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

Journal of Cell and 

•Notch in apoptosis 

Molecular Biology 

Volume 9 · No 1 · June 2011 

http://jcmb.halic.edu.tr 

Xenobiotic genes and susceptibility to bladder cancer 

MMP1 and MMP2 polymorphisms in TMJ disorder 

Silencing suppressors of PVM 

Bio-database compression using enhanced suffix array



Volume 9 · Number 1 

June 2011 

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Published by 

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Faculty of Arts and Sciences 

Editor 

Nagehan ERSOY TUNALI 

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Aslı BAŞAR 

Editorial Assistance 

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İsmail TÜRKAN, İzmir, Turkey 

Mehmet TOPAKTAŞ, Adana, Turkey 

Meral ÜNAL, İstanbul, Turkey 

Selma YILMAZER, İstanbul, Turkey 

Ziya ZİYLAN, İstanbul, Turkey


CONTENTS Volume 9 · Number 1 · June 2011 

Review Articles 

Decision making mechanism influences the regulatory function of Notch in 

Apoptosis 

1 

A.NAIR 

Polymorphisms in the xenobiotic genes and susceptibility to bladder cancer 

5 

N.ERSOY TUNALI and N.O.TİRYAKİOĞLU 

Research Articles 

Identification of silencing suppressors of potato virus M 

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

Identification of a novel dehydration responsive transcript from tossa jute 

(Corcohrus olitorius L.) 

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

Protective effects of curcumin on cadmium chloride induced colon toxicity in 

Swiss albino mice 

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

In vitro regeneration of Cleome viscosa – an important medicinal herb 

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

Bio-database compression using enhanced suffix array for pairwise sequence 

alignment 

A.KUNTHAVAI and S.VASANTHA RATHNA 

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

and characterization of the recombinant enzyme 

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

Investigation of the MMP1 and MMP3 promoter polymorphisms in 

temporomandibular joint disorder 

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

Lead induced alterations in blood cell counts and hemoglobin during gestation 

and lactation in Swiss albino mice 

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

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

some vital organs in adult Wistar rats 

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

A.O.AYOKA 

Short Communication 

Investigation of the EGFR gene variations in bladder cancer patients using 

INFINITI TM Analyzer 

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

Instructions for Authors 

15 

21 

31 

37 

45 

53 

63 

69 

75 

83 

87

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

Haliç University, Printed in Turkey. 


Decision making mechanism influences the regulatory function of 

Notch in apoptosis 

Ajay NAIR* 

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

9054, New Zealand 

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

Received: 24 January 2011; Accepted: 28 February 2011 

Abstract 

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

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

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

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

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

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

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

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

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

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

Karar verme mekanizması Notch'un apoptozdaki düzenleyici rolünü etkiler 

Özet 

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

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

kesilme olayları sonucunda gerçekleşen Notch intrasellüler bölgesinin nükleer lokalizasyonundan bağımsız 

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

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

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

doğal seleksiyon teorisi aracılığıyla Notch proteininin apoptozda düzenleyici işlevinin muhtemel evrimsel 

önemini göstermeye çalışacaktır. 

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

Introduction 

The Notch protein is a transmembrane receptor 

possessing distinct extracellular and intracellular 

domains. Specific ligand proteins (single pass 

transmembrane proteins belonging to the 

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

the extracellular domain which in turn triggers 

successive proteolytic cleavage events starting with 

that of the extracellular domain, catalyzed by a 

metalloprotease, TACE (Tumor Necrosis Factor 

Alpha Converting Enzyme). This is then followed 

by the cleavage of the remaining notch protein 

catalyzed by γ-secretase (Perumalsamy et al., 

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

intracellular domain and migrates to the nucleus to 

influence gene expression. This is the conventional 

Notch signaling pathway. Apart from the canonical 

pathway of Notch signaling there also an

2 Ajar NAIR 

alternative pathway which culminates in the release 

of anti-apoptotic factors and the inactivation of proapoptotic 

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

2004). However, this unorthodox pathway avoids 

the usual nuclear localization. Recent research on 

the same has engendered the possibility of a 

membrane-tethered or cytoplasm- localized form, 

something that has been a subject of intense 

research since then. Now the question regarding the 

membrane or cytoplasmic localization of the Notch 

intracellular domain during its anti-apoptotic non 

canonical signal transduction is certainly puzzling. 

But what is utmost intriguing is that the structural 

composition of NICD doesn’t completely and 

substantially support a membrane tethered or 

cytosolic form separately. This assertion can be 

made more explicit with the following findings 

from various works done in this regard: 

1. The existence of a nuclear localization sequence, 

intact in the NICD. 

2. Notch intracellular domain construct with a 

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

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

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

full-length Notch intracellular domain is 

abbreviated as NICD. While investigating the 

regulatory function of Notch-1 on apoptosis, one 

anti-apoptotic protein in particular is significantly 

upregulated in NICD-expressing Jurkat cells: the 

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

three-fold increase of XIAP protein was detected in 

NICD-expressing T cells. Over expression of the 

full-length Notch intracellular domain also resulted 

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

3. Notch increases XIAP stability through 

downregulation of proteasome-mediated protein 

degradation and upregulation of XIAP by Notch 

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

Dan et al., 2004). 

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

terminal, then followed by six repeats of Ankyrin 

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

Transactivation domain (TAD) and the NICD 

structure ends at the C terminal, by a PEST domain 

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

domains, do not affect the binding of NICD to 

XIAP. Truncation of the TAD from NICD on the 

other hand abolishes its association with His-XIAP, 

indicating that TAD is the XIAP binding region on 

NICD. NICD directly binds XIAP, where the Cterminal 

domain on NIC and the RING-finger 

domain of XIAP are critical for such interaction 

(Liu et al., 2007). 

5. NICD interacts with XIAP in the cytoplasm; 

which was confirmed by the finding that coexpression 

of XIAP decreases NIC-directed HES5 

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

et al., 2007). 

6.TAD dictates the transcription activity of NICD. 

Partial deletion of TAD domain retains most of the 

NICD transcription activity, shown by activation of 

the HES5 promoter, yet completely eliminating the 

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

al. 2007). If there is an independent membrane 

tethered form, then the canonical pathway must not 

get influenced by any means. 

Figure 1. Structural makeup of NICD. RAM 

domain at the N terminal is followed by six ANK 

repeats, a nuclear localization sequence, a TAD 

(Trans-activation domain) and a PEST domain at 

the C terminal. Truncation of the TAD affects 

NICD’s association with XIAP; altering NICD’s 

anti-apoptotic function.

The aforesaid, thus, strongly suggests that there 

is more to be added to complete the puzzle. From 

the above points it seems that there exists a 

decision making mechanism, within the cytoplasm 

which decides the fate of the NICD, which either 

complies with its usual nuclear retention or NCID 

gets stabilized in the cytoplasm. And certainly this 

point out to the possibility of an alternate 

modification of the NICD, other than those 

mentioned above, that perhaps answers for such a 

diversity seen in Notch, in terms of its action. 

Figure 2. Activation of transcription, via NICD’s 

usual nuclear retention in a cell. A) activated notch 

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

XIAP. D) NICD with truncated or deleted TAD; 

NICD fails to bind XIAP. E) HES5; NICD 

localizes to nucleus and associates with HES-5 to 

turn on transcription. 

An evolutionary rationale 

This perplexing behavioral change exhibited by 

Notch receptors and its signaling cascade may 

perhaps be explained in an alternate fashion, by 

observing it from an angle that concentrates on 

reasoning out the advantage of a T cell that may 

hold when urged to respond to an environmental 

stimulus like neglect or etoposide or DNA damage 

induced death signals. It’s recognized that energy is 

consumed for two central processes in any 

biological cell; one is for reproduction and the other 

for cell-maintenance. Although indirectly, the notch 

canonical pathway is responsible for meeting the 

requirements pertaining to development, 

Processing of Notch rescues cells from apoptosis 3 

reproduction etc. But a situation like neglect or 

other antagonist stimulus poses an unfavorable 

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

switch to a mode which would momentarily 

conserve much of the cells free energy for cell 

maintenance by promoting cell survival signaling 

pathways rather than promoting the customary 

transcriptional activities (developmental and 

metabolic); for it is utterly uneconomical to spend 

the same energy on reproducing the cells in an 

attempt to revitalize the cell population. Therefore, 

notch signaling in entirety is bound to have a 

decision making mechanism from the time of its 

activation. But does notch directly respond to the 

demand or the environmental stimulus. Perhaps 

there is more than a straight intrusion from Notch. 

Notch interactors, other than the canonical and noncanonical, 

are mostly playing a pivotal role in 

answering this question which still stands 

challengeable inspite of the various breakthroughs 

made in the context of Notch and its regulatory 

function on apoptosis. The question that continues 

to seek an answer is, why does a mechanism like 

the Notch signaling pathway, which is one of the 

highly conserved cell signaling systems present in 

most multicellular organisms in terms of involving 

gene regulation mechanisms that control multiple 

cell differentiation processes, incorporate a 

different role such as anti apoptosis? Because notch 

holds the potential to influence cell fate decisions, 

it is imperative that there be a decision making 

mechanism that can modulate this omnipotent 

transmembrane protein and its actions, depending 

upon the need of the hour from a toll of billion 

deaths (Neglect or DNA damage induced). 

Probably cells have earned and evolved towards 

this non conventional Notch pathway, that rescues 

cells from self flattening. This postulation finds 

support from a concept called- The Selfish Gene 

Theory or Gene Selection Theory, according to 

which “adaptations are the phenotypic effects of 

genes to maximize their own representation and 

continued survival in the future generations”. 

Genes build vehicles to meet their mutual interests 

of jumping into the next generation of vehicles 

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

genes. So it is important that there exist a sequential 

set of events which end up modulating the NICD in 

response to an apoptotic stimulus; this change will 

ensure cell survival and as a consequence, promote 

the perpetuation of genes. 

Neglect like situation, (known to activate antiapoptotic 

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

4 Ajar NAIR 

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

the cell’s energy. The cell prepares itself for 

apoptosis; apoptosis promoting factors are 

activated. Such an event forces mitochondria 

towards an immediate fragmentation which marks 

the onset of apoptosis; fragmentation induces 

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

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

apoptotic distress signal, such as the one mentioned 

above, prevents the usual processing of NICD, 

which causes the partial or complete deletion of its 

trans-activation domain; in so doing prevent its 

nuclear localization and its ability to stimulate 

transcription by associating with transcription 

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

mechanism in the form of an additional physical 

modification of NICD, in the cytoplasm, changes 

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

to respond to the induced stress. Besides activating, 

anti-apoptotic factors like XIAP, this tailored NICD 

may well be acting upstream to the factors which 

promote mitochondrial connectivity (for e.g. 

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

activation, cytochrome-c release and eventually halt 

cell demolition (Figure 3). 

Figure 3. Proposed alteration in notch activity 

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

stress leads to, a shift in the energy metabolism, 

and the release pro-apoptotic factors (shown as 

blue circles); this results in mitochondrial 

fragmentation. C) This might act as an alarm 

upstream notch to stop its usual course to the 

nucleus; HES5 (shown in blue inside the nucleus) 

remains unbound to trigger transcription. D) 

Cleaved NICD binds to XIAP via its intact TAD. 

E) This interaction might activate other antiapoptotic 

factors and also release factors (for e.g. 

Mfn) to restore mitochondrial connectivity. 

Conclusion 

For each and every phenomenon or process that 

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

occurrence in terms of the benefit it provides to a 

cell. This leads to a specific adaptation with respect 

to the cell’s requirement at a particular instance 

(such as the long term survival of Memory T cells 

due to the regulatory function of Notch-1 on 

apoptosis). Through rigorous selective pressures on 

a cell, several individual molecular processes are 

scrutinized. The one that makes a cell evermore 

specialized with respect to the immediate demand, 

is selected by natural selection. It is this long 

evolutionary history that, in all probabilities, 

endowed notch with this non-canonical 

mechanism. Hence, notions that speculate a 

discrete membrane localized form for Notch, do 

not seem to hold up. 

References 

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

inhibits apoptosis by direct interference with 

XIAP ubiquitination and degradation. The 

EMBO Journal. 26: 1660–1669, 2007. 

Perumalsamy LR, Nagala M, Banerjee P and Sarin 

A. A hierarchical cascade activated by noncanonical 

Notch signaling and the mTOR– 

Rictor complex regulates neglect-induced death 

in mammalian cells. Cell Death and 

Differentiation. 16: 879-889, 2009. 

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

Effect of Notch-1 Requires p56lck-dependent 

Akt/PKB-mediated Signaling in T Cells, The 

Journal of Biological Chemistry. 279: 2937– 

2944, 2004. 

Suen DF, Norris KL and Youle RJ. Mitochondrial 

dynamics and apoptosis. Genes & Development. 

22: 1577-1590, 2008.

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



Polymorphisms in the xenobiotic genes and susceptibility to 

bladder cancer 

Nagehan ERSOY TUNALI* and Necip Ozan TİRYAKİOĞLU 

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

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

Received: 13 May 2011; Accepted: 20 June 2011 

Summary 

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

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

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

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

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

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

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

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

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

with special emphasis on the last two enzyme group. 

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

polymorphism 

Ksenobiyotik genlerdeki polimorfizmler ve mesane kanserine yatkınlık 

Özet 

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

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

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

enzimlerdeki polimorfizmler maruz kalınan karsinojelerin metabolize edilmesinde kişisel 

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

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

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

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

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

enzim grubuna özel vurgu yaparak tanıtacağız. 

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

yatkınlık, genetik polimorfizm 

Introduction 

Cancer is regarded as a multifactorial disorder 

affected both by genetic and environmental factors, 

which culminate in dysregulated cell cycle. 

Although environmental risk factors are considered 

as the most important among others, 

epidemiological studies have shown that a certain 

part of the population is in fact more prone to 

develop cancers despite being exposed to the same 

type and amount of environmental risks. This

6 Nagehan ERSOY TUNALI and Necip Ozan TİRYAKİOĞLU 

finding has led the molecular epidemiology studies 

to focus on predisposition to cancer via genetic 

factors. It has been speculated that either the 

polymophisms in the DNA repair genes or those in 

carcinogen metabolizing genes should primarily be 

questioned as possible candidates of genetic 

modifiers. The polymorphisms in the DNA repair 

genes may alter the function of the related enzyme 

and thereby affect the repair capacity of the 

individual. This kind of alteration in the DNA 

repair mechanism may get the individual more 

sensitive to carcinogens and more susceptible to 

cancer formation (Franekova et al., 2008). 

Likewise, polymorphisms in the genes coding for 

the xenobiotic-metabolizing enzymes may cause 

individual variations in metabolizing the exposed 

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

individuals with a certain genetic background may 

become more susceptible to develop cancer. 

Xenobiotic metabolism 

Xenobiotic metabolism (from the Greek xenos 

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

composed of several metabolic pathways that 

modify the chemical structure of xenobiotics, e.g. 

drugs, carcinogens, poisons. Upon their intake by 

an organism, xenobiotics undergo several metabolic 

Metabolising genes and susceptibility to bladder 

Cancer 

Polymorphisms in the xenobiotic-metabolising 

genes are pivotal to the above-mentioned 

alterations and therefore have been intensively 

investigated in many cancer types. Foremost of 

Figure 1. Activation pathways of carcinogens 

conversions to facilitate their solubility in order to 

expedite their removal from the organism. These 

so-called biotransformation processes consists of 

two consecutive steps; an initiation phase, where 

carcinogens are modified to accept reactive groups 

(Phase I) via the action of cytochrome P450 

oxidases; and a second phase where they are 

converted into more easily extractable molecules by 

conjugation reactions (Phase II) via transferase 

enzymes. The xenobiotic metabolism can be 

regarded as a detoxification mechanism consisting 

of two phases, however, conversion reactions 

catalyzed by xenobiotic-metabolising enzymes can 

also activate potential carcinogens (Figure 1.).The 

reason for this is the vastly diverse chemical 

structures of carcinogens which can not be 

compensated by the limited number of conversion 

reactions catalyzed by xenobiotic enzymes. The 

capability of the xenobiotic metabolism to either 

activate or detoxify carcinogens constitute a fragile 

balance between activation and detoxification of 

carcinogens, which defines the amount of DNA 

damage accumulated in the cells. Alterations 

affecting enzyme activity shifts this balance 

towards either detoxification or activation of 

carcinogens. 

these cancers is the bladder cancer, for which the 

well-known etiological risk factors are occupational 

exposure to certain carcinogens and cigarette 

smoking. In fact, smoking is proved to be the 

primary cause of approximately 70% of the bladder 

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

interaction between genetic and environmental

factors accounts for the different levels of 

susceptibilities to the development of bladder 

cancer. Bladder cancer incidence varies highly 

between distinct regions worldwide, with 

developed countries having the highest incidence 

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

bladder cancer incidence in developed countries 

despite increased awareness against risk factors 

indicates the existence of genetic factors affecting 

bladder cancer susceptibility. Thereby, 

polymorphisms with possible association to bladder 

cancer have been widely investigated. Many 

metabolizing genes and their polymorphisms have 

been identified as responsible for individual 

susceptibility to cause BC. 

This review is written as an attempt to 

demonstrate the association between xenobioticmetabolising 

gene polymorphisms and bladder 

cancer. Among metabolizing enzymes, we will 

introduce N-acetyltransferases (NAT), Glutathione- 

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

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

Methylenetetrahydrofolate reductase (MTFR), 

Cytochrome P-450 (CYP) and Aldo-Ketoreductases 

(AKR) with special emphasis on the last two 

enzyme group and related polymorphisms. 

N-acetyltransferases 

In humans, genetic locus for the two isoenzymes of 

NAT, NAT1 and NAT2, resides at chromosome 

8p22. NAT1 and NAT2 are polymorphic and both 

catalyze N- and O-acetylation of aromatic and 

heterocyclic amines (Franekova et al., 2008). 

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

the bio-conversion of heterocyclic amines to 

electrophilic nitrenium ions. NAT2 catalyzes the 

activation and deactivation of various aromatic 

amines, heterocyclic amines and hydrazin drugs. 

Polymorphisms in the NAT2 gene have been 

strongly associated with bladder cancer. Based on 

the 13 SNPs, 30 different NAT2 alleles have been 

defined 

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

T.html). Depending on the effects of the 

polymorphisms, NAT2 alleles have been 

categorised in two groups as slow and fast 

acetylators. Individuals who lack both of the two 

functional NAT2 alleles have been designated as 

slow acetylators. A meta-analysis with combined 

results from 31 different studies has shown that 

NAT2 slow acetylation alleles increase the risk of 

Genetic susceptibility to bladder cancer 7 

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

Closas et al., 2005). 

NAT1 enzyme transfers one acetyl group from 

acetyl-coA to arylamine and hydrazin substrates. 

Many NAT1 variants have been defined to date 

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

T.html), however studies have revealed conflicting 

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

significance could not be fully identified yet. 

Since aromatic amines are present in cigarette 

smoke are the major risk factors for bladder cancer 

and are metabolized by NAT enzymes, it is for sure 

that the polymorphisms of the NAT genes have 

important roles in predisposing the individuals to 

bladder cancer (Franekova et al., 2008). 

Glutathione-S-transferases 

Cytosolic GSTs belong to a superfamily of Phase II 

enzymes, which contain sub-families and 

polymorphic isoenzymes. The genetic 

polymorphisms of these enzymes affect the 

individual susceptibility to cancer, cardiovascular 

and respiratory diseases. GSTs facillitate the 

detoxification of electrophilic compounds by 

gluthathione conjugation. They interact with a wide 

variety of substrates including polycyclic aromatic 

hydrocarbon (PAH) epoxides and oxidative stress 

by-products. GSTM1 detoxifies carcinogenic PAHlike 

benzopyrene. A common deletion 

polymorphism in GSTM1 gene causes the loss of 

enzyme activity and has been shown to be strongly 

associated with increased risk for bladder cancer 

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

According to a meta-analysis conducted to show 

the overall effect of this polymorphism, an odds 

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

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

smaller reactive hydrocarbons, like ethylene oxide. 

GSTT1 deletions, which have high frequency in 

Caucasians, have been associated with increased 

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

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

conjugation and detoxification of many 

xenobiotics. The results of the studies investigating 

GSTT1 and GSTP1 gene polymorphisms are 

generally conflicting (Brockmoller et al., 1996; 

Lee et al., 2002.). 

In summary, polymorphisms of GSTM1, 

GSTT1 and GSTP1 constitute risk factors for many 

cancer types. Especially the null alleles of GSTM1

8 Nagehan ERSOY TUNALI and Necip Ozan TİRYAKİĞLU 

and GSTT1 are risk factors for bladder cancer in 

chain smokers. 

Sulphotransferases 

SULTs are encoded by a supergene family and 

catalyze the sulphonation of many xenobiotic 

compounds, including drugs and carcinogens. 

SULT enzymes play role as one of the main 

detoxification systems in adults and in developing 

human fetuses. This gene family is not very 

polymorphic, except SULT1A1. SULT1A1 

Arg213His polymorphism, which causes a decrease 

in enzyme activity and thermal stability, has been 

suggested as a protective factor for bladder cancer 

(Zheng et al., 2003). 

NAD(P)H dehidrogenase, Quinone 1 

NQO1 enzyme protects the cell against 

electrophilic and oxidizing metabolites of 

xenobiotic and endogenous quinone compounds. 

The gene coding for the NQO1 enzyme harbors 

several polymorphisms affecting enzyme activity. 

A187C transition results in the replacement of 

proline with serine and decreases enzyme activity. 

Accordingly, Ser187 allele has been shown to 

increase bladder cancer risk (Franekova et al., 

2008). 

Methylenetetrahydrofolate reductases 

MTHFR converts 5,10-methylenetetrahydrofolate 

(a methyl donor in deoxythymidine monophosphate 

synthesis) to 5-methyltetrahydrofolate. Folate 

deficiency is related to breakage of DNA strands 

and binding of urasil to DNA. If an MTHFR variant 

causes a decrease in the folate levels by decreasing 

the enzyme activity; one can expect a tendency to 

DNA strand breakage and cancer formation. On the 

other hand, variant MTHFR activity alters the 

amount of methyl donors and thereby involves in 

bladder cancer formation by changing the status of 

promotor methylation. MTHFR 677C>T and 

1298A>C polymorphisms are proved to decrease 

enzyme activity. However, the relationship between 

the mentioned polymorphisms, enzyme activity and 

susceptibility to bladder cancer should be more 

extensively studied to seek for confirming results 

(Karagas et al., 2005). 

Cytochrome P-450 genes 

CYP enzymes catalyze Phase I reactions and 

belong to the microsomal enzyme super family. 

CYP enzyme system consists of over 20 highly 

polymorphic CYP enzymes 

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

enzyme is expressed in epithelium and plays a key 

role in the activation of many procarcinogens 

including PAHs and aromatic amines arising from 

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

located in heme-binding domain and MspI (m1) 

mutation has been shown to increase enzyme 

activity. White blood cells of smokers harboring a 

Ile-Val mutation have been shown to contain more 

PAH-DNA adducts in comparison to non-smokers. 

Expression of CYP1A1 has been shown to elevate 

in bladder cancer patients in correlation with 

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

and m2 mutations, on the other hand, are directly 

associated with increased risk for lung cancer 

(Aynacioglu et al., 1998). CYP1A1 mutations 

exhibit variations among different ethnic groups. In 

contrast to Causians, prevalances of m1 and m2 are 

high among Asian populations. M3 mutation was 

identified only in Africans. In molecular and 

epidemiologic studies, CYP1A1*2B allele, 

harboring m1 and m2 mutations, was found to be 

correlated with increased risk for lung cancer. 

Figure 2 shows the localisation of these four 

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

gene. Human CYP1B1 gene is located at 

chromosome 2p21 and contains three exons in a 

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

enzyme (Figure 3). It was found to be 

overexpressed in many cancer types including, 

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

lower expression in normal somatic tissues (Thier 

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

and heterocyclic amines. It also catalyzes the 

conversion of benzo[a]pyrene to carcinogenic 

metabolite diol epoxide-2, which indicates its 

significant role in the activation of tobacco 

carcinogens. Studies conducted so far indicate that 

CYP1B1 polymorphisms, especially codon 432 

polymorphism, change enzyme activity and 

contribute to carcinogenesis. CYP1B1 codon 432 

polymorphism increases risk for smokers for head 

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

CYP2D6 gene codes for debrisoquine 

hydroxylase. Its subtrates include aromatic amines 

and tobacco nitrosamines. The existence of 

homozygous recessive mutations in this gene 

results in the inability of metabolising certain 

compounds. Individuals with these mutations

compromise nearly 5-10% of the Caucasian 

population which are categorised as poor 

metabolizers (PM) because of their inability to 

metabolise certain compunds. The PM phenotype is 


associated with increased susceptibility to cancer, 

indicating a possible role for this enzyme in the 

conversion of procarcinogens to active carcinogens. 

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

Most common mutations responsible for the PM 

phenotype have been identified. G/A transition 

located on exon3-intron4 conjunction creates an 

early stop codon resulting in defective mRNA. This 

CYP2E1 metabolizes many procarcinogens, e.g. 

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

and other volatile nitrosamines, 

which are inhaled via cigarette smoking. 

Inter-individual expression variants of human 

CYP2E1 gene have been identified. The most 

frequently studied RFLPs on the CYP2E1 gene are 

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

proved to alter enzyme activity. Among them, 

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

(mutant allele: CYP2E1*6) polymorphisms. These 

two polymorphisms together constitute the 

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

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

linkage disequilibrium with eachother. CYP2E1*6 

allele variant is present in nearly 10% of the 

European population. Recently, Haufroid et al. 

(2002) have shown that people with at least one 

CYP2E1*6 allele have lower chlorzoxazone than 

homozygous wild type individuals. There are ethnic 

variations in CYP2E1*5A allele frequencies among 

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

mutation accounts for 80% of the PM phenotype. 

While the deletion of the whole CYP2D6 gene 

accounts for the 15% of PM phenotypes (Sobti et 

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

Asians and Europeans. Europeans are 5% 

heterozygous; Asians are 37% heterozygous and 

6% homozygous for this allele. Because of the 

certain inter-ethnic variations of the CYP2E1*5A 

allele, genetic polymophisms, enzyme expression 

and alterations in the chemical metabolism are still 

not clear and the studies are conflicting each other 

(Bolt et al., 2003). 

Aldo-Ketoreductases 

AKRs catalyze the conversion of carbonyl groups 

to alcohol derivatives. Their most common 

endogenic substrates are lipid aldehydes, steroids 

and prostoglandines. They also activate prodrugs 

and polycyclic aromatic hydrocarbons (PAHs). In 

addition, they have been shown to induce resistance 

against chemotherapeutic agents like cysplatine and 

doxorubicin. The essential function of AKRs is to 

convert aldehydes to primary and secondary 

alcohols by reduction (Flynn and Green 1993; 

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


conjugation reactions and therefore aldoketoreductases 

are considered as Phase I enzymes. 

Human AKRs interact with various substrates 

including drugs, carcinogens and reactive 

aldehydes. Their important role in the metabolism 

of these substrates signifies them as key enzymes in 

designating the carcinogenicity of these substrates. 

There are 15 AKR families discovered so far. 

Human AKRs are categorised into 3 families as 

AKR1, AKR6 and AKR7. 8 of the 13 discovered 

human AKRs belong to the AKR1 family 

(AKR1A1, AKR1B1, AKR1B10, AKR1C1, 

AKR1C2, AKR1C3, AKR1C4 and AKR1D1). 

Most of the AKR enzymes are 34-37 kDa 

monomeric proteins with a catalytic tetrade region, 

conserved among family members. Their substrate 

binding domains, on the other hand, show great 

variety, representing the diversity of their substrates 

(Jin and Penning, 2007). 

AKRs and activation of PAHs 

There are three different pathways of PAH 

activation. These are P-450 peroxidase induced 

formation of radical cations, P-450 induced 

formation of diol-epoxides and AKR induced 

formation of reactive o-quinones. The latter are 

highly reactive molecules which can bind to most 

macrobiomolecules including DNA, RNA and 

proteins preemminently (Jin and Penning, 2007). 

The formation of o-quinones generates reactive 

oxygen species as by-products, which activate RAS 

targets by inducing the expression of antioxidant 

response element (ARE) containing genes. Since 

RAS pathway is responsible for transmitting the 

extracellular growth signals to nucleus, o-quinones 

produced by AKRs are considered to effect both the 

induction and progress of carcinogenesis (Jin and 

Penning, 2007). 

Function and tissue specific expression of Aldo- 

Ketoreductases 

Human AKR1C family members; AKR1C1, 

AKR1C2, AKR1C3 and AKR1C4; show great 

homology to each other. They all contain 9 exons 

and are located on chromosome 10. The expression 

of AKR1C genes are regulated by AREs located in 

their promoters. 

AKR1C1 and AKR1C2 have been shown to be 

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

2001). To confirm these results Palackal and 

colleagues have measured the expression levels of 

AKR1C1, AKR1C2 and AKR1C3 isoforms in lung 

adenocarcinoma cells and obtained similar results 

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

conducted by Dozmorov et al. have shown that 

increased AKR1C3 expression induce angiogenesis 

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

.AKR polymorphisms 

Even though the structural difference between 

AKR1C1 and AKR1C2 arises only from 7 different 

aminoacids, they have vastly different functions. 

Nonhomologous aminoacids are T38V, R47H, 

L54V, C87S, V151M, R170H and Q1721 which 

are highly polymorphic. Common polymorphisms 

in the AKR1C1 gene are T38A, T38I and R47H, 

whilst H47R, S87C, L172Q and V38A are common 

polymorphisms in AKR1C2. There have been 17 

polymorphisms identified in the coding regions of 

AKR1C3. Nine of these polymorphisms results in 

amino acid changes while the other eight are 

synonymous (Table 1.). 

One of the nonsynonymous polymorphisms, the 

G106T polymorphism, has been shown to produce 

truncated protein; and another nonsynonymous 

polymorphism, A230G, has been associated with 

decreased cholesterol levels in the blood 

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

polymorphism is located on the C-terminal and has 

been shown to affect substrate binding specifity 

(Kume et al., 1999). 

There are a few studies conducted to investigate 

the association between the AKR polymorphisms 

and cancer. AKR polymorphisms have been 

investigated in Non-Hodgkin lymphoma and 

AKR1A1 rs2088102 polymorphism has been 

shown to be associated with increased risk (Lan et 

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

between prostate cancer and AKR1C3 

polymorphism have been investigated has shown an 

increased risk for rs7741 polymorphism 

(Cunningham et al., 2007). 

Studies concerning rs12529 polymorphism gave 

conflicting results. rs12529 has been shown to be 

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

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

confer protection for bladder cancer (Lan et al., 

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

Figuerova et al.(2008) investigated 8 

polymorphisms located in two regions of AKR1C3 

gene, which are likely to affect bladder cancer risk 

(Table2).


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

Exon Polymorphism Function Nucleotide change Aminoacid change Allele 

3 rs7741 synonymous G90A P30 AKR1C3*1A 

4 rs12387 synonymous G312A K104 AKR1C3*1B 

6 rs1131124 synonymous G495A V165 AKR1C3*1C 

3 rs1804061 synonymous A117G K39 AKR1C3*1D 

6 rs1937839 synonymous G459A K153 AKR1C3*1E 

8 rs7342072 synonymous C705T L235 AKR1C3*1F 

6 rs17849485 synonymous C546T L182 AKR1C3*1G 

3 rs28943574 synonymous A231G E77 AKR1C3*1H 

1 rs12529 nonsynonymous C15G Q5H AKR1C3*2 

6 rs1131132 nonsynonymous C525G M175I AKR1C3*3 

3 rs1804062 nonsynonymous G106T E36term AKR1C3*4 

9 rs1804059 nonsynonymous G879T M293I AKR1C3*5 

4 rs4987102 nonsynonymous G316A A106T AKR1C3*6 

3 rs11551177 nonsynonymous A230G E77G AKR1C3*7 

9 rs11551178 nonsynonymous G883T A295S AKR1C3*8 

5 rs28943579 nonsynonymous G434A C145Y AKR1C3*9 

5 rs28943580 nonsynonymous C772T R258C AKR1C3*10 

In this study, rs3763676, rs12775701 and 

rs1937843 polymorphisms were found to be 

associated with increased bladder cancer risk; 

rs1937845, rs12529, rs4881400 and rs4641368 

polymorphisms were found to confer protection 

against bladder cancer. Recently, rs12529 

polymorphism has been shown to be associated 

with decreased bladder cancer risk (Tiryakioğlu, 

2011). According to this retrospective study, 

Conclusion 

homozygous GG variant genotype protects against 

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

which supports the findings of Figuerova et 

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

Upon adjustment of these figures according to 

important risk factors in bladder cancer, namely, 

sex and cigarette smoking status, Tiryakioğlu 

(2001) have shown that rs12529 GG genotype still 

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

Table 2. AKR1C3 polymorphisms associated with bladder cancer 

Polymorphism Genotype Odds Ratio 

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

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

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

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

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

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

The genetic variations in the human genome is truly 

a powerful source for the investigation of the 

complex disesases where genetic and 

environmental factors interact to characterize the 

final outcome. Over the past ten years, researchers 

agreed on that rare genetic defects imputed to 

certain populations are not usually responsible for 

ever increasing cancer cases; rather polymorphic


variations in the DNA sequences confer individual 

susceptibilities. This notion has been accepted for 

many cancer promoting mechanisms, including 

bladder cancer (Kim and Quan, 2005). Bladder 

cancer still conserves its importance regarding 

genetic and epidemiologic studies, since 

environmental factors, mainly cigarette smoking 

and occupational exposure to certain carcinogens 

play major roles in the formation of this cancer 

type. There are reasons for the high importance of 

this cancer type in terms of molecular research. In 

the first place, genetic polymorphisms in the 

metabolizing genes may alter the functions of the 

related enzymes, which will result in abnormalities 

in the xenobiotic functions of the cell. As an 

outcome, the environmental carcinogens may not 

be detoxified properly and hence cause bladder 

cancer. Identification of the polymorphisms in the 

xenobiotic genes and their functional relevance to 

bladder cancer formation will certainly highlight 

the importance of inter-individual genetic 

variations and the phenomenon of genetic 

susceptibility, which can be adapted easily to other 

genetic conditions. Looking at the same view from 

another direction we can still come up with the 

importance of genetic polymorphisms in defining 

disease risks. Since bladder cancer is caused mainly 

by environmental factors, people living at the same 

standards in terms of cigarette smoking, diet and 

other environmental exposures can be investigated 

for the frequency of occurrence of bladder cancer. 

Keeping the environmental factors constant, the 

only remaining factor will be the genetic variations. 

Comparing the frequencies of the genetic 

polymorphisms among bladder cancer cases and 

healthy controls exposed to the same environments, 

genetic polymorphims responsible for the bladder 

cancer formation can be identified. There are for 

sure hundreds of genetic loci to investigate, 

however, it will be wise to begin with the genes 

coding for the xenobiotic-metabolizing enzymes. 

On the other hand, the above-mentioned studies 

should be performed for each ethnic and 

geographic population in order to delineate 

genotypic distributions and to define genetic 

susceptibility loci. Using the accumulated data, 

researchers can establish diagnostic and even presymptomatic 

criteria on the basis of genetic 

polymorphisms. Until now, the prognostic and 

diagnostic value of the current accumulated data 

prove to be problematic to utilize. The main reason 

for this is the conflicting results in addition to 

overlooked environmental and demographic 

factors. Another reason is the small scope of the 

most studies, focusing on a few genes instead of 

taking the whole metabolic systems into account. 

Also, sample sizes of the studies are one of the 

major problems; wider retrospective research 

studies including hundreds of samples should be 

constructed. With the ever reducing time and 

money costs of whole genome association studies, 

it becomes rapidly easier to perform wide-range 

studies, paving the way for more rewarding 

research. 

References 

Abdel-Rahman SZ, Anwar Wa, Abdel-Aal WE, 

Mostafa HM and Au WW. GSTM1 and GSTT1 

genes are potential risk modifiers for bladder 

cancer. Cancer Detection and Prevention 

22:129-138, 1998. 

Aynacıoglu AS, Cascorbi I, Mrozikiewicz PM and 

Roots I. High frequency of CYP1A1 mutations 

in a Turkish population. Arch. Toxicol. 72: 215- 

218, 1998. 

Bartsch H, Nair U, Risch A, Rojas M, Wikman H 

and Alexandrov K. Genetic polymorphism of 

CYP genes, alone or in combination, as a risk 

modifier of tobacco-related cancers. Cancer 

Epidemio. Biomarkers Prev. 9: 3-28, 2000. 

Bolt HM, Roos PH and Thier R. The cytochrome 

P-450 isoenzyme CYP2E1 in the biological 

processing of industrial chemicals: 

consequences for occupational and 

environmental medicine. Int. Arch Occup 

Environ Health. 76: 174-185, 2003. 

Brockmoller J, Cascorbi I, Kerb R and Roots I. 

Combined analysis of inherited polymorphisms 

in arylamine N-acetyltransferase 2, glutathione 

S-transferase M1 and T1, microsomal epoxide 

hydrolase and cytochrome P450 enzymes as 

modulators bladder cancer risk. Cancer 

Research. 56: 3915-3925, 1996. 

Cascorbi I, Brockmoller J and Roots I. A C4887A 

polymorphism in exon 7 of human CYP1A1: 

population frequency, mutation linkages, and 

impact on lung cancer susceptibility. Cancer 

Research. 4965-4969, 1996.

Cunningham JM. SJ Hebbring SJ, McDonnell SK, 

Cicek MS, Christensen GB, Wang L, Jacobsen 

SJ, Cerhan JR, Blute ML, Schaid DJ and 

Thibodeau SN. Evaluation of genetic variations 

Dozmorov MG, Azzarello JT, Wren JD, Fung KM, 

Yang Q, Davis JS, Hurst RE, Culkin DJ, 

Penning TM and Lin HK. Elevated AKR1C3 

expression promotes prostate cancer cell 

survival and prostate cell-mediated endothelial 

cell tube formation: implications for prostate 

cancer progression. BMC Cancer.10: 672, 2010. 

Febbo PG, Kantoff PW, Giovannucci E, Brown M, 

Chang G, Hennekens CH and Stampfer M. 

Debrisoquine Hydroxylase (CYP2D6) And 

Prostate Cancer. Cancer Epidemiol Biomarkers 

Prev. 7: 1075-1078, 1998. 

Figuerova JD, Malats N, Garci-Closas M, Real FX, 

Silverman D, Kogevinas M, Chanock S, Welch 

R, Dosemeci M, Lan Q, Tardón A, Serra C, 

Carrato A, García-Closas R, Castaño-Vinyals G 

and Rothman N. Bladder cancer risk and 

genetic variation in AKR1C3 and other 

metabolizing genes. Carcinogenesis. 

29(10):1955-1962, 2008. 

Flynn TG and Green NC. The aldo-keto reductases: 

an overview. Adv Exp Med Biol. 328:251-7, 

1993. 

Franekova M, Halasova E, Bukovska E, Luptak J 

and Dobrota D. Gene polymorphisms in bladder 

cancer. Urologic Oncology. 26(1):1-8, 2008. 

García-Closas M, Malats N, Silverman D, 

Dosemeci M, Kogevinas M, Hein DW, Tardón 

A, Serra C, Carrato A, García-Closas R, Lloreta 

J, Castaño-Vinyals G, Yeager M, Welch R, 

Chanock S, Chatterjee N, Wacholder S, 

Samanic C, Torà M, Fernández F, Real FX and 

Rothman N. NAT2 slow acetylation, GSTM1 

null genotype, and risk of bladder cancer: 

results from the Spanish Bladder Cancer Study 

and meta-analyses. Lancet. 366(9486):649-59, 

2005. 

Hein DW, Boukouvala S, Grant DM, Minchin RF 

and Sim E. Human Arylamine N- 

Acetyltransferase Gene Nomenclature. 

Retrieved August 21, 2009 from 

http://louisville.edu/medschool/pharmacology/N 

AT.html 


in the androgen and estrogen metabolic 

pathways as risk factors for sporadic and 

familial prostate cancer. Cancer Epidemiology, 

Biomarkers and Prevention. 16: 969–978. 2007. 

Hsu NY, Ho HC, Chow KC, Lin TY, Shih CS, 

Wang LS and Tsai CM. Overexpression of 

dihydrodiol dehydrogenase as a prognostic 

marker of non-small cell lung cancer. Cancer 

Res. 61(6):2727-31, 2001. 

Hung RJ, Boffetta P, Brennan P, Malaveille C, 

Hautefeuille A, Donato F, Gelatti U, Spaliviero 

M, Placidi D, Carta A, Scotto di Carlo A and 

Porru S. GST, NAT, SULT1A1, CYP1B1 

genetic polymorphisms, interactions with 

environmental exposures and bladder cancer 

risk in a high-risk population. International 

Journal of Cancer. 110(4):598-604, 2004. 

Hyndman D and Penning TM. Single Nucleotide 

Polymorphisms in Human AKR Genes. 

Retrieved October 19, 2010 from 

http://www.med.upenn.edu/akr/polymorphisms. 

shtml 

Jakobsson J. Palonek E, Lorentzon M, Ohlsson C, 

Rane A and Ekström L. A novel polymorphism 

in the 17beta-hydroxysteroid dehydrogenase 

type 5 (aldo-keto reductase 1C3) gene is 

associated with lower serum testosterone levels 

in caucasian men. The Pharmacogenomics 

Journal. 7: 282–289, 2007. 

Jiang Z, Li C and Wang X. Glutathione Stransferase 

M1 polymorphism and bladder 

cancer risk: a meta-analysis involving 33 

studies. Exp Biol Med. 236(6):723-8, 2011. 

Jin Y and Penning TM. Aldo-keto reductases and 

bioactivation/detoxication. Annual Reviews 

Pharmacology and Toxicology. 47:263-92, 

2007. 

Karagas MR, Park S, Nelson HH, Andrew AS, 

Mott L, Schned A and Kelsey KT. 

Methylenetetrahydrofolate reductase (MTHFR) 

variants and bladder cancer: a population-based 

case-control study. Int J Hyg Environ Health. 

208: 321-327, 2005. 

Kim WJ and Quan C. Genetic and epigenetic 

aspects of bladder cancer. J Cell Biochem. 95: 

24-33, 2005.

14 

Kume T, Iwasa H, Shiraishi H, Yokoi T, 

Nagashima K, Otsuka M, Terada T, Takagi 

T, Hara A and Kamataki T. Characterization 

of a novel variant (S145C/L311V) of 

3alpha-hydroxysteroid/dihydrodiol 

dehydrogenase in human liver. 

Pharmacogenetics. 9(6):763-71, 1999. 

Lan Q, Mumford JL, Shen M, Demarini DM, 

Bonner MR, He X, Yeager M, Welch R, 

Chanock S, Tian L, Chapman RS, Zheng T, 

Keohavong P, Caporaso N and Rothman N. 

Oxidative damage-related genes AKR1C3 

and OGG1 modulate risks for lung cancer 

due to exposure to PAH-rich coal 

combustion emissions. Carcinogenesis. 

25:2177–2181, 2004. 

Lan Q, Zheng T, Shen M, Zhang Y, Wang SS, 

Zahm SH, Holford TR, Leaderer B, Boyle P 

and Chanock S. Genetic polymorphisms in 

the oxidative stress pathway and 

susceptibility to non-Hodgkin lymphoma. 

Human Genetics. 121:161–168, 2007. 

Lee SJ, Cho SH, Park SK, Kim SW, Park MS, 

Choi HY, Choi JY, Lee SY, Im HJ, Kim JY, 

Yoon KJ, Choi H, Shin SG, Park TW, 

Rothman N, Hirvonen A and Kang D. 

Combined effect of glutathione S-transferase 

M1 and T1 genotypes on bladder cancer 

risk. Cancer Letters. 177(2):173-9, 2002. 

Longuemaux S, Delomenıe C, Gallou C, 

Mejean A, Vincent-viry M, Bouvier R, Droz 

D, Krishnamoorthy R, Galteau MM, Junien 

C, Beroud C and Dupret JM. Candidate 

genetic modifiers of individual susceptibility 

to renal cell carcinoma: a study of 

polymorphic human xenobioticmetabolizing 

enzymes. Cancer Res. 59: 

2903-2908, 1999. 

Palackal NT, Lee H, Harvey R, Blair I, and 

Penning TM. Activation of polycyclic 

aromatic hydrocarbon trans-dihydrodiol 

proximate and their functional 

overexpression in human lung carcinoma 

(A549) cells. Journal of Biological 

Chemistry. 277(27):24799-24808, 2002. 

Parkin DM, Whelan S, Ferlay J, Leppo L and 

Thomas DB. Cancer incidence in five 

continents. IARC Publication, Lyon, 2002. 

Parkin DM. The global burden of urinary 

bladder cancer. Scandinavian Journal of 

Urology and Nephrology. 218:12-20, 2008. 

Penning TM, Jin Y, Steckelbroeck S, Lanisnik 

Rizner T and Lewis M. Structure-function of 

human 3 alpha-hydroxysteroid 

dehydrogenases: genes and proteins. Mol 

Cell Endocrinol. 215(1-2):63-72, 2004. 

Salinas-Sánchez AS, Sánchez-Sánchez F, 

Donate-Moreno MJ, Rubio-Del-Campo A, 

Gimenez-Bachs JM, Lorenzo-Romero JG, 

Serrano-Oviedo L and Escribano J. 

Polymorphic deletions of the GSTT1 and 

GSTM1 genes and susceptibility to bladder 

cancer. BJU Int. 107(11):1825-32, 2011. 

Silverman DT, Rothman N, Devesa SS, Syrigos 

KN and Skinner DG. Bladder cancer: 

biology, diagnosis, and management. 

Oxford University Press, New York, 1999. 

Sobti RC, Al-badran, AI Sharma, S Sharma, SK 

Krishan A and Mohan H. Genetic 

polymorphisms of CYP2D6, GSTM1, and 

GSTT1 genes and bladder cancer risk in 

North India. Cancer Genet. Cytogenet. 156: 

68-73, 2005. 

Srivastava DS, Kumar A, Mittal B and Mittal 

RD. GSTM1 and GSTT1 genes in bladder 

cancer: A study from North India. Archives 

of Toxicology 78:430-434, 2004. 

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

Cytochrome P450 1B1, a new keystone in 

gene-environment interactions related to 

human head and neck cancer? Toxicol. 76: 

249-256, 2002. 

Tiryakioğlu O. Investigation of aldoketoreductase 

1C3 rs12529 polymorphism in 

bladder cancer patients. MSc Thesis. Haliç 

University, 2011. 

Zheng L, Wang Y, Schabath MB, Grossman 

H.B and Wu X. Sulfotransferase 1A1 

(SULT1A1) polymorphism and bladder 

cancer risk: A case-control study. Cancer 

Letters. 202:61-69, 2003.

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




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

ISKAKOV 1 

1 

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

Biochemistry, Almaty, Kazakhstan 

2 

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

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

Received: 10 May 2010; Accepted: 20 January 2011 

Abstract 

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

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

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

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

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

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

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

activity. 

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

16C line 

Patates virüsü M’nin susturucu baskılayıcılarının tayini 

Özet 

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

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

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

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

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

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

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

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

benthamiana 16C hattı 

Introduction 

Recently a new defense system was detected that 

does function in plants, namely post-transcriptional 

gene silencing (PTGS) or RNA interference 

(RNAi). In response to virus infection, this 

“adaptive immune-system” is induced based on the 

recognition of double-stranded (ds) RNA or 

excessive quantities of RNA (Jorgensen, 2003). 

RNAi is an evolutionary conserved mechanism in 

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

aberrant endogenous or exogenous RNA molecules 

(Sontheimer, 2005; Voinnet, 2005). 

Due to this silencing defense system, plant 

viruses are thought to need a counter defense 

mechanism to successfully infect a plant. 

Depending on the virus, such PTGS suppression 

can be a secondary function of almost any type of 

viral protein. In plants, RNA silencing has been 

demonstrated to be one of the most important

16 

Ruslan KRYLDAKOV et al. 

antiviral mechanisms (Li and Ding, 2001; Ding et 

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

antiviral RNA silencing pathway, plant viruses 

have evolved specific suppressor proteins. Many of 

these proteins were previously described as 

virulence factors or pathogenicity determinants. It 

has been reported that many plant viruses encode 

suppressor proteins to combat against RNA 

silencing (Voinnet, Pinto and Baulcombe, 1999; Li 

and Ding, 2001). Identification of silencing 

suppressors of different viruses is a crucial step in 

direction of development of resistance against viral 

infection (Ding and Voinnet, 2007). 

Silencing suppressors can interfere with several 

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

to produce siRNAs, destabilization of siRNAs, 

binding of siRNAs and siRNA usage as part of 

RISC, which is required for target RNA 

elimination. Silencing suppressors are usually 

multifunctional viral proteins and in different virus 

families different viral proteins gain suppressor 

function as an additional property. Interference 

with suppressor function would inhibit the viral 

counter-defense system. 

The research and identification of silencing 

suppressors of PVM were carried out. Potato is one 

of the most important crops, and PVM, together 

with PVY, is responsible for 80% of all viruscaused 

losses of potato yields, especially in 

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

identification of PVM’s silencing suppressors 

points to the role of gene silencing as a natural 

antiviral defense system in plants. 

Materials and methods 

RNA isolation 

A PVM isolate was maintained in Nicotiana 

tabacum plants. Leaves of N. tabacum were 

harvested and frozen in liquid nitrogen, ground to a 

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

extracted once with 1/5 volume of chloroform. 

RNA was precipitated with the addition of an equal 

volume of isopropanol and incubated at +4°C for 

30 min. RNA was washed with 70% ethanol and 

dissolved in water. 

Construction of PVM vectors 

The last 5 of 6 protein sequences of PVM were 

obtained with using SuperScript II Reverse 

Transcriptase according to manufacture protocol. 

PVM cDNA fragments encoding the 25kDa, 

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

PCR-amplified from cDNA and individually cloned 

using Gateway cloning kit (Invitrogen). First, the 

PCR products were cloned into pDONR207 vector 

using BP clonase, then they were re-cloned into 

pMDC32 destination vector harbouring double 35S 

promoter. 

Plant material and A. tumefaciens infiltration 

GFP transgenic 16C line of Nicotiana benthamiana 

plants were grown as described previously (Marano 

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

Recombinant Agrobacterium tumefaciens strain 

C58C1, carrying different constructs, were grown 

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

tetracycline and 50 μg/ml kanamycin for two days, 

then transferred to 18 ml of fresh LB medium with 

appropriate antibiotics and were grown overnight at 

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

10 min. The pellet was resuspended in 

agroinfiltration solution (10mM MgCl2, 10 mM 

MES and 100 μM acetosyringone) up to desired 

optical density (OD600). The bacteria solution was 

left at room temperature for overnight before 

infiltration. Infiltration carried on underside leaves 

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

equal volumes of indicated A. 

tumefaciens cultures (OD600=1) were mixed before 

infiltration. After 5-10 days post infiltration, plants 

were observed for silencing effect. 

GFP Imaging 

Visual detection of GFP fluorescence in whole 

plant was performed using a 100W hand-held longwave 

ultraviolet lamp. Plants were photographed 

with a yellow filter. 

Results 

Potato virus M belongs to the genus Carlavirus of 

the Flexiviridae family. The PVM genome is a 

single positive-strand RNA of 8534 nucleotides. 

The sequence contains six open reading frames 

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

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

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

(Zavriev et al., 1991).

Identification of silencing suppressors of PVM 17 

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

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

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

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

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

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

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

Table 1. Proteins of PVM and their sizes 

Symbol of protein Protein Start 

End 

Length 

(nt) 

(nt) 

(bp) 

ORF 1 polyprotein 74 5980 1968 

ORF 2 25kDa protein 6019 6708 229 



ORF 5 coat protein 7225 8139 304 


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

2006), the protein 11K is most putative silencing 

suppressor. The aim of our investigation was the 

check of suppressor activity of all PVM proteins 

except first one, largest polyprotein due to its size. 

The primers for cloning were designed using 

the GeneRunner programme version 3.0 

(ORF2sense 5 ’ - 

gggacaagtttgtacaaaaaagcaggctatggatgtgattgtagatttg- 

3 ’ and ORF2antisense 5 ’ - 

ggggaccactttgtacaagaaagctgggtctcaggcggcggtgtaagt 

gg-3 ’ for ORF2; ORF3sense 5 ’ - 

ggggacaagtttgtacaaaaaagcaggctatgccacttacaccgccgc 

c-3 ’ and ORF3antisense 5 ’ - 

ggggaccactttgtacaagaaagctgggtctcatgtgtgtgagcccgca 

c-3 ’ for ORF3; ORF4sense 5 ’ - 

ggggacaagtttgtacaaaaaagcaggctatgatagtgtatgtacttgta 

g-3 ’ and ORF4antisense 5 ’ - 

ggggaccactttgtacaagaaagctgggtcttatgacctaaaggaacca 

cac-3 ’ for ORF4; ORF5sense 5 ’ - 

ggggacaagtttgtacaaaaaagcaggctatgggagattcaacgaaga 

aag-3 ’ and ORF5antisense 5 ’ - 

ggggaccactttgtacaagaaagctgggtctcattttctattagactttac 

at-3 ’ for ORF5; ORF6sense 5 ’ - 

ggggacaagtttgtacaaaaaagcaggctatgaaggacgtaaccaag 

gtggctt-3 ’ and ORF6antisense 5 ’ - 

ggggaccactttgtacaagaaagctgggtcctactctcgcttgttgatga 

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

used for cloning. The DNA of the different PVM 

ORFs were obtained by RT-PCR, and cloned into 

the Gateway-compatible binary T-DNA destination 

vector pMDC32. Each of these constructs were 

electroporated into Agrobacterium tumefaciens 

C58C1 strain according to the manufacturer’s 

instructions. All constructs described above were 

verified by nucleotide sequencing. 

The Nicotiana benthamiana plant, constitutively 

expressing GFP transgene (line 16C; a gift from 

David Baulcombe), and the Agrobacterium 

infiltration operation have been described 

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

benthamiana line 16C were cultured in growth 

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

For coinfiltration, equal volumes of individual 

Agrobacterium cultures (OD600= 1) were mixed

18 


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

P19 of tombusviruses were used as a positive 

control. GFP fluorescence was observed under 

long-wavelength UV light and photographed using 

a digital camera with a yellow filter. 

GFP expression reached the highest level in all 

leaves infiltrated with GFP, as well as GFP plus 

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

shown by the enhanced green fluorescence in the 

infiltrated patches. The green fluorescence intensity 

remained strong in the patches coinfiltrated with 

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

with ORF 6 also resulted in GFP fluorescence over 

a similar period of observations, but not as strong 

and clear as above described samples (Figure 2). 

On the other hand, GFP was silenced upon 

inoculation with OFR 2 and ORF 4. 

Figure 2. Effects of different ORFs of PVM on 

gene silencing in GFP transformants of Nicotiana 

benthamiana. Infiltrated leaves are shown. 

Silencing of GFP is manifested in red fluorescent. 

Green spots indicate suppression of silencing. 

Photographs were taken of UV-illuminated plants. 

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

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

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

protein; 7: ORF6, 11K protein 

According to these data the most promising 

candidates as silencing suppressors are ORF 3 (12K 

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

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

while ORF 2 and ORF 4 revealed no suppressor 

activity. We have repeated this experiment three 

times which yielded the same results. 

Discussion 

Most of the viruses have some sort of silencing 

suppressor system as an evolutionary counterresponse 

to the antiviral activity of the plant gene 

silencing defence. Gene silencing suppressors can 

inhibit silencing of transgenes or endogenous 

genes. Test for silencing suppression was based on 

a previously described experimental system 

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

benthamiana line 16C plants were agroinfiltrated 

with an A. tumefaciens C58C1 strain carrying the 

construct 35S GFP, the strong green fluorescent 

signal disappeared under UV light due to GFP 

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

silencing suppressor are co-agroinfiltrated, the 

fluorescent signal does not disappear and the area 

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

the inhibition of gene silencing caused by that 

suppressor. 

Thus, the result of experiments shows that 

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

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

protein) of potato virus M has a suppressor activity. 

It corresponds to the results of recent articles about 

suppression activity of virus proteins responsible 

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

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

10K-16K proteins with a conserved amino acid 

sequence motif encoded by diverse filamentous 

viruses (Chiba et al., 2006). 

The 12K protein encoded by the second ORF of 

PVM triple gene block (TGB) which involved in 

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

virus. This protein possesses conserved 

hydrophobic domain and plays significant role in 

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

viruses have evolved special 

mechanisms to suppress RNA silencing (Voinnet et 

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

The potex-like Carlavirus PVM requires the 

coat protein for virus cell-to-cell movement 

(Morozov and Solovyev, 2003; Verchot-Lubicz, 

2005). In RNA silencing assays, plant viral 

suppressors differ by their ability to suppress 

intracellular and/or intercellular silencing (Li and 

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

Spreading of silencing in plants occurs through 

plasmodesmata and results from a cell-to-cell 

movement of a short-range silencing signal, most 

probably 21-nt short interfering RNAs (siRNAs), 

that are produced by one of the plant Dicer 

enzymes (Kalantidis et al., 2008). 

11K protein had been shown to be a nonspecific 

nucleic acid-binding protein (Gramstat, 

Courtpozanis and Rohde, 1990), similar to p10 

which is silencing suppressor of Grapevine virus A 

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

to be translated from the CP subgenomic RNA by a 

(-1) frameshift within the A-rich region

AAUAGAAAAUGA preceding the 12K AUG 

translational start codon (underlined) and 

representing the 3’-terminus of the CP coding 

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

Courtpozanis and Rohde, 1990). 

This article describes the identification of viral 

suppressors of RNA silencing encoded by a natural 

pathogen. An exact role for any of the identified 

PVM suppressors in host infection remains to be 

established. 

The potential of using viral suppressors to help 

understand the mechanism of RNA silencing in 

plants is largely unexplored, and these studies 

promise to be a fertile area of research. Viral 

suppressors of silencing also provide unique tools 

to understand the mechanism of RNA silencing as 

well as interaction between virus and their hosts. 

Moreover, it became an extremely useful technique 

for molecular biology and very powerful 

biotechnological tool. 

Acknowledgement 

This work has been supported by funds from 

INTAS, Young Scientist Fellowship Nr. 06- 

1000014-6200. 

References 

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

and Baulcombe DC. Viral pathogenicity 

determinants are suppressors of transgene 

silencing in Nicotiana benthamiana. The 

EMBO Journal. 17: 6739-6746, 1998. 

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

Mawassi M, Koonin EV, Carrington JC and 

Dolja VV. Diverse suppressors of RNA 

silencing enhance agroinfection by a viral 

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

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

silencing: a conserved antiviral immunity of 

plants and animals. Virus Research. 102: 109- 

115, 2004. 

Ding SW and Voinnet O. Antiviral Immunity 

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

2007. 

Gramstat A, Courtpozanis A and Rohde W. The 12 

kDa protein of potato virus M displays 

properties of a nucleic acid-binding regulatory 

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

Identification of silencing suppressors of PVM 19 

Hamilton A, Voinnet O, Chappell L and Baulcombe 

D. Two classes of short interfering RNA in 

RNA silencing. The EMBO Journal. 21: 4671- 

4679, 2002. 

Jorgensen RA. RNA silencing in plants: 

mechanisms, methods, and applications. Plant 

& Animal Genomes XI Conference, USA. 

2003. 

Kalantidis K, Schumacher HT, Alexiadis T and 

Helm JM. RNA silencing movement in plants. 

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

Li WX and Ding SW. Viral suppressors of RNA 

silencing. Current Opinion in Biotechnology. 

12: 150-154, 2001. 

Marano MR and Baulcombe D. Pathogen-derived 

resistance targeted against the negative-strand 

RNA of tobacco mosaic virus: RNA strandspecific 

gene silencing? Plant Journal. 13: 

537-546, 1998. 

Morozov SYu and Solovyev AG. Triple gene block: 

modular design of a multifunctional machine 

for plant virus movement. Journal of General 

Virology. 84: 1351-1366, 2003. 

Roth BM, Pruss GJ and Vance VB. Plant viral 

suppressors of RNA silencing. Virus Research. 

102: 97-108, 2004. 

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

Fedorkin O, Morozov SYu and Atabekov JG. 

Movement protein-derived resistance to triple 

gene block-containing plant viruses. Journal of 

General Virology. 78: 1241-1246, 1997. 

Sontheimer EJ. Assembly and function of RNA 

silencing complexes. Nature Reviews. 6: 127- 

138, 2005. 

Sozinova L, Abay A, Ramankulov E, Nachmias A 

and Loebenstein G. Reducing Virus Incidence 

in Potato Seed Tubers by Rapid Propagation: A 

Pilot Scheme in Kazakhstan, The 28 th Congress 

of the Israeli Phytopathological Society, The 

Volcani Center, Israel. 2007. 

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

Model for Potexviruses. Molecular Plant- 

Microbe Interactions Journal. 18: 283-290, 

2005. 

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

Systemic spread of sequence-specific transgene 

RNA degradation is initiated by localized

20 


introduction of ectopic promoterless DNA. 

Cell. 95: 177-187, 1998. 

Voinnet O, Pinto YM and Baulcombe DC. 

Suppression of gene silencing: A general 

strategy used by diverse DNA and RNA 

viruses of plants. Proceedings of the National 

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

Voinnet O, Lederer C and Baulcombe DC. A viral 

movement protein prevents spread of the gene 

silencing signal in Nicotiana benthamiana. 

Cell. 103: 157-167, 2000. 

Voinnet O. RNA silencing as a plant immune 

system against viruses. Trends in Genetics. 17: 

449-459, 2001. 

Voinnet O. Induction and suppression of RNA 

silencing: insights from viral infections. Nature 

Reviews Genetics. 6: 206-220, 2005. 

Zavriev SK, Kanyuka KV and Levay KE. The 

genome organization of potato virus M RNA. 

Journal of General Virology. 72: 9-14, 1991. 

Zhou ZSh, Dell’Orco M, Saldarelli P, Turturo C, 

Minafra A and Martelli GP. Identification of an 

RNA-silencing suppressor in the genome of 

Grapevine virus A. Journal of General 

Virology. 87: 2387-2395, 2006.




Identification of a novel dehydration responsive transcript from 

tossa jute (Corcohrus olitorius L.) 

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

KABIR 3 , Arzuba AKTER 1 1, * 

and Haseena KHAN 

1 

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

2 

Bangladesh Jute Research Institute, Dhaka 1207, Bangladesh 

3 

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

δ 

Contributed equally to the study. 

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

Received: 18 January 2011; Accepted: 23 April 2011 

Abstract 

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

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

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

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

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

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

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

plant species. 

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

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

tanımlanması 

Özet 

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

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

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

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

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

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

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

bulunamamıştır. 

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

Introduction 

Unlike their mobile animal counterparts, sessile 

plants have to adjust their gene expression pattern 

as well as metabolite profile to cope with altered 

local environmental conditions (Kulheim et al. 

2002). A number of genes are involved in these 

pathways which can either be up-regulated (Hannah 

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

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

22 Sazia SHARMIN et al. 

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

function. Identification of these genes may lead to 

the development of commercial crops, which are 

better suited to grow in a particular unfavourable 

condition. Due to cross-species transferability of 

gene functions, this improvement can be achieved 

in non-source plant species as well. 

Jute (Corchorus olitorius L.) is the most 

important bast fibre producing plant (Kundu, 1956). 

It is the major fibre producing plant of Bangladesh 

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

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

The production quality and yield of this 

economically important crop is affected by several 

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

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

abiotic factors e.g. salinity, submergence and low 

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

2010). Thus, development of jute varieties with 

increased resistance to both biotic and abiotic stress 

conditions have considerable economic importance. 

In spite of its enormous commercial value, only 

a few genes or part of genes from jute have been 

identified or sequenced (Ahmed et al. 2009; Alam 

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

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

we report a novel transcript from Corchorus 

olitorus var. 9897 responsive to dehydration stress. 

Search for known homologues failed to identify 

functionally characterized counterparts in any other 

plant species. 


Stress treatment of plant seedlings 

Plant seeds were incubated at room temperature in 

the absence of light for two days in petri dishes in 

the presence of water only. Germinated plant 

seedlings were subjected to different stress 

conditions such as low temperature, dehydration, 

fungus and abscisic acid (ABA) beginning third 

day of their germination according to methods 

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

2010). Fungus treatment was done by spraying 

suspension of Macrophomina phaseolina on the 

plate with germinated seedlings (Alam et al. 2010; 

Mahmood et al. 2010). For dehydration treatment, 

the seedlings were treated with 50 mM manitol to 

create an environment similar to water deficit 

condition. The concentration of manitol was 

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

Salt stress was applied by adding 50 mM NaCl to 

the plate on day 3. The concentration was increased 

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

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

treatment was carried out by growing the seedlings 

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

2010). 

Isolation of DNA and RNA 

Genomic DNA from different Corchorus species 

was isolated using the CTAB method (Murray and 

Thompson, 1980). RNA was isolated using 

TRIZOL reagent (Invitrogen) according to the 

provided manual. For tissue specific RNA isolation, 

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

liquid N2. RNA was isolated from ground tissue 

using TRIZOL reagent (Invitrogen) as per 

manufacturer’s instruction 

Identification of the dehydration responsive 

transcript 

The transcript was identified as an additional band 

while carrying out RT (Reverse Transcriptase)- 

PCR of a cold stress responsive gene, LDLP (Low 

density lipoprotein like protein) from C. olitorius 

using the primer RevP (unpublished result). RT- 

PCR was carried out using RNA isolated from C. 

olitorius grown under normal condition. This 

additional band was extracted from agarose gel 

using QIAGEN MinElute Gel Extraction Kit and 

sequenced (1 st Base PTE Singapore) using ForP 

and RevP as primers. Gene specific primers 

(GSPF1, GSPR1 and GSPR2) for the transcript was 

designed using Primer3Plus (Untergasser et al. 

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

These primers were used to sequence partial CDS 

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

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

trilocularis. 

Table 1. Primers used in the study 

Primer Sequence 

ForP 5’- ATACTTATGGGAGAATGAAAG -3’ 

RevP 5’- TACAGGTAAAATGTCACCAA -3’ 

GSP-R1 5’- GAGGCTTTTCTTGGAAC -3’ 

GSP-R2 5’- GTCCCGATCCCTTGAATTAC -3’ 

GSP-F1 5’- CTTAATCGGAGCAGGGTGAG -3’

Semiquantitative RT-PCR 

Total RNA was isolated from jute seedlings and 

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

RNA was isolated from total RNA using mRNA 

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

and quantified. First strand cDNA was synthesized 

from 50 ng of mRNA using gene specific primer 

GSP-R2. PCR amplification of the cDNA was 

carried out for 25 cycles using primers GSP-F1 and 

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

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

as the internal standard for reverse transcriptase 

polymerase chain reaction (Alam et al. 2010). 

5′ RACE 

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

determined by 5′- Rapid Amplification of cDNA 

Ends (RACE) protocol. The first strand cDNA was 

prepared from mRNA using the primer GSPR1. 

Purification of first strand cDNA and TdT tailing 

was carried out as recommended by the 

manufacturer (5′ RACE system, Invitrogen 

Corporation, Carlsbad, CA). First round PCR of 

target cDNA was carried out using the primer 

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

min, and 72°C for 2 min). 

Southern blot 

15 μg DNA was digested with EcoRI and SacI and 

size-fractionated on 1% agarose gel and then 

transferred onto a positively charged nylon 

membrane (Amersham Hybond-N). Probe was 

synthesized from RT-PCR product using primer 

GSP-F1 and GSPR2 and Dig nucleic acid labeling 

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

guideline for highly stringent hybridization and 

detection conditions was followed (probe 10 

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

SDS). 

Northern blot 

10 μg RNA was separated by electrophoreses in 1X 

MOPS and transferred to a positively charged 

nylon membrane (Amersham Hybond-N). Probe 

was synthesized from RT-PCR product by using 

primer GSPF and GSPR2 and Dig nucleic acid 

labelling and detection kit (Roche, Cat No. 

11175033910). Manufacturer’s guideline for highly 

stringent hybridization and detection conditions 

Novel dehydration responsive transcript from jute 23 

was followed (probe 10 ng/mL, stringency washes 

with 0.1X SSC, 0.1% SDS). 

Sequence assembly and analysis 

Sequences from gel extracted band and 5′ RACE 

was assembled using CAP3 (Huang and Madan, 

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

Coding sequences were identified using ESTScan2 

(Iseli et al. 1999). Phylogenetic tree was 

constructed using MEGA4 by UPGMA method 

with default parameters (Tamura et al. 2007). 

Multiple sequence alignment was done using 

Clustalx (Larkin et al. 2007). Protein secondary 

structure was predicted from NPS@ webserver 

(Combet et al. 2000). 

Results 

Identification of the transcript 

The dehydration responsive transcript was 

identified by sequencing of additional band 

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

sequenced transcript failed to show significant 

similarity to any of the so far functionally 

characterized proteins in blast search at NCBI 

database. An additional 277 bp sequence at the 5′ 

end was determined by 5′ RACE. Partial CDS of 

drp from five other Corchorus spp (C. capsularis, 

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

trilocularis) were obtained using gene specific 

primer (GSP) pairs. Sequence information was 

deposited to DDBJ (Accession numbers 

AB571589-AB571594). 

Copy number determination 

To determine the copy number of the gene within 

the C. olitorius genome, isolated genomic DNA 

was digested EcoRI and SacI. Southern blot with 

both enzyme digests showed four distinct bands 

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

genome has four copies of drp. 

Expression study: Tissue and stress specific 

expression 

Reverse transcriptase PCR (RT-PCR) from RNA 

isolated from leaf, stem and root suggested that drp 

was expressed in all three tissues (Figure 2). 

Semiquantitative RT-PCR showed reduced 

expression under dehydration stressed condition


(Figure 3). Similar expression pattern was observed 

in Northern blot of C. olitorius transcript with a 

cDNA probe specific for the sequence (Figure 4). 

The transcript also showed somewhat reduced 

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

SacI (b) digestion. Arrows indicate bands to which 

probe was hybridized. 

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

and actin control (bottom) under different stress 

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

Experimental, Low temperature, Salt, Dehydration, 

Fungal and ABA treatment, respectively). 

Comparative analysis of drp from different 

Corchorus spp. 

Translated protein sequence alignment showed a 

highly conserved segment of 32 amino acid 

expression in low temperature and fungus treated 

plants (Figures 3 and 4). 

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

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

respectively). 

Figure 4. Northern blot of drp under different 

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

Normal, Experimental, Low temperature, Salt, 

Dehydration, Fungal and ABA treatment, 

respectively). rRNA is shown below as a control 

for equal loading. 

residues (Figure 5a). Secondary structure prediction 

suggested that the region is mainly composed of 

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

phylogenetic tree (Figure 6) of drp nucleotide 

sequence from different Corchorus species was 

constructed using MEGA4 (Tamura et al. 2007).

A 

Corchorus_olitorius SAPKARQRCVADGKQVNIPAPSYDAMGGRIAE 

Corchorus_capsularis SAPKARQRCVADGKQVNIPAPSYDAMGGRIAE 

Corchorus_tridens SAPKARQRCVADGKQVNIPAPSYDAMGGRIAE 

Corchorus_pseudo-olitorius SAPKARQRCVADGKQVNIPAPSYDAMGGRIAE 

Corchorus_trilocularis SAPKARQRCVADGKLVNIPAPSYSAMGGRIAE 

Corchorus_aestuans SAPKARQRCVADGKQVNIPAPSYNAMGGRIAE 

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

B 

10 20 30 

| | | 

UNK_136050 SAPKARQRCVADGKQVNIPAPSYDAMGGRIAE 

DPM ctchhhhhhehtcccecccccccchhcchhcc 

DSC ccccceeeeecccccccccccccccccccccc 

GOR1 hhhhhheeeeettteeeeeeceeeehhhhhhh 

GOR3 cccchhheehhccceecccccccccccceehh 

HNNC cccccccccccccccccccccccccccccccc 

MLRC cccccceeeecccccecccccccccccccccc 

PHD ccccccchhhhccccccccccccccccccccc 

Predator ccchhhhhhhcccccccccccccccccccccc 

SOPM ccccccheeecttccccccccccchhtchhhh 

Sec.Cons. ccccc?heeecccccccccccccccccccccc 

Sequence length : 32 


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

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

secondary structure from different methods of prediction. 

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

consensus secondary structure. 

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

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

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

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

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

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

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

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

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

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

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


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

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

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

Discussion 

Acquisition of dehydration tolerance is one of the 

traits that enabled land-plants to settle into 

terrestrial habitats (Richardt et al. 2007). 

Dehydration stress is a specific form of water 

deficit stress that results in alteration of plants at 

the cellular, morphological and developmental 

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

pathway can be classified into two broad categories 

viz. ABA dependent and ABA independent 

pathway (Ramanjulu and Bartels, 2002). Individual 

pathways are discussed in detail elsewhere 

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

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

Yamaguchi-Shinozaki and Shinozaki, 2006). 

The newly identified transcript, namely drp, 

was found to be expressed in all three tissues tested 

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

Analysis of the expression pattern after stress 

treatments suggests decreased expression in 

dehydration stress, but increased expression in 

ABA stress condition (Figures 3 and 4). 

Comparative analyses of water deficit stressed 

transcriptome of a number of plants identified plant 

genes showing altered expression in either 

dehydration stress alone or in combination with any 

other water deficit stress or ABA treatment (Bray, 

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

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

identified single transcript showing opposite 

expression patterns in water deficit stress and ABA 

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

transcript also shows somewhat reduced expression 

in low temperature treated plants. Previous studies 

on dehydration stress responsive genes also found 

similar correlation with low temperature response 

pathways (Shinozaki and Yamaguchi-Shinozaki, 

2000). 

Comparative analysis of different experimental 

methods of water deficit stress treatment revealed 

that three transcripts (At2g06850, At1g72610 and 

At5g20630) are down-regulated in Arabidopsis 

irrespective of the method (filter paper, mannitol or 

soil water deficit) applied (Bray, 2004). 

Interestingly all the three genes are either known or 

hypothesized to function in plant cell wall 

modification pathways that control growth (Bray, 

2004). Both semi-quantitative RT (reverse 

transcriptase) -PCR and northern blot suggests 

reduced expression of drp in fungus treated plants. 

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

2000) revealed that a significant portion of fungal 

inoculation responsive genes in Arabidopsis are 

associated with cell wall synthesis and 

modification. Thus, drp might also play a role in 

defence pathways against fungal infection in jute 

by playing a role in cell wall synthesis and/or 

modification. 

Southern blot of the transcript suggests that C. 

olitorius genome has four copies of drp gene 

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

copies of the drp gene are under different 

regulatory mechanisms and the observed 

expression patterns are of different drp genes which

are indistinguishable in the region selected for 

analyses. 

The newly identified transcript failed to show 

significant similarity to any of the so far 

functionally characterized proteins. It is to be noted 

that plant specific nucleotide blast in NCBI 

database shows significant similarity to a 

hypothetical Poplus trichocarpa protein 

(XM_002337327.1) and other plant transcripts. 

Paralogues of the gene were found to be present in 

all tested Corchorus species. Phylogenetic analysis 

of drp gene and its homologues suggests that C. 

capsularis is the closest neighbour of C. olitorius in 

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

dicots grouped into same clade in the tree (Figure 

6). 

Multiple sequence alignment of protein 

sequences from different Corchorus spp. identified 

a highly conserved segment of 32 amino acid 

residues. Secondary structure prediction at NPS@ 

webserver (Combet et al. 2000) suggested these 

amino acids are most likely to form random coils 

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

specifically coils add flexibility to the protein 

structure (Buxbaum, 2007). Our literature survey 

revealed an interesting relationship between 

random coils and their possible role in dehydration 

response. It has been long known that different 

organisms release osmolytes when subjected to 

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

and co-workers (1998) identified that osmolytes 

cause contraction of random coils leading to 

decrease of conformational entropy and subsequent 

stabilization of protein folding. 

The present study has identified a novel 

dehydration responsive transcript from different 

jute (Corchorus) species. Further studies are 

required to elucidate the detailed mechanism of 

action of this transcript. 


This research was supported by the United States 

Department of Agriculture (USDA). We are 

thankful to Bangladesh Jute Research Institute 

(BJRI) for providing seeds of different jute species. 

References 

Agarwal P and Jha B. Transcription factors in 

plants and ABA dependent and independent 

abiotic stress signalling. Biol Plant. 54:201-212, 

2010. 


Ahmed S, Nabi MZ, Alam MM, Islam MS, Samira 

R, Moosa MM and Khan H. A computational 

and experimental approach for developing jute 

ESTs from genomic clones. Aust J Crop Sci. 

3:322-328, 2009. 

Alam M, Sharmin S, Nabi Z, Mondal S, Islam M, 

Nayeem S, Shoyaib M and Khan H. A Putative 

Leucine-Rich Repeat Receptor-Like Kinase of 

Jute Involved in Stress Response. Plant Mol 

Biol Rep. 28:394-402, 2010. 

Basu A, Ghosh M, Meyer R, Powell W, Basak SL 

and Sen SK. Analysis of genetic diversity in 

cultivated jute determined by means of SSR 

markers and AFLP profiling. Crop Science. 

44:678-685, 2004. 

Buxbaum E. Protein structure. Fundamentals of 

protein structure and function. Springer 

ScienceBusiness Media, LLC, New York. 12- 

37, 2007. 

BBS. Statistical pocket book of Bangladesh 

statistics division, Bangladesh Bureau of 

Statistics, Ministry of Planning, the People's 

Republic of Bangladesh. 2004. 

Bray EA. Molecular responses to water deficit. 

Plant Physiol. 103:1035-1040, 1993. 

Bray EA. Genes commonly regulated by waterdeficit 

stress in Arabidopsis thaliana. J Exp Bot. 

55:2331-2341, 2004. 

Combet C, Blanchet C, Geourjon C, and Deléage 

G. NPS@: Network Protein Sequence Analysis. 

Trends in Biochemical Science. 25:147-150, 

2000. 

Deléage G and Roux B. An algorithm for protein 

secondary structure prediction based on class 

prediction. Protein Eng. 1:289-294, 1987. 

Frishman D and Argos P. Incorporation of nonlocal 

interactions in protein secondary structure 

prediction from the amino acid sequence. 

Protein Eng. 9:133-142, 1996. 

Garnier J, Osguthorpe DJ and Robson B. Analysis 

of the accuracy and implications of simple 

methods for predicting the secondary structure 

of globular proteins. J Mol Biol. 120:97-120, 

1978. 

Geourjon C and Deléage G. SOPM: a selfoptimized 

method for protein secondary


structure prediction. Protein Eng. 7:157-164, 

1994. 

Ghosh T. Hand book on jute. Food and Agriculture 

Organization of the United Nations (FAO):115- 

116, 1983. 

Gibrat JF, Garnier J and Robson B. Further 

developments of protein secondary structure 

prediction using information theory: New 

parameters and consideration of residue pairs. J 

Mol Biol. 198:425-443, 1987. 

Guermeur Y. Combinaison de classifieurs 

statistiques, Application a la prediction de 

structure secondaire des proteines., p 169, 

Paris: University of Paris 06, 1997. 

Guermeur Y, Geourjon C, Gallinari P and Deléage 

G. Improved performance in protein secondary 

structure prediction by inhomogeneous score 

combination. Bioinformatics. 15:413-421, 1999. 

Hannah MA, Heyer AG and Hincha DK. A Global 

Survey of Gene Regulation during Cold 

Acclimation in Arabidopsis thaliana. PLoS 

Genetics. 1:e26, 2005. 

Holappa LD and Walker-Simmons MK. The Wheat 

Abscisic Acid-Responsive Protein Kinase 

mRNA, PKABA1, Is Up-Regulated by 

Dehydration, Cold Temperature, and Osmotic 

Stress. Plant Physiol. 108:1203-1210, 1995. 

Hossain MB, Awal A, Rahman MA, Haque S and 

Khan H. Distinction between cold-sensitive and 

-tolerant Jute by DNA polymorphism. Journal 

of Biochemistry and Molecular Biology. 

36:427-432, 2003. 

Huang X and Madan A. CAP3: A DNA Sequence 

Assembly Program. Genome Research. 9:868- 

877, 1999. 

Ingram J and Bartels D. The molecular basis of 

dehydration tolerance in plants. Annual Review 

of Plant Physiol and Plant Molecular Biology. 

47:377-403, 1996. 

Iseli C, Jongeneel CV and Bucher P. ESTScan: a 

program for detecting, evaluating, and 

reconstructing potential coding regions in EST 

sequences. In: Proceedings / International 

Conference on Intelligent Systems for 

Molecular Biology pp 138-148, 1999. 

Islam AS, Taliaferro M, Lee CT, Ingram C, 

Montalvo RJ, Ende Gvd, Alam S, Siddiqui J 

and Sathasivan K. Preliminary progress in Jute, 

Corchorus species. genome analysis. Plant 

Tissue Cult Biotechnol. 15:145-156, 2005. 

Keka SI, Shamsuzzaman M, Pahloan MU, Pervin 

S, Rahman MM and Khan H. Identifying simple 

sequence repeat (SSR) marker linked to mite 

tolerance in jute species. Bangladesh Journal of 

Botany. 37:161-171, 2008. 

King RD and Sternberg MJE. Identification and 

application of the concepts important for 

accurate and reliable protein secondary structure 

prediction. Protein Sci. 5:2298-2310, 1996. 

Kulheim C, Agren J and Jansson S. Rapid 

Regulation of Light Harvesting and Plant 

Fitness in the Field. Science, 297:91-93, 2002. 

Kundu B. Jute — world’s foremost bast fibre, II. 

Technology, marketing, production and 

utilization. Economic Botany. 10:203-240, 

1956. 

Larkin MA, Blackshields G, Brown NP, Chenna R, 

McGettigan PA, McWilliam H, Valentin F, 

Wallace IM, Wilm A, Lopez R, Thompson JD, 

Gibson TJ and Higgins DG. Clustal W and 

Clustal X version 2.0. Bioinformatics. 23:2947- 

2948, 2007. 

Mahmood N, Azam MS, Islam MS, Sharmin S, 

Moosa MM, Hossain MA and Khan H. 

Differentially expressed transcripts of wild and 

cultivated jute (Corchorus spp.) varieties upon 

fungal, Macrophomina phaseolina. infection. 

Annals of Biological Research. 1:120-127, 

2010. 

Murray MG and Thompson WF. Rapid isolation of 

high molecular weight plant DNA. Nucleic 

Acids Res. 8:4321-4326, 1980. 

Nakashima K, Ito Y and Yamaguchi-Shinozaki K. 

Transcriptional Regulatory Networks in 

Response to Abiotic Stresses in Arabidopsis and 

Grasses. Plant Physiol. 149:88-95, 2009. 

Perriere G, Combet C, Penel S, Blanchet C, 

Thioulouse J, Geourjon C, Grassot J, Charavay 

C, Gouy M, Duret L and Deleage G. Integrated 

databanks access and sequence/structure 

analysis services at the PBIL. Nucleic Acids 

Res. 31:3393-3399, 2003. 

Qu Y, Bolen CL, and Bolen DW. Osmolyte-driven 

contraction of a random coil protein. 

Proceedings of the National Academy of 

Sciences of the United States of America. 

95:9268-9273,1998.

Rabbani MA, Maruyama K, Abe H, Khan MA, 

Katsura K, Ito Y, Yoshiwara K, Seki M, 

Shinozaki K and Yamaguchi-Shinozaki K. 

Monitoring Expression Profiles of Rice Genes 

under Cold, Drought, and High-Salinity 

Stresses and Abscisic Acid Application Using 

cDNA Microarray and RNA Gel-Blot Analyses. 

Plant Physiol. 133:1755-1767, 2003. 

Ramanjulu S and Bartels D. Drought- and 

desiccation-induced modulation of gene 

expression in plants. Plant, Cell & 

Environment. 25:141-151, 2002. 

Richardt S, Timmerhaus G, Lang D, Qudeimat E, 

Corrêa L, Reski R, Rensing S and Frank W. 

Microarray analysis of the moss Physcomitrella 

patens reveals evolutionarily conserved 

transcriptional regulation of salt stress and 

abscisic acid signalling. Plant Molecular 

Biology. 72:27-45, 2007. 

Rost B and Sander C. Prediction of Protein 

Secondary Structure at Better than 70% 

Accuracy. J Mol Biol. 232:584-599, 1993. 

Samira R, Moosa MM, Alam MM, Keka SI and 

Khan H. In silico analysis of jute SSR library 

and experimental verification of assembly. 

Plant Omics Journal. 3:57-65, 2010. 

Schenk PM, Kazan K, Wilson I, Anderson JP, 

Richmond T, Somerville SC and Manners JM. 

Coordinated plant defense responses in 

Arabidopsis revealed by microarray analysis. 

Proceedings of the National Academy Science 

of the United States of America. 97:11655- 

11660, 2000. 

Seki M, Ishida J, Narusaka M, Fujita M, Nanjo T, 

Umezawa T, Kamiya A, Nakajima M, Enju A, 

Sakurai T, Satou M, Akiyama K, Yamaguchi- 

Shinozaki K, Carninci P, Kawai J, Hayashizaki 

Y and Shinozaki K. Monitoring the expression 

pattern of around 7,000 Arabidopsis genes 

under ABA treatments using a full-length 

cDNA microarray. Functional & Integrative 

Genomics. 2:282-291, 2002. 

Shinozaki K and Yamaguchi-Shinozaki K. 

Molecular responses to dehydration and low 

temperature: differences and cross-talk between 

two stress signalling pathways. Current Opinion 

in Plant Biology. 3:217-223, 2000. 

Talame V, Ozturk NZ, Bohnert HJ and Tuberosa R. 

Barley transcript profiles under dehydration 

shock and drought stress treatments: a 


comparative analysis. J Exp Bot. 58:229-240, 

2007. 

Tamura K, Dudley J, Nei M and Kumar S. 

MEGA4: Molecular Evolutionary Genetics 

Analysis, MEGA. Software Version 4.0. Mol 

Biol Evol. 24:1596-1599, 2007. 

Umezawa T, Fujita M, Fujita Y, Yamaguchi- 

Shinozaki K and Shinozaki K. Engineering 

drought tolerance in plants: discovering and 

tailoring genes to unlock the future. Curr Opin 

Biotechnol. 17:113-122, 2006. 

Untergasser A, Nijveen H, Rao X, Bisseling T, 

Geurts R and Leunissen JAM. Primer3Plus, an 

enhanced web interface to Primer3. Nucleic 

Acids Res. 35:W71-74, 2007. 

Wazni MW, Islam AS, Taliaferro JM, Anwar N 

and Sathasivan K. Novel ESTs from a Jute, 

Corchorus olitorius L). cDNA library. Plant 

Tissue Cult Biotechnol. 17:173-182, 2007. 

Yamaguchi-Shinozaki K and Shinozaki K. 

Transcriptional regulatory netwirks in cellular 

responses and tolerance to dehydration and cold 

stress. Annu Rev Plant Biol, 57:781-803, 2006. 

Yan S, Tang Z, Su W and Sun W. Proteomic 

analysis of salt stress-responsive proteins in rice 

root. Proteomics. 5:235-244, 2005. 

Yancey P, Clark M, Hand S, Bowlus R and Somero 

G. Living with water stress: evolution of 

osmolyte systems. Science. 217:1214-1222, 

1982.




Protective effects of curcumin on cadmium chloride induced colon 

toxicity in Swiss albino mice 

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

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

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

Received: 26 October 2010; Accepted: 29 April 2011 

Abstract 

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

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

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

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

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

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

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

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

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

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

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

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

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

areas, suppression of mucosa was significantly reduced. 

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

Swiss albino farelerde zerdeçalın kadmiyum klorür ile indüklenmiş kolon 

toksisitesine koruyucu etkileri 

Özet 

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

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

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

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

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

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

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

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

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

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

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

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

azalması ile zerdeçalın koruyucu rolünü anlaşılır bir biçimde açıklar. 

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

32 Preeti SINGH et al. 

Introduction 

Cadmium, an omnipresent, hazardous, heavy metal 

is recognized to produce severe toxic effects in 

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

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

for Toxic Substances and Disease Registry 

(ATSDR, 1989) in Atlanta, Georgia has listed 

cadmium as number 7 in its top 20 list of hazardous 

substances. Cadmium will invariably be present in 

our society, either in useful products in the form of 

nickel-cadmium batteries, dyes, plastics, 

electrochemistry, paint pigments or in controlled 

wastes as a major source of pollution, in water and 

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

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

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

been found to produce wide range of biochemical 

and physiological dysfunctions as manifested in the 

forms of various diseases viz. Itai-itai, kidney 

malfunction, inflammation, Parkinsons disorder, 

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

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

Ikeda et al.,2000).Various modes and 

methodologies are being devised to combat 

cadmium-induced toxicity with a focus on herbal 

formulations. The use of herbal medicine increases 

every day and still finds a wide use worldwide. 

Traditional herbs have more acceptance than 

prescription drugs in many cultures. One of such 

drug is curcumin derived from Curcuma longa the 

dried powered form of which is known as Turmeric 

and is a mandatory condiment of every Indian 

kitchen. Turmeric extract containing the ingredient 

curcumin is marked widely in the western world as 

a dietary supplement for the treatment and 

prevention of a variety of disorder, including 

inflammation, arthritis and other proposed uses of 

curcumin is in Alzheimer's Disease, cancer 

prevention, cataract prevention, chronic, high 

cholesterol, liver protection, menstrual pain, 

multiple sclerosis, osteoarthritis, rheumatoid 

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

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

reported to have antibacterial, antiamoebic, 

antifungal, antiviral and anti HIV activities 

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

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

been shown to be 300 times more potent than 

vitamin E. However no concrete evidence in 

scientific field is available pertaining to the colonic 

protective effects of curcumin on cadmium chloride 

induced toxicity. Hence, the aim of the present 

research was to fulfill this lacuna and to determine 

the efficacy of curcumin in rendering protection 

against cadmium chloride induced colonic toxicity 

at the cellular level. 


In the present experiment the following chemical 

reagents were used. Cadmium chloride: CdCl2. 

H2O was procured from Glaxo company (India), 

with following features: Molecular Weight = 

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

Crystalline: C21H20O6 was procured from Loba 

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

Weight = 368.39 Minimum assay (acidimetric) 

99%, Melting point= 170- 180°C. 

The present study was conducted after taking 

prior approval by Indian Animal Ethical Committee 

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

Swiss albino mice 32-50 days old and weighing 

around to 30-40g were maintained in plastic cages 

under controlled lighting conditions (12:12 light: 

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

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

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

mice per experiment were taken and treated with 

CdCl2 and curcumin. The doses were prepared 

daily in distilled water and were administered by 

gastric gavage method. 

The dose protocol was as follows: Group-I 

Control group (a) were treated with only vehicle 

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

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

days; Group-III Experimental batch were 

administered CdCl2 50mg/kg/animal for a single 

day; Group-IV Experimental batch with were pretreated 

with curcumin 10mg/animal/day for 15 days 

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

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

administration of the last dose, the control and the 

experimental animal were sacrificed. Their colon 

was excised and subsequently fixed in Bouins 

solutions. After fixation, wax block and paraffin 

sections of the colons were prepared, processed and 

differentially stained in hematoxyline and eosin for 

general histopathological assessment. The sections 

were stained in bromophenol blue for assessment of 

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

glycoproteins. They were then observed under a 

light microscope and the gross histopathological 

profile was assessed.

Results 

The colonic histopathological profile of control 

group mice revealed no aberrations the colonic 

mucosa was distinct with no lacerations and the 

goblet cells present were filled. Mucin production 

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

Similarly, no significant variations were observed 

in curcumin treated mice, which showed a similar 

structural profile as that of control group mice 

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

animals treated with CdCl2 was blunt and 

suppressed, clearly delineating histopathological 

Protective effects of curcumin on colon toxicity 33 

damage. Emptied goblet cells, lacerated mucosa, 

destruction of surface epithelium, marked decline 

in mucin production and hemorrhagic areas in 

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

animals pretreated with curcumin and subsequently 

treated with CdCl2 the histopathology of colon 

clearly delineates the protection accorded by 

curcumin as the induced damage by CdCl2 was 

negligible and the number of empty goblet cells, 

hemorrhagic areas, suppression of mucosa was 

significantly reduced (Figure 1e and 1f). 

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

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

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

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

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

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

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

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

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

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

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

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

magnification).


Discussion 

Cadmium is an omnipresent heavy metal which 

enters the biological systems from natural sources, 

such as volcanic emissions, weathering of rocks, 

mining processes as well as from industrial 

applications, agricultural practices and human 

usages. Cadmium emissions into the environment 

are normally continuous between the three main 

environmental compartments air, water and soil. 

The majority of cadmium exposure arises from 

ingestion of food substances due to uptake of 

cadmium by plants from fertilizers, sewage sludge, 

manure and atmospheric deposition (Lee and 

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

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

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

uptake of cadmium is mainly through cigarette 

smoking and food intake. In vegetarian diet, 

mushrooms, cocoa powder, potatoes, fruits, wheat, 

grains, bran, sugerbeet fiber, carrot, dried seaweeds 

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

non-vegetarian diets shellfish, mussel, meat and 

fish are rich in cadmium. These cadmium rich 

foods can greatly increase cadmium concentration 

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

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

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

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

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

orally ingested cadmium is absorbed in the 

intestines, but individual values range from less 

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

that humans do not have effective pathways for 

cadmium elimination, hence cadmium shows the 

phenomenon of bioaccumulation. Once absorbed 

by an organism, it remains resident for many years. 

Where cadmium toxicity is concerned, colon is of 

prime importance, for of the total cadmium that 

enters the gastrointestinal tract 90-95% is excreted 

out and hence colonic cells are exposed to cadmium 

in fecal matter as well as to cadmium present in the 

circulation. Hence in the present experiment we 

aimed to assess the toxic effects of cadmium 

chloride as well as to monitor the protective effects 

of curcumin in colon of Swiss albino mice on the 

basis of histopathological observations. The 

integrity of colon depends upon the balance 

between the hostile factors, one of them being 

cadmium, which damages the mucosa ,goblet cells 

etc. and the protective factors such as certain 

internal secretions like mucin and certain external 

agents like curcumin which render protection 

against cadmium toxicity. Pertaining to cadmium 

toxicity our results are similar to the results of 

various studies where cadmium has been reported 

to cause variations in histoarctecture of colon. 

These reports revealed that oral exposure to 

cadmium caused severe necrosis, hemorrhage and 

ulcers in the colonic epithelium as well induced 

decreased body weight and muscle atrophy (Barret 

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

1974 and ATSDR, 1999). These reports give 

validity to our experiment, where we have also 

discerned similar cadmium induced aberrations in 

colon. In contrast to the injurious activity of 

cadmium, curcumin serves as protective agent 

where it has been shown to decrease congestion and 

inflammation in stagnant mucus membranes and no 

negative side effects have been associated with 

curcumin supplementation (Mukhopadhyay et 

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

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

Curcumin has also been reported to have proposed 

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

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

protectively coat esophagus, stomach, intestinal 

mucus membrane and reduce acid secretion. 

However no information is available pertaining to 

protective effect of curcumin in relation to 

cadmium induced toxicity. Hence, this study clearly 

delineates the protective effect of curcumin against 

CdCl2 induced colonic toxicity. 

References 

Anderson O, Nielsen JB and Svendsen E. Oral 

cadmium chloride intoxication in mice: Effects 

of dose on tissue damage, intestinal absorption 

and relative organ distribution. Toxicology. 

48:225-236, 1988. 

ATSDR (Agency for Toxic Substances and Disease 

Registry). Toxicological profile for cadmium. 

ATSDR/U.S. Public Health Service, 

Atlanta/TP-88/08, 1989. 

ATSDR (Agency for Toxic Substances and Disease 

Registry). Toxicological profile for Cadmium. 

ATSDR / US department of Health and Human 

Services, Atlanta/, US, 1999. 

Ammon HPT, Anazodo M I, Safayhi H and 

Dhawan BN. Curcumin a potent inhibitor of 

leukotriene B4 formation in rat

polymorphonuclear neutrophils (PMNL). 

Planta Med. 67: 1-6, 1992. 

Azuine M and Bhide S. Chemo preventive effect of 

turmeric against stomach and skin tumors 

induced by chemical carcinogens in Swiss mice. 

Nutr Cancer. 17: 77-83, 1992. 

Barret HM, Irwin DA and Semmons E. Studies on 

the toxicity of inhaled cadmium. The acute 

toxicity of cadmium oxide by inhalaltion. J Ing 

Hyg Toxicol. 29: 279, 1947. 

Bernard A, Lauwery R, and Amor AO. Loss of 

glomerular polyanion correlated with 

albuminuria in experimental cadmium 

nephropathy. Arch Pathol. 66:272-278, 1992. 

Chen CJ and Wang CJ. Ecological correlation 

between arsenic level in well water and age 

adjusted mortality from malignant neoplasms. 

Cancer Res. 50: 5470 - 5474, 1990. 

Chen YC, Kuo TC, Lin-Shiau SY and Lin J K. 

Induction of HSP70 gene expression by 

modulation of Ca(+2) ion and cellular p53 

protein by curcumin in colorectal carcinoma 

cells. Mol Carcinogen. 17: 224-234, 1996. 

Deodhar SD, Sethi R and Srimal RC. Preliminary 

study on antirheumatic activity of curcumin 

(diferuloyl methane). Indian J Med Res. 71:632- 

634, 1980. 

Dikshit M, Rastogi L, Shukla R and Srimal RC. 

Prevention of ischaemia-induced biochemical 

changes by curcumin and quinidine in the cat 

heart. Indian J Med Res. 101: 31 - 35, 1995. 

Friberg L, Piscator MA, Nordberg G and 

Kjellstrom T. Cadmium in the Environment, 

Vol. II: A Toxicological and Epidemiological 

Appraisal, Exposure, Dose and Metabolism, 

Cleveland, OH, CRC Press. 1974. 

Friberg L, Piscator MA, Nordberg G and 

Kjellstrom T. Cadmium in Environment. 2nd.Ed 

CRC Press, Boca Raton, Florida, 1975. 

Friberg L, Kjellstrom T, Nordberg G and Piscator 

A. Cadmium and Health: Toxicological and 

Epidemiological Appraisal, US Environment 

Protection Agency, Washington DC, 1985a. 

Protective effects of curcumin on colon toxicity 35 

Friberg L, Elinder CG, Kjellstrom T and Nordberg 

G. Cadmium and Health: A Toxicological and 

Epidemiological Appraisal, CRC Press, Baca 

Raton, Florida. 23 – 63, 1985b. 

Eigner D and Scholz D. Ferula asafetida and 

Curcuma longa in traditional medicinal 

treatment and diet in Nepal. J Ethnopharmacol. 

67: 1-6, 1999. 

Egan ME, Pearson M, Weiner SA, Rajendran V, 

Rubin D, Glockner-Pagel J, Canny S, Du K, 

Lukacs GL and Caplan MJ. Curcumin a major 

constituent of turmeric, corrects cystic fibrosis 

defects. Science. 23; 304 (5670): 600-2, 2004. 

Goyer R, Amdur MO, Doull JD and Klaassen CD. 

Toxicology: Toxic effects of metals. 4th ed. Eds. 

Casarett and Doull's. Progamone Press, New 

York. 623 -680, 1991. 

Hotz P, Buchet JP, Bernard A, Lison D and 

Lauwerys R. Renal effects of low-level 

environmental cadmium exposure: 5-year 

follow-up of a subcohort from the Cadmibel 

Study, Lancet. 354:1508, 1989. 

Iwata K, Saito H, Moryiama M and Nakano A. 

Follow up study of renal tubular dysfunction 

and mortality in residents of an area polluted 

with cadmium. B r J Ind Med. 49: 736 - 737, 

1992. 

Ikeda M, Zang Z W, Moon C S, Shimbo S, 

Watanabe T, Nakatsuka H, MatsudaInoguchi N 

and Higashikawa K. Possible effects of 

environmental cadmium exposure on kidney 

function in the Japanese general population. Int 

Arch Occup Environ Health. 73: 15 - 25, 2000. 

Jarup L, Alfven T, Persson B, Toss G and Elinder 

CG. Cadmium may be a risk factor for 

osteoporosis. Occup Environ Med. 55: 435- 

439, 1998a. 

Lee JS and White KL. A review of health effects of 

cadmium. Am J Ind Med. 1: 307-317, 1980. 

Lauwerys R, Bernard A and Buchet JP. Does 

environmental exposure to cadmium represent a 

health risk? Conclusions from the Cadmibel 

Study. Acta Clin Belg. United States 

Environmental Protection Agency. 46: 219 - 

225, 1991.


Mukhopadhyay A, Basu N and Ghatak N. Antiinflammatory 

and irritant activities of curcumin 

analogues in rats. Agents Actions. 12: 508 - 515, 

1982. 

Mortellini R, Foresti R, Bassi R and Green CJ. 

Curcumin, an antioxidant and anti-inflammatory 

agent, induces heme oxygenase-1 and protects 

endothelial cells against oxidative stress. Free 

Radic Biol Med. 28:1303-1312, 2000. 

Nogawa K, Ishizaki A and Evrin PE. Urinary 

beta2-microglobulin excretion among people 

exposed to cadmium in the general 

environment: An epidemiological study in 

cooperation between Japan and Sweden. 

Environ Res. 13(2):318-344, 1977. 

Richardson ME, Fox MRS, and Fry BE. 

Pathological changes produced in Japanese 

quail by ingestion of cadmium. J Nutr. 104:323, 

1974. 

Rao TS, Basu N and Siddiqui H. Antiinflammatory 

activity of curcumin analogues. 

Indian J Med Res. 75:574-8, 1982. 

Risk Assessment Information System (RAIS). 

Toxicity summary for cadmium. Chemical 

Hazard Evaluation and Communication Group, 

Biomedical and Environmental Information 

Analysis Section, Health and Safety Research 

Division. 1991. 

Ruby A J and Kuttan G. Anti-tumour and 

antioxidant activity of natural curcuminoids. 

Cancer Lett .94: 79 - 80, 1995. 

Singh P, Chaudhary S, Patni A and Sankhla V. 

Effect of cadmium chloride induced 

genotoxicity in bone marrow chromosomes of 

Swiss albino mice and subsequent protective 

effects of Emblica officinalis and vitamin C. J 

of Herbal Medicine and Toxicology. 1 (2): 67- 

71, 2007. 

Stawe H D, Wilson M and Goyer R A. Clinical and 

morphological effects of oral cadmium toxicity 

in rabbits. Arch Pathol. 94: 389 - 405, 1972. 

Shukla Y, Arora A and Taneja P. Antimutagenic 

potential of curcumin on chromosomal 

aberrations in Wistar rats. Mutat Res. 515: 197- 

202, 2002. 

Vahter M, Bergland M, Nermall B and Akesson A. 

Bioavailability of cadmium from shellfish and 

mixed diet in women. Toxicol Appl Pharmacol. 

136: 332 -341, 1996. 

World Health Organization (WHO). Environmental 

Health Criteria 134. Cadmium International 

Programme on Chemical Safety (IPCS) 

Monograph, 2000.




In vitro regeneration of Cleome viscosa – an important medicinal 

herb 

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

Soundarapandian KANNAN 2 

1 

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

2 

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

Coimbatore, India. 

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

Received: 30 August 2010; Accepted: 26 May 2011 

Abstract 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Özet 

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

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

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

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

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

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

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

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

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

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

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

yapılabilmesi için temel bir araç olarak kullanılabilir. 

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

38 Jeyaraj ANBURAJ et al. 

Introduction 

Cleome viscosa Linn. (Capparidaceae) is 

commonly known as Tickweed, wild mustard or 

Spider plant (English), Hurhur (Hindi) and 

Hurhuria (Bengali). It occurs in woodland and 

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

roadsides and wasteland, often occurring on sandy 

soils, but sometimes on calcareous and rocky soils. 

It is a widely distributed herb with yellow flowers 

and long slender pods containing seeds. The whole 

plant is sticky in nature and has a strong odour 

resembling asafoetida. It is found in both seasonal 

dry and humid conditions, from mean sea level up 

to an altitude of 1000 m. The seeds have no 

dormancy and germinate readily after shedding. 

Plants start flowering 3–4 weeks after germination 

and the lifecycle is about 3 months. Cleome viscosa 

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

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

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

included in the Capparaceae family, which 

comprises 427 species occurring in tropical and 

subtropical regions of the world (Brummit, 1992). 

In Asia and Africa the leaves and seeds are used 

as a rubefacient and vesicant and to treat infections, 

fever, rheumatism and headache. The whole herb is 

used in the treatment of ringworm, flatulence, colic, 

dyspepsia, constipation, cough, bronchitis, cardiac 

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

2000). Leaves are used as the external application 

to inflammation of the middle ear and applied on 

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

hepatoprotective activity (Gupta and Dixit, 2009). 

Traditionally, this plant is used in various disorders 

such as diarrhoea, fever, inflammation, liver 

diseases, bronchitis, skin diseases, and malarial 

fever. Its juice is useful in piles, lumbago and 

earache (Rukmini, 1978). The analgesic, antipyretic 

and anti-diarrhoeal activities of the extract have 

been reported by researchers. A decoction is used 

as an expectorant and digestive stimulant and the 

vapour from a steaming decoction of the whole 

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

seeds and its oil have anti-helminthic properties but 

they are ineffective in treating roundworm 

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

anti-convulsant effect in Swiss albino mice (Mishra 

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

rheumatism (Rukmini, 1978). An aqueous seed 

extract displayed significant analgesic activity in 

mice and local anaesthetic activity in guinea pigs 

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

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

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

of the extracts have been confirmed. Leaves and 

young shoots are cooked as a vegetable 

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

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

pickled or used as a mustard substitute in curries 

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

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

Oil obtained from the seeds is used for cooking 

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

in liver diseases, chronic painful joints and mental 

disorders (Chatterjee and Pakrashi, 1991). 

The induction of callus growth and subsequent 

differentiation and organogenesis is accomplished 

by the differential application of growth regulators 

and the control conditions in the culture medium. 

With the stimulus of endogenous growth substances 

or by addition of exogenous growth regulators to 

the nutrient medium cell division, cell growth and 

tissue differentiation are induced (Tripathi and 

Tripathi 2003). Organogenesis permits the 

manipulation of large numbers of high quality plant 

material within a short period of time. The vast 

usage of the Cleome viscosa in medicinal purpose, 

based on the above reason, it is increasing 

commercial demand of Cleome viscosa. The 

present study was undertaken to develop a more 

efficient protocol for rapid in vitro multiplication of 

Cleome viscosa using leaf explants as an initial 

plant material. 


The plants were collected from in and around 

Virudhunagar district, Tamil Nadu, India (Figure 

1). Small young leaves were collected from healthy 

plants of Cleome viscosa L defoliated and sectioned 

into many segments. They were washed under 

continuous flashing of running tap water for 30 

minutes and then with distilled water three times. 

Leaves were treated with 0.1% mercury chloride 

for 1.5 minutes and washed with sterilized double 

distilled water for 3 times. The small washed 

explants were treated with 3% hydrogen peroxide 

for 1.5 minutes and then washed with sterilized 

double distilled water for 3 times. At final the 

explants were treated with 70% ethanol for 0.5 

minutes and then washed with sterilized double 

distilled water for 3 times in laminar air flow 

cabinet.

Figure 1. Natural habitat of Cleome 

(Linn.). 

Culture media and growth condition 

viscosa 

The medium comprised of macro and 

micro 

elements according to Murashige and Skoog (1962) 

with myoinositol (100mg/l), thamine 

HCl 

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

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

agar. The plant growth regulators used were 

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

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

(NAA) and Benzyl aminopurine (BAP). All 

experiments were carried out in culture tubes 

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

The pH of the medium was adjusted to 5.8 prior to 

autoclaving at 121ºC for 20 minutes. Cultures were 

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

Callus induction and shoot regeneration 

The small segments of leaf explants were cultured 

on MS medium fortified with different 

concentration of auxins such as, IAA, NAA and 

IBA ranging from 0.5 – 4.0mg/l for callus 

initiation. Explants were found to give profuse 

callusing and when callusing was observed in entire 

explant, the callus was transferred to MS medium 

supplemented with various concentrations of BAP 

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

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

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

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

40 explants was used for each experiment and was 

repeated 5 times. Data on percent of response, 

number of shoots and shoot length formation per 

explant was recorded after 30 days of culture. 

Callus Growth Measurement 

In order to minimize the differences in growth that 

may have been the result of variation in inoculum 

Regeneration of Cleome viscosa 39 

size, a growth index was used to evaluate the callus 

growth after 30 days of culture. Dry weight of calli 

was evaluated by drying fresh calli in an air forced 

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

determined according to the formula: 

Regeneration of roots and development of complete 

plantlets 

For rooting individual microshoots (1.5-2.5mm 

length) were isolated from callus clumps and 

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

different auxins such as, IBA, IAA and NAA to 

induce of root formation. A total of 40 plantlets 

were taken for each experiment and were repeated 

5 times. Percent of rooting, number of root 

formations and root length was recorded 40 days 

after root initiation. All the rooted plants were 

carefully removed from culture tubes and washed 

in tap water to remove agar gels and transferred to 

plastic pots containing soil mixture (sterile soil, 

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

vitro regenerated plants covered with polythene 

bags to maintain high humidity. These were 

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

photoperiod and watered regularly. After 3 – 4 

weeks, the polythene bags were removed and 

established plantlets were transplanted to earthen 

pots in a greenhouse. 

Statistical analysis 

All results reported are the means of five replicate. 

The result are expressed as mean values ± standard 

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

P≤0.05 levels using one way analysis of variance 

(ANOVA). Mean comparisons were carried out by 

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

analysis (SPSS package, Version 17). 

Results 

The fresh smaller size of explants provide less 

chance of contamination, as well as, longer leaves 

showed, total loss of morphogenic potential. 3% 

hydrogen peroxide, 0.1% mercuric chloride and 

70% ethanol was used to sterilize the leaves.


Surface of the leaves were sterilized with 0.1% 

mercuric chloride for 1.5minutes, 3% hydrogen 

peroxide for 1.5minutes and 70% ethanol for 

0.5minutes reported the maximum growth without 

contamination. 

Interactive effects of culture medium and 

explant type were statistically significant (P≤0.05) 

for calli fresh weight, and calli growth index (Table 

1). Calli were initiated from leaf explants of 

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

of culture on MS medium fortified with different 

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

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

mean for calli growth index, and calli fresh weight 

of leaf explant belonged to the MS culture medium, 

supplemented with 2mg/L IAA. Leaf explant was 

superior to the nodal explants regarding the growth 

index, and calli fresh weight. About 81% callusing 

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

fresh mass and calli growth index 2.3±0.07 

(P≤0.05) was observed. The optimum concentration 

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

with 122±0.54mg fresh mass was observed in leaf 

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

callusing response and callus growth index 

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

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

mass was observed in leaf explants cultured on 

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

The callus cultured on MS medium with 3.0% 

sucrose and 0.8% agar medium supplemented with 

hormones such as, BAP, KN and BAP+KN 

produced microshoots. High frequency (95%) 

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

number of shoots and an average length of 

4.10±0.18cm shoots in MS medium supplemented 

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

cultured on cytokinin combination BAP and KN 

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

microshoots. About 85% moderate shooting 

response with 7.2±0.83 number of shoots and an 

average length of 3.62±0.15cm shoots from leaf 

callus. Low frequency (69%) multiple shoot 

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

shoots and an average length of 3.15±0.28cm 

shoots in MS medium supplemented with 1.0mg/l 

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

The healthy shoots with two to three leaves 

were isolated from shoot clumps and cultured on 

root induction medium to induce adventitious 

rooting. MS medium fortified with different 

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

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

individual plantlets cultured on MS medium 

supplemented with NAA (0.1mg/l) fortified 

medium induced high frequency (91%) rooting 

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

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

no significant compared with concentration of 

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

medium containing other hormones such as IBA 

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

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

2 I). The healthy rooted plants were carefully 

removed from the culture tubes and washed in 

running tap water to remove agar gels and 

transferred to plastic pots containing soil mixture. 

Finally, the regenerated plants were successfully 

transferred to earthen pot containing soils. Almost 

all (90%) the in vitro regenerated plants 

successfully survived in green house condition 

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

Figure 2. A) Callus initiation in Cleome viscosa 

from leaf explants in MS media containing 2mg/l 

IBA. B) Callus initiation in Cleome viscosa from 

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

C) Callus induction in Cleome viscosa from leaf 

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

Kinetin MS media. H) Shoot regeneration from 

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

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

vitro regeneration of complete plantlets of Cleome 

viscosa from leaf explants. K) Plants growing in 

Callus induction in Cleome viscosa from leaf 

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

Callus induction in Cleome viscosa from leaf 

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

Shoot regeneration from callus on 2mg/l BAP MS 

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

earthen pots containing soil under green house 

condition.


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

30days. 

Growth regulators Callusing Callus growth Callus growth 

Callus 

(mg/l) 

IAA 

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

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

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

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

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

4 

IBA 

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

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

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

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

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

4 

NAA 

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

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

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


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


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

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

induction from leaf of Cleome viscosa. 

Growth regulators Callusing 

Number of 

Shoot length 

(mg/l) 

Bap 

response % total shoots) 

(cm) 

0.5 60 

5.4±0.54ac 

3.50±0.24a 

1 80 

6.2±0.83bd 

3.60±0.16b 

2 95 

7.6±0.54ac 

4.10±0.18abc 

3 55 

2.6±0.89ab 

3.20±0.25c 

4 50 

2.2±0.45cd 

3.10±0.18b 

Kn 

Bap+Kn 

0.5 

1 

2 

3 

4 

60 

69 

54 

45 

40 

5.2±0.45a 

5.6±0.55b 

5.1±0.84c 

4.8±0.95d 

4.2±0.84e 

2.85±0.21a 

3.15±0.28b 

3.08±0.15c 

3.20±0.25d 

3.00±0.13e 

0.5 

1 

2 

3 

4 

60 

45 

75 

80 

85 

5.4±0.89a 

4.2±0.84bc 

6.0±0.71c 

4.4±0.89d 

7.2±0.83abd 

2.95±0.31a 

2.63±0.18b 

3.25±0.15bc 

2.80±0.14c 

3.62±0.15abc 

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


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

viscosa. 

Growth regulators 

(mg/l) 

NAA 

0.1 

0.2 

0.5 

1 

IBA 

IAA 

0.1 

0.2 

0.5 

1 

0.1 

0.2 

0.5 

1 

Callusing 

response % 

91 

80 

55 

40 

85 

70 

40 

35 

65 

40 

35 

25 

Number of 

total shoots) 

3.4±0.89ac 

3.2±0.45bd 

2.6±0.89ab 

2.4±0.54cd 

3.2±0.83a 

3.0±0.71b 

2.2±0.84ab 

1.6±0.54ab 

2.8±0.84ab 

1.6±0.89a 

1.4±0.55b 

1.2±0.44a 

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

Discussion 

The fresh smaller size of explants provide less 

chance of contamination, longer leaves showed a 

total loss of morphogenic potential as well. A 

similar report has been published in Rauvolfia 

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

shoot regeneration from small leaf explants of 

Rauvolfia serpentine L. The surface sterilization of 

explants is very essential for establishment, as well 

as for optimum induction of callus and plantlet 

regeneration in vitro condition (Yeoman and 

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

peroxide, 0.1% mercuric chloride and 70% ethanol 

was used to sterilize the leaves, resulted the 

maximum growth without contamination. 

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

cultured explants showed more than 80% 

contamination free cultures when treated with 0.1% 

HgCl2 for surface sterilization. 

The MS medium was the most effective for 

callusing of leaf explants. The explants cultured on 

MS medium supplemented with different auxins of 

IBA, NAA and IAA showed varied response for 

callusing. Among three types of auxins, IBA and 

IAA were previously found to be very effective 

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

Shoot length 

(cm) 

3.00±0.25abc 

2.50±0.28a 

2.45±0.23b 

2.30±0.23c 

2.10±0.23ac 

1.85±0.41b 

1.80±0.19a 

1.53±0.18bc 

2.00±0.18ac 

1.85±0.12b 

1.65±0.16a 

1.50±0.20bc 

investigation calli were initiated from leaf explants 

of Cleome after two weeks of culture on MS 

medium fortified with different auxins IBA (0.5-4 

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

About 81% callusing response with maximum of 

178mg fresh mass was observed in leaf explants 

cultured on MS medium supplemented with IAA 

(2mg/l). 

In this present research that the callus cultured 

on MS medium, hormones such as BAP, KN and 

BAP+KN produced microshoots. High frequency 

(95%) multiple shoot induction with 7.6±0.54 

number of shoots and an average length of 

4.10±0.18 shoots in MS medium supplemented 

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

two cytokinins used BAP induced significantly 

higher percentage of shoot initiation and mean 

number of shoot, whereas higher mean shoot length 

was obtained in the shoots obtained on media 

supplement with KN (Gokhale and Bansal 2009). 

The healthy shoots were isolated from shoot 

clumps and cultured on root induction medium to 

induce adventitious rooting. MS medium was 

fortified with different concentrations of auxins 

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

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

MS medium supplemented with NAA (0.1mg/l) 

fortified medium induced high frequency (91%) 

rooting with 3.4±0.89 number of microroots and an 

average length of 3.00±0.25 roots. When cultured 

on the MS medium containing other hormones such 

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

showed lower response than that of 0.1mg/l of 

NAA. A similar report has been published by Amin 

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

induced in in vitro regenerated shoots by culturing 

them on half strength of MS medium with 0.1- 

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

types of auxins, NAA was found to be most 

effective at different concentrations tested for root 

production. The findings are in agreement with 

those observed in other plant species. Although 

Gokhale and Bansal (2009) reported that the three 

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

raised shoots of O. indicum, yet NAA responded 

best for all parameters of rooting. For shoots with 

highest percentage of root induction, maximum 

number of elongated roots was developed on MS 

medium containing NAA. Long, branched healthy 

roots were produced in this rooting media. 

The in vitro derived plants acclimated better 

under ex vitro conditions when they were 

transferred on specially made plastic trays 

containing coco-peat as potting mix and moistened 

uniformly at periodic intervals taking special care 

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

followed from this stage up to their establishment 

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

present investigation the healthy rooted plants were 

carefully removed from the culture tubes and 

washed in running tap water to remove agar gels 

and transferred to plastic pots containing soil 

mixture. Finally, the regenerated plants were 

successfully transferred to earthen pot containing 

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

plants successfully survived at green house 

conditions. Similarly, the success of transplantation 

was 85% when plantlets were sufficiently healthy 

with new growth.They were subsequently 

transferred to larger pots and gradually acclimated 

to outdoor conditions. The protocol reported here is 

reproducible, it has a potential for being utilized to 

conserve the germplasm and allowing at the same 

time, a large scale micropropagation of this 

important medicinal plants (Jeyakumar and 

Jayabalan, 2002). 

In conclusion, a significant progress has been 

made in the in vitro regeneration system of this 


medicinally important Cleome viscosa. Using this 

technique, it is possible to produce healthy and 

disease free clones which could be released to their 

natural habitat in large scale. It may also be useful 

for gene manipulation for this important medicinal 

species. 

Acknowledgements 

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

head at Department of Biotechnology, Ayya Nadar 

Janaki Ammal College, Sivakasi, India for his 

stimulating guidance, invaluable suggestions and 

critical and persistent encouragement. I wish to 

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

for helping me to carry out my project work in 

Ayya Nadar Janaki Ammal College, Sivakasi, 

India. 

References 

Amin MN, Rahman MM, Manik MS. In vitro 

clonal propagation of Paederia foetida L.- A 

medicinal plant of Bangaladesh. Plant tissue 

Cult. 13:117-123, 2003. 

Brummit RK. Vascular plants families and genera. 

Kew: Royal Botanic Gardens; 1992. 

Chatterjee A, Pakrashi SC. The Treatise on Indian 

Medicinal Plants. Vol.1, PID, CSIR, New 

Delhi, P.215, 1991. 

Chopra RN, Nayar SL, Chopra IC. Glossary of 

Indian Medicinal Plants. Council of Scientific 

and Industrial Research, New Delhi, 1986. 

CSIR, The wealth of India: A dictionary of Indian 

raw materials and industrial products raw 

materials. Council of Scientific and Industrial 

Research, New Delhi, India, Vol. 2, pp 427, 

1950. 

Devi BP, Boominathan R, Mandal SC. Evaluation 

of anti-diarrheal activity of Cleome viscosa L. 

extract in rats. Phytomedicine. 9: 739–742, 

2002. 

Devi BP, Boominathan R, Mandal SC. Evaluation 

of antipyretic potential of Cleome viscosa 

Linn. (Capparidaceae) extract in rats. Journal 

of Ethnopharmacology. 87: 11–13, 2003. 

Facciola S. Cornucopia- A source Book of Edible 

Plants. Kampong Publications, ISBN 0- 

9628087-0-9, 1990.


Gokhale M, Bansal YK. Direct In vitro 

regeneration of a medicinal tree Oroxylum 

indicum (L.) Vent. through tissue culture. 

African Journal of Biotechnology. 8: 3777- 

3781, 2009. 

Gupta NK, Dixit VK. Evaluation of 

hepatoprotective activity of Cleome viscose 

Linn. extract. Indian J Pharmacol. 41: 36-40, 

2009 

Jeyakumar M, Jeyabalan N. In vitro plant 

regeneration from cotyledonary node of 

Psoralea corylifolia L. Plant Tissue Cult. 

12:125-129, 2002. 

Kirtikar KR, Basu BD. Indian medicinal plants. 2nd 

ed. Vol. 1. Periodical Experts, New Delhi: p. 

183-5, 1975. 

Manandhar NP. Plants and people of Nepal. 

Timber Press. Oregon. ISBN 0-88192-527-6, 

2002. 

Mishra A, Mishra AK, Jain SK. Anticonvulsant 

activity of cleome viscosa seed extracts in 

Swiss albino mice. International Journal of 

Pharmacy and Pharmaceutical Sciences. 2: 

177-181, 2010. 

Murashige T, Skoog F. A revised medium for rapid 

growth and bioassays with tobacco tissue 

cultures. Plant Physiol. 15:473-497, 1962. 

Parimala DB, Boominathan R, Mandal SC. Studies 

on psychopharmacological effects of Cleome 

viscosa Linn. Extracts in rats and mice. 

Phytotherapy Research. 18:169-172, 2004. 

Parimaladevi B, Boominathan R, Mandal SC. 

Studies on analgesic activity of Cleome 

viscosa in mice. Fitoterapia. 74: 262–266, 

2003. 

Rukmini C. Chemical, nutritional and toxicological 

evaluation of the seed oil of Cleome viscose. 

Indian Journal of Medical Research. 67: 604– 

607, 1978. 

Sairkar P, Chandravanshi MK, Shukla NP, 

Mehrotra NN. Mass production of an 

economically important medicinal plant 

Stevia rebaudiana using in vitro propagation 

techniques. Journal of Medicinal Plant 

Research. 3:266-270, 2009. 

Saxena BR, Koli MC, Saxena RC. Preliminary 

ethnomedical and phytochemical study of 

Cleome viscosa L. Ethnobotany. 12: 47–50, 

2000. 

Singh P, Singh A, Shukla AK, Singh L, Pande V, 

Nailwal TK. Somatic embryogenesis and in 

vitro regeneration of an endangered medicinal 

plant Sarpagandha(Rauvolfia serpentiana L.). 

Life Science Journal. 6:57-62, 2009. 

Singh PDA, West ME. Pharmacological 

investigations of sticky viscome extract 

(Cleome viscosa L.) in rats, mice and guineapigs. 

Phytotherapy Research. 5: 82–84, 1991. 

Tripathi L, Tripathi JN. Role of biotechnology in 

medicinal plants. Tropical Journal of 

Pharmaceutical Research. 2:243-253, 2003. 

Yeoman MM, Macleod AJ. Tissue (callus) culture 

technique. Plant tissue and cell culture. 1:31- 

59, 1977.




Bio-database compression using enhanced suffix array for pairwise 

sequence alignment 

Arumugam KUNTHAVAI *1 and Somasundaram VASANTHA RATHNA 2 

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

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

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

Received: 19 February 2011; Accepted: 10 June 2011 

Abstract 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

by them. 

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

Çiftli dizi sıralaması için kuvvetlendirilmiş sonek dizilişi kullanarak biyo-veritabanı 

sıkıştırması 

Özet 

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

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

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

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

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

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

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

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

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

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

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

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

karşılaştırıldığında yarı zamanda çok benzer sonuçlar elde edildiğini gösterir. 

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

46 Arumugam KUNTHAVAI and Somasundaram VASANTHA RATHNA 

Introduction 

Bioinformatics is the field of analyzing the 

biological information using computers and 

statistical techniques; the science of developing and 

utilizing computer databases and algorithms to 

accelerate and enhance biological research. The 

primary goal of bioinformatics is to increase the 

understanding of biological processes. Common 

activities of bioinformatics include mapping and 

analyzing DNA and protein sequences, aligning 

different DNA and protein sequences to compare 

them and creating and viewing 3-D models of 

protein structures. It has been successfully applied to 

predict the function, structure and evolution of 

biological sequences. It can reveal biological 

relationship among organisms, for example, finding 

evolutionary information, determining causes and 

cures of diseases. 

Sequence comparison is a basic operation of the 

sequencing problem. Sequences can be aligned 

across their entire length (global alignment) or only 

in certain regions (local alignment). Local sequence 

alignment plays a major role in the analysis of DNA 

and protein sequences (GusFeild, 1997). This paper 

describes the pair wise local alignment, which is the 

basic step for many other applications like detecting 

homology, finding protein structure and function, 

deciphering evolutionary relationships, etc. Smith 

Waterman developed a dynamic programming 

approach to sequence alignment problem that is 

widely used (Smith and Waterman, 1999). BLAST 

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

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

is two commonly used programs for similarity 

searching on biological sequences. The methods 

widely used are based on heuristic paradigms and 

have relatively a fast execution time, they do not 

produce optimal alignments sought by entirely 

sequenced. This reality presents the need for 

comparing long DNA or protein sequences, which is 

a challenging task due to its high demands for 

computational requirements (power and memory). 

One important feature of BLAST is its ability to 

compare a query with a database of sequences. 

Considering the rapid growth of database sizes, this 

problem demands ever-growing computation 

resources, and remains as a computational challenge. 

Divya in Sequence Comparison Tool - SCT (Divya 

et al., 2006) developed sequence similarity in a 

database instead of pair wise sequence alignment. 

This tool preprocesses the database to create a 

special generalized suffix tree from the sequences in 

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

one of the most important data structures in string 

processing and comparative genomic. The space 

consumption of the suffix tree is a bottleneck in 

large-scale applications such as genome analysis. To 

overcome this bottleneck, in this paper suffix tree is 

replaced with suffix arrays enhanced with the Lcptable. 

Every algorithm that uses a suffix tree as data 

structure can systematically be replaced with an 

algorithm that uses an enhanced suffix array 

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

problem in the same time complexity but with 

improved space complexity. The generic name 

enhanced suffix array (Manber and Myers, 1993) 

stands for data structures consisting of the basic 

suffix array enhanced with additional tables. 

Generalized suffix array (GSArray) is formed for 

the query sequence and longest common 

subsequence (LCS) is obtained and is compared 

against the database; sequences, which consist of 

LCS, are identified from the database. Sequences are 

ranked with respect to the number of significant 

patterns they share with the query sequence. Finally 

database is compressed by selecting only a given 

number of sequences with topmost ranks. 


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

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

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

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

and also assumed that the special symbol $ is an 

element of ∑ (which is larger than all other 

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

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

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

character at position i and ending with the character 

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

the pair of positions (i, j). 

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

rooted directed tree with exactly n+1 leaves 

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

root, has at least two children and each edge is 

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

edges out of a node can have edge-labels beginning 

with the same character. The key feature of the suffix 

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

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

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

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

< n. The space and time complexity of this suffix 

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

shown in Figure 1 for the string S = acaaacatat. 

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

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

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

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

S2. 

However, the space consumption of the suffix tree 

is a major problem when comparing large genomes. 

Figure 1. The suffix tree for S = acaaacatat. 

Suffix array 

Suffix array is designed for efficient searching of a 

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

input character) in its basic form. Searching a text 

can be performed by binary search using the suffix 

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

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

arrays. Suffix arrays will prove to be better than 

suffix trees for many applications. The suffix array 

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

integers in the range 0 to n, specifying the 

lexicographic ordering of the n + 1 suffixes of the 

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

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

ascending lexicographic order as shown in Table 1. 

Suffix array generation 

Creating suffix array requires time O (nlogn) and 

searching for a pattern in it requires time O 

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

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

2002), the authors provides a systematic way of 

replacing string processing algorithm based on a 

bottom-up traversal of a suffix tree by a 

corresponding algorithm that is based on an 

enhanced suffix array. The advantages of this 

approach are better search, more space efficient 

Bio-database compression using enhanced suffix array 47 

than suffix tree since the implementation of the 

enhanced suffix array requires only 5n bytes and 

the running times of the algorithms are much better 

than those based on the suffix tree and the 

algorithm is easier to implement on enhanced suffix 

arrays than on suffix trees. 

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

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

0 2 0 aaacatat$ 

1 3 2 aacatat$ 

2 0 1 acaaacatat$ 

3 4 3 acatat$ 

4 6 1 atat$ 

5 8 2 at$ 

6 1 0 caaacatat$ 

7 5 2 catat$ 

8 7 0 tat$ 

9 9 1 t$ 

10 10 0 $ 

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

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

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

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

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

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

maximal if it is left and right maximal. A substring 

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

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

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

repeat that never occurs as a substring of any other 

maximal 

repeat. 

The suffix array suftab is an array of integers in 

the range 0 to n, specifying the lexicographic 

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

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

of suffixes of S$ in ascending lexicographic order, 

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

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

requires 

4n bytes. 

The Lcp-interval table Lcptab is an array of 

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

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

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

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

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

by-product during the construction of the suffix


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

shown in Figure 2. 

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

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

is called an Lcp-interval 

of Lcp-value ℓ 

(denoted by ℓ-[i...j]) if 

Lcptab[i] < ℓ 

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

Lcptab[k] = ℓ for at least one k with 

i+1 ≤ k ≤ j 

Lcptab [j + 1] < ℓ 

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

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

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

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

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

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

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

on the analogy between the suffix array and the 

suffix tree, it is desirable to enhance the suffix array 

with additional information to determine, for any ℓ- 

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

using enhancing the suffix array with two tables. 

Enhanced Suffix array 

The new data structure consists of the suffix array, 

the Lcp-interval table, and an additional 

table: the 

child-table cldtab shown in Table 2. 

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

from 0 to n and each entry contains three values: 

up, down, and nextℓIndex. Each of these three 

values requires 4 bytes in the worst case. The 

values of each cldtab-entry are defined as follows 

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

1. cldtab[i].up = 

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

and for all k Є [q + 1..i - 1] : 

Lcptab[k] ≥ Lcptab[q]} 

2. cldtab[i].down 

= 

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

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

Lcptab[q]} 

3. cldtab[i].next ℓ Index = 

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

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

Lcptab[i]} 

The child-table stores the parent-child 

relationship of Lcp-intervals. For an ℓ -interval 

[i...j] whose ℓ -indices are i1 < i2

with a highly conserved region. It is calculated 

based on the length of the pattern and frequency 

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

given pattern p is classified as significant if it 

satisfies the following constraints: 

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

significant pattern must be sufficiently long 

to carry important biological information. 

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

significant pattern 

must have a sufficiently 

high score. 

GSArray is constructed for the entire sequences 

in the database. While constructing GSArray, at 

each node i, the length and the 

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

pi in the database) is stored for the corresponding 

pattern pi this frequency is incremented for every 

new node. Then the score function 

is 

calculated using the equation (1) 

(1) 

Where = Function value of the pattern, 

= frequency of the node, 

= length of the label of prefix, 

i = node number, 

|DB| = Database size. 

The score function is used as a measure 

to determine whether a particular pattern is 

significant to be included in the post-processing for 

evaluation of sequences, to determine the closest 

set of sequences. Then the query sequence Q and 

number of sequences to be selected from the 

database is read from the user, temporarily added 

onto GSArray. This enables to determine which 

suffixes of the query are shared by the sequence in 

the database. The query sequence is only 

temporarily added to the tree so that GSAlign is not 

affected for future sequence searches. Initially all 

the nodes in the GSArray are 0. When the query 

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

set to 1. This expedites the search for common 

patterns within the GSArray because only those 

paths in the tree for patterns that contain substrings 

of the query sequence are examined. In depth-first 

manner, starting at the root all the nodes are visited 

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

child whose value is 1, then the search backtrack to 

its parent node. During this traversal all the 

significant patterns are collected. The sequence 

may have other common patterns that are not 

significant. An optimal alignment between these 

two sequences in an ideal case contains all 

significant patterns. After this process the query 


sequence from GSArray is deleted. Top ten 

significant patterns are selected and stored. The 

sequence that contains significant patterns is 

extracted and stored. Reverse check is made to 

obtain the accuracy of the results; it computes how 

many chosen patterns are being shared by each of 

the sequences extracted already. Higher the number 

(weight), greater will be the similarity of the 

corresponding sequence to the query. Based on this 

weight 

the sequences are ranked. 

A top n sequences are transferred to new 

database. Algorithm for 

BioDB Compression Tool 

is shown in Figure 3. 

//Input: Set of DNA and Protein Sequences 

// Output: Compressed set of DNA and Protein sequences 

S1: Read DNA or Protein database; 

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

=0; 

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

label length, frequency at nodes and sequences traversing 

through the branches; 

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

patterns in the form of prefixes; 

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

select from the user; 

S6: Temporarily add the suffixes of query to the generalized 

GSArray; 

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

are traversed by the query sequence; 

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

query Sequence, which lies above a defined threshold on 

the 

function-value; 

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

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

in the subset; 

S9: Rank the sequences according to these weights; 

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

database; 

Figure 3. Algorithm for BioDB Compression Tool 

Cross validation 

The real world DNA (GSS, EST) and Protein 

sequence (Protein C, S) databases from Oryza 

sativa group is extracted from NCBI website and 

enhanced suffix array is formed for all the 

sequences in the database. Based on the user given 

query sequence, the significant patterns are 

generated. The sequences in the DNA or Protein 

database are given weights according to the number 

of patterns they contain. The compressed database 

is formed by selecting top ‘n’ sequences with 

highest 

ranks and written into a new database. 

The latest data mining technique, 7-fold cross 

validation is used to validate the results obtained 

from BioDB Compression Tool and WU-BLAST.


The main idea of 7-fold cross validation approach 

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

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

data are used for training data set and the remaining 

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

Tool is applied on training data set and then on 

WU_BLAST for single user given query sequence. 

For the same query sequence WU-BLAST alone is 

applied on the testing data set. The average of this 

seven runs is computed for analysis. In this paper 

such seven queries are taken and analyzed. 49 

sequences from Oryza sativa GSS gi: 288881557 to 

gi: 288881606 are taken as a database set, 42 

sequences are used for training data set and 7 

sequences are used for test data set. Single query 

sequence is applied first on BioDB Compression 

Tool, data base is compressed. WU-BLAST is 

performed on the new data base and the same query 

sequence. Then for same sequence WU-BLAST 

alone is applied. The sequences in the training and 

test data set are interchanged and the above steps 

are repeated until every fold is used for training. 

Average of the result from 7 runs are calculated and 

stored. This is experimented for 7 different queries. 

The objective is to test whether the results are 

consistent for all the queries on a particular 

database in terms of computational time. 

Results 

Compressed database from BioDB Compression 

Tool is cross validated using 7-fold cross validation 

approach. The cross validation results of seven 

different queries for DNA database set GSS gi: 

288881557 to gi: 288881606 are shown in Figure 4 

and EST gi: 288886142 to gi: 288886191 are 

shown in Figure 5 and the results of seven different 

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

Figure 4. 

Results of cross validation for GSS DNA 

dataset 

Figure 5. Results of cross validation for EST DNA 

dataset 

The idea is to test whether the results are 

consistent for all queries on a particular database in 

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

is inferred that the developed BioDB Compression 

Tool performs much better than WU-BLAST. 

Computational complexity of using developed 

BioDB Compression Tool is reduced compared to 

WU-BLAST because; compression takes place 

along sequence alignment. The Enhanced suffix 

array algorithm used in BioDB Compression Tool 

requires 5n bytes character where SCT requires 20n 

bytes which uses suffix tree. 

Figure 6. Results of cross validation for Protein C 

Hence it is proved that space complexity is 

approximately five times less than SCT. 

Implementation results show that the running time 

of developed algorithm BioDB Compression Tool 

using enhanced suffix array is much better than 

SCT which uses suffix tree.

Figure 7. Results of cross validation for Protein S 

Discussion 

In this paper the BioDB Compression Tool using 

enhanced suffix array has been developed. BioDB 

Compression Tool pre-processes the database to 

create a generalized enhanced suffix array and 

extended by adding frequency and length 

information for the patterns. BioDB Compression 

Tool distinguishes patterns by computing 

significance-scores. A pattern is regarded as 

significant if it is long enough, and it appears 

frequently enough in the database. The scoring 

function takes into account a pattern's length and 

frequency, the given threshold values, and 

determines if a pattern is significant. Using these, 

for a given query sequence BioDB Compression 

Tool compresses the database to only a few 

sequences that share the most significant patterns 

with the query. This compression in database size 

speeds-up the local alignment of the query 

sequence against the database. Experimental results 

have shown that BioDB Compression Tool 

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

reduce the time of a database search to nearly five 

times originally taken by WU-BLAST. Results 

from WU-BLAST have shown that this method is 

experimentally effective, as the results obtained by 

BioDB Compression Tool produces accurate 

alignment. Combined with the extended suffix 

array, BioDB Compression Tool has the advantage 

of using WU-BLAST to do the local sequence 

alignment. Latest Data Mining technique, 7-fold 

cross validation is applied to attain a greater 

accuracy in the results and the results are 

satisfactory. The Enhanced suffix array algorithm 

used in BioDB Compression Tool requires 5n bytes 

/character where SCT requires 20n bytes which 

uses suffix tree. So, space complexity is 

approximately five times more than SCT. 

Experimental results show that the running time of 


BioDB Compression Tool is much better than SCT 

which uses suffix tree. 

In this paper, a small domain of sequences have 

been selected from the DNA and Protein database 

and experimentally proved that, enhanced suffix 

array reduces space complexity by five times. 

BioDB Compression Tool can also be applied to 

Global Sequence Alignment and Multiple Sequence 

Alignment. This work can also be applied on 

databases of protein sequences for alignment. 

References 

Abouelhoda MI, Kurtz S and Ohlebusch E. The 

enhanced suffix array and its applications to 

genome analysis. Proc. Workshop on 

Algorithms in Bioinformatics. Springer- Verlag, 

Berlin. 2452: 449- 463, 2002. 

Abouelhoda MI, Stefan Kurtz and Enno Ohlebusch. 

Replacing suffix trees with enhanced suffix 

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

2004. 

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

Lipmann D. Basic Local Alignment Search 

Tool. Journal of Molecular Biology. 215:403 - 

410, 1990. 

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

White O and Salzberg SL. Alignment of Whole 

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

1999. 

Divya RS, Abdullah NA and Xindong Wu. Using 

an extended suffix Treeto speed-up sequence 

alignment. IADIS International Conference 

Applied Computing. 655-660, 2006. 

Gus Field D. Algorithms on Strings, Trees and 

Sequences. Cambridge University Press. 1997. 

Basic Local Sequence Alignment Tool. Retrieved 

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

Manber U and Myers EW. Suffix Arrays: A New 

Method for On-Line String Searches. SIAM 

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

Matthew Bellgard, Thomas Gamble, Mark 

Reynolds, Adam Hunter, Ed Trifonov, and Ross 

Taplin. Gap apping: a paradigm for aligning two 

sequences. Applied Bioinformatics. 2(3 Suppl): 

31-35, 2003. 

Pearson WR. Flexible sequence similarity 

searching with the FASTA3 program package. 

Methods in Molecular Biology.132: 185-219,


2000. 

Smith TF and Waterman MS. Identification of 

common molecular subsequences. Journal of 

Molecular Biology. 147:195-197, 1981




Cloning and expression of Lentinula edodes cellobiohydrolase gene 

in E. coli and characterization of the recombinant enzyme 

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

Amangeldy BISSENBAEV 1, * 

1 

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

050038, Almaty,al-Farabi 71, Kazakhstan 

2 

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

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

Received: 11 March 2011; Accepted: 10 June 2011 

Abstract 

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

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

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

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

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

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

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

50 0 C and pH 7. 

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

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

recombinant enzimin karakterizasyonu 

Özet 

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

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

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

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

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

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

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

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

Introduction 

Cellulose, consisting of glucose units linked 

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

abundant carbohydrate in the biosphere. An 

estimated rate of cellulose synthesis is 

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

solution for problems of energy, chemicals, 

and food, cellulose is the most promising renewable 

carbon source that is available in large quantities 

(Murai et al., 1998). 

Full hydrolysis of cellulose requires synergistic 

action of three major types of enzymatic activity: 

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

(CBH; EC 3.1.2.91) and βglucosidases 

(EC 3.2.1.21). CBHs hydrolyze

54 Sabira TAIPAKOVA et al. 

crystalline cellulose by initiating their action from 

the ends of the cellulose chains and producing 

primarily cellobiose. Endoglucanases catalyze 

hydrolysis internally in the cellulose chain and 

attack the amorphous regions in cellulose, 

providing new chain ends for CBHs. β- 

glucosidases hydrolyze cellobiose to glucose. These 

enzymes are collectively known as cellulases and 

act in a synergistic manner to facilitate complete 

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

(Teeri, 1997) 

CBHs are key components in the multi-enzyme 

cellulose complexes. Most of them belong to 

glycosyl hydrolase families 6 or 7. Cellulolytic 

fungus generally produces two different CBHs, 

CBHI and CBHII. These two types of enzymes 

which are classified based on sequence identity and 

can achieve complete, although slow, solubilization 

of cellulose crystals even without help of 

endoglucanases (Teeri, 1997). 

Lentinula edodes, commonly referred to as the 

Shiitake mushroom, is the most popular and 

economically important edible mushroom in the 

world because of its taste, nutritional and medicinal 

properties (Mizuno, 1995). L. edodes has 

traditionally been grown on freshly cut logs 

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

decays for nutrients all the major polymers 

(cellulose, hemicelluloses, and lignin) found in 

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

edodes produces a wide variety of enzymes that 

may have high activities against cellulosic biomass. 

Several papers reported that L. edodes has a 

strong ability to produce thermostable cellulases, 

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

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

least two forms of cellobiohydrolase (CEL7A and 

CEL6B). Genes encoding each of these enzymes 

were cloned from L. edodes grown on a wood 

substrate using a PCR based strategy with 

degenerate primers directed at the cellulose-binding 

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

nucleotide sequence analysis it was shown that 

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

belongs to glycosyl hydrolase family 7 and has 

sequence similarities to CBHI genes from other 

fungi. Whereas cel6B gene encodes a 444 aa 

protein that belongs to glycosyl hydrolase family 6 

and has sequence similarities to CBHII genes from 

other fungi. However, both fungal CBHs genes 

have not been previously expressed and 

characterized. 

In this paper we report the cloning of a L. 

edodes cel7A gene and its successful expression in 

a heterologous host, E. coli. 


Materials 

Restriction enzymes, T4 DNA ligase and Taq DNA 

polymerase were purchased from Fermentas Life 

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

was isolated using High Pure Plasmid Isolation Kit 

and DNA fragments were purified using Agarose 

Gel DNA Extraction Kit (Roche Diagnostics 

GmbH. Mannheim, Germany). All other chemicals 

and reagents were analytical grade and were 

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

USA). The expression vector pET11d was from 

Invitrogen, USA. 

Strains and culture conditions 

L. edodes strain N127 was obtained from collection 

of Kazakh National Agrarian University (Almaty, 

Kazakhstan). L. edodes strain N127 was cultured at 

room temperature on poplar sawdust blocks. From 

sawdust blocks the mycelium was harvested with a 

scalpel. The mycelium samples were pooled and 

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

further processing. Escherichia coli DH5a were 

used as a host for plasmid propagation. E.coli 

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

host for expression of the recombinant CEL7A. 

Total RNA extraction and RT-PCR 

Total RNA extraction of L.edodes and RT-PCR 

was performed as described (Sambrook et al., 

1989). Total RNA isolated as described above was 

used as a template for RT reactions. Using the 

sequence of the L. edodes cel7A cDNA available in 

the GenBank database (GenBank accession number 

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

GATCACCATGGTCCGAACAGCAGCTCTCCT 

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

CTAGGATCCCTACAAACATTGACTGTAGTA 

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

underlined bases in primers are the sites of 

restriction enzymes NcoI and BamHI, respectively.

Construction of expression vector and protein 

expression 

The PCR product and pET11d vector were doubledigested 

by NcoI and BamHI at 37 0 C according to 

the protocol. The digested products were ligated by 

T4 ligase at 4 0 C for overnight. The recombinant 

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

Rosetta (DE3) competent cells and the 

transformants were selected on LB plates with 

ampicillin. The different resistant transformants 

were picked and inoculated into 20 mL LB medium 

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

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

the incubation was continued in 1L LB medium. 

When the culture grown at 30°C reached an 

OD600=0.4–0.6, isopropyl-D-thiogalactopyranoside 

(IPTG) was added to a final concentration of 0.2 

mM to induce expression. After incubation cells 

were harvested by centrifugation at 6000 rpm for 5 

min at 4°C. Cells were resuspended in storage 

buffer containing 20 mM HEPES-KOH pH 7.6, 40 

mM NaCl. The cells were lysed by successive 

cycles of freezing (liquid nitrogen) and thawing by 

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

clear cell lysate was obtained. This mix was used as 

crude protein extracts or alternatively the sonicated 

mixture was centrifuged at 14 000 g for 10 min to 

remove cell debris, and this cleared supernatant 

fluid was used as crude proteins extracts. The crude 

protein extracts and cellular fractions regarded as 

crude enzyme extracts were used for identifying 

CEL7A expression by SDS-PAGE, Western 

blotting and enzyme analysis. 

Preparation of anti-CEL7A antibodies and Western 

blotting 

The enzyme sample (preincubated with Complete 

EDTA-free Protease Inhibitor Cocktail) was 

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

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

with Coomassie Brillant Blue R-250 and the major 

protein band was excised from the gel and used as 

antigen. The gel piece was homogenized with 

liquid nitrogen and emulsified in Freund’s complete 

adjuvant (Sigma) and injected subcutaneously into 

a rabbit. The same amount of booster injections 

was given every two weeks using incomplete 

Freund’s adjuvant. The rabbit was bled before the 

first injection (preimmune serum) and then one 

week after the last booster to obtain immune serum 

(anti-CEL7A antibodies). Western blots were 

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

performed by standard protocols using 1:400 anti- 

CEL7A polyclonal antibody diluted in blocking 

buffer and 1:10000 horseradish peroxidaseconjugated 

secondary antibody. 

Mass spectrometry 

The target bands on SDS-PAGE gels were excised 

and subjected to in-gel digestion with trypsin 

followed by peptide mass fingerprinting by matrix 

assisted laser desorption ionization time-of-flight 

mass spectrometry (MALDI-TOF) using a 

MALDI-TOF-TOF spectrometer, Ultraflex II 

(Bruker). Spectra from all experiments were 

converted to DTA files and merged to facilitate 

database searching using the Mascot search 

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

against the non-redundant protein sequences of 

GenBank (National Center for Biotechnology 

Information). 

Protein and enzyme assays 

The protein content was determined by Bradford 

method (Bradford, 1976) Enzyme activity was 

assayed with Avicel, filter paper, 

carboxymethylcellulose (CMC) as a substrate by 

measuring the amount of reducing sugar according 

to the Nelson-Somogyi method (Somogyi, 1952; 

Nelson, 1944). The reaction mixture consisted of 

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

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

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

The generated reducing sugar was quantified at 540 

nm and using D-glucose as a standard for 

spectrophotometric estimation. One unit of 

cellobiohydrolase activity was defined as the 

amount of enzyme that releases 1 μM of reducing 

sugar per minute per mg of total protein under the 

assay conditions. 

When p-Nitrophenyl β-D-lactopyranoside 

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

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

performed in 500 μl reaction volume in 50 mM 

sodium phosphate buffer (pH 7.0) containing 8 mM 

of pNP-Cel or pNP-Lac with appropriate amounts 

of enzyme. The reaction was terminated by the 

addition of 2 ml of 1 M Na2CO3 after 1 h 

incubation. One unit of enzyme activity was 

defined as the amount of the enzyme that produced 

the equivalence of 1 μM pNP at the optimal 

conditions in 1 min per 1 mg of total protein.


Results 

To isolate a gene coding CEL7A, we designed two 

oligonucleotide primers based on the previously 

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

was done using genomic DNA of L. edodes N127 

as a template. PCR product showed a clear band 

about 2000 bp (Figure 1B). 

In the following experiments we amplified 

cel7A cDNA by reverse transcriptase (RT) PCR 

starting from total RNA. High quality RNA was 

isolated from mycelium of L. edodes grown on the 

poplar sawdust using the CTAB-based extraction 

method. RNA examined by electrophoresis on 

0.8% agarose gel showed two bands corresponding 

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

RNA degradation occurred during the isolation. 

The A260/A280 and A260/A230 absorbance ratios 

were 1.9 and 2.0, respectively. This indicates low 

contamination by protein substances and secondary 

metabolites in the isolated RNA samples. In 

general, the RNA obtained was of high quality and 

integrity. Total RNA prepared as above was 

subjected to RT-PCR with the same primers. A 

single DNA band about 1551 bp in size was 

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

was ligated into pET11d vector and propagated in 

E.coli. The clones were sequenced from both 

directions. 

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

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

Determination of the nucleotide sequence 

revealed complete coincidence with the nucleotide 

sequence of the cel7A gene L. edodes strain 

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

2001). 

The blastp results in Table 1 showed that the 

deduced amino acid sequence of 516 amino acids is 

identical with L. edodes cellobiohydrolase I amino 

acid sequence and showed high homology to other 

fungal CBHI enzymes belonging to glycosyl 

hydrolases family 7: 99% identity to cellulase of 

Irpex lacteus, 96% to cellobiohydrolase I of 

Schizophyllum commune and 95% to 

cellobiohydrolase I–II of Volvariella volvacea. 

Results in this study show that amino acid 

sequence deduced from the nucleotide sequence of 

cel7A is homologous to other CBHs. However, 

until now the cel7A gene of L. edodes 

cellobiohydrolase has not been expressed in 

bacterial and yeast systems. Therefore, it was 

needed to be determined if it really codes a CBH. 

In order to confirm that the cel7A gene encodes 

a cellobiohydrolase we used E. coli Rosetta (DE3) 

expression system. The synthesized by RT-PCR 

1551bp fragment was digested with restriction 

endonucleases NcoI and BamHI and cloned in the 

pET11d plasmid with retention of the reading 

frame. The inserted fragment was tested for the 

absence of mutations by sequencing. The resulting

plasmid pET11d/cel7A was then used to transform 

E. coli Rosetta (DE3). 

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


Genbank accession Organism and gene Score E value Identity 

AAK95563.1 

Lentinula edodes cellulase CEL7A 

mRNA 

1043 1043 100% 

BAA76365.1 Irpex lacteus cellulase mRNA 639 639 99% 

AAX55505.1 

Schizophyllum commune H4-8 

glycoside hydrolase family 7 protein 

586 625 96% 

AAT64007.1 

Volvariella volvacea 

cellobiohydrolase I-II mRNA 

The transformation of competent E.coli cells 

with the ligation products of the pET11d vector and 

the L. edodes cel7A gene amplicon yielded over 47 

colonies, among which 4 individual clones were 

selected. All the selected colonies were assayed by 

PCR and restriction analysis for the presence of the 

recombinant plasmids carrying the cel7A gene. 

Plasmid DNAs were isolated and purified 

according to the protocol of High Pure Plasmid 

Isolation Kit. Plasmid DNAs were digested by NcoI 

and BamHI, followed by agarose gel 

electrophoresis. From Figure 2 we can see that 

plasmid DNAs was cut into two fragments, one 

Figure 2. Verification of the recombinant plasmid 

pET11d/cel7A digested by Nco I and BamHI M - 

Marker (bp); 1-4 Clones 

647 681 95% 

about 1551 bp corresponding to cel7A gene, and the 

other about 5674 bp corresponding to the vector. 

We amplified the cel7A gene by PCR, using 

transformant plasmid DNA as a template and gene 

specific primers. The fragment detected by agarose 

gel electrophoresis corresponded in length to the 

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

product corresponding in length to the cloned cel7A 

gene sequence indicated that the analyzed colonies 

contained plasmids carrying the relevant gene. As a 

result of screening, we identified four colonies 

carrying the recombinant plasmids. 

Figure 3. Verification of the recombinant plasmid 

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


Synthesis of CEL7A protein was assessed in 

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

expressing the cel7A gene was analyzed by SDS- 

PAGE and Western blotting using polyclonal anti- 

CEL7A antibodies. 

After induction with IPTG for 4-12 hours the 

cells were lysed and the protein samples were 

prepared for SDS-PAGE by boiling in 2x sample 

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

crude extracts of cells harboring the plasmid 

pET11b/cel7A were shown on SDS-PAGE (Figure 

4A), whereas no similar protein bands from crude 

extracts of cells harboring the plasmid pET11d 

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

PAGE gel was transferred to a PVDF membrane 

and probed with anti-CEL7A polyclonal antibody. 

Western blot analysis revealed a major protein 

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

extracts of the cells harboring the plasmid 

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

validate the identity of these proteins to CEL7A, 

the polypeptides were excised from the gel, 

subjected to trypsin digestion and MALDI-TOF- 

MS analysis. MASCOT search results revealed a 

top score of 659 for CBHI, where probability based 

mowse score > 44 are significant and indicate 

identity or extensive homology (p


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

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

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

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

Crude extract of an E. coli expressing the cel7A 

gene was examined for its ability to hydrolyze 

various cellulosic substrates at pH 7 and 50 0 C 

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

pNP-Lac and pNP-Cell, but activity towards 

carboxymethylcellulose (CMC) was much lower 

compared to Avicel and Filter paper. These results 

strongly indicate that CEL7A has cellobiohydrolase 

activity. 

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

Substrate 

Activity 

(U/mg total protein) 

Avicel 21.4 ± 1.4 

Filter paper 23.2 ± 0.3 

CMC 2.9 ± 0.5 

pNP-Lac 4.1 ± 0.08 

pNP-Cell 3.8 ± 0.1


Discussion 

CBH enzymes are key components in fungal 

cellulase systems, and their functional activity is 

critical for consolidated bioprocessing for 

bioethanol production. For example, CBHs make 

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

and CBH plays a particularly important role, 

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

2007). A number of cellulase genes from bacteria 

and fungus have been cloned and expressed in yeast 

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

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

endoglucanase (EG) production in recombinant 

strains was more successful than CBH production 

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

considering that EG enzymes usually have specific 

activities of 2 to 3 orders higher in magnitude on 

synthetic and amorphous cellulose substrates, such 

as carboxymethylcellulose (CMC), in comparison 

to CBHs. Thus it is easier to measure the presence 

of even small amounts of heterologous EG 

compared to CBHs. 

Expression of fungal cellulases in E. coli as 

non-glycosylated forms has been attempted with 

limited success. Only a few cellulose genes, such as 

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

were expressed in E. coli, but the productivity of 

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

In this study, we have successfully expressed the 

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

of the T7 promoter. SDS–PAGE and Western 

blotting analysis showed that CEL7A constitutes a 

major protein produced in E. coli with a molecular 

weight of 53,5 kDa, which agrees with the 

predicted size from the deduced amino acid 

sequence. Amino acid sequencing of the putative 

recombinant protein by MALDI-TOF and its 

analysis using NCBI BLAST indicated that the 

enzyme contained putative conserved domains of 

glycosyl hydrolase family 7. 

As shown in Figure 4A plasmid vector 

pET11d/cel7A expresses two additional bands 

which in SDS-PAGE migrate as 53 kDa and 49 

kDa proteins. Since the difference in molecular 

weight between these two proteins is approximately 

5 kDa we may suggest that the CEL7A protein was 

partially degraded possibly during cell lysis. The 

cel7A enzyme contains 430 aa catalytic domain and 

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

and serine/threonine-rich linker sequence (Lee et 

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

degradation of 57 aa C-terminal CBD and linker 

sequence results in truncated 49 kDa protein. 

Indeed, western blot analysis confirms the presence 

of low molecular weight CEL7A proteins which 

migrate below the full-length recombinant protein 

in SDS-PAGE suggesting proteolytic degradation 

of CEL7A during expression and/or cell lysis. 

Furthermore we hypothesize that the linker region 

of E. coli-expressed CEL7A might be more 

susceptible to proteolysis as compared to 

glycosylated form of this protein expressed in yeast 

cells (unpublished observation). It seems likely that 

glycosylated linker region in native CEL7A of L. 

edodes protects the enzyme from proteolytic 

degradation nevertheless further investigations are 

required. 

The substrate specificities indicated that CEL7A 

contains cellobiohydrolase activity since it could 

hydrolyze crystalline cellulose (Avicel, Filter 

paper). Activity of cloned enzyme toward CMC 

was much lower than to Avicel and filter paper. 

These data are consistent with the established 

results that fungal CBH proteins have very limited 

action on substituted cellulose such as CMC or 

hydroxyethylcellulose (Basiria and Mishra, 1989; 

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

cellulases are glycoproteins, the modification of the 

native protein by glycosylation has been reported to 

play an important role in synthesis, secretion, and 

stability of extracellular cellulases (Basiria and 

Mishra, 1989). Here we have demonstrated that 

CEL7A synthesized in E. coli without 

glycosylation has an enzymatical activity. This 

indicates that glycosylation is not necessary for 

enzymatic activity of CEL7A. 

In summary, a gene encoding CEL7A was 

successfully isolated from L. edodes strain N127 

using RT-PCR technique. The determined 

nucleotide sequence is completely corresponded to 

the nucleotide sequence of the cel7A gene of L. 

edodes strain Stamets CS-2 which was described 

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

successfully expressed in E. coli in an active form. 

Enzymatic properties of CEL7A were also 

determined. The optimal temperature for enzyme 

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


This work was supported by grant from National 

Centre for Biotechnology of the Republic of 

Kazakhstan.

References 

Basiria VS and Mishra S. Regulation aspects of 

cellulose biosynthesis and secretion. CRC Crit 

Rev Biotechnol. 9: 61-162, 1989. 

Bradford MM. A rapid and sensitive method for the 

quantitation of microgram quantities of protein 

using the principle of protein–dye binding. 

Anal Biochem. 72: 248-252, 1976. 

Ekino KK, Goto M and Furukawa K. Heterologous 

expression and characterization of 

endoglucanase I (EGI) from Trichoderma 

viride HK-75. Biosci Biotechnol Biochem. 63 

(10): 1714-1720, 1999. 

Hong J, Tamaki H, Akiba S, Yamamoto K and 

Kumagai H. Cloning of a gene encoding a 

highly stable endo-b-1,4-β-glucanase from 

Aspergillus niger and its expression in yeast. J 

Biosci Bioeng. 92: 434-441, 2001. 

Hong J, Tamaki H, Yamamoto K and Kumagai H. 

Cloning of a gene encoding thermo-stable 

endo-b-1,4-glucanase from Thermoascus 

aurantiacus and its expression in yeast. 

Biotechnol Lett 25: 657–661, 2003. 

Kanokratana P, Chantasingh D, Champreda V, 

Tanapongpipat S, Pootanakit K and 

Eurwilaichitr L. Identification and expression 

of cellobiohydrolase (CBHI) gene from an 

endophytic fungus, Fusicoccum sp. 

(BCC4124) in Pichia pastoris. Protein Expr 

Purif. 58: 148-153, 2008. 

Leatham GF. Extracellular enzymes produced by 

the cultivated mushroom Lentinus edodes 

during degradation of a lignocellulosic 

medium. Appl Environ Microbiol. 50: 859-867, 

1985. 

Lee CC, Wong DWS and Robertson GH. Cloning 

and characterization of two cellulase genes 

from Lentinula edodes. FEMS Microbiology 

Letters. 205: 355-360, 2001. 


Mizuno T. Shiitake, Lentinus edodes: functional 

properties for medicinal and food purposes. 

Food Rev Int. 11: 111-128, 1995. 

Murai T, Ueda M, Kawaguchi T, Arai M and 

Tanaka A. Assimilation of 

cellooligosaccharides by a cell surfaceengineered 

yeast expressing beta -glucosidase 

and carboxymethylcellulase from Aspergillus 

aculeatus. Appl Environ Microbiol. 64: 4857- 

4861, 1998. 

Nelson NJ. A photometric adaptation of the 

Somogyi method for the determination of 

glucose. J Biol Chem. 153: 375-380, 1944. 

Pereira Júnior JA, Correia MJ and Oliveira NT. 

Cellulase activity of a Lentinula edodes (Berk.) 

Pegl. strain grown in media containing 

carboximethylcellulose or microcrystalline 

cellulose. Brazilian Archives of Biology and 

Technology. 46: 333-337, 2003 

Qiao Y, Mao AJ, He YZ, Liu WF and Dong ZY. 

Secreted expression of Trichoderma reesei 

endo-glucanase II gene in Pichia pastoris and 

analysis of enzymatic properties. Mycosystema. 

23(3): 388–396, 2004. 

Sambrook et al. Molecular Cloning: a Laboratory 

Manual (2 nd ed.). Cold Spring Harbor 

Laboratory Press. 1989. 

Somogyi M. Notes on sugar determination. J Biol 

Chem. 195: 19-23, 1952. 

Teeri TT. Crystalline cellulose degradation: new 

insight into the function of cellobiohydrolases. 

Trends in Biotechnology. 15: 160-167, 1997. 

van Zyl WH, Lynd LR, den Haan R and McBride 

JE. Consolidated bioprocessing for bioethanol 

production using Saccharomyces cerevisiae in 

biofuels. Adv Biochem Eng Biotechnol. 108: 

205-235, 2007.




Investigation of the MMP1 and MMP3 promoter polymorphisms in 

temporomandibular joint disorder 

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

KARATAS 3 and Teoman AKCAY 5 

1 

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

2 

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

3 

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

Turkey. 

4 

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

5 

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

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


Abstract 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

MMP3 promotor polymorphism, orofacial pain. 

Temporomandibular bağlantı bozukluklarında MMP1 ve MMP3 promotor polimorfizmlerinin 

incelenmesi 

Özet 

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

iyonlarına bağımlı endopeptidazlardır ve temporomandibular bağlantı bozukluklarında (TBB) hücrelerarası 

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

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

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

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

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

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

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

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

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

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

yapılması ile daha anlamlı sonuçların alınacağına inanmaktayız.

64 Necati TASKIN et al. 

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

polimorfizmi, MMP3 promotor polimorfizmi, orofasyal ağrı 

Introduction 

Temporomandibular joint disorders (TMJD) are the 

orofacial problems involving masticatory 

musculature, the temporomandibular joint (TMJ), 

and related structures. This disorder frequently 

causes chronic pain in the orofacial region 

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

cartilage, capsule, ligaments and synovial 

membrane are the major clinical outcomes of TMJ. 

These degenerative changes in the tissues are slow 

at the beginning but with the progression of the 

disease, irreversible changes occur in the joint 

(Stegenga, 2001). 

The main characteristic change in TMJ is the 

alteration of the equilibrium between synthesis and 

degradation of extracellular matrix (ECM) 

collagens, especially by proteolysis (Planello et al., 

2011). Accumulation of the hydrolyzed proteins, 

catabolites formed by proteolytic activities, 

inflammatory modulators like cytokines and nitric 

oxide, matrix metalloproteinases (MMPs) found in 

the synovial fluid are the reason of inflammatory 

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

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

MMPs are considered to be the key proteolytic 

enzymes for the destruction of ECM in TMJ 

(Wadhwa and Kapila, 2008). 

MMPs are metal dependent endopeptidases that 

are capable of cleaving most, if not all of the 

constituents of the extracellular matrix including 

collagen, fibronectin, and proteoglycans (Astolfi et 

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

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

activated after the cleavage of N- terminal domain 

(Visse and Negasse, 2003) by other proteinases. 

Also several promotor polymorphisms were 

reported to regulate transcription activity and these 

have been associated with degenerative diseases 

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

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

influence the basal and inducible levels of MMP 

expression. An insertion/ deletion variation of 

MMP1 promotor at position -1607 creates two 

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

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

MMP3 also has a functional variation at -1612 

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

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

wide range of extracellular matrix proteins and 

should be regarded as a potential interest in DNA 

polymorphism- TMJ studies. 

Of the big family of MMPs, MMP1 and MMP3 

are synthesized and exocytosed in fibroblasts and 

chondrocytes, increasing the possibility of their 

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

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

MMP3 polymorphisms in TMJ formation. 


Study Group 

35 TMJ patients and 50 healthy subjects with no 

history of any genetically based disease were 

enrolled to the study. All the participants were 

informed in detail about the study. The participants 

were unrelated Turkish people from the eastern 

region of Turkey. The TMJ degeneration group 

included 30 individuals of both genders and 

undergone MRI scans in Marmara University, 

Faculty of Dentistry, Department of Oral and 

Maxillofacial Surgery. The individuals who have 

shown at least one sign of degenerative process in 

any of the mandibular condyles were included to 

the TMJ group, according to their MRI scans. 

Amplification and Genotyping of SNPs 

Genomic DNA isolated from peripheral blood by 

using a commercial kit (Roche, Germany) by 

following the instruction guide. For MMP1 

analysis, PCR reactions were carried out in a 

volume of 50 µl containing 50–100 ng DNA 

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

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

Taq DNA polymerase (Fermentas, Germany) and 

1.0 mM of each primer previously described 

(Rutter 98). The conditions of PCR amplification 

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

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

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

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

PCR reactions were carried out in a volume of 50

µl containing 50–100 ng DNA template in 10 mM 

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

mM each of dNTPs, 1.0 U Taq DNA polymerase 

(Fermentas, Germany) and 1.0 mM of each primer 

previously described (Ye et al., 1996) The 

conditions of PCR amplification were as follows: 

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

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

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

4˚C. 

In order to complete genotype analyses, amplicons 

MMP1 and MMP3 promotor polymorphisms in TMJ disorder 65 

of MMP1 and MMP3 were digested by XmnI (New 

England Biolabs) and Tth111I (New England 

Biolabs) restriction enzymes, respectively, as 

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

allele of MMP1 was digested to 89 and 29 bp 

whereas 2G allele was not digested and remained in 

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

MMP3 yielded 97 and 33 bp products by digestion, 

6A allele was not digested and maintained the 

original length of 130 bp. All the visualization 

processes were carried out by 12% polyacrylamide 

gel stained with etidium bromide (Figure 1) 

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

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

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

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

Statistical analysis 

Statistical analysis for identifying the differences 

between TMJ and control groups was performed 

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

was accepted as statistically significant whereas 

values higher than 0,05 was not. 

Results 

For the MMP1 genotypes, 8 of the 35 patients and 

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

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

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

polymorphisms and p- values were summarized in 

Table 1. When we compare two groups for MMP1 

genotypes, we could not find any statistically 

significant association, all of the p- values are 

0,5%. 

For the MMP3 genotypes, of the 50 controls, 8 

were 5A/5A, 23 were 5A/6A and 19 were 6A/6A, 

respectively. 6 of the 35 patients were 5A/5A, 14 

were 5A/6A and 15 were 6A/6A. Genotypes of 

MMP1 polymorphisms were summarized in Table 

2. Like in MMP1 genotypes, we could not find any 

statistically significant association in patient and 

control group, the p- values are 0,5%.


Discussion 

Table 1. Genotype distribution and OR of MMP1 polymorphism in control and TMJ groups 

Genotypes 

Control Group 

(n=50) 

TMJ Group 

(n=35) 

p- value 

OR (95% CI) 

Control/TMJ Group 

1G/1G 11 8 0,56 0,952 (0,338-2,678) 

1G/2G 28 14 0,14 1,909 (0,794-4,589) 

2G/2G 11 13 0,1 0,541 (0,206-1,422) 

Table 2. Genotype distribution and OR of MMP3 polymorphism in control and TMJ groups 

Genotypes 

Control Group 

(n=50) 

TMJ Group 

(n=35) 

p- value 

OR (95% CI) 

Control/TMJ Group 

5A/5A 8 6 0,889 0,921 (0,289-2,935) 

5A/6A 23 14 0,659 1,278 (0,532-3,067) 

6A/6A 19 15 0,653 0,817 (0,339-1,970) 

MMPs are secreted by pro- enzymes and activated 

after the removal of their N- terminal domain 

(Visse and Negase, 2003). For MMP1 and MMP3 

enzymes, there is an additional activation process, 

transcriptional regulation. Promoter 1G/2G 

polymorphism at the -1607 position of MMP1 gene 

and 5A/6A promoter polymorphism of MMP3 were 

reported to influence gene expression levels of 

related MMPs, and associate these SNPs with 

degenerative diseases (Ye et al., 1996; Barlas et al., 

2009; Astolfi et al., 2006). 

Rutter et al. reported that 2G allele of 

MMP1gene increases the gene expression and 

mRNA levels of MMP1 gene (Rutter et al., 1998). 

This allele creates a binding site for Ets 

transcription factor family, which promotes 37- 

fold in vitro increase of transcription activity. Also 

another study, in transgenic mice, showed the in 

vivo effect of 2G allele on transcription activity 

(Coon et al., 2009). 

We could not observe a significant difference in 

1G/2G SNP of MMP1 gene between TMJ patients 

and control group. Our findings are in accordance 

with the study reporting no association between 

MMP1 -1607 polymorphism and rheumatoid 

arthritis progression, degenerative disease including 

systemic joints. Although there are some reports 

indicating the molecular difference of systemic 

joints and TMJ, the exact mechanism of 

degenerative effect, in which MMP1 is in charge, is 

still unclear (Wadhwa and Capila, 2008). There are 

also other studies that our findings are not in 

agreement. These studies reported the association 

of 2G allele and certain types of cancer (Peng et al., 

2010), periodontitis (de Souza et al., 2003) and 

TMJ (Planello et al., 2011). The latter associated 

the 2G allele and TMJ degeneration and showed 

that 2G/2G individuals have a 2,47 time higher 

probability of developing TMJ when compared to 

1G/1G and 1G/2G individuals. At the same study, 

authors couldn’t find any allelic association, 

claiming only 2G/2G individuals are in risk of 

developing TMJ, individuals without 2G allele are 

protected from the disease. Small sample size, 

heterogenetic nature of genetic diseases and 

probable multifactorial background of TMJ may be 

the reasons of our results that are not in agreement 

with previous mentioned studies. 

Like MMP1, MMP3 has an SNP at position - 

1612 which shows variation in enzyme activity. 5A 

allele in this position of the gene has a greater 

activity to 6A allele, in vitro (Ye at al., 1996). 

Allele 5A is associated to increase the risk of acute 

myocardial infarction (Terashima et al., 1999) and 

in another study, 5A allele has been suspected to 

atherosclerosis (Ye, 2000). MMP3 has an 

accumulative activity during the initial phase of 

TMJ, degrades various types of matrix proteins and 

activates other MMPs like MMP1, MMP8 and 

MMP13, leading to an increase in tissue 

degradation (Planello et al., 2011; Fujita et al., 

2009). We couldn’t find any association with the 

polymorphism and TMJ, like Planello et al (2011). 

The exact activity of MMP3 in tissue degradation is 

not clear and may be the SNP examined has a 

limited influence on the enzyme activity.

There are number of factors which affect 

unexpected results on polymorphism- disease 

association studies. Ethnicities of the races, 

different allele frequencies between populations, 

classifications of patient groups are only some of 

them. Also the metabolism or the activity processes 

of the examined molecules are important. In our 

study, MMP1 and MMP3, they are regulated not 

only by transcription, but also activation of the pro- 

enzyme and inhibitions of enzymes by their tissue 

inhibitors (TIMPs) (Astolfi et al., 2006). There are 

other factors that influence secretion, cell surface 

localization and clearance of the degraded MMPs. 

These regulation factors may help us to explain 

why our results didn’t support the information on 

the literature. Astolfi et al. (2006) also supported 

the idea that increase in mRNA levels may not 

necessarily lead to increased levels of MMPS. 

References 

Astolfi CM, Shinohara AL, da Silva RA, Santos 

MC, Line SR, de Souza AP. Genetic 

polymorphisms in the MMP-1 and MMP-3 gene 

may contribute to chronic periodontitis in a 

Brazilian population. J Clin Periodontol. 33: 

699–703, 2006. 

Barlas IO, Sezgin M, Erdal ME, Sahin G, Ankarali 

HC, Altintas ZM, Turkmen E. Association of (- 

1,607) 1G/2G polymorphism of matrix 

metalloproteinase-1 gene with knee 

osteoarthritis in the Turkish population (knee 

osteoarthritis and MMPs gene polymorphisms). 

Rheumatol Int. 29: 383– 388, 2009. 

Coon CI, Fiering S, Gaudet J, Wyatt CA, 

Brinckerhoff CE. Site controlled transgenic 

mice validating increased expression from 

human matrix metalloproteinase (MMP-1) 

promoter due to a naturally occurring SNP. 

Matrix Biol. 28: 425–431, 2009. 

De Maat MP, Jukema J, Ye S. Effect of 

stromelysin-1 promotor on efficacy of 

pravastatin in coronary atherosclerosis and 

restenosis. Am. J. of Cardiology. 83:852-856, 

1999. 

de Souza AP, Trevilatto PC, Scarel-Caminaga RM, 

Brito RB, Line SR. MMP-1 promoter 

polymorphism: association with chronic 

periodontitis severity in a Brazilian population. 

J Clin Periodontol. 30: 154–158, 2003. 

MMP1 and MMP3 promotor polymorphisms in TMJ disorder 67 

Dijkgraaf LC, de Bont LG, Boering G, Liem RS. 

The structure, biochemistry, and metabolism of 

osteoarthritic cartilage: a review of the 

literature. J Oral Maxillofac Surg. 53: 1182– 

1192, 1995. 

Dworkin SF, Tuner JA, Mancl L, Wilson L, 

Massoth D, Huggins KH, Le Reshe L, Truelove 

E. A randomized clinical trial of a tailored 

comprehensive care treatment program for 

temporomandibular disorders. J Orafac 

Pain.16:259–276, 2002. 

Fujita H, Morisugi T, Tanaka Y, Kawakami T, 

Kirita T, Yoshimura Y. MMP-3 activation is a 

hallmark indicating an early change in TMJ 

disorders, and is related to nitration. Int J Oral 

Maxillofac Surg. 38: 70–78, 2009. 

Kubota E, Kubota T, Matsumoto J, Shibata T, 

Murakami KI. Synovial fluid cytokines and 

proteinases as markers of temporomandibular 

joint disease. J Oral Maxillofac Surg. 56: 192– 

198, 1998. 

Peng B, Cao L, Wang W, Xian L, Jiang D, Zhao J, 

Zhang Z, Wang X, Yu L. Polymorphisms in the 

promoter regions of matrix metalloproteinases 1 

and 3 and cancer risk: a meta- analysis of 50 

case-control studies. Mutagenesis. 25: 41–48, 

2010. 

Planello AC, Campos MIG, Meloto CB, Secolin R, 

Rizatti-Barbosa CM, Line SRP, de Souza AP. 

Association of matrix metalloproteinase gene 

polymorphism with temporomandibular joint 

degeneration. Eur J Oral Sci. 119: 1–6, 2011. 

Rutter JL, Mitchell TI, Buttice G, Meyers J, 

Gusella JF, Ozelius LJ, Brinckerhoff CE. A 

single nucleotide polymorphism in the matrix 

metalloproteinase-1 promoter creates an Ets 

binding site and augments transcription. Cancer 

Res. 58: 5321–5325, 1998. 

Srinivas R, Sorsa T, Tjaderhane L, Niemi E, 

Raustia A, Pernu H, Teronen O, Salo T. Matrix 

metalloproteinases in mild and severe 

temporomandibular joint internal derangement 

synovial fluid. Oral Surg Oral Med Oral Pathol 

Oral Radiol Endod. 91: 517–525, 2001. 

Stegenga B. Osteoarthritis of the 

temporomandibular joint organ and its 

relationship to disc displacement. J Orofac 

Pain. 15: 193–205, 2001. 

Terashima M, Akita H, Kanazawa K, Inoue N,


Yamada S, Ito K, Matsuda Y, Takai E, Iwai C, 

Kurogane H, Yoshida Y, Yokoyama M. 

Stromelysin promoter 5A/6A polymorphism is 

associated with acute myocardial infarction. 

Circulation. 99: 2717–2719, 1999. 

Visse R, Nagase H. Matrix metalloproteinases and 

tissue inhibitors of metalloproteinases: 

structure, function, and bio-chemistry. Circ Res. 

92: 827–839, 2003. 

Wadhwa S, Kapila S. TMJ disorders: future 

innovations in diagnostics and therapeutics. J 

Dent Educ. 72: 930–947, 2008. 

Ye S, Eriksson P, Hamsten A, Kurkinen M, 

Humphries SE, Henney AM. Progression of 

coronary atherosclerosis is associated with a 

common genetic variant of the human 

stromelysin-1 promoter which results in reduced 

gene expression. J Biol Chem. 271: 13055– 

13060, 1996. 

Ye S. Polymorphism in matrix metalloproteinase 

gene promoters: implication in regulation of 

gene expression and susceptibility of various 

diseases. Matrix Biol. 19: 623– 629, 2000.

Journal of Cell and Molecular Biology 9(1):69-74, 2011 Research Article 69 



Lead induced alterations in blood cell counts and hemoglobin 

during gestation and lactation in Swiss albino mice 

Isha BARBER, Ragini SHARMA*, Sheetal MOGRA, Khushbu PANWAR and Umesh 

GARU 

Environmental and Developmental Toxicology Research Lab, Department of Zoology, M. L. S. University, 

Udaipur- 313001 Rajasthan (INDIA) 

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

Received: 31 March 2011; Accepted: 20 June 2011 

Abstract 

Although a number of studies in animal models have shown changes in hemoglobin content and blood cell 

counts after lead administration during gestation and lactation, lead induced hematological changes are not 

well established. In the present study, hemoglobin content and blood cells of normal and lead exposed 

pregnant and lactating Swiss mice were compared in which selected pregnant females were treated with lead 

acetate by gavages (266.66, 533.33, 1066.66 mg/kg BW) during gestation and lactation. Animals were 

exposed to heavy metal lead orally from day 10 of gestation to 3 rd weeks of lactation. Hemoglobin content 

and blood cell counts were examined on 17 th day of gestation and on 1 st , 7 th , 14 th and 21 st day after birth. The 

results indicated that in lead intoxicated pregnant females, hemoglobin content and red blood cell (RBC) 

counts decreased and white blood cell (WBC) counts increased. From the results of above study it can be 

concluded that high levels of lead exposure during gestation and lactation can severely damage heme 

synthesis and alter the number of RBC and WBC. 

Keywords: Lead acetate, Swiss albino mice, hemoglobin, RBC, WBC. 

Swiss albino farelerde gebelik ve süt verme süresince kurşun ile tetiklenen 

hemoglobin ve kan hücreleri sayılarındaki değişimler 

Özet 

Pek çok hayvan modeli çalışmalarının gebelik ve süt verme boyunca kurşun yüklemesi sonrasında kan 

hücrelerinin sayıları ve hemoglobin miktarlarındaki değişiklikleri göstermesine rağmen, kurşun ile tetiklenen 

hematolojik değişiklikler iyi belirlenememiştir. Bu çalışmada normal ve kurşuna maruz kalan hamile ve süt 

veren Swiss farelerin hemoglobin miktarı ve kan hücreleri gebelik ve emzirme sırasında gavajla kurşun asetat 

(266.66, 533.33, 1066.66 mg/kg BW) uygulanan seçilmiş hamile dişilerinkiyle karşılaştırılmıştır. Hayvanlar 

gebeliğin 1. gününden süt vermenin 3. haftasına kadar oral olarak ağır metal kurşuna maruz bırakılmıştır. 

Hemoglobin miktarı ve kan hücresi sayıları gebeliğin 17. günü ve doğumdan sonraki 1., 7., 14., ve 21. 

günlerde incelenmiştir. Sonuçlar, kurşun ile zehirlenmiş gebe dişilerde hemoglobin miktarının ve kırmızı kan 

hücresi (RBC) sayılarının azaldığını ve beyaz kan hücrelerinin (WBC) arttığını göstermiştir. Yukarıda 

belirtilen sonuçlardan, gebelik ve süt verme sırasında yüksek miktarda kurşuna maruz kalmanın hem grubu 

sentezine ciddi zarar verebileceği ve RBC ve WBC sayılarını değiştireceği sonucuna varılabilir. 

Anahtar Sözcükler: Kurşun asetat, Swiss albino fare, hemoglobin, RBC, WBC.

70 Isha BARBER et al. 

Introduction 

In the modern society, thousands of hazardous 

chemicals and heavy metals are being produced and 

used in a wide variety of work places all over the 

world. Heavy metals are trace metals that are at 

least five times denser than water and are taken into 

body via inhalation, ingestion and skin absorption. 

It should be noted that most of the pathological 

conditions in body arise as a result of the exposure 

to these injurious substances. 

Lead and other heavy metals create reactive 

radicals which damage cell structure including 

DNA and cell membrane (Flora et al., 2008). Lead 

poisoning can cause a variety of symptoms and 

signs which vary depending on the individual and 

the duration of lead exposure (Karri et al., 2008; 

Kosnett, 2005). Gestational lead exposure has many 

adverse effects on development; a few of them may 

be most pronounced during the first trimester 

(Mogra et al., 2009) 

Many investigators studied the lower as well as 

higher exposure levels to lead. According to 

Escribano et al., (1997) 140 mg/ kg BW dose was 

an approximate environmental daily-exposure 

level. This dose was used as the minimum dose. 

Gurber et al. (1997) studied the higher 

concentration of lead that was 1000 mg/kg BW 

exposure in industrial areas. 

The amount of lead in blood and tissues, as well 

as the time course of exposure, determines the level 

of toxicity (Pearson and Schonfeld, 2003). Blood 

often shows pathological changes before the 

external signs of poisoning become apparent. 

The absorbed lead enters the blood stream 

where over 90 percent of it is bound to the red cells 

with a biological half life of 25-28 days (Azar et 

al., 1975). Toxicological effects of lead have their 

origin in perturbation in cell function of various 

organ systems. The major biochemical effect of 

lead is its interference with heme synthesis which 

leads to hematological damage (Awad and William, 

1997). Despite several published accounts on 

pathophysiological alterations of lead toxicity and 

the cure of lead poisoning by sequestering agents 

(Royce and Rosenberg, 1993), the approaches are 

limited in scope. Therefore the present 

investigation was focused to evaluate the changes 

in the number of blood cells and Hb content during 

pregnancy and lactation followed by comparison of 

this data with the high levels of lead intoxication 

during gestation and lactation. 


Random breed Swiss albino mice were used for the 

present study. Sexually mature male and females 

weighing 28-30 gm were put in breeding cages in 

the ratio of 1:4 and provided standard diet and 

water ad libitum. The cages were checked every 

day in the morning and females showing vaginal 

plug were isolated. The selected pregnant females 

were divided into 4 groups and exposed orally by 

lead acetate (266.66, 533.33 and 1066.66 mg/kg 

BW) with the help of canula. All the experimental 

work was approved by the institutional animal 

ethics committee (No.CS/Res/07/759). 

(1) Group 1- Control (distilled water only) 

(2) Group 2- Exposure of lead acetate (266.66 

mg/kg BW) from 10 th day of gestation up to 21 st 

day of lactation. 







During the respective tenure of experiment, 

hemoglobin, RBC and WBC counts of female 

Swiss mice were recorded on 17 th day of gestation 

and on 1 st , 7 th , 14 th and 21 st days of lactation. Blood 

samples for hemoglobin and blood cell counts were 

obtained from the tail of each mouse. The tip of the 

tail was cleaned with spirit before being cut with a 

sharp blade and was not squeezed to avoid dilution 

of blood by tissue fluid. The first few drops of 

blood were discarded and the blood was diluted, for 

cell counting with the help of haemocytometer. The 

hemoglobin was estimated by hemoglobinometer. 

Number of (RBCs) and (WBCs) were estimated 

with haemocytometer adopting the method 

described by Dacie and Lewis (1975). The 

statistical analysis was performed by using analysis 

of variance (ANOVA) for the comparison of data 

between different experimental groups. 

Results 

Introducing lead acetate to female Swiss mice 

during gestation and lactation period produced a 

significant decrease in the hemoglobin content and 

number of RBC. Exposure during the gestation 

period produced a significant decrease in WBC 

counts at the time of birth whereas number of cells 

increase during lactation period.

Data regarding changes in hemoglobin, RBC 

and WBC counts among different experimental 

groups and the results obtained for hemoglobin, 

RBC and WBC are summarized in Tables 1-3. It is 

evident from the tables that control animals have 

12.5 g/dL Hb, 9.21×10 6 RBC/mm 3 and 8.22×10 3 

WBC/mm 3 in their blood on 17 th day of gestation. 

As compared to controls, hemoglobin content, RBC 

and WBC counts of lead- treated female mice were 

significantly decreased at the time of birth, after 

introduction of 266.66, 533.33 and 1066.66 mg/kg 

BW of lead acetate. 

Lead induced hematological changes 71 

The results collected for the hemoglobin is 

summarized in Table 1. 

It is evident that control animals have 11.5, 

12.0, 13.1 and 14.0 g/dL Hb in their blood on 1 st , 

7 th , 14 th and 21 st day of lactation, respectively. 

Introduction of 266.66, 533.33 and 1066.66mg/kg 

BW doses of lead acetate produced a significant 

decrease (p


Table 3. WBC count (×10 3 cells/mm 3 ) in pregnant and lactating female mice treated with lead acetate. 

Treatment group 

Prenatal 

Days 

Postnatal 

17 1 7 14 21 

Control 8.22±3.92 8.07±3.30 8.33±4.30 8.56±3.56 8.86±3.47 

Lead acetate (266.66 mg/kg BW) 8.62±5.00* 8.48±1.79* 8.70±5.77 8.71±7.50 9.18±1.25 

Lead acetate (533.33 mg/kg BW) 8.98±9.46* 8.62±2.95* 9.10±1.47* 9.20±2.04* 9.31±2.09* 

Lead acetate (1066.66 mg/kg BW) 9.26±1.70* 9.00±1.73* 9.17±8.66* 9.35±1.68* 9.46±1.43* 

Values were expressed as means ± S.D., values are significantly different (p

elated to major structural and functional changes 

of the intimate relation between fetus and mother. 

These structural changes are related to implantation 

and placentation; and the functional changes to 

increasing hypoxia in the crowded condition of the 

gravid uterus (Robert and Rosenberg, 1993). 

In conclusion, the present study indicates that 

lead adversely affects hemoglobin and blood cell 

counts. Dose dependent significant decrease in 

hemoglobin and RBC counts were observed after 

administration of 266.66, 533.33 and 1066.66 

mg/kg/body weight doses of lead acetate. However, 

WBC counts showed increase in their number. 

The results observed in this paper can be used 

as a background information for the evaluation of 

reproductive toxicity induced by lead during 

gestation and lactation in Swiss mice. Moreover, 

the mice seem to be a useful animal model for 

investigating the mechanisms of alterations in the 

blood cells and hemoglobin content which is 

reported to occur during pregnancy and lactation in 

mice. 

References 

Angle CR, McIntire MS and Swanson MS. 

Erythrocyte nucleotides in children increased 

blood lead and cytidine triphosphate. Pediatr 

Res 16:331-334, 1982. 

Awad M and William Jr. Textbook of biochemistry 

with clinical correlation. John Wiley and 

Sons, INC, Newyork. 1997. 

Azar A, Snee RD, Habibi K. Lead toxicity in 

mammalian blood. In: Griffin TB, Knelson 

JH (Ed) Lead. Academic, Newyork. 254, 

1975. 

Banu R, Sharma R and Qureshi N. Amelioration of 

lead induced alterations in body weight and 

blood cell counts by antioxidant vitamins. 

Journal of Herbal Medicine and Toxicology 

1(2): 59-66, 2007. 

Dacie JV and Lewis SM. In: Practical Hematology, 

5th Edn. J. and A. Churchill Ltd. London, 

1975. 

De Rijk EP, van Esch E and Flik G. Pregnancy 

dating in the rat: placental morphology and 

maternal blood parameters. Toxicol Pathol 

30:271–282, 2002. 

Lead induced hematological changes 73 

EPA, U.S. Air Quality Criteria for Lead. EPA 

600/8-83-028F. Washington, DC: U.S. 

Environmental Protection Agency, 1986. 

Escribano,A.,Revilla, M., Herna’ndez, E.R., Seco, 

C., Gonza’lez-Riola, J., Villa, L.F. and Rico, 

H. Effect of lead on bone development and 

bone mass: a morphometric, densitometric, 

and histomorphometric study in growing 

rats. Calcif Tissue Int. 60: 200-203, 1997. 

Flora Sj, Mittal M and Mehta A. Heavy metal 

induced oxidative stress & its possible 

reversal by chelation therapy. The Indian 

Journal of Medical Research 128 (4): 501– 

23, 2008. 

Fruhman GJ. Blood formation in the pregnant 

mouse. Blood J. Hematol. 31: 242-248, 

1968. 

Gruber, H.E., Gonic, H.C., Khalil-Manesh, F., 

Sanchez, T.V., Matsinger, S., Meyer, M. and 

Sharp, C.F. Osteopenia induced by longterm, 

low and high-level exposure of the 

adult rat of lead. Miner. Electrolyte Metab., 

23, 65-33,1997. 

Harkness JE and Wagner JE. Biologia e clínica de 

coelhos e roedores. 3ª ed. São Paulo: Roca, 

1993. 

Howells MR, Jones SE, Napier JA, Saunders K and 

Cavill I. Erythropoiesis in pregnancy. Br J 

Haematol 64:595–599, 1986. 

Karri Sk, Saper Rb and Kales Sn. Lead 

encephalopathy due to traditional medicines. 

Current Drug Safety 3 (1): 54–9, 2008. 

Kosnett MJ. Lead. In Brent, J. Critical Care 

Toxicology: Diagnosis and Management of 

the Critically Poisoned Patient. Gulf 

Professional Publishing. 2005. 

Mercelina-Roumans PE, Ubachs JM and van 

Wersch JW. Leukocyte count and leukocyte 

differential in smoking and non-smoking 

females during pregnancy. Eur J Obstet 

Gynecol Reprod Biol 55:169–173,1994. 

Mogra S, Sharma R and Qureshi N. Effects of 

maternal lead acetate exposure on prenatal 

development of Swiss albino mice. Asian J 

Environ. Sci. 4(2):216-220, 2009. 

Paglia DE, Valentine WN and Dahigren JG. Effects 

of low-level lead exposure on pyrimidine 5'nucleotidase 

and other erythrocyte enzymes.


Possible role of pyrimidine 5'-nucleotidase 

in the pathogenesis of lead-induced anemia. 

J Clin Invest 56:1164-1169, 1975. 

Paglia DE, Valentine WN and Fink K. Lead 

poisoning. Further observations on 

erythrocyte pyrimidine-nucleotidase 

deficiency and intracellular accumulation of 

pyrimidine nucleotides. J Clin Invest. 

60:1362-1366, 1977. 

Paprika MV and Sharma BB. Journal of Cell and 

Tissue Research 3(1):12-17, 2003. 

Pearson HA, and Schonfeld DJ.. Lead. In Rudolph, 

C.D.. Rudolph'sPediatrics, 21st edition. 

McGraw-Hill Professional. 2003 

Robbins SL and Angell M. In: Basic pathology, 

2nd Edn. W.B. Saunders Co. Philadelphia, 

3-30, 1976. 

Robert R and Csilla S. The effect of pregnancy on 

peripheral blood in the mouse. Biol. Bull., 

136: 454-460, 1969. 

Royce S and Rosenberg J. Chelation therapy in 

workers with lead exposure. Western J Med 

158: 371-375, 1993. 

Rugh R and Somogyi C. Pre-and postnatal normal 

mouse blood cell counts. Proc Soc Exp Biol 

Med 127, 1267-1271, 1968. 

Yu MH. Soil and water pollution: Environmental 

metals and metalloids. Environmental 

Toxicology: Biological and Health Effects of 

Pollutants. CRC Press. 2005. 

Zarrow MX. Gestation, In: Edgar Allen, Ed., Sex 

and Internal Secretion, Volume II. Williams 

and Wilkins Co., Baltimore. 958-1031, 1961.




The effects of aqueous and ethanolic leaf extracts of Vernonia 

amygdalina on some vital organs in adult Wistar rats 

Spencer NWANGWU 1 , David Adesanya OFUSORI 2,* , Sunday JOSIAH 1 , Osasere Frank 

AMEGOR 3 , Helen NJOYA 1 and Abiodun Oladele AYOKA 4 

1 Department of Biochemistry, School of Basic Medical Sciences, Igbinedion University, Okada, Edo-State, 

Nigeria 

2 Department of Anatomy and Cell Biology, Faculty of Basic Medical Sciences, Obafemi Awolowo University, 

Ile-Ife, Osun-State, Nigeria 

3 Department of Medical Laboratory Science, College of Health Sciences, Igbinedion University, Okada, Edo 

State, Nigeria 

4 Department of Physiological Sciences, Faculty of Basic Medical Sciences, Obafemi Awolowo University, 

Ile-Ife, Osun-State, Nigeria 

(* author for correspondence; davidofus234@yahoo.com 

Received: 15 February 2011; Accepted: 20 June 2011 

Abstract 

The aim of this study was to comparatively determine the effects of aqueous and ethanolic extracts of 

Vernonia amygdalina (Asteraceae) leaves, adopting histological procedures on the liver, stomach and kidney 

in adult wistar rats.There were four groups of animals: A, B, C and D. Groups A, B and C served as the 

treated animals while group D served as the control animals. Each of the groups was further subdivided into 

two i.e A1 & A2; B1 & B2; C1 & C2 and D1 & D2. Animals in groups A1, B1 and C1 were administered 

orally with aqueous extract of V. amygdalina 100, 200 and 300 mg/kg respectively while animals in groups 

A2, B2 and C2 were administered orally with ethanolic extract of V. amygdalina 100, 200 and 300 mg/kg 

respectively. The control groups, D1 and D2, received equal volume of normal saline. There were no 

significant derangement in the cytoarchitecture of the stomach, liver and kidney. Rather, the cytoarchitecture 

of the animals treated with both the ethanolic and aqueous extracts (300mg/kg) of V. amygdalina were better 

organized when compared with the control. It was therefore concluded that both ethanolic and aqueous leaf 

extracts of V. amygdalina are non-toxic and may possess cytoprotective potential. 

Keywords: Vernonia amygdalina, histological procedures, liver, stomach, kidney 

Vernonia amygdalina yaprağının sulu ve etanolik özütlerinin erişkin Wistar 

sıçanlarının bazı hayati organlarına etkileri 

Özet 

Çalışmanın amacı, erişkin Wistar sıçanlarda Vernonia amygdalina (Asteraceae) yapraklarının sulu ve 

etanolik özütlerinin karaciğer, mide ve böbrek üzerindeki etkilerini histolojik yöntemlerle karşılaştırmalı 

olarak belirlemektir. Çalışmada dört grup hayvan bulunmaktadır: A, B, C ve D. D grubu kontrol hayvanları 

olarak belirlenirken; A, B ve C grupları muamele edilmiş hayvanları göstermektedir. Her bir grup ayrıca iki 

alt gruba bölünmüştür; A1 & A2; B1 & B2; C1 & C2, D1 & D2. A1, B1 ve C1 gruplarındaki hayvanlara 

sırasıyla 100, 200 ve 300 mg/kg V.amygdalina sulu özütü oral olarak uygulanırken A2, B2 ve C2’ye ise 

sırasıyla 100, 200 ve 300 mg/kg V.amygdalina etanolik özütü oral olarak uygulanmıştır. Kontrol grupları 

olan D1 ve D2 eşit hacimde normal tuz almışlardır. Mide, karaciğer ve böbreğin hücresel yapısında belirgin 

bir bozulma bulunmamıştır. Bilakis hem sulu, hem de etanolik V. amygdalina (300mg/kg) özütü uygulanan

76 Spencer NWANGWU et al. 

hayvanların hücresel yapıları kontrollerle karşılaştırıldığında daha iyi organizedir. Bu nedenle sulu ve 

etanolik V. amygdalina yaprak özütlerinin her ikisinin de toksik olmadığı ve hücre koruyucu potansiyele 

sahip olabileceği sonucuna varılmıştır. 

Anahtar Sözcükler: Vernonia amygdalina, histolojik süreçler, karaciğer, mide, böbrek. 

Introduction 

Vernonia amygdalina (Del. Asteraceae) 

commonly known as “bitter leaf” is a valuable 

shrub that is widespread in East and West Africa. 

It is 2-5 m tall with petiolate green leaves of about 

6 mm diameter (Ojiako and Nwanjo, 2006). In 

Nigeria the stem is used as chew-sticks, while the 

leaves are being used as a popular vegetable for 

soups particularly among the Igbos of Southern 

Nigeria, Africa (Ojiako and Nwanjo, 2006). Its 

medicinal values for fever, laxative, pile 

(haemorrhoids) and gastro-intestinal troubles have 

been investigated and reported by the following 

authors: (Oliver, 1960; Ainslie, 1973; Kupcham, 

1971; Akah and Okafor, 1992; Abosi and 

Raseroka, 2003; Huffman, 2003; Izevbigie et al., 

2004). In fact all parts of the plant have been 

known to be pharmacologically useful. Oral 

administration of the aqueous leaf extract of the 

plant was found to relieve pain (Tekobo et al., 

2002) and to lower body temperature (Tekobo et 

al., 2002). Atangwho et al. (2010) reported 

instantaneous reduction of blood glucose and a 

variation in blood glucose similar to that of 

insulin-treated rats in animals administered with 

ethanol extract of V. amygdalina. 

Nutritionally, V. amygdalina is used mainly in 

soup making in the tropics and also as an 

appetizer and febrifuge (Ijeh et al., 1996; Iwu, 

1996, Ojiako and Nwanjo, 2006) and has proven 

to be a successful supplement in weaning foods 

(Ojiako and Nwanjo, 2006). In Nigeria, as in 

other tropical countries of Africa where the daily 

diet is dominated by starchy staple foods, 

vegetables are the cheapest and most readily 

available sources of important proteins, vitamins, 

minerals and essential amino acids (Okafor, 1983, 

Ojiako and Nwanjo, 2006). The importance of V. 

amygdalina in animal nutrition in Nigeria has also 

been well documented (Onwuka et al., 1989; 

Aregheore et al., 1998; Ojiako and Nwanjo 2006). 

Despite these beneficial uses of the plant, there 

has been conflicting reports on its exact 

toxicological potentials on some visceral organs. 

For instance Aregheore et al., (1998) reported the 

presence of toxic phytochemicals. There are also 

reports of actual hepatotoxicity in mice (Igile et 

al., 1995), also, there was a report on 

hepatoprotective effects in rats (Babalola et al., 

2001). Ojiako and Nwanjo (2006) reported that V. 

amygdalina leaves may be toxic (just like several 

other vegetables) if consumed in very large 

quantities but the potential danger is not higher 

than has been observed for other common 

vegetables that are routinely consumed in Africa 

in even larger quantities. 

In view of these conflicting reports, we 

therefore set to comparatively determine the 

effects of aqueous and ethanolic leaf extracts of V. 

amygdalina, adopting histological procedures on 

the stomach, liver and kidney in adult wistar rats. 


Experimental Site 

This research was conducted in the Department of 

Biochemistry, School of Basic Medical Sciences, 

Igbinedion University, Nigeria. 

Experimental Animals 

A total of forty healthy wistar rats obtained from a 

private farm in Benin City were used for the 

experiment. These animals were acclimatized for 

two weeks before the commencement of the 

study. The animals were treated in accordance 

with the “Guide for the Care and Use of 

Laboratory Animals” prepared by the National 

Academy of Sciences and published by the 

National Institutes of Health (NIH, 1985) . 

Experimental Design 

There were four groups of animals: A, B, C and 

D. Groups A, B and C served as the treated 

animals while group D served as the control 

animals. Each of the groups (n=10) was further 

subdivided into two i.e A: A1 & A2; B: B1 & B2;

C: C1 & C2 and D: D1 & D2. Animals in groups 

A1, B1 and C1 were administered orally with 

aqueous extract of V. amygdalina 100, 200 and 

300 mg/kg respectively while animals in groups 

A2, B2 and C2 were administered orally with 

ethanolic extract of V. amygdalina 100, 200 and 

300 mg/kg respectively. The control groups D1 

and D2 received equal volume of normal saline. 

Collection of plant materials 

Fresh but matured leaves of Vernonia amygdalina 

was procured from a local market in Okada, Edostate. 

They were authenticated in the Botany 

Department, Igbinedion University, Nigeria. The 

leaves were rinsed severally with clean tap water 

to remove dust particles and debris and thereafter 

allowed to completely drain. 

Preparation of plant extracts 

Plant materials were separately chopped into bits 

with a knife on a chopping board. The leaves 

were then air dried and one kilogram (1kg) V. 

amygdalina was reduced to powder with an 

electric blender. The powder was divided into two 

portions. One of the portions was percolated with 

80% ethanol while the other was percolated with 

distilled water. The mixtures were allowed for 48 

h in the refrigerator at 4 0 C for thorough extraction 

of the plants active components. These were then 

filtered with cheesecloth and later with Whatman 

No. 1 filter paper to obtain a homogenous filtrate. 

These filtrates were then concentrated in vacuo at 

low temperature (37- 40 0 C) to about one tenth the 

original volume using a rotary evaporator. The 

concentrates were allowed open in a water bath 

(40 0 C) for complete dryness for both ethanol and 

aqueous extracts of V. amygdalina. The extracts 

(26.7% yield) were then refrigerated at 2- 8 0 C 

until use. 

Histological Procedure 

Histological study was carried out using the 

method of Carleton (1967). These procedures 

Effects of Vernonia amygdalina extracts on vital organs 77 

involved dehydration of the liver, kidney and 

spleen tissues with graded ethanol concentrations 

(50%, 70%, 90% and 100%, respectively), 

clearing in xylene, followed by infiltration in 

paraffin wax for 2 h at 56 o C and embedding in 

paraffin wax for 48 h. Sections (5 μm thick) were 

then obtained, using a rotary microtome, 

subjected to haematoxylin and eosin (H & E) 

staining procedure and examined under a light 

microscope. Permanent photomicrographs of the 

observations were taken, using an Olympus 

Research Microscope (model BX51). 

Results 

There were no significant derangement in the 

cytoarchitecture of the stomach, liver and kidney. 

Rather, the cytoarchitecture of the animals treated 

with both the ethanolic and aqueous extract 

(300mg/kg) of V. amygdalina were better 

organized when compared with the control 

(Figures 1-3). In the stomachs of treated animals, 

there were no disruptions of surface epithelium 

and no presence of submucosal edema as well as 

leucocytes infiltration as compared with control 

groups (Figure 1). There were no hypertrophy of 

the liver and no necrosis of the hepatocytes in the 

treated animals as compared with control groups 

(Figure 2). Also, the kidneys of animals in the 

treated groups presented well preserved renal 

corpuscles and Bowman’s spaces as compared 

with control groups (Figure 3). Animals in both 

the treated and control groups showed no physical 

changes in their appearances. The present study 

demonstrated that oral administration of both 

ethanolic and aqueous extracts (300 mg kg -1 ) 

preserved the hepatic, renal and gastric integrity 

as compared to animals administered with lesser 

dose (200 and 100 mg kg -1 ) and only distilled 

water (Figures 1-3). There were no significant 

differences between the cytoprotective abilities of 

the animals treated with ethanolic or aqueous 

extracts compared to the animals treated with 

control.


A1 A2 

C1 

B1 B2 

Figure 1. Histological section of gastric mucosa treated with A: aqueous extract V. amygdalina (A1-100, B1- 

200, C1-300mg/kg); B: ethanolic extract of V. amygdalina (A2-100, B2-200, C2-300mg/kg). Note that there 

were no disruptions of surface epithelium. No presence of submucosal edema and leucocytes infiltration as 

compared with control groups D1 and D2 (H and E stain, 100x). 

C2 

D1 D2


Figure 2. Histological section of hepatic parenchyma treated with A: aqueous extract V. amygdalina (A1- 

100, B1-200, C1-300mg/kg); B: ethanolic extract of V. amygdalina (A2-100, B2-200, C2-300mg/kg). Note 

that there were no hypertrophy of the liver and no necrosis of the hepatocytes as compared with control 

groups D1 and D2 (H and E stain, 100x).


Figure 3. Histological section of renal parenchyma treated with A: aqueous extract V. amygdalina (A1-100, 

B1-200, C1-300mg/kg); B: ethanolic extract of V. amygdalina (A2-100, B2-200, C2-300mg/kg). Note that 

the renal corpuscles and Bowman’s spaces were well preserved. Also, there were no evidence of cellular 

necrosis as compared with control groups D1 and D2 (H and E stain, 100x). 

Discussion 

The histopathological changes observed in the 

liver, kidney, stomach of aqueous and ethanolic 

extracts of V. amygdalina on rats showed no 

evidence of lesions in the treated animals when 

compared with control. Our histological findings 

further revealed that the cyto-architecture of the 

liver parenchyma was better organized in the 

groups treated with 300mg/kg of both ethanolic and 

aqueous extracts of V. amygdalina. The functions 

of the liver which includes detoxification, 

metabolic activities, synthesis of plasma protein 

and destruction of spent red blood cells among 

other functions will be better enhanced based on

histological evidences from our study. This shows 

that administration of ethanolic and aqueous 

extracts of V. amygdalina provides a protective role 

for the liver which is the first organ susceptible to 

any injurious substances in case of toxicity. This is 

in line with previous work (Ofusori et al. 2008) 

who investigated the effect of Croton zambesicus 

(euphorbiaceae) on the liver. The renal parenchyma 

showed no evidence of vacuolations or distortion of 

any kind rather, renal corpuscles and Bowman’s 

spaces were well preserved as compared with 

control groups in all the experimental groups. It 

was evident on the photomicrograph that the 

tubules constitute the bulk of the renal parenchyma 

with different shapes, diameters and staining 

intensities. This points to the fact that V. 

amygdalina may have a vital role to play in 

osmoregulation and excretion. Also, the gastric 

mucosa of the treated groups were better organized 

when compared with the control (Figure 1). There 

was no evidence of ulceration in the surface 

epithelium, no presence of submucosal edema and 

leucocytes infiltration. Studies have shown that 

anti-oxidants significantly strengthen the gastric 

walls and protect tissue from oxidative damage 

(Marins, 1996). 

All these evidences revealed that V. amygdalina 

is a very important plant that can be exploited for 

cyto-protective purposes. The cyto-protective 

mechanism may be by moping up free radical there 

by protecting the cells of the organs from any type 

of assaults. Several other studies have shown that 

the aqueous and ethanolic leaf extracts of V. 

amygdalina is non-hazardous, even when taken for 

some time. The potency of these extracts may have 

been amplified on account of increased 

concentration or build up of specific 

phytochemicals, although the chemistry is yet 

unknown. Several findings showed that V. 

amygdalina has strong antioxidant activity 

corresponding to mitigation of the generation of 

hydroxyl radicals. Yeh et al., (2003) and Battell et 

al., (1999) postulated that this antioxidant activity 

may provide possible rationale for the observed 

therapeutic effects of V. amygdalina. 

Based on the results obtained, it can be 

concluded that ethanolic and aqueous leaf extracts 

of V. amygdalina are non toxic and may possess 

cytoprotective potential. 


References 

Abosi AO and Raseroka BH. In vivo antimalarial 

activity of Vernonia amygdalina. Br. J. 

Biomedical Sci. 60(2): 89-91, 2003. 

Ainslie JR. List of plants used in native medicine in 

Nigeria. Imperial Forestry Institute, Oxford 

Publishing. 42, 1973. 

Akah PA and Okafor CI. Hypoglycaemic effect of 

Vernonia amygdalina Del, in experimental 

rabbits. Plant Med. Res. 1: 6-10, 1992. 

Aregheore EMK, Makkar HPS and Becker K. Feed 

value of some browse plants from the central 

zone of Delta State, Nig. Trop. Sci. 38(2): 97- 

104, 1998. 

Babalola OO, Anetor JI and Adeniyi FA. 

Amelioration of carbon tetrachloride-induced 

hepatotoxicity by terpenoid extract from leaves 

of Vernonia amydgalina. Afr. J. Med. Sci. 30 (1- 

2): 91-93, 2001. 

Battell ML, Rodrigues B, Yuen VG and McNeill 

JH. Treatment and pharmacological 

interventions in streptozotocin diabetes. In: 

Experimental Models of Diabetes. (McNeill, J. 

H., Ed.), pp.195-216. CRC Press LLC, U.S.A, 

1999. 

Huffman MA. Animal self-medication and ethnomedicine: 

exploration and exploitation of the 

medicinal properties of plants. Proceedings of 

the Nutr. Society. 62(2):371-81, 2003. 

Igile GO, Oleszek W, Jurzysta M, Burda S, Fafunso 

M and Fasanmade AA. Nutritional assesment of 

Vernonia amygdalina leaves in growing mice. 

J. Agric. Food Chem. 43, 2162-2166, 1995. 

Ijeh I, Nwugo VO and Obidoa O. Comparative 

studies on the nutritive Phyto-chemical and 

antimicrobial properties of two varieties of 

Vernomia amygdalina. Plant Pros. Res. Comm. 

1: 71-75, 1996. 

Izevbigie EB, Bryant JL and Walker A. A novel 

natural inhibitor of extracellular signalregulated 

kinases and human breast cancer cell 

growth. Experimental Biol. Med. (Maywood) 

229(2): 163-169, 2004. 

Iwu MM, Okunji E, Akah PA, Tempesta MS, and 

Carley DG. Dioscoretine: The hypoglycaemic


principles of Dioscorea dumentorium. Plant 

Med. 56: 119-126, 1996. 

Kupcham SM. Drugs from Natural products. Plant 

source in drugs discovery, science and 

development. Am. Chem. Soc. 6: 311-318, 1971. 

Martin A. The use of antioxidants in healing. 

Dermatol. Surg., 22: 156-160, 1996. 

Ofusori DA, Adelakun AE and Ayoka AO. 

Histological Investigation of the Effects of 

Croton zambesicus on the Liver of Swiss albino 

mice. The Int. J. Gastroenterology. (7)1: 2008. 

Ojiako OA and Nwanjo H. Is Vernonia amygdalina 

hepatotoxic or hepatoprotective? Response from 

biochemical and toxicity studies in rats. Afr J. 

Biotech. 5 (18): 1648-1651, 2006. 

Okafor JC. Horticulturally promising indigenous 

wild plant species of the Nigerian forest zone. 

Acta Hort. 123: 165-176, 1983. 

Oliver B. Medicinal plants in Nigeria. Nig. Coll. Sci 

Techn. 50-51, 1960. 

Onwuka CFI, Akinsoyinu AO and Tewe OO. Feed 

value of some Nigerian browse plants: chemical 

composition and in vitro digestibility. East 

African Agri. Forestry J. 54: 157 – 163, 1989. 

Tekobo AM, Onabanjo AO, Amole OO and Emeka 

PM . Analgesic and antipyretic effects of the 

aqueous extract of Vernonia amygdalina. West 

Afri. J. Pharm. 16: 68-74, 2002. 

Yeh GY, Eisenberg DM, Kaptcuk TJ and Philips 

RS. System review of herbs and dietary 

supplements for glycemic control in diabetes. 

Diab. Care 26 (4), 1277 – 1294, 2003.

Journal of Cell and Molecular Biology 9(1): 83-86, 2011 Short communication 83 



Investigation of the EGFR gene variations in bladder cancer 

patients using INFINITI TM Analyzer 

Necip Ozan TİRYAKİOĞLU 1 , Özlem KURNAZ 1 , Ömer Onur ÇAKIR 2 , Nagehan ERSOY 

TUNALI 1,* 

1 

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

Turkey 

2 

T.R. Ministry of Health, Bağcılar Training and Research Hospital, Istanbul, Turkey 

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


Abstract 

Dysregulation of epidermal growth factor receptor (EGFR), which leads to increased proliferation, is a 

common event in most cancers. Since its recognition as a keyplayer in carcinogenesis, several anti-EGFR 

agents have been developed as chemotherapeutic agents. Studies investigating the effect of EGFR mutations 

on the activity of anti-EGFR agents resulted in the discovery of a number of cancer tissue-specific mutations 

which either increase or decrease sensitivity to tyrosine kinase inhibitors (TKI). Determination of these 

mutations is essential to prognostic processes, but is limited to tissue availability. Being able to determine 

tumour specific EGFR mutations by using blood sample as the genotyping material will eliminate the need 

for invasive techniques. This study is the first step of a wider research with the final goal of matching 

mutations in tumour tissues with that of normal somatic cells from the same individuals. All the known exon 

18-21 mutations were screened in 48 bladder cancer patients with INFINITI TM analyzer using leukocytes as 

the DNA source. However, none of the investigated mutations have been detected in the genomic DNA 

samples of bladder cancer patients. 

Keywords: INFINITI TM , epidermal growth factor receptor, tyrosine kinase inhibitor, drug sensitivity, bladder 

cancer. 

Mesane kanseri hastalarında EGFR geni varyantlarının INFINITI TM ile incelenmesi 

Özet 

Artmış çoğalmaya sebep olan epidermal büyüme faktörü reseptörünün (EGFR) düzenlenmesindeki 

bozukluklara çoğu kanser türünde rastlanır. Karsinogenezde oynadığı kilit rol farkedildiğinden beri anti- 

EGFR etkili bir çok kemoterapötik ajan geliştirilmiştir. EGFR mutasyonlarının anti-EGFR ajanlarının 

aktivitesine etkisinin incelendiği araştırmalarda tirozin kinaz inhibitörüne hassasiyetini artıran veya azaltan 

bir çok kanser dokusuna özgün mutasyon belirlenmiştir. Bu mutasyonların belirlenmesi tedavi aşamasının 

temelini oluşturur, fakat mutasyonların belirlenmesi kanser dokusuna erişebilirlik ile sınırlıdır. Tümör 

spesifik EGFR mutasyonlarının genotipleme aracı olarak kan örnekleri kullanılarak belirlenebilmesi invaziv 

tekniklere olan gerekliliği ortadan kaldıracaktır. Bu çalışma nihai olarak olarak tümör dokusu 

mutasyonlarının normal somatik hücre mutasyonları ile eşlenmesinin amaçlandığı daha geniş bir araştırmanın 

ilk aşaması olarak gerçekleştirilmiştir. Bilinen tüm exon 18-21 mutasyonları 48 mesane kanseri hastasında 

INFINITI TM cihazı ile DNA kaynağı olarak lökositler kullanılarak taranmıştır. Ancak, araştırılan hiçbir 

mutasyon mesane kanseri hastalarının genomik DNA’larında belirlenememiştir. 

Anahtar Sözcükler: INFINITI TM , epidermal büyüme faktörü reseptörü, tirozin kinaz inhibitörü, ilaç 

hassasiyeti, mesane kanseri.

84 Necip Ozan TİRYAKİOĞLU et al. 

Introduction 

Epidermal growth factor receptor (EGFR) is a cell 

surface receptor that activates signalling cascades 

which mainly induce proliferation and cell survival. 

Human genome codes for four members of the 

EGFR family, EGFR/ErbB1, HER2/ErbB2/neu-2, 

HER3/ErbB3 and HER4/ErbB4. All the family 

members consists of a ligand-binding, a 

transmembrane and a cytoplasmic domain with 

tyrosine kinase activity (Gschwind et al., 2004). 

EGFR members reside on the cell membrane as 

monomers which dimerize upon phosphorylation 

by ligand binding. Eight different molecules have 

been identified as EGFR/ErbB1 ligands. All the 

EGFR ligands, except Crypto, are synthesized as 

precursor molecules and become functional only 

after proteolytic clevage. In addition, EGFR family 

members have been shown to be activated 

indirectly by chemokines, cell adhesion molecules 

and G-protein coupled receptors (Yarden and 

Sliwkowski, 2001). The dimerisation of EGFR 

members can occur as homodimerisation or as 

heterodimerisation with another member of the 

family. Following dimerisation, 

autophosphorylation in homodimers and 

transphosphorylation in heterodimers occurs. 

During this process five tyrosine residues, Y992, 

Y1045, Y1068,Y1148 and Y1173, located on Cterminal 

are phosphorylated. Consequently, EGFRs 

activate a multitude of transducer and adapter 

proteins including HP-2, GRB2, PI3K and Akt. 

Activation of these proteins induce a variety of 

cytoplasmic pathways, mainly MAPK and JNK, 

which trigger cellular events like DNA synthesis 

and proliferation (Oda et al., 2005). 

Dysregulation of EGFRs are mainly caused by 

overexpression of the receptor itself, their ligands 

or both. In addition to overexpression, mutations in 

the tyrosine kinase domain causing consistent 

activation of the receptor account for another major 

mechanism affecting EGFR signalisation. Such 

mutations have been identified in lung, stomach 

and breast cancer tissues, but not in normal tissues. 

Dysregulation of EGFR can also be the result of 

HER2/ErB2 heterodimerisation. HER2 does not 

have ligand binding activity, instead it increases 

the ligand binding affinity of the protein it 

dimerises with. Thus, EGFR/HER2 dimerisation 

leads to a more active receptor and increased 

activity of the EGFR pathways (Herbst, 2004). 

Upon discovery of disturbed EGFR functions in 

multiple cancers, abundant number of studies have 

been conducted in order to develop drugs targeting 

EGFR. Inhibitory molecules targeting EGFR can be 

classified in three main categories; monoclonal 

antibodies, chemical inhibitors and antisense 

oligonucleotides. 

Monoclonal antibodies exert their inhibitory 

effect by binding to the ligand binding domain and 

disrupt activation upon ligand binding. Monoclonal 

antibodies against EGFR have been showed to 

inhibit cell growth and induce apoptosis. However, 

since they exert their effect only on ligand binding 

domain, EGFRs with tyrosine domain mutations 

are prone to be irresponsive to the inhibitory effects 

of monoclonal antibodies. In contrast to 

monoclonal antibodies, chemical inhibitors target 

tyrosine kinase domain and inhibit EGFR by 

blocking ATP from interacting with the tyrosine 

kinase domain. Antisense nucleotides, on the other 

hand, prevent translation of the EGFRs and their 

ligands and carry out their inhibitory effect at a 

much essential level. 

90% of the somatic EGFR mutations are located 

in the tyrosine kinase domain, namely exons 18-21 

(Table 1). Since tumor cells depend on the 

activating mutations on tyrosine kinase domain of 

EGFR for proliferative signals, it has been shown 

that tumor cells harboring such mutations are more 

sensitive to thymidine kinase inhibitors (TKI). 

Thus, determination of the EGFR mutations has 

both prognostic value as an indicator of drug 

response and diagnostic value as a biomarker 

(Lynch et al. , 2004). 

Since the determination of EGFR mutations 

requires tumour samples and collecting a tumour 

sample is only possible through invasive 

techniques, EGFR mutation surveys are limited to 

tissue availability. Being able to scan for the EGFR 

mutations by using whole blood as an alternative 

DNA source may provide information about the 

genotype of the original tumour tissue without the 

need for invasive techniques. Therefore, the study 

presented here is initiated as the first step of a wider 

research with the final goal of matching mutations 

in tumour tissues with that of normal somatic cells 

from the same individuals. 

Table 1. Common EGFR Mutations 

Mutations Frequency Effect 

G719A/C/S 3% Increased sensitivity 

Ex 19 Deletion 48% Increased sensitivity 

Ex20 Deletion 4% Decreased sensitivity 

T790M 5% Decreased sensitivity 

L858R 43% Increased sensitivity 

L861Q 2% Increased sensitivity

EGFR variations in bladder cancer 85 


Ethical Committee for Clinical Research. DNA was 

As the first step, genomic DNA extracted from the 

leukocytes of 48 bladder cancer patients have been 

surveyed for the total of 50 mutations identified to 

date in exons 18-21 of the EGFR gene. The 

selected study population consisted of 40 males and 

8 females with bladder cancer. Tumour tissue and 

blood samples were obtained from each induvidial 

and categorised according to their tumour stage and 

grade. Tumour samples were collected in tubes 

containing RNase Later (Invitrogen) solution, while 

blood samples were collected in tubes containing 

K2EDTA. Ethical approval for his study was 

obtained from Istanbul University Medical Faculty, 

extracted from blood samples by using QiaAmp 

DNA Blood Mini kit (Qiagen) as described in the 

package insert. 

Extracted DNA samples were surveyed for a 

total of 50 mutations in exons 18-21 by 

INFINITI Analyzer (Table 2). INFINITI 

Analyzer has different protocols for different 

assays, but the five essential steps are common to 

all; 1) multiplex PCR amplification of DNA, 2) 

fluorescent label incorporation using analyte 

specific primer extension (ASPE), 3) hybridization 

of the ASPE primers to a microarray followed by 

washing, 4) scanning of the microarray and 5) 

signal detection and analysis. 

Table 2. Scanned EGFR Mutations 

Exon 18 Exon 19 Exon 20 Exon 21 

Glu709 Lys Lys739_Ile744dup Glu762insEAFQ Asn826Ser 

Glu709 Gln Lys745_Glu746del Glu762ins His835Leu 

Glu709 Ala Glu746_Ala750del Val769Met Leu858Arg 

Glu709 Gly Glu746_Thr751delins Val769Leu Leu858Met 

Glu709 Val Glu746_Thr751delins Asp770ins Leu858Arg 

Gly719 Ser Glu746_Ala750delins Ala767_Val769dup Leu861Gln 

Gly719 Arg Glu746_Ala750del Asp770fs Leu861Arg 

Gly719 Cys Glu746 Lys Pro772Arg 

Gly719 Ala Glu746_Ala750delins Ser768_Asp770dup 

Ser720 Phe Glu746_Pro753delins His773Arg 

Glu746_Ser752del His773Leu 

Glu746_Ser752delins Asn771_His773dup 

Glu746_Thr751delins Val774Met 

Glu746_Thr751delins Arg776Cys 

Glu746_Ser752delins Gly779Phe 

Glu746_Thr751delins Thr790Met 

Glu746_Ser752delins 

Leu747_Glu749del 

Leu747_ Thr751delins 

Leu747_Ser752del 

Leu747_Ser752delins 

Leu747_Pro753delins 

Glu746_Glu749del 

Leu747_Glu749del 

Glu746_Thr751del 

Leu747_ Thr751del 

Leu747_ Thr751delins 

Leu747_ Lys754delins 

Leu747_Pro753delins 

Leu747_Ala750del 

Leu747_ Thr751del 

Arg748_Thr751delins 

Glu749_Thr751del 

Thr751_Ile759delins 

Ser752_Ile759del

86 Ozan TİRYAKİOĞLU et al. 

Results 

INFINITI Analyzer detects EGFR mutations 

according to the ratio of the specific fluorescence 

signal emitted from the analyte, represented in 

Relative Fluorescence Unit (RFU), to the 

fluorescence signal of the non-specific 

oligonucleotide background control spots. A ratio 

of 2 and higher indicates the existence of the 

particular EGFR mutation and a ratio lower than 2 

indicates the absence of the particular EGFR 

mutation. 

According to specific RFUs for each mutation, 

none of the 48 DNA samples extracted from blood 

carry inherited EGFR mutations within the tyrosine 

kinase domain (exons 18-21) of EGFR. 

Discussion 

The absence of any mutations in the surveyed 

specimens might be attributed to the rarity of EGFR 

mutations in bladder cancer cases as shown by 

other studies before (Blehm et al., 2006; Villares et 

al., 2007). However, it might also arise from the 

fact that using whole blood as a source of 

information for tumour genotype is an inaccurate 

method. The underlying reason can only be 

identified upon the completion of the second step of 

the study, which will provide us the genotype data 

obtained directly from tumour tissues and thus 

enable to perform a cross-comparison between 

tumour tissue and leukocyte genotype. 

Acknowledgements 

This study was supported by Haliç University and 

CDK Tıbbi Malzeme Tic. Ltd. Şti. (Istanbul, 

TURKEY). 

References 

Gschwind A, Fischer OM and Ullrich A. The 

discovery of receptor tyrosine kinases: targets 

for cancer therapy. Nat Rev Cancer. 4 . 361– 

370, 2004. 

Blehm KN, Spiess PE, Bondaruk JE, Dujka ME, 

Villares GJ, Zhao YJ, Bogler O,Aldape KD, 

Grossman HB, Adam L, McConkey DJ, 

Czerniak BA, Dinney CP and Bar-Eli M. 

Mutations within the kinase domain and 

truncations of the epidermal growth factor 

receptor are rare events in bladder cancer: 

implications for therapy. Clin Cancer Res. 

12(15):4671-7, 2006. 

Herbst RS. Review of epidermal growth factor 

receptor biology. International Journal of 

Radiation Oncology .59(2): S21-S26, 2004. 

Oda K, Matsuoka Y, Funahashi A and Kitano H. 

Comprehensive pathway map of epidermal 

growth factor receptor signaling. Mol Syst Biol. 

1: 20050010, Epub 2005, May25. 

Lynch TJ, Bell DW, Sordella R, Gurubhagavatula 

S, Okimoto RA, Brannigan BW,Harris PL, 

Haserlat SM, Supko JG, Haluska FG, Louis 

DN, Christiani DC, Settleman J and Haber DA. 

Activating mutations in the epidermal growth 

factor receptor underlying responsiveness of 

non-small-cell lung cancer to gefitinib. N Engl J 

Med. 350(21):2129-39, 2004. 

Villares GJ, Zigler M, Blehm K, Bogdan C, 

McConkey D, Colin D, Bar-Eli M. Targeting 

EGFR in bladder cancer.. EGFR in bladder 

cancer. World J Urol. 25(6):573-9. 2007 

Yarden Y and Sliwkowski MX. Untangling the 

ErbB signalling network. Nat Rev Mol Cell 

Biol. 2(2):127-37,2001.

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Redford IR. Evidence for a general relationship 

between the induced level of DNA double 

strand breakage and cell killing after Xirradiation 

of mammalian cells. Int J Radiat 

Biol. 49: 611- 620, 1986. 

Tccioli CE, Cottlieb TM, Blund T. Product of the 

XRCCS gene and its role in DNA repair and 

V(D)J recombination. Science. 265: 1442-1445, 

1994 

Ohlrogge JB. Biochemistry of plant acyl carrier 

proteins. The Biochemistry of Plants: A 

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EE (Ed). Academic Press, New York. 137-157, 

1987. 

Brown LA. How to cope with your supervisor. PhD 

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Web document with no author: Leafy seadragons 

and weedy seadragons 2001. retrieved 

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windspeed.net.au/jenny/seadragons/ 

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Volume 9 · No 1 · June 2011 

Review Articles 


Decision making mechanism influences the regulatory function of Notch in Apoptosis 

A.NAIR 

Polymorphisms in the xenobiotic genes and susceptibility to bladder cancer 

N. ERSOY TUNALI and N. O. TİRYAKİOĞLU 

Research Articles 


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

Identification of a novel dehydration responsive transcript from tossa jute (Corcohrus olitorius L.) 

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

Protective effects of curcumin on cadmium chloride induced colon toxicity in Swiss albino mice 

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

In vitro regeneration of Cleome viscosa – an important medicinal herb 

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

Bio-database compression using enhanced suffix array for pairwise sequence alignment 

A.KUNTHAVAI and S.VASANTHA RATHNA 

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

the recombinant enzyme 

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

Investigation of the MMP1 and MMP3 promoter polymorphisms in temporomandibular joint 

disorder 

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

Lead induced alterations in blood cell counts and hemoglobin during gestation and lactation in Swiss 

albino mice 

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

The effects of aqueous and ethanolic leaf extracts of Vernonia amygdalina on some vital organs in 

adult Wistar rats 

S. NWANGWU, D. A. OFUSORI, S. JOSIAH, O. F. AMEGOR, H. NJOYA and A. O. OYAKA 

Short Communication 

Investigation of the EGFR gene variations in bladder cancer patients using INFINITI TM Analyzer 

N. O. TİRYAKİOĞLU, Ö. KURNAZ, Ö. O. ÇAKIR and N. ERSOY TUNALI 

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