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‹STANBUL

1998

VOLUME 2 • NO. 1 • 2003 • 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. Ahmet YÜKSEL

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, Patros, 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

Zihni DEM‹RBA⁄, Trabzon, Turkey

Mustafa DJAMGOZ, London, UK

Aglika EDREVA, Sofia, Bulgaria

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

Kürflat ÖZD‹LL‹

Damla BÜYÜKTUNÇER

Özge EM‹RO⁄LU

Mehmet Ali TÜFEKÇ‹

Merve ALO⁄LU

Asl› BAfiAR

Ünal EGEL‹, Bursa, Turkey

Candan JOHANSEN, ‹stanbul, Turkey

As›m KADIO⁄LU, Trabzon, Turkey

Valentine KEFEL‹, 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 2/2003

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. Ahmet YÜKSEL

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, Patros, 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

Zihni DEM‹RBA⁄, Trabzon, Turkey

Mustafa DJAMGOZ, London, UK

Aglika EDREVA, Sofia, Bulgaria

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

Kürflat ÖZD‹LL‹

Damla BÜYÜKTUNÇER

Özge EM‹RO⁄LU

Mehmet Ali TÜFEKÇ‹

Merve ALO⁄LU

Asl› BAfiAR

Ünal EGEL‹, Bursa, Turkey

Candan JOHANSEN, ‹stanbul, Turkey

As›m KADIO⁄LU, Trabzon, Turkey

Valentine KEFEL‹, 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 2, No.1, 2003

Dedication

Review articles

Polyamines in plants: An overview

Bitkilerde poliaminler: Genel bir bak›fl

R. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston 1-12

Phenolic cycle in plants and environment

Bitkilerde fenolik döngü ve çevre

V. I. Kefeli, M. V. Kalevitch, B. Borsari 13-18

Research papers

The short-term effects of single toxic dose of citric acid in mice

Farelerde sitrik asidin tek toksik dozunun k›sa süreli etkileri

T. Aktaç, A. Kabo¤lu, E. Bakar, H. Karakafl 19-23

Characterisation of RRPPPP77 mutant lines of the col-5 ecotype of AArraabbiiddooppssiiss tthhaalliiaannaa

Arabidopsis thaliana’n›n col-5 ekotipinden elde edilen mutant hatlardan RPP7

geninin karakterizasyonu

C. Can, M. Özaslan, E. B. Holub 25-30

The effect of mmeettaa-topolin on protein profile in radish cotyledons

Meta-topolinin turp kotiledonlar›nda protein profiline etkisi

S. Ça¤, N. Palavan-Ünsal 31-34

The effect of electromagnetic fields on oxidative DNA damage

Elektromanyetik alan›n oksidatif DNA hasar› üzerindeki etkisi

S. ‹fller, G. Erdem 35-38

Chromosomes of a balanced translocation case evaluated with atomic force

microscopy

Dengeli translokasyon vakas›nda kromozomlar›n atomik güç mikroskobu ile

de¤erlendirilmesi

Z. Y›lmaz, M. A. Ergun, E. Tan 39-42

Effect of epirubicin on mitotic index in cultured L-cells

Epirubisinin kültürdeki L-hücrelerinde mitotik indekse etkisi

G. Özcan Ar›can, M. Topçul 43-48

Letter to editor 49-51

Book reviews 53

Instructions to authors 55-56


This issue is dedicated to

P rof. Dr. Arthur W. Galston

for his invaluable contribution to plant biology


Arthur W. Galston, Curriculum Vitae

Born: April 21, 1920 Eaton Professor of Botany, Emeritus, Department of

Molecular, Cellular and Developmental Biology,

Education: B.S. Cornell University, 1940; Yale University, New Haven, CT 06520-8103,

M.S. University of Illinois, 1942; Ph. D. 1943 Tel. (203) 432-6161; e-mail arthur.galston@yale.edu

Honors: Elected to Phi Beta Kappa; Phi Kappa Phi; Sigma Xi; American Academy of Arts and Sciences, National

Sigma Xi Lecturer, 1966; National Phi Beta Kappa Visiting Scholar, 1972-1973; Award of the New York Academy

of Sciences, 1979; William Clyde De Vane Medal for lifelong teaching and scholarship, Yale University, 1994;

Honorary LL.D, 1980 Iona; Honorary Ph. D., Hebrew University of Jerusalem, 1992.

Experience: Plant Physiologist, Emergency Rubber Project, California Institute of Technology 1943-1944; Instuctor

in Botany, Yale University, 1946-1947; Senior Research California Institute of Technology, 1947-1950; Associate

Professor of Biology, California Institute of Technology, 1951-1955. Professor of Plant Physiology, Department of

Botany, Yale University 1955-1961; Chairman, Department of Botany, 1961-1962; Director, Division of Biological

Sciences, Yale University, 1965-1966; Professor of Biology, 1962-1973; Eaton Professor of Botany, 1973-;

Chairman, Department of Biology 1985-1988; Eaton Professor Emeritus, 1990.

Fellow of the John Simon Guggenheim Memorial Foundation, Stockholm and Sheffield, 1950-1951; Fulbright

Fellow, Canberra, Australia, 1960-1961; National Science Foundation Faculty Fellow, London 1967-1968; Albert

Einstein Fellow and Visiting Professor, Hebrew University of Jerusalem, 1980; Visiting Fellow Wolfson College,

Cambridge, England, 1983; Visiting Scientist, RIKEN Institute, Wako, Saitama, Japan, 1988-1989.

Secretary, American Society of Plant Physiologists, 1955-1957; Vice President, 1957-1958; President, 1962-1963.

Secretary-Treasurer, International Association for Plant Physiology, 1962-1967. Vice-President

Botanical Society, 1967-1968; President 1968-1969; Award, 1970. Member, Commitee on Space Biology and

Medicine, National Research Council; Member Life Sciences Advisory Committee, NASA; also Long Range

Strategic Planning Committee in Life Sciences Advisory Committee, NASA; Member, NASA Disciplinary Working

Group for CELLS (Controlled Ecological Life Support Sytem).

P resent of past Editorial Board Member: Plant Growth Regulation, Pesticide Physiology and Biochemistry,

Environment, Chemical and Engineering News, Science Year, Plant Physiology, Phsiology, Phytochemistry,

American Journal of Botany, Lloydia. Formerly regular columnist, Natural History Magazine.

Former Member: Metabolic and Regulatory Biology Panels, National Science Foundation; Executive Committee,

Growth Society; Life Science Advisory Committee, NASA; and Governing Boards, Biological Sciences Curriculum

Study, Commission on Undergraduate Education in the Biological Sciences and AIBS.

First American scientist to visit the People’s Rebuplic of China, 1971.

Books: ‘Principles of Plant Physiology’ (with J. Bonner), Freeman, 1952. ‘Life of the Green Plant’, Prentice Hall,

1961, 2 nd Ed. , 1964, 3 rd Ed. , 1980 (with P. J. Davies and R. L. Satter). ‘Control Mechanisms in Plant Development’, (with

P. J. Davies), Prentice Hall, 1970. ‘Daily Life in People’s China’, Crowell, 1973; Simon and Schuster, 1975. ‘Green

Wisdom’ Basic Books, Inc. NY, 1981; Putnam, 1983. ‘Life Processes in Plants’, Freeman (Scientific American

Library), 1994. ‘New Dimensions in Bioethics’, Arthur W. Galston and Emily G. Shurr, eds. Kluwer Academic

Publishers, Boston/Dordrecht/London, 2001.

More than 320 articles in referred scientific journal; approximately 60 general articles on problems of science and

society.


Journal of Cell and Molecular Biology 2: 1-12, 2003.

Haliç University, Printed in Turkey.

Polyamines in plants: An overview

Ravindar Kaur-Sawhney 1 *, Antonio F. Tiburcio 2 , Teresa Altabella 2 , and Arthur W. Galston 1

1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT,

06520-8103, USA; 2 Laboratori de Fisiologia Vegetal, Facultad de Farmacia, Universitat de Barcelona,

Spain (* author for correspondence)

Received 21 October 2002; Accepted 10 November 2002

Abstract

This article presents an overview of the role of polyamines (PAs) in plant growth and developmental processes. The

PAs, putrescine, spermidine and spermine are low molecular weight cations present in all living organisms. PAs and

their biosynthetic enzymes have been implicated in a wide range of metabolic processes in plants, ranging from cell

division and organogenesis to protection against stress. Because the PA pathway has now been molecularly and

biochemically elucidated, it is amenable to modulation by genetic approaches. Genes for several key biosynthetic

enzymes namely, arginine decarboxylase, ornithine decarboxylase and S-adenosyl methionine decarboxylase have

been cloned from different plant species, and antibodies to some genes are now available. Both over-expressed and

antisense transgenic approaches to PA biosynthetic genes have provided further evidence that PAs are required for

plant growth and development. However, molecular mechanisms underlying PA effects on these processes remain

unclear. Analysis of gene expression by using DNA microarray genomic techniques should help determine the precise

role of these compounds. The potential of proteomics to unravel the role of PAs in particular cellular processes has

also been examined. The extensive use of the two-hybrid system and other proteomic approaches will provide new

insights into the role of PAs in signal transduction. Furthermore, there is evidence that proteomics provides an

excellent tool for determining supramolecular organizations of PA metabolic enzymes which may help in

understanding homeostatic control of this metabolic pathway.

KKeeyy wwoorrddss:: Polyamines, mutants, transgenic plants, genomics, proteomics

Bitkilerde poliaminler: Genel bir bak›fl

Özet

Bu makalede poliaminlerin (PA) bitki büyüme ve geliflme olaylar›ndaki rolüne genel bir bak›fl yap›lmaktad›r. PA ler

putresin, spermidin ve spermin, düflük molekül a¤›rl›kl› ve tüm canl› organizmalarda mevcut olan maddelerdir. PA

lerin ve bunlar›n biyosentetik enzimlerinin bitkileri strese karfl› korumaya yönelik olarak hücre bölünmesinden

organogeneze kadar de¤iflen genifl bir metabolik olaylar zincirinde yer ald›¤› ortaya konmufltur. Günümüzde PA yolu

moleküler ve biyokimyasal yönden aç›kl›¤a kavufltu¤u için genetik yaklafl›mlarla düzenlenmeye uygundur. Çeflitli

anahtar biyosentez enzimleri, arginin dekarboksilaz, ornitin dekarboksilaz ve S-adenozil metiyonin dekarboksilaz›n

genleri farkl› bitki türlerinde klonlanm›flt›r ve günümüzde baz› genlerin antikorlar›n› elde etmek mümkündür. PA

biyosentezi genlerine hem over-ekspres ve hem de antisens transgenik yaklafl›mlar PA lerin bitki büyüme geliflmesi

için gereklili¤ini daha da ortaya koymufltur. Bununla birlikte bu olaylardaki PA etkilerinin moleküler mekanizmas›

hala aç›kl›¤a kavuflmam›flt›r. DNA mikroarray genom teknikleri kullan›larak yap›lan gen ekspresyon analizleri bu

bilefliklerin rollerini kesin olarak belirlemeye yard›mc› olacakt›r. PA lerin özellikle hücresel olaylardaki rolünü ortaya

koymaya yönelik olarak proteomi¤in potansiyeli de araflt›r›lm›flt›r. ‹ki-hibrit sistemi ve di¤er proteomik yaklafl›mlar›n

yo¤un kullan›m›, PA lerin sinyal iletimindeki rolüne yeni bir bak›fl aç›s› getirecektir. Bundan baflka proteomi¤in, PA

metabolik yolunun homeostatik kontrolünü anlamaya yard›mc› olabilecek, PA metabolizma enzimlerinin

supramoleküler organizasyonunun belirlenmesinde çok önemli bir araç oldu¤u konusunda veriler mevcuttur.

AAnnaahhttaarr ssöözzccüükklleerr:: Poliaminler, mutantlar, transgenik bitkiler, genomik, proteomik

1


2 Ravindar Kaur-Sawhney et al.

1. Introduction

Polyamines (PAs) are low molecular weight

polycations found in all living organisms (Cohen,

1998). They are known to be essential for growth and

development in prokaryotes and eukaryotes (Tabor and

Tabor, 1984; Tiburcio et al., 1990). In plant cells, the

diamine putrescine (Put), triamine spermidine (Spd)

and tetramine spermine (Spm) constitute the major

PAs. They occur in the free form or as conjugates

bound to phenolic acids and other low molecular

weight compounds or to macromolecules such as

proteins and nucleic acids. As such, they stimulate

DNA replication, transcription and translation. They

have been implicated in a wide range of biological

processes in plant growth and development, including

senescence, environmental stress and infection by

fungi and viruses. Their biological activity is attributed

to their cationic nature. These findings have been

discussed in several recent review articles (Tiburcio et

al., 1993; Galston et al.,1997; Bais and Ravishankar,

2002).

The use of PA biosynthesis inhibitors has shown a

causal relationship between changes in endogenous PA

levels and growth responses in plants. These

observations led to further studies into undestanding

the mode of PA action. Some of the important

observations suggest that PAs can act by stabilizing

membranes, scavenging free radicals, affecting nucleic

acids and protein synthesis, RNAse, protease and other

enzyme activities, and interacting with hormones,

phytochrome, and ethylene biosynthesis (reviewed in

Slocum et al., 1984; Galston and Tiburcio, 1991).

Because of these numerous biological interactions of

PAs in plant systems, it has been difficult to determine

their precise role in plant growth and development.

In recent years, however, investigations into

molecular genetics of plant PAs have led to isolation of

a number of genes encoding PA biosynthetic enzymes

and development of antibodies to some of the genes.

Furthermore mutants and transgenic plants with altered

PA metabolism have also been developed. Genomic

and proteomic approaches are being used to further

gain an understanding into the role of PAs in plant

developmental processes. These findings will hopefully

lead to a better understanding of their specific functions

in plants. Several useful reviews on these aspects have

been published (Galston et al., 1997; Walden et al.,

1997; Malmberg et al., 1998; Martin-Tanguy, 2001;

Bais and Ravishankar, 2002).

This article presents an overview of the role of PAs

in plants with particular emphasis on recent

investigations using molecular and genetic

approaches.

2. Polyamine biosynthesis

The PA biosynthetic pathway in plants has been

thoroughly investigated and reviewed in detail (Evans

and Malmberg, 1989; Tiburcio et al., 1990; Slocum,

1991a; Martin-Tanguy, 2001). Briefly, PAs are

synthesized from arginine and ornithine by arginine

decarboxylase (ADC) and ornithine decarboxylase

(ODC) as illustrated in Figure 1. The intermediate

agmatine, synthesized from arginine, is converted to

Put, which is further transformed to Spd and Spm by

successive transfers of aminopropyl groups from

decarboxylated S-adenosylmethionine (dSAM)

catalysed by specific Spd and Spm synthases. The

aminopropyl groups are derived from methionine,

which is first converted to S-adenosylmethionine

(SAM), and then decarboxylated in a reaction

catalyzed by SAM decarboxylase (SAMDC). The

resulting decarboxylated SAM is utilized as an

aminopropyl donor. SAM is a common precursor for

both PAs and ethylene, and SAMDC regulates both

biosynthetic pathways as illustrated in Figure 1.

A number of investigators have used PA inhibitors

to modulate the cellular PA titer in order to determine

their role in various plant processes. Four commonly

used inhibitors of PA synthesis are: 1.

Difluoromethylornithine (DFMO), an irreversible

inhibitor of ODC (reviewed in Bey et al., 1987); 2.

Difluoromethylarginine (DFMA), an irreversible

inhibitor of ADC (Bitonti et al., 1987); 3. Methylglyoxyl-bis

guanylhydrazone (MGBG), a competitive

inhibitor of S-adenosyl-methionine decarboxylase

(SAMDC) (Williams-Ashman and Schenone,1972);

and 4. Cyclohexylamine (CHA), a competitive

inhibitor of spermidine synthase (Hibasami et al.,

1980). Common oxidases are diamine oxidase and

polyamine oxidase (PAO), as reviewed by Smith and

Marshall (1988). Each PA has been found to be

catabolized by a specific oxidase.

Several investigations have dealt with localization

of PAs and their biosynthetic enzymes in plants

(reviewed by Slocum, 1991b). However, paucity of

information regarding the exact cellular and

subcellular localization of these entities remains one of


Methionine

S - adenosylmethionine

AVG

ACC

Ethylene

SAMDC

ACC synthase

ACC oxidase

MGBG

the obstacles in understanding their biological role.

Recent studies have shown that PAs are present in the

cell wall fractions, vacuole, mitochondria and

chloroplasts (Torrigiani et al., 1986; Slocum, 1991b;

Tiburcio et al., 1997). The biosynthetic enzymes,

ODC, SAMDC, and Spd synthase have been reported

to be localized in the cytoplasm, whereas ADC is

localized in the thylakoid membrane of chloroplast

(Borrell et al., 1996; Tiburcio et al., 1997) and PAO in

the cell wall (Kaur-Sawhney et al., 1981). ODC

activity has also been observed in the nucleus

(Slocum, 1991b). However, these findings have to be

interpreted with caution because various procedural

problems can mask the results. Despite these advances

in understanding the metabolic processes involving

PAs and their localization in plant cells, the precise

role of PAs in plant morphogenesis remains elusive.

DFMO

Ornithine Arginine

dSAM

ODC

Polyamines in plants 3

ADC

DFMA

Agmatine

Putrescine

Spdsynthase

Spermidine

Spermine

Spmsynthase

Figure 1: Polyamine biosynthetic pathway and its linkage to ethylene biosynthetis. Biosynthetic enzymes are ADC, ODC and

SAMDC and the inhibitor DFMA, DFMO and MGBG.

3. Polyamines in plant growth and development

The availability of specific inhibitors of PA

biosynthesis has helped in investigating the

mechanisms involved in PA interactions to some extent,

providing a partial understanding of their physiological

role in plant growth and development. Clearly, PAs are

involved in many plant developmental processes,

including cell division, embryogenesis, reproductive

organ development, root growth, tuberization, floral

initiation and development, fruit development and

ripening as well as leaf senescence and abiotic stresses

(reviewed by Evans and Malmberg, 1989; Galston et

al., 1997; Bais and Ravishankar, 2002; Tiburcio et al.,

2002). Changes in free and conjugated PAs and their

biosynthetic enzymes, namely ADC, ODC, and

SAMDC have been found to occur during these

developmental processes. Earlier experiments had

shown that increases in PAs and their biosynthetic

enzymes are associated with rapid cell division in many

plant systems e.g., carrot embryogenesis (Montague


4 Ravindar Kaur-Sawhney et al.

and Koppenbrink, 1978; Feirer et al., 1984), tomato

ovaries (Heimer and Mizrahi, 1982), tobacco ovaries

(Slocum and Galston, 1985), and fruit development

(reviewed in Kakkar and Rai, 1993). Similar results

have been reported for many other plant species

(reviewed in Bais and Ravishankar, 2002). In contrast,

several other studies have suggested that correlations

between PAs and their biosynthetic enzymes and plant

growth processes, especially somatic embryogenesis,

are not universal and may be species specific (reviewed

in Evans and Malmberg, 1989; Galston et al., 1997;

Bais and Ravishankar, 2002).

In general, cells undergoing division contain high

levels of free PAs synthesized via ODC, and cells

undergoing expansion and elongation contain low

levels of free PAs synthesized via ADC (see review by

Galston and Kaur-Sawhney, 1995). High levels of

endogenous PAs and their conjugates have also been

found in apical shoots and meristems prior to

flowering (Cabbane et al., 1981) and flower parts of

many plants (Martin-Tanguy, 1985). Our experiments

using callus cultures derived from thin layer explants

of pedicels from tobacco inflorescence show that

endogenous Spd increased more rapidly than other

PAs in floral buds than in vegetative buds. Addition of

CHA, an inhibitor of Spd synthesis, to the culture

medium reduced flower formation in a dose dependent

manner and such inhibition was correlated with a

switch to initiation of vegetative instead of flower

buds. This inhibition was reversed by the addition of

exogenous Spd (Kaur-Sawhney et al., 1988). More

recently, we have found that higher levels of

endogenous PAs occur in flowers and siliques when

compared with their levels in leaves and bolts of

certain strains of Arabidopsis. Addition of the PA

biosynthetic inhibitors, DFMA and CHA to the culture

medium, at time of seed germination, inhibited bolting

and flower formation and this was partially reversed

by addition of exogenous Spd (Applewhite et al.,

2000). These results clearly show that Spd is involved

in flower initiation and development. Similar results

have been reported in other plants also (reviewed by

Galston et al.,1997; Bais and Ravishankar, 2002).

Many plant growth and development processes

known to be regulated by plant hormones, such as

auxins, 2,4-D, GA and ethylene, have also been

correlated with changes in PA metabolism. These

changes occur in both endogenous levels of PAs and

their biosynthetic enzymes and appear to be tissue

specific (reviewed by Galston and Kaur-

Sawhney,1995). Thus, PAs which may or may not be

mobile in plants (Young and Galston, 1983; Bagni and

Pistocchi, 1991) can serve as intracellular mediators of

hormone actions (Galston and Kaur-Sawhney, 1995).

Supporting evidence for this hypothesis has been

obtained in experiments using specific inhibitors of PA

biosynthesis (Bagni et al., 1981; Egea-Cortines and

Mizrahi, 1991; reviewed in Galston et al., 1997; Bais

and Ravishankar, 2002).

Of the major plant hormones, ethylene has been

most intensively investigated with respect to PA

metabolism. The two metabolites, PAs and ethylene,

play antagonistic roles in plant processes. While PAs

inhibit senescence of leaves (Kaur-Sawhney et al.,

1982), cell cultures of many monocot and dicot species

(Muhitch et al., 1983) and fruit ripening (Kakkar and

Rai, 1993), ethylene promotes these processes. The

most commonly held view is that PAs and ethylene

regulate each other’s synthesis, either directly or

through metabolic competition for SAM, a common

precursor for their biosynthesis (Figure 1). PAs inhibit

ethylene biosynthesis, perhaps by blocking the

conversion of SAM to ACC and of ACC to ethylene

(Apelbaum et al., 1981; Suttle, 1981; Even-Chen et al.,

1982; Furer et al., 1982). Ethylene, on the other hand,

is an effective inhibitor of ADC and SAMDC, key

enzymes in PA biosynthetic pathway (Apelbaum et al.,

1985; Icekson et al., 1985). Thus, PAs may affect

senescence and fruit ripening by modulating PA and

ethylene biosynthesis.

Apparently, PAs are essential members of an array

of internal metabolites required in many plant

developmental processes, but their precise role in these

processes has yet to be established. Whereas, specific

PAs at specific concentrations may be required at

critical stages of growth and morphogenetic events, no

definitive data are available to establish their role as

plant hormones.

4. Manipulation of the polyamine pathway

The PA pathway is ubiquitous in living organisms and

is relatively short (see Section 2) in terms of the

number of enzymes involved. Most of the genes

coding for enzymes involved in the pathway have been

cloned from different sources (Kumar et al., 1997;

Walden et al., 1997; Galston et al., 1997; Tiburcio et

al., 1997; Malmberg et al., 1998; Kumar and Minocha,

1998; Panicot et al., 2002b). Thus, the PA pathway


epresents an excellent model to test various

hypotheses and to answer fundamental biological

questions derived from pathway manipulation (Thu-

Hang et al., 2002; Bhatnagar et al., 2002).

Initially, approaches to manipulate the PA pathway

made use of suicide inhibitors, but the effects of

DFMO and DFMA on ODC and ADC respectively, are

variable in different plant systems, ranging from

inhibition to stimulation or no effect and depending on

the concentration, plant system tested and the

existence of compensatory mechanisms (Slocum and

Galston, 1987). Therefore, alternative approaches to

manipulate polyamine metabolism have been

developed during the recent years.

4.1. Mutants

Mutants deficient in PA biosynthesis have been

isolated from several biological systems. Hafner et al.

(1979) isolated PA mutants in Escherichia coli

showing decreased growth and increased sensitivity to

paraquat (Milton et al., 1990). Yeast mutants

presenting ODC as the sole pathway, show reduced

growth and altered sporulation on PA deficient

medium (Cohn et al., 1980; Whitney and Morris,

1978). Chinese hamster ovary cells lacking ODC

activity do not grow in medium lacking PA (Steglich

and Scheffler, 1983) and a moderately reduced brood

size was observed in a Caenorhabditis elegans ODC

deletion mutant (Macrae et al., 1995). Mutations in

genes affecting Spd and Spm biosynthesis have also

been isolated in yeast. The spe3 Spd synthase mutation

causes a growth arrest, which can be complemented

with externally added Spd (Hamasaki-Katagiri et al.,

1997), while the yeast spe4 mutant is defective in Spm

biosynthesis (Hamasaki-Katagiri et al., 1998).

Less is known about mutants affecting PA

metabolism in plants. Mutants with high levels of

ADC activity have been identified in petunia because

of their abnormal morphology (Geerats et al., 1988),

but the basis of the mutation is still not known.

Screening for resistance to the SAMDC inhibitor

MGBG (Malmberg and Rose, 1987) or to inhibitory

concentrations of Spm (Mirza et al., 1997), yielded

mutants that showed reduced sensitivity to the

respective agent, but these mutants have not been

further exploited for the analysis of PA function.

Watson et al. (1998) isolated EMS mutants of A.

thaliana that are reduced in ADC activity. The mutants

fall into two complementation groups, spe1 and spe2,

which may correspond to the two gene copies

encoding ADC, ADC1 and ADC2 (Watson et al.,

1998). The mutations have not been mapped and

therefore it cannot be excluded that other functions,

i.e. regulatory elements, are affected (Soyka and

Heyer, 1999). More recently, Hanzawa et al. (2000)

reported that the inactivation of the Arabidopsis

ACAULIS5 (ACL5) gene causes a defect in the

elongation of stem internodes by reducing cell

expansion. It was suggested that ACL5 encodes a Spm

synthase, but the possibility that ACL5 may exhibit

broad amine substrate specificities and be involved in

the synthesis of other polyamines could not be

excluded (Hanzawa et al., 2000).

Thus far the only well characterized plant

polyamine biosynthetic mutant has been generated by

using reverse genetics. The availability of mutant

collections generated either by transposon or T-DNA

tagging now facilitates the identification of knockouts

in any gene of interest using PCR-based mutant

screening techniques (Ferrando et al., 2002). By using

these techniques, Soyka and Heyer (2000) isolated an

Arabidopsis thaliana mutant line carrying an insertion

of the En-1 transposable element at the ADC2 locus

which should be regarded as a complete loss-offunction

or knockout mutation. The ADC2 knockout

mutant shows no obvious phenotype change under

normal growth conditions, but is completely devoid of

ADC induction by osmotic stress. As ADC1 gene

expression was not affected in the mutant, it was

concluded that ADC2 is the gene responsible for

induction of ADC and PA biosynthesis under osmotic

stress (Soyka and Heyer, 2000). More recently, Pérez-

Amador et al. (2002) have shown that ADC2 gene

expression is induced in response to mechanical

wounding and methyl jasmonate treatment in

Arabidopsis thaliana. All these observations appear to

indicate that ADC2 is a key gene involved in the PA

response to abiotic stress in Arabidopsis. We envisage

that the extensive use of functional genomics and

reverse genetic studies will facilitate the isolation of

novel knock-out mutants affected in other PA

biosynthetic genes.

4.2. Transgenic plants

Polyamines in plants 5

With the availability of most of the genes involved in

PA metabolism, it has become possible to manipulate

this metabolic pathway using sense and antisense

transgenic approaches. Thus, cellular PA content has


6 Ravindar Kaur-Sawhney et al.

been modulated by overexpression or down regulation

of the key genes ODC, ADC or SAMDC (Kumar et al.,

1997; Walden et al., 1997; Malmberg et al., 1998;

Kumar and Minocha, 1998; Capell et al., 1998; Rajam

et al.,1998; Roy and Wu, 2001; Bhatnagar et al., 2002).

Most of the studies have used the constitutive 35S

promoter, but only few of them were successful in

using either inducible (Masgrau et al., 1997; Panicot et

al., 2002a; Mehta et al., 2002) or tissue-specific

promoters (Rafart-Pedros et al., 1999). Overexpression

of heterologous ODC or ADC cDNAs generally causes

the production of high levels of Put (DeScenzo and

Minocha, 1993; Bastola and Minocha, 1995; Masgrau

et al., 1997; Capell et al., 1998; Bhatnagar et al., 2002;

Panicot et al., 2002a), but in most cases only a small

increase or even no change in Spd and Spm has been

observed. This indicates that elevated levels of Put

resulting from genetic manipulation of a single step

located upstream of the PA biosynthetic pathway (i.e.

ODC or ADC) are not accompanied by an increase in

subsequent biosynthetic reactions (i.e. Spd and Spm

biosynthesis) (Bhatnagar et al., 2002). In contrast,

overexpression of genes located downstream of the

pathway (i.e. SAMDC or SPDS) generally lead to

increased levels of Spd or Spm or both (Thu-Hang et

al., 2002; Mehta et al., 2002). Taken together these

results suggest that the levels of Spd and Spm in the

cells are under a tight homeostatic regulation

(Bhatnagar et al., 2002), which possibly could be

related to a supramolecular organization of some of

these enzymes (see Section 5).

Discrepancies observed among different studies

may have several causes. These include: transgene

source, positional effects, recipient plant system, plant

material analyzed and type of promoter used. A

hierarchical accumulation of polyamines in different

transgenic tissues/organs has been observed (Lepri et

al., 2001). In general, less metabolically active tissues

accumulate higher levels of polyamines (Lepri et al.,

2001). These results are in line with experiments in

which metabolites such as vitamin A and

pharmaceutical antibodies accumulate at high levels in

seeds of different species. It is reasonable to assume

that dormant or less metabolically active tissues

provide a conducive environment for the accumulation

of transgenic products (Thu-Hang et al., 2002). In this

regard, it should be stressed that the most remarkable

results have been obtained by controlled expression of

transgenes using inducible or tissue-specific

promoters. For example, tissue-specific expression of

SAMDC gives rise to smaller potato tubers without

affecting tuber yield (Rafart-Pedros et al., 1999). The

distribution of tuber weights is of agronomic

importance, and generally a reduction of tuber-size

variation is economically advantageous, so that more

tubers fall into a given size grade either for seed or

ware (Rafart-Pedros et al., 1999). Similarly, fruitspecific

expression of heterologous SAMDC in tomato

resulted in ripening-specific accumulation of Spd and

Spm which led to an increase in lycopene, prolonged

vine life, and enhanced fruit juice quality (Mehta et al.,

2002). Besides the agronomic interest of this finding,

this latter study constitutes one of the most striking

evidence regarding the in vivo involvement of

polyamines in a particular developmental process, i.e.

fruit ripening (Mehta et al., 2002).

5. Understanding the role of polyamines

Phenotypic analyses of mutants and transgenic plants

with altered PA levels gives further support to the

previous physiological studies (see Section 3) with

regard to the involvement of these compounds in

several plant processes (reviewed by Tiburcio et al.,

2002). These include somatic embryogenesis (Bastola

and Minocha, 1995), stem elongation and flowering

(Gerats et al., 1988; Masgrau et al., 1997; Hanzawa et

al., 2000; Panicot et al., 2002a), root growth (Watson

et al., 1998; Cordeiro et al., unpublished), tuber

development (Kumar et al., 1996; Rafart-Pedrós et al.,

1999), fruit ripening (Mehta et al., 1997; 2002), abiotic

stresses (Minocha and Sun, 1997; Soyka and Heyer,

1999; Roy and Nu, 2001). However, most of these

mutants and transgenic plants have not been further

exploited for the analysis of PA function. Application

of advanced genomic and proteomic approaches will

help to elucidate the role of PA in particular plant

processes.

5.1. Genomic approaches

The availability of complete genome sequences

permits the use of approaches to explore gene

expression variations on a large genome scale. Either

cDNAs or large oligonucleotide collections are

attached on surfaces to create a microarray. The

hybridisation of the microarray with fluorescent

labelled RNA or cDNA yields an overall image of gene

expression or ‘transcriptome’ (Lockhart and Winzeler,


2000). The global examination of gene expression

should reveal the coincidence of spatial and temporal

transcript expression profiles that may reflect a

requirement of co-ordinated gene product expression

in response to different type of signals. The technology

developed for the Arabidopsis genome has been

accelerated in the recent years both by public funding

through the Arabidopsis Functional Genomics

Consortium in the USA and the GARNet in the UK,

and also by private initiatives like Monsanto,

Affymetrix or Synteny/InCyte (Wisman and Ohlrogge,

2000).

Although there are already many examples in the

literature showing the utility of this approach for

unraveling complex plant responses and signal

transduction processes (Schena et al., 1995; Schaffer et

al., 2000), the use of this technology in our field is

unfortunately in its infancy. So far, DNA microarray

analysis has been used to reveal the induction of ADC

genes during drought stress (Ozturk et al., 2002) or in

response to wounding and methyl jasmonate treatment

(Sasaki et al., 2001; Pérez-Amador et al., 2002).

We envisage that global analysis of gene

expression in well characterized mutant and transgenic

plants with altered polyamine metabolism will provide

novel clues in the near future for understanding the

molecular mechanisms underlying polyamine effects

on plant growth and development.

5.2. Proteomic approaches

Proteomics’ uses biochemical approaches aimed at

systematically characterizing the ‘proteome’ or the

‘protein complement of the genome’ (Wasinger et al.,

1995) in a given organism, tissue, cell or subcellular

compartment. The means of proteome characterization

include protein localization, expression and most

importantly protein interaction maps. A plethora of

innovative procedures has been employed in recent

years for the large-scale analysis of protein signalling

pathways, including the yeast two-hybrid system

(Fields and Song, 1989), protein purification methods

linked to detection by mass spectrometry (Neubauer et

al., 1997; Verma et al., 2000); protein localization

(Ferrando et al., 2000; 2001; Farràs et al., 2001), and

protein microarray techniques (Zhu et al., 2001).

The yeast two-hybrid system is a genetic tool to

describe in vivo protein interactions using the yeast

cell as a test tube. Each separated module of the GAL4

transcription factor, either the DNA binding domain

Polyamines in plants 7

(DBD) or the transcriptional activation domain (AD),

is translationally fused to proteins of interest X or Y,

generating respectively the hybrid proteins X-DBD

(bait) and Y-AD (prey). A powerful aspect of the yeast

molecular genetics involves the facility to isolate the

corresponding cDNAs coding for proteins X or Y,

introduced in the form of plasmid DNA. This latter

feature immediately favored the use of this system to

identify interacting partners for a given bait protein X

using cDNA libraries as a prey (reviewed by Walhout

et al., 2000). The number of studies that have used

proteomics in our field is still scanty. Here we will

provide two examples that demonstrate the potential of

these techniques to (i) unravel the role of PA in

transcription; and (ii) to identify PA metabolons (see

below).

Although the potential role of PAs in affecting gene

expression had already been reported, the molecular

mechanisms underlying their effects were unknown

(Wang et al., 2002). The identification of a polyamine

responsive element and corresponding transacting

protein factors that respond to polyamines has opened

up an exciting new area to study the function of these

compounds in transcription (Wang et al., 1999). By

using the two-hybrid system, it was recently found that

the human homologue of the Arabidopsis subunit

COP9 signalosome complex binds to such transacting

protein factors with the potential to directly affect gene

expression (Wang et al., 2002). Remarkably, the COP9

signalosome proteins were first identified in

Arabidopsis and have been demonstrated to form a

regulatory complex involved in light-activated

development and playing a role in intracellular

signalling (Deng et al., 2000). We envisage that similar

type of experiments will be performed in the plant PA

field that hopefully will provide new insights into the

role of PAs in plant signal transduction.

Increasing number of reports document that many

metabolic reactions are catalysed by complexes of

sequentially acting enzymes that show highly ordered

structural organization (reviewed in Srere, 1987). In

such multienzyme complexes the metabolites pass

from one active enzyme site to the next through a

process termed ‘substrate channeling’. The

supramolecular arrangement of enzymes involved in

such metabolic reactions is referred to as ‘metabolon’.

Metabolons are multienzyme complexes in both

prokaryotes and eukaryotes that represent highly

organized assemblies of sequential enzymes in a

metabolic pathway and are thought to provide


8 Ravindar Kaur-Sawhney et al.

increased metabolic efficiency and higher substrate

selectivity. Metabolons may also help to coordinate the

activities of enzymes by sharing intermediates in a

given pathway, as well as to ensure protection of labile

substrates and sequestration of toxic intermediates

(Sugumaran et al., 2000). In addition, the formation of

multienzyme metabolon complexes may enhance

enzyme stability, improve enzymatic performance and

provide a means for adaptation to alterations of input

of metabolic reactions, especially during demanding

physiological conditions (Abadjieva et al., 2001).

The relevant information about intrinsic properties

of ‘metabolon’ formation can be acquired by studies of

protein-protein interactions using modern proteomic

approaches (Ferrando et al., 2002). In this regard, our

laboratory has recently analyzed possible interactions

between the SPDS and SPMS enzymes of polyamine

biosynthetic pathway in the yeast two-hybrid system

(Panicot et al., 2002b). Using the Arabidopsis

spermidine synthase as bait, two similar proteins were

identified to interact with SPDS2 that were named

SPDS1 and SPMS. Yeast and bacterial mutant

complementation tests revealed that SPDS1 encodes a

novel spermidine synthase, whereas SPMS displays

spermine synthase activity. The heterodimerization

capabilities of enzymes catalyzing the two last steps of

polyamine biosynthesis were also demonstrated in vivo

by co-immunoprecipitation using epitope tagged

SPDS1, SPDS2 and SPMS proteins (Ferrando et al.,

2000; Ferrando et al., 2001). Immunoaffinity

purification and size fractionation of SPDS and SPMS

enzymes labeled with different HA and c-Myc

epitopes revealed that the SPDS and SPMS proteins

co-purify with large multiprotein complexes of 650 to

750 kDa. Further analysis of subunits of isolated

SPDS-SPMS metabolon(s) by mass spectrometry is

expected to yield important information about yet

unknown regulatory subunits of SPDS-SPMS

metabolon in the PA biosynthesis pathway. The

available data support the conclusion that Spd

synthesized by SPDS is effectively channeled to

SPMS to control the formation of the end-product Spm

thereby regulating the synthesis of high molecular

weight polyamines (Panicot et al., 2002b).

6. Conclusions

Considerable evidence indicates that polyamines are

involved in a wide array of plant processes, including

DNA replication, transcription of genes, cell division,

organ development, fruit development and ripening,

leaf senescence and abiotic stresses. Despite ample

evidence of their involvement in these processes, their

precise role in these specific processes remains to be

established. Recent developments of PA-deficient

mutants and transgenic plants as well as of

molecular genetic investigations should further our

understanding of their role in plant growth and

development.

The polyamine pathway is now amenable to

modulation by genetic approaches because it has been

elucidated molecularly and biochemically in plants.

Reverse genetics has identified an Arabidopsis

knockout mutation of ADC2 gene which reveals

inducibility by osmotic stress. Extensive use of

functional genomics and reverse genetics studies will

facilitate the isolation of novel knockout mutants

affected in other polyamine metabolic genes. Sense

and antisense transgenic approaches have revealed the

feasibility of modulating cellular PA contents.

Generally, genetic manipulation of single steps located

upstream of the PA pathway (i.e. ODC or ADC) lead to

elevated levels of Put, but no changes occur in the

higher PAs, Spd and Spm. By contrast, overexpression

of genes located downstream of the pathway (i.e.

SAMDC or Spd synthase) generally leads to increased

levels of Spd and Spm, indicating that the levels of Spd

and Spm are under a tight homeostic cellular control.

Phenotypic analyses of mutants and transgenic plants

affected in polyamine metabolism further support

previous physiological evidence, but the molecular

mechanisms underlying PA effects on plant growth and

development remain to be elucidated. Global analysis

of gene expression by using the available DNA

microarray genomic techniques will help to understand

the role of these compounds. The potential of

proteomics to unravel the role of polyamines in

particular cellular processes is also examined. We

envisage that the extensive use of the two-hybrid

system and other proteomic approaches will provide

new insights into the role of PAs on plant signal

transduction. Furthermore, we provide evidence that

proteomics is an excellent tool to unravel

supramolecular organizations of PA metabolic

enzymes which may help to understand homeostatic

control of this metabolic pathway.


Acknowledgements

AFT acknowledges the grants from Ministerio de

Ciencia y Tecnología BIO-99-453 and BIO-2002-

04459-C02-02.

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Journal of Cell and Molecular Biology 2: 13-18, 2003.

Haliç University, Printed in Turkey.

Phenolic cycle in plants and environment

Valentine I. Kefeli 1 , Maria V. Kalevitch 2 * and Bruno Borsari 3

1 Slippery Rock Watershed Coalition, 3016 Unionville Rd., Cranberry Twp., PA 16066, USA; 2 Robert

Morris University, 881 Narrows Run Rd., Moon Township PA 15108, USA; 3 Slippery Rock University,

101 Eisenberg Bldg., Slippery Rock PA 16057, USA (* author for correspondence)

Received 30 October 2002; Accepted 15 November 2002

Abstract

Phenolic substances are synthesized in plants and in the soil. They exist in the form of polymers and monomers. The

latter group of phenolics is assembled within the chloroplasts of plant cells, whereas soil phenolics are associated

with the process of humus formation on the alumino-silicate matrix of the soil micelle. As plants grow, phenolics

accumulate in cell vacuoles, or polymerize into lignin, which strengthens the secondary cell walls. In addition to this,

phenolics possess also some physiological functions as they regulate cell elongation. When they are excreted from

plant root systems they exert inhibitory growth function within adjacent rhizospheres. This work presents the latest

experimental evidence of phenolic synthesis and transformation in the environment, while providing an

understanding of their effect in plant-soil relations.

KKeeyy wwoorrddss:: Allelopathy, chloroplasts, humus, phenolics, soil micelle

Bitkilerde fenolik döngü ve çevre

Özet

Fenolik maddeler bitkilerde ve toprakta sentezlenir. Bunlar polimerler ve monomerler fleklinde bulunurlar.

Fenoliklerin monomer grubu bitki hücresinin kloroplastlar›nda biraraya gelirken, toprak fenolikleri toprak

misellerinin alumino-silikat matriksi üzerinde humus oluflum olay› ile uyumluluk gösterir. Bitki büyürken hücre

vakuollerinde fenolikler birikir veya sekonder hücre çeperlerine sa¤laml›k kazand›ran ligninlere polimerize olurlar.

Bunlara ilave olarak fenolikler hücre uzamas›n› düzenleyerek baz› fizyolojik ifllevlere de sahiptirler. Bitki kök

sistemlerinden sal›nd›klar› zaman hemen yak›n›ndaki rizosferlerde büyümeyi inhibe edici etki meydana getirirler. Bu

çal›flma fenolik sentezlerinin en son deneysel verilerini ve çevredeki dönüflümlerini sunarken, bitki-toprak

iliflkilerindeki etkilerini anlamam›za yard›m etmektedir.

AAnnaahhttaarr ssöözzccüükklleerr:: Allelopati, kloroplastlar, humus, fenolikler, toprak miseli

Introduction

Phenolics are very stable products in plant organisms.

Generally, they are characterized by a benzene ring

and one hydroxyl group (-OH). They can be converted

into lignin which is the main phenolic polymer in

plants. Microorganisms break down these molecules

and their fragments contribute to the mineralization of

soil nitrogen and humus formation. Thus, humus

participates actively in fulfilling plants nutritional

needs and growth. Light enhances the biosynthesis of

phenolic substances in plant chloroplasts and these

constitute in addition to soil micelles (humus) a second

formation site for this diverse group of organic

13


14 Valentine I. Kefeli et al.

molecules. It should be mentioned however, that

phenolics tend to accumulate in plant vacuoles in

relatively high amounts, or they deposit in the

secondary cell wall as lignin.

Chloroplasts as centers of phenolics biosynthesis

Experiments with chloroplasts of willow (Salix spp.)

leaves showed that the synthesis of phenol-carboxylic

acids and flavonoids is strongly stimulated by light

exposure. Metabolic inhibitors that depress

photosynthetic activity (simazine, diurone,

chloramphenicol), affect negatively the biosynthesis of

flavonoids. Leaves chloroplasts have the capability to

localize phenol compounds, some of which are

specific to these organelles only. The chloroplasts of

spring willow leaves contain more phenols than the

chloroplasts of the same leaves in the autumn. Light is

a mandatory condition to initiate phenolics synthesis

and this is indicated also by the lack of such molecules

in the protoplastids of etiolated willow shoots (Kefeli

and Kalevitch, 2002). Light appears also to induce

flavonols synthesis in the chloroplasts and cytoplasm.

Chalcone and phenolcarbonic acid present in etiolated

willow shoots can be considered metabolic precursors

of light-synthesized flavonols. In certain cell

compartments (vacuoles and cell wall) phenols are

contained in significant amounts (Lewis and

Yamamoto, 1990). However, it is not clear yet how

phenols are translocated within plant cells and how

they affect the function of cell organelles such as

ribosomes and mitochondria. Phenolic substances that

inhibit plant growth (hydroxy derivatives of cinnamic

acid, coumarin and naringenin) are synthesized

similarly to other phenolics. The synthesis of growth

inhibitor derivatives of hydroxycinnamic acids follows

the pathway: shikimic acid-chorismic acid-prephenic

acid-cinnamic acid and p-coumaric acid. A theory of

metabolism bifurcation among phenolic substances,

some of which can inhibit growth and synthesis of

indolic compounds has been proposed. According to

this new approach, indolil-3-acetic acid (IAA)

becomes the main natural auxin (Kefeli, 1978; Kefeli

and Dashek, 1994; Kefeli and Kalevitch, 2002).

Therefore, indole auxins (IAA, indoleacetonitrile) as

well as phenolic inhibitors (p-coumaric acid,

coumarin, naringenin and others) are derived from the

common precursors, shikimic and chorismic acids

(Figure 1).

COOH

CHO

I

C— O

HCOH

I

CH2 Phosphoenolpyruvic

acid

HCOH

CH2O Dehydroquinic acid

5-Dehydroshikimic acid Erythrose-4-phosphate

2-keto-

3-desoxy-

COOH

7-phospho-

D-Araboheptonic acid

OH OH

Shikimic acid

OH

HOOC CH2-CO-COOH Chorismic acid

COOH

OH

Prephenic acid

CH 2-CH-COOH

Phenylalanine

CH-CH-COOH

OH

p-Coumaric acid

Anthranilic acid

Muzafarov and collaborators (1992) investigated

the functions of some phenolics in chloroplasts. They

assumed that the essence of the relationship between

photosynthesis and phenolics biosynthesis is that

phenolics exert a direct and an indirect effect on the

process of solar accumulation itself. From our point of

view, flavonoids as polyfunctional compounds in

green plastids fulfill three major functions as:

• substrates (use polyphenols and their catabolic

products for other kinds of biosynthesis);

• energy sources (electron and proton transport, ion

exchange and membrane potential, radicals

formation);

• regulators (involvement in enzyme reactions as

inhibitors or activators).

During photosynthesis under light, flavonoids

change the rate of electron transport and

photophosphorylation, bringing about the change of

ATP/NADPH ratio. In the reactions of carbon

metabolism they can shift the dynamic equilibrium of

pentosephosphate reduction cycle to enhance the

synthesis of certain metabolites both due to the change

in energy substrate intake and to the interaction with

enzymes of the cycle. Additionally, flavonoids

N

NH 2

CHOH-CHOH-CH 2O

Indolylglycerophosphate

N

I

H

CH 2-CO-COOH

Indolyl-acetic acid

IAA

Figure 1: Phenol-propanoids in metabolic bifurcation.


exercise a feedback control over their own

biosynthesis, although this phenomenon is not clearly

understood. This questionable situation remains as the

biosynthesis of the entire flavonoid structure within

plastids has not been explained, nor the complete

enzymatic package of their biosynthesis has been

discovered yet. Lack of direct eveidence of flavonoids

transport within the cell and through the whole plant

constitues another challenge to a more accurate

description of their functions. Noneless, a variety of

phenolic compounds, present simultaneously within

cells appear to be capable of influencing the rate and

direction of plants metabolic activities. Thus, any

change in the flavonoid structure, or qualitative

composition of the phenol complex result in a change

of the mechanism of its effect upon the processes of

cell energy exchange.

Chalcone and phenolcarbonic acids present in

etiolated willow shoots can be viewed as the potential

precursors of light synthesized flavonoids. However,

the use of paper chromatography to investigate

isosalidpurposide transformation products did not

reveal the presence of any flavonols sensitive to

conventional reagents. Therefore, the transformation

of chalcone (isosalpurposide) in lightless vitro appears

to terminate at a second stage. The synthesis of

eriodyctiol and luteolin that occurred in willow leaves

evidently took place in vivo and under light exposure.

It should be pointed out however, that phloridzin and

isosalipurposide were decomposed from aglycone and

that phloridzin and phloretin produced yellow stains

on the chromatogram as well as flavonoids. It is known

that flavonoid glycosides are revealed as dark spots on

chromatograms exposed to UV light. Therefore, our

yellow stains were classified as flavanones, since they

did not react with AlCl3, nor Na2CO3 like flavonols,

that also form yellow spots. At the same time, similar

to chalcones and aurones, these floridzin

transformation products are yellow colored and they

turn into orange-pink when exposed to Na2CO3 or

NH4OH. Relatively easy transformations of

isosalipurposide and phloridzin into compounds of

other classes (flavanones, chalcones, or aurones)

evidenced the role of these products in the general

metabolism of flavonoids (Figure 2).

OH

Phenolic cycle 15

HO


5´ 1´




C

O

O

CH = CH

O6H10O5 1 4 ´OH

6 5

Isosalipurposide

1

2´, 4´, 6´, 4-tetroxychalcone-

-2´-glucoside of chalconarin-

HO

genin (chalcone)

O

H

2´ 3´

8 1 C

7

2 1´ 4´ OH

6´ 5´

6

3

5 4 CH2 C

O

O

C6H10O3 Salipurposide

naringenin-5-glucoside

2

(Flavanone-glycoside)

HO 7

8

O H

1 C 2

2´ 3´

1´ 4´ OH

6

5

OH

3

4 CH2 C

O

6´ 5´

3

Naringenin

(Flavanone)

Eriodictyol

(Flavanone)

4

Luteolin

(Flavone)

Phenolic substances secreted by roots and leached

from leaves

Plants contain and secrete a diverse group of growth

inhibiting substances that may affect other plants

development, if grown in their vicinity (allelopathy).

Leaf exudates of willow species such as Salix rubra or

Salix viminalis, contain phenolic inhibitors like

naringenin derivative isosalipurposide. Other species

instead like apple trees (Malus spp.) contain

phloridzin, which is a strong respiratory inhibitor.

Roots and leaves of the wild plant Nanaphyton native

to semi-desert regions of Mongolia contain also strong

phenolic inhibitors. Seed as well may secrete

allelochemicals. Tobacco seed (Nicotiana tabacum)

for example suppress germination of its own seed

when leachates come in contact with the seeds (Kefeli

and Kalevitch, 2002). Although the inhibition of

germination was observed at various levels of

intensity, this phenomenon demonstrates the

selectivity of these natural excreta, similar to the effect

of synthetic herbicides. Therefore, increasing evidence

indicates that phenolics and alkaloids play the role of

selective agents. Secondary compounds can be

modified in transgenic plants and genetic mutants.

2 3

Figure 2: Flavonoid biosynthesis.


16 Valentine I. Kefeli et al.

Hence, molecular genetics becomes a tool, which may

help to regulate the level of secondary metabolites in

plants. Therefore, there is a need continue the search

for botanical herbicides as a rise of ecological

concerns has clearly identified the environmental

impact of herbicides of synthesis.

Root exudates affect the germination of seeds of

different crops: monocots and dicots (Table 1 and

Table 2). However, it must be pointed out that only

some phenolics were studied in the exudates of willow

roots (1) which have no analogues in the roots (2) and

leaves found among the common allelopathogens.

Although some of these substances could be retained

by willow roots, others where excreted into an external

medium. Chromatography of these water exudates and

a subsequent investigation of their chromatograms

with UV-B light showed that most of these substances

are polyphenols such as coumarin, or phenolic acids.

The phenolic substances retained by cells had different

chemical properties than those located in the root

exudates. Thus, the data confirm the hypothesis that

Table 1: Effect of root exudation on germination of crop seeds (Non-concentrated exudates).

excreted substances had an allelopathic nature and

were involved in developing ecological relationships

with adjacent plants of different species.

During the composting process water extracts

contain many inhibiting substances that might form

toxic exudates (Kefeli et al., 2001). Paper

chromatography reveals the presence of phenolic acids

and coumarins in water extracts. The highest

concentrations of these inhibitors was measured in

abscised leaves of red maple (Acer rubrum L.). One

gram of dry leaves was mixed in 29 ml of water to

prepare the extracts. The pH of the solution was

between 5.4 and 5.6 and the extracts were incubated

for a week at room temperature while the pH raised to

7.2. Further observations revealed that during

composting the amount of phenolics was drastically

reduced. Seed germination tests were performed with

these water extracts and pure water (control) on lettuce

and wheat seeds. Germination rate and seedling

lengths were measured to demonstrate that phenolics

decreased inhibiting properties after dilution, or after

Variant % to tap water (control)

wheat clover lettuce mustard

Tap water 100 100 100 100

Spider plants (Chlorophytum) exudates 54 93 75 100

Willow (Salix vitaminalis) exudates 58 79 74 138

Stem length (5 tallest plants, mm)

Tap water 29 23 18 25

Spider plants (Chlorophytum) exudates 15 21 14 2

Willow (Salix vitaminalis) exudates 7 18 13.5 3.5

Table 2: Biological activity of willow root exudates after paper chromatography (Biological activity in % to control (water)).

Clover Lettuce

Rf Colour in Germination Stem length Germination Stem length

UV-B light

0 Blue 91 76 90 64

0.14 Blue 94 68 98 58

0.3 Violet 86 80 93 76

0.5 Blue 56 52 71 76

0.67 Yellow 87 68 89 88

0.88 Yellow 52 56 63 64


Secondary

substances

Active secretion

from roots

into the

water and soil

Microorganisms

Composting process

Transformed phenolics and

other secondary substances

Photosynthesis

Plant biomass

in living plants

Biomass of

dead plants

Composting process

(phenolics and N-sources)

To alumo-silicate

matrix

Humus formation

Figure 3: Secondary substances, plant biomass accumulation

and humus formation during allelopathic effects.

contact with fungi. Therefore, the whole process of

allelopathogens formation in the environment could be

tightly connected with the formation of secondary

substances and plant biomass accumulation (Figure 3).

Soil-microbial complex for phenolic decomposition

Phenolic substances are the most resistant metabolites

produced by plants. They undergo further

transformation in the soil, forming humus molecules,

strongly linked to the alumino-silicate matrix. Humus

is more or less a stable fraction of soil organic matter;

it adsorbs mineral elements that serve as important

nutrients for plant growth and development (Kefeli,

2002). The alumino-silicate matrix and humus form

primary soil units. Humus is formed by carbonnitrogen

interaction. Potential sources of carbon

include cellulose and polyphenols from plant leaves,

or transformed lignin polymers.

In order to verify the efficacy of microbial activity

during the humification process, four different soil

horizons in a Grashem soil at the Macoskey Center of

Slippery Rock University of Pennsylvania, USA were

investigated. The presence and number of colonies of

heterotrophic soil microflora were determined in each

Phenolic cycle 17

horizon (TSA (triple-soya-agar, 48 hours, room

temperature). The topsoil (horizon A, 0-28 cm) was

dark gray in color, sandy, high organic matter content

(5.6%), with slightly alkaline pH=7.5. This horizon

was also high in potassium, low in available nitrogen,

and medium in phosphates content, while very high

was the microbial activity. Horizon E (28-52 cm) was

ochric in color, it contained more loam, less organic

matter, lower microbial activity and pH=7.7. Horizon

B (52-62 cm) had no organic matter, microbial activity

was the lowest and pH=7.8. Water permeability was

also measured for each horizon to evaluate penetration

times. The fastest penetration rate was measured in

horizon A (11 minutes), whereas it took 47 minutes for

horizon B and longer (more than 6 hours) below

horizon B. Soil fertility conditions were also assessed

with a wheat/clover germination test. A sand substrate

was used as control, which yielded 30-50%

germination. Horizon A had a germination of 80-82%,

horizon B 40-60%, horizon E/B (with lowest microbial

activity) yielded 30-70% germination rate (Kalevitch

et al., 2002). The results of these experiments appear to

indicate that topsoil (for its highest microbial activity)

Figure 4: Phenolic cycle.


18 Valentine I. Kefeli et al.

is an effective medium usable to facilitate composting

of maple and sumac leaves, contaning nature phenolic

compounds.

Conclusion

Microorganisms have the capability to decompose

phenolic compounds to their monomers, being

deglicosidation of phenolic molecules, followed by

lignin decomposition the biochemical pathways of the

process. Leaves become a primary substrate for soil

microorganisms, while woody materials and sawdust

serve as secondary type of biomass and these

substrates play a major role in humus formation

(Figure 4).

The biosynthesis of phenolic substances within

chloroplasts and its further transformation on the

alumino-silicate matrix of soil micelles led us to

conclude about the existence of phenolics cycle in the

plant-soil system. Although many aspects remain

unknown, the ecological relevance of phenolic

substances in the environment has been amply

demonstrated as this cycle embrace lithosphere,

microsphere and biosphere.

These emerging concepts facilitate the

understanding of complexity within our living systems

and their physical habitat while reinforcing the idea of

interconnectedness among living species and

ecosystems.

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inhibiting compounds from the leaves of the red maple

(Acer rubrum L.) for the germination and growth of

lettuce seeds (Lactuca sative L.) NE-Annual Meet of

ASPP J Plant Phys Abstr. 433.

Kefeli VI and Kalevitch MV. Natural Growth Inhibitors and

Phytohormones in Plant and Environment. Kluwer Acad

Publ. 1-310, 2002. In press.

Muzafarov EN and Zolotareva EV. Uncoupling effect of

hydrocinnamic acid derivatives in pea chloroplasts.

Biochem Physiol Pflanzen. 184: 363-369, 1989.

Yamamoto T, Yokotani-Tomita K, Kosemura S, Yamada K

and Hasegava K. Allelopathic substances exuded from a

serious weed. J Plant Growth Reg. 18: 65-67, 1999.


Journal of Cell and Molecular Biology 2: 19-23, 2003.

Haliç University, Printed in Turkey.

The short-term effects of single toxic dose of citric acid in mice

Tülin Aktaç 1 *, Ayflegül Kabo¤lu 1 , Elvan Bakar 1 and Hamiyet Karakafl 2

1University of Trakya, Faculty of Arts and Sciences, Department of Biology, 22080, Edirne-Turkey;

2University of Trakya, Faculty of Medicine, Department of Biochemistry, 22080, Edirne-Turkey

(* author for correspondence)

Received 12 April 2002; Accepted 03 July 2002

Abstract

The effects of LD25 (480 mg/kg.bw.) dose of citric acid, a food preservative, were investigated on body weight, organ

weights (liver, kidney, spleen), creatin kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT)

and aspartate aminotransferase (AST) enzymes in the blood serum, and the liver tissue of mice after 10 days. Citric

acid (to experimental groups) and physiological saline (to control groups) were given intraperitoneally. The results

of enzyme activities were evaluated using autoanalyzer as IU/L. Even though significant decreases in the body

weights were noted when compared to those of the control group (p0.05, kidney: p>0.05, spleen: p>0.05) and serum enzyme levels

(CK: p>0.05, LDH,: p>0.05, ALT: p>0.05, AST: p>0.05). Microscopical examination of the liver showed

histopathological changes depending on the citric acid. These changes were tissue degeneration, cytoplasmic

vacuolisations, nuclear membrane invaginations, picnotic nucleus and necrosis of the hepatocytes.

KKeeyy wwoorrddss:: Citric acid, food preservative, enzymes, mouse, liver

Farelerde sitrik asidin tek toksik dozunun k›sa süreli etkileri

Özet

Bir besin koruyucu olan sitrik asidin LD25 (480 mg/kg.va.) dozu farelere intraperitoneal yolla uyguland›. 10 gün

sonra hayvanlar›n vücut a¤›rl›klar›, organ a¤›rl›klar› (karaci¤er, böbrek, dalak), kreatin kinaz (CK), laktat

dehidrogenaz (LDH), alanin aminotransferaz (ALT) ve aspartat aminotransferaz (AST) enzimlerinin serum düzeyleri

ile, karaci¤er dokusu üzerinde sitrik asidin etkileri araflt›r›ld›. Otoanalizörde tayin edilen enzim aktiviteleri U/L

olarak de¤erlendirildi. Çal›flmada vücut a¤›rl›klar›nda kontrol grubuna k›yasla anlaml› bir azalma gözlenmesine

ra¤men (p0.05 , böbrek: p>0.05, dalak: p>0.05) ve enzim aktivitelerinde

(CK: p>0.05, LDH: p>0.05, ALT: p>0.05, AST: p>0.05) anlaml› olmayan bir art›fl gözlendi. Karaci¤erin mikroskopik

incelenmesinde doku dejenerasyonu, sitoplazmik vakuolizasyon, nükleer zar çöküntüleri, piknotik nukleuslar ve

hepatositlerde nekroz gibi histopatolojik de¤ifliklikler gözlendi.

AAnnaahhttaarr ssöözzccüükklleerr:: Sitrik asit, besin koruyucu, enzimler, fare, karaci¤er

Introduction

Humans are exposed daily to complex mixtures of

chemical compounds in their food. One of these

substances are antioxidants which are used as food

preservatives. However, peroxides of saturated fats

and their secondary oxidation products, can be toxic

and impair food quality (Würtzen, 1990). Thus despite

19


20 Tülin Aktaç et al.

their economic importance, they can have negative

effects on living organisms. Xenobiotics entering the

organism are held by intestine, kidney and liver cells

for detoxification. These cells contain important

detoxification enzymes. During the detoxification of

xenobiotics, free radicals are produced in

oxidation/reduction reactions, and these radicals can

have destructive effects on tissues.

The toxic effects of many food preservatives on

living organisms have been studied by many

researchers (Makoveç and Sindelar, 1984; Daniel,

1986; Cabel et al., 1988; Kagan et al., 1990; Jung et

al.,1992; Nijhoff and Peters, 1992; Fujitani, 1993;

Weemaes et al., 1997; Mc Farlene et al., 1997; Safer

and Nughamish, 1999; Kabo¤lu and Aktaç, 2002;

Aktaç et al., 2002). Although the citric acid and metal

salts (sodium or potassium citrat) are widely used in

food industry, there is no report on more detailed

effects of citric acid (or its salts) in liver. In addition,

soft drinks, cosmetics and drugs, in which citric acid is

approved for use, are consumed by most of humans

every day.

A way of analysing harmfull effects of foreign

materials entered to organism is to determine the

effects of the chemicals on the enzymes. Enzymes

have a very important role in the metabolical process

since they are biological catalysts. Thus, their abnormal

serum levels indicates various diseases. Among these

enzymes are, creatine kinase (CK), lactate

dehydrogenase (LDH), alanine aminotransferase (ALT)

and aspartate aminotransferase (AST) which are the

most important. Therefore, we studied short term

treatment of citric acid (10 days) in mice. In these

experiments, firstly we tested total body weigths,

organ weights (liver, kidney, spleen), and determined

the serum levels of creatin kinase (CK), lactate

dehydrogenase (LDH), alanin aminotransferase (ALT)

and aspartat aminotransferase (AST), and secondly the

liver tissue was investigated histopathologically.

Material and Methods

Male mice (Balb/C albino) weighing 25-30 g were

used in our experiments. Five mice were used control

group and ten mice were used the citric acid-treated

group. Animals were fed by pellet baits and water.

LD25 dose (480 mg/kg.bw.) of citric acid (Merck; in

physiological saline) were injected intraperitoneally to

experiment group mice, and the same amount of

physiological saline to control group mice. 10 days

after the injection, the mice were killed by cervical

dislocation and then the necessary studies were

commenced. The livers, kidneys and spleens dissected

out, weighed, liver samples were seperated for

microscopical examination. Blood samples were also

taken for enzyme assays. The serum levels of enzymes

were determined using a Merck Mega 600

autoanalyser with the aid of Diasis Kits. Data were

analyzed by M.Whitney U test for multiple

comparisons for the differences between the control

and treated groups. For histological examination, liver

samples were fixed with 10% buffered formalin,

processed and stained hematoxylin-eosin.

Results

The effects of citric acid injection on the body weight

and liver, kidney, spleen weights was shown Table 1

and 2. Although the liver, spleen and kidney weights

were not changed significantly (p>0.05), the body

weights were decreased significantly (p0.05) as shown in Table 2. The results

of the microscopic investigation showed that liver

of mice treated with citric acid has necrotic

changes, compare to the control group (Figure 1-6).

These changes were slightly degeneration of tissue

(Figure 2), cytoplasmic vacuolisation, nuclear

membran invaginations (Figure 3, 4) and picnotic nuclei

(Figure 5). In addition, we observed degeneration of the

blood vessel endothelium (Figure 6).

Discussion

The effects of xenobiotics in living organisms can

investigate in various ways. Among these are, shortterm

toxicity tests which are used very commonly. In

these methods, many parameter are used to test the

effects of xenobiotics. Some of these parameters are

body weight, organ weights, blood profile, and

histopathological examination. In this study, the shortterm

effects of citric acid applied intraperitoneally

were investigated. It was reported that the body weight

decreases in mouse (Würtzen, 1990), and in rats

(Nijhoff and Peters, 1992) by the effects of phenolic

antioxidant butylated hydroxytoluene (BHT) and

butylated hydroxyanisole (BHA) in chronic studies. In


Figure 1: The control group of the liver tissue, bar

representes 20 µm.

Figure 3: Citric acid group. Nuclear invaginations (arrows),

vacuolisation (v), and damaged nucleus (n) in necrotic cells,

bar representes 4 µm.

Figure 5: Citric acid group. Picnotic nuclei (arrows) in

hepatocytes, bar representes 10 µm.

contrast, any significant change was seen in body

weight in F344 rats (0.2, 2.5 and 3.0 % of sodium

benzoate) and in B6C3F1 mice (1.81, 2.09 and 2.4 %

Short-term effects of citric acid 21

Figure 2: Citric acid group. Distortion of general

histological structure of the liver, v: blood vessel, bar

representes 10 µm.

Figure 4: Citric acid group. Invaginations of hypertrophic

cell nucleus (arrow), bar representes 4 µm.

Figure 6: Citric acid group. Degenerated endothelium

(arrows) of blood vessel (v), bar representes 10 µm.

of sodium benzoate) for ten days by Fujitani (1993).

Similarly, Kabo¤lu and Aktaç (2002) were determined

that a significant decrease obtained at 3.0 and 4.0 % of


22 Tülin Aktaç et al.

Table 1: Effect of citric acid on body weight in mice.

sodium benzoate. Also, at the present study we

determined a significant decrease of body weight in

mice by the effect of citric acid (Table 1).

Some autors have shown that food preservatives

had increasing effects to organ weight. The effects of

BHT and BHA on the increasing of the liver and

thyroid weights were demonstrated in mice by

Würtzen (1990). Similarly, the effects of BHT on

increasing of the liver weight in rats was also shown

by Mc Farlene et al. (1997) and Safer and Nughamish

(1999). Fujitani (1993) was also obtained significant

increasing of the liver and kidney by the effects of

sodium benzoate in male rats. In our previous studies,

increasing of the total liver weight were seen oral

treatment of sodium benzoate (Kabo¤lu and Aktaç,

2002) and citric acid (Aktaç et al., 2002) but it was not

significant. Additionally, in the present study, we could

not find any significant change the liver, kidney and

spleen weights by the intraperitoneal injection of citric

acid (Table 2). According to our results, serum CK,

LDH, AST and ALT levels in the treated animals were

Body weight (g)

Before experiment Post experiment

(1. day) (10. days)

Citric acid (LD25 dose) 27.54 ± 0.813 24.76 ± 1.05 *

Control 26.04 ± 1.18 26.58 ± 2.65 **

Values are mean ± SD for ten mice of experiment group and five mice of control group.

(*) significant (p0.05).

Table 2: Effects of citric acid on the organ weights and serum enzyme levels in mice.

Control Citric acid treated

Organ weight

Liver (g) 1.278 ± 0.085 1.257 ± 0.043 *

Kidney (g) 0.2260 ± 0.033 0.1910 ± 0.089 *

Spleen (g) 0.1620 ± 0.036 0.1250 ± 0.014 *

Serum enzyme levels

CK (IU/L) 572 ± 122 1050 ± 255 *

LDH (IU/L) 1296 ± 100 2245 ± 321 *

ALT (IU/L) 695 ± 6.84 101.0 ± 19 *

AST (IU/L) 177.8 ± 3.2 307.2 ± 46.6*

Abbrevations : CK = creatin kinase; LDH = lactate dehydrogenase; ALT = alanine aminotransferase; AST = aspartate aminotransferase.

Values are mean ± SD for ten mice of experiment group and five mice of control group.

(*) not significant (p>0.05).

not significant to compare with the control. These

results were similar with findings obtained in F344 rats

and B6C3F1 mice by Fujitani (1993).

Although the organ weights and serum levels of

enzymes were not changed significantly, the

examination by light microscopy revealed

pathological changes in liver of mice, such as

vacuolisation and glassy cytoplasm in the hepatocyte,

nuclear membrane invaginations, picnotic nuclei.

Similarly, with the effect of sodium benzoate in the

rats and mice, high vacuolisation and glassy

appearance in hepatocyte cytoplasm was explained

(Fujitani, 1993). Again, similar findings were obtained

in the rats with oral treatment of BHT (Mc Farlene et

al., 1997; Safer and Nughamish, 1999), and with

sodium benzoate, benzoic acid and citric acid in mice

(Kabo¤lu and Aktaç, 2002; Aktaç et al., 2002). The

results of present study suggested that citric acid has

hepatotoxic effects and long term exposure may

induce severe damage in liver of mice. However, the

mechanism of damaging effects of citric acid need to


e clarified by more detailed studies.

Finally, we can conclude that consumption of the

foodstuffs containing preservatives is important for the

human health.

References

Aktaç T, Kabo¤lu A, Ertan F, Ekinci F, Hüseyinova G. The

effects of citric acid (antioxidant) and benzoic acid

(antimicrobial agent) on the mouse liver: Biochemical

and histopathological study. Biologia Bratislava. 57(6):

2002. In press.

Cabel MC, Waldroup PW, Shermer WD, Calabotta DF

Effects of ethoxyquin feed preservative and peroxide

level on broiler performance. Poultry Science. 67: 1725-

1730, 1988.

Daniel JW. Metabolic aspects of antioxidants and food

preservatives. Xenobiotica. 16: 10-11, 1986.

Fujitani T. Short-term effect of sodium benzoate in F344 rats

and B6C3F1 mice. Toxicol Lett. 69: 171-179, 1993.

Jung R, Cojocel C, Müller W, Böttger D, Lück E. Evaluation

of the genotoxic potential of sorbic acid and potassium

sorbate. Food Chem Toxicol. 30: 1-7, 1992.

Kabo¤lu A. and Aktaç T.A study of the effects of the sodium

benzoate on the mouse liver. Biologia Bratislava. 57(3):

373-380, 2002.

Kagan VE, Serbinova EA, Packer L. Generation and

recycling of radicals from phenolic antioxidants. Arc

Biochem Biophysiol. 280: 33-39, 1990.

Makoveç P. and Sindelar L. The effect of phenolic

compounds on the activity of respiratory chain enzymes

and on respiration and phosphorylation activities of

potato tuber mitochondria. Biol Plant. 26: 415-422, 1984.

McFarlane M, Price SC, Cottrel S, Grasso P, Bremme JN,

Bomhard ME, Hinton HR. Hepatic and associated

response of rats to pregnancy, lactation and simultaneous

treatment with butylated hydroxytoluene. Food Chem

Toxicol. 35: 753-767, 1997.

Nijhoff WA and Peters WHM. Induction of rat hepatic and

intestinal glutathion S-transferases by butylated

hydroxyanisole. Biochem Pharmacol. 44: 596-600, 1992.

Safer AM and Nughamish AJ. Hepatotoxicity induced by the

antioxidant food additive butylated hydroxytoluene

(BHT) in rats: An electron microscopical study. Histol

Histopathol. 14: 391-406, 1999.

Weemaes CA, De-Cordt SV, Ludikhuyze LR, Van Den

Broeck I, Hendrickx ME, Tobback PP. Influenze of pH,

benzoic acid, EDTA, and glutathione on the pressure

and/or temperature inactivation kinetics of mashroom

polyphenoloxidase. Biotechnol Prog. 13: 25-32, 1997.

Würtzen G. Short comings of current strategy for toxicity

testing of food chemicals: Antioxidants. Food Chem

Toxicol. 28: 743-745, 1990.

Short-term effects of citric acid 23


Journal of Cell and Molecular Biology 2: 25-30, 2003.

Haliç University, Printed in Turkey.

Characterisation of RRPPPP77 mutant lines of the col-5 ecotype of

AArraabbiiddooppssiiss tthhaalliiaannaa

Canan Can 1 , Mehmet Özaslan 1 *, Eric B. Holub 2

1 University of Gaziantep, Faculty of Science & Arts, Department of Biology, 27310 Gaziantep; 2 Plant

Genetics and Biotechnology Department, Horticulture Research International, Wellesbourne, Warwick,

CV35 9EF, England (* author for correspondance)

Received 21 May 2002; Accepted 20 November 2002

Abstract

In this study, phenotypic characterization of RPP7 that confers resistance to Hiks1 isolate of Peronospora parasitica,

deficient mutant lines of Col-5 ecotype of Arabidopsis thaliana was investigated. The Col-5 plants that exposed to

Fast Neutron (FN) were inoculated with 8 different P. Parasitica isolates and symptom development was

investigated. A total of 4 mutant lines were analyzed. It was found that the RPP7 gene present in the Col-5 ecotype

is a unique gene different from the other RPP genes present in Col-5.

KKeeyy wwoorrddss:: Arabidopsis thaliana, Col-5, Hiks-1, Peronospora parasitica

AArraabbiiddooppssiiss tthhaalliiaannaa’n›n Col-5 ekotipinden elde edilen mutant hatlardan RRPPPP77 geninin

karakterizasyonu

Özet

Bu çal›flmada, Arabidopsis thaliana’n›n Col-5 ekotipinde bulunan ve Peronospora parasitica’n›n Hiks-1 izolat›na

karfl› dayan›kl›l›¤› sa¤layan RPP7 geninde mutasyon içeren hatlar›n fenotipik olarak belirlenmesi üzerinde

araflt›rmalar gerçeklefltirilmifltir. Fast Nötron (FN) uygulamalar› ile mutasyon meydana getirilmifl Col-5

tohumlar›ndan geliflen bitkiler 8 farkl› P. parasitica izolat› ile inokule edilerek semptom geliflimleri incelenmifltir.

Toplam olarak 4 mutant hatta gerçeklefltirilen analizlerde, RPP7 geninin Col-5 ekotipinde bulunan ve farkl›

P. parasitica izolatlar›na karfl› dayan›kl›l›¤› sa¤layan genlerden ba¤›ms›z olarak fonksiyon gösteren bir gen oldu¤u

belirlenmifltir.

AAnnaahhttaarr ssöözzccüükklleerr:: Arabidopsis thaliana, Col-5, Hiks-1, Peronospora parasitica

Introduction

Following the isolation of the Pseudomonas syringae

resistance genes (R-gene) from tomato, the research on

isolation and characterization of R-genes against plant

pathogens has been improved (Hammond-Kosack and

Jones, 1997). Recently many R-genes conferring

resistance to fungi, bacteria, nematode and viruses in

rice wheat, tomato, pepper and some other important

crops are isolated and the mechanism of resistance is

determined (Richter and Ronald, 2000). The mutant

lines with lack of R-genes have a potential importance

in this type of work (Mc-Dowel et al., 1998).

Arabidopsis thaliana is a member of cruciferae

family and it is known to have a small genome size of

120 Mb. It is a flowering plant and is a best model for

25


26 Canan Can et al.

the working on genome analyses, growth regulation,

hormons, flowering, disease resistance and

embryogenesis. Arabidopsis and tomato were used to

determine the mechanisms of disease resistance

(Thomas et al., 1997; Botella et al., 1998). It is also the

host of many pests which attacks to crop plants. Many

genes that provides resistance to bacteria and fungi

disease have been isolated and characterized from A.

thaliana (Dangl and Jones, 2001; Feys and Parker,

2000).

Peronospora parasitica is a causal agent of mildew

disease in the genus cabbage, turnip etc. of cruciferae

family. R-genes that determines resistance to P.

parasitica (RPP) were isolated and characterized

(Holub and Beynon, 1997; Parker et al., 1997; Botelli

et al., 1998; McDowell et al., 1998; Bittner-Eddy et al.,

2000). The researches on RPP genes have shown that

these genes are at the specific regions at certain places

of each chromosome called as “Major recognition

complexes-MRC” (Can, 1997; Holub and Beynon,

1997).

RPP7 gene is present in Col-5 ecotype and

recognized by the Hiks-1 isolate of P. parasitica. This

gene was placed onto the first chromosome between

the markers M421 and M213 by using the hybrid lines

of Col-5 and Nd1 ecotypes (Tor et al., 1994; Can et al.,

1995; Can, 1997). The Hiks1 isolate also recognizes

the RPP1 gene which is present in Nd-1 ecotype, and

has an epistatic effect on the RPP7 gene (Tor et al.,

1994).

The mutant lines that lack the R-genes were

studied in detail and has a wide area of interest such as

molecular and classical genetics. However, in order to

study the relationships between A. thaliana and the

RPP genes and to investigate the genome

organizations, some mutant lines were used (Parker et

al., 1996). The mutant lines lacking the RPP genes

were obtained from Ws-O that contain RPP14 gene

exhibiting resistance to No-Co2 isolate by using Ethyl

Methane Sulfate (EMS). The lines were then used to

separate the RPP10 and RPP1 genes, which were

allelic to RPP14 that is on the third chromosome. It

was found that the WsEDS line was susceptible to all

P. parasitica isolates tested and that the WsEDS locus

was necessary for the function of the RPP genes

(Parker et al., 1996; Bittner-Eddy and Beynon, 2001;

Falk et al., 1999). The npr (Non expressor of PR

protein) mutant lines of A. thaliana synthesize the

proteins which are related with pathogenesis. So,

systemic resistance is not seen following inoculation

with many isolates (Century et al., 1995; Aarts et al.,

1998). Similarly, in (Ethylene Intensitive) mutant lines

do have the ethylene synthesis. But it was found that

the P. syringae f.s.p. tomato resistance continued in

this mutant lines. This study showed that ethylene was

not important for A. thaliana and bacteria relationships

(Bent et al., 1994; Dong, 1998). Lsd (Lesions

Simulating Disease resistance response) and acd

(Accelerated Cell Death) mutant lines produce

Hypersensitive Resistance (HR) like symptoms

without a pathogen infection. These symptoms are

formed by the influence of external factors like heat

and light (Lam et al., 1999). So, it is accepted that lsd

and acd loci are negative regulators for HR formation

(Dietrich et al., 1994). In general, the presence of

different resistance mechanisms in A. thaliana which

are directed by RPP genes was found by the

characterization of mutant lines (Glazebrook et al.,

1997; McDowell et al., 2000).

In this study, the mutant lines of the Col-5 ecotype

of A. thaliana were characterized, to understand the

mechanisms of RPP7 gene that confers resistance to

Hiks-1 isolate of P. parasitica.

Material and methods

Plant and fungus material

In this study, Col-5 ecotype of A. thaliana lines having

MRC-B, MRC-C and MRC-H regions (Can, 1997;

Holub and Beynon, 1997) were used as wild type

ecotype. The Fast Neutron (FN) applied mutant lines

were obtained commercially and the selections of

mutant lines were performed by using the Hiks-1

isolate of P. parasitica. The Hiks-1 isolate recognizes

the RPP7 gene which is in the MRC-B region of wild

Col-5 ecotype, and 7 days after inoculation it induces

a resistance which is defined with HR. Four mutant

lines were used in this study denoted as FN3922,

FN3928, FN3929 and FN3930. The HR does not occur

in mutant lines, and the pathogen completes its life

cycle by sexual and asexual sporulation.

Regeneration of Hiks-1 isolate from oospore

population

The Hiks-1 isolate was regenerated by using oospore

population. To do this, the seeds of A. thaliana that

were susceptible to the Hiks-1 isolate were sown into


little plastic pots containing 4:1:1 (torf: perlit: sand) of

mixture for 40-50 seeds each. The pots were irrigated

to wet the seeds and 1-2 x 10 5 oospore/ ml were added

to the pots. The containers were held at 4 °C for 1-2

weeks to break the dormancy. Following this, the

containers were placed into the climated room at 18-20

°C, 10 h light and 14 hour dark period. Within 10-15

days following seed germination, some seedlings

having sporulation was collected and placed into

eppendorf tubes containing 200 µl dH2O. The

eppendorf tubes were shaked gently to allow the

conidia to pass to water. The conidia suspension was

used to inoculate 7 days old seedlings of EBH3529 and

Ksk-1 ecotypes, and the plants were placed into the

climate room. By this way, the regeneration of the

Hiks-1 isolate was done by subculturing 3-4 times. The

conidia were stored at –20 °C and were used when

needed.

The same procedure were applied for, Ahco-1,

Ahco-2, Ahco-7, Wand-1, Cand-5, Hind-2 and Hind-4

isolates using Nd-1 and Col-5 ecotypes (Can, 1997).

Characterization of P. parasitica isolates by using

different A. thaliana ecotypes

Regenerated P. parasitica isolates were inoculated into

Col-5, Ksk-1, Nd-1, Ws-3, Tsu-1, Ler-1, Oy-1 and

Wei-1 ecotypes in order to do phenotypic

characterization. The A. thaliana ecotypes were

obtained from Dr. Eric Holub (HRI- UK)

The conidia suspension was adjusted to 4-5 x 10 4

conidia/ml concentration for plant inoculations. The

cotyledons of 7-8 days of the A. thaliana ecotypes

were inoculated in such a way that it would be one

drop to each cotyledon. The plants were placed into

climated room with 18-20 °C, 10 h light, 14 h darkness

conditions after the inoculation and the plants were

checked at the end of 3. and 7. days. The evaluation

was done regarding the pathogen sporulation and

hypersensitive reaction types (the interaction

phenotypes). Phenotypic reactions were examined

under the fine group as; pitting with no pathogen

sporulation (PN), flecking with no pathogen

sporulation (FN), flecks with delate and moderate

pathogen sporulation, 1-20 sporangiophorus per each

cotyledon (DM), flecking with delate pathogen

sporulation, 5-10 sporangiophorus per each cotyledon

(FDL), early and heavy pathogen sporulation, 20>

sporangiophores per cotyledon (EH), (Holub et al.,

1994).

Microscopic analysis

Fungal development in plant tissue was examined

under light and fluorescence microscope. The infected

leaves were taken and put absolute methanol for 5-6

hours followed by saturated chloral hydrate solution

for 4-5 hours. Then, tissues were placed in 50 %

glycerol solution for microscopic analyses.

DNA analysis

Total plant genomic DNA was isolated with some

modifications by using the methods of Ausubel et al.,

(1994). Five to eight grams of plant material was

grounded in N2 and transferred to the tubes containing

15 ml buffers (100 mM Tris-HCl, 50 mM EDTA, 500

mM NaCl, 10 mM Mercaptoethanol, %25 SDS) with

100 mg/lt proteinase K. The solution was kept at 55 °C

for 1 hour. At the end of this time period, 5 ml of 5 M

potassium acetate was added and held in ice for 20

minutes, and the solution was centrifuged at 17000

rpm for 25 minutes. The supernatant was mixed with

0,6 volume of isopropanol and held at -20 °C for

minutes and the DNA was precipitated. Phenolchloroform

was used to wash the DNA and a second

precipitation was done. The DNA was dissolved in

dH2O and stored at -20 °C. The isolated DNA was

diluted in such to 50-100 ng/µl to use in polimerase

chain reactions (PCR). For PCR reactions, the closest

marker to the RPP7 gene was used (Can, 1997). To do

this, the solution which contains 0.05 mm primer, 2

mm dNTPs, 25 mm MgCl2, 1 x Taq buffer and IU Taq

DNA polymerase was completed to 25 ml volume. The

PCR reactions were performed at 94 °C for 5 min

followed by 94 °C for 1 minute, 56 °C for 1 min, 72 °C

for 13 minute (35 cycles) and 72 °C for 10 min. The

samples were electrophoresed at 80 W for 4 hours.

Results and discussions

Characterisation of RPP7 gene 27

Characterization of P. parasitica isolates

In order to determine the changes at the RPP7 locus in

the mutant lines, Ahco-1, Ahco-2, Ahco-7, Wand-1,

Cand-5, Hind-2 and Hind-4 isolates were used. Ahco-

1, Ahco-2, Ahco-7 recognize MRC-B region which is

located at the first chromosome in the Nd-1 ecotype

(Can, 1997), and these isolates were presumed to

recognize RPP7 allele of Nd-1 ecotype. Wand-1,


28 Canan Can et al.

Table 1: Interaction phenotypes of different P. parasitica isolates on the A. thaliana ecotypes.

Cand-5, Hind-2 and Hind-4 isolates recognize MRC-B

and MRC-C region which present at the second

chromosome in the Col-5 ecotype. The isolates

regenerated from the oospore populations were

inoculated on different A. thaliana ecotypes (Col-5,

Ksk-1, Nd-1, Ws-3, Ler-1, Oy-1 and Wei-1) to

determine if they were original. The results are shown

in Table1.

As it could be seen in Table 1, the isolates

generated from the oospore populations were found to

be as original, and there was no variation (Can, 1997).

Therefore these isolates were used to inoculate the

mutant lines recovered through inoculation with the

Hiks-1 isolate.

Interaction phenotypes on different A. thaliana ecotypes*

P. parasitica isolates Col-5 Ksk-1 Nd-1 Ws-3 Ler-1 Oy-1 Wei-1

Hiks-1 FN EH PN PN FN DM FDL

Ahco-1 DM FN FDL FN FDL FN FDL

Ahco-2 DM FN FDL FN FN FN DM

Ahco-7 DM FN FDL FN FDL FN EH

Wand-1 FN FN EH FN FN FN DM

Cand-5 FN EH EH FN CN FDL DL

Hind-2 FN FN EH PN FN FN EH

Hind-4 FR FN EH PN EH FN EH

*Necrotic pits (PN), necrotic flecks (FN), cavities (CN), flecks with delate and moderate pathogen sporulation, 1-20

sporangiophorus per each cotyledon (DM), flecking with delate pathogen sporulation, 5-10 sporangiophorus per each cotyledon

(FDL), early and heavy pathogen sporulation, 20> sporangiophores per cotyledon (EH).

Phenotypic characterization of mutant lines

P. parasitica isolates were used to inoculate the Col-5

lines. The results were shown in Table 2.

As indicated in Table 1, DM and EH phenotypes

developed, following inoculation of FN3922, FN3928,

FN3929 and FN3930 mutant lines with the Hiks-1

isolate. These results revealed that the RPP7 gene is

not present in the mutant lines. However, Ahco-1,

Ahco-2 and Ahco-7 isolates exhibited the EH

phenotype compared to DM in the wild Col-5 ecotype.

This result showed that absence of the RPP7 gene

increased susceptibility. The important point in here

was that mutation of one R-gene could effect the

resistance in same plant to other isolate. The result

Table 2: Interaction phenotypes exhibited by Col-5 mutant lines following inoculation with different P. parasitica isolates.

The interaction phenotypes on wild type and mutant lines*

P. parasitica isolates Col-5 Nd-1 Ksk-1 FN3922 FN3928 FN3929 FN3930

Hiks-1 FN PN EH EH DM EH EH

Ahco-1 DM FDL FN EH EH EH EH

Ahco-2 DM FDL FN EH EH EH EH

Ahco-7 DM FDL FN EH EH EH EH

Wand-1 FN EH FN EH FN FN FDL

Cand-5 FN EH EH EH FN FN FN

Hind-2 FN EH FN FN FN FN FN

Hind-4 FR EH FN EH FN FN FN

*Necrotic pits (PN), necrotic flecks (FN), flecks with moderate and late pathogen sporulation, 1-20 sporangiophorus per each

cotyledon (DM), flecking with delate pathogen sporulation, 5-10 sporangiophorus per each cotyledon (FDL), early and heavy

pathogen sporulation, 20> sporangiophores per cotyledon (EH).


Figure 1: Microscopic reactions occurred in Col-5 ecotype

following inoculation with the Hiks-1 isolate. (A) Six hours

after inoculation. a. Haustorium formed in the mesofil cells.

b. Cell death and (B) Twelve hours after inoculation.

Hypersensitive reaction and cell death.

750 bp →

500 bp →

Figure 3: Distribution of g2a markers in mutant lines.

M indicates the 1 kb DNA marker.

may also show that when the RPP genes are allelic

they do function in coordination.

Cand-5 isolate recognizes MRC-B region which is

present in Col-5 and determined with FN interaction

phenotype. FN3922 mutant line exhibited the EH

phenotype with Cand-5 and FN with other isolates

tested (Table 2). This result may show that FN3922

may be a Col-5 contamination. On the other word it is

possible that in this line, many RPP genes present in

the MRC-B loci (Can, 1997) could be mutated.

Therefore, FN3922 may not be a specific mutant of the

RPP7 gene. As it is known the Fast Neutron

application results point mutations, so that at MRC-B

region in FN3922, many point mutations or deletions

may have been occurred.

Inoculations of the FN3928, FN3929 and FN3930

mutant lines with Wand-1, Hind-2, Hind-4 resulted FN

phenotype. This results shows that the RPP7 gene has

no correlation with the RPP genes present in seconder

chromosome at the MRC-C region (Table 2).

Microscopic characterization of mutant lines

The fungal development in plant tissue was examined.

Following inoculations at the first, third and seven

days the cotyledons from the mutant lines and the wild

type Col-5 were taken and prepared as described

before. At the first six hour of inoculation of Col-5

with the Hiks-1 isolate cell death was observed. The

susceptible genotypes and the mutant lines allowed the

penetration and mycelial development without any cell

(Figure 1, 2). Fungal development in the mutant lines

supported the macroscopic results (Mc Dowell et al.,

2000).

DNA analyses of mutant lines

Characterisation of RPP7 gene 29

Figure 2: Microscopic reactions occured the FN3929

mutant line following inoculation with the Hiks-1 isolate.

Formation of fungal haustorium in the mesophyl cell (a) and

hif development (b), twelve hours after inoculation.

nga280 and g2a markers present at the lower arm of

the first chromosome of A. thaliana were detected to

give less recombination with the RPP7 loci (Can,

1997). Therefore the mutant lines were subjected to

PCR analyses with these markers. Results are given in

Figure 3.

The distributions of the bands in mutant lines were

similar to those in the wild type Col-5. This result may

suggest that no deletions may occurred in the mutant

lines and that the loss of function could be due to a

point mutation.


30 Canan Can et al.

References

Aarts N, Metz M, Holub E, Staskawicz BJ, Daniels M and

Parker JE. Differential requirements for EDS1 and

NDR1 by disease resistance genes define at least two R

gene-mediated pathways in Arabidopsis. Proc Natl Acad

Sci USA. 95: 10306-10311, 1998.

Ausubel F, Brent R, Kingston RE, Moore DA, Seiddman JG,

Smith JA and Struhl K. Current Protocols in Molecular

Biology. John Wiley and Sons. 1994.

Bent AF, Kunkel BN, Dahlbeck D, Brown KL, Schmidt R,

Giraudat J, Leung J and Staskawicz BJ. RPS2 of

Arabidopsis thaliana, A leucine-rich repeat class of plant

disease resistance genes. Science. 265 (5180): 1856-

1860, 1994.

Bittner-Eddy PD, Crute IR, Holub EB and Beynon JL.

RPP13 is a simple locus in Arabidopsis thaliana for

alleles that specify downy mildew resistance to different

avirulence determinants in Peronospora parasitica. The

Plant Journal. 21(2): 177-188, 2000.

Bittner-Eddy PD and Beynon JL. The Arabidopsis downy

mildew resistance gene, RPP13-Nd, functions

independently of NDR1 and EDS1 and does not require

the accumulation of salicylic acid. Mol Plant-Microbe

Interactions. 14: 416-421, 2001.

Botella MA, Parker JE, Frost LN, Bittner-Eddy PD,

Beynon JL, Daniels MJ, Holub EB and Jones JDG.

Three genes of the Arabidopsis RPP1 complex

resistance locus recognize distinct Peronospora

parasitica avirulence determinants. Plant Cell. 10: 1847-

1860, 1998.

Can C. Bittner-Eddy P, Tör M, Williams K, Gunn N, Bakht S,

Atkinson L, Debener T, Chimot P, Crute I, Beynon J and

Holub EB. Revealing the organization of RPP loci in the

Arabidopsis thaliana genome, Poster abstract in 6 th

International Conference on Arabidopsis Research,

Medison, Winconsin. 7-11 June, 1995.

Can C. Genomic organisation of pathogen recognition genes

in Arabidopsis thaliana to Peronospora parasitica,

Ph. D. Thesis, University of London, Wye Collage, UK.

1997.

Century KS, Holub EB and Staskawicz BJ. NDR1, a locus of

Arabidopsis thaliana that is required for disease

resistance both a bacterial and a fungal pathogen. Proc

Natl Acad Sci USA. 92 (14): 6597-6601, 1995.

Dangl JL and Jones JDG. Plant pathogens and integrated

defense responses to infection. Nature. 411: 826-833,

2001.

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Dangl JL. A novel zinc finger protein is encoded by the

Arabidopsis LSD1 gene and functions as a negative

regulator of plant cell death. Cell. 88: 685-694, 1994.

Dong X. SA, JA, ethylene, and disease resistance in

plants. Curr Opin Plant Biol. 1: 316-323, 1998.

Falk A, Feys BJ, Frost LN, Jones JDG, Daniels MJ and

Parker JE. EDS1, an essential component of R genemediated

disease resistance in Arabidopsis has

homology to eukaryotic lipases. Proc Natl Acad Sci

USA. 96: 3292-3297, 1999.

Feys BJ and Parker JE. Interplay of signaling pathways in

plant disease resistance. Trends Genet. 16: 449-455,

2000.

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characterisation of interactions between isolates of

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Mc-Dowel JM, Dhandaydham M, Long TA, Aarts MGM,

Goff S, Holub EB and Dangl JL. Intragenic

recombination and diversifying selection contribute to

the evolution of downy mildew resistance at the RPP8

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Holub EB. Downy mildew (Peronospora parasitica)

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requirements for NDR1, EDS1, NPR1 and salicylic acid

accumulation, The Plant Journal. 22 (6): 523-529, 2000.

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Daniels MJ. Characterization of eds1, a mutation in

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Plant Cell. 8 (11): 2033-2046, 1996.

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der Biezen EA, Moores T, Dean C, Daniels MJ and

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Mol Plant-Microbe Interac. 7 (2): 214-222, 1994.


Introduction

Journal of Cell and Molecular Biology 2: 31-34, 2003.

Haliç University, Printed in Turkey.

The effect of mmeettaa-topolin on protein profile in radish cotyledons

Serap Ça¤ 1 and Narçin Palavan-Ünsal2 *

1Istanbul University, Department of Biology, Botany Section, Süleymaniye 34460, Istanbul-Turkey;

2Haliç University; Department of Molecular Biology and Genetics, F›nd›kzade 34280, Istanbul-Turkey

(* author for correspondance)

Received 27 September 2002; Accepted 30 November 2002

Abstract

Meta-topolin (mT) has been established as an active aromatic cytokinin recently. The present investigation assessed

the effects of mT on radish cotyledon growth and protein content. 0.05 to 1 mM mT increased the cotyledon growth

about 2 fold in fresh weight basis. mT at 0.1, 0.25 and 0.5 mM concentrations caused an increase in soluble protein

levels compared to the control cotyledons almost in the same ratio by 3 %. Compared to control cotyledons analysis

of the soluble proteins displayed different electrophoretic pattern in mT treated cotyledons.

KKeeyy wwoorrddss:: Cotyledon growth, meta-topolin, protein

MMeettaa-topolinin turp kotiledonlar›nda protein profiline etkisi

Özet

Son y›llarda meta-topolin (mT) aktif aromatik sitokinin olarak saptand›. Bu araflt›rma da mT’in turp kotiledonlar›n›n

büyüme ve protein içeri¤ine etkisi araflt›r›ld›. 0.05-1 mM mT kotiledon büyümesini taze a¤›rl›k baz›nda yaklafl›k 2

kat kadar teflvik etti. 0.05, 0.1 ve 0.25 mM mT çözünür protein düzeylerini kontrole oranla yaklafl›k % 3 oran›nda

artt›rd›. Çözünür proteinlerin analizleri, mT uygulanan kotiledonlarda kontrole oranla farkl› bir elektroforetik dizilim

gösterdi.

AAnnaahhttaarr ssöözzccüükklleerr:: Kotiledon büyümesi, meta-topolin, protein

Cytokinins, N 6 -substituted adenine derivatives are a

class of plant hormones that were first identified as

factors that promoted cell division (Miller et al., 1955;

1956) and have been implicated in many other aspects

of plant growth and development including shoot

initiation and growth, apical dominance, senescence

and photomorphogenetic development (Letham,

1971; Thimann, 1980; Mok and Mok, 1994).

Although the physiological effects of cytokinins have

been well documented, the molecular mechanisms

underlying cytokinin action remain obscure (Mok and

Mok, 1994; Binns, 1994).

Bioassays are used to establish the relative

biological activity of plant hormones compared with

others. The cytokinin bioassays used most frequently

depend on growth of tissues in sterile culture (Letham

1967). Such methods are extremely sensitive but it

needs at least 3 weeks to get final results. Letham

(1971) described a rapid bioassay for cytokinins based

on the ability of these compounds to promote

markedly the expansion of radish cotyledons excised

soon after seed germination.

To date the effects of common cytokinins i.e.

kinetin, benzyladenine (BA) and its riboside have been

31


32 Serap Ça¤ and Narçin Palavan-Ünsal

documented in radish cotyledons. A new active

aromatic cytokinin meta-topolin (mT) have been

determined by Strnad et al. (1997) in poplar. The

sensitivity of the radish cotyledon bioassay to mT has

been established by us before (Palavan-Ünsal et al.,

2002). This study will focus on the effect of mT on

soluble protein contents in radish cotyledons that has

not been studied before.

Material and methods

Plant material and bioassay

Radish (Raphanus sativus L.) seeds were germinated in

darkness for 4 days at 25 °C on moist filter paper in 5

cm petri dishes. Cotyledons were excised excluding

petiole tissues and four cotyledons were placed in each

petri dish after measuring the fresh weight. The

cotyledons were placed with their adaxial sides down

on the paper. They were incubated in a growth chamber

at 25°C ± 2°C and 12 h light-dark photoperiods. Three

ml mT was applied per petri dish at 0.05, 0.1, 0.25, 0.5

and 1.0 mM concentrations. Cotyledon growth was

measured by determining fresh and dry weights 3 days

after the application (Letham, 1971) and the data

presented here representative of 15 experiments.

Measurement of soluble protein content

Soluble protein content was determined as in Bradford

(1976) using bovine serum albumin as standard. Each

experiment was repeated four times and each treatment

included three replicates.

Electrophoresis for proteins

Sodium dodecylsulphate (SDS)-polyacrylamide slab

gel electrophoresis was performed according to

Laemmli (1970). Gel containing 3.0 % (stacking gel)

and 10.0 % (separation gel) acrylamide were prepared

from a stock solution of 30.0 % of acrylamide and 0.8

% N, N’-bis methylene acrylamide. The gels were

polymerized chemically by the addition of ammonium

persulphate. The mixture was completely dissociated

by immersing the samples for 3 min in boiling water.

Electrophoresis was carried out with a current of 150 V

per gel until the bromophenol blue marker reached the

bottom of the gel. The proteins were stained in the gel

with Coomassie brilliant blue solution for overnight at

room temperature. The gels were diffusion-destained

by repeated washing in the solution containing 7.5 %

acetic acide, 5 % methanole and 87.5 % distilled water.

Results and discussion

The early observations revealed that cytokinins exert

parallel effects in maintain protein or nucleic acid

levels while inhibiting senescence. Cytokinins

stimulate both structural and enzymatic protein

synthesis. They are selectively increasing the levels

of certain enzymes associated generally with

photosynthetic process (Feierabend, 1969). It is not

clear whether the enhanced activity is due to greater

synthesis, inhibition of degradation or activation of the

enzymes.

We already observed that new aromatic cytokinin

mT at 0.25 to 1 mM concentration range delayed

the senescence in excised wheat leaf segments

(Palavan et al., 2002). This concentration range was

high for radish cotyledon growth therefore lower

concentrations were examined (0.05 to 1 mM) in

addition.

Cotyledon growth increased with the treatments of

mT significantly (Figure 1). Stimulation of cotyledon

growth was closely related with increasing

concentrations of mT; 0.05 to 1 mM mT increased the

cotyledon growth about two fold in fresh weight basis

(p


Figure 2: The effect of meta-topolin on soluble protein content

during the growth of radish cotyledons. Values are average

of 4 experiments.

Figure 3: SDS-PAGE analysis of soluble proteins from

meta-topolin treated radish cotyledons. Gel was stained with

Coomassie blue. Lane 1: Control, Lane 2: 0.05 mM mT,

Lane 3: 0.1 mM mT, Lane 4: 0.25 mM mT, Lane 5: 0.5 mM

mT, Lane 6: 1 mM mT treatments. Molecular mass (kDa) of

markers are indicated on left hand margin.

from cell enlargement during cotyledon growth.

Cytokinin treatment promotes additional cell

expansion with no increase in the dry weight of the

treated cotyledons (Huff and Ross, 1975).

Letham (1971) reported the ability of cytokinins to

promote markedly the expansion of radish cotyledons

and explained this response by the promotion of cell

enlargement. mT also as a most active aromatic

meta-topolin effect on protein 33

cytokinin as reported by Strnad et al. (1997) caused to

cotyledon growth markedly as shown in Figure 1.

mT was found to increase the soluble protein

contents of radish cotyledons. Treatments with 0.1, 0.25

and 0.5 mM mT resulted an increase in soluble protein

content in the same ratio (by 3,4 and 3 % respectively)

compared to the control cotyledons (Figure 2).

These findings correlated with electrophoretic

determinations (Figure 3). Soluble proteins of mT

treated radish cotyledons were analyzed using SDS-

PAGE technique in order to test whether and

significant amount of difference in protein profile

occurred with mT treatments. Analysis of the soluble

proteins displayed different electrophoretic pattern in

mT treated cotyledons compared to control. Protein

bands were very sharp and dark in 0.05, 0.1 and 0.25

mM mT treated samples and their molecular masses

ranges between 66 to 45 kDa’s. Molecular mass of 45

to 29 kDa’s were weak in 0.5 and 1 mM mT treated

and in control cotyledons also. On the other hand

protein bands were very sharp and dark in 0.05, 0.1

and 0.25 mM mT treated cotyledons comparing with

0.5 and 1 mM mT treated and control cotyledons.

Obvious bands were also observed around 30 kDa in

cotyledons treated with 0.05, 0.1 and 0.25 mM mT.

Besides these there were additional bands in mT

treated samples different from controls and these

bands were weak in 0.5 and 1.0 mM mT treated

samples compared to the other applications around 24

kDa.

There is good evidence that cytokinins play a role

in regulating protein synthesis (Tepfer and Fosket,

1978). Cytokinins can not only increase the rate of

protein synthesis, but also change the spectrum of

proteins produced by plant tissues.

Results obtained in this study showed that, total

soluble protein content in radish cotyledons not

effected from exogenously applied mT. On the other

hand, when protein profile was examined

electrophoretically additional bands were observed in

mT treated samples. These can be explained by the fact

that mT stimulate new protein synthesis without

effecting total protein content.

In conclusion, natural aromatic cytokinin mT has

an important role in the control of cotyledon growth

and this response closely associated with protein

profile. The results of this research are exhibited mTas

a promising plant growth regulators in physiological

studies.


34 Serap Ça¤ and Narçin Palavan-Ünsal

Acknowledgement

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

generous gift of aromatic cytokinins and to Damla

Büyüktunçer for technical assistance. This study was

supported by Istanbul University Research Fund

(Project number: B-430/13042000).

References

Binns AN. Cytokinin accumulation and action: biochemical,

genetic and molecular approaches. Ann Rev Plant

Physiol Plant Mol Biol. 45: 173-196,1994.

Bradford AM. A rapid and sensitive method for the

quantification of microgram quantities of protein

utilizing the principle of protein-dye binding. Anal

Biochem. 72: 248-254, 1976.

Feierabend J. Der Einfluss von Cytokinin auf die Bildung

von Photosyntheseenzyme im Roggenkeimlingen.

Planta. 84: 11-29, 1969.

Huff AK, Ross CW. Promotion of radish cotyledon

enlargement and reducing sugar content by zeatin and

red light. Plant Physiol. 56: 429-433, 1975.

Laemmli UK. Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature.

227: 680-685, 1970.

Letham DS. Chemistry and physiology of kinetin-like

compounds. Ann Rev Plant Physiol. 18: 349-364, 1967.

Letham DS. Regulators of cell division in plant tissues. XII.

A cytokinin bioassay using excised radish cotyledons.

Physiol Plant. 25: 391-396, 1971.

Miller CO, Skoog F, Von Saltza, MH, Strong F. Kinetin a cell

division factor from deoxyribonucleic acid. J Am Chem

Soc. 77: 1392-1293, 1955.

Miller CO, Skoog F, Okomura FS, von Saltza MH,

Strong FM. Isolation, structure and synthesis of kinetin a

substance promoting cell division. J Am Chem Soc.

78: 1345-1350, 1956.

Mok DWS, Mok MC. Cytokinins: Chemistry, Activity and

Function. CRC Press, Boca Raton. 1994.

Palavan-Ünsal N, Ça¤ S, Çetin E. Growth responses of

excised radish cotyledons to meta-topolin. Canadian J

Plant Sci. 82: 191-194, 2002.

Strnad M, Hanus J, Vanek T, Kaminek M, Ballantine JA,

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

aromatic cytokinin from poplar leaves (Populus x

canadensis Moench., cv. Robusta). Phytochemistry.

45: 213-218, 1997.

Tepfer DA, Fosket DE. Hormone-mediated translational

control of protein synthesis in cultured cells of Glycine

max. Dev Biol. 62: 486-497, 1978.

Thimann KV. Senescence in Plants. 85-115. CRC Press,

Boca Raton. 1980.


Journal of Cell and Molecular Biology 2: 35-38, 2003.

Haliç University, Printed in Turkey.

The effect of electromagnetic fields on oxidative DNA damage

Serkan ‹fller 1 and Günhan Erdem 2 *

1 Department of Biology, Institute of Applied Sciences, Çanakkale Onsekiz Mart University, Çanakkale,

Turkey; 2 College of Health, Çanakkale Onsekiz Mart University, Çanakkale, Turkey (*author for

correspondence)

Received 30 September 2002; Accepted 26 December 2002

Abstract

Many recent studies have focused on the investigation of the biological effects of electromagnetic field. Although

the several types of biological effects of electromagnetic fields have been shown, the molecular mechanisms of these

effects have not been explained yet. Some epidemiological studies have suggested that exposure to ambient, lowlevel

50-60 Hz electromagnetic fields increase risk of disease including cancer such as leukemia among children who

live close to power lines or among men whose jobs expose them to electromagnetic field, while others have

suggested that electromagnetic fields exposure could increase both the concentration of free radicals and oscillating

free radicals. Electromagnetic fields are known to affect radical pair recombination and they may increase the

concentration of oxygen free radicals in living cells. In this study, oxidative stress was formed by the oxidation of

ascorbic acid and the effect of 50 Hz, 0.3 mT electromagnetic fields on the oxidative DNA damage has been

investigated. The results of the study showed that extremely low-frequency electromagnetic fields enhanced the

effect of oxidative stress on DNA damage and supported the idea obtained from the previous studies on an increasing

effect of electromagnetic fields on the concentration and the life-time of free radicals.

KKeeyy wwoorrddss:: Electromagnetic fields, DNA damage, ascorbic acid, vitamin C, oxidative stress

Elektromanyetik alan›n oksidatif DNA hasar› üzerindeki etkisi

Özet

Günümüzdeki birçok çal›flma, elektromanyetik alan›n biyolojik etkilerinin araflt›r›lmas› üzerinde odaklanm›flt›r.

Elektromanyetik alan›n biyolojik etkilerinin baz› türlerinin gösterilmifl olmas›na ra¤men, bu etkilerin moleküler

mekanizmalar› henüz aç›klanamam›flt›r. Baz› epidemiyolojik çal›flmalar, 50-60 Hz dolay›ndaki düflük düzeyli

elektromanyetik alana maruz kalman›n yüksek gerilim hatlar›na yak›n yaflamakta olan çocuklarda veya

elektromanyetik alana maruz kalarak çal›flanlarda görülen lösemi gibi kanser vakalar›n› kapsayan hastal›klara iliflkin

riski art›rd›¤›n› öne sürerken, baz› çal›flmalar ise elektromanyetik alan maruziyetinin serbest radikal

konsantrasyonunu ve serbest radikallerin izlenebilirli¤ini art›rabilece¤ini ileri sürmüfltür. Elektromanyetik alan›n

radikal çifti rekombinasyonunu etkiledi¤i bilinmektedir ve bu da, hücrelerdeki oksijene dayal› serbest radikal

konsantrasyonunu art›rabilir. Bu çal›flmada, askorbik asit oksidasyonu ile oksidatif stres oluflturulmufl ve 50 Hz, 0.3

mT düzeyindeki elektromanyetik alan›n, oksidatif DNA hasar› üzerindeki etkisi araflt›r›lm›flt›r. Bu çal›flman›n

sonuçlar›, oldukça düflük frekansl› elektromanyetik alan›n, oksidatif stresin DNA hasar› üzerindeki etkisini art›rd›¤›n›

göstermifl ve önceki araflt›rmalardan elde edilen, elektromanyetik alan›n serbest radikal konsantrasyonu ve yar› ömrü

üzerindeki art›r›c› etkisine dair düflünceleri desteklemifltir.

AAnnaahhttaarr ssöözzccüükklleerr:: Elektromanyetik alan, DNA hasar›, askorbik asit, C vitamini, oksidatif stres

35


36 Serkan ‹fller and Günhan Erdem

Introduction

There are many reports on the biological effects of

electromagnetic fields (EMF) and there have been

many attempts to develop a theoretical explanation of

this phenomenon. Some epidemiological studies have

suggested that exposure to ambient, low-level 50/60

Hz EMF increases risk of disease including cancer

such as leukemia among children who live close to

power lines or among men whose jobs expose them to

EMF (Wertheimer and Leeper, 1979; Tomenius, 1986;

Savitz et al., 1988; London et al., 1991). EMF firstly

affects the cell membrane. Some ion channels such as

Na-K ATPase have been affected according the level

of EMF. The alteration in the activity of these proteins

causes an increasing or decreasing intracellular

concentration of many ions such as Na + , K + , Mg 2+ and

Ca 2+ which plays very important roles in cell signaling.

Therefore, the biological effects of EMF expand

among the cellular systems (Goodman et al., 1995).

Although the several types of biological effects of

EMF have been shown, the molecular mechanisms of

these effects have not been explained yet. Some

studies have suggested that EMF exposure could be

due to both the increase in the concentration (Jajte,

2000) and oscillating of free radicals (Scaiano et al.,

1995). EMF is known to affect radical pair

recombination and they may increase the

concentration of oxygen free radicals in living cells

(Jajte, 2000). Increasing the concentration of free

radicals creates oxidative stress and some biological

reactions such as DNA damage occur under this

condition. Metabolic energy production or effects of

chemicals and radiation can form oxidative stress.

In this study, oxidative stress was formed by the

oxidation of ascorbic acid with Cu 2+ ions and the effect

of 50 Hz, 0.3 mT EMF on the oxidative DNA damage

was investigated.

Materials and methods

DNA isolation

High molecular weight (app. 10 kb) human genomic

DNA was isolated from the white blood cells with the

modified method of Poncz et al. (1982) by using MBI

Fermentas genomic DNA isolation system. Molecular

weight and purity of DNA samples were controlled by

agarose gel electrophoresis. The concentration of

DNA samples was spectrophotometrically determined.

All DNA samples were free from proteins, RNAs and

solvents used for extraction.

Oxidative DNA cleavage reactions

Cleavage reactions were carried out in a medium

containing 0.5 µg DNA, 20 mM Tris-HCl (Sigma) pH

7.8, 0.25 mM ultra-pure ascorbic acid (Merck) and

CuCl2 (Sigma) in the final concentrations of 2.5, 5, 7.5

and 10 µM, in a final volume 10 µl. Other antioxidants

(glutathion, cystein and dithiothreitol, Sigma) and

metal chelator (EDTA, Merck) were added to the

reaction mixtures at a final concentration of 0.5 mM.

The mixtures were incubated at room temperature for

10, 20 and 30 minutes. Adding EDTA at a final

concentration of 25 mM stopped reactions. DNA

cleavages in the reaction mixtures were analyzed on

the 1% agarose gel (Promega) electrophoresis.

EMF exposure system

Electromagnetic fields were applied by using the

Helmholtz coil. The coil system was constructed by

using the polyester sphere that was surrounded by

copper wire with 0.75 cm diameter (Galt et al., 1995).

The diameter and height of the sphere were 16 and 26

cm, respectively. 50 Hz, 4.5 V electricity was applied

to coil system. As a result, 0.3 mT EMF was generated

at the center of the coil system which includes handles

for the sample tubes.

Results and discussion

The oxidative DNA damage was induced by the

concentration of cupric ions (Figure 1). In the constant

ascorbate concentration (0.25 mM), oxidative DNA

breakage was started in the presence of 2.5 µM

copper(II) ions and EMF also induced the DNA

breakage at this condition (Figure 1, lanes 4 and 10).

Therefore, main DNA band in the lane 10 of Figure 1

is thinner than the lane 4. In the presence of high

cupric ions concentration, excess scission of DNA

molecules occurred at the EMF when compared to

normal conditions (Figure 1, lanes 6 and 12).

Electromagnetic fields did not have an effect on the

oxidation of ascorbic acid in the absence of cupric ions

(specific data was not shown, but the sample in Figure

3, lane 12 had reflected this result, because EDTA was


1 2 3 4 5 6 7 8 9 10 11 12

Figure 1: The effect of EMF and Cu(II) concentrations on

oxidative DNA damage. All lanes include 0.5 µg DNA. The

DNA samples in the lanes from 1 to 6 were incubated under

normal condition, 7 to 12 were incubated in EMF at room

temperature in the presence of 0.25 mM ascorbic acid

except the 1 and 7 which were control lanes. Cu(II)

concentrations were 1.25 µM in 2 and 8, 2.5 µM in 3 and 9,

5 µM in 4 and 10, 7.5 µM in 5 and 11, 10 µM in 6 and 12

lanes. Incubation time was 30 min for all samples.

chelating cupric ions and eliminated their oxidative

effects).

The results of this study showed that the oxidative

DNA damage depends on the incubation time (Figure 2).

DNA breakages could be observed at the 20 th minute of

incubation time (Figure 2, lanes 6 and 8). EMF

exposure enhances the oxidative DNA damage after

the 20 th minute (Figure 2, lanes 2 and 4).

1 2 3 4 5 6 7 8 9 10 11 12

Figure 3: The effect of some antioxidants (glutathione,

cystein, dithiothreitol) and metal chelator (EDTA) on

excessive oxidative DNA damage in EMF. All lanes include

0.5 µg DNA. Ascorbic acid and Cu(II) concentrations were

0.25 mM and 7.5 µM in all lanes except the control DNA

lanes 1 and 7, respectively. Lanes 2 and 8 were scission

controls. Glutathione (3 and 9), cystein (4 and 10),

dithiothreitol (5 and 11) and EDTA (6 and 12)

concentrations were 0.5 mM. The samples in 1 to 6 were

incubated at normal condition. The others were incubated in

EMF. Incubation time was 30 min for all samples.

EMF and oxidative DNA damage 37

1 2 3 4 5 6 7 8 9 10 11 12

Figure 2: The effect of incubation time on oxidative DNA

damage depends in electromagnetic fields. All lanes include

0.5 µg DNA. Incubation times were 30 min from 1 to 4, 20

min from 5 to 8 and 10 min from 9 to 12. Ascorbic acid

concentration were 0.25 mM in all lanes. Cu(II)

concentrations were 2.5 µM in 1, 3, 5, 7, 9 and 11 while

5 µM in 2, 4, 6, 8, 10 and 12. The samples in 1, 2, 5, 6, 9 and

10 were incubated in EMF. Other samples were incubated at

normal conditions.

In the presence of EDTA as a cationic metal

chelator, oxidative DNA damage was not observed.

This result showed that ascorbate oxidation and

oxidative DNA damage depend on cupric ions as an

oxidizing agent (Figure 3, lanes 6 and 12). As an

antioxidant, cystein did not block the oxidative DNA

damage (Figure 3, lanes 4 and 10). Glutathione

reduced the oxidative stress. Therefore, the DNA

damage was formed as aggregation rather than

fragmentation in the presence of glutathione (Figure 3,

lanes 3 and 9). Dithiotreitol (DTT) was the most

effective antioxidant of all investigated but EMF

exposure inhibited the effectiveness of DTT (Figure 3,

lanes 5 and 11).

The oxidative species produced by ascorbate

oxidation in the presence of copper(II) ions damage

the DNA molecules (Figure 1). Previously DNA

damage depending on ascorbate oxidation had been

studied (Erdem et al., 1994; Zareie et al., 1996).

Oxidative DNA damage was observed as

fragmentation or aggregation. The degree of oxidative

DNA damage varies in the levels and reactivity of free

radicals produced in the reaction medium. In the

presence of oxygen, the hydroxyl and peroxyl radicals

such as superoxide anion and hydroperoxyl radical are

produced by the reaction between radical form of

ascorbic acid (ascorbyl radical) and molecular oxygen

(Fuchs et al., 1990).

These radicals attack to electrophilic nuclei on the

targets and create secondary carbon radicals. At the


38 Serkan ‹fller and Günhan Erdem

high level or high reactivity of these radicals, excess

formation of secondary carbon radicals on the same

DNA molecule causes a reaction between each other

and then the DNA damage occurs as fragmentation.

Therefore, DNA size became smaller and gave the

smeared patterns on gel electrophoresis (lanes 5 and 6

in Figure 1) However, at the low level or low reactivity

of oxygen species, the oxidative DNA damage results

in aggregation of the DNA molecules with the

intermolecular reaction of the secondary carbon

radicals. Thus, the DNA samples became heavier and

were retarded on the gel electrophoresis (lanes 3 and 9

in Figure 3).

Our results showed that extremely low-frequency

EMF enhanced the effect of oxidative stress on DNA

damage and supported the idea obtained from previous

studies on an increasing effect of EMF on the

concentration and the life-time of free radicals (Jajte,

2000; Scaiano et al., 1995; Jajte and ZmySlony, 2000).

Especially the comparisons of lane 2 to lane 4 in

Figure 2 and lane 5 to lane 11 in Figure 3, indicate that

the degree of the oxidative stress under the EMF is

greater than the normal condition.

In the brain cells of rats, an increase in DNA

single- and double-strand breaks had been found after

acute exposure to a sinusoidal 60 Hz magnetic field.

When the experiment was carried out in the presence

of melatonin or a radical scavenger compound N-tertbutyl-alpha-phenylnitrone

(PBN), the effect of

magnetic fields on brain cell DNA was not observed

(Lai and Singh, 1997). Melatonin is a neurohormone

and it is also an antioxidant and a free radical

scavenger. Therefore, this hormone could protect

biological systems against oxidative damage. The

increasing effect of EMF on the concentration of free

radicals has been suggested that melatonin suppression

in humans may increase the probability of mutagenic

and carcinogenic risk (Jajte and ZmySlony, 2000).

EMF (≥1 mT) increases the concentration of free

radicals that escape from the alkyl sulphate and

sulphonate micelles. The effect of extremely lowfrequency

EMF on the radicals formed from singlet

precursors is larger than triplet precursors. Some

radicals such as hydroxyl and peroxyl radicals

generated in the biological reactions are formed from

singlet precursors (Eveson et al., 2000).

In conclusion, the results obtained from our study

suggest that the effects of extra low frequency EMF on

the concentration of free radicals and the

recombination of radical pairs might trigger the

carcinogenesis in the populations living close to the

overhead electric power distribution lines.

References

Erdem G, Öner C, Önal AM, K›sakürek D and Ö¤üfl A. Free

radical mediated interaction of ascorbic acid and

ascorbate/Cu(II) with viral and plasmid DNAs. J Biosci.

19: 9-17, 1994.

Eveson RW, Timmel CR, Brocklehurst B, Hore PJ and

McLauchland KA. The effects of weak magnetic fields

on radical recombination reactions in micelles. Int J

Radiation Biol. 76: 1509-1522, 2000.

Fuchs J, Mehlhron RJ and Packer L. Assay for free radical

reductase activity in biological tissue by electron spin

resonance spectroscopy. Methods in Enzymology.

186: 670-674, 1990.

Galt S, Whalstrom J, Hamnerius Y, Holmqvist D and

Johannesson T. Study of effects of 50 Hz magnetic fields

on chromosome aberration and growth-related enzyme

ODC in human amniotic cells. Bioelectrochemistry and

Bioenergetics. 36: 1-8, 1995.

Goodman EM, Greenebaum B and Marron MT. Effects of

electromagnetic fields on molecules and cells. In: Int

Rew Cytology, A Survey of Cell Biology. Jean KW and

Jarvik J (Ed). Academic Press. 158: 279-338, 1995.

Jajte J and ZmySlony M. The role of melatonin in the

molecular mechanism of weak, static and extremely low

frequency (50 Hz) magnetic fields (ELF). Medycyna

Pracy. 51: 51-57, 2000.

Jajte JM. Programmed cell death as a biological function of

electromagnetic fields at a frequency of (50/60 Hz).

Medycyna Pracy. 51: 383-389, 2000.

Lai H and Singh NP. Melatonin and N-tert-butyl-alphaphenylnitrone

block 60-Hz magnetic field-induced DNA

single and double strand breaks in rat brain cells.

J Pineal Res. 22: 152-62, 1997.

London SJ, Thomas DC, Bowman JD, Sobel E, Cheng TC

and Peters JM. Exposure to residential electric and

magnetic fields and risk of childhood leukemia. Am J

Epidemiol. 134: 923-37, 1991.

Poncz M, Solowiejczyk D, Harpel B, Mory Y, Schwartz E

and Surrey S. Construction of human gene libraries from

small amounts of peripheral blood: Analysis of ß-like

globin genes. Hemoglobin. 6: 27-36, 1982.

Savitz DA, Wachtel H, Barnes FA, John EM and Tvrdik JG.

Case-control study of childhood cancer and exposure to

60-Hz magnetic fields. Am J Epidemiol. 128: 21-38, 1988.

Scaiano JC, Cozens FL and Mohtat N. Development of a

model and application of the radical pair mechanism to

radicals in micelles. Photochemistry and Photobiology.

62: 818-829, 1995.

Tomenius L. 50-Hz electromagnetic environment and the

incidence of childhood tumors in Stockholm County.

Bioelectromagnetics. 7: 191-207, 1986.

Wertheimer N and Leeper E. Electrical wiring configurations

and childhood cancer. Am J Epidemiol. 109: 273-284,

1979.

Zareie MH, Erdem G, Öner C, Öner R, Ö¤üfl A and Piflkin E.

Investigation of ascorbate-Cu (II) induced cleavage of

DNA by scanning tunneling microscopy. Int J Biol

Macromol. 19: 69-73, 1996.


Journal of Cell and Molecular Biology 2: 39-42, 2003.

Haliç University, Printed in Turkey.

Chromosomes of a balanced translocation case evaluated with

atomic force microscopy

Zerrin Y›lmaz 1 *, Mehmet Ali Ergun 2 , Erdal Tan 3

1 Department of Medical Biology and Genetics, Baskent University, Faculty of Medicine, 06570,

Maltepe, Ankara, Turkey; 2 Department of Medical Biology and Genetics, Gazi University, Faculty of

Medicine, 06510, Besevler, Ankara, Turkey; 3 Materials Research Department, Ankara Nuclear Research

and Training Center, 06100, Besevler, Ankara, Turkey (*author for correspondence)

Received 2 December 2002; Accepted 30 December 2002

Abstract

A couple was referred to our genetics department for cytogenetic analysis because of two previous abortions. The

cytogenetic analysis of the male was found as 46, XY and the female revealed a balanced translocation; 46, XX,t

(7;12) (p21;q14) and also she had 14 cenh+ as her mother. Atomic force microscopy (AFM) is a useful method for

detecting detailed structures of chromosomes. With the help of this new technique the surface topography of human

chromosomes can be examined. We used AFM in order to analyse the surface topography of derivative chromosomes

of the patients, and found a 0.6 µm gap region. In this study, we aimed to examine the differences between the images

of the derivative chromosomes detected by light and atomic force microscopy analyses.

KKeeyy wwoorrddss:: Balanced translocation, chromosome polymorphism, atomic force microscopy

Dengeli translokasyon vakas›nda kromozomlar›n atomik güç mikroskobu ile

de¤erlendirilmesi

Özet

Ardarda iki gebelik kayb› nedeniyle departman›m›za yönlendirilen çiftin sitogenetik analizleri yap›lm›flt›r. Erkekte

normal kromozom kuruluflu 46, XY saptanm›fl ancak kad›nda dengeli translokasyonla birlikte

14. kromozoma ait sentromer art›fl› saptanm›flt›r; 46, XX, t (7;12) (p21;q14), 14 cenh+. Proband›n ailesinde yap›lan

sitogenetik çal›flma ile ayn› kromozom kuruluflunun proband›n annesinden kal›t›ld›¤› saptanm›flt›r. Atomik güç

mikroskobu kromozomlar›n yap›sal olarak detayl› incelenmesinde kullan›lmaktad›r. Bu yeni tekni¤in yard›m›yla

insan kromozomlar›n›n yüzey topografisi incelenebilmektedir. Biz de atomik güç mikroskobunu kullanarak derivatif

kromozomun yüzey topografisini araflt›rd›k ve 0.6 µm’lik bir gap bölgesi saptad›k. Bu çal›flmada probanda ait

derivatif kromozom yap›s›n› hem ›fl›k mikroskobu hem de atomik güç mikroskobu ile ayr› ayr› de¤erlendirerek

sonuçlar›m›z› karfl›laflt›rd›k.

AAnnaahhttaarr ssöözzccüükklleerr:: Dengeli translokasyon, kromozom polimorfizmi, atomik güç mikroskobu

Introduction

Cytogenetics is the study of genetic material at the

cellular level. Human cytogenetics is almost always

concerned with light microscope studies of

chromosomes. Staining procedures which provide a

39


40 Zerrin Y›lmaz et al.

uniform unbanded appearance to chromosomes are

referred to solid or covential staining. They can,

however, be useful for studies on chromosome

breakage as scoring gaps and breaks can be difficult in

lightly stained chromosome bands. Giemsa banding

(G-banding) has become the most widely used

technique for the routine staining of human

chromosomes. The chromosome banding patterns

obtained reflect both the structural and functional

composition of chromosomes. Consititutive

heterochromatin is the structural chromosomal

material seen as dark staining material in interphase as

well as during mitosis. It includes both repetetive

DNA, satellit DNA and some non-repetetive DNA.

C-banding can be used to demonstrate the repetetive

DNA (Benn and Perle, 1992).

Atomic force microscopy (AFM) is a diagnostic

tool for detecting detailed structures of the

chromosomes and the surface topography of human

chromosomes can be examined using this new

technique (Binning et al., 1986; Musio et al., 1997).

AFM could be considered as a tool for further

chromosomal studies.

In our previous studies using AFM, we showed

that, unbanded human metaphase chromosomes

displayed a banding pattern similar to G-bands, and for

the first time we have provided an AFM imaging of

chromosomes in trisomy 13, 21 and Klinefelter

Syndrome patients (Ergun et al., 1999). Besides, G and

C-banding patterns of chromosomes were also

investigated (Sahin et al., 2000; Tan et al., 2001).

In this study, we used AFM in order to analyse the

surface topography of derivative chromosomes of a

female patient whose daughter was referred to our

Genetics department with the chief complaints of

abortions.

Materials and methods

Case presentation

In this study we evaluated the chromosomes of a

family. This family was referred to our genetics

department for cytogenetic analysis because of two

previous abortions during the first trimester. They had

no live-born children after a marriage of 5 years. The

male was 37 years old and healthy, and his nonconsanguineous

wife was 36 years old. Her physical

examination revealed no abnormalities in

genitourinary, endocrinological and other organ

systems; also laboratory findings were normal.

Light microscopy analysis

Metaphase chromosome preparation was obtained

from peripheral blood lymphocytes using standard

techniques (Verma and Babu, 1995). Conventional

cytogenetic analysis was carried out using GTGbanding

and C-banding techniques (Benn and Perle,

1992). The chromosome images were captured by

computer imaging (Cytovision system, Image

analysis, Applied Imaging, Saunderland, UK).

For each patient we analysed 20 metaphases, and

C-banding procedure was performed while

investigating 14 cenh+.

Atomic force microscopy and analysis

The AFM used in this study was TopoMetrix

TMX2000 Explorer, operating in contact mode and air.

Throughout the surface analysis, we have used

standard pyramidal tip (1520-00) with the radius of

curvature of approximately 1000 A°. During the

surface analysis, the metaphase region was primarily

determined and addressed by light microscopy. Later,

the region under consideration was scanned via AFM

at various scan ranges changing from 150 µm down to

10 µm or less to image the chromosomes in a good

manner. The applied force and the image resolution

were between 1 and 3 nN and 400x400 pixels (or

higher) respectively for each image acquisition. The

raw data gathered were analysed by using the software

of the microscopy system in two or three-dimensional

patterns.

In our study, the chromosomes of the patient were

spread on glass surface. Then, the metaphase spreads

were analysed by AFM. Line measure analysis was

performed on derivative chromosomes.

Results

The karyotype of the male revealed 46, XY, while the

cytogenetic analysis of his wife was karyotyped as

46, XX, t (7;12) (p 21;q 14); a balanced translocation.

She also had 14cenh+. In order to understand the origin

of this translocation chromosome, her mother was

karyotyped and she was also found to be a translocation

carrier; 46, XX, t (7;12) (p 21; q14) and 14 cenh+.


Figure 1: The partial karyotype of the daughter (a) and the

mother (b); derivative chromosomes 7 and 12 and

chromosomes 14 with C-banding images are shown.

Figure 2: AFM image of derivative chromosome 7 and line

measure analysis of the gap region.

The karyotypes of the mother and the daughter

were similar; 46, XX, t (7;12) (p 21; q 14). The partial

karyotype of the daughter (Figure 1a) and the mother

(Figure 1b) with the derivative chromosomes 7, 12 and

chromosomes 14 with C-banding images are shown.

We performed detailed measurements on the

derivative chromosomes of the mother and detected

the attached chromosome fragment from derivative

chromosome 12 to the derivative chromosome 7. Our

measurements revealed a 0.6 µm gap region (Figure 2).

Besides we also analysed the satellite region of

chromosome 14 (Figure 3) of the mother.

Discussion

AFM in chromosome evaluation 41

Figure 3: AFM image of chromosome 14 indicated by an

arrow.

In this study we evaluated the detailed structures of

translocated chromosomes of the mother with AFM.

Line measure analysis revealed a gap region on the

derivative chromosome 7, which was measured as 0.6

µm. It was equivalent to a mid-sized G-band region

(Figure 2).

In the previous studies, it was reported that these

gap regions correspond to narrowing and grooving

regions on chromosomes, and are considered as

negative G- band regions (Uehara et al., 1996). The

gap regions were also observed in fragile X (Harrison

et al., 1983) and in radiation exposed chromosomes

(Mullinger and Johnson, 1987). It was thought that,

these regions correspond to high-order structural

aberrations resulting from an incomplete or irregular

composition of chromatid fibres induced by a

translocation of a chromosomal fragment. The gap

regions were the results of chromosomal

rearrangements (Uehara et al., 1996).

Our second analysis was on the satellite region of

the chromosome 14 of the mother. First of all, GTGbanding

revealed an increase in the heterochromatin

region of the short arm of chromosome 14. Then, we

performed C-banding procedure to understand if this

region was belonging to a constitutive heterochromatin

region, or to an extra banding region. The results of

C-banding confirmed that these regions were

belonging to heterochromatin region. Our 3dimensional

AFM analysis for the satellite region

showed an augmentation on the short arm of this

chromosome (Figure 3). These heterochromatin

regions are polymorphic regions, and they are highly


42 Zerrin Y›lmaz et al.

repetitive regions that are located on the centromeres

of chromosomes 1, 9, and 16 and on the distal arm of

Y chromosome (Burkholder and Duczek, 1980; Cook,

1995). Our AFM image also helps us to understand

that these regions were not belonging to G- banding

regions, as there was not a banding pattern (Tan et al.,

2001).

AFM can be considered as a novel technique for

analysing detailed structures of chromosomes for its

line measure analysis and 3-D image capture

capabilities. Reflecting these capabilities, AFM helped

us to investigate the gap region on the derivative

chromosome and this study is also novel by making

new implementations on the mechanism of

translocation. As a conclusion, the capability of AFM

for detecting chromosomal abnormalities will reflect

light into further studies.

References

Benn PA and Perle MA. Chromosome staining and banding.

In: Human Cyotgenetics. A Practical Approach.

Rooney DE and Czepulowski BH (Ed). New York.

Oxford University Press. 1: 91-118, 1992.

Binning G, Rohrer H and Gerber C. Atomic force

microscopy. Phys Rev Lett. 56: 930- 933, 1986.

Burkholder GD and Duczek LL. Proteins in chromosome

banding. II. Effect of R- and C-banding treatments on the

proteins of isolated nuclei. Chromosoma. 79: 43-51, 1980.

Cook PR. A chromomeric model for nuclear and

chromosome structure. Journal of Cell Science.

108: 2927-2935, 1995.

Ergun MA, Tan E, Sahin FI and Menevse A. Numerical

chromosome abnormalities detected by atomic force

microscopy. Scanning. 21: 182-186, 1999.

Harrison CJ, Jack EM, Allen TD and Harris R. The fragile

X: A scanning electron microscope study. J Med Genet.

20: 280-5, 1983.

Mullinger AM and Johnson RT. Scanning electron

microscope analysis of structural changes and

aberrations in human chromosomes associated with the

inhibition and reversal of inhibition of ultraviolet light

induced DNA repair. Chromosoma. 96: 39-44, 1987.

Musio A, Mariani T, Frediani C, Ascoli C and Sbrana I.

Atomic force microscopy imaging of chromosome

structure during G-banding treatments. Genome.

40: 127-131, 1997.

Sahin FI, Ergun MA, Tan E and Menevse A. The mechanism

of G- banding detected by atomic force microscopy.

Scanning. 22: 24-27, 2000.

Tan E, Sahin FI, Ergun MA, Ercan I and Menevse A.

C-banding visualised by AFM. Scanning. 23: 32-35,

2001.

Uehara S, Sasaki H, Takabayashi T and Yajima A. Structural

aberrations of metaphase derivative chromosomes from

reciprocal translocations as revealed by scanning

electron microscopy. Cytogenet Cell Genet. 74: 76-79,

1996.

Verma RS and Babu A Banding techniques. In: Human

Chromosomes Principles and Techniques. Verma RS and

Babu A (Ed). McGraw-Hill Inc. New York. 72-133,1995.


Journal of Cell and Molecular Biology 2: 43-48, 2003.

Haliç University, Printed in Turkey.

Effect of epirubicin on mitotic index in cultured L-cells

Gül Özcan Ar›can* and Mehmet Topçul

‹stanbul University, Faculty of Science, Department of Biology, 34459 Vezneciler, ‹stanbul, Turkey

(*author for correspondence)

Received 9 December 2002; Accepted 30 December 2002

Abstract

Cancer chemotherapy is an additional application to surgical operations and radiotherapy in the treatment of

widespread tumors. An anthracycline-derived antibiotic, epirubicin (EPI) is one of the clinically used antineoplastic

drugs. In this study the cytotoxic effects of EPI in transformed mouse fibroblasts (L-cell) were examined. EPI

concentrations of 0.001 µg/ml, 0.01 µg/ml and 0.1 µg/ml were applied to the cells for 2, 4, 8, 16 and 32 hours. The

results showed that EPI diminished mitotic index of L-cells depends upon time and applied concentrations. This

decrease was found statistically significant in each treatment group when compared to control (p


44 Gül Özcan Ar›can and Mehmet Topçul

Skladonowski and Konopa, 1994). In addition,

topoisomerase-II has also been shown to be inactivated

by EPI (Robert and Gianni, 1993; Haldane et al.,

1993).

In vitro studies showed that EPI possesses

cytotoxicity at least equivalent to that of doxorubicin

against a variety of animal and human tumor cell lines

including those derived from breast, liver, lung,

gastric, colorectal, squamous cell, cervical, bladder,

ovarian carcinomas, neuroblastoma and leukaemia

(Bagnara et al., 1987; Zhang et al., 1992; Bartkowiak

et al., 1992).

EPI is a cell cycle phase non-specific

anthracycline, with maximal cytotoxic effects in the S

and G2 phases. Preliminary in vitro studies were

carried out on HeLa cells. The first tests demonstrated

that EPI and doxorubicin gave essentially the same

inhibition of HeLa cell colony formation (Di marco et

al., 1976). Similarly, EPI was as active as doxorubicin

on mouse embryo fibroblast proliferation (Di marco et

al., 1977), but was taken up in greater amount than

doxorubicin by L1210 leukemia cells in vitro (Wilson

et al., 1981).

There have been few studies about the effect of EPI

on mitotic index of rapidly proliferating cells. In this

study, we have therefore studied the effect at EPI,

employed in the concentrations of 0.001 µg/ml,

0.01 µg/ml and 0.1 µg/ml for a period of 2 to 32 hours,

on proliferation of transformed L-cells in culture

which was investigated by measuring mitotic index in

order to investigate the effectiveness of this drug in

chemotherapy.

Material and methods

Chemical

EPI (4’-epidoxorubicin), an anthracycline antibiotic, is

a doxorubicin stereoisomer, possessing the L-arabino

instead of the L-lyxo configuration of the sugar moiety

(Figure 1). In EPI therefore the hydroxyl group on the

sugar moiety, possessing the stable 1 C4 conformation,

has an equatorial orientation (Plosker and Faulds,

1993).

Cell line

The cells used in this study were derived from mouse

fibroblast by in vitro malign transformation (Earle,

1943). Transformed L-cells obtained from mouse

subcutaneous connective tissue in 1943. They were

supplied by Dr. P.P. Dendy of Department of

Radiotherapeutics, Cambridge University, in 1975.

The cells were grown in Medium-199 (M-199, Gibco

lab.) containing 10% foetal bovine serum (FBS, Gibco

lab.), 100 µg/ml streptomycin and 100 IU/ml

penicillin, and were passaged twice a week in

appropriate number of 25 cm 2 flasks and the volume of

the complete medium in each flask was completely to

12 ml. Cells were removed from the surface of culture

flasks by addition of 0.25% trypsine (Gibco lab.) and

centrifuged for 3 minutes at 1500 cycle/min.

Following the addition of M-199 on the cell

precipitate, the cells became ready for the experiment.

Cell doubling time (Tc) of L-cells was 22.8 hours

(Özcan and R›dvano¤ullar›, 1996). L-cells were

cultured on the cover-slips as 3.10 4 cells/ml in petri

dishes and incubated for 24 hours with 95% air and 5%

CO2 containing medium at 37°C with pH 7.2 in a

dessicator. At the end of this incubation medium was

removed, replaced with medium containing EPI

concentrations.

Drug application

Figure 1: Structural formulae of EPI.

Epirubicin (Farmorubicin, Carlo Erba) was dissolved

immediatedly before use in sterile medium (M-199) to

give the required concentration. We used 0.001 µg/ml,

0.01 µg/ml and 0.1 µg/ml concentrations of EPI. Cells

were treated with these doses for 2, 4, 8, 16 and 32

hours.


Mitotic index analysis

Mitotic index were studied by the methods of Feulgen.

Before the cells were treated with Feulgen, they were

prepared with 1 N HCl at room temperature for 1

minute and then hydrolized with 1 N HCl for 10.5

minutes at 60°C. After slides were treated with

Feulgen, they were rinsed for few minutes in distilled

water and stained with 10% Giemsa stain solution pH

6.8, for 3 minutes and washed twice in phosphate

Figure 2: Mitosis in L-cells under the light microscope

(3.3x100).

buffer. After staining, the slides were rinsed in distilled

water. And then the slides were air dried. At last

mitotic index were calculated by counting metaphases,

anaphases and telophases for each tested drug

concentration and control (Figure 2). At least three

thousands cells were examined from each slide for

mitotic index.

Statistical analysis

Mitotic index values which obtained from experiments

were calculated to evaluate the statistical analysis. The

differences between the percentage distrubition of M

phase of the various treatment groups and control were

compared by the Student-t test (n=25).

Results

Epirubicin effect on mitotic index 45

The effect of EPI on mitotic index of L-cells

in culture was investigated. EPI concentrations of

0.001 µg/ml, 0.01 µg/ml and 0.1 µg/ml were applied to

the cells for time periods of 2, 4, 8, 16 and 32 hours.

In this study, EPI diminished the mitotic index of Lcells

with increasing both treatment time and drug

concentration compared to controls (untreated group).

From the value of 2 hours treatment, we saw that all

EPI concentrations had a rapid effect. In subsequent

hours, this effect seemed to continue. The values of

mitotic index reached a minimum at EPI concentration

of 0.1 µg/ml with increasing drug concentration. Table

1 reveals that treatments of EPI decreased the

percentage of the cells at M phase. With increasing

time the differences among the effects of various drug

concentrations tended to be lower being very small at

2 to 8 hours applications. The inhibition of mitosis was

higher in 16 and 32 hours applications than those in 2,

4 and 8 hours EPI applications in Table 1 especially

with EPI concentration of 0.1 µg/ml. However, in the

treatment of 0.1 µg/ml concentration, mitotic

inhibition reached a maximum at 32 hours application.

The values of mitotic index of the cells treated with

EPI for 32 hours showed that mitotic index decreased

as drug concentrations were increased.

Table 1: Mitotic index values in cultures of L-cells treated with various concentrations of EPI, given in mean ± Standard deviation

(SD).

Mitotic index (%)

EPI 2 hours 4 hours 8 hours 16 hours 32 hours

concentrations

Control 1.44 ± 0.12 SD

0.001 µg/ml 1.35 ± 0.09 a

0.01 µg/ml 1.29 ± 0.11 a

0.1 µg/ml 0.94 ± 0.02 c

a : p < 0.05, b : p < 0.01, c : p < 0.001

1.93 ± 0.14 3.04 ± 0.08 3.39 ± 0.15 3.84 ± 0.21

1.80 ± 0.10 a

2.72 ± 0.07 a

2.96 ± 0.04 b

3.10 ± 0.30 b

1.79 ± 0.05 b

2.61 ± 0.06 b

2.70 ± 0.13 b

2.99 ± 0.16 b

1.04 ± 0.01 c

1.77 ± 0.09 c

1.85 ± 0.08 c

1.02 ± 0.22 c


46 Gül Özcan Ar›can and Mehmet Topçul

EPI significantly decreased the mitotic index in

cultures of L-cells. The results show that EPI

decreased the mitotic index at significant level p


seem to be concordant with the above mentioned

studies suggesting that cytotoxic effects of EPI might

occur in the G1 and M phases at higher drug

concentration.

In our study, decreases in the mitotic index of cells

with increasing both treatment time and EPI

concentration have confirmed that EPI is an effective

inhibitor of mitosis.

In conclusion, the results of this study declared the

cell kinetics and cytotoxic effects of the anticancer

drug, EPI, in treated cultures of L-cell line. Although

EPI has less systemic and cardiac toxicity than

doxorubicin and other anthracyclines with an

equivalent spectrum of antitumor action, it still has

cytotoxic effects.

Acknowledgment

We would like to thank Prof. Dr. Atilla Özalpan for his

kind help and critics.

References

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epirubicin on human normal and leukemic hemopoietic

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the cytokinetic and cytotoxic effects of epirubicin in

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Cantoni O, Sestili P, Cattabeni F. Comparative effects of

doxorubicin and 4’-epidoxorubicin on nucleic acid

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epirubicin. In: Advances in Anthracycline Chemotherapy:

Epirubicin. Bonadonna G (Ed). Masson, Milano-Italy.

31-40, 1984.

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In: Cancer: Principles and Practice of Oncology. De vita

VT (Ed). AJF Lippincott, Philadelphia. 374-385, 1993.

Di marco A, Casazza AM, Gambetta R, Supino R, Zunino F.

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stereochemistry of daunorubicin and adriamycin

derivates. Cancer Res. 36: 1962-1966, 1976

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Rivolta P, Velcich A, Zaccara A, Zunino F. Changes of

activity of daunorubicin, adriamycinand stereoisomers

following the introduction or removal of hydroxyl

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Epirubicin effect on mitotic index 47

Di marco A. Epirubicin: Mechanism of action at the cellular

level. In: Advances in Anthracycline Chemotherapy:

Epirubicin. Bonadonna G (Ed). Masson, Milano-Italy.

41-47, 1984.

Earle WR. Production of malignancy in vitro. IV. The mouse

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El-Mahdy Sayed Othman O. Cytogenetic effect of the

anticancer drug epirubicin on Chinese hamster cell line

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Haldane A, Finlay GJ, Baguley BC. A comparison of the

effects of aphidicolin and other inhibitors on

topoisomerase II-directed cytotoxic drugs. Oncol Res.

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kinetic effects of doxorubicin and 4’-epidoxorubicin

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Lollini PL, De Giovanni C, Del Re B. Myogenic

differentiation of human rhabdomyosarcoma cells

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Giannaarelli D, Vaccaro A, Dottovio AM, Terzoli E.

Phase II study of epirubicin and vinorelbine eith

granulocyte colony-stimulating factor: A high-activity,

dose-dense weekly regimen for advanced breast cancer.

Ann Oncol. 10 (8): 937-942, 1999.

Özcan G and R›dvano¤ullar› M. The effect of epirubicin on

the cell cycle of L-cells. 13 th National Congress of

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Özcan FG, Topçul MR, Y›lmazer N, R›dvano¤ullar› M.

Effect of epirubicin on 3 H-thymidine labelling index in

cultured L-strain cells. J Exp Clin Cancer Res. 16(1): 23-

27, 1997.

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pharmacodynamic and pharmacokinetic properties, and

therapeutic use in cancer chemotherapy. In: Drugs.

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Robert J and Gianni L. Pharmacokinetics and metabolism of

anthracyclines. Cancer Surv. 17: 219-252, 1993.

Rocchi P, Ferreri AM, Simone G. Epirubicin-induced

differentiation of human neuroblastoma cells in vitro.

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crosslinking induced by anthracyclines in tumour cells.

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Huan S, Soltys K, Prosser A, Davies RA. Pilot study of

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48 Gül Özcan Ar›can and Mehmet Topçul

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nuclear RNA synthesis in cultured mouse leukemia

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with other cytotoxic and anti-emetic drugs. Anticancer

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intermediate- and high-grade Non-Hodgkins’s

lymphomas with an intensive epirubicin-containing

regimen. Blood. 82 (12): 3564-3573, 1993.


Letter to editor

P rostate cancer and importance of

tumor marker studies

P rostat kanseri ve tümor mark›rlar› ile

ilgili çal›flmalar›n önemi

Prostate cancer is the most commonly new diagnosed

noncutaneous malignancy in men in USA. In the year

2002, according to the health statistics 189,000 men in

the United States are expected to be diagnosed with the

disease and 30,200 men are expected to die of it.

Incidence varies greatly, with African Americans

having the highest incidence in the world (224 cases

per 100,000 population). The incidence of prostate

cancer in African Americans stands in stark contrast to

the incidence in white Americans (150 per 100,000)

and that in men in Western Europe (39.6 per 100,000),

Japan (8.5 per 100,000), and China (1.1 per 100,000).

Tumor markers are biological molecules that

indicate the presence of malignancy. They are

potentially useful in cancer screening, aiding

diagnosis, assessing prognosis, predicting in advance a

likely response to therapy, and monitoring patients

before and after diagnosis. Because of low prevalance

of most cancers in the general population and the

limited sensivity and spesificity of avaible markers,

these tests alone are generally of little value in

screening for cancer in healthy subjects. Currently,

however, prostate spesific antigen (PSA) in

combination with digital rectal examination (DRE) are

undergoing evaluation as screening modalities for

prostate cancer. Because of a lack of sensitivity and

spesificity markers are rarely of use in early diagnosis

of cancer. Also they can be used as monitoring disease

evaluation with therapy. The goal of future research

should be that development of the most specific, cheap

and easy markers for common cancer types as prostate

cancer.

P rostate spesific antigen: Screening prostate cancer

provides a dilemma unique among cancer sites. The

best strategy is determination of the ratio of the

prostate serum antigen (PSA) to the volume of the

prostate gland in prostate cancer diagnosis.

Determination of the free PSA (i.e., the percentage of

PSA that is unbound to serum proteins) has also been

suggested as a means of distinguishing malignancy

from benign hyperplasia. PSA has revolutionized the

management of prostate cancer since its development

in the 1980s. For unclear reasons, PSA derived from

malignant epithelial cells tends to bind more avidly to

serum proteins. Thus, in men with an elevated serum

PSA level, cancer is more likely to be present when the

percentage of free PSA is low. Because the relative

sensitivity versus specificity varies, depending on the

free PSA cutoff, the optimal cutoff value for free PSA

is still under debate. Prostate specific antigen (PSA)

represents the best serum marker for prostatic

carcinoma and is considered as most perfect tumor

marker available today. Nevertheless, the use of PSA

to detect prostate cancer is clinically imprecise since

benign and malignant prostate disease can cause

elevations in PSA. It is sensitive but spesificity is not

good to show tumor agressiveness, and so does not

benign prostatic hypertropy from invasive cancer.

Age-spesific cut-offs have been suggested to improve

spesificity, but there is still substantial overlap

between normals and those with cancer. Further

markers of tumor agressiviness, either measured in

serum or needle biopsy specimens are needed to

determine which patients are in need of curative

treatment.

Serum acid phosphatase: Serum acid phosphatase

(ACP) served as the only serum tumor marker for

prostate cancer between the 1930s and 1980s. more

sensitive serum tumor marker in detection of localized

disease and in monitoring response to therapy. In the

past two decades, the use of ACP has diminished

because of problems with lack of sensitivity and

specificity and because of the discovery of prostatespecific

antigen (PSA), a is an independent predictor

of biochemical recurrence in men who undergo

surgery. ACP level is independently predictive of

biochemical recurrence following radical retropubic

prostatectomy (RRP), when adjusted for other

predictive variables.

Granins: The nomenclature for chromogranin-A

continues to evolve; for simplicity,it is referred as

49


50

granin-A (GRN-A). GRN-A is a 49-kilodalton protein

that is produced exclusively by endocrine and

neurondocrine (NE) cells. It is costored and cosecreted

with the resident hormones of these cells, such as

catecholamines and calcitonin (CT). Although the

function of GRN-A is not known, it can serve as a

tissue and serum marker for a variety of endocrine

cells and tumors. There are several major cancer types

are characterized by NE differentiation. Recently, the

importance of NE differentiation and the attendant

expression of chromogranin-A has become

appreciated for prostate cancer. Clinical and basic

roles of chromogranin-A in human prostate cancer are

still studied. Although the function of GRN-A is not

known, several theories have emerged about its role:

(1) that it participates and perhaps regulates the storage

and secretion of its coresident hormones in secretory

vesicles; (2) that it inhibits proteolytic cleavage

enzymes; (3) that it binds calcium and thus regulates

the biologic effects of this ion; and (4) that it is a

precursor for peptides that have unique biologic effects

on the function and growth of its resident cells.

Function notwithstanding, the production of GRN-A in

NE prostate cancers has resulted in the availability of

a new serum and tissue marker for the tumors. The

clinical potential of GRN-A as a serum and tumor

marker in prostate cancer. It is now wellestablished

that GRN-A can be a marker for advanced disease.

More importantly, GRN-A may be a marker for early

and recurrent disease, even in the absence of abnormal

PSA. GRN-A serum levels may also have prognostic

significance, especially for androgen-independent

prostate cancer.

E-cadherin: In attempts to determine which cancers

of patients with clinically localized disease who

undergo radical prostatectomy will recur, the most

well-characterized and accepted predictors are model

equations that take into account preoperative serum

prostate-specific antigen (PSA), final Gleason score,

and final pathologic stage. Prediction of regression for

the individual patient using these statistical models,

however, is still not precise, and these models could

still be improved on. Thus, additional markers are

needed to more accurately target high-risk patients for

inclusion in clinical trials involving investigational

therapies for locally advanced prostate carcinoma.

Several other approaches show promise in this regard,

including nuclear morphometry, where the results have

been quite consistent. Other more controversial

markers include DNA ploidy and other biomarkers,

such as the amount of tumor angiogenesis, and

immunohistochemical levels of various markers,

including Ki-67, Bcl-2, p53, and E-cadherin. Ecadherin

as a biomarker to predict prognosis in

patients at risk of disease recurrence after radical

prostatectomy is warranted.

Serum total homocystein: Homocystein (Hcy) as a

tumor marker targets to reveal chemotherapy effects

on patients. It is largely derived from cellular

methionine, an essential amino acid drawn from

dietary intake. Intracellular homocysteine is normally

secreted extracellularly, at rapid rates. In the

circulating blood, the majority of the homocysteine

binds to albumin, forming a disulfide linkage.

Approximately 10% to 20% of the Hcy also exists as a

mixed disulfide with cysteine or with homocysteine

itself . Very little Hcy is present in the circulating

blood in a free reduced form (approximately

1%).Elevated serum tHcy (total homocysteine, free

and protein-bound) are detectable in patients with

malignant diseases. Finding increased circulating tHcy

in tumor cells may also be related to the so-called

‘‘methionine dependency’’ of many, but not all, tumor

cells. Many tumor cells are methionine dependent

because of their inability to convert homocysteine

(Hcy) to methionine by way of the remethylation

reaction. On the other hand, normal cells have no

problem obtaining methionine from homocysteine.

Folate is critical to the remethylation reaction. Any

folate deficiency will result in the impairment of

function of the remethylation reaction, causing

accumulation of Hcy. Therefore, it was generally

believed that the rapid proliferation rate of tumor cells,

such as in prostate cancer and in the so-called

methionine dependency of tumor cells, was due to the

depletion of folate by the rapid growing tumor cells

and changing levels of fLV (a form of folate) in 24 h

after therapy. In other words, with a better

understanding of the effects of various drugs, the rise

and fall of circulating tHcy could be used as a new

tumor marker to monitor cancer patients during

therapy, complementing commonly used tumor

markers. The general impression that elevated tHcy is

detectable in cancer patients derives from the fact that

many cancer patients take anti-folate drugs such as

methotrexate. It is important to know that the level of

tHcy reflects the tumor cell proliferation rate.

Regardless of the folate status, it is very likely given

our results and others that the rapid proliferation of

tumor cells is one of the major reasons that elevated


circulating tHcy can be detected in cancer patients.

Conceivably, circulating tHcy could very well be used

as a marker to monitor cancer patients during therapy,

complementing the currently used tumor markers.

Choline kinase: Choline kinase (ChoK) is the first

enzyme in the Kennedy pathway, responsible for the

de novo synthesis of phosphatidylcholine (PC), one of

the basic lipid components of membranes. ChoK is

responsible of the generation of phosphorylcholine

(PCho) from its precursor, choline. Both ChoK and its

product, PCho, have been recently reported as

essential molecules in cell proliferation and

transformation. Generation of Pcho from ChoK

activity has been described as an essential event in

growth factor-induced mitogenesis in fibroblasts and

has been found to cooperate with several mitogens.

Furthermore, overexpression of several oncogenes

induces increased levels of ChoK and the intracellular

levels of PCho. A strong correlation can be established

between ChoK activity and cancer onset at least in

some human tumors. Additional evidence gives

support for a role of ChoK in the generation of human

tumors, since studies using nuclear magnetic

resonance (NMR) techniques have demonstrated

elevated levels of PCho in human tumoral tissues with

respect to the normal ones, including breast, prostate

carcinomas. ChoK is overexpressed with high

incidence in both, tumor- derived cell lines and

tumoral tissues, these results indicate the putative use

of ChoK as a tumor marker, potentially useful in

diagnosis and screening of the progression of tumors.

The recent findings show that overexpression of

the polycomb group transcriptional represor enhancer

zeste Gene (EZH2) in prostate cancer raises the

possibility that transcriptional regulation at the

chromatin level play a role in the development of the

metabolic phenotype and suggest new exploration

prespective on patient stratification, therapeutics and a

tumor marker identity.

Also proliferation markers in biopsies such as K67,

expression levels of mRNA and/or proteins for bcl2,

p53, p27 etc. and molecular changes in tumor

supressor genes such as PTEN or mutations in genes or

mutations in genes can be candidate markers. This is

urgently needed since radical surgery carries a high

morbidity leading to impotence and/or incontinence.

Serdar Ar›san

fiiflli Etfal State Hospital

1. Urology Clinics, fiiflli, ‹stanbul

51


Book reviews

Çetin ALGÜNEfi, Radyasyon Biyofizi¤i, Trakya

Üniversitesi Yay›nlar›, Edirne, 135 sayfa, ISBN: 975-

374-051-4, 2002.

Kitapta atom ve çekirde¤inin yap›s›, karars›z

çekirdekler, iyonizan radyasyon tipleri ve özellikleri,

radyasyonun madde ile etkileflmesi ve radyasyon

birimleri, iyonizasyona sebep olmayan radyasyonlar,

iyonizan radyasyonlar›n biyolojik etkileri ve

radyasyondan korunma konular› tart›fl›lm›flt›r.

Bölümlerin ayr›nt›l› incelenmesinde, kitab›n

diziliminin geleneksel tarzda, flekil ve tablolar›n

geçtikleri yerlerde metin aras›nda verildi¤i

görülmektedir. Bütün konular, aç›klay›c› flekil, tablo ve

örneklerle desteklenmifltir.

Bu kitab›n radyasyon biyofizi¤i konusunda önemli

bilgiler kazand›rmas› aç›s›ndan çok yararl› bir rehber

olaca¤› kan›s›nday›m. Ayr›ca Türkiye’de bu alanlarda

kaynak oluflturacak Türkçe eserlerin say›lar› da son

derece s›n›rl› oldu¤u için, bu kitab› biyologlara,

radyobiyologlara, fizikçilere, radyasyon etkileri ile

ilgilenen ziraatç› ve veterinerlere öneririm.

Atilla ÖZALPAN

Haliç Üniversitesi,

Moleküler Biyoloji ve Genetik Bölümü

Çetin ALGÜNEfi, Radiation Biophysics, Published

by Trakya University, Edirne, 135 pp, ISBN: 975-374-

051-4, 2002.

In the book, atomic and nuclear structure, unstable

nuclei, types and properties of ionizing radiations,

interaction of radiation with matter, radiation units,

non-ionizing radiations, biological effects of ionizing

radiations and radiation protection are discussed.

In detail, the layout of the book has a traditional

format in that figures and tables have been integrated

into the text at appropriate places. All statement are

supported with a plenty of explenatory figures, tables

and examples.

53

The book is a valuable guide of radiation

biophysics. On the other hand, this book is a good

document because there are very limited Turkish

publication in this area. For this reason, I

recommended this book for the biologists, physisists,

agriculturists and veterinarians who apply radiation on

living organisms for several purposes.

Atilla ÖZALPAN

Haliç University,

Department of Molecular Biology and Genetics


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Tables and Figures

1. Each table should be typed on a separate sheet,

numbered with Arabic numerals and accompanied

by a short instructive title line plus an explanatory

caption at the top. Indicate footnotes within tables


56

by superscript small letters and type footnotes

below the table. Each table must be referred to in

the text.

2. Fine drawings can either submitted as original

drawings ready for print or as clean and high

contrast glossy black and white photographs.

3. Photographs must be supplied as black and white,

high contrast, glossy prints, trimmed at right angles.

4. Captions for figures should be typed

double-spaced, on a separate sheet. Each caption

should be identified as Figure 1 etc. and be

complete, clean and concise, so that each figure

and its caption could be understood without

reference to the text. Do not give magnification on

scales in the figure titles; instead draw bar scales

directly on the figures.

5. Each illustration should have the title of the paper

and the figure number written on the back in soft

pencil. The top of the figure should also be

indicated on the back.

6. The approximate position of the tables and figures

should be indicated in the margin of the manuscript.

Units, abbreviations and scientific names

1. Only SI units should be used. Current abbreviations

can be used without explanation. Other must be

explained. In case of doubt always give an

explanation.

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

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 2, No. 1, 2003

Dedication

Review articles

Polyamines in plants: An overview

Bitkilerde poliaminler: Genel bir bak›fl

R. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston 1-12

Phenolic cycle in plants and environment

Bitkilerde fenolik döngü ve çevre

V. I. Kefeli, M. V. Kalevitch, B. Borsari 13-18

Research papers

The short-term effects of single toxic dose of citric acid in mice

Farelerde sitrik asidin tek toksik dozunun k›sa süreli etkileri

T. Aktaç, A. Kabo¤lu, E. Bakar, H. Karakafl 19-23

Characterisation of RPP7 mutant lines of the col-5 ecotype of Arabidopsis thaliana

Arabidopsis thaliana’n›n col-5 ekotipinden elde edilen mutant hatlardan RPP7

geninin karakterizasyonu

C. Can, M. Özaslan, E. B. Holub 25-30

The effect of meta-topolin on protein profile in radish cotyledons

Meta-topolinin turp kotiledonlar›nda protein profiline etkisi

S. Ça¤, N. Palavan-Ünsal 31-34

The effect of electromagnetic fields on oxidative DNA damage

Elektromanyetik alan›n oksidatif DNA hasar› üzerindeki etkisi

S. ‹fller, G. Erdem 35-38

Chromosomes of a balanced translocation case evaluated with atomic force microscopy

Dengeli translokasyon vakas›nda kromozomlar›n atomik güç mikroskobu ile

de¤erlendirilmesi

Z. Y›lmaz, M. A. Ergun, E. Tan 39-42

Effect of epirubicin on mitotic index in cultured L-cells

Epirubisinin kültürdeki L-hücrelerinde mitotik indekse etkisi

G. Özcan Ar›can, M. Topçul 43-48

Letter to editor 49-51

Book reviews 53

Instructions to authors 55-56

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