of the

lower part of ileum was removed, cut free of

mesentery and sectioned into pieces of 1 cm length.

Each ileal strip was washed to remove any remaining

food material using a hypodermic syringe filled with

Krebs’ solution (Gurib and Subratty, 2002). The aorta

of the rat was then removed, stripped of adhering

tissue and its lumen was washed with Krebs’ solution

containing 2 ml heparin (5000 IU/L). The ileal and

aortal strips were immersed in Krebs’ solution

(bubbled with carbogen, i.e., 95% O2 and 5% CO2)

containing 2ml heparin (5000 IU/L), until mounted in

the organ bath. Before mounting each strip, the glassjacketed

organ bath was filled with Krebs’ solution (25

ml), which was bubbled with carbogen for five

minutes. The organ bath solution was maintained at

37°C with a thermostatted water pump (Lauda, model-

1) and the pH of the solution adjusted to 7.4. Each

organ strip was mounted in the organ bath according to

the method described in literature (Gurib and Subratty,

2002; Subratty and Moonsamy, 1998). Isometric

contractile responses were recorded via a forcedisplacement

transducer connected to a multipen

recorder (Rikadenki Model R50, Japan). Each

mounted strip was adjusted to a resting tension of 1.5g

and allowed to equilibrate in the organ bath for about

15 mins until a baseline tone was achieved. Following

stabilization, the organ strip was challenged with 100,

200, 300, 400, 500, 600, 700, 800, 900 and 1000 µL of

the test plant extract. Contractile responses were

recorded as increases in the baseline tone while

relaxation responses were recorded as decreases in the

baseline tone taking into account the fact that during

stabilization, a tension of about 1.5 g was already

applied on the strips.

Preparation of plant extracts for challenging aortal

and ileal strips

Dried plant extract (132 mg) was dissolved in

methanol (1 ml). Addition of this solution (100 µL) to

the organ bath already containing Krebs’ solution (25

ml) afforded a final bath concentration of 0.528 mg/ml

(528 ppm) plant extract. For each series of

experiments, a control strip was included and

challenged with pure methanol (100 µL addition).


18 Fawzia Bibi Narod et al.

Table 1: MIC values obtained with ciprofloxacin and amoxycillin (mg/ml).

Results

Results obtained for the antimicrobial tests performed

E.coli P.aeruginosa S.typhimurium S.aureus

Ciprofloxacin 2.44 x10 -5 3.13 x10 -2 1.95 x 10 -4 3.13 x 10 -3

Amoxycillin >31.25 Not active 1.59 x 10 -3 3.16 x 10 -2

Table 2: Minimum Inhibitory Concentration (MIC-mg/ml) values of plant extracts and fractions.

Plant extracts Test organisms

E.coli P.aeruginosa S.typhimurium S.aureus A.niger C.albicans

1. A. madagascariense Lam.

Crude water extract (leaf and stem) 8 8 8 4 - -

Hexane fraction (stem) - 32 - 16 - -

Methanol: chloroform (1:1) fraction (stem) - 16 - 16 16 16

Methanol fraction (stem) 8 8 8 8 - -

Hexane fraction (leaf) - - - - 16 -

Methanol: chloroform (1:1) fraction (leaf) - 32 - - 16 -

Methanol fraction (leaf) 8 8 8 2 16 -

2. F. flexuosa (Lam.) C. Jeffrey

Crude water extract (leaf and stem) 4 8 4 4 - -

Hexane fraction (stem) - - - 16 - -

Methanol: chloroform (1:1) fraction (stem) 32 8 8 8 16 -

Methanol fraction (stem) - - - 8 - -

Hexane fraction (leaf) - - - - 16 -

Methanol: chloroform (1:1) fraction (leaf) 8 8 8 4 16 -

Methanol fraction (leaf) 8 8 8 4 16 -

3. T. asiatica (L.) Lam.

Crude water extract (leaf and stem) - 8 - 8 - -

Hexane fraction (stem) - - - - - -

Methanol: chloroform (1:1) fraction (stem) - 4 - 2 8 -

Methanol fraction (stem) - - - - 16 -

Hexane fraction (leaf) - 8 - 8 4 8

Methanol: chloroform (1:1) fraction (leaf) 16 16 16 16 16 -

Methanol fraction (leaf) - 8 - 16 16 -

4. V. lanceolata (Lam.) G. Don

Crude water extract (leaf and stem) - - - - - -

Hexane fraction (stem) - 32 - 32 - -

Methanol: chloroform (1:1) fraction (stem) - 16 - 16 4 -

Methanol fraction (stem) - - - - 4 16

Hexane fraction (leaf) - - - - - -

Methanol: chloroform (1:1) fraction (leaf) - - - 16 32 -

Methanol fraction (leaf) - - - - 16 -

Key - : no antimicrobial activity.

on extracts of A. madagascariense Lam., F. flexuosa

(Lam.) C. Jeffrey, T. asiatica (L.) Lam. and V.

lanceolata (Lam.) G. Don are presented in Table 2,


Table 3: Antimicrobial activity of herbal teas investigated (MIC values in mg/ml).

Herbal tea Test organisms

while Table 3 highlights the antimicrobial properties of

the herbal teas screened. The effects of plant extracts

obtained from these endemic plants on isolated rat ileal

and aortal strips in vitro are presented in Table 4.

Our results showed that twenty (71.4%) out of the

twenty-eight plant extracts screened possessed

antibacterial activity against at least one of the four test

bacteria with sample standard deviation 7.97,

Investigations in medicinal plants 19

E.coli P.aeruginosa S.typhimurium S.aureus A.niger C.albicans

A. madagascariense Lam. - 16 16 8 - -

T. asiatica (L.) Lam. - - 16 - 16 16

V. lanceolata (Lam.) G. Don - - - - - -

Key - : no antimicrobial activity. The figures give the MIC values in mg/ml

Table 4: Results of in vitro assays performed on rat ileal and aortal strips.

Plant fractions Responses induced in

Rat ileal strip Rat aortal strip

1. A. madagascariense Lam.

Methanol fraction of stem Weak contractile responses No response was induced in the rat

followed by strong sustained ones. aortal strips.

2. F. flexuosa (Lam.) C. Jeffrey

Methanol fraction of stem No observable response induced. Strong sustainable contractile responses.

Methanol fraction of leaf No observable response. Very strong sustainable contractile responses.

Chloroform: methanol (1: 1) Contractile responses initially Data not available.

fraction of leaf unsustained followed by

sustainable ones.

3. T. asiatica (L.) Lam.

Methanol fraction of leaf Strong contractile responses Strong sustainable contractile responses.

followed by weak ones.

Chloroform: methanol (1:1) No significant response induced. Data not available.

fraction of stem

4. V. lanceolata (Lam.) G. Don

Methanol fraction of stem Weak contractile responses Contractile responses of non-sustainable type.

followed by stronger and

sustained contraction.

Chloroform: methanol (1: 1) No significant contractile responses. No significant response induced.

fraction of stem

population standard deviation 7.89 and an arithmetical

mean of 11.38 for the MIC values. Only 17 extracts

(60.7%) were found to be active against at least one of

the test fungi with sample standard deviation 6.29,

population standard deviation 6.13 and an arithmetical

mean of 14.2 for the MIC values. Our results have

shown that the screened plant extracts were more

active against the test bacteria than the test fungi.


20 Fawzia Bibi Narod et al.

Though plant fractions obtained separately from leaves

and stems of V. lanceolata (Lam.) G. Don were found

to possess antibacterial and antifungal properties

(Table 1), curiously enough a crude water extract of

the plant (stems and leaves together) as well as the

herbal tea containing this same plant did not display

any antimicrobial or antifungal property.

Discussion

Both A. madagascariense Lam. and F. flexuosa

(Lam.) C. Jeffrey are known to possess astringent

properties and are used by the local population against

dysentery (Gurib-Fakim et al., 1995,1996). Dysentery

is classified as an infectious disease since it is

normally caused by microorganisms (Bloom, 1994;

Weller & Wells, 1990). Thus the observed

antimicrobial activity of A. madagascariense Lam. and

F. flexuosa (Lam.) C. Jeffrey (Table 2) might justify

their use against dysentery since extracts obtained

from both plants have been shown to possess the

ability to inhibit growth of both Gram-positive and

Gram-negative bacteria. It is also known that

contraction and relaxation of intestinal smooth

muscles play an important role in symptoms

accompanying dysentery (Bloom, 1994; Weller and

Wells, 1990). Hence the fact that extracts obtained

from A. madagascariense Lam. and F. flexuosa (Lam.)

C. Jeffrey have been shown to exhibit contractile

properties on rat ileal smooth muscles (Table 4) could

further validate the effectiveness of these plants in the

treatment of dysentery.

T. asiatica (L.) Lam. is used to relieve fever;

furthermore, a herbal tea obtained from leaves of T.

asiatica (L.) Lam., Cymbopogon citratus (Poaceae)

and a piece of ginger (Zingiber officinale,

Zingiberaceae) is also reported to alleviate fever

(Gurib-Fakim et al., 1997). Fever is very often the

symptom accompanying infections by bacteria or

viruses (Bloom, 1994). Both these ethnobotanical data

concerning T. asiatica (L.) Lam. have been validated

by our present investigations, since extracts of the

plant as well as the above-mentioned herbal tea have

exhibited antimicrobial activity (Tables 2 and 3).

In the literature, it would appear that T. asiatica

(L.) Lam. and V. lanceolata (Lam.) G. Don are

effective in the treatment of asthma (Gurib-Fakim et

al., 1997). Contraction of airway smooth muscles is

one of the cardinal features of bronchial asthma

(Pedersen et al., 1999). Our present investigations

have shown that extracts obtained from these plants

exhibit contractile properties on both rat ileal and

aortal smooth muscles in vitro (Table 4). It is arguable

whether with such contractile properties on isolated rat

smooth muscles, T. asiatica (L.) Lam. and V.

lanceolata (Lam.) G. Don could be effective against

asthma. Furthermore, the properties on aorta should

also not be overlooked. Nevertheless, the present work

can be extended to evaluate the possible effectiveness

of extracts and infusions of T. asiatica (L.) Lam. and V.

lanceolata (Lam.) G. Don on pre-contracted airway

smooth muscles mounted in vitro in view of studying

their effects on the airway muscles.

In conclusion, results of our present biological

investigations have shown that the 4 endemic

medicinal plants studied do possess interesting

biological activities and have to some extent validated

the reported ethnobotanical uses of the plants and their

herbal teas. Besides, our findings also highlight the

risks of use or abuse of medicinal plants; some of the

plant extracts tested induced contraction on rat aortal

strips (Table 4). Contraction on the aorta will lead

possibly to a hypertensive state and in the long run to

the development of cardiovascular accidence.

Therefore use of these plants by persons who may

have coronary artery diseases should be discouraged.

Acknowledgement

The authors are grateful to the Tertiary Education

Commission of Mauritius and the University of

Mauritius for financial support.

References

Bloom SR. Toohey’s Medicine – A Textbook for Students in

the Health Care Professions; 15 th Ed n . Longman Group

Ltd. 1-377, 1994.

Gurib-Fakim A, Guého J, Bissoondoyal MD. Plantes

Médicinales de Maurice. Tome 1. Editions de l’Ocean

Indien, Rose-Hill, Mauritius. 1- 492, 1995.

Gurib-Fakim A, Guého J, Bissoondoyal MD. Plantes

Médicinales de Maurice. Tome 2. Editions de l’Ocean

Indien, Rose-Hill, Mauritius. 1- 532, 1996.

Gurib-Fakim A, Guého J, Bissoondoyal MD. Plantes

Médicinales de Maurice. Tome 3. Editions de l’Ocean

Indien, Rose-Hill, Mauritius. 1- 472, 1997.

Gurib FBH, Subratty AH. Effect of genistein, a tyrosine

kinase inhibitor, on TAME-esterase induced contractions


in rat aorta in vitro. Indian J Exp Biol. 40: 1191-1194,

2002.

Mitscher LA, Leu RP, Bathala MS, Wu WN, Beal JL.

Antimicrobial agents from higher plants. I. Introduction,

rationale and methodology. Lloydia. 35: 157-166, 1972.

Pedersen O, Gurib-Fakim A, Subratty AH, Adersen A.

Pharmacological properties of seven medicinal plants of

the Rubiaceae from Mauritius. Pharmaceutical Biology.

36 (5): 1-6, 1999.

Subratty AH, Moonsamy J. Is TAME a potent constrictor of

non-airway smooth muscles? Indian J Exp Biol. 36: 618-

621, 1998.

Weller BF, Wells RJ. Baillière’s Nurses’ Dictionary; 21 st Ed n .

Baillière Tindall Ltd. 1-594,1990.

Investigations in medicinal plants 21


Journal of Cell and Molecular Biology 3: 23-31, 2004.

Haliç University, Printed in Turkey.

The role of mmeettaa-topolin in senescence of wheat leaf segments

Narçin Palavan-Ünsal 1 *, Serap Ça¤ 2 and Ergül Çetin 2

1 Haliç University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics,

F›nd›kzade 34280, Istanbul-Turkey; 2 Istanbul University, Faculty of Science, Department of Biology,

Süleymaniye 34460, Istanbul-Turkey (*author for correspondence)

Received 24 September 2003; Accepted 13 January 2004

Abstract

Numerous reports ascribe a stimulatory or inhibitory function of cytokinins in different developmental processes

such as root growth and branching, control of apical dominance in shoot, chloroplast development and leaf

senescence. Recently Strnad et al. (1997) discovered a new aromatic cytokinin, namely meta-topolin and they

suggested this substance as a potential alternative of benzyladenin.

In our previous study we have determined that a rapid breakdown of chlorophyll and proteins in excised wheat

leaf segments during the senescence period prevented by aromatic cytokinin meta-topolin . In this study, peroxidase

activity was increased and senescence delayed compared to control leaves in meta-topolin treated leaf segments, and

total chlorophyll content also exhibited similar results. Application of mT at high concentrations (0.5-1 mM) caused

a decrement in polyamine content, but at low concentration (0.25 mM) it reasoned increment. Besides these, metatopolin

decreased the loss of total chlorophyll and it was estimated similar trends for total nitrogen content. In

summary, according to the results of our study it can be concluded that meta-topolin has a contributing mechanism

of the antisenescence action.

KKeeyy wwoorrddss:: meta-topolin, nitrogen, peroxidase, polyamine, senescence

MMeettaa-topolinin bu¤day yaprak segmentlerinin senesensindeki rolü

Özet

Birçok araflt›rma sitokininlerin farkl› geliflim olaylar›nda örne¤in kök büyümesi ve dallanmas›nda, gövdede apikal

dominansinin kontrolünde, kloroplast gelifliminde ve yaprak senesensinde teflvik edici veya ket vurucu ifllevlere

sahip oldu¤unu ortaya koymufltur. Son y›llarda Strnad ve ark. (1997) meta-topolin ad› verilen yeni bir aromatik

sitokinin keflfetmifller ve bu maddenin benziladenine potansiyel bir alternatif oldu¤unu ileri sürmüfllerdir.

Biz daha önce bu¤day yaprak segmentlerinde senesens s›ras›nda klorofil ve proteinin h›zl› y›k›m›n›n aromatik

sitokinin meta-topolin taraf›ndan önlendi¤ini gösterdik. Bu araflt›rmada meta-topolin uygulanan yaprak

segmentlerinde peroksidaz aktivitesi kontrol yapraklar›na k›yasla artt›, senesens gecikti ve total klorofil içeri¤inde

de benzer bulgular elde edildi. Ayr›ca, mT in yüksek konsantrasyonda (0.5-1 mM) uygulanmas› poliamin içeri¤inde

azal›fla, düflük konsantrasyonda ise (0.25 mM) art›fla sebep oldu. Bundan baflka, meta-topolin total klorofil kayb›n›

azaltt› ve benzer e¤ilim total azot içeri¤inde de saptand›. Özetle meta-topolin’in antisenesens aktivitede yönlendirici

bir mekanizmaya sahip oldu¤u sonucuna var›labilinir.

AAnnaahhttaarr ssöözzccüükklleerr:: meta-topolin, azot, peroksidaz, poliamin, senesens

23


24 Narçin Palavan-Ünsal et al.

Introduction

Plant senescence is initiated and accompanied by a

series of derivative events. In leaves senescence is

correlated with a sharp decrease in nucleic acid and

protein content, followed by disintegration of

chloroplast structure and ultimately chlorophyll loss

(Thimann, 1980; Stoddart and Thomas, 1982).

Cytokinins have been regarded as the most potent

senescence-retarding hormones in plants and play a

significant role in the regulation of leaf senescence

(Richmond and Lang, 1957; Thimann, 1980).

Retardation of senescence by cytokinin, including

benzyladenin (BA) in excised leaves and cotyledons

have been reported in several plant species and this

synthetic growth regulator has little or no effect as a

retardant of senescence in attached organs (Kraus et

al., 1993; Gilbert et al., 1980).

A new family of endogenous aromatic cytokinin

have been discovered by Strnad et al. (1997) and

named as meta-topolin (6-[3-hydroxylbenzylamino]purine)

(mT). mT was put forwarded by these

researchers as an alternative of BA.

The activities of protease and peroxidase (POD)

have been reported to show an increase with the

advancement of senescence (Grover and Sinha, 1985).

There are several studies showing that POD activity

increases during senescence of detached leaves or leaf

discs (Parish, 1968; Mukherjee and Rao, 1993).

Increases in the activity of POD isoenzymes with the

physiological age of the leaves have been reported

(Parish, 1968; Ford and Simon, 1972). Parish (1968)

also suggested that the increase in the activity of POD

is one of the most reliable indicators of maturity and

senescence. However Srivastava et al. (1983) found no

difference in POD activity between young and mature

leaves of barley.

The polyamines (PAs), spermidine (Spd) and

spermine (Spm) and the related diamines putrescine

(Put) and cadaverine (Cad) are polycations

synthesizing in most living cells (Bachrach, 1973) and

have been implicated as an essential growth factors for

plants (Galston and Kaur-Sawhney, 1990). PAs applied

exogenously are potent inhibitors of senescence of oat

leaf protoplasts (Altman et al., 1977; Kaur-Sawhney et

al., 1980) and of leaves and storage tissue from several

plants (Kaur-Sawhney and Galston, 1979). PAbiosynthetic

enzyme activities and titers decreased in

senescing attached and detached oat leaves incubated

in dark (Kaur-Sawhney et al., 1982), therefore these

observations suggest that PAs are involved in the

control of plant growth and senescence.

One of the early events in leaf senescence is the

well-documented rise in protease activity (Thimann,

1980). Exogenous application of PAs retard

senescence and the increment in protease activity is

one of the early occurrences of senescence (Kaur-

Sawhney et al., 1982).

It was established in previous study that

application of mT to wheat leaf segments retarded

senescence by decreasing protease activity and

chlorophyll loss by resembling to PAs (Palavan-Ünsal

et al., 2002b). The aim of this study was to establish

the effect of mT on POD activity and nitrogen content

and their relation with PA metabolism during the

senescence of excised wheat leaf segments.

Materials and methods

Plant material

Six first leaf segments (3 cm each) from 10 days old

wheat (Triticum aestivum) seedlings were floated in

various concentrations (0.25, 0.5 and 1.0 mM) of mT

for 10 days in plant growth chamber (12 h light, 12 h

dark photoperiod and 25±2°C). Distilled water was

used for control.

Measurement of chlorophyll content

For chlorophyll determination, six leaf segments were

homogenized in 80 % acetone. The samples were

centrifuged at 3000 rpm for 5 min and the optical

density of the supernatant was read at 663 and 645 nm

with a spectrophotometer according to the Arnon

(1949).

Peroxidase activity

POD activity was analysed in a reaction volume of 3

ml containing 0.1M K-phosphate buffer, pH 5.8, 15

mM guaiacol and 5 mM H2O2. The reaction was started

by the addition of an appropriate volume of the crude

homogenate and the formation of tetraguaiacol (at 470

nm) was followed continuously for 2 min in

spectrophotometer (Shimadzu UV 160) (Birecka et al.,

1973).


Electrophoresis for isoperoxidases

Polyacrylamide gel electrophoresis was conducted as

described by Liu (1973). A separating gel of 8%

acrylamide was used. The enzyme extract in 50%

glycerol with 1 % bromophenol blue was applied to

the gel. Electrophoresis was conducted in a cold at 4°C

using reservoir buffer (14.1 g glycine and 3 g tris per

liter, pH 8.3) at 10 mA. For analysis of peroxidase

isoenzymes, the gel incubated for 20 min in the

solution containing 0.1 M sodium phosphate buffer,

pH 6.5 mM guaiacol, 5 mM H2O2 and then it was

stored in 50% methanol.

Polyamine determinations

The PAs were extracted with 5% HClO4, separated and

detected after dansylation as described by Seiler and

Wiechman (1967) using silica gel G plates with

cyclohexane ethylacetate (3:2, v/v) as the solvent.

Fluorescence was measured with spectrofluorimeter

(Shimadzu RF 5000) (emission 500 nm, excitation 360

nm) and the results were compared with dansylated

standards.

Nitrogen determination

The nitrogen content in the digested plant samples was

determined by the procedure modified from Middleton

(1960). Nitrogen content was determined by digest with

Nessler’s reagent. 1.0 ml of diluted digest solution, 4.0

ml H2O, 1.0 ml of 10 N NaOH, 1.0 ml of 0.6 N sodium

tartrate, 1.0 ml 2% (w/v) gum arabic and 2.0 ml

Nessler’s solution were added. The arabic gum and

Nessler’s solution were filtered through the filter paper

before use. The intensity of the resulting brownishyellow

colour was measured in spectrophotometer.

Standard nitrogen samples containing glycine were

digested and determined with each analysis.

Initial values of each analysis were measured in

leaf segments at the start of each experiment. All

results were expressed and discussed according to the

final controls that were incubated in distilled water for

10 days.

Results and discussion

In the present study it was determined that fresh

weight of wheat leaf segments increased gradually

meta-topolin in senescence 25

Table 1: Fresh weight changes in excised wheat leaf

segments after 10 days incubation in meta-topolin. Values

are average of 10 replicates.

Treatment Fresh Weight (mg)

Initial control 81.20 ± 1.7

Final control 85.09 ± 1.7

0.25 mM mT 88.72 ± 1.3

0.5 mM mT 90.22 ± 1.0

1.0 mM mT 92.80 ± 1.2

with increasing concentrations of mT when compared

to final control leaves (Table 1). There was no

significant difference between initial and final control

fresh weight values. 0.25, 0.5 and 1.0 mM mT

increased the fresh weight by 4, 6 and 9 %

respectively. We previously reported that natural

aromatic cytokinin mT is active in moderating radish

cotyledon growth (Palavan-Ünsal et al., 2002a).

When chlorophyll losses were compared between

initial and final control leaf segments, 46, 47 and 55 %

losses in chlorophyll a, chlorophyll b and total

chlorophyll contents were found respectively. Figure 1

shows the loss of chlorophyll decrement by the

application of mT in wheat leaf segments and the loss

of total chlorophyll, chlorophyll a and chlorophyll b in

the senesced final control leaves were greater than that

of mT treated leaf segments. It was found that the most

effective concentration of mT was 1 mM, which

inhibited the loss of chlorophyll. Total chlorophyll

content was 36 % more in 1 mM mT treated leaves

compared to final control leaves on 10 th day of

incubation. As seen in Figure 1 there were similar

establishments in chlorophyll a content but these

differences were not so remarkable for chlorophyll b.

Many researchers (Thimann, 1980; Stoddart and

Thomas, 1982; Chen and Kao, 1991) have reported

that exogenously applied cytokinins retarded the loss

of photosynthetic pigments during the senescence of

leaves and cotyledons.

The changes of POD activity in excised wheat leaf

segments treated with mT or distilled water were

presented in Figure 2. POD activity increased 48 % in

final control leaf segments compared to initial control.

It was also increased gradually in mT treated leaf

segments compared to final control condition on 10 th

day of incubation. POD activities were estimated as

17, 72, 90 % higher in 0.25, 0.5 and 1 mM mT applied

leaf segments, respectively. These results showed that


26 Narçin Palavan-Ünsal et al.

Figure 1: Effect of meta-topolin on chlorophyll content of wheat leaf segments on 10 th day of incubation. Vertical bars represent

standard errors. Each value is average of 4 experiments.


meta-topolin in senescence 27

Figure 2: Effects of meta-topolin on peroxidase activity of wheat leaf segments on 10 th day of incubation. Vertical bars represent

standard errors. Each value is average of 5 experiments.

Figure 3: Effects of meta-topolin on the number of

peroxidase isoenzymes of excised wheat leaf segments on

10 th day of incubation. The gel was stained for POD activity

using 0.1 M sodium phosphate buffer, pH 6, 5 mM guaiacol,

and 5 mM H2O2. The direction of electrophoretic migration

was from top (-) to bottom (+). Lane 1: Control; Lane 2:

0.25 mM mT; Lane 3: 0.5 mM mT, Lane 4: 1 mM mT.

there was a correlation between chlorophyll contents

and POD activity; the increasing ratio in POD activity

was in a larger scale than that of the chlorophyll

content depending on increasing concentration of mT.

Changes in the contents of POD isoenzymes in

excised wheat leaf segments that were treated with mT

and distilled water were also determined. In gel

analyses a difference in the number of POD

isoenzymes were observed in leaves treated with 0.5

mM mT compared to final control and 0.25 and 1.0

mM mT treatments (Figure 3). In addition, the effects

of 0.25 and 0.5 mM mT on the activity of POD were

found spectrophotometrically higher than that of final

control and of 1 mM mT treatments.

There are several reports showing that POD

activity increases during the senescence of excised

leaves. Increase in the activity of this enzyme with

physiological age of the leaves has also been reported

(Ford and Simon, 1972). Parish (1968) suggested that

the increase in the activity of POD is one of the most

reliable indicators of maturity and senescence.

Whereas, Ford and Simon (1972) established several

fold increment in POD activity when senescence was

delayed in cucumber cotyledons confirming our

results. It seems possible that POD has no functional

significance during the senescence process of leaf

segments of wheat.


28 Narçin Palavan-Ünsal et al.

Figure 4: Effects of meta-topolin on polyamine content of wheat leaf segments on 10 th day of incubation. Vertical bars represent

standard errors. The data presented here represent of 5 experiments and each measurement was done in duplicate.

Figure 4 shows the effect of mT on endogenous PA

contents after 10 days of incubation of wheat leaf

segments. Decline in Spm contents in final control

comparing to initial control leaves were established. It


was also estimated about 10 % increments in Put, Spd

an Spm contents only with 0.25 mM treatment

compared to final control. Application of higher

concentrations (0.5-1.0 mM) of mT resulted in

inhibition of PA levels during the senescence of wheat

leaf segments.

Among their other physiological effects, PAs are

involved in the control of several stress-related

phenomena such as senescence, wounding, heat and

salinity both in plant organs and isolated cells and

tissues. Thus exogenous application of PAs and related

precursors retarded leaf senescence, stabilizing them

against lysis. It was demonstrated that PAs retard

chlorophyll loss and prevent the rise of RNase and

proteases (Altman, 1982; Kaur-Sawhney and Galston,

1979).

It has also been reported that BA and kinetin (K)

promote cotyledon growth and this response is

accompanied by an increase in arginine decarboxylase

activity and Put titer (Cho, 1983). Besides, Walker et

al. (1988) reported that application of cytokinin to

excised cotyledon resulted in a large increase in

chlorophyll and Put levels. In addition, Srivastava et

al. (1981) established that K delayed senescence and

meta-topolin in senescence 29

Figure 5: Effects of meta-topolin on total nitrogen content of wheat leaf segments on 10 th day of incubation. Vertical bars

represent standard errors. Each value is average of 4 experiments.

retarded the decrease in the activity of diamino oxidase

and Spd and Spm levels. POD isoenzymes and PAs

may play an important role in protecting plants against

injury of oxidative factors. These findings are in

agreement with only 0.25 mM mT treatment in this

study.

In addition, the changes in the contents of total

nitrogen in leaf segments, which were treated with

water or mT on 10 th day of incubation, were analysed.

There was no significant difference in nitrogen content

between final control and 0.25 mM mT treatments

(Figure 5). However, mT at 0.5 and 1.0 mM

concentrations have been found to be effective in

increasing total nitrogen content while retarding the

senescence.

Feller et al. (1977) have established the role of leaf

proteases in the nitrogen economy of developing

cereals and have determined a close relationship

between depletion of protein and the built up of certain

proteolytic activities in the leaf. Decreasing protease

activity and increasing soluble protein content in

senescing leaf segments on 10 th day of incubation in

mT solutions were established in a former study

(Palavan-Ünsal et al. 2002b). In the same experimental


30 Narçin Palavan-Ünsal et al.

system, increased total nitrogen content with the

application of a new aromatic cytokinin mT was also

found. Total nitrogen content was lower in senesced

leaf segments than that of mT treated ones. Generally,

decreased total nitrogen content during the senescence

was shown in attached leaf or cotyledons (Storey and

Beevers, 1977). In our instance no significant

differences were determined, but there were some

trends in the same direction.

Actually, some authors suggest that PA increment

may represent only the tip of huge nitrogen

metabolism that is affected all stress conditions. In

agreement with this suggestion, it was established in

this study that both nitrogen and PAs which are also

nitrogenous substances increases by the application of

mT which is a new antisenescence agent. From all

these results it is possible to conclude that increased

POD activity, PA and nitrogen contents in mT treated

excised leaf segments was a contributing mechanism

to the overall antisenescence action of this new

aromatic cytokinin.

Acknowledgement

We thank to Dr. M. Strnad and his colleagues for the

generous gift of aromatic cytokinins. We also thankful

to Damla Büyüktunçer for technical assistance. This

study is supported by Istanbul University Research

Fund (project number: B-432/13042000).

References

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Altman A, Kaur-Sawhney R and Galston AW. Stabilization

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1977.

Arnon DI. Copper enzymes in chloroplasts,

polyphenoloxidase in Beta vulgaris. Plant Physiol. 24:

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Bachrach U. Involvement of polyamines in plant growth and

senescence. In: Adv Polyamine Res. Caldarera CM,

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Birecka H, Briber A and Catalfamo JL. Comparative studies

on tobacco pith and sweet potato root isoperoxidases in

relation to injury, indolacetic acid and ethylene effects.

Plant Physiol. 52: 43-49, 1973.

Chen CT and Kao CH Senescence of rice leaves XXX, levels

of endogenous polyamines and dark-induced senescence

of rice leaves. Plant Cell Physiol. 32: 935-941, 1991.

Cho SC. Effects of cytokinin and several inorganic cations

on the polyamine content of lettuce cotyledons. Plant

Cell Physiol. 24: 27-32, 1983.

Feller UK, Soong T and Haeman RH. Leaf proteolytic

activities and senescence during grain development of

field-grown corn (Zea mays L.). Plant Physiol. 59: 290-

294, 1977.

Ford TW and Simon EW. Peroxidase and glucose-6

phosphate dehydrogenase levels in cotyledons of

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Galston AW and Kaur-Sawhney R. Polyamines in plant

physiology. Plant Physiol. 94: 406-410, 1990.

Gilbert ML, Thompson JE and Dumbroff EB. Delayed

cotyledon senescence following treatment with

cytokinin, an effect at the level of membranes. Can J

Bot. 58: 1797-1803, 1980.

Grover A and Sinha SK. Senescence of detached leaves in

pigeon pea and chick pea: Regulation by developing

pods. Physiol Plant. 65: 368-507, 1985.

Kaur-Sawhney R, Flores HE and Galston AW. Polyamineinduced

DNA synthesis and mitosis in oat leaf

protoplasts. Plant Physiol. 65: 368-371, 1980.

Kaur-Sawhney R and Galston AW. Interaction of polyamines

and light biochemical processes involved in leaf

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Kaur-Sawhney R, Shih L, Flores HE and Galston AW.

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senescence in oat leaves. Plant Physiol. 69: 405-410,

1982.

Kraus TE, Hofstra G and Fletcher RA. Regulation of

senescence by benzylaminopurine and uniconazole in

intact and excised soybean cotyledons. Plant Physiol

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Retardation of senescence by meta-topolin in wheat

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Spermin Und Spermidin als I-dimethylamino-naphtalin

5 sulfon saure Derivative. Hoppe Seyler’s Z Physiol

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Srivastava SK, Raj ADS and Naik BI. Polyamine

metabolism during aging and senescence of pea leaves.

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Srivastava SK, Washi DJ and Naik BL. Control of

senescence by polyamines and guanidines in young and

mature barley leaves. Phytochem. 22: 2151-2154, 1983.

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plants. In: Encyclopedia of Plant Physiology. Boulter D

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Storey R and Beevers L. Proteolytic activity in relationship

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Fussell B and Hanke DE. Meta-topolin, a highly active

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meta-topolin in senescence 31


Journal of Cell and Molecular Biology 3: 33-37, 2004.

Haliç University, Printed in Turkey.

Characterization of BBaacciilllluuss species by numerical analysis of their SDS-

PAGE protein profiles

Ismet Berber

Department of Biology, Faculty of Art and Science, Yüzüncü Y›l University, 65080, Van, Turkey

Received 1 December 2003; Accepted 26 January 2004

Abstract

In the present study nine reference Bacillus strains were characterized by whole-cell protein profiles using sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A numerical classification of the protein profiles

revealed two distinct clusters at 47% similarity level. Cluster 1 comprised four strains belongs to B. subtilis and B.

megaterium species at similarity levels changed between 67 and 85%, while cluster 2 consisted five strains belongs

to various Bacillus species at similarity levels changed between 50 and 70%. The strains of the cluster 2 were clearly

separated from that of cluster 1 by numerical analysis. Our results indicates that SDS-PAGE method combined with

computerized analysis of cellular protein profiles provide an effective approach to investigate of taxonomic

relationships within Bacillus species.

KKeeyy wwoorrddss:: Characterization, Bacillus, SDS-PAGE, numerical taxonomy

BBaacciilllluuss türlerinin SDS-PAGE protein profillerinin nümerik analizle karakterizasyonu

Özet

Bu çal›flmada, dokuz referans Bacillus ›rk› sodyum dodesil sülfat poliakrilamid jel elektroforezi kullan›larak elde

edilen toplam hücresel protein profillerine göre karakterize edildi. Protein profil sonuçlar› esas al›narak yap›lan

nümerik s›n›fland›rma %47 benzerlik düzeyinde iki ayr› grup (cluster) oluflturdu. Grup 1 benzerlik düzeyi %67-85

aras›nda de¤iflen B. subtilis ve B. megaterium türlerine ait dört ›rk›; grup 2 benzerlik seviyesi % 50-70 aras›nda

de¤iflen farkl› Bacillus türlerine ait befl ›rk› içermektedir. Nümerik analizde grup 1’e ait ›rklar grup 2’den aç›kça

ayr›lmaktayd›. Bizim sonuçlar›m›z, SDS-PAGE yöntemiyle elde edilen hücresel protein profillerinin bilgisayar

analizleriyle birlefltirilmesinin Bacillus türlerinin taksonomik iliflkilerinin incelenmesinde etkili bir yaklafl›m

sa¤lad›¤›n› gösterdi.

AAnnaahhttaarr ssöözzccüükklleerr:: Karakterizasyon, Bacillus, SDS-PAGE, nümerik taksonomi

Introduction

The genus Bacillus are generally defined as grampositive,

aerobic or facultative anaerobic, motile,

peritrichous flagella and endospore-forming rodshaped

microorganisms (Claus and Berkeley, 1986).

This diversity was apparent even with classical

phenotypic characterization based primarily on

morphology, nutrition, growth characteristics; and

various substrate utilization and physiological

assessments (Slepecky and Hemphill, 1992). Although

physiological reactions are generally used to determine

the species of the genus, inconsistencies in test results

can make identification difficult (Ash et al., 1991).

33


34 Ismet Berber

Description of the genus has been improved by using

information obtained from DNA base composition and

DNA-DNA hybridization studies which was listed in

Bergey’s Manual of Systematic Bacteriology 40 (Claus

and Berkeley, 1986). However, in the literature there

are newly identified species which were shown to be

genetically and phenotypically distinct from other

Bacillus species and have not been described in

Bergey’s Manual (Slepecky and Hemphill, 1992).

A number of different methods have been used for

typing Bacillus species follow: Serotyping,

bacteriophage typing, bacteriocin activities,

antibiogram and biotyping, plasmid typing, analysis of

cellular fatty acid content, native-PAGE, small-subunitribosomal

RNA sequencing and genome analysis (Ash

et al., 1991; Berber and Cokmus, 2001). Although these

methods have been used for identification of Bacillus

species, characterization of these microorganisms is

still not well defined (Ivanova et al., 1999).

A second level information for a cell, other than

sequencing of bacterial genome, can be obtained from

cellular protein profiles. Different types of

electrophoresis were used to explore the profiles. The

protein profiles produced by SDS-PAGE of whole cell

extract have been found correlates closely with DNA-

DNA hybridization results suggests it could be

appropiate to use SDS-PAGE for rapid bacterial

identification (Vauterin et al., 1990; Niemi et al., 1993;

Berber et al., 2003).

Combination of polyacrylamide gel electrophoresis

(PAGE) of proteins with computerized analysis of

profiles provided an effective approach to investigate

the taxonomic relationships among many bacterial

species (Kersters, 1985; Costas, 1992). This paper

describes PAGE results of nine reference Bacillus

species. The aim of the study was to evaluate the

usefulness of the technique as a taxonomic tool in this

genus.

Materials and methods

Bacteria and growth conditions

The test bacteria used in our study have been provided

from Prof Dr. Cumhur Cokmus (Department of

Biology, Faculty of Sciences, Ankara University

Ankara TURKEY). All cultures were grown at 30°C

for 24 h on NYSM (Difco, DETRO‹T)) agar and

propagated at least twice before use.

Preparation of whole-cell proteins

For each culture, a loopful of overnight growth on

NYSM agar plate was suspended in 15 ml NYSM

broth, and incubated on rotated incubator for 48 h (at

30°C, 150 rpm). Samples were then transferred into

1.3 ml eppendorf tubes, centrifuged for 3 minutes at

12,100 rpm, and the collected cells were washed three

times with distilled water. The washed cells were

stirred after adding 25 µl SDS-samples buffer on (0.06

M Tris-HCl, 2.5% Glycerol, 0.5% SDS, 1.25% ßmercaptoethanol)

and the proteins were denatured by

boiling the tubes for 5 minutes (Laemmli, 1970).

SDS-PAGE

Solubilized proteins were subjected to SDS-PAGE in

gel slabs of 1 mm thickness (3.5cm, 4% stacking and

16.5cm, 12.5% resolving gels) as described by

Laemmli (1970). Electrophoresis was performed with

a discontinuous buffer system in a BRL gel apparatus

model V16-2BRL Gaithersburg MD, USA. The gel

was run at 30 mA until the bromophenol blue marker

had reached to the bottom of the gel. Gels were then

stained with Coomassie Brillant Blue R-250.

Data analysis

Gels were examined by naked eyes directly and the

protein profiles were recorded as binary data, that is, 1

or 0. The resultant data were typed into the MINITAB

(Version 13.1) program. This is a program for data

input and analysis of binary data and is run on IBM

computer. The similarity and relationship between the

protein traces of test strains were expressed in a

dendrogram derived by using the Pearson productmoment

correlation coefficient and unweighted pair

group method with arithmetic averages algorithm.

Results

Figure 1 shows the whole-cell protein profiles of

Bacillus species obtained by sodium dodecyl sulphate

polyacrylamide gel electrophoresis. There were

considerable differences in protein profiles of Bacillus

species at 20.000-66.000 kDa region. Most of Bacillus

species, except B. megaterium DSM 32 and B.

megaterium ATCC 1842, contain the protein band

(marked by 1) at the top of each lane. B. subtilis and B.


Figure 1: SDS-PAGE of whole-cell proteins of Bacillus

species. Lines: A, B. subtilis; B, B. subtilis DSM 10; C, B.

megaterium DSM 32; D, B. megaterium ATCC 1842; E, B.

licheniformis; F, B. megaterium; G, B. sphaericus MRS

400; H, B. thuringiensis var. israelensis; I, B. cereus ATCC

7064; M, Molecular weight marker (x10 3 kDa).

subtilis DSM 10 strains had similar protein patterns for

the bands marked by 10 and 12 (lines A and B).

However, B. subtilis DSM 10 strain distinguished from

other strain with the presence of a band (marked by

16). B. megaterium DSM 32 (Fig 1, line C), B.

megaterium ATCC 1842 (Fig 1, line D) and B.

megaterium (Fig 1, line F) had crucial differences in

protein bands, although they belong to the strains of

same specie. These strains were separated from each

other in the presence or absent of some protein bands

marked by 3, 4, 6, 11, 13 and 15. B. licheniformis and

B. thuringiensis var. israelensis obviously differed

from other Bacillus species (Fig 1, lines E and H).

Moreover, some variations were observed between

protein profiles of two species (B. sphaericus MRS

400 and B. cereus 7064) and other Bacillus species

(Fig 1, lines G and I). Protein pattern of B. cereus 7064

strain was slightly similar to B. sphaericus MRS 400

strain, although, strain was distinguished by the

presence of four darkly protein bands (marked as 5, 8,

9, 14).

A dendrogram produced after numerical analysis of

the whole-cell protein profiles using the Pearson

product-moment correlation coefficient and

unweighted pair group method with arithmetic

averages algorithm (UPGMA) is shown in Figure 2.

Numerical analysis revealed clearly two distinct

clusters at a similarity level of 47% as shown in the

dendrogram (Fig 2). Cluster 1 comprised four strains

at the similarity levels changed between 67 and 85%

belongs to B. subtilis and B. megaterium species. Two

members of the cluster 1 (B. subtilis and B. subtilis

DSM 10), formed the cluster at over 85 % of similarity

level, had a characteristic two pairs of banding pattern

at the 20.000-29.000 kDa in the molecular weight

region (Fig 1). Cluster 2 included five strains related to

different Bacillus species. The similarity level of

members of the cluster 2 changed between 50 and

70%. The strains of the cluster 2 were clearly separated

from cluster 1 by numerical analysis.

Discussion

Characterization of Bacillus species 35

47 65 82 100

Percentage Similarity

B. sphaerious

MRS 400

B. cereus

ATCC 7064

B. thuringiensis

var israelensis

B. megaterium

B. licheniformis

B. megaterium

DSM 32

B. megaterium

ATCC 1842

B. subtilis

DSM 10

B. subtilis

Figure 2: Electrophoretic protein patterns and dendrogram

based on unweighted pair group method with arithmetic

averages algorithm (UPGMA) of the protein patterns of

whole-cell of Bacillus species.

Gram-positive, rod-shaped, aerobic or facultative

anaerobic spore-forming bacteria have been assigned

to the genus Bacillus. The genus Bacillus is

phenotypically heterogeneous with its members

exhibiting an extremely wide range of nutritional

requirements, growth conditions, metabolic diversity

and DNA base composition (Claus and Berkeley,

1986). In addition, the results of 16S rRNA sequence

analysis reconfirm the insufficient defined genera on

Cluster 2

Cluster 1


36 Ismet Berber

the basis of phenotypic criteria (Woese, 1987).

Protein electrophoresis has been of great value for

delineation of numerous bacterial taxa (Vauterin et al.,

1990; Costas, 1992). Each of the different

electrophoretic techniques has its own discrimination

level and field of application. It is also widely

acknowledged that the electrophoretic separation of

cellular proteins is a sensitive technique which mainly

provides information on the similarity of the strains at

and below the species level. In addition, it is also

generally accepted that the objective comparison of

electrophoretic protein patterns provides a reliable

measure of genomic inter-relationship.

Our results have showed that electrophoretic

methods can provide valuable information which may

be used in identification of Bacillus strains. These

results are in good agreement with previous researches

(Lewis et al., 1987; Cokmus and Yousten, 1994; Zheng

and Slavik, 1999; Berber and Cokmus, 2001). It is

known that protein profiles of whole-cell and

extracellular proteins are good enough to distinguish

most of bacterial genera at species level (Elliott and

Facklam, 1993; Sacilik et al., 1998; Berber et al.,

2003). Some researchers (Costas et al., 1993; Cokmus

and Yousten, 1994; Atalan et al., 2000) also

differentiated the strains of Proteus species, strains of

Bacillus sphaericus and strains of Streptomyces

species by whole-cell proteins using SDS-PAGE at the

subspecies level.

The conventional tests based on the phenotypic

characteristics can clearly lead to misclassification in

some bacterial taxa. Recently, it has been reported that

the elecrophoretic technique, as a practical method, is

necessary for integrated use of phenotypic characters

in identification of bacterial genera at all level (Murray

et al., 1990). In our results, numerical analysis of onedimensional

SDS-PAGE of the protein patterns of

whole-cell of Bacillus species provides a useful

approach towards clarifying relationship within the

Bacillus species. As each species cluster had

characteristically distinctive protein band patterns, we

suggest that simple visual comparison of the principal

bands provides a rapid means of identifying isolates

from various sources, by combination of cellular

protein patterns. We conclude that numerical analysis

of SDS-PAGE of whole-cell proteins is an extremely

useful in taxonomic assesment in studing Bacillus

species.

Acknowledgement

I would like to thank Prof. Dr. Cumhur COKMUS

(Department of Biology, Faculty of Sciences, Ankara

University, Tando¤an 06100 Ankara-TURKEY) for

bacterial strains.

References

Atalan E, Manfio GP, Ward AC, Kroppestedt RM. and

Goodfellow M. Biosystematic studies on novel

Streptomycetes from soil. Antonie Van Leeuwenhoek.

77: 337-353, 2000.

Ash E, Farrow JAE, Wallbanks S and Collins MD.

Phylogenetic heterogeneity of the genus Bacillus

revealed by comparative analysis of small-subunitribosomal

RNA sequences. Letters in Applied

Microbiolgy. 13: 2002-2006, 1991.

Berber I and Cokmus C. Characterization of Bacillus

sphaericus strains by Native-PAGE. Bull of Pure and

Appl Sci. 20 (1): 17-21, 2001.

Berber I, Cokmus C and Atalan E. Characterization of

Staphylococcus species by SDS-PAGE of Whole-Cell

and Extracellular Proteins. Microbiology. 72 (1): 42-47,

2003.

Claus D and Berkeley CW. The genus Bacillus. In: Bergey’s

Manual of Systematic Bacteriology. Vol 2. Sneath PHA

(Ed). Williams, Wilkins, Baltimore. 34: 1105-1139, 1986.

Cokmus C and Yousten AA. Characterization of Bacillus

sphaericus strains by SDS-PAGE. J Invertebr Pathol.

64: 267-268, 1987.

Costas M, Holmes B, Frith KA, Riddle C and Hawkey PM.

Identification and typing of Proteus penneri and Proteus

vulgaris biogroups 2 and 3, from clinical sources, by

computerized analysis of electrophoretic protein

patterns. J of Applied Bacteriology. 75: 489-498, 1993.

Costas M. Classification, identification and typing of

bacteria by the analysis of their one-dimensional

polyacrylamide gel electrophoretic protein patterns. In:

Advances in Electrophoresis. Vol 5. Chrambach A,

Dunn NJ and Radola BJ (Ed). 351-408, 1992.

Elliott JA and Facklam RR. Identification of Leuconostoc

spp. by Analysis of Soluble Whole-Cell Protein Patterns.

J Clin Microbiol. 31 (5): 1030-1033, 1993.

Ivanova PE, Vysotskii MV, Svetashev VI, Nedashkovskaya

OI, Gorshkova NM, Mikhailov VV, Yumoto N,

Shigeri Y, Taguchi T and Yoshikawa S. Characterization

of Bacillus strains of marine origin. International

Microbiol. 2: 267-271, 1999.

Kersters K. Numerical methods in the classification of

bacteria by protein electrophoresis. In: Computer Assisted

Bacterial Systematic. Goodfellow M, Jones D and

Priest FG (Ed). London: Academic Pres. 337-368, 1985.


Laemmli UK. Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature

(London). 227: 680-685, 1970.

Lewis LO, Yousten AA and Murray RGE. Characterization

the surface protein layers of the mosquito pathogenic

strains of Bacillus sphaericus. J Bacteriol. 169 (1): 72-

79, 1987.

Murray RGE, Brenner DJ, Colwell RR, Devos P,

Goodfellow M, Grimont PAD, Pfennig, N, Stackebrant E

and Zavarzin GA. Report of the ad hoc commite on

approches to taxonomy within the preteobacteria.

International Journal of Systematic Bacteriology. 40:

213-215, 1990.

Niemi RM, Niemela SI, Bamford DH, Hantula J, Hyvarinen

T, Forsten T and Raateland A. Presumptive fecal

Streptococci in environmental samples characterized by

one-dimensional sodium dodecyl sulfate-polyacrilamide

gel electrophoresis. App and Env Microbiol. 59 (7):

2190-2196, 1993.

Sacilik SC, Osmanagaoglu O, Berber I and Cokmus C. A

new method for characterization of gram positive cocci

by SDS-PAGE of extracellular proteins. 8 th International

Symposium on the Genetics of Industrial

Microorganisms. Jerusalem, Israel. 65, 1998.

Slepecky RA and Hemphill HE. The Genus Bacillus

Nonmedical in “The Prokaryotes”. Vol II. Balows A,

Trüper HG, Drowkin M, Tno WH, Schleifer KH (Ed).

Springer-Verlag, New-York, Berlin, Heidelberg, London,

Paris, Tokyo, Hong-Kong, Barcelona, Budapest. 48,

1697-1745, 1992.

Vauterin L, Vantomme R, Pot B, Hoste B, Swings J and

Kersters K. Taxonomic analysis of Xhantomonas

campestris pv. begonidae and X. campestris pv.

pelargonii by means of phythopathological, phenotypic,

protein electrophoretic and DNA hybridization methods.

Systematic and Applied Bacteriol. 13: 166-167, 1990.

Woese CR. Bacterial Evolution. Microbiological Reviews.

51: 221-271, 1987.

Zheng G and Slavik MF. Isolation, partial purification and

characterization of a bacteriocin produced by a newly

isolated Bacillus subtilis strain. Letters in Applied

Microbiology. 28: 363-367, 1999.

Characterization of Bacillus species 37


Journal of Cell and Molecular Biology 3: 39-44, 2004.

Haliç University, Printed in Turkey.

A kinetic model for iinn--vviittrroo intestinal uptake of LL--tyrosine and D (+)glucose

across rat everted gut sacs in the presence of MMoommoorrddiiccaa

cchhaarraannttiiaa, a medicinal plant used in traditional medicine against

diabetes mellitus

Mohamad Fawzi Mahomoodally 1 , Ameenah-Gurib Fakim 2 , Anwar Hussein Subratty 1 *

1 Department of Health and Medical Sciences, 2 Department of Chemistry, Faculty of Science, University

of Mauritius, Reduit, Mauritius (*author for correspondence)

Received 10 December 2003; Accepted 19 January 2004

Abstract

Momordica charantia (MC) is a traditional antidiabetic medicinal plant used in many parts of the world, including

Mauritius. An everted rat gut sac technique was used to investigate the effect of MC on kinetic parameters of D (+)glucose

and L-tyrosine. Everted guts were mounted in a gut sac bath and aqueous extract of MC fruit was added to

the mucosal medium (3.62 mg/mL) at varying substrate concentrations. Michaelis-Menten constant (Km) and

maximal velocity (Vmax) were calculated in the presence and absence of MC fruit extract. It was observed that MC

significantly reduced Vmax of D-(+)- glucose uptake by 0.09 mM hr -1 , whereas Km remained unaltered suggested a

non-competitive type of inhibition was present. L-Tyrosine uptake in the presence of MC fruit extract did not fit to

a relatively simple kinetic model.

KKeeyy wwoorrddss:: Momordica charantia, kinetics, diabetes mellitus, traditional medicine

Diabetes mellitus’a karfl› kullan›lan geleneksel t›bbi bitki MMoommoorrddiiccaa cchhaarraannttiiaa

varl›¤›nda ters yüz edilmifl s›çan ba¤›rsa¤›ndan LL--tirozin ve D(+)-glukoz geçifli için

iinn vviittrroo kinetik model

Özet

Momordica charantia, (MC) Mauritus’da dahil dünyan›n birçok yerinde geleneksel antidiyabetik t›bbi ilaç olarak

kullan›lmaktad›r. Ters yüz edilmifl ba¤›rsak tekni¤i MC nin D(+)-glukoz ve L-tirozinin kinetik parametrelerine olan

etkilerini araflt›rmak için kullan›ld›. Ters yüz edilmifl ba¤›rsaklar ba¤›rsak banyosuna kondu ve de¤iflik

konsanrasyonda substrat içeren mukoza medyumuna MC meyvesinin sulu ekstresi eklendi (3.62mg/mL). Michaelis-

Menten sabit (Km) ve maksimum h›z (Vmax) MC meyve ekstresinin mevcudiyetinde ve yoklu¤unda hesap edildi. MC

nin kayda de¤er bir flekilde D(+)-glukoz al›n›fl›n›n Vmax’›n› azaltt›¤› (0.09 mM hr -1 ), buna karfl›n Km’nin de¤iflmedi¤i

ve bunun da rekabet etmeyen bir inhibisyon tipini ortaya koydu¤u belirlendi. MC meyve ekstresi varl›¤›nda L-tirozin

al›n›fl› nisbeten basit olan kinetik modele uygunluk göstermedi.

AAnnaahhttaarr ssöözzccüükklleerr:: Momordica charantia, kinetik, diyabet, geleneksel t›p

39


40 Mohamad Fawzi Mahomoodally et al.

Introduction

Diabetes mellitus (DM) is a debilitating and often life

threatening disease with increasing incidence in rural

populations throughout the world. It was postulated

that DM is the most common chronic disorder affected

more than 176 million people worldwide, and this

global figure has been set to double by the year 2030

(Tiwari and Madhusudana, 2002). DM does not only

kill, but also is one of the major causes of adult

blindness, kidney failure, gangrene, neuropathy, heart

attack, and stroke (Bransome, 1992). In Mauritius, DM

is becoming a devastating scourge with more than

100,000 cases and with 4.6 % death rate. Nephritic

syndromes, nephrosis and amputations were

prominent prior to the deaths in 2000 (Ministry of

Health & Quality of Life, 2003).

Before the introduction of insulin in 1922, the

treatment of DM relied heavily on dietary measures,

which included the use of traditional plant therapies

(Alison and Flatt, 1998). The Papyrus Ebers of 1550

BC had recommended a high-fiber diet of wheat grains

and orche (Bailey and Day, 1989). More than 1200

species of organisms have been used

ethnopharmacologically or experimentally to treat

symptoms of DM, and several reviews on plants with

known antidiabetic activity or with traditional use as

antidiabetic remedies have been published

(Fransworth and Segelman, 1971; Ajganonkar, 1979;

Oliver-Bever, 1980; Bailey and Day, 1988;

Winkelman, 1989). They described more than 725

genera in 183 families, extending phylogenetically all

the way from marine algae and fungi to advanced

plants such as composites (Marles and Farnsworth,

1995). It would thus appear that traditional antidiabetic

plants might provide a useful source for

developing new oral hypoglycemic compounds as

pharmaceutical entities or simple dietary adjuncts to

the exiting therapies. Although an orally active

botanical substitute to endogenous insulin seems

unlikely, developing new phytochemical molecules,

which may stimulate endogenous insulin biosynthesis

and secretion (and promote insulin action) is a realistic

possibility (Bailey and Day, 1998). In other words, as

several investigators suggested, studying such

traditional medicines might offer an alternative and

natural key to unlock diabetologists’ pharmacy.

On the other hand, suggested mechanisms

describing therapeutic effects of several traditional

medicinal plant systems are holistic (Handa et al.,

1989). Most of the reported hypoglycemic plants are

anecdotal, and only few have received adequate

scientific evaluation. The fundamental mechanisms of

these medicinal systems are still unexplainable using

modern tools (Rahman and Zaman, 1989). It is

claimed that most medicinal preparations in traditional

medicines contain a variety of synergistically acting

phytochemicals that are thought to act on a variety of

targets by various modes and mechanisms (Tiwari and

Madhusudana, 2002).

In accordance with the recommendation of the

World Health Organisation expert committee on DM

(Alison and Flatt, 1991), investigation of

hypoglycemic agents from plants, which have been

used as traditional medicines seems of paramount

importance. In this study, we investigated the effect of

Momordica charantia (MC), a well-documented

hypoglycemic plant (Chatterjee, 1994; Singh, 1986;

Ng et al., 1986), on the kinetic uptake of D (+)-glucose

and L-tyrosine in in-vitro everted gut sac model as

described by Subratty (2003).

Materials and methods

Preparation of the crude extracts from Momordica

charantia fruit

A powdered mixture of MC dry fruit (10g) was

extracted with 50 mL water at 90°C for 5 hours using

Soxhlet apparatus. The water was evaporated under

vacuum at 50°C, and the precipitate was transferred

into 10 mL distilled water. Percentage yield was

calculated, and the paste-like material was diluted in

distilled water to use in experiments.

Experimental design and surgical procedure

Adult male Swiss albino rats weighing 100-150 g and

housed at temperature 25 ± 2 °C were used in this

study. Animals were maintained on commercial feed

and tap water ad libitum. Before each experiment, the

animals were starved for 12 hours but allowed for tap

water ad libitum use. Rats were sacrificed by severe

blow on the head against a hard surface. The abdomen

was opened by a midline incision. The entire small

intestine was removed quickly by cutting across the

upper end of the duodenum and the lower end of the

ileum, and by stripping the mesentery manually

(Barthe et al., 1998). The small intestine was then

washed out with normal saline solution (0.9% w/v

NaCl) using a syringe equipped with blunt end.


Figure 1: Set up used to assess kinetic parameters in rat

everted gut sac (adapted from Kooshapur and Chaideh,

1999).

Preparation of everted gut sacs

Intestinal segments (10±2 cm) were everted according

to the method described by Wilson & Wiseman. After

being blotted with a piece of filter paper, a 1 g glass

weight was fixed and tied to the end of the everted gut

segment to make an empty gut sac. This was important

to prevent peristaltic muscular contractions, which

may otherwise alter the shape and internal volume of

the sac. The 1 g glass weight was the minimum weight

to secure the above-mentioned conditions and to

prevent the sac septum to become thin. A scheme

describing the gut sac bath system used in the study

was shown in Figure 1.

After weighing, the empty sac was filled with 1 mL

of Krebs-Henseleit bicarbonate buffer (KHB). The

composition of the buffer was (mM/L): NaHCO3 25;

NaCl 118; KCl 4.7; MgSO4 1.2; NaH2PO4 1.2; CaCl2

1.2; and Na 4EDTA 9.7 mg/L. Glucose (2g/L) was

D (+)-glucose uptake and Momordica charantia 41

added to the medium just before the start of the

appropriate experiment. The pH was maintained at 7.4.

The sac was filled with a blunted-ended syringe and

then the needle was slipped off carefully, and the loose

ligature on the proximal end was tightened. The filled

and distended sac was weighted and the difference in

weight was taken as the measure of the initial serosal

volume. The compartment containing the buffer in the

sac was named serosal fluid compartment.

The distended sacs was placed inside a 50 mL KHB

bath (mucosal fluid compartment) and mounted as

described by Khoshapur and Chaideh (1999). This gut

sac bath was surrounded by a water jacket maintained

at 37-40 °C. The mucosal fluid compartment was

continuously mixed with air bubbles using the mixture

of 95 % O2 and 5% CO2, and pH maintained at 7.5.

Effects of Momordica charantia on the uptake of

glucose and tyrosine transport

For studying the effect of the plant extract on the

uptake of glucose and tyrosine (substrates), glucose

and tyrosine at varying concentrations were added into

mucosal compartment fluid. The plant extract was also

added in the same compartment (3.62 mg/mL).

Measurements of final volume

At the end of the incubation period (30 min), the sacs

were removed from the gut sac bath, blotted by a

standardized procedure as described above and

weighted. The serosal fluid was drained through a

small incision into a test tube. The emptied sac was

shaken gently to remove the adhered fluid and the

tissue was weighted. The final serosal volume was

determined by subtracting (after incubation) the

weight of the empty sac from that of the filled sac. The

mucosal fluid transfer was expressed in the terms of

diminution of fluid volume in the mucosal

compartment during the course of experiment. The

serosal fluid transfer was reflected as an increase in the

volume of serosal compartment inside the sac. The gut

fluid uptake was determined by measuring an increase

in the volume of fluid in the gut wall.

Tyrosine concentrations in both compartments

were determined by a spectrophotometric method

(Subratty, 2003). Glucose concentrations were

measured using a commercially available glucose

oxidase kit (Boehringer Mannheim, Mannheim,

Germany). The amount of tyrosine and glucose


42 Mohamad Fawzi Mahomoodally et al.

transported from the mucosal compartment was

characterized as ‘uptake’ while the serosal gain of the

substances is treated as ‘release’. Uptake and release of

glucose and tyrosine were expressed as mM/g tissue

wet weight/h. All chemicals were procured from

Sigma (UK). Glucose and tyrosine experiments were

performed separately.

Control experiments

In each serie of experiments, control everted gut sacs

derived from the same rat in a buffer containing no

substrate were run in parallel. The controls were run

either with or without MC and results were corrected

accordingly.

Data analysis

In terms of enzyme kinetics, the amount of L-tyrosine

and glucose transported per hour were analogue to the

velocity of transfer, in other words, to the

concentration difference of the tyrosine and glucose

between compartments at the beginning and end of an

experiment (Subratty, 2003). The Michaelis - Menten

constant (Km), which is the affinity of the transferring

enzyme for the substrate, and maximal velocity (Vmax),

which is the rate of transfer reaction, in the presence as

well as in the absence of MC were determined from

the differences of uptake and release values using the

Michaelis-Menten and Lineweaver-Burk Plots in

Microsoft Excel 2000. Comparison of difference

between the controls and experimental groups were

examined using One-Way Analysis of Variance

(ANOVA) test for mean ± SEM of Km and Vmax. Any

difference with p values less than 0.05 were considered

as statistically significant. Mean Km and Vmax were

presented as single entities in tables.

Results

Table 1 lists the biochemical parameters of D (+)glucose

transport across rat everted small intestines invitro.

The Km and Vmax were calculated in the absence

as well as in the presence of MC fruit extract in the

mucosal solution. Data analysis revealed that Vmax for

glucose uptake decreased (P < 0.05) by 0.09 mM hr -1

in the presence of the aqueous MC fruit extract. In

contrast, the apparent Km (22.2 Mm) remained

unaltered (P>0.05) in the presence of the fruit extract.

Table 1: Biochemical parameters obtained for the effect of

MC fruit extract on the transport of D (+)-glucose at

different concentrations (4-10 mM) across the rat everted

gut sacs. N= number of sacs used. The everted gut sacs were

incubated in Krebs- Henseleit buffer (pH= 7.4) at 37°C.

Experiments Vmax Km

(mM hr –1 ) (mM)

Control (N=7) 0.17 22.2

Momordica charantia 0.08 22.2

fruit extract

(3.62 mg/ml) (N=7)

Table 2 depicts the biochemical parameters of Ltyrosine

uptake across rat everted small intestines invitro.

Incubation of gut sacs in MC containing mucosal

solutions with varying concentration of tyrosine did

not follow a simple kinetic model. Negative values for

Vmax (-0.01 mM hr –1 ) and Km (-4.0 mM ) were obtained

for the tyrosine uptake across the everted gut sacs.

Table 2: Biochemical parameters obtained for the effect of

MC fruit extract on the transport of L-Tyrosine at different

concentrations (0.5- 2.0 mM) across the rat everted gut sacs.

N= number of sacs used. The everted intestines were

incubated in Krebs-Henseleit buffer (pH= 7.4) at 37°C.

Experiments Vmax Km

(mM hr –1 ) (mM)

Control (N=7) 0.03 0.075

Momordica charantia -0.01 -4.0

fruit extract

(3.62 mg/ml) (N=7)

Discussion

Progress in understanding the metabolic staging of

DM over the past few years has led to significant

advances in regimen for treatment of this devastating

disease. The most challenging goal in the management

of patients with DM is to achieve blood glucose level

as close to normal as possible. Unfortunately, postprandial

hyperglycaemia (PPHG) or hyperinsulinaemia

are independent risk factors for the development of

vascular complications in DM patients (Tiwari and

Madhusudana, 2002). Starting from the very beginning


of carbohydrate metabolism, mechanisms playing role

in release and transport of glucose across the intestinal

brush border membrane down to the blood stream have

attracted much attention recently as potential targets to

control PPHG. In this category, majority of recent

studies reported the potential use of antidiabetic

medicinal plants on inhibition of glucose transport.

Drugs that reduce PPHG by suppressing the absorption

of carbohydrate are effective in prevention and

treatment of non-insulin dependent DM. Recently,

Matsuda et al. (1998) studied the effect of plant

saponins on the transport of glucose in-vitro.

Our findings would tend to indicate that glucose

transport was significantly decreased in the presence

of the crude extract of Momordica fruits, which caused

a decrease in the Vmax by 0.09 mM hr -1 . However, the

Km (22.2 mM) remained unaltered in the presence as

well in the absence of the fruit extract (Table 1). This

indicates that MC act by bringing a non-competitive

type of inhibition of glucose at the level of the small

intestine. Therefore, it is most probable that active

phytochemicals in the fruit of MC binds on the glucose

transporters thus may lead to wash out of glucose from

the body. The latter may be responsible for the

hypoglycemic phenomena after MC fruit juice was

administered noted by various investigators in animals

or subjects (Leatherdale et al., 1981; Aktar, 1982).

Based on this data generated in this study, regarding

the mechanism of action, we propose that MC extracts

may possess hypoglycaemic properties by inhibiting

the glucose transport at the site of intestinal brush

border membranes.

In simple kinetic model, an amino acid ‘A’ interacts

with a membrane component or “carrier-site” ‘X’ to

form a binary complex. Investigation of the directional

influx of tyrosine from the mucosal membranes into

the epithelium here indicated that the transfer process

did not follow reasonably well the simple kinetic

model. The Vmax and Km values for tyrosine in the

presence of the fruit extract were negative (Table 2),

similar to our previous observation (Subratty, 2003).

We have proposed that the negative values for the

kinetic parameters could be due to reversal of the

normal sodium and potassium gradient across the

brush border membrane, which in turn translocated

tyrosine back to the mucosal surface. Phytochemicals

in the extract (saponins and glycosides) may alter

sodium-potassium gradient on the membrane. The

negative values might mean lack of transfer rather than

pumping non-existing tyrosine in serosal compartment

D (+)-glucose uptake and Momordica charantia 43

back to mucosal compartment, and may arise due to

technical limitations. Under these conditions, sodium

required to form ternary complex in the relevant

binding protein, which otherwise possesses

dislocation, may derive from the serosal fluid, and this

conformational change in the protein may reverse the

transport (Subratty, 2003). However, a clear picture for

the mechanism is not clear. Further work needs to be

undertaken to investigate the exact nature of possible

carriers, which may involve in tyrosine transport in the

presence of the fruit extract.

In conclusions, our study provides evidence for a

biochemical mechanism which carries blood glucose

lowering effect of Momordica charantia fruit in

intestine via non-competitive inhibition. However,

further kinetic data on carrier-mediated transport of D

(+)-glucose and L-tyrosine in the presence of MC fruit

extract is needed.

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Alison and Flatt. World Health Organization. Guidelines for

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traditional medicines. WHO/TRM/91.4. WHO.

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Bailey CJ and Day C. Traditional plant medicines as

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44 Mohamad Fawzi Mahomoodally et al.

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and Yoshikawa M. Antidiabetic principles of natural

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Journal of Cell and Molecular Biology 3: 45-50, 2004.

Haliç University, Printed in Turkey.

P rostatic intraepithelial neoplasm: Retrospective results of clinical,

histopathological approaches

Osman Nuri Akbulut, Soner Guney, ‹brahim Duman, ‹lker Çömez, Serdar Arisan*,

Erbil Ergenekon

fiiflli Etfal Research and Training Hospital, 1 st Urology Clinics, fiiflli-Istanbul, Turkey (*author for

correspondence)

Received 12 December 2003; Accepted 24 December 2003

Abstract

Prostate cancer is the main problem in advanced aged population and it is the major reason causing death in western

countries. Frequently the tumor metastasis or locally invades tissues when it is diagnosed. The main target should be

the early diagnosis of the tumor. Today, the most important precursor lesion of prostate cancer is prostate

intraepithelial neoplasm (PIN) in pathological analysis. This study aims to discuss PIN diagnostic value. This study

comprimised on retrospectical determination of prostate specific antigen (PSA) levels and/or digital rectal

examination (DRE) and sextant biopsies with the guide of transrectal ultrasonography (TRUS). Between April 2001

and September 2003, 166 patients were included in the study. Follow- up period was 31 months. Pathological results

of prostate cancer, high grade PIN (hgPIN), low grade PIN (lgPIN) BPH and age, average serum PSA levels, DRE

findings, Gleason Score and Gleason Grade results for prostate cancer patients were compared. In conclusion, the

patients who had hgPIN lesions with BPH adviced for periodic follow up for prostate specific antigen (PSA) and

digital rectal examination (DRE) and in necessary conditions, rebiopsy.

KKeeyy wwoorrddss:: PIN, prostate cancer, BPH, Gleason score, PSA

P rostat intraepitel neoplazi: Klinik ve histopatolojik retrospektif sonuçlar

Özet

Prostat kanseri ileri yafl grubu erkeklerde en s›k görülen ve en önemli ölüm sebeplerinden biridir. Prostat kanserinin

tan›s› kondu¤unda, genellikle kanser ya lokal invazyon göstermifl ya da metastatik hale gelmifltir. Günümüzde prostat

kanseri için prekürsor lezyon kavram›na uyan en önemli antite Prostatik Intraepitelyal Neoplazi'dir (PIN). Çal›flma,

prostat kanserinin erken tan›s› amac›yla de¤erlendirme kapsam›na al›nan, serum PSA (Prostatik Spesifik Antijen)

ve/veya PRM (Parmakla Rektal Muayene) sonucu TRUS (Transrektal Ultrasonografi) eflli¤inde sextant prostat

biyopsileri yap›lan hastalar›n, klinik ve histopatolojik kay›tlar›n›n retrospektif olarak incelenmesini içermektedir.

Nisan 2001 - Eylül 2003 tarihleri aras›ndaki 31 ayl›k sürede, poliklini¤imize baflvuran ve prostat kanseri flüphesiyle

TRUS eflli¤inde prostat biyopsisi uygulanan 166 hastan›n biyopsi materyallerinin histopatolojik bulgular›

retrospektif olarak incelendi. Prostat kanseri, yüksek grade (yg) PIN, düflük grade (dg) PIN ve BPH patolojik

sonuçlar›; yafl, ortalama serum PSA de¤erleri, PRM bulgular› ve prostat kanserli hastalann Gleason grade ve Gleason

skor de¤erleriyle karfl›laflt›r›ld›. Bu nedenle YG PIN bulgular› saptanan BPH'li olgular›n periyodik PSA ölçümleri ve

PRM ile takip edilmesini, klinik de¤iflkenlerin varl›¤› durumunda biyopsi tekrar› için yeniden de¤erlendirilmesini

tavsiye ediyoruz.

AAnnaahhttaarr ssöözzccüükklleerr:: PIN, prostat kanseri, BPH, Gleason skoru, PSA

45


46 Osman Nuri Akbulut et al.

Introduction

Prostate cancer is the first reason of men deaths from

cancer in advanced age. The incidence of prostate

cancer is directly increased with age. Another reason

for increased incidence of illness is early diagnosis and

advanced techniques in diagnosis. This cancer is

silently develops and its beginning and processing in

the body can not be easy detectable. Because signs of

prostate cancer appears in the late phase. Therefore,

generally diagnosis can be done in cancer phase or

locally invasive with metastatic lesions. Today, local

cancer type of prostate can be cured, but late phase of

neoplasms can not be cured easily. The early diagnosis

of prostate cancer has been facilitated by the

development of serum prostate-specific antigen (PSA)

testing and evolution in transrectal ultrasound-guided

biopsy of the prostate. Over a decade has passed since

the initial recommendations for systematic sextant

sampling of the prostate to increase the accuracy of

cancer detection (Montironi and Schulman, 1986).

Subsequently, variations in the number and location of

biopsies have been proposed to maximize prostate

cancer detection and obtain more complete

information regarding tumor grade, tumor volume, and

local stage (Mc Neal and Bostwick, 1986; Epstein et

al., 1995; Wiley et al., 1997; Bostwick, 1996).

Although current biopsy strategies provide a wide

sampling of the prostate gland, biopsy histology may

not be conclusive for either the presence or absence of

adenocarcinoma. Prostatic intraepithelial neoplasia

(PIN) is found in a significant fraction of patients

undergoing transrectal prostate biopsies. In this article,

we discuss the significance of prostatic intraepithelial

neoplasia and other abnormal histology findings and

current evidence addressing the presence of cancer and

need for additional prostate biopsies.

Table 1: Low grade and high grade PIN comparison.

PIN is characterized by architecturally benign

prostatic acini lined by atypical cells, as defined by

cytologic criteria (Kellokumpu-Lehtinen, 1980;

Babanian et al., 1991). Nuclear changes are similar to

those seen in prostate cancer, with nuclear enlargement

and atypia, and prominent nucleoli. Accompanying

architectural findings may include flat, tufting,

papillary, and cribiform patterns. High-grade prostatic

intraepithelial neoplasia (hgPIN) encompasses both

grade 2 and grade 3 dysplasia with increasing

hyperchromatism and size of nucleoli. Low-grade PIN

(lgPIN), formerly grade 1 PIN, also exhibits crowding

and irregularity of the secretory epithelial layer with

less marked cytologic atypia; elongated

hyperchromatic nuclei and small nucleoli are present

yet not prominent. Table 1 summarizes features of

lgPIN and hgPIN. This study comprised on patients

group who have sextant biopsies with clinical history

and indications. All specimens investigated

pathologically and compared in eachother. We aim to

show the importance of PIN findings in prostate cancer

early diagnosis.

Material and methods

Low-grade PIN High-grade PIN

Between April 2001 and September 2003 total 166

patients in 31 months were investigated in our clinics

for suspicious indicators for prostate cancer. Biopsy

tissues were obtained with transurethral

ultrasonography (TRUS) and all specimens were

investigated histopathologically. Especially study

group was chosen from lower urinary track syndrome

patients and suspicious for prostate cancer whose PSA

levels were over 4 ng per ml.

The average age of study group was 67.5 (range

45-92). All specimens were investigated by one

Cytology

Nuclei Enlarged, pleiomorphic Enlarged, pleiomorphic

Chromatin Normal Increased density, clumping

Nucleoli Rarely prominent Frequently large, prominent, and multiple

Architecture Epithelial cell crowding and More crowding and stratification 4 patterns:

stratification Irregular spacing tufting, cribiform, micropapillary, flat

Basal cell layer Intact May show disruption

Basement membrane Intact Intact


Table 2: The Study group specimens histopathological characteristics.

PCA BPH

Table 3: Comparison of clinical and histopathological results of BPH cases.

BPH+hgPIN BPH+lgPIN BPH

pathologist according to TNM classification and WHO

system. In this study, prostate cancer, hgPIN, lgPIN

and BPH pathological results were compared with

serum PSA levels, DRE results. Only prostate cancer

patient specimens were compared with Gleason grade

to Gleason scores. PSA levels of study group were also

determined before biopsy application. DRE results

were evaluated as positive and negative for all

patients. TRUS with 6.5 MHz probe was done

transrectal (Shinokara et al., 1989). All biopsy

materials were obtained with disposable 18G

TRUCUT needle biopsy with automatic biopsy gun.

The presence of basal cells was apparent by

haematoxyline and eosin staining and was confirmed

by immunohistochemistry. The intraductal and

invasive signet ring cells were mucin negative and

were immunoreactive for PSA.

All specimens were kept in 10% formaline solution

for fixation. Prostate cancer diagnosis was obtained

from Gleason grade and score system. PIN diagnosis

was done according to McNeal and Bostwick (1986).

Pathological isolated hgPIN and lgPIN determined in

tissues followed up with DRE and PSA levels and if

needed again biopsy treatment adviced to patients.

Clinically and histopathological results were

investigated with SPSS statistical software version 11.0

and Kruskal-Wallis one way ANOVA and Chi-Square

variability methods were applied. Smaller than 0.05

value is accepted significant for probability analysis.

Results

PIN and prostate cancer 47

PCa+hg PIN PCa+lg PIN PCa+BPH PCa BPH+hg PIN BPH+lg PIN BPH Total

Case number 34 4 5 8 21 28 66 166

Total 51 115

Case number 21 28 66

Average age (year) 66.30±8.50 (49-84) 67.17±10.01 (51-86) 63.80±8.65 (45-82)

Average PSA ng/ml 12.27±8.68 11.32±6.71 7.77±3.49

DRE (%) n % n % n %

(-) 8 44 9 33 34 51

(+) 10 56 19 67 32 49

All patients characteristics were showed on Table 1. In

this study, 51 patients (% 31.3) were prostate cancer,

115 patients (% 33.3) were BPH. 38 of 51 prostate

cancer patients had also PIN lesions (74.5 %). 34 of 51

prostate cancer patients (66.6%) were also hgPIN and

4 of them (7.8%). 5 of the prostate cancer patients had

also BPH focal points (9.8%) (Figure 1). In another

investigation it was obtained that 8 of prostate cancer

patients had only cancer findings histopathologically.

46 of 115 non cancer patients was BPH (40%) and

also PIN. Isolated PIN which was not seen with cancer

findings were also investigated pathological. 21 hgPIN

(18.3%), 28 lgPIN (24.3%) was investigated in 46

patients (Figure 2). 66 of patients (57.4%) had no PIN

and cancer findings histopathologically (Table2).

Biopsy again treated for 6 of 21 (28.6%) isolated

hgPIN patients and results gave neoplastic cells

in this followups. Table 2 represents prostate

cancer PCa, PCa+BPH, PCa+hgPIN and PCa+lgPIN

histopathological results with average PSA levels and

DRE findings besides Gleason grade and Gleason

scores.

115 non cancer patients’ characteristics such as

PSA levels, DRE and percentages were described on

Table 2. PSA levels of 46 isolated PIN patients was

12.2 ng per mg and their BPH findings and PSA levels

correlation was highly meaningful (p


48 Osman Nuri Akbulut et al.

Figure 1: Prostate cancer and BPH cases according to PIN

Grade.

87 of of study group (52.6%) had PIN findings and 34

of 55 hgPIN findings (61.8%) had neoplastic focal

areas at the same time. 21 of the same group (38.2%)

was determined as isolated PIN. 32 lgPIN specimens

were investigated pathologically and 4 of them

(12.5%) had also cancer lesions, 28 of them (87.5%)

determined as isolated type PIN. PIN grade

classification, prostate cancer and BPH results

represented in Table 3.

7 different histopathological classifications when

compare to ages of the patients were not statistically

meaningful. Comparisons of PSA levels and DRE

results with 7 different classified groups were not

meaningful. Histopathologic results of patients

indicated that PSA levels and DRE were important

prostate cancer, but not significant within the age and

age groups. When isolated PIN patients investigated in

both hgPIN and ldPIN groups were not statistically

significant for age, PSA levels and DRE results.

Subgroups with cancer diagnosis in biopsied

patterns compared in each other for Gleason grade and

Gleason score and prostate cancer isolated group with

prostate cancer + hgPIN and lgPIN + prostate cancer

was statistically significant. Isolated prostate cancer

and hgPIN + prostate cancer subgroups had higher

statistical meaning for their Gleason scores (p


Prostate cancer diagnosis rate is increased last

years. Clinical studies are able to show early

precursors of illness successfully (Cooner et al., 1990).

Dispersed basal layer and PIN grade has a multiple

relation. 56% and 0.7% damaged basal layer showed

for hgPIN and lgPIN. McNeal and Bostwick (1986)

established that PIN rate for BPH was 43% and

prostate cancer cases were 82%. In this study PIN

grade was 42.6% for BPH and 74.5% for prostate

cancer patients.

When isolated hgPIN cases followed up, their

rebiopsy material showed 33-100% prostate cancer

diagnosis. Therefore PIN histopathological results can

be predictive factor for cancer occurence. 21 of hgPIN

patient adviced for re-biopsy and 18 of them accepted.

6 of 18 re biopsied material (33.3%) investigated by

one pathologist and diagnosed with prostate cancer.

Keetch et al. (1995) established that PSA levels of

lgPIN patients who diagnosed with prostate cancer

was similar in each other. Our study group who has

isolated lgPIN followed up for PSA levels and DRE

results. 10 of the cases had higher PSA level, so

adviced re biopsy. However 8 of them accepted our

suggestion and 2 of them (7.2%). Rest of them had

lgPIN pathological results. Trancoso et al. (1989)

compared PIN grade and PIN with prostate cancer

cases and they found that 21% of lgPIN cases and 54%

hgPIN cases were showing cancer indications. This

study results were 7.9% for lgPIN and 66.7% for

hgPIN results.

PIN and PSA relation in literature is well studied

and reports showed that average PSA levels of patients

who have PIN between BPH and prostate cancer

indicator values (Davidson et al., 1995; Brawer, 1989).

Our results were higher than BPH patients. However

these data were not statistically significant.

PIN with prostate cancer when investigated for

Gleason Grade and Gleason Score, findings indicate

that isolated PIN lesions in biopsy material will gain

more clinical importance in the following years.

Troncoso et al. (1989) explained this situation in

prostatectomy materials for PIN localization,

diversion, PIN grade and Gleason Grade and Gleason

Scores of prostate cancer cases histopathologically.

They also showed different results for DNA ploidy in

these cases. PIN and cancer grades are correlated in

each other and when PIN grade increment directly

correlated with prostate volume. However this

correlation could not be established between PIN

volume and cancer volume. 11 patients who are

classified as isolated prostate cancer had higher

Gleason Grade and Gleason Score values than prostate

cancer with hgPIN and prostate cancer with lgPIN.

However prostate cancer with hgPIN had more

Gleason Grade and Gleason Score, the differences

were not significant (p>0.05).

More expanded studies with prostate cancer

patients and prostatectomy materials will be useful to

understand PIN and prostate cancer biological relation.

Nowadays, localized prostate cancer treatments

chosen and patient survival rates are depends on

histopathological grade and scoring systems. If lgPIN

described low grade and scored cancer, follow up

procedure of lgPIN patients will be under discussion.

Deprivation of androgens, PIN and other diplastic

evaluations established that hormone dependency. If

antiandrogen treatments and effects on PIN explained

more detailed, how prophylactic or contraceptive

therapies can be continued with chemotherapeutic

agents will be clear. Neither patient age, PSA levels or

nor DRE are not enough for detemination of BPH,

hgPIN with prostate cancer and lgPIN for prostate

cancer.

Conclusion

This study comprised for discuss the PIN outcome

with what kind of prostatic illness. Tha data obtained

from this study established that hgPIN comes with

prostate cancer more than lg PIN. However, lgPIN

generally seen with BPH. Therefore, hgPIN with BPH

cases have to follow up with periodical PSA level

determination and DRE results. If the clinical

parameter have indicated changes, re biopsy should be

adviced to patients.

References

PIN and prostate cancer 49

Andriole GL and Catalona Wl. Using PSA to screen for

prostate cancer: The Washington University Experience.

Urol Clin North Am. 20: 647-649, 1993.

Babaian RJ, Miyashita H and Evans RB. Early detection

program for prostate cancer: Results and identification of

high risk patient population. Urology. 193: 37-39, 1991.

Bigler SA, Deering RE and Brawer MK. Comparison of

microscopic vascularity in benign and malignant prostate

tissue. Hum Path. 24: 220-223, 1993.

Bostwick DG. Progression of prostatic intraepithelial

neoplasia to early invasive adenocarcinoma. Eur Urol.


50 Osman Nuri Akbulut et al.

30: 145-148, 1996.

Botswick DG and Brawer MK. Prostatic intraepithelial

neoplasia and early invasion in prostate cancer. Cancer.

59: 788-793, 1987.

Brawer MK. Prostatic intraepithelial neoplasia and prostate

specific antigen. Urology. (Suppl). 34: 62-66, 1989.

Cooner WH, Mosley BR and Rutherford CL Jr. Prostate

cancer detection in a clinical urological practise by

ultrasonography, digital rectal examination, and prostate

specific antigen. J Urol. 143: 1146-1154, 1990.

Davidson D, Bostwick DG, Qian J, Wollan PC, Oesterling

JE, Rudders RA, Siroky M and Stilmant M. Prostatic

intraepithelial neoplasia is a risk factor for

adenocarcinoma: predictive accuracyn needle biopsies.

J Urol. 154: 1295-1298, 1995.

Epstein JI. Pathological cansiderations for the practicing

urologist. In: Cancer of the Prostate. Current Practice,

Future Directions. Wein AJ, Malkowicz SB (Ed).

Philadelphia, CoMed Communications. 15-31, 1989.

Epstein JI, Grignon DJ, Humphrey PA, McNeal JE,

Sesterhenn IA, Troncoso P and Wheeler TM.

Interobserver reproducibility in the diagnosis of prostatic

intraepitelial neoplasia. Am J Sur Path. 19: 873-886, 1995.

Greene DR, Wheeler TM and Egana S. A comparison of the

morphological features of cancer arising in the transition

zone and in the peripheral zone of the prostate. J Urol.

146: 1069-1076, 1991.

Keech DW, Humphery P, Stahl D, Smith DS, Catalona Wl.

Morphometric analysis and clinical follow-up of isolated

prostatic intraepithelial neoplasia in needle biopsy of the

prostate. J Urol. 154: 347-349, 1995.

Kellokumpu-Lehtinen P. The histochemical localization of

acid phosphatase in human fetal urethra and prostatic

epithelium. Invest Urol. 17: 435-438, 1980.

Kovi J, Mostofi FK, Heshmat MY, Enterline JP. Large acinar

atypical hyperplasia and cardilama of the prostate.

Cancer. 61: 555-558, 1988.

McNeal JE and Bostwick DG. Intraductal dysplasia: a

premalignant lesion of the prostate. Hum Path. 17: 64-

67, 1986.

Montironi R and Schulman CC. Precursors of prostatic

cancer: Progression, regression and chemoprevention.

Eur Urol. 30: 133-139, 1996.

Sark WA, Haas GP and Grignon DJ. High grade prostatic

intraepithelial neoplasia and prostatic adenocarcinoma

between the ages 20-69: An autopsy study of 249 cases.

Modern Path. 6: 68-71, 1993.

Shinohara K, Wheeler TM and Scardino PT. The appearence

of prostate cancer in transrectal ultrasonography:

Correlation of imaging and pathological examinations.

J Urol. 14: 76-82, 1989.

Troncoso P, Babaian RJ, Ro JY and Grignon DJ. Prostatic

intraepithelial neoplasia and invasive prostatic

adenocarcinoma in cystoprostatectomy specimens.

Urology (Suppl). 34: 52-55, 1989.

Weinstein MH, Epstein JI. Significance of high grade

prostatic intraepithelial neoplasia on needle biopsy. Hum

Path. 24: 624-628, 1993.

Wiley EL, Davidson P, Melntire DD and Sagalowskiy AI.

Risk of concurrent prostate cancer in cystoprostatectomy

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Journal of Cell and Molecular Biology 3: 51-119, 2004.

Haliç University, Printed in Turkey.

Bladder tumor antigen sensitivity in bladder cancer patients

Soner Güney, Serdar Arisan*, ‹brahim Duman, Turhan Çaflkurlu, Cem Sönmez,

Erbil Ergenekon

fiiflli Etfal Research and Training Hospital, 1 st Urology Clinics, fiiflli-Istanbul, Turkey (*author for

correspondence)

Received 19 December 2003; Accepted 26 January 2004

Abstract

With its incidence continuing to increase, bladder cancer is now the fifth most common cancer in the world.

Approximately half of these patients will have muscle-invasive disease at diagnosis and have distant metastasis

within 2 years and 60% die within 5 years despite treatment. Therefore, rapid and sensitive urine based markers are

still under investigation. In this study, we aim to find the BTA stat results for 259 Turkish patients (200 patients with

and 59 without bladder cancer) whose voided urine samples were samples were available before cystoscopy or other

surgical intervention. Histopathological results compared with BTA results. The mean age of 200 patients included

in the study was 67 years and range was 25 to 81. 164 of 200 patient was follow up patients and the rest of them was

first diagnosed group. In our study groups BTA stat test’s sensitivity and specificity was 75% and 80% respectively.

However it is not enough specific and more variability of obtained data is not reliable. Besides of these, healthy

donors’ urine tests were 40% positive. This means BTA stat have specificity problems. Therefore BTA stat test is not

a sufficient diagnostic test.

KKeeyy wwoorrddss:: BTA stat, bladder cancer, urine based tests

Mesane kanseri hastalar›nda mesane tümör antijen hassasiyeti

Özet

Mesane kanseri artan görülme s›kl›¤› ile dünyada 5. s›rada yer almaktad›r. Hastalar›n yaklafl›k yar›s›nda kasa

invazyon vard›r ve bunlar›n %60’› 2 y›l içerisinde metastaz gösterip 5 y›l içerisinde ölmektedir. Bundan ötürü çabuk

ve hassas idrar testleri halen araflt›r›lmaktad›r. Bu çal›flmada 259 Türk hastan›n (200 hasta ve 59 kontrol) idrar

örnekleri incelendi. Histopatoloji sonuçlar› BTA sonuçlar› ile karfl›laflt›r›ld›. 200 hastan›n ortalama yafllar› 67 olup

25-81 yaflalar› aras›nda idi. BTA hassasiyeti ve spesifitesi %75 ve %80 olarak saptand›. Ancak elde edilen datalar

çok varyasyona sahip oldu¤u için güvenilir de¤ildir. Bunun yan›s›ra sa¤l›kl› hastalarda BTA testi %40 olarak

saptanm›flt›r. Bu da BTA stat’›n spesifite problemlerinin oldu¤unu göstermektedir. BTA stat testi etkili bir

diyagnostik test de¤ildir.

AAnnaahhttaarr ssöözzccüükklleerr:: BTA stat, mesane kanseri, idrar testleri

Introduction

With its incidence continuing to increase, bladder

cancer is now the fifth most common cancer in US,

with an estimated 56,500 new cases predicted for

2002, of which 12,600 patients are expected to die of

the disease (Buhiyan et al., 2003). The three main

types of cancers that affect the bladder are urothelial

carcinoma (transitional cell carcinoma or TCC),

squamous cell carcinoma, and adenocarcinoma. TCC,

by far the most common form of bladder cancer

accounting for more than 90% of these cancers in US,

51


52 Soner Güney et al.

is the second most common malignancy of

genitourinary tract and third most common cause of

death among genitourinary tumors (Arisan, 2003).

Bladder cancer is currently diagnosed using

cystoscopy and cytology in patients with suspicious

signs and symptoms. Most patients with bladder

cancer are diagnosed upon a gross or microscopic

hematuria (Buhiyan et al., 2003; Arisan, 2003; Konety

and Getzenberg, 2001). Approximately 75-80% of

newly diagnosed bladder neoplasms are confined to

the urothelium or invade the lamina propria (Ta and

T1) and can be managed with transurethral resection

(TUR) and intravesical therapy. The recurrence rate of

these neoplasms are high with progression (Simon et

al., 2003). Patients having high-grade disease have a

worse prognosis. Approximately half of these patients

will have muscle-invasive disease at diagnosis and

have distant metastasis within 2 years and 60% die

within 5 years despite treatment. Systemic treatment

options for bladder cancer include surgery,

chemotherapy, radiation, and immunotherapy (Konety

and Getzenberg, 2003).

This situation indicates the importance of exact and

sensitive diagnosis at initial level of illness. Current

follow-up procedure after initial presentation typically

includes cystoscopy and urine cytology every 3

months for 1-2 years, every 6 months for an additional

2-3 years and then annually, assuming no recurrence

(Simon et al., 2003).

Cystoscopy is a relatively short, minimally

traumatic office procedure performed with topical

anesthesia that identifies nearly all papillary and

sessile lesions. However, despite the introduction of

the flexible cystoscope, it is still an invasive

procedure, and causes some discomfort. Cystoscopy

aided by cytology is the mainstay for diagnosing

bladder cancer (Konety et al., 1999). Voided urine

cytology has been used since 1945 as a screening test

for this condition (Papanicolau and Marshall, 1945).

Cytology has a high sensitivity and specificity for the

detection of high-grade tumors. However its

sensitivity for low-grade tumor is poor and also

expensive. It also requires a highly trained

cytopathologist, who may not be available in all areas.

On the other hand, cystoscopy is invasive and

relatively expensive too, and it may also be

inconclusive at times. Especially in many patients with

an indwelling catheter or active inflammation,

cystoscopy may not be definitive due to the grossly

abnormal appearance of the bladder mucosa. The

deficiencies of cytology and the invasiveness of

cystoscopy again render each test suboptimal for

tumor surveillance. Therefore, a noninvasive method

for detecting and monitoring bladder cancer would be

objective, noninvasive, and easy to administer and

interpret, and have high sensitivity and specificity

(Wiener et al., 1998; Malik and Murphy, 1999).

Other urological malignancies such as prostate

cancer have an existing serum based marker that has

been well studied or they are not likely to release

potential marker substances into the urine, making

urine based testing less useful (Saad et al., 2001;

Bhuiyan et al., 2003). There are many investigations to

find the most sensitive, useful for clinics and cheap

marker for bladder cancer. However some Food and

Drug Administration (USA) proved tests or still under

research ones are not sufficient and enough for urine

based diagnosis.

These tests detect human complement factor H

related protein. This protein can be isolated from urine

samples of bladder cancer patients. BTA stat is a

spectrochromatographic assay and it is really rapid and

single step test (Malkowicz, 2000; D'Hallewin and

Baert, 1995). Its sensitivity rate is 75% for carcinoma

in situ patients and its specificity is over 85%. This test

takes only 5-7 min for each specimen. Easy applicable

test can advice office usage without trained laboratory

technician and equipment (Arisan, 2003; Konety and

Getzenberg, 2001). However BTA stat test, under

benign genitourinary conditions, particularly

hematuria, may yield false positive results (Messing et

al., 1995). Therefore it is not a golden standard for

diagnosis.

This study designed for showing BTA stat test

sensitivity rates in Turkish bladder cancer patients in

expanded patient population with other diagnostic

features. These results may be useful for clinic

diagnosis of bladder cancer in Turkish population.

Materials and methods

Patients

Total 259 patients (200 patients with and 59 without

bladder cancer) whose voided urine samples were

samples were available before cystoscopy or other

surgical intervention at the Sisli Etfal Research and

Training Hospital, 1st urology clinics between

March 1998-October 2002, histopathological results


compared with BTA results. Table 1 showed the details

for the study group patients. All the tumor specimens

were histological diagnosed as transitional cell

carcinomas. A half of the patients with bladder cancer

had a past history of the disease. The patients without

bladder cancer included 59 with benign urological

diseases such as urolithiasis and benign prostate

hyperplasia and 19 who had a past treatment history of

bladder cancer and were followed up with no evidence

of recurrence.

Study procedure

Before any manipulation, an urine specimen from

spontaneous miction was collected from the patient.

The specimen was divided into two different samples;

one for the BTA stat test and the other analysis, with

the pathologist unaware of the test results. Next,

cystoscopy was performed. Endoscopic resection and

histopathological analysis were performed on those

patients diagnosed with vesical cancer by at least one

of the three tests, with cancer staging per the TNM

classification and cancer grading as per the World

Health Organization criteria.

Histopathological analysis

Tumors were classified by the criteria of the World

Health Organization and staged according to TNM

system by one pathologist who was unaware the study.

Also sensitivity and specificity of the test were

compared with these data and applied SPSS statistical

software version 11.0. p value less than 0.05 were

considered significant.

BTA test

BTA stat test in bladder carcinoma 53

Table 1: Characteristics of 259 patients whose voided urine was evaluated by urinary cytology and the Bard bladder tumor

antigen (BTA) test.

Patients With bladder cancer Without bladder cancer

Age 67 60

Male/Female 129/71 28/22

Past medical history of bladder cancer 164 19

The BARD BTA test (Bard Diagnostic Sci. C.R. Bard,

Redmond, WA, USA) is a latex aggregation assay that

qualitatively detects basement membrane complexes

Table 2: Relationship between BTA test results and histopathological findings for 200 patients with bladder cancer.

Stage

Patients number Test Sensitivity (%) 95% Confidence Interval

Ta 41 BTA stat test 40 32-62

Cytology 29

T1 120 BTA stat test 85 72-96

Cytology 74

T2-4 39 BTA stat test 91 89-95

Cytology 86

Grade

G1 54 BTA stat test 43 40-51

Cytology 30

G2 86 BTA stat test 70 67-76

Cytology 53

G3 60 BTA stat test 95 85-90

Cytology 89


54 Soner Güney et al.

of the bladder wall in the urine samples. This test was

performed in a blinded fashion by an impartial

observer on all patient specimens in accordance with

the manufacturer instructions. It is a one-step

qualitative assay that recognizes a tumor antigen of the

complement factor H family associated with

transitional cell carcinoma. Five drops of fresh or

previously frozen urine are placed on the entrance pool

of a small immunochromatographic environment. The

urine reacts with a colloidal gold-conjugate

antibladder tumor associated antigen antibody.

Antigen complexes are captured by another

antibladder tumor-associated antigen antibody. A

positive result is indicated by a red line after 5 minutes.

A second line indicates an internal control zone.

Excluded from the study were patients who had

undergone surgical treatment or instrumentation

within 14 days of testing, as well as patients active

urinary tract infections.

Statistical analysis

Statistical values within the data were analyzed by

SPSS statistical software (version 11.0).

Results

The mean age of 200 patients included in the study

was 67 years and range was 25 to 81. 164 of 200

patient was follow up patients and the rest of them was

first diagnosed group. In our study groups BTA stat

test’s sensitivity and specificity was 75% and 80%

respectively. The BTA stat test results were decreased

in low staged and graded patients. Table 2 explains the

relationship between histological findings for bladder

cancer and cytology and BTA test results for 200

patients with and 40 without bladder cancers. The

histopathological diagnoses of the patients with

bladder cancer were available and related with BTA

test results. Positive BTA test results were found

showed in Table 3 with cytological results. With BTA

stat test, we had 24 false positive (40%) results

These data is not statistically significant. Table 2

demonstrates the relationship between histopathological

results and BTA test results for 200 patients with bladder

cancer.

Diagnostic categories enabled us to calculate the

sensitivities and specificities of clinical outcomes

based on different decision thresholds or decision

levels such as pathological investigations. In every

clinical finding for diagnosis has different sensitivity

and specificity rate.

Discussion

Various predominantly monoclonal antibodies directed

against tumor associated antigens are receiving

increased attention for the primary diagnosis and

follow up of bladder cancer. Immunocytochemical

methods using voided urine specimens have the

advantage of being noninvasive. Several soluble

markers have been previously described. The bladder

tumor antigen (BTA Trak), nuclear matrix protein

(NMP22), and urinary bladder antigen (UBC) have

been investigated as diagnostic methods to substitute

for voided urine cytology examination (Rife et al.,

1979; Simon et al., 2003; Lokeshwar and Soloway,

2001). Unfortunately these techniques need upper

degree laboratory supply and they are not providing

office use strip test. Also Food and Drug

Administration proved and under preclinical

researches type tests have extremely variable

sensitivity.

The first generation of the BTA test consisted of a

latex agglutination test conducted in several steps that

detected basement membrane complexes in the urine.

The test provided better results than those provided by

cytologic analysis. Its range is between 40.4% and

78% against a cytologic sensitivity of 16% and 39.3%.

However Lee et al. (2000) demonstrated a greater

sensitivity in cytologic analysis (54%). There are no

superior results for BTA in published studies (Ian et al.,

2001; Konety and Getzenberg, 2001).

The characteristics of the patients who underwent

diagnostic test in our hospital did not differ from the

series of other investigators, with a similar mean age

and range. Ravery et al. (1998) and Sarosdy et al.

(1995) reported sensitivity higher than that of

cytological analysis in the different stages. (45% Ta,

85% T1, 75% T2-4) for BTA stat test. When they

compared the grades for BTA stat test, 38% G1, 64%

G2 and 71% G3 were obtained.

In our study group, the sensitivity of test 40% in

Ta, 85% in T1 and 91% in T2-4. When grades

investigated for BTA stat test results, 43% sensitivity

for G1, 70% for G2 and 95% for G3 was determined

(Table 2). The sensitivity of the BTA test is better than

cytological experiments. However it is not enough


specific and more variability of obtained data is not

reliable. Besides of these, healthy donors’ urine tests

were 40% positive. This means BTA stat have

specificity problems.

In conclusion, urine tests for bladder cancer should

have the highest sensitivity and also must be easy

applicable and cheap. Therefore more valuable urine

tests or such biological response tests should be

investigated for exact diagnosis. BTA stat test is not a

gold standard for diagnosis.

References

Arisan S. The importance of molecular tumor markers for

bladder cancer. Turk J of Cancer. 33: 171-176, 2003.

Bhuiyan J, Akhter J, O’Kane DJ. Performance characteristics

of multiple urinary tumor markers and sample collection

techniques in the detection of transitional cell carcinoma

of the bladder. Cli Chem Acta. 4: 69-77, 2003.

D'Hallewin MA, Baert L. Initial evaluation of the bladder

tumor antigen test in superficial bladder cancer. J Urol.

155: 475-476, 1995.

Konety BR, Getzenberg RH. Urine based markers of

urological malignancy. J of Urol. 93: 600-611, 2001.

Konety BR, Metro MJ, Melham MF and Salup RR.

Diagnostic value of voided urine and bladder barbotage

cytology in detecting transitional cell carcinoma of the

urinary tract. Urol Int. 62: 26-30, 1999.

Lee MY, Tsou MH, Cheng MH, Chang DS, Yang AL and

Ko JS. Clinical application of nmp22 and urinary

cytology in patients with hematuria or a history of

urothelial carcinoma. World J Urol. 18: 401-405, 2000.

Lokeshwar V and Soloway M. Current bladder tumor tests:

does their projected utility fulfil clinical necessity?

J Urol. 165: 1067-1077, 2001.

Malik SN and Murphy WM. Monitoring patients for bladder

neoplasms: What can be expected of urinary cytology

consultations in clinical practice? Urology. 54: 62-66,

1999.

Malkowicz SB. The application of human complement

factor h-related protein (bta trak) in monitoring patients

with bladder cancer. Urol Clin North Am. 27: 63-73,

2000.

Messing EM, Young TB and Hunt VB. Hematuria home

screening: repeat testing results. J Urol. 154: 57-61, 1995.

Papanicolau GN and Marshall VF. Urine sediment smears as

a diagnostic procedure in cancers of the urinary tract.

Sci. 101: 519-523, 1945.

Ravery V, Meulemans A, Ishak L and Boccon-Gibod L.

Detection of bladder cancer using the BTA test. Review

and personal experience. Immunoanalyse & Biologie

Spécialisée. 13: 157-160, 1998.

BTA stat test in bladder carcinoma 55

Rife CC, Farrow GM and Utz DC. Urine cytology of

transitional cell neoplasms. Urol Clin North Am. 3: 599-

605, 1979.

Saad A, Hanbury DC and McNicholas TA. Boustead GB,

Woodman AC. The early detection and diagnosis of

bladder cancer: A critical review of the options. Eur

Urol. 39: 619-633, 2001.

Sarosdy MF, DeVere RW and Soloway MS. Results of a

multicenter trial using the BTA test to monitor for and

diagnose recurrent bladder cancer. J Urol. 154: 579-584,

1995.

Simon M, Vinata BL and Soloway M. Current bladder cancer

tests: Unnecessary or beneficial? Crit Rew in Onc/Hema.

11: 352-368, 2003.

Walsh IK, Keane PK, Ishak LM and Flessland KA. The BTA

stat test: A tumor marker for the detection of upper tract

transitional cell carcinoma. Urology. 58: 532-535, 2001.

Wiener HG, Mian C, Haitel A, Pycha A, Schatzl G,

Marberger M. Can urine bound diagnostic tests replace

cystoscopy in the management of bladder cancer?

J Urol. 159: 1876-1880, 1998.

Wiener HG, Vooijs GP and Hof-Grootenboer van't B.

Accuracy of urinary cytology in the diagnosis of primary

and recurrent bladder cancer. Acta Cytol. 37: 163-169,

1993.


Book reviews

Meral ÜNAL, Bitki (Angiosperm) Embriyolojisi,

Mart Matbaas›, 304 Sayfa, ISBN: 975-400-040-9, 2004.

Bitki embriyosunun geliflimini inceleyen bir bilim

dal› olan bitki embriyolojisi; bitki sistemati¤i, genetik

ve fizyoloji ile birlikte biyoloji biliminin temelini

oluflturmaktad›r. Bu kitapta bitki embriyolojisinin

tarihçesi, çiçek, mikrosporangiyum, erkek gametofit,

megasporangiyum, difli gametofit, tozlaflma, döllenme,

efley uyuflmazl›¤›, endosperma, embriyo, poliembriyoni,

apomiksis ve tohum bölümleri yer almaktad›r.

Son y›llarda embriyoloji bilimi deneysel alanda da

çok önemli ilerlemeler kaydetmifltir. Bu kitap özellikle

biyologlar, ziraatçiler, bitki biyoteknologlar› ve

ormanc›lar için önemli bir kaynak oluflturmaktad›r. Bu

alanlarda lisans ve lisansüstü e¤itim yapan ö¤rencilerin

yararlanabilece¤i önemli bir kaynakçad›r.

Bu kitap hem klasik bulgular› ve hem de son

geliflmeleri içerdi¤inden bu alanda araflt›rma

yapanlar›n yararlanaca¤› flekildedir, bu amaçla da her

bölüm sonunda kaynakça verilmifltir. Ayr›ca, son

derece karmafl›k konular flekil ve foto¤raflarla kolay

anlafl›labilir hale getirilmifltir. Ülkemizde bu alanda

yeterli kaynak olmad›¤› göz önüne al›nd›¤›nda bu

kitab›n önemi daha da ortaya ç›kmaktad›r. Ayr›ca bu

kitab› araflt›rma kütüphanelerine önemli bir katk›

olarak tavsiye ederim.

Narç›n Palavan-Ünsal

Haliç Üniversitesi,

Moleküler Biyoloji ve Genetik Bölümü

57

Meral ÜNAL, Plant (Angiosperm) Embryology,

Mart Matbaas›, 304 PP, ISBN: 975-400-040-9, 2004.

Plant embryology investigates the development

of plant embryo which is the basis of botany

science with plant taxonomy, genetics and physiology

altogether. This book includes history of embryology,

flower microsporangium, male gametophyte,

megasporangium, female gametophyte, pollination,

fertilization, self-incompatibility endosperm, embryo,

poliembryony, apomixis and seed.

In recent years, embryology science has recorded

very important developments in experimental area.

This publication is an important source particularly for

biologists, agronomists, plant biotechnologists and

forestry engineers. Besides, this source is also an

important reference book for undergraduate and

graduate students studying at this area. Since, this

book contains classic data and recent developments in

plant embryology, It is also useful for scientists

studying plant embryology. For this purpose an

invaluable reference list was presented at the end of

each section. Furthermore, extremely complex topics

were introduced with and illustrations and photographs

to easy understanding.

If one can consider the availability of limited

document at this area in our country, the importance

and necessity of this book become more evident.

Finally, I recommend this book to lecturer and to a

major academic and research libraries as an important

addition to their collection of plant biology literature.

Narç›n Palavan-Ünsal

Halic University,

Department of Molecular Biology and Genetics


58

Instructions for authors

General

1. Journal of Cell and Molecular Biology

is published biannually (January and July) and

covers all aspects relating to cell biology, molecular

biology, genetics, microbiology and related topics.

2. Manuscripts must be written in English.

Particular attention should be given to

consistency in the use of technical terms

and abbreviations.

3. Manuscripts (in triplicate) should be sent to:

The Editorial Office

Journal of Cell and Molecular Biology,

Haliç Üniversitesi,

Fen-Edebiyat Fakültesi,

Ahmet Vefik Pafla Cad. No :1

34280, F›nd›kzade,

‹stanbul-Turkey

4. Each submitted manuscript will be assessed by a

member of the editorial board and by two expert

referees. Authors will be consulted if the paper is

considered suitable for publication but alterations

are tought desirable. After these alterations have

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final.

5. The author is the only responsible person from the

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Three types of paper will be published, these are

original research papers, review articles and letters to

the editor. Book reviews are also welcome.

1. Original research papers: Only original

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and ideas will be accepted. Manuscripts should not

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3. The first page should indicate the title of the

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below), Tables (see below), Figure legends (see

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1. Only SI units should be used. Current abbreviations

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2. Latin names should be underlined or typed in italics.

References:

1. Citation in the text should take the form: Smith and

Robinson (1990) or (Smith and Robinson, 1990). If

several papers by the same author in the same

year are cited, 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) or (Smith et al., 1990).

2. 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 X-irradiation of

mammalian cells. Int J Radiat Biol. 49: 611- 620,

1986.

Taccioli CE, Cottlieb TM and Blund T. Ku 80: 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. In: The Biochemistry of Plants: A

Comprehensive Treatise. Stumpf PK and Conn EE

(Ed). Academic Press, New York. 137-157, 1987.

Weaver RF. Molecular Biology. WCB/Mc

Graw-Hill.1999.

2. Only papers published or in press should be cited in

the literature list. Unpublished results, including

submitted manuscripts and those in preparation,

should be cited as unpublished in the text.

3. The list of literature must be typed double space

throughout and checked thoroughly before

submission.

P roofs and offprints

59

1. Page proofs will be sent to the corresponding

author for checking before publication. Corrected

proofs should be sent back to the Editor within

three days of receipt, otherwise Editor reserves the

rights to correct the proofs himself and to send the

material for publication.

2. Contributors receive 25 offprints of their articles

free of charge.


Journal of Cell and Molecular Biology

CONTENTS Volume 3, No.1, 2004

Dedication

Review articles

Gene therapy for the haemoglobinopathies

Hemaglobinopati için gen terapisi

A. Athanassiadou 1-7

Systemic acquired resistance: Characterization of genes associated with plant

defence response

Sonradan sistemik olarak elde edilmifl direnç: Bitki savunma cevab› ile ilgili

genlerin karakterizasyonu

‹. Tiryaki, H. Tunaz 9-14

Research papers

Biological investigations into Antidesma madagascariense Lam. (Euphorbiaceae),

Faujasiopsis flexuosa (Lam.) C. Jeffrey (Asteraceae), Toddalia asiatica (L.) Lam.

and Vepris lanceolata (Lam.) G. Don (Rutaceae)

Antidesma madagascariense Lam. (Euphorbiaceae), Faujasiopsis flexuosa (Lam.)

C. Jeffrey (Asteraceae), Toddalia asiatica (L.) Lam. and Vepris lanceolata (Lam.)

G. Don (Rutaceae) da biyolojik araflt›rmalar

F. B. Narod, A. Gurib-Fakim, A. H. Subratty 15-21

The role of meta-topolin in senescence of wheat leaf segments

Meta-topolinin bu¤day yaprak segmentlerinin senesensindeki rolü

N. Palavan-Ünsal, S. Ça¤, E. Çetin 23-31

Characterization of Bacillus species by numerical analysis of their SDS-PAGE

protein profiles

Bacillus Türlerinin SDS-PAGE protein profillerinin nümerik analiziyle karakterizasyonu

‹. Berber 33-37

A kinetic model for in-vitro intestinal uptake of L-tyrosine and D (+)-glucose across

rat everted gut sacs in the presence of Momordica charantia, a medicinal plant used

in traditional medicine against diabetes mellitus

Diabetes mellitus’a karfl› kullan›lan geleneksel t›bbi bitki Momordica charantia varl›¤›nda ters

yüz edilmifl s›çan ba¤›rsa¤›ndan L-tirozin ve D(+)-glukoz geçifli için in vitro kinetik model

M. F. Mahomoodally, A.Gurib-Fakim, A. H. Subratty 39-44

Prostatic intraepithelial neoplasm: Retrospective results of clinical,

histopathological approaches

Prostat intraepitel neoplasm: Klinik ve histopatolojik retrospektif sonuçlar

O. N. Akbulut, S. Güney, ‹. Duman, ‹. Çömez, S. Arisan, E. Ergenekon 45-50

Bladder tumor antigen sensitivity in bladder cancer patients

Mesane kanseri hastalar›nda mesane tumör antijen hassasiyet

S. Güney, S. Arisan, ‹. Duman, T. Çaflkurlu, C. Sönmez, E. Ergenekon 51-55

Book Reviews 57

Instructions to authors 58-59

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