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Journal of Cell andMolecular Biology

VOLUME 2 • NO. 1 • 2003 • ISSN 1303-3646

HAL‹Ç UNIVERSITYFACULTY OF ARTS AND SCIENCES

‹STANBUL1998

JJoouurrnnaall ooff CCeellll aanndd MMoolleeccuullaarr BBiioollooggyy

PPuubblliisshheedd bbyy HHaalliiçç UUnniivveerrssiittyy

FFaaccuullttyy ooff AArrttss aanndd SScciieenncceess

EEddiittoorr

Atilla ÖZALPAN

AAssssoocciiaattee EEddiittoorr

Narç›n PALAVAN ÜNSAL

EEddiittoorriiaall BBooaarrdd

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

AAddvviissoorryy BBooaarrdd

Igor ALEXANDROV, Dubna, RussiaÇetin ALGÜNEfi, Edirne, TurkeyAglaia ATHANASSIADOU, Patros, Greecefiehnaz BOLKENT, ‹stanbul, TurkeyNihat BOZCUK, Ankara, Turkey‹smail ÇAKMAK, ‹stanbul, TurkeyAdile ÇEV‹KBAfi, ‹stanbul, TurkeyBeyaz›t ÇIRAKO⁄LU, ‹stanbul, TurkeyAyfl›n ÇOTUK, ‹stanbul, TurkeyZihni DEM‹RBA⁄, Trabzon, TurkeyMustafa DJAMGOZ, London, UKAglika EDREVA, Sofia, Bulgaria

Ünal EGEL‹, Bursa, TurkeyCandan JOHANSEN, ‹stanbul, TurkeyAs›m KADIO⁄LU, Trabzon, TurkeyValentine KEFEL‹, Pennsylvania, USAGöksel OLGUN, Edirne, TurkeyU¤ur ÖZBEK, ‹stanbul, TurkeyZekiye SULUDERE, Ankara, Turkey‹smail TÜRKAN, ‹zmir, TurkeyMehmet TOPAKTAfi, Adana, TurkeyMeral ÜNAL, ‹stanbul, TurkeyMustafa YAT‹N, Boston, USAZiya Z‹YLAN, ‹stanbul, Turkey

Haliç UniversityFaculty of Arts and Sciences


FFoouunnddeerrProf. Dr. Gündüz GED‹KO⁄LU

President of Board of Trustee

RRiigghhttss hheelldd bbyyProf. Dr. Ahmet YÜKSEL

Rector

Correspondence Address:TThhee EEddiittoorriiaall OOffffiiccee

JJoouurrnnaall ooff CCeellll aanndd MMoolleeccuullaarr BBiioollooggyyHaliç Üniversitesi, Fen-Edebiyat Fakültesi,

Ahmet Vefik Pafla Cad., No: 1, 34280,F›nd›kzade, ‹stanbul-Turkey

Phone: 90 212 530 50 24Fax: 90 212 530 35 35

E-mail: [email protected]

JJoouurrnnaall ooff CCeellll aanndd MMoolleeccuullaarr BBiioollooggyy iissiinnddeexxeedd iinn EEBBSSCCOO ddaattaabbaassee..

Summaries of all articles in this journal areavailable free of charge from www.halic.edu.tr

ISSN 1303-3646

printed at yaflar printing house


VVoolluummee 22//22000033


‹stanbul-TURKEY


PPuubblliisshheedd bbyy HHaalliiçç UUnniivveerrssiittyy

FFaaccuullttyy ooff AArrttss aanndd SScciieenncceess

EEddiittoorr

Atilla ÖZALPAN

AAssssoocciiaattee EEddiittoorr

Narç›n PALAVAN ÜNSAL

EEddiittoorriiaall BBooaarrdd

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

AAddvviissoorryy BBooaarrdd

Igor ALEXANDROV, Dubna, RussiaÇetin ALGÜNEfi, Edirne, TurkeyAglaia ATHANASSIADOU, Patros, Greecefiehnaz BOLKENT, ‹stanbul, TurkeyNihat BOZCUK, Ankara, Turkey‹smail ÇAKMAK, ‹stanbul, TurkeyAdile ÇEV‹KBAfi, ‹stanbul, TurkeyBeyaz›t ÇIRAKO⁄LU, ‹stanbul, TurkeyAyfl›n ÇOTUK, ‹stanbul, TurkeyZihni DEM‹RBA⁄, Trabzon, TurkeyMustafa DJAMGOZ, London, UKAglika EDREVA, Sofia, Bulgaria

Ünal EGEL‹, Bursa, TurkeyCandan JOHANSEN, ‹stanbul, TurkeyAs›m KADIO⁄LU, Trabzon, TurkeyValentine KEFEL‹, Pennsylvania, USAGöksel OLGUN, Edirne, TurkeyU¤ur ÖZBEK, ‹stanbul, TurkeyZekiye SULUDERE, Ankara, Turkey‹smail TÜRKAN, ‹zmir, TurkeyMehmet TOPAKTAfi, Adana, TurkeyMeral ÜNAL, ‹stanbul, TurkeyMustafa YAT‹N, Boston, USAZiya Z‹YLAN, ‹stanbul, Turkey



FFoouunnddeerrProf. Dr. Gündüz GED‹KO⁄LU

President of Board of Trustee

RRiigghhttss hheelldd bbyyProf. Dr. Ahmet YÜKSEL

Rector

Correspondence Address:TThhee EEddiittoorriiaall OOffffiiccee

JJoouurrnnaall ooff CCeellll aanndd MMoolleeccuullaarr BBiioollooggyyHaliç Üniversitesi, Fen-Edebiyat Fakültesi,

Ahmet Vefik Pafla Cad., No: 1, 34280,F›nd›kzade, ‹stanbul-Turkey

Phone: 90 212 530 50 24Fax: 90 212 530 35 35

E-mail: [email protected]

JJoouurrnnaall ooff CCeellll aanndd MMoolleeccuullaarr BBiioollooggyy iissiinnddeexxeedd iinn EEBBSSCCOO ddaattaabbaassee..

Summaries of all articles in this journal areavailable free of charge from www.halic.edu.tr

ISSN 1303-3646

printed at yaflar printing house


CCOONNTTEENNTTSS VVoolluummee 22,, NNoo..11,, 22000033

DDeeddiiccaattiioonn

RReevviieeww aarrttiicclleess

PPoollyyaammiinneess iinn ppllaannttss:: AAnn oovveerrvviieewwBitkilerde poliaminler: Genel bir bak›flR. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston 1-12

PPhheennoolliicc ccyyccllee iinn ppllaannttss aanndd eennvviirroonnmmeennttBitkilerde fenolik döngü ve çevreV. I. Kefeli, M. V. Kalevitch, B. Borsari 13-18

RReesseeaarrcchh ppaappeerrss

TThhee sshhoorrtt--tteerrmm eeffffeeccttss ooff ssiinnggllee ttooxxiicc ddoossee ooff cciittrriicc aacciidd iinn mmiicceeFarelerde sitrik asidin tek toksik dozunun k›sa süreli etkileriT. Aktaç, A. Kabo¤lu, E. Bakar, H. Karakafl 19-23

CChhaarraacctteerriissaattiioonn ooff RRPPPP77 mmuuttaanntt lliinneess ooff tthhee ccooll--55 eeccoottyyppee ooff AArraabbiiddooppssiiss tthhaalliiaannaaArabidopsis thaliana’n›n col-5 ekotipinden elde edilen mutant hatlardan RPP7geninin karakterizasyonuC. Can, M. Özaslan, E. B. Holub 25-30

TThhee eeffffeecctt ooff mmeettaa--ttooppoolliinn oonn pprrootteeiinn pprrooffiillee iinn rraaddiisshh ccoottyylleeddoonnssMeta-topolinin turp kotiledonlar›nda protein profiline etkisiS. Ça¤, N. Palavan-Ünsal 31-34

TThhee eeffffeecctt ooff eelleeccttrroommaaggnneettiicc ffiieellddss oonn ooxxiiddaattiivvee DDNNAA ddaammaaggeeElektromanyetik alan›n oksidatif DNA hasar› üzerindeki etkisiS. ‹fller, G. Erdem 35-38

CChhrroommoossoommeess ooff aa bbaallaanncceedd ttrraannssllooccaattiioonn ccaassee eevvaalluuaatteedd wwiitthh aattoommiicc ffoorrcceemmiiccrroossccooppyyDengeli translokasyon vakas›nda kromozomlar›n atomik güç mikroskobu ilede¤erlendirilmesiZ. Y›lmaz, M. A. Ergun, E. Tan 39-42

EEffffeecctt ooff eeppiirruubbiicciinn oonn mmiittoottiicc iinnddeexx iinn ccuullttuurreedd LL--cceellllssEpirubisinin kültürdeki L-hücrelerinde mitotik indekse etkisiG. Özcan Ar›can, M. Topçul 43-48

LLeetttteerr ttoo eeddiittoorr 49-51

BBooookk rreevviieewwss 53

IInnssttrruuccttiioonnss ttoo aauutthhoorrss 55-56

This issue is dedicated to

PP rrooff.. DDrr.. AArrtthhuurr WW.. GGaallssttoonn

for his invaluable contribution to plant biology

HHoonnoorrss:: Elected to Phi Beta Kappa; Phi Kappa Phi; Sigma Xi; American Academy of Arts and Sciences, NationalSigma Xi Lecturer, 1966; National Phi Beta Kappa Visiting Scholar, 1972-1973; Award of the New York Academyof 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.

EExxppeerriieennccee:: Plant Physiologist, Emergency Rubber Project, California Institute of Technology 1943-1944; Instuctorin Botany, Yale University, 1946-1947; Senior Research California Institute of Technology, 1947-1950; AssociateProfessor of Biology, California Institute of Technology, 1951-1955. Professor of Plant Physiology, Department ofBotany, Yale University 1955-1961; Chairman, Department of Botany, 1961-1962; Director, Division of BiologicalSciences, 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; FulbrightFellow, Canberra, Australia, 1960-1961; National Science Foundation Faculty Fellow, London 1967-1968; AlbertEinstein 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 andMedicine, National Research Council; Member Life Sciences Advisory Committee, NASA; also Long RangeStrategic Planning Committee in Life Sciences Advisory Committee, NASA; Member, NASA Disciplinary WorkingGroup for CELLS (Controlled Ecological Life Support Sytem).

PP rreesseenntt ooff ppaasstt EEddiittoorriiaall BBooaarrdd MMeemmbbeerr:: 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.

FFoorrmmeerr MMeemmbbeerr:: Metabolic and Regulatory Biology Panels, National Science Foundation; Executive Committee,Growth Society; Life Science Advisory Committee, NASA; and Governing Boards, Biological Sciences CurriculumStudy, Commission on Undergraduate Education in the Biological Sciences and AIBS.First American scientist to visit the People’s Rebuplic of China, 1971.

BBooookkss:: ‘Principles of Plant Physiology’ (with J. Bonner), Freeman, 1952. ‘Life of the Green Plant’, Prentice Hall,1961, 2nd Ed., 1964, 3rd Ed., 1980 (with P. J. Davies and R. L. Satter). ‘Control Mechanisms in Plant Development’, (withP. J. Davies), Prentice Hall, 1970. ‘Daily Life in People’s China’, Crowell, 1973; Simon and Schuster, 1975. ‘GreenWisdom’ Basic Books, Inc. NY, 1981; Putnam, 1983. ‘Life Processes in Plants’, Freeman (Scientific AmericanLibrary), 1994. ‘New Dimensions in Bioethics’, Arthur W. Galston and Emily G. Shurr, eds. Kluwer AcademicPublishers, Boston/Dordrecht/London, 2001.

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

BBoorrnn:: April 21, 1920 Eaton Professor of Botany, Emeritus, Department of Molecular, Cellular and Developmental Biology,

EEdduuccaattiioonn:: 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 [email protected]

AArrtthhuurr WW.. GGaallssttoonn,, CCuurrrriiccuulluumm VViittaaee

Journal of Cell and Molecular Biology 22: 1-12, 2003.Haliç University, Printed in Turkey.

1

PPoollyyaammiinneess iinn ppllaannttss:: AAnn oovveerrvviieeww

Ravindar Kaur-Sawhney1*, Antonio F. Tiburcio2, Teresa Altabella2, and Arthur W. Galston1

1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT,06520-8103, USA; 2Laboratori de Fisiologia Vegetal, Facultad de Farmacia, Universitat de Barcelona,Spain (* author for correspondence)

Received 21 October 2002; Accepted 10 November 2002

AAbbssttrraacctt

This article presents an overview of the role of polyamines (PAs) in plant growth and developmental processes. ThePAs, putrescine, spermidine and spermine are low molecular weight cations present in all living organisms. PAs andtheir biosynthetic enzymes have been implicated in a wide range of metabolic processes in plants, ranging from celldivision and organogenesis to protection against stress. Because the PA pathway has now been molecularly andbiochemically elucidated, it is amenable to modulation by genetic approaches. Genes for several key biosyntheticenzymes namely, arginine decarboxylase, ornithine decarboxylase and S-adenosyl methionine decarboxylase havebeen cloned from different plant species, and antibodies to some genes are now available. Both over-expressed andantisense transgenic approaches to PA biosynthetic genes have provided further evidence that PAs are required forplant growth and development. However, molecular mechanisms underlying PA effects on these processes remainunclear. Analysis of gene expression by using DNA microarray genomic techniques should help determine the preciserole of these compounds. The potential of proteomics to unravel the role of PAs in particular cellular processes hasalso been examined. The extensive use of the two-hybrid system and other proteomic approaches will provide newinsights into the role of PAs in signal transduction. Furthermore, there is evidence that proteomics provides anexcellent tool for determining supramolecular organizations of PA metabolic enzymes which may help inunderstanding homeostatic control of this metabolic pathway.

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

BBiittkkiilleerrddee ppoolliiaammiinnlleerr:: GGeenneell bbiirr bbaakk››flfl

ÖÖzzeett

Bu makalede poliaminlerin (PA) bitki büyüme ve geliflme olaylar›ndaki rolüne genel bir bak›fl yap›lmaktad›r. PA lerputresin, spermidin ve spermin, düflük molekül a¤›rl›kl› ve tüm canl› organizmalarda mevcut olan maddelerdir. PAlerin ve bunlar›n biyosentetik enzimlerinin bitkileri strese karfl› korumaya yönelik olarak hücre bölünmesindenorganogeneze kadar de¤iflen genifl bir metabolik olaylar zincirinde yer ald›¤› ortaya konmufltur. Günümüzde PA yolumoleküler ve biyokimyasal yönden aç›kl›¤a kavufltu¤u için genetik yaklafl›mlarla düzenlenmeye uygundur. Çeflitlianahtar biyosentez enzimleri, arginin dekarboksilaz, ornitin dekarboksilaz ve S-adenozil metiyonin dekarboksilaz›ngenleri farkl› bitki türlerinde klonlanm›flt›r ve günümüzde baz› genlerin antikorlar›n› elde etmek mümkündür. PAbiyosentezi genlerine hem over-ekspres ve hem de antisens transgenik yaklafl›mlar PA lerin bitki büyüme geliflmesiiçin 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 bubilefliklerin rollerini kesin olarak belirlemeye yard›mc› olacakt›r. PA lerin özellikle hücresel olaylardaki rolünü ortayakoymaya yönelik olarak proteomi¤in potansiyeli de araflt›r›lm›flt›r. ‹ki-hibrit sistemi ve di¤er proteomik yaklafl›mlar›nyo¤un kullan›m›, PA lerin sinyal iletimindeki rolüne yeni bir bak›fl aç›s› getirecektir. Bundan baflka proteomi¤in, PAmetabolik yolunun homeostatik kontrolünü anlamaya yard›mc› olabilecek, PA metabolizma enzimlerininsupramoleküler organizasyonunun belirlenmesinde çok önemli bir araç oldu¤u konusunda veriler mevcuttur.

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

11.. IInnttrroodduuccttiioonn

Polyamines (PAs) are low molecular weightpolycations found in all living organisms (Cohen,1998). They are known to be essential for growth anddevelopment in prokaryotes and eukaryotes (Tabor andTabor, 1984; Tiburcio et al., 1990). In plant cells, thediamine putrescine (Put), triamine spermidine (Spd)and tetramine spermine (Spm) constitute the majorPAs. They occur in the free form or as conjugatesbound to phenolic acids and other low molecularweight compounds or to macromolecules such asproteins and nucleic acids. As such, they stimulateDNA replication, transcription and translation. Theyhave been implicated in a wide range of biologicalprocesses in plant growth and development, includingsenescence, environmental stress and infection byfungi and viruses. Their biological activity is attributedto their cationic nature. These findings have beendiscussed in several recent review articles (Tiburcio etal., 1993; Galston et al.,1997; Bais and Ravishankar,2002).

The use of PA biosynthesis inhibitors has shown acausal relationship between changes in endogenous PAlevels and growth responses in plants. Theseobservations led to further studies into undestandingthe mode of PA action. Some of the importantobservations suggest that PAs can act by stabilizingmembranes, scavenging free radicals, affecting nucleicacids and protein synthesis, RNAse, protease and otherenzyme activities, and interacting with hormones,phytochrome, and ethylene biosynthesis (reviewed inSlocum et al., 1984; Galston and Tiburcio, 1991).Because of these numerous biological interactions ofPAs in plant systems, it has been difficult to determinetheir precise role in plant growth and development.

In recent years, however, investigations intomolecular genetics of plant PAs have led to isolation ofa number of genes encoding PA biosynthetic enzymesand development of antibodies to some of the genes.Furthermore mutants and transgenic plants with alteredPA metabolism have also been developed. Genomicand proteomic approaches are being used to furthergain an understanding into the role of PAs in plantdevelopmental processes. These findings will hopefullylead to a better understanding of their specific functionsin plants. Several useful reviews on these aspects havebeen 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 PAsin plants with particular emphasis on recentinvestigations using molecular and geneticapproaches.

22.. PPoollyyaammiinnee bbiioossyynntthheessiiss

The PA biosynthetic pathway in plants has beenthoroughly investigated and reviewed in detail (Evansand Malmberg, 1989; Tiburcio et al., 1990; Slocum,1991a; Martin-Tanguy, 2001). Briefly, PAs aresynthesized from arginine and ornithine by argininedecarboxylase (ADC) and ornithine decarboxylase(ODC) as illustrated in Figure 1. The intermediateagmatine, synthesized from arginine, is converted toPut, which is further transformed to Spd and Spm bysuccessive transfers of aminopropyl groups fromdecarboxylated S-adenosylmethionine (dSAM)catalysed by specific Spd and Spm synthases. Theaminopropyl groups are derived from methionine,which is first converted to S-adenosylmethionine(SAM), and then decarboxylated in a reactioncatalyzed by SAM decarboxylase (SAMDC). Theresulting decarboxylated SAM is utilized as anaminopropyl donor. SAM is a common precursor forboth PAs and ethylene, and SAMDC regulates bothbiosynthetic pathways as illustrated in Figure 1.

A number of investigators have used PA inhibitorsto modulate the cellular PA titer in order to determinetheir role in various plant processes. Four commonlyused inhibitors of PA synthesis are: 1.Difluoromethylornithine (DFMO), an irreversibleinhibitor of ODC (reviewed in Bey et al., 1987); 2.Difluoromethylarginine (DFMA), an irreversibleinhibitor of ADC (Bitonti et al., 1987); 3. Methyl-glyoxyl-bis guanylhydrazone (MGBG), a competitiveinhibitor of S-adenosyl-methionine decarboxylase(SAMDC) (Williams-Ashman and Schenone,1972);and 4. Cyclohexylamine (CHA), a competitiveinhibitor of spermidine synthase (Hibasami et al.,1980). Common oxidases are diamine oxidase andpolyamine oxidase (PAO), as reviewed by Smith andMarshall (1988). Each PA has been found to becatabolized by a specific oxidase.

Several investigations have dealt with localizationof PAs and their biosynthetic enzymes in plants(reviewed by Slocum, 1991b). However, paucity ofinformation regarding the exact cellular andsubcellular localization of these entities remains one of

2 Ravindar Kaur-Sawhney et al.

the obstacles in understanding their biological role.Recent studies have shown that PAs are present in thecell wall fractions, vacuole, mitochondria andchloroplasts (Torrigiani et al., 1986; Slocum, 1991b;Tiburcio et al., 1997). The biosynthetic enzymes,ODC, SAMDC, and Spd synthase have been reportedto be localized in the cytoplasm, whereas ADC islocalized in the thylakoid membrane of chloroplast(Borrell et al., 1996; Tiburcio et al., 1997) and PAO inthe cell wall (Kaur-Sawhney et al., 1981). ODCactivity has also been observed in the nucleus(Slocum, 1991b). However, these findings have to beinterpreted with caution because various proceduralproblems can mask the results. Despite these advancesin understanding the metabolic processes involvingPAs and their localization in plant cells, the preciserole of PAs in plant morphogenesis remains elusive.

33.. PPoollyyaammiinneess iinn ppllaanntt ggrroowwtthh aanndd ddeevveellooppmmeenntt

The availability of specific inhibitors of PAbiosynthesis has helped in investigating themechanisms involved in PA interactions to some extent,providing a partial understanding of their physiologicalrole in plant growth and development. Clearly, PAs areinvolved in many plant developmental processes,including cell division, embryogenesis, reproductiveorgan development, root growth, tuberization, floralinitiation and development, fruit development andripening as well as leaf senescence and abiotic stresses(reviewed by Evans and Malmberg, 1989; Galston etal., 1997; Bais and Ravishankar, 2002; Tiburcio et al.,2002). Changes in free and conjugated PAs and theirbiosynthetic enzymes, namely ADC, ODC, andSAMDC have been found to occur during thesedevelopmental processes. Earlier experiments hadshown that increases in PAs and their biosyntheticenzymes are associated with rapid cell division in manyplant systems e.g., carrot embryogenesis (Montague

Polyamines in plants 3

Methionine

S - adenosylmethionine

ACC synthaseAVG

ACC oxidase

ACC

Ethylene

MGBG

SAMDC

dSAM

DFMODFMA

ADCODC

Ornithine Arginine

Agmatine

Putrescine

Spermidine

Spermine

Spmsynthase

Spdsynthase

FFiigguurree 11:: Polyamine biosynthetic pathway and its linkage to ethylene biosynthetis. Biosynthetic enzymes are ADC, ODC andSAMDC and the inhibitor DFMA, DFMO and MGBG.

and Koppenbrink, 1978; Feirer et al., 1984), tomatoovaries (Heimer and Mizrahi, 1982), tobacco ovaries(Slocum and Galston, 1985), and fruit development(reviewed in Kakkar and Rai, 1993). Similar resultshave been reported for many other plant species(reviewed in Bais and Ravishankar, 2002). In contrast,several other studies have suggested that correlationsbetween PAs and their biosynthetic enzymes and plantgrowth processes, especially somatic embryogenesis,are not universal and may be species specific (reviewedin Evans and Malmberg, 1989; Galston et al., 1997;Bais and Ravishankar, 2002).

In general, cells undergoing division contain highlevels of free PAs synthesized via ODC, and cellsundergoing expansion and elongation contain lowlevels of free PAs synthesized via ADC (see review byGalston and Kaur-Sawhney, 1995). High levels ofendogenous PAs and their conjugates have also beenfound in apical shoots and meristems prior toflowering (Cabbane et al., 1981) and flower parts ofmany plants (Martin-Tanguy, 1985). Our experimentsusing callus cultures derived from thin layer explantsof pedicels from tobacco inflorescence show thatendogenous Spd increased more rapidly than otherPAs in floral buds than in vegetative buds. Addition ofCHA, an inhibitor of Spd synthesis, to the culturemedium reduced flower formation in a dose dependentmanner and such inhibition was correlated with aswitch to initiation of vegetative instead of flowerbuds. This inhibition was reversed by the addition ofexogenous Spd (Kaur-Sawhney et al., 1988). Morerecently, we have found that higher levels ofendogenous PAs occur in flowers and siliques whencompared with their levels in leaves and bolts ofcertain strains of Arabidopsis. Addition of the PAbiosynthetic inhibitors, DFMA and CHA to the culturemedium, at time of seed germination, inhibited boltingand flower formation and this was partially reversedby addition of exogenous Spd (Applewhite et al.,2000). These results clearly show that Spd is involvedin flower initiation and development. Similar resultshave been reported in other plants also (reviewed byGalston et al.,1997; Bais and Ravishankar, 2002).

Many plant growth and development processesknown to be regulated by plant hormones, such asauxins, 2,4-D, GA and ethylene, have also beencorrelated with changes in PA metabolism. Thesechanges occur in both endogenous levels of PAs andtheir biosynthetic enzymes and appear to be tissuespecific (reviewed by Galston and Kaur-

Sawhney,1995). Thus, PAs which may or may not bemobile in plants (Young and Galston, 1983; Bagni andPistocchi, 1991) can serve as intracellular mediators ofhormone actions (Galston and Kaur-Sawhney, 1995).Supporting evidence for this hypothesis has beenobtained in experiments using specific inhibitors of PAbiosynthesis (Bagni et al., 1981; Egea-Cortines andMizrahi, 1991; reviewed in Galston et al., 1997; Baisand Ravishankar, 2002).

Of the major plant hormones, ethylene has beenmost intensively investigated with respect to PAmetabolism. The two metabolites, PAs and ethylene,play antagonistic roles in plant processes. While PAsinhibit senescence of leaves (Kaur-Sawhney et al.,1982), cell cultures of many monocot and dicot species(Muhitch et al., 1983) and fruit ripening (Kakkar andRai, 1993), ethylene promotes these processes. Themost commonly held view is that PAs and ethyleneregulate each other’s synthesis, either directly orthrough metabolic competition for SAM, a commonprecursor for their biosynthesis (Figure 1). PAs inhibitethylene biosynthesis, perhaps by blocking theconversion 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, keyenzymes in PA biosynthetic pathway (Apelbaum et al.,1985; Icekson et al., 1985). Thus, PAs may affectsenescence and fruit ripening by modulating PA andethylene biosynthesis.

Apparently, PAs are essential members of an arrayof internal metabolites required in many plantdevelopmental processes, but their precise role in theseprocesses has yet to be established. Whereas, specificPAs at specific concentrations may be required atcritical stages of growth and morphogenetic events, nodefinitive data are available to establish their role asplant hormones.

44.. MMaanniippuullaattiioonn ooff tthhee ppoollyyaammiinnee ppaatthhwwaayy

The PA pathway is ubiquitous in living organisms andis relatively short (see Section 2) in terms of thenumber of enzymes involved. Most of the genescoding for enzymes involved in the pathway have beencloned from different sources (Kumar et al., 1997;Walden et al., 1997; Galston et al., 1997; Tiburcio etal., 1997; Malmberg et al., 1998; Kumar and Minocha,1998; Panicot et al., 2002b). Thus, the PA pathway


represents an excellent model to test varioushypotheses and to answer fundamental biologicalquestions derived from pathway manipulation (Thu-Hang et al., 2002; Bhatnagar et al., 2002).Initially, approaches to manipulate the PA pathwaymade use of suicide inhibitors, but the effects ofDFMO and DFMA on ODC and ADC respectively, arevariable in different plant systems, ranging frominhibition to stimulation or no effect and depending onthe concentration, plant system tested and theexistence of compensatory mechanisms (Slocum andGalston, 1987). Therefore, alternative approaches tomanipulate polyamine metabolism have beendeveloped during the recent years.

4.1. Mutants

Mutants deficient in PA biosynthesis have beenisolated from several biological systems. Hafner et al.(1979) isolated PA mutants in Escherichia colishowing decreased growth and increased sensitivity toparaquat (Milton et al., 1990). Yeast mutantspresenting ODC as the sole pathway, show reducedgrowth and altered sporulation on PA deficientmedium (Cohn et al., 1980; Whitney and Morris,1978). Chinese hamster ovary cells lacking ODCactivity do not grow in medium lacking PA (Steglichand Scheffler, 1983) and a moderately reduced broodsize was observed in a Caenorhabditis elegans ODCdeletion mutant (Macrae et al., 1995). Mutations ingenes affecting Spd and Spm biosynthesis have alsobeen isolated in yeast. The spe3 Spd synthase mutationcauses a growth arrest, which can be complementedwith externally added Spd (Hamasaki-Katagiri et al.,1997), while the yeast spe4 mutant is defective in Spmbiosynthesis (Hamasaki-Katagiri et al., 1998).

Less is known about mutants affecting PAmetabolism in plants. Mutants with high levels ofADC activity have been identified in petunia becauseof their abnormal morphology (Geerats et al., 1988),but the basis of the mutation is still not known.Screening for resistance to the SAMDC inhibitorMGBG (Malmberg and Rose, 1987) or to inhibitoryconcentrations of Spm (Mirza et al., 1997), yieldedmutants that showed reduced sensitivity to therespective agent, but these mutants have not beenfurther exploited for the analysis of PA function.Watson et al. (1998) isolated EMS mutants of A.thaliana that are reduced in ADC activity. The mutantsfall into two complementation groups, spe1 and spe2,

which may correspond to the two gene copiesencoding ADC, ADC1 and ADC2 (Watson et al.,1998). The mutations have not been mapped andtherefore it cannot be excluded that other functions,i.e. regulatory elements, are affected (Soyka andHeyer, 1999). More recently, Hanzawa et al. (2000)reported that the inactivation of the ArabidopsisACAULIS5 (ACL5) gene causes a defect in theelongation of stem internodes by reducing cellexpansion. It was suggested that ACL5 encodes a Spmsynthase, but the possibility that ACL5 may exhibitbroad amine substrate specificities and be involved inthe synthesis of other polyamines could not beexcluded (Hanzawa et al., 2000).

Thus far the only well characterized plantpolyamine biosynthetic mutant has been generated byusing reverse genetics. The availability of mutantcollections generated either by transposon or T-DNAtagging now facilitates the identification of knockoutsin any gene of interest using PCR-based mutantscreening techniques (Ferrando et al., 2002). By usingthese techniques, Soyka and Heyer (2000) isolated anArabidopsis thaliana mutant line carrying an insertionof the En-1 transposable element at the ADC2 locuswhich should be regarded as a complete loss-of-function or knockout mutation. The ADC2 knockoutmutant shows no obvious phenotype change undernormal growth conditions, but is completely devoid ofADC induction by osmotic stress. As ADC1 geneexpression was not affected in the mutant, it wasconcluded that ADC2 is the gene responsible forinduction of ADC and PA biosynthesis under osmoticstress (Soyka and Heyer, 2000). More recently, Pérez-Amador et al. (2002) have shown that ADC2 geneexpression is induced in response to mechanicalwounding and methyl jasmonate treatment inArabidopsis thaliana. All these observations appear toindicate that ADC2 is a key gene involved in the PAresponse to abiotic stress in Arabidopsis. We envisagethat the extensive use of functional genomics andreverse genetic studies will facilitate the isolation ofnovel knock-out mutants affected in other PAbiosynthetic genes.

4.2. Transgenic plants

With the availability of most of the genes involved inPA metabolism, it has become possible to manipulatethis metabolic pathway using sense and antisensetransgenic approaches. Thus, cellular PA content has


been modulated by overexpression or down regulationof 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; Rajamet al.,1998; Roy and Wu, 2001; Bhatnagar et al., 2002).Most of the studies have used the constitutive 35Spromoter, but only few of them were successful inusing either inducible (Masgrau et al., 1997; Panicot etal., 2002a; Mehta et al., 2002) or tissue-specificpromoters (Rafart-Pedros et al., 1999). Overexpressionof heterologous ODC or ADC cDNAs generally causesthe production of high levels of Put (DeScenzo andMinocha, 1993; Bastola and Minocha, 1995; Masgrauet al., 1997; Capell et al., 1998; Bhatnagar et al., 2002;Panicot et al., 2002a), but in most cases only a smallincrease or even no change in Spd and Spm has beenobserved. This indicates that elevated levels of Putresulting from genetic manipulation of a single steplocated upstream of the PA biosynthetic pathway (i.e.ODC or ADC) are not accompanied by an increase insubsequent biosynthetic reactions (i.e. Spd and Spmbiosynthesis) (Bhatnagar et al., 2002). In contrast,overexpression of genes located downstream of thepathway (i.e. SAMDC or SPDS) generally lead toincreased levels of Spd or Spm or both (Thu-Hang etal., 2002; Mehta et al., 2002). Taken together theseresults suggest that the levels of Spd and Spm in thecells are under a tight homeostatic regulation(Bhatnagar et al., 2002), which possibly could berelated to a supramolecular organization of some ofthese enzymes (see Section 5).

Discrepancies observed among different studiesmay have several causes. These include: transgenesource, positional effects, recipient plant system, plantmaterial analyzed and type of promoter used. Ahierarchical accumulation of polyamines in differenttransgenic tissues/organs has been observed (Lepri etal., 2001). In general, less metabolically active tissuesaccumulate higher levels of polyamines (Lepri et al.,2001). These results are in line with experiments inwhich metabolites such as vitamin A andpharmaceutical antibodies accumulate at high levels inseeds of different species. It is reasonable to assumethat dormant or less metabolically active tissuesprovide a conducive environment for the accumulationof transgenic products (Thu-Hang et al., 2002). In thisregard, it should be stressed that the most remarkableresults have been obtained by controlled expression oftransgenes using inducible or tissue-specificpromoters. For example, tissue-specific expression of

SAMDC gives rise to smaller potato tubers withoutaffecting tuber yield (Rafart-Pedros et al., 1999). Thedistribution of tuber weights is of agronomicimportance, and generally a reduction of tuber-sizevariation is economically advantageous, so that moretubers fall into a given size grade either for seed orware (Rafart-Pedros et al., 1999). Similarly, fruit-specific expression of heterologous SAMDC in tomatoresulted in ripening-specific accumulation of Spd andSpm which led to an increase in lycopene, prolongedvine 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 strikingevidence regarding the in vivo involvement ofpolyamines in a particular developmental process, i.e.fruit ripening (Mehta et al., 2002).

55.. UUnnddeerrssttaannddiinngg tthhee rroollee ooff ppoollyyaammiinneess

Phenotypic analyses of mutants and transgenic plantswith altered PA levels gives further support to theprevious physiological studies (see Section 3) withregard to the involvement of these compounds inseveral plant processes (reviewed by Tiburcio et al.,2002). These include somatic embryogenesis (Bastolaand Minocha, 1995), stem elongation and flowering(Gerats et al., 1988; Masgrau et al., 1997; Hanzawa etal., 2000; Panicot et al., 2002a), root growth (Watsonet al., 1998; Cordeiro et al., unpublished), tuberdevelopment (Kumar et al., 1996; Rafart-Pedrós et al.,1999), fruit ripening (Mehta et al., 1997; 2002), abioticstresses (Minocha and Sun, 1997; Soyka and Heyer,1999; Roy and Nu, 2001). However, most of thesemutants and transgenic plants have not been furtherexploited for the analysis of PA function. Applicationof advanced genomic and proteomic approaches willhelp to elucidate the role of PA in particular plantprocesses.

5.1. Genomic approaches

The availability of complete genome sequencespermits the use of approaches to explore geneexpression variations on a large genome scale. EithercDNAs or large oligonucleotide collections areattached on surfaces to create a microarray. Thehybridisation of the microarray with fluorescentlabelled RNA or cDNA yields an overall image of geneexpression or ‘transcriptome’ (Lockhart and Winzeler,


2000). The global examination of gene expressionshould reveal the coincidence of spatial and temporaltranscript expression profiles that may reflect arequirement of co-ordinated gene product expressionin response to different type of signals. The technologydeveloped for the Arabidopsis genome has beenaccelerated in the recent years both by public fundingthrough the Arabidopsis Functional GenomicsConsortium 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 theliterature showing the utility of this approach forunraveling complex plant responses and signaltransduction processes (Schena et al., 1995; Schaffer etal., 2000), the use of this technology in our field isunfortunately in its infancy. So far, DNA microarrayanalysis has been used to reveal the induction of ADCgenes during drought stress (Ozturk et al., 2002) or inresponse to wounding and methyl jasmonate treatment(Sasaki et al., 2001; Pérez-Amador et al., 2002).

We envisage that global analysis of geneexpression in well characterized mutant and transgenicplants with altered polyamine metabolism will providenovel clues in the near future for understanding themolecular mechanisms underlying polyamine effectson plant growth and development.

5.2. Proteomic approaches

Proteomics’ uses biochemical approaches aimed atsystematically characterizing the ‘proteome’ or the‘protein complement of the genome’ (Wasinger et al.,1995) in a given organism, tissue, cell or subcellularcompartment. The means of proteome characterizationinclude protein localization, expression and mostimportantly protein interaction maps. A plethora ofinnovative procedures has been employed in recentyears for the large-scale analysis of protein signallingpathways, including the yeast two-hybrid system(Fields and Song, 1989), protein purification methodslinked to detection by mass spectrometry (Neubauer etal., 1997; Verma et al., 2000); protein localization(Ferrando et al., 2000; 2001; Farràs et al., 2001), andprotein microarray techniques (Zhu et al., 2001).

The yeast two-hybrid system is a genetic tool todescribe in vivo protein interactions using the yeastcell as a test tube. Each separated module of the GAL4transcription factor, either the DNA binding domain

(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 yeastmolecular genetics involves the facility to isolate thecorresponding cDNAs coding for proteins X or Y,introduced in the form of plasmid DNA. This latterfeature immediately favored the use of this system toidentify interacting partners for a given bait protein Xusing cDNA libraries as a prey (reviewed by Walhoutet al., 2000). The number of studies that have usedproteomics in our field is still scanty. Here we willprovide two examples that demonstrate the potential ofthese techniques to (i) unravel the role of PA intranscription; and (ii) to identify PA metabolons (seebelow).

Although the potential role of PAs in affecting geneexpression had already been reported, the molecularmechanisms underlying their effects were unknown(Wang et al., 2002). The identification of a polyamineresponsive element and corresponding transactingprotein factors that respond to polyamines has openedup an exciting new area to study the function of thesecompounds in transcription (Wang et al., 1999). Byusing the two-hybrid system, it was recently found thatthe human homologue of the Arabidopsis subunitCOP9 signalosome complex binds to such transactingprotein factors with the potential to directly affect geneexpression (Wang et al., 2002). Remarkably, the COP9signalosome proteins were first identified inArabidopsis and have been demonstrated to form aregulatory complex involved in light-activateddevelopment and playing a role in intracellularsignalling (Deng et al., 2000). We envisage that similartype of experiments will be performed in the plant PAfield that hopefully will provide new insights into therole of PAs in plant signal transduction.

Increasing number of reports document that manymetabolic reactions are catalysed by complexes ofsequentially acting enzymes that show highly orderedstructural organization (reviewed in Srere, 1987). Insuch multienzyme complexes the metabolites passfrom one active enzyme site to the next through aprocess termed ‘substrate channeling’. Thesupramolecular arrangement of enzymes involved insuch metabolic reactions is referred to as ‘metabolon’.Metabolons are multienzyme complexes in bothprokaryotes and eukaryotes that represent highlyorganized assemblies of sequential enzymes in ametabolic pathway and are thought to provide


increased metabolic efficiency and higher substrateselectivity. Metabolons may also help to coordinate theactivities of enzymes by sharing intermediates in agiven pathway, as well as to ensure protection of labilesubstrates and sequestration of toxic intermediates(Sugumaran et al., 2000). In addition, the formation ofmultienzyme metabolon complexes may enhanceenzyme stability, improve enzymatic performance andprovide a means for adaptation to alterations of inputof metabolic reactions, especially during demandingphysiological conditions (Abadjieva et al., 2001).

The relevant information about intrinsic propertiesof ‘metabolon’ formation can be acquired by studies ofprotein-protein interactions using modern proteomicapproaches (Ferrando et al., 2002). In this regard, ourlaboratory has recently analyzed possible interactionsbetween the SPDS and SPMS enzymes of polyaminebiosynthetic pathway in the yeast two-hybrid system(Panicot et al., 2002b). Using the Arabidopsisspermidine synthase as bait, two similar proteins wereidentified to interact with SPDS2 that were namedSPDS1 and SPMS. Yeast and bacterial mutantcomplementation tests revealed that SPDS1 encodes anovel spermidine synthase, whereas SPMS displaysspermine synthase activity. The heterodimerizationcapabilities of enzymes catalyzing the two last steps ofpolyamine biosynthesis were also demonstrated in vivoby co-immunoprecipitation using epitope taggedSPDS1, SPDS2 and SPMS proteins (Ferrando et al.,2000; Ferrando et al., 2001). Immunoaffinitypurification and size fractionation of SPDS and SPMSenzymes labeled with different HA and c-Mycepitopes revealed that the SPDS and SPMS proteinsco-purify with large multiprotein complexes of 650 to750 kDa. Further analysis of subunits of isolatedSPDS-SPMS metabolon(s) by mass spectrometry isexpected to yield important information about yetunknown regulatory subunits of SPDS-SPMSmetabolon in the PA biosynthesis pathway. Theavailable data support the conclusion that Spdsynthesized by SPDS is effectively channeled toSPMS to control the formation of the end-product Spmthereby regulating the synthesis of high molecularweight polyamines (Panicot et al., 2002b).

66.. CCoonncclluussiioonnss

Considerable evidence indicates that polyamines areinvolved 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 ampleevidence of their involvement in these processes, theirprecise role in these specific processes remains to beestablished. Recent developments of PA-deficientmutants and transgenic plants as well as ofmolecular genetic investigations should further ourunderstanding of their role in plant growth anddevelopment.

The polyamine pathway is now amenable tomodulation by genetic approaches because it has beenelucidated molecularly and biochemically in plants.Reverse genetics has identified an Arabidopsisknockout mutation of ADC2 gene which revealsinducibility by osmotic stress. Extensive use offunctional genomics and reverse genetics studies willfacilitate the isolation of novel knockout mutantsaffected in other polyamine metabolic genes. Senseand antisense transgenic approaches have revealed thefeasibility of modulating cellular PA contents.Generally, genetic manipulation of single steps locatedupstream of the PA pathway (i.e. ODC or ADC) lead toelevated levels of Put, but no changes occur in thehigher PAs, Spd and Spm. By contrast, overexpressionof genes located downstream of the pathway (i.e.SAMDC or Spd synthase) generally leads to increasedlevels of Spd and Spm, indicating that the levels of Spdand Spm are under a tight homeostic cellular control.Phenotypic analyses of mutants and transgenic plantsaffected in polyamine metabolism further supportprevious physiological evidence, but the molecularmechanisms underlying PA effects on plant growth anddevelopment remain to be elucidated. Global analysisof gene expression by using the available DNAmicroarray genomic techniques will help to understandthe role of these compounds. The potential ofproteomics to unravel the role of polyamines inparticular cellular processes is also examined. Weenvisage that the extensive use of the two-hybridsystem and other proteomic approaches will providenew insights into the role of PAs on plant signaltransduction. Furthermore, we provide evidence thatproteomics is an excellent tool to unravelsupramolecular organizations of PA metabolicenzymes which may help to understand homeostaticcontrol of this metabolic pathway.


AAcckknnoowwlleeddggeemmeennttss

AFT acknowledges the grants from Ministerio deCiencia y Tecnología BIO-99-453 and BIO-2002-04459-C02-02.

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13

PPhheennoolliicc ccyyccllee iinn ppllaannttss aanndd eennvviirroonnmmeenntt

Valentine I. Kefeli1, Maria V. Kalevitch2* and Bruno Borsari3

1Slippery Rock Watershed Coalition, 3016 Unionville Rd., Cranberry Twp., PA 16066, USA; 2RobertMorris University, 881 Narrows Run Rd., Moon Township PA 15108, USA; 3Slippery Rock University,101 Eisenberg Bldg., Slippery Rock PA 16057, USA (* author for correspondence)

Received 30 October 2002; Accepted 15 November 2002

AAbbssttrraacctt

Phenolic substances are synthesized in plants and in the soil. They exist in the form of polymers and monomers. Thelatter group of phenolics is assembled within the chloroplasts of plant cells, whereas soil phenolics are associatedwith the process of humus formation on the alumino-silicate matrix of the soil micelle. As plants grow, phenolicsaccumulate 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 fromplant root systems they exert inhibitory growth function within adjacent rhizospheres. This work presents the latestexperimental evidence of phenolic synthesis and transformation in the environment, while providing anunderstanding of their effect in plant-soil relations.

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

BBiittkkiilleerrddee ffeennoolliikk ddöönnggüü vvee ççeevvrree

ÖÖzzeett

Fenolik maddeler bitkilerde ve toprakta sentezlenir. Bunlar polimerler ve monomerler fleklinde bulunurlar.Fenoliklerin monomer grubu bitki hücresinin kloroplastlar›nda biraraya gelirken, toprak fenolikleri toprakmisellerinin alumino-silikat matriksi üzerinde humus oluflum olay› ile uyumluluk gösterir. Bitki büyürken hücrevakuollerinde 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 köksistemlerinden sal›nd›klar› zaman hemen yak›n›ndaki rizosferlerde büyümeyi inhibe edici etki meydana getirirler. Buçal›flma fenolik sentezlerinin en son deneysel verilerini ve çevredeki dönüflümlerini sunarken, bitki-toprakiliflkilerindeki etkilerini anlamam›za yard›m etmektedir.

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

IInnttrroodduuccttiioonn

Phenolics are very stable products in plant organisms.Generally, they are characterized by a benzene ringand one hydroxyl group (-OH). They can be convertedinto lignin which is the main phenolic polymer inplants. Microorganisms break down these molecules

and their fragments contribute to the mineralization ofsoil nitrogen and humus formation. Thus, humusparticipates actively in fulfilling plants nutritionalneeds and growth. Light enhances the biosynthesis ofphenolic substances in plant chloroplasts and theseconstitute in addition to soil micelles (humus) a secondformation site for this diverse group of organic

molecules. It should be mentioned however, thatphenolics tend to accumulate in plant vacuoles inrelatively high amounts, or they deposit in thesecondary cell wall as lignin.

CChhlloorrooppllaassttss aass cceenntteerrss ooff pphheennoolliiccss bbiioossyynntthheessiiss

Experiments with chloroplasts of willow (Salix spp.)leaves showed that the synthesis of phenol-carboxylicacids and flavonoids is strongly stimulated by lightexposure. Metabolic inhibitors that depressphotosynthetic activity (simazine, diurone,chloramphenicol), affect negatively the biosynthesis offlavonoids. Leaves chloroplasts have the capability tolocalize phenol compounds, some of which arespecific to these organelles only. The chloroplasts ofspring willow leaves contain more phenols than thechloroplasts of the same leaves in the autumn. Light isa mandatory condition to initiate phenolics synthesisand this is indicated also by the lack of such moleculesin the protoplastids of etiolated willow shoots (Kefeliand Kalevitch, 2002). Light appears also to induceflavonols synthesis in the chloroplasts and cytoplasm.Chalcone and phenolcarbonic acid present in etiolatedwillow shoots can be considered metabolic precursorsof light-synthesized flavonols. In certain cellcompartments (vacuoles and cell wall) phenols arecontained in significant amounts (Lewis andYamamoto, 1990). However, it is not clear yet howphenols are translocated within plant cells and howthey affect the function of cell organelles such asribosomes and mitochondria. Phenolic substances thatinhibit plant growth (hydroxy derivatives of cinnamicacid, coumarin and naringenin) are synthesizedsimilarly to other phenolics. The synthesis of growthinhibitor derivatives of hydroxycinnamic acids followsthe pathway: shikimic acid-chorismic acid-prephenicacid-cinnamic acid and p-coumaric acid. A theory ofmetabolism bifurcation among phenolic substances,some of which can inhibit growth and synthesis ofindolic compounds has been proposed. According tothis new approach, indolil-3-acetic acid (IAA)becomes the main natural auxin (Kefeli, 1978; Kefeliand Dashek, 1994; Kefeli and Kalevitch, 2002).Therefore, indole auxins (IAA, indoleacetonitrile) aswell as phenolic inhibitors (p-coumaric acid,coumarin, naringenin and others) are derived from thecommon precursors, shikimic and chorismic acids(Figure 1).

Muzafarov and collaborators (1992) investigatedthe functions of some phenolics in chloroplasts. Theyassumed that the essence of the relationship betweenphotosynthesis and phenolics biosynthesis is thatphenolics exert a direct and an indirect effect on theprocess of solar accumulation itself. From our point ofview, flavonoids as polyfunctional compounds ingreen 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 andphotophosphorylation, bringing about the change ofATP/NADPH ratio. In the reactions of carbonmetabolism they can shift the dynamic equilibrium ofpentosephosphate reduction cycle to enhance thesynthesis of certain metabolites both due to the changein energy substrate intake and to the interaction withenzymes of the cycle. Additionally, flavonoids

14 Valentine I. Kefeli et al.

COOHIC— OICH2

CHO

HCOH

HCOH

CH2OPhosphoenol-pyruvic acid

Dehydroquinic acid5-Dehydroshikimic acid Erythrose-4-phosphate

2-keto-3-desoxy-7-phospho-D-Araboheptonic acid

COOH

OH

OH

OH

COOH

NH2

CH2-CO-COOH

CHOH-CHOH-CH2O

HOOC

OH

CH2-CH-COOH

CH2-CO-COOHCH-CH-COOH

OH

Shikimic acid

Chorismic acid

Phenylalanine

Prephenic acid Anthranilic acid

p-Coumaric acid

Indolylglycerophosphate

Indolyl-acetic acidIAA

NIH

N

FFiigguurree 11:: Phenol-propanoids in metabolic bifurcation.

exercise a feedback control over their ownbiosynthesis, although this phenomenon is not clearlyunderstood. This questionable situation remains as thebiosynthesis of the entire flavonoid structure withinplastids has not been explained, nor the completeenzymatic package of their biosynthesis has beendiscovered yet. Lack of direct eveidence of flavonoidstransport within the cell and through the whole plantconstitues another challenge to a more accuratedescription of their functions. Noneless, a variety ofphenolic compounds, present simultaneously withincells appear to be capable of influencing the rate anddirection of plants metabolic activities. Thus, anychange in the flavonoid structure, or qualitativecomposition of the phenol complex result in a changeof the mechanism of its effect upon the processes ofcell energy exchange.

Chalcone and phenolcarbonic acids present inetiolated willow shoots can be viewed as the potentialprecursors of light synthesized flavonoids. However,the use of paper chromatography to investigateisosalidpurposide transformation products did notreveal the presence of any flavonols sensitive toconventional reagents. Therefore, the transformationof chalcone (isosalpurposide) in lightless vitro appearsto terminate at a second stage. The synthesis oferiodyctiol and luteolin that occurred in willow leavesevidently took place in vivo and under light exposure.It should be pointed out however, that phloridzin andisosalipurposide were decomposed from aglycone andthat phloridzin and phloretin produced yellow stainson the chromatogram as well as flavonoids. It is knownthat flavonoid glycosides are revealed as dark spots onchromatograms exposed to UV light. Therefore, ouryellow stains were classified as flavanones, since theydid not react with AlCl3, nor Na2CO3 like flavonols,that also form yellow spots. At the same time, similarto chalcones and aurones, these floridzintransformation products are yellow colored and theyturn into orange-pink when exposed to Na2CO3 orNH4OH. Relatively easy transformations ofisosalipurposide and phloridzin into compounds ofother classes (flavanones, chalcones, or aurones)evidenced the role of these products in the generalmetabolism of flavonoids (Figure 2).

PPhheennoolliicc ssuubbssttaanncceess sseeccrreetteedd bbyy rroooottss aanndd lleeaacchheeddffrroomm lleeaavveess

Plants contain and secrete a diverse group of growthinhibiting substances that may affect other plantsdevelopment, if grown in their vicinity (allelopathy).Leaf exudates of willow species such as Salix rubra orSalix viminalis, contain phenolic inhibitors likenaringenin derivative isosalipurposide. Other speciesinstead like apple trees (Malus spp.) containphloridzin, which is a strong respiratory inhibitor.Roots and leaves of the wild plant Nanaphyton nativeto semi-desert regions of Mongolia contain also strongphenolic inhibitors. Seed as well may secreteallelochemicals. Tobacco seed (Nicotiana tabacum)for example suppress germination of its own seedwhen leachates come in contact with the seeds (Kefeliand Kalevitch, 2002). Although the inhibition ofgermination was observed at various levels ofintensity, this phenomenon demonstrates theselectivity of these natural excreta, similar to the effectof synthetic herbicides. Therefore, increasing evidenceindicates that phenolics and alkaloids play the role ofselective agents. Secondary compounds can bemodified in transgenic plants and genetic mutants.

Phenolic cycle 15

OH

HO

CH = CH

O O6H10O5

´OH

Isosalipurposide2´, 4´, 6´, 4-tetroxychalcone--2´-glucoside of chalconarin-genin (chalcone)

1

HO OHCO

O

C6H10O3

H

CH2

2

Salipurposidenaringenin-5-glucoside(Flavanone-glycoside)

HO OHCO

OH

H

CH2

3

4

Naringenin(Flavanone)

Luteolin(Flavone)

Eriodictyol(Flavanone)

CO

CO

CO

6´1´

2´3´4´5´ 1

2 3

456

1´

2´ 3´

4´5´6´

1

4

23

76

8

5

1

4

23

76

8

5

1´

2´ 3´

4´5´6´

FFiigguurree 22:: Flavonoid biosynthesis.

Hence, molecular genetics becomes a tool, which mayhelp to regulate the level of secondary metabolites inplants. Therefore, there is a need continue the searchfor botanical herbicides as a rise of ecologicalconcerns has clearly identified the environmentalimpact of herbicides of synthesis.

Root exudates affect the germination of seeds ofdifferent crops: monocots and dicots (Table 1 andTable 2). However, it must be pointed out that onlysome phenolics were studied in the exudates of willowroots (1) which have no analogues in the roots (2) andleaves found among the common allelopathogens.Although some of these substances could be retainedby willow roots, others where excreted into an externalmedium. Chromatography of these water exudates anda subsequent investigation of their chromatogramswith UV-B light showed that most of these substancesare polyphenols such as coumarin, or phenolic acids.The phenolic substances retained by cells had differentchemical properties than those located in the rootexudates. Thus, the data confirm the hypothesis that

excreted substances had an allelopathic nature andwere involved in developing ecological relationshipswith adjacent plants of different species.

During the composting process water extractscontain many inhibiting substances that might formtoxic exudates (Kefeli et al., 2001). Paperchromatography reveals the presence of phenolic acidsand coumarins in water extracts. The highestconcentrations of these inhibitors was measured inabscised leaves of red maple (Acer rubrum L.). Onegram of dry leaves was mixed in 29 ml of water toprepare the extracts. The pH of the solution wasbetween 5.4 and 5.6 and the extracts were incubatedfor a week at room temperature while the pH raised to7.2. Further observations revealed that duringcomposting the amount of phenolics was drasticallyreduced. Seed germination tests were performed withthese water extracts and pure water (control) on lettuceand wheat seeds. Germination rate and seedlinglengths were measured to demonstrate that phenolicsdecreased inhibiting properties after dilution, or after


TTaabbllee 11:: Effect of root exudation on germination of crop seeds (Non-concentrated exudates).

Variant % to tap water (control)

wheat clover lettuce mustard

Tap water 100 100 100 100Spider plants (Chlorophytum) exudates 54 93 75 100Willow (Salix vitaminalis) exudates 58 79 74 138

Stem length (5 tallest plants, mm)

Tap water 29 23 18 25Spider plants (Chlorophytum) exudates 15 21 14 2Willow (Salix vitaminalis) exudates 7 18 13.5 3.5

TTaabbllee 22:: 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 lengthUV-B light

0 Blue 91 76 90 640.14 Blue 94 68 98 580.3 Violet 86 80 93 760.5 Blue 56 52 71 760.67 Yellow 87 68 89 880.88 Yellow 52 56 63 64

contact with fungi. Therefore, the whole process ofallelopathogens formation in the environment could betightly connected with the formation of secondarysubstances and plant biomass accumulation (Figure 3).

SSooiill--mmiiccrroobbiiaall ccoommpplleexx ffoorr pphheennoolliicc ddeeccoommppoossiittiioonn

Phenolic substances are the most resistant metabolitesproduced by plants. They undergo furthertransformation in the soil, forming humus molecules,strongly linked to the alumino-silicate matrix. Humusis more or less a stable fraction of soil organic matter;it adsorbs mineral elements that serve as importantnutrients for plant growth and development (Kefeli,2002). The alumino-silicate matrix and humus formprimary soil units. Humus is formed by carbon-nitrogen interaction. Potential sources of carboninclude cellulose and polyphenols from plant leaves,or transformed lignin polymers.

In order to verify the efficacy of microbial activityduring the humification process, four different soilhorizons in a Grashem soil at the Macoskey Center ofSlippery Rock University of Pennsylvania, USA wereinvestigated. The presence and number of colonies ofheterotrophic soil microflora were determined in each

horizon (TSA (triple-soya-agar, 48 hours, roomtemperature). The topsoil (horizon A, 0-28 cm) wasdark gray in color, sandy, high organic matter content(5.6%), with slightly alkaline pH=7.5. This horizonwas also high in potassium, low in available nitrogen,and medium in phosphates content, while very highwas the microbial activity. Horizon E (28-52 cm) wasochric in color, it contained more loam, less organicmatter, lower microbial activity and pH=7.7. HorizonB (52-62 cm) had no organic matter, microbial activitywas the lowest and pH=7.8. Water permeability wasalso measured for each horizon to evaluate penetrationtimes. The fastest penetration rate was measured inhorizon A (11 minutes), whereas it took 47 minutes forhorizon B and longer (more than 6 hours) belowhorizon B. Soil fertility conditions were also assessedwith a wheat/clover germination test. A sand substratewas 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 microbialactivity) yielded 30-70% germination rate (Kalevitchet al., 2002). The results of these experiments appear toindicate that topsoil (for its highest microbial activity)

Phenolic cycle 17

Photosynthesis

Secondarysubstances Plant biomass

in living plants

Biomass ofdead plants

Composting process(phenolics and N-sources)

To alumo-silicatematrix

Humus formation

Active secretionfrom roots

into thewater and soil

MicroorganismsComposting process

Transformed phenolics andother secondary substances

FFiigguurree 33:: Secondary substances, plant biomass accumula-tion and humus formation during allelopathic effects.

FFiigguurree 44:: Phenolic cycle.

is an effective medium usable to facilitate compostingof maple and sumac leaves, contaning nature phenoliccompounds.

CCoonncclluussiioonn

Microorganisms have the capability to decomposephenolic compounds to their monomers, beingdeglicosidation of phenolic molecules, followed bylignin decomposition the biochemical pathways of theprocess. Leaves become a primary substrate for soilmicroorganisms, while woody materials and sawdustserve as secondary type of biomass and thesesubstrates play a major role in humus formation(Figure 4).

The biosynthesis of phenolic substances withinchloroplasts and its further transformation on thealumino-silicate matrix of soil micelles led us toconclude about the existence of phenolics cycle in theplant-soil system. Although many aspects remainunknown, the ecological relevance of phenolicsubstances in the environment has been amplydemonstrated as this cycle embrace lithosphere,microsphere and biosphere.

These emerging concepts facilitate theunderstanding of complexity within our living systemsand their physical habitat while reinforcing the idea ofinterconnectedness among living species andecosystems.


Kalevitch MV, Kefeli VI, Borsari B and Liguory A. Soil microflora and fabricated soils. American Society for Microbiology. 103rd General Meeting. Washington DC. 2003 (In press).

Kefeli VI. Fabricated soil for landscape restoration. SME Ann Meet. 02-142, 2002.

Kefeli VI and Dashek WV. Non hormonal stimulators and inhibitors. Biol Rev Cambrige. 59: 273-288, 1984.

Kefeli VI, Borsari B and Welton S. The isolation of 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.



19

TThhee sshhoorrtt--tteerrmm eeffffeeccttss ooff ssiinnggllee ttooxxiicc ddoossee ooff cciittrriicc aacciidd iinn mmiiccee

Tülin Aktaç1*, Ayflegül Kabo¤lu1, Elvan Bakar1 and Hamiyet Karakafl2

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

AAbbssttrraacctt

The effects of LD25 (480 mg/kg.bw.) dose of citric acid, a food preservative, were investigated on body weight, organweights (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. Citricacid (to experimental groups) and physiological saline (to control groups) were given intraperitoneally. The resultsof enzyme activities were evaluated using autoanalyzer as IU/L. Even though significant decreases in the bodyweights were noted when compared to those of the control group (p<0.001), generally an insignificant increase wereobserved in the organ weights (liver: p>0.05, 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 showedhistopathological changes depending on the citric acid. These changes were tissue degeneration, cytoplasmicvacuolisations, nuclear membrane invaginations, picnotic nucleus and necrosis of the hepatocytes.

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

FFaarreelleerrddee ssiittrriikk aassiiddiinn tteekk ttookkssiikk ddoozzuunnuunn kk››ssaa ssüürreellii eettkkiilleerrii

ÖÖzzeett

Bir besin koruyucu olan sitrik asidin LD25 (480 mg/kg.va.) dozu farelere intraperitoneal yolla uyguland›. 10 günsonra hayvanlar›n vücut a¤›rl›klar›, organ a¤›rl›klar› (karaci¤er, böbrek, dalak), kreatin kinaz (CK), laktatdehidrogenaz (LDH), alanin aminotransferaz (ALT) ve aspartat aminotransferaz (AST) enzimlerinin serum düzeyleriile, karaci¤er dokusu üzerinde sitrik asidin etkileri araflt›r›ld›. Otoanalizörde tayin edilen enzim aktiviteleri U/Lolarak de¤erlendirildi. Çal›flmada vücut a¤›rl›klar›nda kontrol grubuna k›yasla anlaml› bir azalma gözlenmesinera¤men (p<0.001), organ a¤›rl›klar›nda (karaci¤er: p>0.05 , 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 mikroskopikincelenmesinde doku dejenerasyonu, sitoplazmik vakuolizasyon, nükleer zar çöküntüleri, piknotik nukleuslar vehepatositlerde nekroz gibi histopatolojik de¤ifliklikler gözlendi.

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


Humans are exposed daily to complex mixtures ofchemical compounds in their food. One of these

substances are antioxidants which are used as foodpreservatives. However, peroxides of saturated fatsand their secondary oxidation products, can be toxicand impair food quality (Würtzen, 1990). Thus despite

their economic importance, they can have negativeeffects on living organisms. Xenobiotics entering theorganism are held by intestine, kidney and liver cellsfor detoxification. These cells contain importantdetoxification enzymes. During the detoxification ofxenobiotics, free radicals are produced inoxidation/reduction reactions, and these radicals canhave destructive effects on tissues.

The toxic effects of many food preservatives onliving organisms have been studied by manyresearchers (Makoveç and Sindelar, 1984; Daniel,1986; Cabel et al., 1988; Kagan et al., 1990; Jung etal.,1992; Nijhoff and Peters, 1992; Fujitani, 1993;Weemaes et al., 1997; Mc Farlene et al., 1997; Saferand Nughamish, 1999; Kabo¤lu and Aktaç, 2002;Aktaç et al., 2002). Although the citric acid and metalsalts (sodium or potassium citrat) are widely used infood industry, there is no report on more detailedeffects of citric acid (or its salts) in liver. In addition,soft drinks, cosmetics and drugs, in which citric acid isapproved for use, are consumed by most of humansevery day.

A way of analysing harmfull effects of foreignmaterials entered to organism is to determine theeffects of the chemicals on the enzymes. Enzymeshave a very important role in the metabolical processsince they are biological catalysts. Thus, their abnormalserum levels indicates various diseases. Among theseenzymes are, creatine kinase (CK), lactatedehydrogenase (LDH), alanine aminotransferase (ALT)and aspartate aminotransferase (AST) which are themost important. Therefore, we studied short termtreatment of citric acid (10 days) in mice. In theseexperiments, firstly we tested total body weigths,organ weights (liver, kidney, spleen), and determinedthe serum levels of creatin kinase (CK), lactatedehydrogenase (LDH), alanin aminotransferase (ALT)and aspartat aminotransferase (AST), and secondly theliver tissue was investigated histopathologically.

MMaatteerriiaall aanndd MMeetthhooddss

Male mice (Balb/C albino) weighing 25-30 g wereused in our experiments. Five mice were used controlgroup and ten mice were used the citric acid-treatedgroup. Animals were fed by pellet baits and water.LD25 dose (480 mg/kg.bw.) of citric acid (Merck; inphysiological saline) were injected intraperitoneally toexperiment group mice, and the same amount of

physiological saline to control group mice. 10 daysafter the injection, the mice were killed by cervicaldislocation and then the necessary studies werecommenced. The livers, kidneys and spleens dissectedout, weighed, liver samples were seperated formicroscopical examination. Blood samples were alsotaken for enzyme assays. The serum levels of enzymeswere determined using a Merck Mega 600autoanalyser with the aid of Diasis Kits. Data wereanalyzed by M.Whitney U test for multiplecomparisons for the differences between the controland treated groups. For histological examination, liversamples were fixed with 10% buffered formalin,processed and stained hematoxylin-eosin.

RReessuullttss

The effects of citric acid injection on the body weightand liver, kidney, spleen weights was shown Table 1and 2. Although the liver, spleen and kidney weightswere not changed significantly (p>0.05), the bodyweights were decreased significantly (p<0.001). CK,LDH, ALT and AST activities were not changedstatistically (p >0.05) as shown in Table 2. The resultsof the microscopic investigation showed that liverof mice treated with citric acid has necroticchanges, compare to the control group (Figure 1-6).These changes were slightly degeneration of tissue(Figure 2), cytoplasmic vacuolisation, nuclearmembran invaginations (Figure 3, 4) and picnotic nuclei(Figure 5). In addition, we observed degeneration of theblood vessel endothelium (Figure 6).

DDiissccuussssiioonn

The effects of xenobiotics in living organisms caninvestigate in various ways. Among these are, short-term toxicity tests which are used very commonly. Inthese methods, many parameter are used to test theeffects of xenobiotics. Some of these parameters arebody weight, organ weights, blood profile, andhistopathological examination. In this study, the short-term effects of citric acid applied intraperitoneallywere investigated. It was reported that the body weightdecreases in mouse (Würtzen, 1990), and in rats(Nijhoff and Peters, 1992) by the effects of phenolicantioxidant butylated hydroxytoluene (BHT) andbutylated hydroxyanisole (BHA) in chronic studies. In

20 Tülin Aktaç et al.

contrast, any significant change was seen in bodyweight in F344 rats (0.2, 2.5 and 3.0 % of sodiumbenzoate) and in B6C3F1 mice (1.81, 2.09 and 2.4 %

of sodium benzoate) for ten days by Fujitani (1993).Similarly, Kabo¤lu and Aktaç (2002) were determinedthat a significant decrease obtained at 3.0 and 4.0 % of

Short-term effects of citric acid 21

FFiigguurree 11:: The control group of the liver tissue, barrepresentes 20 µm.

FFiigguurree 22:: Citric acid group. Distortion of generalhistological structure of the liver, v: blood vessel, barrepresentes 10 µm.

FFiigguurree 33:: Citric acid group. Nuclear invaginations (arrows),vacuolisation (v), and damaged nucleus (n) in necrotic cells,bar representes 4 µm.

FFiigguurree 55:: Citric acid group. Picnotic nuclei (arrows) inhepatocytes, bar representes 10 µm.

FFiigguurree 44:: Citric acid group. Invaginations of hypertrophiccell nucleus (arrow), bar representes 4 µm.

FFiigguurree 66:: Citric acid group. Degenerated endothelium(arrows) of blood vessel (v), bar representes 10 µm.

sodium benzoate. Also, at the present study wedetermined a significant decrease of body weight inmice by the effect of citric acid (Table 1).

Some autors have shown that food preservativeshad increasing effects to organ weight. The effects ofBHT and BHA on the increasing of the liver andthyroid weights were demonstrated in mice byWürtzen (1990). Similarly, the effects of BHT onincreasing of the liver weight in rats was also shownby Mc Farlene et al. (1997) and Safer and Nughamish(1999). Fujitani (1993) was also obtained significantincreasing of the liver and kidney by the effects ofsodium benzoate in male rats. In our previous studies,increasing of the total liver weight were seen oraltreatment of sodium benzoate (Kabo¤lu and Aktaç,2002) and citric acid (Aktaç et al., 2002) but it was notsignificant. Additionally, in the present study, we couldnot find any significant change the liver, kidney andspleen weights by the intraperitoneal injection of citricacid (Table 2). According to our results, serum CK,LDH, AST and ALT levels in the treated animals were

not significant to compare with the control. Theseresults were similar with findings obtained in F344 ratsand B6C3F1 mice by Fujitani (1993).

Although the organ weights and serum levels ofenzymes were not changed significantly, theexamination by light microscopy revealedpathological changes in liver of mice, such asvacuolisation and glassy cytoplasm in the hepatocyte,nuclear membrane invaginations, picnotic nuclei.Similarly, with the effect of sodium benzoate in therats and mice, high vacuolisation and glassyappearance in hepatocyte cytoplasm was explained(Fujitani, 1993). Again, similar findings were obtainedin the rats with oral treatment of BHT (Mc Farlene etal., 1997; Safer and Nughamish, 1999), and withsodium benzoate, benzoic acid and citric acid in mice(Kabo¤lu and Aktaç, 2002; Aktaç et al., 2002). Theresults of present study suggested that citric acid hashepatotoxic effects and long term exposure mayinduce severe damage in liver of mice. However, themechanism of damaging effects of citric acid need to

22 Tülin Aktaç et al.

TTaabbllee 11:: Effect of citric acid on body weight in mice.

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 (p<0.001).(**) not significant (p>0.05).

TTaabbllee 22:: 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 levelsCK (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).

be clarified by more detailed studies.Finally, we can conclude that consumption of the

foodstuffs containing preservatives is important for thehuman health.


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


25

CChhaarraacctteerriissaattiioonn ooff RRPPPP77 mmuuttaanntt lliinneess ooff tthhee ccooll--55 eeccoottyyppee ooffAArraabbiiddooppssiiss tthhaalliiaannaa

Canan Can1, Mehmet Özaslan1*, Eric B. Holub2

1University of Gaziantep, Faculty of Science & Arts, Department of Biology, 27310 Gaziantep; 2PlantGenetics and Biotechnology Department, Horticulture Research International, Wellesbourne, Warwick,CV35 9EF, England (* author for correspondance)

Received 21 May 2002; Accepted 20 November 2002

AAbbssttrraacctt

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 toFast Neutron (FN) were inoculated with 8 different P. Parasitica isolates and symptom development wasinvestigated. A total of 4 mutant lines were analyzed. It was found that the RPP7 gene present in the Col-5 ecotypeis 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’’nn››nn CCooll--55 eekkoottiippiinnddeenn eellddee eeddiilleenn mmuuttaanntt hhaattllaarrddaann RRPPPP77 ggeenniinniinnkkaarraakktteerriizzaassyyoonnuu

ÖÖzzeett

Bu çal›flmada, Arabidopsis thaliana’n›n Col-5 ekotipinde bulunan ve Peronospora parasitica’n›n Hiks-1 izolat›nakarfl› dayan›kl›l›¤› sa¤layan RPP7 geninde mutasyon içeren hatlar›n fenotipik olarak belirlenmesi üzerindearaflt›rmalar gerçeklefltirilmifltir. Fast Nötron (FN) uygulamalar› ile mutasyon meydana getirilmifl Col-5tohumlar›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¤ubelirlenmifltir.

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


Following the isolation of the Pseudomonas syringaeresistance genes (R-gene) from tomato, the research onisolation and characterization of R-genes against plantpathogens has been improved (Hammond-Kosack andJones, 1997). Recently many R-genes conferringresistance to fungi, bacteria, nematode and viruses in

rice wheat, tomato, pepper and some other importantcrops are isolated and the mechanism of resistance isdetermined (Richter and Ronald, 2000). The mutantlines with lack of R-genes have a potential importancein this type of work (Mc-Dowel et al., 1998).

Arabidopsis thaliana is a member of cruciferaefamily and it is known to have a small genome size of120 Mb. It is a flowering plant and is a best model for

the working on genome analyses, growth regulation,hormons, flowering, disease resistance andembryogenesis. Arabidopsis and tomato were used todetermine the mechanisms of disease resistance(Thomas et al., 1997; Botella et al., 1998). It is also thehost of many pests which attacks to crop plants. Manygenes that provides resistance to bacteria and fungidisease have been isolated and characterized from A.thaliana (Dangl and Jones, 2001; Feys and Parker,2000).

Peronospora parasitica is a causal agent of mildewdisease in the genus cabbage, turnip etc. of cruciferaefamily. R-genes that determines resistance to P.parasitica (RPP) were isolated and characterized(Holub and Beynon, 1997; Parker et al., 1997; Botelliet al., 1998; McDowell et al., 1998; Bittner-Eddy et al.,2000). The researches on RPP genes have shown thatthese genes are at the specific regions at certain placesof each chromosome called as “Major recognitioncomplexes-MRC” (Can, 1997; Holub and Beynon,1997).

RPP7 gene is present in Col-5 ecotype andrecognized by the Hiks-1 isolate of P. parasitica. Thisgene was placed onto the first chromosome betweenthe markers M421 and M213 by using the hybrid linesof Col-5 and Nd1 ecotypes (Tor et al., 1994; Can et al.,1995; Can, 1997). The Hiks1 isolate also recognizesthe RPP1 gene which is present in Nd-1 ecotype, andhas an epistatic effect on the RPP7 gene (Tor et al.,1994).

The mutant lines that lack the R-genes werestudied in detail and has a wide area of interest such asmolecular and classical genetics. However, in order tostudy the relationships between A. thaliana and theRPP genes and to investigate the genomeorganizations, some mutant lines were used (Parker etal., 1996). The mutant lines lacking the RPP geneswere obtained from Ws-O that contain RPP14 geneexhibiting resistance to No-Co2 isolate by using EthylMethane Sulfate (EMS). The lines were then used toseparate the RPP10 and RPP1 genes, which wereallelic to RPP14 that is on the third chromosome. Itwas found that the WsEDS line was susceptible to allP. parasitica isolates tested and that the WsEDS locuswas 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 PRprotein) mutant lines of A. thaliana synthesize theproteins 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 linesdo have the ethylene synthesis. But it was found thatthe P. syringae f.s.p. tomato resistance continued inthis mutant lines. This study showed that ethylene wasnot important for A. thaliana and bacteria relationships(Bent et al., 1994; Dong, 1998). Lsd (LesionsSimulating Disease resistance response) and acd(Accelerated Cell Death) mutant lines produceHypersensitive Resistance (HR) like symptomswithout a pathogen infection. These symptoms areformed by the influence of external factors like heatand light (Lam et al., 1999). So, it is accepted that lsdand acd loci are negative regulators for HR formation(Dietrich et al., 1994). In general, the presence ofdifferent resistance mechanisms in A. thaliana whichare directed by RPP genes was found by thecharacterization of mutant lines (Glazebrook et al.,1997; McDowell et al., 2000).

In this study, the mutant lines of the Col-5 ecotypeof A. thaliana were characterized, to understand themechanisms of RPP7 gene that confers resistance toHiks-1 isolate of P. parasitica.

MMaatteerriiaall aanndd mmeetthhooddss

Plant and fungus material

In this study, Col-5 ecotype of A. thaliana lines havingMRC-B, MRC-C and MRC-H regions (Can, 1997;Holub and Beynon, 1997) were used as wild typeecotype. The Fast Neutron (FN) applied mutant lineswere obtained commercially and the selections ofmutant lines were performed by using the Hiks-1isolate of P. parasitica. The Hiks-1 isolate recognizesthe RPP7 gene which is in the MRC-B region of wildCol-5 ecotype, and 7 days after inoculation it inducesa resistance which is defined with HR. Four mutantlines were used in this study denoted as FN3922,FN3928, FN3929 and FN3930. The HR does not occurin mutant lines, and the pathogen completes its lifecycle by sexual and asexual sporulation.

Regeneration of Hiks-1 isolate from oosporepopulation

The Hiks-1 isolate was regenerated by using oosporepopulation. To do this, the seeds of A. thaliana thatwere susceptible to the Hiks-1 isolate were sown into

26 Canan Can et al.

little plastic pots containing 4:1:1 (torf: perlit: sand) ofmixture for 40-50 seeds each. The pots were irrigatedto wet the seeds and 1-2 x 105 oospore/ ml were addedto the pots. The containers were held at 4 °C for 1-2weeks to break the dormancy. Following this, thecontainers were placed into the climated room at 18-20°C, 10 h light and 14 hour dark period. Within 10-15days following seed germination, some seedlingshaving sporulation was collected and placed intoeppendorf tubes containing 200 µl dH2O. Theeppendorf tubes were shaked gently to allow theconidia to pass to water. The conidia suspension wasused to inoculate 7 days old seedlings of EBH3529 andKsk-1 ecotypes, and the plants were placed into theclimate room. By this way, the regeneration of theHiks-1 isolate was done by subculturing 3-4 times. Theconidia were stored at –20 °C and were used whenneeded.

The same procedure were applied for, Ahco-1,Ahco-2, Ahco-7, Wand-1, Cand-5, Hind-2 and Hind-4isolates using Nd-1 and Col-5 ecotypes (Can, 1997).

Characterization of P. parasitica isolates by usingdifferent A. thaliana ecotypes

Regenerated P. parasitica isolates were inoculated intoCol-5, Ksk-1, Nd-1, Ws-3, Tsu-1, Ler-1, Oy-1 andWei-1 ecotypes in order to do phenotypiccharacterization. The A. thaliana ecotypes wereobtained from Dr. Eric Holub (HRI- UK)

The conidia suspension was adjusted to 4-5 x 104

conidia/ml concentration for plant inoculations. Thecotyledons of 7-8 days of the A. thaliana ecotypeswere inoculated in such a way that it would be onedrop to each cotyledon. The plants were placed intoclimated room with 18-20 °C, 10 h light, 14 h darknessconditions after the inoculation and the plants werechecked at the end of 3. and 7. days. The evaluationwas done regarding the pathogen sporulation andhypersensitive reaction types (the interactionphenotypes). Phenotypic reactions were examinedunder the fine group as; pitting with no pathogensporulation (PN), flecking with no pathogensporulation (FN), flecks with delate and moderatepathogen sporulation, 1-20 sporangiophorus per eachcotyledon (DM), flecking with delate pathogensporulation, 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 examinedunder light and fluorescence microscope. The infectedleaves were taken and put absolute methanol for 5-6hours followed by saturated chloral hydrate solutionfor 4-5 hours. Then, tissues were placed in 50 %glycerol solution for microscopic analyses.

DNA analysis

Total plant genomic DNA was isolated with somemodifications by using the methods of Ausubel et al.,(1994). Five to eight grams of plant material wasgrounded in N2 and transferred to the tubes containing15 ml buffers (100 mM Tris-HCl, 50 mM EDTA, 500mM NaCl, 10 mM Mercaptoethanol, %25 SDS) with100 mg/lt proteinase K. The solution was kept at 55 °Cfor 1 hour. At the end of this time period, 5 ml of 5 Mpotassium acetate was added and held in ice for 20minutes, and the solution was centrifuged at 17000rpm for 25 minutes. The supernatant was mixed with0,6 volume of isopropanol and held at -20 °C forminutes and the DNA was precipitated. Phenol-chloroform was used to wash the DNA and a secondprecipitation was done. The DNA was dissolved indH2O and stored at -20 °C. The isolated DNA wasdiluted in such to 50-100 ng/µl to use in polimerasechain reactions (PCR). For PCR reactions, the closestmarker to the RPP7 gene was used (Can, 1997). To dothis, the solution which contains 0.05 mm primer, 2mm dNTPs, 25 mm MgCl2, 1 x Taq buffer and IU TaqDNA polymerase was completed to 25 ml volume. ThePCR reactions were performed at 94 °C for 5 minfollowed by 94 °C for 1 minute, 56 °C for 1 min, 72 °Cfor 13 minute (35 cycles) and 72 °C for 10 min. Thesamples were electrophoresed at 80 W for 4 hours.

RReessuullttss aanndd ddiissccuussssiioonnss

Characterization of P. parasitica isolates

In order to determine the changes at the RPP7 locus inthe 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 islocated at the first chromosome in the Nd-1 ecotype(Can, 1997), and these isolates were presumed torecognize RPP7 allele of Nd-1 ecotype. Wand-1,

Characterisation of RPP7 gene 27

Cand-5, Hind-2 and Hind-4 isolates recognize MRC-Band MRC-C region which present at the secondchromosome in the Col-5 ecotype. The isolatesregenerated from the oospore populations wereinoculated on different A. thaliana ecotypes (Col-5,Ksk-1, Nd-1, Ws-3, Ler-1, Oy-1 and Wei-1) todetermine if they were original. The results are shownin Table1.

As it could be seen in Table 1, the isolatesgenerated from the oospore populations were found tobe as original, and there was no variation (Can, 1997).Therefore these isolates were used to inoculate themutant lines recovered through inoculation with theHiks-1 isolate.

Phenotypic characterization of mutant lines

P. parasitica isolates were used to inoculate the Col-5lines. The results were shown in Table 2.

As indicated in Table 1, DM and EH phenotypesdeveloped, following inoculation of FN3922, FN3928,FN3929 and FN3930 mutant lines with the Hiks-1isolate. These results revealed that the RPP7 gene isnot present in the mutant lines. However, Ahco-1,Ahco-2 and Ahco-7 isolates exhibited the EHphenotype compared to DM in the wild Col-5 ecotype.This result showed that absence of the RPP7 geneincreased susceptibility. The important point in herewas that mutation of one R-gene could effect theresistance in same plant to other isolate. The result

28 Canan Can et al.

TTaabbllee 11:: Interaction phenotypes of different P. parasitica isolates on the A. thaliana ecotypes.

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 FDLAhco-1 DM FN FDL FN FDL FN FDLAhco-2 DM FN FDL FN FN FN DMAhco-7 DM FN FDL FN FDL FN EHWand-1 FN FN EH FN FN FN DMCand-5 FN EH EH FN CN FDL DLHind-2 FN FN EH PN FN FN EHHind-4 FR FN EH PN EH FN EH

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

TTaabbllee 22:: 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 EHAhco-1 DM FDL FN EH EH EH EHAhco-2 DM FDL FN EH EH EH EHAhco-7 DM FDL FN EH EH EH EHWand-1 FN EH FN EH FN FN FDLCand-5 FN EH EH EH FN FN FNHind-2 FN EH FN FN FN FN FNHind-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 eachcotyledon (DM), flecking with delate pathogen sporulation, 5-10 sporangiophorus per each cotyledon (FDL), early and heavypathogen sporulation, 20> sporangiophores per cotyledon (EH).

may also show that when the RPP genes are allelicthey do function in coordination.

Cand-5 isolate recognizes MRC-B region which ispresent in Col-5 and determined with FN interactionphenotype. FN3922 mutant line exhibited the EHphenotype with Cand-5 and FN with other isolatestested (Table 2). This result may show that FN3922may be a Col-5 contamination. On the other word it ispossible that in this line, many RPP genes present inthe MRC-B loci (Can, 1997) could be mutated.Therefore, FN3922 may not be a specific mutant of theRPP7 gene. As it is known the Fast Neutron

application results point mutations, so that at MRC-Bregion in FN3922, many point mutations or deletionsmay have been occurred.

Inoculations of the FN3928, FN3929 and FN3930mutant lines with Wand-1, Hind-2, Hind-4 resulted FNphenotype. This results shows that the RPP7 gene hasno correlation with the RPP genes present in seconderchromosome 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 sevendays the cotyledons from the mutant lines and the wildtype Col-5 were taken and prepared as describedbefore. At the first six hour of inoculation of Col-5with the Hiks-1 isolate cell death was observed. Thesusceptible genotypes and the mutant lines allowed thepenetration and mycelial development without any cell(Figure 1, 2). Fungal development in the mutant linessupported the macroscopic results (Mc Dowell et al.,2000).

DNA analyses of mutant lines

nga280 and g2a markers present at the lower arm ofthe first chromosome of A. thaliana were detected togive less recombination with the RPP7 loci (Can,1997). Therefore the mutant lines were subjected toPCR analyses with these markers. Results are given inFigure 3.

The distributions of the bands in mutant lines weresimilar to those in the wild type Col-5. This result maysuggest that no deletions may occurred in the mutantlines and that the loss of function could be due to apoint mutation.

Characterisation of RPP7 gene 29

FFiigguurree 11:: Microscopic reactions occurred in Col-5 ecotypefollowing inoculation with the Hiks-1 isolate. (A) Six hoursafter inoculation. a. Haustorium formed in the mesofil cells.b. Cell death and (B) Twelve hours after inoculation.Hypersensitive reaction and cell death.

FFiigguurree 22:: Microscopic reactions occured the FN3929mutant line following inoculation with the Hiks-1 isolate.Formation of fungal haustorium in the mesophyl cell (a) andhif development (b), twelve hours after inoculation.

FFiigguurree 33:: Distribution of g2a markers in mutant lines.M indicates the 1 kb DNA marker.

750 bp →

500 bp →


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 Peronosporaparasitica 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 6th

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.

Dietrich RA, Richberg MA, Schmidt R, Dean C andDangl 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 gene-mediated disease resistance in Arabidopsis has

homology to eukaryotic lipases. Proc Natl Acad SciUSA. 96: 3292-3297, 1999.

Feys BJ and Parker JE. Interplay of signaling pathways in plant disease resistance. Trends Genet. 16: 449-455, 2000.

Glazebrook J, Rogers EE and Ausubel FM. Use of Arabidopsis for genetic dissection of plant defense responses. Annu Rev Genet. 31: 547-569, 1997.

Hammond-Kosack KE and Jones JDG. Plant disease resistance genes. Annu Rev Plant Physiol Plant Mol Biol. 48: 575-607, 1997.

Holub EB, Beynon J and Crute I. Phenotypic and genotypic characterisation of interactions between isolates of Peronospora parasitica and accessions of Arabidopsis thaliana. Mol Plant-Microbe Interacts. 7 (2): 223-239, 1994.

Holub EB. and Beynon J. Symbiology of mouse-ear cress (Arabidopsis thaliana) and oomycetes. Adv Bot Res.24: 227-273, 1997.

Lam E, Pontier D and Pozo O. Die and let live-programmed cell death in plants. Curr Opin in Plant Biol. 2: 502-507, 1999.

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 locus of Arabidopsis. Plant Cell. 10: 1861-1874, 1998.

McDowel JM, Cuzick A, Can C, Beynon J, Dangl JL and Holub EB. Downy mildew (Peronospora parasitica) resistance genes in Arabidopsis vary in functional requirements for NDR1, EDS1, NPR1 and salicylic acid accumulation, The Plant Journal. 22 (6): 523-529, 2000.

Parker JE, Holub EB, Frost LN, Falk A, Gunn ND and Daniels MJ. Characterization of eds1, a mutation in Arabidopsis supressing resistance to Peronospora parasitica specified by several different RPP genes. Plant Cell. 8 (11): 2033-2046, 1996.

Parker JE, Coleman MJ, Szabo V, Frost LN, Schmidt R, Van der Biezen EA, Moores T, Dean C, Daniels MJ and Jones JDG. The Arabidopsis downy mildew resistance gene RPP5 shares similarity to the Toll and Interleukin-1receptors with N and L6. Plant Cell. 9: 879-894, 1997.

Richter TE and Ronald PC. The evolution of disease resistance genes. Plant Molecular Biology. 42: 195-204, 2000.

Thomas CM, Jones DA, Parniske M, Harrison K, Balint-Kurti P, Hatzixanyhis K and Jones JDG. Characterization of the tomato Cf-4 gene for resistance to Cladosporium fulvum identifys sequences that determine recognitional specificity in Cf-4 and Cf-9. Plant Cell. 9: 2209-2224, 1997.

Tor M, Holub EB, Brose E, Mussker R, Gunn N, Can C, Crute I and Beynon JL. Map positions of three loci in Arabidopsis thaliana associated with isolate-specific recognition of Peronospora parasitica (downy mildew). Mol Plant-Microbe Interac. 7 (2): 214-222, 1994.

30 Canan Can et al.


Cytokinins, N6-substituted adenine derivatives are aclass of plant hormones that were first identified asfactors that promoted cell division (Miller et al., 1955;1956) and have been implicated in many other aspectsof plant growth and development including shootinitiation and growth, apical dominance, senescenceand photomorphogenetic development (Letham,1971; Thimann, 1980; Mok and Mok, 1994).Although the physiological effects of cytokinins havebeen well documented, the molecular mechanismsunderlying cytokinin action remain obscure (Mok and

Mok, 1994; Binns, 1994).Bioassays are used to establish the relative

biological activity of plant hormones compared withothers. The cytokinin bioassays used most frequentlydepend on growth of tissues in sterile culture (Letham1967). Such methods are extremely sensitive but itneeds at least 3 weeks to get final results. Letham(1971) described a rapid bioassay for cytokinins basedon the ability of these compounds to promotemarkedly the expansion of radish cotyledons excisedsoon after seed germination.

To date the effects of common cytokinins i.e.kinetin, benzyladenine (BA) and its riboside have been


31

TThhee eeffffeecctt ooff mmeettaa--ttooppoolliinn oonn pprrootteeiinn pprrooffiillee iinn rraaddiisshh ccoottyylleeddoonnss

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

AAbbssttrraacctt

Meta-topolin (mT) has been established as an active aromatic cytokinin recently. The present investigation assessedthe effects of mT on radish cotyledon growth and protein content. 0.05 to 1 mM mT increased the cotyledon growthabout 2 fold in fresh weight basis. mT at 0.1, 0.25 and 0.5 mM concentrations caused an increase in soluble proteinlevels compared to the control cotyledons almost in the same ratio by 3 %. Compared to control cotyledons analysisof the soluble proteins displayed different electrophoretic pattern in mT treated cotyledons.

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

MMeettaa--ttooppoolliinniinn ttuurrpp kkoottiilleeddoonnllaarr››nnddaa pprrootteeiinn pprrooffiilliinnee eettkkiissii

ÖÖzzeett

Son y›llarda meta-topolin (mT) aktif aromatik sitokinin olarak saptand›. Bu araflt›rma da mT’in turp kotiledonlar›n›nbüyüme ve protein içeri¤ine etkisi araflt›r›ld›. 0.05-1 mM mT kotiledon büyümesini taze a¤›rl›k baz›nda yaklafl›k 2kat kadar teflvik etti. 0.05, 0.1 ve 0.25 mM mT çözünür protein düzeylerini kontrole oranla yaklafl›k % 3 oran›ndaartt›rd›. Çözünür proteinlerin analizleri, mT uygulanan kotiledonlarda kontrole oranla farkl› bir elektroforetik dizilimgösterdi.

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

documented in radish cotyledons. A new activearomatic cytokinin meta-topolin (mT) have beendetermined by Strnad et al. (1997) in poplar. Thesensitivity of the radish cotyledon bioassay to mT hasbeen established by us before (Palavan-Ünsal et al.,2002). This study will focus on the effect of mT onsoluble protein contents in radish cotyledons that hasnot been studied before.


Plant material and bioassay

Radish (Raphanus sativus L.) seeds were germinated indarkness for 4 days at 25 °C on moist filter paper in 5cm petri dishes. Cotyledons were excised excludingpetiole tissues and four cotyledons were placed in eachpetri dish after measuring the fresh weight. Thecotyledons were placed with their adaxial sides downon the paper. They were incubated in a growth chamberat 25°C ± 2°C and 12 h light-dark photoperiods. Threeml mT was applied per petri dish at 0.05, 0.1, 0.25, 0.5and 1.0 mM concentrations. Cotyledon growth wasmeasured by determining fresh and dry weights 3 daysafter the application (Letham, 1971) and the datapresented 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. Eachexperiment was repeated four times and each treatmentincluded three replicates.

Electrophoresis for proteins

Sodium dodecylsulphate (SDS)-polyacrylamide slabgel electrophoresis was performed according toLaemmli (1970). Gel containing 3.0 % (stacking gel)and 10.0 % (separation gel) acrylamide were preparedfrom a stock solution of 30.0 % of acrylamide and 0.8% N, N’-bis methylene acrylamide. The gels werepolymerized chemically by the addition of ammoniumpersulphate. The mixture was completely dissociatedby immersing the samples for 3 min in boiling water.Electrophoresis was carried out with a current of 150 Vper gel until the bromophenol blue marker reached thebottom of the gel. The proteins were stained in the gelwith Coomassie brilliant blue solution for overnight at

room temperature. The gels were diffusion-destainedby repeated washing in the solution containing 7.5 %acetic acide, 5 % methanole and 87.5 % distilled water.

RReessuullttss aanndd ddiissccuussssiioonn

The early observations revealed that cytokinins exertparallel effects in maintain protein or nucleic acidlevels while inhibiting senescence. Cytokininsstimulate both structural and enzymatic proteinsynthesis. They are selectively increasing the levelsof certain enzymes associated generally withphotosynthetic process (Feierabend, 1969). It is notclear whether the enhanced activity is due to greatersynthesis, inhibition of degradation or activation of theenzymes.

We already observed that new aromatic cytokininmT at 0.25 to 1 mM concentration range delayedthe senescence in excised wheat leaf segments(Palavan et al., 2002). This concentration range washigh for radish cotyledon growth therefore lowerconcentrations were examined (0.05 to 1 mM) inaddition.

Cotyledon growth increased with the treatments ofmT significantly (Figure 1). Stimulation of cotyledongrowth was closely related with increasingconcentrations of mT; 0.05 to 1 mM mT increased thecotyledon growth about two fold in fresh weight basis(p<0.05), while dry weights of cotyledons during thegrowth were not effected by mT application (Palavan-Ünsal et al., 2002).

Cytokinins promote cell enlargement in certaintissues and organs. This effect is most clearly seen incotyledons. The expansion of cotyledons is resulted

32 Serap Ça¤ and Narçin Palavan-Ünsal

FFiigguurree 11:: The effect of meta-topolin on cotyledon growth inradish (Values are average of 30 cotyledons).

from cell enlargement during cotyledon growth.Cytokinin treatment promotes additional cellexpansion with no increase in the dry weight of thetreated cotyledons (Huff and Ross, 1975).

Letham (1971) reported the ability of cytokinins topromote markedly the expansion of radish cotyledonsand explained this response by the promotion of cellenlargement. mT also as a most active aromatic

cytokinin as reported by Strnad et al. (1997) caused tocotyledon growth markedly as shown in Figure 1.

mT was found to increase the soluble proteincontents of radish cotyledons. Treatments with 0.1, 0.25and 0.5 mM mT resulted an increase in soluble proteincontent in the same ratio (by 3,4 and 3 % respectively)compared to the control cotyledons (Figure 2).

These findings correlated with electrophoreticdeterminations (Figure 3). Soluble proteins of mTtreated radish cotyledons were analyzed using SDS-PAGE technique in order to test whether andsignificant amount of difference in protein profileoccurred with mT treatments. Analysis of the solubleproteins displayed different electrophoretic pattern inmT treated cotyledons compared to control. Proteinbands were very sharp and dark in 0.05, 0.1 and 0.25mM mT treated samples and their molecular massesranges between 66 to 45 kDa’s. Molecular mass of 45to 29 kDa’s were weak in 0.5 and 1 mM mT treatedand in control cotyledons also. On the other handprotein bands were very sharp and dark in 0.05, 0.1and 0.25 mM mT treated cotyledons comparing with0.5 and 1 mM mT treated and control cotyledons.Obvious bands were also observed around 30 kDa incotyledons treated with 0.05, 0.1 and 0.25 mM mT.Besides these there were additional bands in mTtreated samples different from controls and thesebands were weak in 0.5 and 1.0 mM mT treatedsamples compared to the other applications around 24kDa.

There is good evidence that cytokinins play a rolein regulating protein synthesis (Tepfer and Fosket,1978). Cytokinins can not only increase the rate ofprotein synthesis, but also change the spectrum ofproteins produced by plant tissues.

Results obtained in this study showed that, totalsoluble protein content in radish cotyledons noteffected from exogenously applied mT. On the otherhand, when protein profile was examinedelectrophoretically additional bands were observed inmT treated samples. These can be explained by the factthat mT stimulate new protein synthesis withouteffecting total protein content.

In conclusion, natural aromatic cytokinin mT hasan important role in the control of cotyledon growthand this response closely associated with proteinprofile. The results of this research are exhibited mT asa promising plant growth regulators in physiologicalstudies.

meta-topolin effect on protein 33

FFiigguurree 22:: The effect of meta-topolin on soluble protein con-tent during the growth of radish cotyledons. Values are aver-age of 4 experiments.

FFiigguurree 33:: SDS-PAGE analysis of soluble proteins frommeta-topolin treated radish cotyledons. Gel was stained withCoomassie 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 mMmT, Lane 6: 1 mM mT treatments. Molecular mass (kDa) ofmarkers are indicated on left hand margin.

AAcckknnoowwlleeddggeemmeenntt

We thank to Dr. M. Strnad and his colleagues for thegenerous gift of aromatic cytokinins and to DamlaBüyüktunçer for technical assistance. This study wassupported by Istanbul University Research Fund(Project number: B-430/13042000).


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.

34 Serap Ça¤ and Narçin Palavan-Ünsal


35

TThhee eeffffeecctt ooff eelleeccttrroommaaggnneettiicc ffiieellddss oonn ooxxiiddaattiivvee DDNNAA ddaammaaggee

Serkan ‹fller1 and Günhan Erdem2*1Department of Biology, Institute of Applied Sciences, Çanakkale Onsekiz Mart University, Çanakkale,Turkey; 2College of Health, Çanakkale Onsekiz Mart University, Çanakkale, Turkey (*author forcorrespondence)

Received 30 September 2002; Accepted 26 December 2002

AAbbssttrraacctt

Many recent studies have focused on the investigation of the biological effects of electromagnetic field. Althoughthe several types of biological effects of electromagnetic fields have been shown, the molecular mechanisms of theseeffects have not been explained yet. Some epidemiological studies have suggested that exposure to ambient, low-level 50-60 Hz electromagnetic fields increase risk of disease including cancer such as leukemia among children wholive close to power lines or among men whose jobs expose them to electromagnetic field, while others havesuggested that electromagnetic fields exposure could increase both the concentration of free radicals and oscillatingfree radicals. Electromagnetic fields are known to affect radical pair recombination and they may increase theconcentration of oxygen free radicals in living cells. In this study, oxidative stress was formed by the oxidation ofascorbic acid and the effect of 50 Hz, 0.3 mT electromagnetic fields on the oxidative DNA damage has beeninvestigated. The results of the study showed that extremely low-frequency electromagnetic fields enhanced theeffect of oxidative stress on DNA damage and supported the idea obtained from the previous studies on an increasingeffect 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

EElleekkttrroommaannyyeettiikk aallaann››nn ookkssiiddaattiiff DDNNAA hhaassaarr›› üüzzeerriinnddeekkii eettkkiissii

ÖÖzzeett

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 molekülermekanizmalar› henüz aç›klanamam›flt›r. Baz› epidemiyolojik çal›flmalar, 50-60 Hz dolay›ndaki düflük düzeylielektromanyetik alana maruz kalman›n yüksek gerilim hatlar›na yak›n yaflamakta olan çocuklarda veyaelektromanyetik alana maruz kalarak çal›flanlarda görülen lösemi gibi kanser vakalar›n› kapsayan hastal›klara iliflkinriski art›rd›¤›n› öne sürerken, baz› çal›flmalar ise elektromanyetik alan maruziyetinin serbest radikalkonsantrasyonunu ve serbest radikallerin izlenebilirli¤ini art›rabilece¤ini ileri sürmüfltür. Elektromanyetik alan›nradikal çifti rekombinasyonunu etkiledi¤i bilinmektedir ve bu da, hücrelerdeki oksijene dayal› serbest radikalkonsantrasyonunu art›rabilir. Bu çal›flmada, askorbik asit oksidasyonu ile oksidatif stres oluflturulmufl ve 50 Hz, 0.3mT düzeyindeki elektromanyetik alan›n, oksidatif DNA hasar› üzerindeki etkisi araflt›r›lm›flt›r. Bu çal›flman›nsonuçlar›, 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› ömrüüzerindeki art›r›c› etkisine dair düflünceleri desteklemifltir.

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


There are many reports on the biological effects ofelectromagnetic fields (EMF) and there have beenmany attempts to develop a theoretical explanation ofthis phenomenon. Some epidemiological studies havesuggested that exposure to ambient, low-level 50/60Hz EMF increases risk of disease including cancersuch as leukemia among children who live close topower lines or among men whose jobs expose them toEMF (Wertheimer and Leeper, 1979; Tomenius, 1986;Savitz et al., 1988; London et al., 1991). EMF firstlyaffects the cell membrane. Some ion channels such asNa-K ATPase have been affected according the levelof EMF. The alteration in the activity of these proteinscauses an increasing or decreasing intracellularconcentration of many ions such as Na+, K+, Mg2+ andCa2+ which plays very important roles in cell signaling.Therefore, the biological effects of EMF expandamong the cellular systems (Goodman et al., 1995).Although the several types of biological effects ofEMF have been shown, the molecular mechanisms ofthese effects have not been explained yet. Somestudies have suggested that EMF exposure could bedue to both the increase in the concentration (Jajte,2000) and oscillating of free radicals (Scaiano et al.,1995). EMF is known to affect radical pairrecombination and they may increase theconcentration of oxygen free radicals in living cells(Jajte, 2000). Increasing the concentration of freeradicals creates oxidative stress and some biologicalreactions such as DNA damage occur under thiscondition. Metabolic energy production or effects ofchemicals and radiation can form oxidative stress.

In this study, oxidative stress was formed by theoxidation of ascorbic acid with Cu2+ ions and the effectof 50 Hz, 0.3 mT EMF on the oxidative DNA damagewas investigated.

MMaatteerriiaallss aanndd mmeetthhooddss

DNA isolation

High molecular weight (app. 10 kb) human genomicDNA was isolated from the white blood cells with themodified method of Poncz et al. (1982) by using MBIFermentas genomic DNA isolation system. Molecularweight and purity of DNA samples were controlled byagarose gel electrophoresis. The concentration of

DNA samples was spectrophotometrically determined.All DNA samples were free from proteins, RNAs andsolvents used for extraction.

Oxidative DNA cleavage reactions

Cleavage reactions were carried out in a mediumcontaining 0.5 µg DNA, 20 mM Tris-HCl (Sigma) pH7.8, 0.25 mM ultra-pure ascorbic acid (Merck) andCuCl2 (Sigma) in the final concentrations of 2.5, 5, 7.5and 10 µM, in a final volume 10 µl. Other antioxidants(glutathion, cystein and dithiothreitol, Sigma) andmetal chelator (EDTA, Merck) were added to thereaction mixtures at a final concentration of 0.5 mM.The mixtures were incubated at room temperature for10, 20 and 30 minutes. Adding EDTA at a finalconcentration of 25 mM stopped reactions. DNAcleavages in the reaction mixtures were analyzed onthe 1% agarose gel (Promega) electrophoresis.

EMF exposure system

Electromagnetic fields were applied by using theHelmholtz coil. The coil system was constructed byusing the polyester sphere that was surrounded bycopper wire with 0.75 cm diameter (Galt et al., 1995).The diameter and height of the sphere were 16 and 26cm, respectively. 50 Hz, 4.5 V electricity was appliedto coil system. As a result, 0.3 mT EMF was generatedat the center of the coil system which includes handlesfor the sample tubes.

RReessuullttss aanndd ddiissccuussssiioonn

The oxidative DNA damage was induced by theconcentration of cupric ions (Figure 1). In the constantascorbate concentration (0.25 mM), oxidative DNAbreakage was started in the presence of 2.5 µMcopper(II) ions and EMF also induced the DNAbreakage at this condition (Figure 1, lanes 4 and 10).Therefore, main DNA band in the lane 10 of Figure 1is thinner than the lane 4. In the presence of highcupric ions concentration, excess scission of DNAmolecules occurred at the EMF when compared tonormal conditions (Figure 1, lanes 6 and 12).Electromagnetic fields did not have an effect on theoxidation of ascorbic acid in the absence of cupric ions(specific data was not shown, but the sample in Figure3, lane 12 had reflected this result, because EDTA was

36 Serkan ‹fller and Günhan Erdem

chelating cupric ions and eliminated their oxidativeeffects).

The results of this study showed that the oxidativeDNA damage depends on the incubation time (Figure 2).DNA breakages could be observed at the 20th minute ofincubation time (Figure 2, lanes 6 and 8). EMFexposure enhances the oxidative DNA damage afterthe 20th minute (Figure 2, lanes 2 and 4).

In the presence of EDTA as a cationic metalchelator, oxidative DNA damage was not observed.This result showed that ascorbate oxidation andoxidative DNA damage depend on cupric ions as anoxidizing agent (Figure 3, lanes 6 and 12). As anantioxidant, cystein did not block the oxidative DNAdamage (Figure 3, lanes 4 and 10). Glutathionereduced the oxidative stress. Therefore, the DNAdamage was formed as aggregation rather thanfragmentation in the presence of glutathione (Figure 3,lanes 3 and 9). Dithiotreitol (DTT) was the mosteffective antioxidant of all investigated but EMFexposure inhibited the effectiveness of DTT (Figure 3,lanes 5 and 11).

The oxidative species produced by ascorbateoxidation in the presence of copper(II) ions damagethe DNA molecules (Figure 1). Previously DNAdamage depending on ascorbate oxidation had beenstudied (Erdem et al., 1994; Zareie et al., 1996).Oxidative DNA damage was observed asfragmentation or aggregation. The degree of oxidativeDNA damage varies in the levels and reactivity of freeradicals produced in the reaction medium. In thepresence of oxygen, the hydroxyl and peroxyl radicalssuch as superoxide anion and hydroperoxyl radical areproduced by the reaction between radical form ofascorbic acid (ascorbyl radical) and molecular oxygen(Fuchs et al., 1990).

These radicals attack to electrophilic nuclei on thetargets and create secondary carbon radicals. At the

EMF and oxidative DNA damage 37

FFiigguurree 11:: The effect of EMF and Cu(II) concentrations onoxidative DNA damage. All lanes include 0.5 µg DNA. TheDNA samples in the lanes from 1 to 6 were incubated undernormal condition, 7 to 12 were incubated in EMF at roomtemperature in the presence of 0.25 mM ascorbic acidexcept 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 12lanes. Incubation time was 30 min for all samples.

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

FFiigguurree 22:: The effect of incubation time on oxidative DNAdamage depends in electromagnetic fields. All lanes include0.5 µg DNA. Incubation times were 30 min from 1 to 4, 20min from 5 to 8 and 10 min from 9 to 12. Ascorbic acidconcentration were 0.25 mM in all lanes. Cu(II)concentrations were 2.5 µM in 1, 3, 5, 7, 9 and 11 while5 µM in 2, 4, 6, 8, 10 and 12. The samples in 1, 2, 5, 6, 9 and10 were incubated in EMF. Other samples were incubated atnormal conditions.

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

FFiigguurree 33:: The effect of some antioxidants (glutathione,cystein, dithiothreitol) and metal chelator (EDTA) onexcessive oxidative DNA damage in EMF. All lanes include0.5 µg DNA. Ascorbic acid and Cu(II) concentrations were0.25 mM and 7.5 µM in all lanes except the control DNAlanes 1 and 7, respectively. Lanes 2 and 8 were scissioncontrols. 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 wereincubated at normal condition. The others were incubated inEMF. Incubation time was 30 min for all samples.

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

high level or high reactivity of these radicals, excessformation of secondary carbon radicals on the sameDNA molecule causes a reaction between each otherand then the DNA damage occurs as fragmentation.Therefore, DNA size became smaller and gave thesmeared patterns on gel electrophoresis (lanes 5 and 6in Figure 1) However, at the low level or low reactivityof oxygen species, the oxidative DNA damage resultsin aggregation of the DNA molecules with theintermolecular reaction of the secondary carbonradicals. Thus, the DNA samples became heavier andwere retarded on the gel electrophoresis (lanes 3 and 9in Figure 3).

Our results showed that extremely low-frequencyEMF enhanced the effect of oxidative stress on DNAdamage and supported the idea obtained from previousstudies on an increasing effect of EMF on theconcentration 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 inFigure 2 and lane 5 to lane 11 in Figure 3, indicate thatthe degree of the oxidative stress under the EMF isgreater than the normal condition.

In the brain cells of rats, an increase in DNAsingle- and double-strand breaks had been found afteracute exposure to a sinusoidal 60 Hz magnetic field.When the experiment was carried out in the presenceof melatonin or a radical scavenger compound N-tert-butyl-alpha-phenylnitrone (PBN), the effect ofmagnetic fields on brain cell DNA was not observed(Lai and Singh, 1997). Melatonin is a neurohormoneand it is also an antioxidant and a free radicalscavenger. Therefore, this hormone could protectbiological systems against oxidative damage. Theincreasing effect of EMF on the concentration of freeradicals has been suggested that melatonin suppressionin humans may increase the probability of mutagenicand carcinogenic risk (Jajte and ZmySlony, 2000).

EMF (≥1 mT) increases the concentration of freeradicals that escape from the alkyl sulphate andsulphonate micelles. The effect of extremely low-frequency EMF on the radicals formed from singletprecursors is larger than triplet precursors. Someradicals such as hydroxyl and peroxyl radicalsgenerated in the biological reactions are formed fromsinglet precursors (Eveson et al., 2000).

In conclusion, the results obtained from our studysuggest that the effects of extra low frequency EMF onthe concentration of free radicals and therecombination of radical pairs might trigger thecarcinogenesis in the populations living close to theoverhead electric power distribution lines.


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-alpha-phenylnitrone block 60-Hz magnetic field-induced DNAsingle 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.

38 Serkan ‹fller and Günhan Erdem


39

CChhrroommoossoommeess ooff aa bbaallaanncceedd ttrraannssllooccaattiioonn ccaassee eevvaalluuaatteedd wwiitthh aattoommiicc ffoorrccee mmiiccrroossccooppyy

Zerrin Y›lmaz1*, Mehmet Ali Ergun2, Erdal Tan3

1Department of Medical Biology and Genetics, Baskent University, Faculty of Medicine, 06570,Maltepe, Ankara, Turkey; 2Department of Medical Biology and Genetics, Gazi University, Faculty ofMedicine, 06510, Besevler, Ankara, Turkey; 3Materials Research Department, Ankara Nuclear Researchand Training Center, 06100, Besevler, Ankara, Turkey (*author for correspondence)

Received 2 December 2002; Accepted 30 December 2002

AAbbssttrraacctt

A couple was referred to our genetics department for cytogenetic analysis because of two previous abortions. Thecytogenetic 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 fordetecting detailed structures of chromosomes. With the help of this new technique the surface topography of humanchromosomes can be examined. We used AFM in order to analyse the surface topography of derivative chromosomesof the patients, and found a 0.6 µm gap region. In this study, we aimed to examine the differences between the imagesof the derivative chromosomes detected by light and atomic force microscopy analyses.

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

DDeennggeellii ttrraannssllookkaassyyoonn vvaakkaass››nnddaa kkrroommoozzoommllaarr››nn aattoommiikk ggüüçç mmiikkrroosskkoobbuu iilleeddee¤¤eerrlleennddiirriillmmeessii

ÖÖzzeett

Ardarda iki gebelik kayb› nedeniyle departman›m›za yönlendirilen çiftin sitogenetik analizleri yap›lm›flt›r. Erkektenormal kromozom kuruluflu 46, XY saptanm›fl ancak kad›nda dengeli translokasyonla birlikte14. kromozoma ait sentromer art›fl› saptanm›flt›r; 46, XX, t (7;12) (p21;q14), 14 cenh+. Proband›n ailesinde yap›lansitogenetik çal›flma ile ayn› kromozom kuruluflunun proband›n annesinden kal›t›ld›¤› saptanm›flt›r. Atomik güçmikroskobu kromozomlar›n yap›sal olarak detayl› incelenmesinde kullan›lmaktad›r. Bu yeni tekni¤in yard›m›ylainsan kromozomlar›n›n yüzey topografisi incelenebilmektedir. Biz de atomik güç mikroskobunu kullanarak derivatifkromozomun yüzey topografisini araflt›rd›k ve 0.6 µm’lik bir gap bölgesi saptad›k. Bu çal›flmada probanda aitderivatif kromozom yap›s›n› hem ›fl›k mikroskobu hem de atomik güç mikroskobu ile ayr› ayr› de¤erlendirereksonuçlar›m›z› karfl›laflt›rd›k.

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


Cytogenetics is the study of genetic material at the

cellular level. Human cytogenetics is almost alwaysconcerned with light microscope studies ofchromosomes. Staining procedures which provide a

uniform unbanded appearance to chromosomes arereferred to solid or covential staining. They can,however, be useful for studies on chromosomebreakage as scoring gaps and breaks can be difficult inlightly stained chromosome bands. Giemsa banding(G-banding) has become the most widely usedtechnique for the routine staining of humanchromosomes. The chromosome banding patternsobtained reflect both the structural and functionalcomposition of chromosomes. Consititutiveheterochromatin is the structural chromosomalmaterial seen as dark staining material in interphase aswell as during mitosis. It includes both repetetiveDNA, satellit DNA and some non-repetetive DNA.C-banding can be used to demonstrate the repetetiveDNA (Benn and Perle, 1992).

Atomic force microscopy (AFM) is a diagnostictool for detecting detailed structures of thechromosomes and the surface topography of humanchromosomes can be examined using this newtechnique (Binning et al., 1986; Musio et al., 1997).AFM could be considered as a tool for furtherchromosomal studies.

In our previous studies using AFM, we showedthat, unbanded human metaphase chromosomesdisplayed a banding pattern similar to G-bands, and forthe first time we have provided an AFM imaging ofchromosomes in trisomy 13, 21 and KlinefelterSyndrome patients (Ergun et al., 1999). Besides, G andC-banding patterns of chromosomes were alsoinvestigated (Sahin et al., 2000; Tan et al., 2001).

In this study, we used AFM in order to analyse thesurface topography of derivative chromosomes of afemale patient whose daughter was referred to ourGenetics department with the chief complaints ofabortions.

MMaatteerriiaallss aanndd mmeetthhooddss

Case presentation

In this study we evaluated the chromosomes of afamily. This family was referred to our geneticsdepartment for cytogenetic analysis because of twoprevious abortions during the first trimester. They hadno live-born children after a marriage of 5 years. Themale was 37 years old and healthy, and his non-consanguineous wife was 36 years old. Her physicalexamination revealed no abnormalities in

genitourinary, endocrinological and other organsystems; also laboratory findings were normal.

Light microscopy analysis

Metaphase chromosome preparation was obtainedfrom peripheral blood lymphocytes using standardtechniques (Verma and Babu, 1995). Conventionalcytogenetic analysis was carried out using GTG-banding and C-banding techniques (Benn and Perle,1992). The chromosome images were captured bycomputer imaging (Cytovision system, Imageanalysis, Applied Imaging, Saunderland, UK).For each patient we analysed 20 metaphases, andC-banding procedure was performed whileinvestigating 14 cenh+.

Atomic force microscopy and analysis

The AFM used in this study was TopoMetrixTMX2000 Explorer, operating in contact mode and air.Throughout the surface analysis, we have usedstandard pyramidal tip (1520-00) with the radius ofcurvature of approximately 1000 A°. During thesurface analysis, the metaphase region was primarilydetermined and addressed by light microscopy. Later,the region under consideration was scanned via AFMat various scan ranges changing from 150 µm down to10 µm or less to image the chromosomes in a goodmanner. The applied force and the image resolutionwere between 1 and 3 nN and 400x400 pixels (orhigher) respectively for each image acquisition. Theraw data gathered were analysed by using the softwareof the microscopy system in two or three-dimensionalpatterns.

In our study, the chromosomes of the patient werespread on glass surface. Then, the metaphase spreadswere analysed by AFM. Line measure analysis wasperformed on derivative chromosomes.

RReessuullttss

The karyotype of the male revealed 46, XY, while thecytogenetic analysis of his wife was karyotyped as46, XX, t (7;12) (p 21;q 14); a balanced translocation.She also had 14cenh+. In order to understand the originof this translocation chromosome, her mother waskaryotyped and she was also found to be a translocationcarrier; 46, XX, t (7;12) (p 21; q14) and 14 cenh+.

40 Zerrin Y›lmaz et al.

The karyotypes of the mother and the daughterwere similar; 46, XX, t (7;12) (p 21; q 14). The partialkaryotype of the daughter (Figure 1a) and the mother(Figure 1b) with the derivative chromosomes 7, 12 andchromosomes 14 with C-banding images are shown.

We performed detailed measurements on thederivative chromosomes of the mother and detectedthe attached chromosome fragment from derivativechromosome 12 to the derivative chromosome 7. Ourmeasurements revealed a 0.6 µm gap region (Figure 2).Besides we also analysed the satellite region ofchromosome 14 (Figure 3) of the mother.


In this study we evaluated the detailed structures oftranslocated chromosomes of the mother with AFM.Line measure analysis revealed a gap region on thederivative chromosome 7, which was measured as 0.6µm. It was equivalent to a mid-sized G-band region(Figure 2).

In the previous studies, it was reported that thesegap regions correspond to narrowing and groovingregions on chromosomes, and are considered asnegative G- band regions (Uehara et al., 1996). Thegap regions were also observed in fragile X (Harrisonet al., 1983) and in radiation exposed chromosomes(Mullinger and Johnson, 1987). It was thought that,these regions correspond to high-order structuralaberrations resulting from an incomplete or irregularcomposition of chromatid fibres induced by atranslocation of a chromosomal fragment. The gapregions were the results of chromosomalrearrangements (Uehara et al., 1996).

Our second analysis was on the satellite region ofthe chromosome 14 of the mother. First of all, GTG-banding revealed an increase in the heterochromatinregion of the short arm of chromosome 14. Then, weperformed C-banding procedure to understand if thisregion was belonging to a constitutive heterochromatinregion, or to an extra banding region. The results ofC-banding confirmed that these regions werebelonging to heterochromatin region. Our 3-dimensional AFM analysis for the satellite regionshowed an augmentation on the short arm of thischromosome (Figure 3). These heterochromatinregions are polymorphic regions, and they are highly

AFM in chromosome evaluation 41

FFiigguurree 11:: The partial karyotype of the daughter (a) and themother (b); derivative chromosomes 7 and 12 andchromosomes 14 with C-banding images are shown.

FFiigguurree 33:: AFM image of chromosome 14 indicated by anarrow.

FFiigguurree 22:: AFM image of derivative chromosome 7 and linemeasure analysis of the gap region.

repetitive regions that are located on the centromeresof chromosomes 1, 9, and 16 and on the distal arm ofY chromosome (Burkholder and Duczek, 1980; Cook,1995). Our AFM image also helps us to understandthat these regions were not belonging to G- bandingregions, as there was not a banding pattern (Tan et al.,2001).

AFM can be considered as a novel technique foranalysing detailed structures of chromosomes for itsline measure analysis and 3-D image capturecapabilities. Reflecting these capabilities, AFM helpedus to investigate the gap region on the derivativechromosome and this study is also novel by makingnew implementations on the mechanism oftranslocation. As a conclusion, the capability of AFMfor detecting chromosomal abnormalities will reflectlight into further studies.


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 fragileX: 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.

42 Zerrin Y›lmaz et al.


43

EEffffeecctt ooff eeppiirruubbiicciinn oonn mmiittoottiicc iinnddeexx iinn ccuullttuurreedd LL--cceellllss

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

AAbbssttrraacctt

Cancer chemotherapy is an additional application to surgical operations and radiotherapy in the treatment ofwidespread tumors. An anthracycline-derived antibiotic, epirubicin (EPI) is one of the clinically used antineoplasticdrugs. In this study the cytotoxic effects of EPI in transformed mouse fibroblasts (L-cell) were examined. EPIconcentrations 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. Theresults showed that EPI diminished mitotic index of L-cells depends upon time and applied concentrations. Thisdecrease was found statistically significant in each treatment group when compared to control (p<0.05 - p<0.001).

KKeeyy wwoorrddss:: Epirubicin, L-cell, transformed fibroblast, mitotic index, in vitro

EEppiirruubbiissiinniinn kküüllttüürrddeekkii LL--hhüüccrreelleerriinnddee mmiittoottiikk iinnddeekkssee eettkkiissii

ÖÖzzeett

Kanser kemoterapisi, yayg›n tümörlerin tedavisinde cerrahi uygulama ve radyoterapinin yan›nda gerçeklefltirilen ekbir uygulamad›r. Antrasiklin türevi bir antibiyotik olan epirubisin (EPI) klinik olarak kullan›lan antineoplastikilaçlardan birisidir. Bu çal›flmada, EPI in sitotoksik etkileri, transforme edilmifl fare fibroblastlar›nda (L-hücreleri)araflt›r›ld›. EPI in 0.001 µg/ml, 0.01 µg/ml ve 0.1 µg/ml konsantrasyonlar› 2, 4, 8, 16 ve 32 saat süresince hücrelereuyguland›. Sonuçlar uygulanan zaman ve konsantrasyona ba¤l› olarak EPI in L-hücrelerinin mitotik indeksde¤erlerini düflürdü¤ünü gösterdi. Bu düflüfl kontrol grubu ile karfl›laflt›r›ld›¤›nda, her bir deney grubunda istatistikolarak anlaml› bulundu (p<0.05 - p<0.001).

AAnnaahhttaarr ssöözzccüükklleerr:: Epirubisin, L-hücreleri, transforme edilmifl fibroblast, mitotik indeks, in vitro


Of the cancer drugs in clinical use, the anthracyclineshave a spectrum of antitumor activity and are clearlythe most useful cancer drugs among the naturalproduct (Chabner and Mayers, 1993).

Epirubicin (EPI) is the epimer of the anthracyclineantibiotic doxorubicin, with inversion of the 4’-hydroxyl group on the sugar moiety, and has been usedalone or in combination with other cytotoxic agents inthe treatment of a variety of malignancies (Young,

1989; Zuckerman et al., 1993). This drug is commonlyused since it has an equivalent spectrum of antitumoraction as doxorubicin but with less systemic andcardiac toxicity (El-Mahdy Sayed Othman, 2000).

The mechanism of anti-tumour action for EPI hasnot been completely elucidated. Various studies haverevealed that EPI enters the cells rapidly and islocalised in nuclei and forms a complex with DNA byintercalation between DNA strands (Di marco, 1984).DNA replication and transcription have been shown tobe inhibited by this intercalation (Lollini et al., 1989;

Skladonowski and Konopa, 1994). In addition,topoisomerase-II has also been shown to be inactivatedby EPI (Robert and Gianni, 1993; Haldane et al.,1993).

In vitro studies showed that EPI possessescytotoxicity at least equivalent to that of doxorubicinagainst a variety of animal and human tumor cell linesincluding 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; Bartkowiaket al., 1992).

EPI is a cell cycle phase non-specificanthracycline, with maximal cytotoxic effects in the Sand G2 phases. Preliminary in vitro studies werecarried out on HeLa cells. The first tests demonstratedthat EPI and doxorubicin gave essentially the sameinhibition of HeLa cell colony formation (Di marco etal., 1976). Similarly, EPI was as active as doxorubicinon mouse embryo fibroblast proliferation (Di marco etal., 1977), but was taken up in greater amount thandoxorubicin by L1210 leukemia cells in vitro (Wilsonet al., 1981).

There have been few studies about the effect of EPIon mitotic index of rapidly proliferating cells. In thisstudy, 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 culturewhich was investigated by measuring mitotic index inorder to investigate the effectiveness of this drug inchemotherapy.


Chemical

EPI (4’-epidoxorubicin), an anthracycline antibiotic, isa doxorubicin stereoisomer, possessing the L-arabinoinstead of the L-lyxo configuration of the sugar moiety(Figure 1). In EPI therefore the hydroxyl group on thesugar moiety, possessing the stable 1C4 conformation,has an equatorial orientation (Plosker and Faulds,1993).

Cell line

The cells used in this study were derived from mousefibroblast by in vitro malign transformation (Earle,

1943). Transformed L-cells obtained from mousesubcutaneous connective tissue in 1943. They weresupplied by Dr. P.P. Dendy of Department ofRadiotherapeutics, Cambridge University, in 1975.The cells were grown in Medium-199 (M-199, Gibcolab.) containing 10% foetal bovine serum (FBS, Gibcolab.), 100 µg/ml streptomycin and 100 IU/mlpenicillin, and were passaged twice a week inappropriate number of 25 cm2 flasks and the volume ofthe complete medium in each flask was completely to12 ml. Cells were removed from the surface of cultureflasks by addition of 0.25% trypsine (Gibco lab.) andcentrifuged for 3 minutes at 1500 cycle/min.Following the addition of M-199 on the cellprecipitate, 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 werecultured on the cover-slips as 3.104 cells/ml in petridishes and incubated for 24 hours with 95% air and 5%CO2 containing medium at 37°C with pH 7.2 in adessicator. At the end of this incubation medium wasremoved, replaced with medium containing EPIconcentrations.

Drug application

Epirubicin (Farmorubicin, Carlo Erba) was dissolvedimmediatedly before use in sterile medium (M-199) togive the required concentration. We used 0.001 µg/ml,0.01 µg/ml and 0.1 µg/ml concentrations of EPI. Cellswere treated with these doses for 2, 4, 8, 16 and 32hours.

44 Gül Özcan Ar›can and Mehmet Topçul

FFiigguurree 11:: Structural formulae of EPI.

Mitotic index analysis

Mitotic index were studied by the methods of Feulgen.Before the cells were treated with Feulgen, they wereprepared with 1 N HCl at room temperature for 1minute and then hydrolized with 1 N HCl for 10.5minutes at 60°C. After slides were treated withFeulgen, they were rinsed for few minutes in distilledwater and stained with 10% Giemsa stain solution pH6.8, for 3 minutes and washed twice in phosphate

buffer. After staining, the slides were rinsed in distilledwater. And then the slides were air dried. At lastmitotic index were calculated by counting metaphases,anaphases and telophases for each tested drugconcentration and control (Figure 2). At least threethousands cells were examined from each slide formitotic index.

Statistical analysis

Mitotic index values which obtained from experimentswere calculated to evaluate the statistical analysis. Thedifferences between the percentage distrubition of Mphase of the various treatment groups and control werecompared by the Student-t test (n=25).

RReessuullttss

The effect of EPI on mitotic index of L-cellsin culture was investigated. EPI concentrations of0.001 µg/ml, 0.01 µg/ml and 0.1 µg/ml were applied tothe cells for time periods of 2, 4, 8, 16 and 32 hours. In this study, EPI diminished the mitotic index of L-cells with increasing both treatment time and drugconcentration compared to controls (untreated group).From the value of 2 hours treatment, we saw that allEPI concentrations had a rapid effect. In subsequenthours, this effect seemed to continue. The values ofmitotic index reached a minimum at EPI concentrationof 0.1 µg/ml with increasing drug concentration. Table1 reveals that treatments of EPI decreased thepercentage of the cells at M phase. With increasingtime the differences among the effects of various drugconcentrations tended to be lower being very small at2 to 8 hours applications. The inhibition of mitosis washigher in 16 and 32 hours applications than those in 2,4 and 8 hours EPI applications in Table 1 especiallywith EPI concentration of 0.1 µg/ml. However, in thetreatment of 0.1 µg/ml concentration, mitoticinhibition reached a maximum at 32 hours application.The values of mitotic index of the cells treated withEPI for 32 hours showed that mitotic index decreasedas drug concentrations were increased.

Epirubicin effect on mitotic index 45

FFiigguurree 22:: Mitosis in L-cells under the light microscope(3.3x100).

TTaabbllee 11:: Mitotic index values in cultures of L-cells treated with various concentrations of EPI, given in mean ± Standard devia-tion (SD).

Mitotic index (%)

EPI 2 hours 4 hours 8 hours 16 hours 32 hoursconcentrations

Control 1.44 ± 0.12 SD 1.93 ± 0.14 3.04 ± 0.08 3.39 ± 0.15 3.84 ± 0.210.001 µg/ml 1.35 ± 0.09 a 1.80 ± 0.10 a 2.72 ± 0.07 a 2.96 ± 0.04 b 3.10 ± 0.30 b

0.01 µg/ml 1.29 ± 0.11 a 1.79 ± 0.05 b 2.61 ± 0.06 b 2.70 ± 0.13 b 2.99 ± 0.16 b

0.1 µg/ml 0.94 ± 0.02 c 1.04 ± 0.01 c 1.77 ± 0.09 c 1.85 ± 0.08 c 1.02 ± 0.22 c

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

EPI significantly decreased the mitotic index incultures of L-cells. The results show that EPIdecreased the mitotic index at significant level p<0.05- p<0.01 for lower drug concentrations 0.01 µg/ml and0.001 µg/ml, at highly significant level p< 0.001 for0.1 µg/ml drug concentration when compared with thecontrol.

In addition, the reductions in mitosis of the cellsfollowing different treatment times (2, 4, 8, 16 and 32hours) with 0.1 µg/ml EPI concentration werestatistically significant (p<0.001) from each other.However, this level of significance for the differenttreatment times was not observed with 0.01 µg/ml and0.001 µg/ml concentrations of EPI, respectively.


Anthracycline antibiotics have been used extensivelyin the treatment of wide variety of malignancies, andare a standard component of many combinationchemotherapy regimens. EPI has been used alone or incombination with other antineoplastic agents in thetreatment of a broad range of neoplasms. Studies inunderstanding the mechanism of EPI have suggestedthat EPI forms a complex with DNA by intercalationbetween DNA strands, thus inhibiting DNA replicationand transcription (Özcan et al., 1997; Stewart et al.,1997), and it increases DNA strand brekage (Cantoni etal., 1990).

EPI induced differentiation of humanneuroblastoma cells in vitro, possibly related to areduction in the growth of surviving cells, thusallowing activation of intrinsic differentiationmechanisms. Following culture of humanneuroblastoma cell lines with EPI 10 or 100 nmol/L for24 hours, outgrowth of neurite processes wasdetectable 3 or 4 days after exposure, and maximalmorphological differentiation was achieved after 5 or 6days (Rocchi et al., 1987).

EPI has also been shown to be effective ininhibiting basement membrane degradation, a propertydeemed necessary to prevent development ofmetastases (Plosker and Faulds, 1993). In addition, EPIhas been shown to inhibit proliferation of allneuroblastoma cell lines by 69 to 78 % relative tocontrols (Rocchi et al., 1987), of a human alveolarrhabdomyosarcoma cell clone (Lollini et al., 1989),and of haemopoietic progenitor cells from severalhuman leukaemic cell lines in liquid culture (Bagnaraet al., 1987).

Although, in vitro studies with antitumour agents,and with anthracyclines in particular, have not shownto predict the antitumour activity in vivo (Sinha andPoliti, 1990; Nistico et al., 1999), in some studies,significant correlations have been detected between thein vitro activity of EPI and other anthracyclines againstvarious tumour specimens, and therapeutic response(Bartkowiak et al., 1992; Plosker and Faulds, 1993).

Anthracyclines, including EPI, appear to result inmaximal cell death in the S and G2 phases of the cellcycle, but cytotoxic effects may occur in the G1 and Mphases at higher drug concentrations (Plosker andFaulds, 1993; Topçul et al., 2002). Maximal lethaleffects of EPI were demonstrated in the S and G2

phases of the cell cycle in murine and human tumourcell lines (Hill and Whelan, 1982).

An important comparison between EPI anddoxorubicin in vitro was carried out by Hill andWhelan (1982). The studies were performed on a widerange of murine and human cell lines: NIL8 (Syriangolden hamster cells); four human tumor lines (COLO-206 and LOVO, derived from colon carcinomas; SCC-T/G, derived from a squamous cell carcinoma from thetongue; CHP 100, derived from a neuroblastoma);L5178Y lymphoblastoid cell sub-lines. In the all celllines tested, both drugs showed comparablecytotoxicity, which increased exponentially with drugconcentration and with duration of exposure. Of highinterest was the study carried out on NIL8 cellssynchronized by mitotic selection and treated for 1hour with the drug. Results showed that maximum cellkill was observed with both drug in the S phase, somekill during early G2, but no kill in G1 and M if lowconcentrations were used. Data from flowmicrofluorimetry analyses and monitoring of mitoticindices suggested population arrest in G2 fordoxorubicin (Casazza and Giuliani, 1984).

In the present study, treating L-cells with variousconcentrations of EPI for 2, 4, 8, 16 and 32 hours,decreased mitosis. The results showed that EPIdiminished mitotic index of L-cells, depending upontime and applied concentrations. When compared tothe control, this decrease was found statisticallysignificant in each group (p<0.05-p<0.001). The valuesof mitotic index reached a minimum at EPIconcentration of 0.1 µg/ml with increasing treatmenttime. Increased concentrations resulted in a decreaseon the values of mitotic index, being statisticallysignificant (p<0.001). Briefly, EPI concentration of 0.1µg/ml showed to possess relatively more effect onproliferation of L-cells. Thus, the results of our study


seem to be concordant with the above mentionedstudies suggesting that cytotoxic effects of EPI mightoccur in the G1 and M phases at higher drugconcentration.

In our study, decreases in the mitotic index of cellswith increasing both treatment time and EPIconcentration have confirmed that EPI is an effectiveinhibitor of mitosis.

In conclusion, the results of this study declared thecell kinetics and cytotoxic effects of the anticancerdrug, EPI, in treated cultures of L-cell line. AlthoughEPI has less systemic and cardiac toxicity thandoxorubicin and other anthracyclines with anequivalent spectrum of antitumor action, it still hascytotoxic effects.

AAcckknnoowwlleeddggmmeenntt

We would like to thank Prof. Dr. Atilla Özalpan for hiskind help and critics.


Bagnara GP, Rocchi P, Bonsi L. The in vitro effects of epirubicin on human normal and leukemic hemopoietic cells. Anticancer Research. 7: 1197-1200, 1987.

Bartkowiak D, Hemmer J, Rottinger E. Dose dependence of the cytokinetic and cytotoxic effects of epirubicin in vitro. Cancer Chemother Pharmacol. 30 (3):189-192, 1992.

Cantoni O, Sestili P, Cattabeni F. Comparative effects of doxorubicin and 4’-epidoxorubicin on nucleic acid metabolism and cytotoxicity in a human tumor cell line. Cancer Chemother and Pharmacol. 27: 47-51, 1990.

Casazza AM and Giuliani FC. Preclinical properties of epirubicin. In: Advances in Anthracycline Chemotherapy: Epirubicin. Bonadonna G (Ed). Masson, Milano-Italy.31-40, 1984.

Chabner BA and Myers CE. Antitumor antibiotics,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. Relationship between activity and aminosugar stereochemistry of daunorubicin and adriamycin derivates. Cancer Res. 36: 1962-1966, 1976

Di marco A, Casazza AM, Dasdia T, Necco A, Pratesi G, Rivolta P, Velcich A, Zaccara A, Zunino F. Changes of activity of daunorubicin, adriamycinand stereoisomers following the introduction or removal of hydroxyl groups in the amino sugar moiety. Chem Biol Interac.19: 291-302, 1977.

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 fibroblast cultures and changes seen in the living cells.J Nat Cancer Inst. 4: 165-212, 1943.

El-Mahdy Sayed Othman O. Cytogenetic effect of the anticancer drug epirubicin on Chinese hamster cell line in vitro. Mutation Res. 468: 109-115, 2000.

Haldane A, Finlay GJ, Baguley BC. A comparison of the effects of aphidicolin and other inhibitors on topoisomerase II-directed cytotoxic drugs. Oncol Res.5 (3): 133-138, 1993.

Hill BT and Whelan RDH. A comparison of the lethal and kinetic effects of doxorubicin and 4’-epidoxorubicinin vitro. Tumori. 68: 29-37, 1982.

Lollini PL, De Giovanni C, Del Re B. Myogenic differentiation of human rhabdomyosarcoma cells induced in vitro by antineoplastic drugs. Cancer Research. 49: 3631-3636, 1989.

Nistico C, Garufic C, Barni S, Frontini L, Galla DA, 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. 13th National Congress of Biology. Istanbul, Turkey. 3: 267-276, 1996.

Özcan FG, Topçul MR, Y›lmazer N, R›dvano¤ullar› M. Effect of epirubicin on 3H-thymidine labelling index in cultured L-strain cells. J Exp Clin Cancer Res. 16(1): 23-27, 1997.

Plosker GL and Faulds D. Epirubicin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in cancer chemotherapy. In: Drugs. Chrisps P (Ed). 0012-6667. 45 (5): 788-856, 1993.

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. Anticancer Research.7: 247-250, 1987.

Sinha BK and Politi PM. Anthracyclines. Cancer chemother apy. Biol Response Modif. 11: 45-57, 1990.

Skladanowski A and Konopa J. Interstrand DNAcrosslinking induced by anthracyclines in tumour cells. Biochem Pharmacol. 47 (12): 2269-2278, 1994.

Stewart DJ, Cripps MC, Dahrouge RGS, Yau J, Tomiak E, Huan S, Soltys K, Prosser A, Davies RA. Pilot study of multiple chemotherapy resistance modulators plus epirubicin in the treatment of resistant malignancies. Cancer Chemother Pharmacol. 41: 1-8, 1997.

Topçul MR, Ar›can Özcan G, Erensoy N, Özalpan A. Effect of epirubicin and tamoxifen on labelling index in FM3Acells. J Cell and Mol Biol. 2: 81-85, 2002

Epirubicin effect on mitotic index 47

Wilson RG, Kalonaros V, King M. Comparative inhibition of nuclear RNA synthesis in cultured mouse leukemia L1210 cells by adriamycin and 4’-epi-adriamycin. Chemico-Biological Interaction. 37: 351-363, 1981.

Young CW. Clinical toxicity of epirubicin. Update on epirubicin. In: Advances in Clinical Oncology. Robustelli della cuna G and Bonadonna G (Ed). Edimes-Pavia, Italy. 29-38, 1989.

Zhang W, Zalcberg JR, Cosolo W. Interaction of epirubicin with other cytotoxic and anti-emetic drugs. Anticancer Drugs. 3 (6): 593-597, 1992.

Zuckerman KS, Case-Dc JR, Gams RA. Chemotherapy of intermediate- and high-grade Non-Hodgkins’s lymphomas with an intensive epirubicin-containing regimen. Blood. 82 (12): 3564-3573, 1993.


PP rroossttaattee ccaanncceerr aanndd iimmppoorrttaannccee ooffttuummoorr mmaarrkkeerr ssttuuddiieess

PP rroossttaatt kkaannsseerrii vvee ttüümmoorr mmaarrkk››rrllaarr›› iilleeiillggiillii ççaall››flflmmaallaarr››nn öönneemmii

Prostate cancer is the most commonly new diagnosednoncutaneous malignancy in men in USA. In the year2002, according to the health statistics 189,000 men inthe United States are expected to be diagnosed with thedisease and 30,200 men are expected to die of it.Incidence varies greatly, with African Americanshaving the highest incidence in the world (224 casesper 100,000 population). The incidence of prostatecancer in African Americans stands in stark contrast tothe 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 thatindicate the presence of malignancy. They arepotentially useful in cancer screening, aidingdiagnosis, assessing prognosis, predicting in advance alikely response to therapy, and monitoring patientsbefore and after diagnosis. Because of low prevalanceof most cancers in the general population and thelimited sensivity and spesificity of avaible markers,these tests alone are generally of little value inscreening for cancer in healthy subjects. Currently,however, prostate spesific antigen (PSA) incombination with digital rectal examination (DRE) areundergoing evaluation as screening modalities forprostate cancer. Because of a lack of sensitivity andspesificity markers are rarely of use in early diagnosisof cancer. Also they can be used as monitoring diseaseevaluation with therapy. The goal of future researchshould be that development of the most specific, cheapand easy markers for common cancer types as prostatecancer.PP rroossttaattee ssppeessiiffiicc aannttiiggeenn:: Screening prostate cancerprovides a dilemma unique among cancer sites. Thebest strategy is determination of the ratio of theprostate serum antigen (PSA) to the volume of theprostate gland in prostate cancer diagnosis.

Determination of the free PSA (i.e., the percentage ofPSA that is unbound to serum proteins) has also beensuggested as a means of distinguishing malignancyfrom benign hyperplasia. PSA has revolutionized themanagement of prostate cancer since its developmentin the 1980s. For unclear reasons, PSA derived frommalignant epithelial cells tends to bind more avidly toserum proteins. Thus, in men with an elevated serumPSA level, cancer is more likely to be present when thepercentage of free PSA is low. Because the relativesensitivity versus specificity varies, depending on thefree PSA cutoff, the optimal cutoff value for free PSAis still under debate. Prostate specific antigen (PSA)represents the best serum marker for prostaticcarcinoma and is considered as most perfect tumormarker available today. Nevertheless, the use of PSAto detect prostate cancer is clinically imprecise sincebenign and malignant prostate disease can causeelevations in PSA. It is sensitive but spesificity is notgood to show tumor agressiveness, and so does notbenign prostatic hypertropy from invasive cancer.Age-spesific cut-offs have been suggested to improvespesificity, but there is still substantial overlapbetween normals and those with cancer. Furthermarkers of tumor agressiviness, either measured inserum or needle biopsy specimens are needed todetermine which patients are in need of curativetreatment. SSeerruumm aacciidd pphhoosspphhaattaassee:: Serum acid phosphatase(ACP) served as the only serum tumor marker forprostate cancer between the 1930s and 1980s. moresensitive serum tumor marker in detection of localizeddisease and in monitoring response to therapy. In thepast two decades, the use of ACP has diminishedbecause of problems with lack of sensitivity andspecificity and because of the discovery of prostate-specific antigen (PSA), a is an independent predictorof biochemical recurrence in men who undergosurgery. ACP level is independently predictive ofbiochemical recurrence following radical retropubicprostatectomy (RRP), when adjusted for otherpredictive variables. GGrraanniinnss:: The nomenclature for chromogranin-Acontinues to evolve; for simplicity,it is referred as

49

LLeetttteerr ttoo eeddiittoorr

granin-A (GRN-A). GRN-A is a 49-kilodalton proteinthat is produced exclusively by endocrine andneurondocrine (NE) cells. It is costored and cosecretedwith the resident hormones of these cells, such ascatecholamines and calcitonin (CT). Although thefunction of GRN-A is not known, it can serve as atissue and serum marker for a variety of endocrinecells and tumors. There are several major cancer typesare characterized by NE differentiation. Recently, theimportance of NE differentiation and the attendantexpression of chromogranin-A has becomeappreciated for prostate cancer. Clinical and basicroles of chromogranin-A in human prostate cancer arestill studied. Although the function of GRN-A is notknown, several theories have emerged about its role:(1) that it participates and perhaps regulates the storageand secretion of its coresident hormones in secretoryvesicles; (2) that it inhibits proteolytic cleavageenzymes; (3) that it binds calcium and thus regulatesthe biologic effects of this ion; and (4) that it is aprecursor for peptides that have unique biologic effectson the function and growth of its resident cells.Function notwithstanding, the production of GRN-A inNE prostate cancers has resulted in the availability ofa new serum and tissue marker for the tumors. Theclinical potential of GRN-A as a serum and tumormarker in prostate cancer. It is now wellestablishedthat GRN-A can be a marker for advanced disease.More importantly, GRN-A may be a marker for earlyand recurrent disease, even in the absence of abnormalPSA. GRN-A serum levels may also have prognosticsignificance, especially for androgen-independentprostate cancer. EE--ccaaddhheerriinn:: In attempts to determine which cancersof patients with clinically localized disease whoundergo radical prostatectomy will recur, the mostwell-characterized and accepted predictors are modelequations that take into account preoperative serumprostate-specific antigen (PSA), final Gleason score,and final pathologic stage. Prediction of regression forthe individual patient using these statistical models,however, is still not precise, and these models couldstill be improved on. Thus, additional markers areneeded to more accurately target high-risk patients forinclusion in clinical trials involving investigationaltherapies for locally advanced prostate carcinoma.Several other approaches show promise in this regard,including nuclear morphometry, where the results havebeen quite consistent. Other more controversialmarkers include DNA ploidy and other biomarkers,

such as the amount of tumor angiogenesis, andimmunohistochemical levels of various markers,including Ki-67, Bcl-2, p53, and E-cadherin. E-cadherin as a biomarker to predict prognosis inpatients at risk of disease recurrence after radicalprostatectomy is warranted.SSeerruumm ttoottaall hhoommooccyysstteeiinn:: Homocystein (Hcy) as atumor marker targets to reveal chemotherapy effectson patients. It is largely derived from cellularmethionine, an essential amino acid drawn fromdietary intake. Intracellular homocysteine is normallysecreted extracellularly, at rapid rates. In thecirculating blood, the majority of the homocysteinebinds to albumin, forming a disulfide linkage.Approximately 10% to 20% of the Hcy also exists as amixed disulfide with cysteine or with homocysteineitself . Very little Hcy is present in the circulatingblood in a free reduced form (approximately1%).Elevated serum tHcy (total homocysteine, freeand protein-bound) are detectable in patients withmalignant diseases. Finding increased circulating tHcyin tumor cells may also be related to the so-called‘‘methionine dependency’’ of many, but not all, tumorcells. Many tumor cells are methionine dependentbecause of their inability to convert homocysteine(Hcy) to methionine by way of the remethylationreaction. On the other hand, normal cells have noproblem obtaining methionine from homocysteine.Folate is critical to the remethylation reaction. Anyfolate deficiency will result in the impairment offunction of the remethylation reaction, causingaccumulation of Hcy. Therefore, it was generallybelieved that the rapid proliferation rate of tumor cells,such as in prostate cancer and in the so-calledmethionine dependency of tumor cells, was due to thedepletion of folate by the rapid growing tumor cellsand changing levels of fLV (a form of folate) in 24 hafter therapy. In other words, with a betterunderstanding of the effects of various drugs, the riseand fall of circulating tHcy could be used as a newtumor marker to monitor cancer patients duringtherapy, complementing commonly used tumormarkers. The general impression that elevated tHcy isdetectable in cancer patients derives from the fact thatmany cancer patients take anti-folate drugs such asmethotrexate. It is important to know that the level oftHcy reflects the tumor cell proliferation rate.Regardless of the folate status, it is very likely givenour results and others that the rapid proliferation oftumor cells is one of the major reasons that elevated

50

circulating tHcy can be detected in cancer patients.Conceivably, circulating tHcy could very well be usedas a marker to monitor cancer patients during therapy,complementing the currently used tumor markers.CChhoolliinnee kkiinnaassee:: Choline kinase (ChoK) is the firstenzyme in the Kennedy pathway, responsible for thede novo synthesis of phosphatidylcholine (PC), one ofthe basic lipid components of membranes. ChoK isresponsible of the generation of phosphorylcholine(PCho) from its precursor, choline. Both ChoK and itsproduct, PCho, have been recently reported asessential molecules in cell proliferation andtransformation. Generation of Pcho from ChoKactivity has been described as an essential event ingrowth factor-induced mitogenesis in fibroblasts andhas been found to cooperate with several mitogens.Furthermore, overexpression of several oncogenesinduces increased levels of ChoK and the intracellularlevels of PCho. A strong correlation can be establishedbetween ChoK activity and cancer onset at least insome human tumors. Additional evidence givessupport for a role of ChoK in the generation of humantumors, since studies using nuclear magneticresonance (NMR) techniques have demonstratedelevated levels of PCho in human tumoral tissues withrespect to the normal ones, including breast, prostatecarcinomas. ChoK is overexpressed with highincidence in both, tumor- derived cell lines andtumoral tissues, these results indicate the putative useof ChoK as a tumor marker, potentially useful indiagnosis and screening of the progression of tumors.

The recent findings show that overexpression ofthe polycomb group transcriptional represor enhancerzeste Gene (EZH2) in prostate cancer raises thepossibility that transcriptional regulation at thechromatin level play a role in the development of themetabolic phenotype and suggest new explorationprespective on patient stratification, therapeutics and atumor 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 tumorsupressor genes such as PTEN or mutations in genes ormutations in genes can be candidate markers. This isurgently needed since radical surgery carries a highmorbidity leading to impotence and/or incontinence.

Serdar Ar›sanfiiflli Etfal State Hospital

1. Urology Clinics, fiiflli, ‹stanbul

51

53

ÇÇeettiinn AALLGGÜÜNNEEfifi,, RRaaddyyaassyyoonn BBiiyyooffiizzii¤¤ii, TrakyaÜniversitesi Yay›nlar›, Edirne, 135 sayfa, ISBN: 975-374-051-4, 2002.

Kitapta atom ve çekirde¤inin yap›s›, karars›zçekirdekler, iyonizan radyasyon tipleri ve özellikleri,radyasyonun madde ile etkileflmesi ve radyasyonbirimleri, iyonizasyona sebep olmayan radyasyonlar,iyonizan radyasyonlar›n biyolojik etkileri veradyasyondan korunma konular› tart›fl›lm›flt›r.

Bölümlerin ayr›nt›l› incelenmesinde, kitab›ndiziliminin geleneksel tarzda, flekil ve tablolar›ngeçtikleri yerlerde metin aras›nda verildi¤igörülmektedir. Bütün konular, aç›klay›c› flekil, tablo veörneklerle desteklenmifltir.

Bu kitab›n radyasyon biyofizi¤i konusunda önemlibilgiler kazand›rmas› aç›s›ndan çok yararl› bir rehberolaca¤› kan›s›nday›m. Ayr›ca Türkiye’de bu alanlardakaynak oluflturacak Türkçe eserlerin say›lar› da sonderece s›n›rl› oldu¤u için, bu kitab› biyologlara,radyobiyologlara, fizikçilere, radyasyon etkileri ileilgilenen ziraatç› ve veterinerlere öneririm.

Atilla ÖZALPANHaliç Üniversitesi,

Moleküler Biyoloji ve Genetik Bölümü

ÇÇeettiinn AALLGGÜÜNNEEfifi,, RRaaddiiaattiioonn BBiioopphhyyssiiccss, Publishedby Trakya University, Edirne, 135 pp, ISBN: 975-374-051-4, 2002.

In the book, atomic and nuclear structure, unstablenuclei, types and properties of ionizing radiations,interaction of radiation with matter, radiation units,non-ionizing radiations, biological effects of ionizingradiations and radiation protection are discussed.

In detail, the layout of the book has a traditionalformat in that figures and tables have been integratedinto the text at appropriate places. All statement aresupported with a plenty of explenatory figures, tablesand examples.

The book is a valuable guide of radiationbiophysics. On the other hand, this book is a gooddocument because there are very limited Turkishpublication in this area. For this reason, Irecommended this book for the biologists, physisists,agriculturists and veterinarians who apply radiation onliving organisms for several purposes.

Atilla ÖZALPANHaliç University,

Department of Molecular Biology and Genetics

BBooookk rreevviieewwss

55

GGeenneerraall

1. JJoouurrnnaall ooff CCeellll aanndd MMoolleeccuullaarr BBiioollooggyyis published biannually (January and July) and covers all aspects relating to cell biology, molecular biology, genetics, microbiology and related topics.

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Three types of paper will be published, these areoriginal research papers, review articles and letters tothe editor. Book reviews are also welcome.1. Original research papers: Only original

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UUnniittss,, aabbbbrreevviiaattiioonnss aanndd sscciieennttiiffiicc nnaammeess

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1. Citation in the text should take the form: Smith andRobinson (1990) or (Smith and Robinson, 1990). Ifseveral papers by the same author in the sameyear are cited, they should be lettered in sequence(1990a), (1990b), etc. When papers are by morethen two authors they should be cited as Smith etal. (1990) or (Smith et al., 1990).

2. In the list, references must be placed in alphabeticalorder. The following models for the reference listcover all situations. The punctuation given must beexactly followed.

Redford IR. Evidence for a general relationshipbetween the induced level of DNA double-strandbreakage and cell killing after X-irradiation ofmammalian 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 andV (D) J recombination. Science. 265: 1442-1445,1994.

Ohlrogge JB. Biochemistry of plant acyl carrierproteins. In: The Biochemistry of Plants: AComprehensive Treatise. Stumpf PK and Conn EE(Ed). Academic Press, New York. 137-157, 1987.

Weaver RF. Molecular Biology. WCB/McGraw-Hill.1999.

2. Only papers published or in press should be cited inthe literature list. Unpublished results, includingsubmitted manuscripts and those in preparation,should be cited as unpublished in the text.

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PP rrooooffss aanndd ooffffpprriinnttss

1. Page proofs will be sent to the correspondingauthor for checking before publication. Correctedproofs should be sent back to the Editor withinthree days of receipt, otherwise Editor reserves therights to correct the proofs himself and to send thematerial for publication.

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56

Journal of Cell and Molecular Biology

CONTENTS Volume 2, No. 1, 2003

Dedication

Review articles

Polyamines in plants: An overviewBitkilerde poliaminler: Genel bir bak›flR. Kaur-Sawhney, A.F. Tiburcio, T. Altabella, A.W. Galston 1-12

Phenolic cycle in plants and environmentBitkilerde fenolik döngü ve çevreV. I. Kefeli, M. V. Kalevitch, B. Borsari 13-18

Research papers

The short-term effects of single toxic dose of citric acid in miceFarelerde sitrik asidin tek toksik dozunun k›sa süreli etkileriT. Aktaç, A. Kabo¤lu, E. Bakar, H. Karakafl 19-23

Characterisation of RPP7 mutant lines of the col-5 ecotype of Arabidopsis thalianaArabidopsis thaliana’n›n col-5 ekotipinden elde edilen mutant hatlardan RPP7geninin karakterizasyonuC. Can, M. Özaslan, E. B. Holub 25-30

The effect of meta-topolin on protein profile in radish cotyledonsMeta-topolinin turp kotiledonlar›nda protein profiline etkisiS. Ça¤, N. Palavan-Ünsal 31-34

The effect of electromagnetic fields on oxidative DNA damageElektromanyetik alan›n oksidatif DNA hasar› üzerindeki etkisiS. ‹fller, G. Erdem 35-38

Chromosomes of a balanced translocation case evaluated with atomic force microscopyDengeli translokasyon vakas›nda kromozomlar›n atomik güç mikroskobu ilede¤erlendirilmesiZ. Y›lmaz, M. A. Ergun, E. Tan 39-42

Effect of epirubicin on mitotic index in cultured L-cellsEpirubisinin kültürdeki L-hücrelerinde mitotik indekse etkisiG. Özcan Ar›can, M. Topçul 43-48

Letter to editor 49-51

Book reviews 53

Instructions to authors 55-56

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