Journal of Cell and Molecular Biology - ResearchGate

jcmb.halic.edu.tr

Journal of Cell and Molecular Biology - ResearchGate

Journal of Cell and

Molecular Biology

•Circadian rhythm genes in cancer

Tunneling nanotubes

Genetic screening of Turkish barley genotypes

Strontium ranelate induces genotoxicity

Volume 9 · No 2 · December 2011

http://jcmb.halic.edu.tr


Journal of Cell and

Molecular Biology

Volume 9 · Number 2

December 2011

İstanbul-TURKEY


Haliç University

Faculty of Arts and Sciences

Journal of Cell and Molecular Biology

Founder

Gündüz GEDİKOĞLU

President of Board of Trustee

Rights held by

A. Sait SEVGENER

Rector

Correspondence Address:

Journal of Cell and Molecular Biology

Haliç Üniversitesi

Fen-Edebiyat Fakültesi,

Sıracevizler Cad. No:29 Bomonti 34381 Şişli

İstanbul-Turkey

Phone: +90 212 343 08 87

Fax: +90 212 231 06 31

E-mail: jcmb@halic.edu.tr

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Editor-in-Chief

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Editorial Board

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Baki

ÖZDİLLİ

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A.Meriç ALTINÖZ, Istanbul, Turkey

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Şehnaz BOLKENT, İstanbul, Turkey

Nihat BOZCUK, Ankara, Turkey

A. Nur BUYRU, İstanbul, Turkey

Kemal BÜYÜKGÜZEL, Zonguldak, Turkey

Hande ÇAĞLAYAN, İstanbul, Turkey

İsmail ÇAKMAK, İstanbul, Turkey

Ayla ÇELİK, Mersin, Turkey

Adile ÇEVİKBAŞ, İstanbul, Turkey

Beyazıt ÇIRAKOĞLU, İstanbul, Turkey

Fevzi DALDAL, Pennsylvania, USA

Zihni DEMİRBAĞ, Trabzon, Turkey

Gizem DİNLER DOĞANAY, İstanbul, Turkey

Mustafa DJAMGÖZ, London, UK

Aglika EDREVA, Sofia, Bulgaria

Ünal EGELİ, Bursa, Turkey

Anne FRARY, İzmir, Turkey

Hande GÜRER ORHAN, İzmir, Turkey

Nermin GÖZÜKIRMIZI, İstanbul, Turkey

Ferruh ÖZCAN, İstanbul, Turkey

Asım KADIOĞLU, Trabzon, Turkey

Maria V. KALEVITCH, Pennsylvania, USA

Nevin Gül KARAGÜLER, İstanbul, Turkey

Valentine KEFELİ, Pennsylvania, USA

Meral KENCE, Ankara, Turkey

Fatma Neşe KÖK, İstanbul, Turkey

Uğur ÖZBEK, İstanbul, Turkey

Ayşe ÖZDEMİR, İstanbul, Turkey

Pınar SAİP, Istanbul, TURKEY

Sevtap SAVAŞ, Toronto, Canada

Müge TÜRET SAYAR, İstanbul, Turkey

İsmail TÜRKAN, İzmir, Turkey

Mehmet TOPAKTAŞ, Adana, Turkey

Meral ÜNAL, İstanbul, Turkey

İlhan YAYLIM ERALTAN, İstanbul, Turkey

Selma YILMAZER, İstanbul, Turkey

Ziya ZİYLAN, İstanbul, Turkey


Journal of Cell and Molecular Biology

CONTENTS

Volume 9 · Number 2 · December 2011

Review Articles

The role of circadian rhythm genes in cancer

Kanserde sirkadiyan ritim genlerinin rolü

H. ATMACA and S. UZUNOĞLU

Tunneling nanotubes – Crossing the bridge

M. McGOWAN

Research Articles

Genetic screening of Turkish barley genotypes using simple sequence

repeat markers

H. SİPAHİ

Strontium ranelate induces genotoxicity in bone marrow and peripheral

blood upon acute and chronic treatment

A. ÇELİK, S. YALIN, Ö. SAĞIR, Ü. ÇÖMELEKOĞLU and D. EKE

Cloning, expression, purification, and quantification of the 17% Nterminal

domain of apolipoprotein b-100

H. M. KHACHFE and D. ATKINSON

Cysteine protease from the malaria parasite, Plasmodium berghei-

purification and biochemical characterization

E. AMLABU, A. J. NOK, H. M. INUWA, B. C. AKIN-OSANAIYE and E.

HARUNA

Optimization of cellulase enzyme production from corn cobs using

Alternaria alternata by solid state fermentation

A. IJAZ, Z. ANWAR , Y. ZAFAR , I. HUSSAIN, A. MUHAMMAD, M.

IRSHAD and S. MEHMOOD

Guidelines for Authors

Front cover image: “The DNA puzzle”, Shutterstock image ID: 1144448

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11

19

27

37

43

51

57


Journal of Cell and Molecular Biology 9(2):1-10, 2011 Review Article 1

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Kanserde sirkadiyan ritim genlerinin rolü

The role of circadian rhythm genes in cancer

Harika ATMACA and Selim UZUNOĞLU *

Celal Bayar University, Faculty of Arts and Sciences, Department of Biology, Manisa, Turkey.

(* author for correspondenceselim@bayar.edu.tr)

Received: 27 October 2011; Accepted: 9 December 2011

Abstract

Circadian (In Latin: Circa=around, Diem=day) rhythm describes the processes of 24 hour oscillations

in the living systems. At the cellular level, circadian rhythm is controlled by a molecular network with

positive and negative feedbacks. The known critical elements in the positive feedback loop are Clock

and Bmal1; the ones in complementary negative feedback are mainly Period and Cryptochrome genes.

In cancer, which is an important health problem today, dysregulation of circadian rhythm is an

important risk factor. In this review, circadian rhythm genes involved in cell proliferation, apoptosis,

DNA repair, metabolism, detoxification and response to DNA damage and their roles in cancer

development are summarized.

Keywords: Circadian rhythm, cancer, period, cryptochrome, molecular clock

Özet

Sirkadiyan (Latince: circa=yaklaşık, diem=gün) ritim canlı sistemlerdeki 24 saatlik dalgalanmalara

maruz olayları tanımlar. Hücresel seviyede bakıldığında sirkadiyan ritim, pozitif ve negatif

geribildirimler içeren moleküler ağ bir tarafından kontrol edilir. Pozitif geribildirim döngüsünde

bilinen kritik elementler Clock ve Bmal1, tamamlayıcı negatif geribildirimde ise Period ve

Cryptochrome genleridir. Günümüzde önemli bir sağlık sorunu olan kanserde sirkadiyan ritmin

bozulması önemli bir risk faktörüdür. Bu derlemede hücre çoğalması, apoptoz, DNA tamiri,

metabolizma, detoksifikasyon ve DNA hasarına cevapla ilişkili sirkadiyan ritim genleri ve kanser

oluşumundaki rolleri özetlenmiştir.

Anahtar Sözcükler: Sirkadiyan ritim, kanser, periyot, kriptokrom, moleküler saat

Kısaltmalar listesi

Clock: Circadian Locomotor Output Cycles Kaput, Bmal1: Brain-muscle-arnt-like 117 protein 1,

CRY: Cryptochrome, PER: Period homolog 1, E-box: Enhancer box, ROR: Retinoid-related orphan

receptor-alpha, REV-ERBα: Nuclear receptor Rev-ErbA-alpha, NONO: Non-POU domain containing,

octamer-binding, DEC: Deleted in esophageal cancer 1, CYP2A5: Cytochrome P450 2A5, CYP2C50:

Cytochrome P450 2C50, CES3: Carboxylesterase 3, MDR1: ATP-binding cassette, sub-family B

(MDR/TAP), member 1, Npas2: Neuronal PAS domain protein 2, Fas: TNF receptor superfamily,

member 6, Bax: BCL2-associated X protein, c-myc: Cell division cycle associated 7, Chk1: CHK1

checkpoint homolog, Chk2: CHK2 checkpoint homolog, Atr1: Ataxia telangiectasia and Rad3 related,

Jak2: Janus kinase 2, ER: Estrogen receptor, Pbef: Pre- B- cell colony- enhancing factor, Akt1: V-akt

murine thymoma viral oncogene homolog 1, Cdk2: Cyclin-dependent kinase 2, TGFβ: Transforming

growth factor, beta, EGF: Epidermal growth factor, CCL5: Chemokine (C-C motif) ligand 5,

BDKRB2: Bradykinin receptor B2, SP100: SP100 nuclear antigen, Wee1: WEE1 homolog, Mdm-2:

Mdm2 p53 binding protein homolog (mouse), Gadd45: Growth arrest and DNA-damage-inducible


2Harika ATMACA and Selim UZUNOĞLU

Giriş

Uzay zamanda gerçekleşen canlılık

fenomenleri, süreç bakımından da kontrole

tabidir. Hücredeki her bir molekül, belirli bir

zamanda sentezlenir, belirli bir süre

fonksiyonunu icra eder ve belli bir sürenin

sonunda da yıkıma maruz kalır. Bu

perspektiften bakıldığında, hücresel olayların

düzenlenmesinde ve kontrolünde zamanlama ve

süreyi kontrol eden genler ve bunların ürünleri

olan proteinler vardır. Zaman ve sürenin ölçüm

ve kontrolünde rol alan biyolojik moleküllerin

ve bunların sentezinden sorumlu genetik

elementlerin anlaşılması kanser başta olmak

üzere birçok hastalığın mekanizmasını

çözümlemede önemlidir.

Zamanlama ve süre perspektifinden canlılık

olayları incelendiğinde, osilasyon, periyot ve

ritimlerin sağlıklı hücresel faaliyetler için kritik

rol oynadığı görülür (Tablo 1).

Tablo 1. Genlerin aktivasyon düzeyleri, süreler, biyolojik fonksiyonlar ve araştırma alanları (Rossi,

2002).

Gen Aktivasyonundaki

Düzeyler

Evrimsel süreçlerde aktif olan

genler

Kalıtım Esnasında aktif olan

genler

Gelişim sırasında aktif olan

genler

Canlılığın devamı için

gerekli/sürekli aktif genler

Sirkadiyan ritime göre aktifleşen

genler

Yaklaşık Süre Temel Fonksiyon Araştırma Alanı

Jeolojik devirler Çeşitliliğin kökeni Evrimsel biyoloji

Nesiller arası

Replikasyon ve

rekombinasyon

Genetik

Bir ömür boyu Büyüme Embriyoloji

Günlük- Haftalık Metabolizma

Sirkadiyan

Sistem fonksiyonlarının

Senkronizasyonu

Fonksiyonel genom

bilimi

Kronobiyoloji

Geç cevapta aktifleşen genler 4-8 saat İmmunite İmmunoloji

Erken cevabın ortalarında aktive

olan genler

Davranışlara cevap olarak aktive

olan genler

Fizyolojik değişikliklere bağlı

olarak aktive olan genler

Erken cevabın başında hızlıca

aktive olan genler

1-2 saat Çevresel cevap

Değişken saatlik

dilimler

Uyanıklık, uyku, ruh

hali

Fiziko-nöroimmunoloji

Psikoloji

Dakikalar-Saatler Hafıza, öğrenme Nöroloji

Saniyeler- Dakikalar Uyarılma, stres Psikobiyoloji


Öyle ki, biyolojik ritimler tek hücreli

organizmalardan memelilere kadar bütün canlılarda

mevcuttur (Waterhouse J, 1999). Örneğin

nörotransmitter ve reseptör sayısındaki

değişiklikler, kan basıncı, vücut sıcaklığındaki

dalgalanmalar, uyku-uyanıklık, hatta DNA

replikasyonunun bile gün içinde değişiklikler

gösteren ritimleri vardır (Waterhouse J, 1999;

Lowrey ve Takahashi, 2004). Bu ritimlerden biri

sirkadiyan ritim (Latince: circa=yaklaşık,

diem=gün) olup, canlılardaki 24 saatlik dilimdeki

dalgalanmalara maruz olayları tanımlar. Sirkadiyan

ritimler hücre, organ, endokrin sistem ve organizma

ölçeğinde gözlenir. Sirkadiyan ritimlerin

kontrolünde hem genetik faktörler hem de çevresel

uyaranlar rol oynar. Çevresel uyaranların

sirkadiyan genlerinin okunmasını nasıl düzenlediği

ise epigenetik faktörler tarafından belirlenir.

Dolayısıyla, hücresel olayların zamanlaması ve

süresinin kontrolünde genetik, epigenetik ve

çevresel uyaranlar birlikte etkileşir.

Sirkadiyan ritmin temel moleküler mekanizması

Memelilerde organizma ölçeğinde gözlenen

sirkadiyan ritmin düzenlenmesinde “Epifiz bezi” ve

“Suprakiazmatik çekirdek” rol oynar (Kondratov ve

ark., 2007). Hücresel ölçekte bakıldığında ise

sirkadiyan ritim, pozitif ve negatif geribildirimler

içeren moleküler ağ bir tarafından kontrol edilir

(Lowrey ve Takahashi, 2004; Ko ve Takahashi,

2006; Son ve ark., 2011). Bu ağın pozitif

geribildirim döngüsündeki moleküler oyuncular

Clock ve Bmal1 isimli transkripsiyon faktörü

kodlayan genlerdir. Her iki transkripsiyon faktörü

de “Basic helix-loop-helix (bHLH)-PAS”

transkripsiyon faktör ailesine aittir. Bunların

özelliği benzer motiflere sahip ortak domeynler

içermeleridir (McGuire ve ark., 1995). Örneğin,

PAS domeyni hem bHLH transkripsiyon

faktörlerinde hem de Drosophila’daki Period isimli

sirkadiyan geninde bulunur. Bu faktörler genellikle

hücre tipi farklılaşmasının düzenlenmesinde ve

çoğalmada görev alan proteinlerin

transkripsiyonundan sorumludur (McGuire ve ark.,

1995). Heterodimer formunda aktifleşen Clock ve

Bmal1 proteinleri, Period ve Cryptochrome gibi

sirkadiyan ritim genlerinin transkripsiyonunu

düzenler. Memelilerde sirkadiyan ritimleri

düzenleyici genlerin transkripsiyonunu

zenginleştirici dizilerden biri E-box cis elementi

olup, Period ve Cryptochrome genlerinin ortak

Sirkadiyan ritim genleri ve kanser 3

özelliği bu diziye sahip olmalarıdır. Clock

ve Bmal1 heterodimeri de bu bölgeye

bağlanarak transkripsiyonu başlatırlar

(Lowrey ve Takahashi, 2004; Ko ve

Takahashi, 2006) (Şekil 1).

Genomun sağlıklı işleyişi ağ tabanlı

moleküler etkileşimlerle düzenlendiğinden,

sirkadiyan ritim genlerinin sentezini kontrol

eden Bmal1 transkripsiyon faktörü de

kontrole tabidir. Bu kontrol retinoik asit

reseptörle ilişkili orphan nükleer reseptörleri

olan ROR ve REV-ERB molekülleri ile

gerçekleştirilir. RORα, Bmal1 geninin

transkripsiyonunu başlatırken, REV-ERB

ise bu transkripsiyonu baskılar (Ko ve

Takahashi, 2006). Diğer bir ifadeyle; ROR

ve REV-ERB molekülleri Bmal1’in

transkripsiyonunu kontrol ederken,

Clock/Bmal1 heterodimeri de bu nükleer

reseptörlerin sentezini kontrol eder (Lowrey

ve Takahashi, 2004; Ko ve Takahashi,

2006).

Bu döngünün negatif geribildirim

oyuncuları ise Period ve Cryptochrome

genleridir. Memelilerde sirkadiyan ritim, bu

genlerin transkripsiyon ve translasyonunun

döngüsel geribildirimi ile düzenlenir. Period

ve Cryptochrome genlerinin

transkripsiyonunu Clock/Bmal1

heterodimeri başlatırken, transkripsiyonunu

kendisi engeller. Açarsak;

Period/Cryptochrome heterodimeri geri

beslemeyle kendi sentezini kontrol eder. Bu

pozitif ve negatif geribildirim

döngülerindeki proteinlerin stabiliteleri ve

nükleer translokasyonları fosforilasyon ve

übikütinlenme işlemleri ile de düzenlenir.

Fosforilasyon işleminde Kazein kinaz 1 ε

(CK1ε), Kazein kinaz 1 δ (CK1δ), NONO

moleküllerinin rol oynadığı gösterilmiştir

(Lowrey ve Takahashi, 2004; Ko ve

Takahashi, 2006) (Şekil 1). Sirkadiyan

osilasyonunun çevrimi, moleküler osilatörler

tarafından transkripsiyon ve translasyonun

otomatik olarak düzenlenmesi neticesinde

gerçekleşir. Bu mekanizmada PER1 ve

PER2 genlerinin promotor bölgesinde

bulunan E-box (CACGT[G/T]) bölgesi

kritik öneme sahiptir. Bmal1 ve Clock

proteinleri kompleks oluşturarak bu bölgeye

bağlanır ve E-box bölgesi içeren PER


4Harika ATMACA and Selim UZUNOĞLU

genlerinin transkripsiyonu ve translasyonu

başlatılır. Sentezlenen PER proteinleri çekirdeğe

aktarılır, burada CRY proteini ile heterodimer

oluşturur. Bu heterodimer ise E-box bölgesine

bağlanarak, transkripsiyonun tekrar başlamasını

engeller. Belli bir süre sonra PER/CRY baskılayıcı

kompleksi yıkılır, Clock/Bmal1 heterodimeri tekrar

E-box bölgesine bağlanır ve transkripsiyon

döngüsü yeniden başlatılır. Yardımcı döngüler

(REV-ERBα, RORα, DEC) de, osilasyon özelliği

gösteren bu temel döngünün düzenlenmesinde rol


oynar (Okamura ve ark., 2010; Son ve ark.,

2011).

Zamanlama ve süreç kontrolü canlılığın

her ölçeğinde hayati bir faktördür.

Zamanlama ve süreç noktasında

gerçekleşecek herhangi bir hata kendi

ölçeğinde sorunlara yol açar. Canlılığın en

temel fonksiyonel birimi olan hücrede ve

çok hücreli organizmalarda sirkadiyan

ritmin bozulması birçok hastalığa zemin

hazırlar.


Şekil 1. Memelilerde saat genlerinin transkripsiyon-translasyonunun döngüsel geri bildirimi.

Günümüzde pek çok insanın ölümüne neden

olan kanserde sirkadiyan ritmin bozulması

önemli bir risk faktörü olmakla beraber,

aralarındaki ilişki net olarak

aydınlatılamamıştır. Hücre çoğalması, apoptoz,

DNA tamiri, metabolizma, detoksifikasyon ve

DNA hasarına cevap gibi hücresel olaylar

sirkadiyan ritim ile kontrol edilir (Mongrain ve

Cermakian, 2009; Rana ve Mahmood, 2010).

İlaçların farmakokinetiği (emilim, dağılım,

metabolizma ve atılım) sirkadiyan saat

tarafından kontrol edilir. Metabolizma ve

detoksifikasyonun ana işlemcisi olan karaciğer,

sirkadiyan ritimler üretebilen bir biyolojik saat

gibidir. Bir çalışmada, sıçan karaciğerinde 3906

genin zamana bağlı ekspresyon profilleri

araştırılmış, 67 genin ekspresyonunun

sirkadiyan ritim gösterdiği bulunmuştur (Ohdo

ve ark., 2011). Sirkadiyan ritim gösteren bu

genlerin transkripsiyonun düzenlenmesinde, ilaç

metabolizmasında, iyon taşınımında, sinyal

iletiminde ve immün cevapta rol alan genler

olduğu gösterilmiştir. Sirkadiyan saat, özellikle

PAR domaini içeren temel lösin fermuar (PAR

bZip) motifli transkripsiyon faktörlerinin

ekspresyon profillerini kontrol eder. Bu

faktörler ise karaciğerde ksenobiyotik

detoksifikasyon sisteminin koordinasyonunda iş

gören enzimlerin (CYP2A5, CYP2C50 ve

CES3) ifadesini düzenler. PAR bZip

transkripsiyon faktörleri mutant olan farelerde

ilaç metabolizmasıyla ilgili enzimlerin

(karboksilesterazlar, sitokrom p450 enzimleri,

glutatyon-s-transferaz enzimleri, p450

oksiredüktaz, sulfotransferazlar gibi)

ekspresyon profillerinin değiştiği gösterilmiştir.

Ayrıca, kanserde çoklu ilaç direncinden

sorumlu P-glikoprotein’in (MDR1a) ifade


profilindeki 24 saatlik değişikliklerin sirkadiyan

saatin moleküler bileşenleri tarafından

düzenlendiği saptanmıştır (Ohdo ve ark., 2011).

Sirkadiyan ritmi düzenleyen genler aynı

zamanda hücre döngüsünde rol alan çeşitli

transkripsiyon faktörlerini, tümör baskılayıcı

genleri ve bazı kaspazların sentezini de kontrol

eder (Rana ve Mahmood, 2010). Bu nedenledir

ki, sirkadiyan ritim genleri hücre çoğalması ve

apoptoz gibi kanserle ilişkili biyolojik olayları

önemli ölçüde etkiler. Ancak moleküler

mekanizmaları detaylı olarak açıklanamamıştır.

Kanserle ilişkili sirkadiyan ritim genleri

Kanserle ilişkili sirkadiyan ritim genleri son

yıllarda tanımlanmaya başlanmıştır (Tablo 2).

Bu genlerden yoğun olarak araştırılan bazılarına

kısaca değinilecektir.

Clock geni

Clock (Circadian Locomotor Output Cycles

Kaput), memelilerde tanımlanan ilk sirkadiyan

ritim genidir (Sehgal, 2004). Clock proteini,

Bmal1 ile dimer formu oluşturduğunda E-box

düzenleyici elementlerine bağlanarak, hedef

genlerin ifadesini artırır (Ko ve Takahashi,

2006; Mongrain ve Cermakian, 2009).

Clock geninin susturulduğu deneysel

çalışmalarda yapılan mikroarray analizleri,

kanserli dokularda karsinogenez ile ilişkili pek

çok molekülde değişiklikler olduğunu ortaya

koymuştur. Bu nedenle Clock geninin onkojenik

karaktere sahip olduğu belirtilmiştir (Sehgal,

2004, Rana ve Mahmood, 2010).

Clock geni uğramış mutasyona farelerle

yapılan çalışmalarda, hücre döngüsü inhibitörü

genlerinin (p21, p27, Chk1, Chk2 ve Atr1)

transkripsiyonunda yüksek düzeyde artış,

proliferasyonda rol oynayanlarda ise (Jak2, ER,

Pbef, Akt1, Cdk2, cyclin D3 ve cyclin E1,

TGFβ, EGF) azalış anlamlı tespit edilmiştir

(Miller ve ark., 2006). Bu veriler Clock geninin

hücre döngüsündeki önemini ortaya

koymaktadır.

441 meme kanseri hastasının doku

örnekleriyle yapılan bir mikroarray

çalışmasında, hücre döngüsü düzenlenmesi ve

meme kanseri progresyonu ile bağlantılı bir gen

olan CCL5’in transkripsiyonunda 2.9 artış kat

bulunmuştur. Epiteliyal meme hücrelerinin

çoğalmasını indükleyen BDKRB2 geninin

transkripsiyonunda 2.1 kat, metastazın


Sirkadiyan ritim genleri ve kanser 5

indüklenmesi, meme kanseri progresyonu ve

kötü prognozla bağlantılı SP100 geninin

transkripsiyonunda 2.3 kat azalma görülmüştür.

Clock geninin kontrol grubuna göre daha fazla

ifade edildiği, östrojen/progesteron reseptör

negatif grupta, pozitif olanlara göre ifadesinin

daha fazla olduğu gösterilmiştir. Ayrıca,

hipermetilasyonla Clock geninin

transkripsiyonunun engellenmesi ile meme

kanseri progresyonundaki yavaşlama arasında

bağlantı bulunmuştur (Hoffman ve ark., 2010a).

Literatürdeki çalışmalarda meme kanseri

(Zhu ve ark., 2005), prostat kanseri (Chu ve

ark., 2008) ve non-Hodgkin lenfoma (Hoffman

ve ark., 2009) örneklerinde sirkadiyan

genlerindeki varyasyonlar gösterilmiştir. Zhu ve

ark. prostat kanserinin malinyitesiyle Clock

geninin intronundaki tek nükleotid polimorfizmi

(rs11133373) arasındaki ilişkiyi göstermiştir

(Zhu ve ark., 2009).

Bmal1 geni

Clock/Bmal1 heterodimeri G2 fazından M

fazına geçişte rol alan Wee1 geninin ifadesini

düzenler. Bunun yanında, Cyclin D1 (G1 den S

fazına geçişte aktif) ve c-Myc (G0 dan G1 fazına

geçişte aktif) genlerinin transkripsiyonunda da

doğrudan etkili olduğu tespit edilmiştir (Zeng

ve ark., 2010; Rana ve Mahmood, 2010). Bmal1

geni inaktif olan insan hücrelerinin DNA hasarı

sonucu aktiflenen p53 mekanizması üzerinden

ölüme gidemediği ve buna bağlı olarak hücre

çoğalmasının durdurulamadığı gösterilmiştir.

Fareler üzerinde yapılan in vivo çalışmalarda,

p21 ekspresyonunun artışına bağlı olarak G1

fazında Bmal1’e bağlı gecikme belirlenmiştir.

Bu verinin aksine, Bmal1 geni olmayan insan

hücrelerinde radyasyonla uyarılan çoğalmanın

engellenmesinin, p53 ve p21 seviyelerindeki

azalmayla uyumlu olduğu gösterilmiştir. Bu

çelişkili bulgular, türler arası varyasyondan

veya in vivo/in vitro deney koşullarından

kaynaklanabilir (Rana ve Mahmood, 2010). Bu

bulgular, sirkadiyan ritim genlerinden olan

Bmal1’in hücre döngüsünün kontrolünde görev

aldığını gösterse de, karsinogenez ile ilişkisinin

daha detaylı çalışmalarla ortaya konmasına

ihtiyaç vardır.

Period genleri

Period geni ilk defa 1971 yılında Konopka ve

Benzer tarafından Drospohila’da tanımlanmıştır


6Harika ATMACA and Selim UZUNOĞLU

(Sehgal, 2004). Daha sonra memelilerde de bu

genin homologu olan üç tane Period geni

(PER1, PER2 ve PER3) tanımlanmıştır. Bu

genlerin hücre çoğalmasında rol oynadıkları ve

tümör baskılayıcı özellik gösterdikleri tespit

edilmiştir (Hua ve ark., 2006; Goodspeed ve

Lee, 2007). PER2 geninin insan meme sağlıklı

epiteliyal hücre kültürlerinde ifade edildiği

ancak meme kanseri hücre kültürlerinde

ifadesinin düşük olduğu belirlenmiştir. Meme

kanserlerinde PER2 geni aktiflendiğinde, hücre

çoğalmasının baskılandığı ve apoptoza giden

hücre sayısında artış olduğu tespit edilmiştir. Bu

durum, PER2 geninin baskılayıcı özelliğine bir

delildir (Rana ve Mahmood, 2010).

Yapılan araştırmalarda, meme ve kolon

kanserlerinde PER1 ve PER2 genlerinde

mutasyon tespit edilmiştir. Akut lösemi, meme,

kolon, endometrial, akciğer ve pankreas

tümörlerinde, normal dokulara göre Period

geninin mRNA ve protein seviyelerinde azalma

tespit edilmiştir (Murga ve ark., 2003; Chen ve

ark., 2005; Ko ve Takahashi, 2006; Winter ve

ark., 2007; Krugluger ve ark., 2007). Bu gende

hem genetik hem de epigenetik (DNA

metilasyonu ve histon asetilasyonu)

değişiklikler gözlenmiştir (Hua ve ark., 2006).

Bu polimorfizmlerin kanser riskini artırmayla

bağlantısı gösterilmişse de, altta yatan

moleküler mekanizmalar net olarak

açıklanamamıştır.

İnsan kolon kanseri hücrelerinde PER2

mutasyonunun intestinal beta katenin

düzeylerini artırdığı, bunun yanında kolonda

polip oluşumunu da artırdığı bildirilmiştir.

Ayrıca bu artışın Cyclin D1 proteininin

sentezinde artışa ve dolayısıyla hücre

çoğalmasında da artışa neden olduğu

gösterilmiştir (Wood ve ark., 2009). PER2 geni

susturulmuş fare modellerinde, Cyclin D1,

Cyclin A, Mdm-2, Gadd45 genlerinin

ekspresyonlarında anlamlı değişiklikler

saptanmıştır (Hua ve ark., 2006 ). Benzer bir

başka çalışmada ise, γ radyasyona maruz

bırakılmış mutant farelerde, kontrol grubuna

göre tümör oluşumunda artış, apoptoza giden

hücrelerde ise azalma tespit edilmiştir.

PER1 (rs885747 ve rs2289591), PER2

(rs7602358) ve PER3 (rs1012477) genlerinde

bulunan tek nükleotid polimorfizmlerinin

prostat kanserine yatkınlıkla ilişkisi olduğu,


bunun yanında, PER1 (rs885747 ve rs2289591)

ve PER3 (rs1012477) genlerindeki tek nükleotid

polimorfizmlerin hastalığın agresifliği ile ilişkili

olduğu saptanmıştır (Zhu ve ark., 2009).

Meme kanseri biyopsi örneklerinde yapılan

çalışmada, PER3 genindeki polimorfizmlerin

menopoz öncesi kadınlarda meme kanseri

riskini arttırdığı ortaya konmuş, dolayısıyla

PER3 geninin bazı polimorfik varyantlarının

potansiyel bir belirteç olabileceği

vurgulanmıştır (Rana ve Mahmood, 2010).

Chen ve ark.’nın yaptığı bir çalışmada,

meme tümörlerinin %95’inde PER1 ve PER2

genlerinin transkripsiyon düzeylerinde normal

hücrelere göre farklılıklar belirlenmiştir. Benzer

şekilde, akciğer kanseri tümörlerinin %70’inde,

akut miyeloid lösemilerin ise %42’sinde normal

doku hücrelerine kıyasla PER1 geninin

transkripsiyonunda azalma tespit edilmiştir

(Chen ve ark., 2005).

Cryptochrome genleri

Sirkadiyan ritmin düzenlenmesinde,

transkripsiyonu baskılayıcı rol oynayan

Cryptochrome (CRY1 ve CRY2) ve Period

(PER1, PER2 ve PER3) en çok çalışılan

genlerdir.

CRY2 geninin ekspresyonundaki değişikliklerin,

DNA hasarı kontrolü ve hücre döngüsünde rol

alan genlerin ekspresyonunu doğrudan

etkilediği gösterilmiştir (Gauger ve Sancar,

2005; Sancar ve ark., 2010). Dolayısıyla,

karsinogenezisle ilişkili pek çok hücresel yolak

CRY2 geninin kontrolü altındadır.

CRY1 ve CRY2 susturulmuş genleri fareler

iyonize radyasyona maruz bırakıldığında

radyasyona bağlı kanser oluşumunda azalmalar

görülmüştür (Hoffman ve ark., 2010b).

Dolayısıyla, CRY genlerinin aktivasyonunun

radyasyona bağlı karsinogenezde kritik rol

oynadığı düşünülmektedir.

CRY2 susturulmuş geni meme kanseri hücre

kültürleri mutajenlerle muamele edildiğinde,

DNA hasarında kontrol hücrelerine oranla

artış anlamlı gözlenmiştir (Antoch ve

Kondratov, 2009). Bu bulgu CRY genlerinin

DNA tamirinde önemli rol oynadığını,

dolayısıyla hücrenin genotoksik strese

duyarlılığını etkilediğini göstermektedir.


Sirkadiyan ritim genleri ve kanser 7

Tablo 2. Kanserde rol alan sirkadiyan ritim genleri (Ohdo ve ark., 2010).

Gen Kanser Tipi Genotip/Gen ifadesi

PER2

(Fare)

PER1,2,3

(İnsan)

PER2

(İnsan)

PER2

(İnsan)

PER1

(İnsan)

CRY2

(İnsan)

CRY1,2

(Fare)

CRY1

(İnsan)

BMAL1

(İnsan)

CK1

Npas2

(İnsan)

DEC2

(İnsan)

(Wu ve

ark., 2011)

Lenfoma Eksik

Meme kanseri

PER1 ve 2

promotorlarının

hipermetilasyonu

/İfadesinde azalma

Akut myeloid lösemi İfadesinde azalma

Kolorektal kanser İfadesinde azalma

Prostat kanseri İfadesinde azalma

Non-Hodgkin

lenfoma

P53 mutant farede

timik lenfoma

Kronik lenfoid

lösemi

B hücreli lenfoma,

akut lenfositik ve

myeloid lösemi

Nörodejeneratif

hastalıklar ve kanser

Non-Hodgkin

lenfoma ve meme

kanseri

Tek nükleotid

polimorfizmi

Eksik

CRY1/PER1 ifade

oranında değişim

İfadesinde artış

İfadesinde azalma

Düzenlenmesinde

bozukluk

Tek nükleotid

polimorfizmi

İfadesinde azalma

Kanser Prognozuna

Etkisi

Tümör büyümesinde

artış

Apoptozda azalma

Tümör büyümesinde

artış

Başlama ve/veya

ilerlemesi

Tümör oluşumunda

Mekanizma

Bmal1 ifadesinde azalma

c-myc represyonunun

engellenmesi

Tümör süpresör ve hücre

döngüsünde rol oynayan genlerin

düzenlenmesindeki bozukluklar

c-erbB2 ifadesinde düzensizlikler

CCAATT/artırıcı dizisine

bağlanan proteinlerdeki azalış

artış Beta kateninde azalma

Tümör büyümesinde

artış

Apoptozda azalma

p53 eksik farede

kanserin ortaya

çıkışında azalma ve

ömür uzaması

Kanserin ortaya

çıkışında gecikme

Kronik lenfoid

löseminin olası

sonuçlarının

öngörülebilir hale

gelmesi

azalış

Kanserde

artış

Kanserde

Kanserde artış

Kanserde artış

Meme kanseri Baskılanmış Apoptozda artış

Androjen reseptör transkripsiyon

aktivitesinin düzenlenmesinde

bozukluklar

İmmun cevabın değişimi

Hepatik sistem gelişimindeki

değişiklikler

Genotoksik strese cevap olarak

p53 mutant hücrelerin apoptoza

duyarlılaştırlması

Hücre döngüsüyle ve DNA hasar

cevabıyla ilişkili genlerin

ifadelerindeki değişiklikler

Büyümenin baskılanması

p53 aktivasyonu üzerinden

çoğalmanın durdurulamaması

Büyümenin aktivasyonu

Sinyal iletim yolaklarında

etkileşim

Bazı hücre döngüsü ve DNA

tamir genlerinin ifadelerinin

baskılanması

Apoptozda rol oynayan

proteinlerde [Fas, Bax,c-Myc,

kaspaz-8, poli (ADP-riboz)

polimeraz (PARP)] değişimler


8Harika ATMACA and Selim UZUNOĞLU

Tartışma ve Sonuç

Hücre ölçeğinden başlayarak organizmaya kadar

sirkadiyan ritmin her düzeyde düzenlenmesi ve

kontrolü organizmanın yaşamı için kritik öneme

sahiptir. Transkriptom makinasının işleyişini

düzenleyen transkripsiyon faktörlerinin bir kısmı,

hem sirkadiyan ritmi oluşturan genlerin

transkripsiyon-translasyon çevrimlerinin

düzenlenmesinde, hem de döngüsel geribildirim

yoluyla transkriptom makinesinin kontrolünde rol

alır (döngüsel geribildirim yoluyla oto-düzenleme).

Biyolojik saatin önemli bir bileşeni olan sirkadiyan

ritim genlerinin ürünleri, hangi genlerin ne zaman

ne kadar süreyle okunacağını düzenleyen kompleks

moleküler sistemin öncül bileşenleridir. Bu

nedenle, pozitif ve negatif geribildirim döngüleriyle

birbirlerinin ekspresyonunu ve aktivasyonunu

kontrol eden sirkadiyan ritim genlerindeki herhangi

bir aksaklık, kontrol mekanizmasının, dolayısıyla

ritmin bozulmasına yol açar.

Memeli genomundaki genlerin %10’unun

sirkadiyan ritim genlerinin kontrolünde olduğu

belirtilmiştir (Son ve ark., 2011). Bu kontrol

genellikle hormonal ve metabolik yolakların üst

düzeylerinde gerçekleştiğinden, buradaki bir

değişiklik kademeli olarak yolağın aşağı

basamaklarını, hedef organ, organel ve molekülleri

etkiler. Enformasyon, enerji ve yapı taşlarının

örgütlenmesi üzerine kurulan yaşamın moleküler

temelinde, bilginin doğru konumda, doğru zamanda

ve sürede kullanılması sağlıklı bir yaşam için

olmazsa olmazdır. Enformasyonun ve

yapıtaşlarının doğru zamanda ve süre içinde

kullanımı ve örgütlenmesi sirkadiyan ritimlerle

gerçekleştirilir. Bu perspektiften, sirkadiyan

ritimlerin kontrolünde rol alan genler birincil

seviyede bütün canlılık olaylarını etkiler. Bunun

anlamı, sirkadiyan ritim genlerinin aktivasyon ve

inaktivasyonundaki bir değişikliğin, hem hücresel

hem de organizma düzeyindeki fizyolojik olayları

etkilemesidir. Örnek verirsek, DNA hasar tamiri,

hücre döngüsünün düzenlenmesi, apoptoz gibi

karsinogenezle ilişkili yolaklarda rol oynayan

genlerin transkripsiyonu ve aktivasyonu sirkadiyan

ritim genlerinin kontrolündedir. Bu genlerin bir

veya birkaçında oluşabilecek yapısal veya

fonksiyonel herhangi bir değişikliğin hücreyi

kansere götürmesi muhtemeldir.

Yukarıda belirtildiği gibi sirkadiyan ritim

genleri, hormonal yolakların işleyişini de kontrol

eder. Özellikle hormona bağlı kanserlerde


sirkadiyan ritimlerin bu rolünü görmek

mümkündür. Örneğin prostat androjene,

meme hücreleri de çoğalmak ve normal

gelişimlerini sürdürebilmek için östrojene

ihtiyaç duyarlar. Sirkadiyan ritim

genlerindeki yapısal veya fonksiyonel bir

değişiklikle bu hormonların aşırı sentezi

gerçekleşirse bu durum hücre çoğalmasını

arttıracak, sonuçta ortamda karsinojen bir

varmış madde gibi tümör oluşumu

gözlenecektir.

Sirkadiyan sistem, gerek kanserin

oluşum ve gelişim mekanizmalarını

çalışmada, gerekse kanserin tedavisini

kolaylaştırmada yeni kronoterapötik

stratejiler geliştirmek için özgün bir

sistemdir. Sirkadiyan saati, hücre döngüsüne

ve metabolizmaya bağlayan moleküler

bağlantılar, son yıllarda ortaya konulmaya

ve anlaşılmaya başlanmıştır. Bu moleküler

bağlantıların kapsamlı şekilde anlaşılması,

şüphesiz belirli kanser türlerinin tedavisine

olumlu katkılar yapacaktır. Sirkadiyan

kontrolün kaybı, organ ve sistemler

seviyesinde ortaya çıkan hastalıkların

oluşumuna ve gelişimine de katkı yapar.

Bunun için sirkadiyan kontrolün kaybına

veya bozulmasına yol açan moleküler

mekanizmaların bilinmesine yönelik yeni

araştırmalara ihtiyaç vardır.

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Journal of Cell and Molecular Biology 9(2): 11-18, 2011 Review Article 11

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Tunneling nanotubes – Crossing the bridge

Marc McGOWAN*

BergenBio AS, Thormohlensgt 51, 5006, Bergen, Norway

(* author for correspondence; mkmcgowan@hotmail.co.uk)

Received: 29 October 2011; Accepted: 22 December 2011

Abstract

Since their discovery and subsequent publication in 2004, tunneling nanotubes (TNTs) have quickly gained

interest in direct cellular communication. Their name is taken from both their original discovery diameter

size being 50-200 nm, and also their ability to move through the extracellular matrix (tunneling) to reach and

couple with other cells. TNTs are the extensions of the cell membrane which houses F-actin in smaller tubes

(0.7 µm diameter) nanotubes. Each year more cell

types have been discovered to form TNTs and traffic cellular components. In recent years TNTs have been

found to traffic viruses, prions along with organelles and surface proteins. With new findings related to viral

hijacking of TNTs and spreading of diseases, TNTs are now demonstrating a capability to spread disease

among cells without activating an immune response. With new research focusing on pathogenesis and disease

spreading, TNTs are now becoming a larger area of intercellular networking and pose great importance to

biomedical research. This review demonstrates some new ideas and research into TNTs.

Keywords: Tunneling nanotubes, intercellular transfer, p53, MAPK, cancer.

Nanotüp Tünelleme- Köprüyü geçmek

Özet

Nanotup tünellemelerin keşfinden ve 2004’te yayınlanmasından bu yana, nanotüp tünellemeler (TNT) direkt

hücresel iletişimde hızlıca ilgi kazanmışlardır. İsimlerini, orijinal keşfedilmiş olarak çaplarının 50-200 nm

oluşundan ve diğer hücrelere ulaşmak ve onlarla bağlantı oluşturmak için ekstrasellüler matriks boyunca

hareket etme (tünelleme) kabiliyetlerinden almışlardır. TNT’ler daha küçük tüplerde (0.7 µm çap) F aktinle birlikte mikrotübül hücre membranı

uzantılarıdır. Her yıl TNT’leri oluşturan ve hücresel bileşenlere geçit sağlayan daha fazla sayıda hücre tipi

keşfedilmektedir. Son yıllarda TNT’lerin organeller ve yüzey proteinleriyle birlikte prion ve virüslere geçit

sağladığı bulunmuştur. viral kaçırılma ve hastalıkların yayılımıyla ilgili yeni bulgularla TNT’lerin bir immun

cevap oluşturmadan hücreler arasında hastalığı yayma yeteneğini gösterilmektedir. Patogenez ve hastalık

yayılımına odaklanan yeni araştırmalarla birlikte, TNT’ler hücreler arası haberleşmenin büyük bir kısmını

oluşturur hale gelmiştir ve biyomedikal araştırmalar için büyük önem taşımaktadır. Bu derleme TNT’lere

yönelik bazı yeni fikirleri ve araştırmaları yansıtmaktadır.

Anahtar Sözcükler: Nanotüp tünelleme, hücreler arası transfer, p53, MAPK, kanser.

Introduction

Direct cell-cell communication is crucial for

multicellular organisms to crosstalk and to pass

information from one cell to another. Until recently,

direct cell-cell communication was only described

via gap junctions and synaptic signalling, this was

assumed to be the only way of passing information

between eukaryotic cells. However, it was not until

a student of Hans-Hermann Gerdes, a researcher at

EMBL Germany, was required to perform a media

change on PC12 cultured rat neural cells that some

strange structures were observed. Neglecting to

perform a routine media change and instead

allowing the cells to remain in old media and

become stressed had allowed these cells to develop

extremely thin tube structures protruding from one

cell and connecting with another. From these initial

discoveries emerged the description of a new


12 Marc McGOWAN

structure termed “tunneling nanotubes” (TNTs)

(Rustom et al., 2004). This structure has now been

documented in a variety of cells types such as

astrocytes, immune cells and cancers to name a few

(Önfelt et al., 2006; Watts et al., 2005; Rustom et

al., 2004). Upon further research these tubes were

found to be intercellular highways for lipid

molecules, surface proteins and calcium ions to be

passed from one cell to an adjacent neighbouring

cell (Wang et al., 2010; Gerdes and Carvalho, 2008;

Rustom et al., 2004). It soon became quite clear

that these TNTs had a place in intercellular

communication and could be described as an

additional mechanism for direct cell-cell

communication together with gap junctions and

synaptic signalling.

This review paper will describe some of the

early findings and characteristics along with new

discoveries and suggestions regarding diseasebearing

mechanics and trafficking by TNTs.

TNT characterization

TNTs were first described in 2004 as “filopodialike

protrusions” and were identified in both

cultured rat pheochromocytoma PC12 and in

human and rat embryonic kidney cells in the first

instance (Rustom et al., 2004). These ultrafine

protrusions were observed to extend from one cell

and connect with its closest neighbouring cell

without being in contact with the substratum

(culture plate) indicating these protrusions were not

relics from previous cell divisions. These TNTs

were further studied to assess their structure and

were found to consist of F-actin (Rustom et al.,

2004). Even though the tubes are small in diameter

they were able to pass small lipid molecules and

organelles from the donor to the recipient cell

(Rustom et al., 2004). It was observed at this time

that these tubes were able to pass signals and

organelles from one cell to another in a

unidirectional manner; meaning only one cell was

able to pass to another and not the other way

around. However, it was recorded in human

macrophage cells that this process was

bidirectional, in that both cells could pass

information to one another due to a larger TNT size

that contained both F-actin and microtubules

(Önfelt et al., 2006). Macrophages were observed

to have two distinct TNT diameters, those that were

0.7 µm diameter. It was

observed that TNTs with diameters less than 0.7

µm thick mainly contained F-actin and those that

were larger than 0.7 µm contained both F-actin and

microtubules. TNTs formed only with F-actin are


able to transport molecules unidirectional, while

those formed with both F-actin and microtubules

are able to transport molecules and lipid organelles

(Mi et al., 2011). TNTs were assessed and found to

be very delicate and easily damaged by mechanical

fixation techniques and prolonged exposure to

light. The TNT’s sensitivity to light made it

difficult to observe under the light microscope for

lengthy periods of time. However, the use of

trypsin-EDTA did not show any damage to TNT

formation resulting in the ability to culture cells

without disturbing this process (Rustom et al.,

2004). TNTs have now been documented in a

variety of cells in vitro: cultured rat

pheochromocytoma PC12 (Rustom et al., 2004),

human embryonic kidney cells (HEK293) (Rustom

et al., 2004), EBV-transformed human B-cell line,

J774 murine macrophage cells human monocytederived

macrophage (Önfelt et al., 2006; Önfelt et

al., 2004), DU 145 human prostate cancer cells

(Vidulescu et al., 2004), THP-1 monocyte (Watkins

and Salter, 2005), hepatic HepG2 (Wüstner, 2007),

TRVb-1 cells (Wüstner, 2007), bovine mammary

gland epithelial cells (Wüstner, 2007), rat astrocyte

primary cell (Zhu et al., 2005), myeloid-lineage

dendritic cells (Watkins and Salter, 2005),

hematopoietic stem and progenitor cells (Freund et

al., 2006). They have also been identified in mouse

corneal cells (Chinnery et al., 2008) and between

cardiomyocytes and cardiofibroblasts (He et al.,

2011) in vivo.

Transfer of molecules

Endosomes, mitochondria, endoplasmic reticulum

(ER), calcium and surface proteins have all been

identified to have the ability to cross TNTs in

various cell types (Gerdes et al., 2007). Some

molecules such as calcium were found to require

TNTs to be coupled to cellular gap junctions in

order to allow passive transport (Wang et al., 2010).

To demonstrate Ca 2+ passive transfer, one cell was

coupled to another via TNTs and a small current

was induced to the donor cell. It was observed that

the donor cell was able to transmit the electrical

signal via the TNT to a recipient cell. This process

was amplified by the addition of a second TNT

coupling to the same cell. Cells that were not

coupled did not show any signs of being

depolarised. It was also documented in the same

article that only cells that were coupled by TNTs

and gap-junctions were able to transmit electrical

signals, and those cells that did not express gapjunction

(e.g. PC12 cells) were unable to complete

this task (Wang et al., 2010). However, it was later


argued that Ca 2+ transfer was more spontaneous

than provoked as TNTs were observed to house ER

and extend it through the TNT, which would allow

Ca 2+ stores to be released within the TNT (Smith et

al., 2011). In addition, TNTs in macrophage could

trap bacteria on the surface of the smaller F-actin

TNT, transport them to the cell which are

subsequently phagocytised (Önfelt et al., 2006), a

term called surfing (Lehmann et al., 2005).

Additional observations were made that

macrophage could interconnect several cells

simultaneously in a large network (Önfelt et al.,

2006). This would increase both communication

and abilities to trap and surf bacteria amongst

several cells.

Nanotube extension – extending a helping

hand or a cry for help?

Research has shown that damaged cells can form

TNTs and extend them to healthy cells (Wang et al.,

2011) (Figure 1), or from healthy to damaged cells

for repair such as stem cells (Yasuda et al., 2011;

Cselenyak et al., 2010) (Figure 2). It was observed

in tissue samples from the cornea cells of mice that

TNTs were present after the cells were subjected to

stress, this was also the first discovery of TNTs in

vivo (Chinnery et al., 2008). Hydrogen peroxide, a

reactive oxygen species (ROS), was added to rat

astrocytes in culture to promote activation of p38

mitogen-activated protein kinase (MAPK). The

increase of ROS and subsequent activation of p38

MAPK demonstrated the increase of TNT

formation between cells highlighting a potential

mechanism of development (Zhu et al., 2005). The

same results of TNT formation were recorded by

use of serum depletion in culture medium (Wang et

al., 2011). It was speculated from this research that

p53 was also expressed in cells that had sustained

stress (by either H2O2 or serum depletion) and that

these cells were responsible for the formation of

TNTs. Cells that had their p53 silenced were unable

to form TNTs (Wang et al., 2011). This is

interesting as p38 MAPK is known to

phosphorylate and activate p53 preventing it from

being targeted by mouse double minute 2 (MDM2)

and ubiquitinated (Lu et al., 2008). However,

silencing either p38 or p53 directly will reduce the

activity of p53, which in theory would reduce

formation of TNTs. However, so far, and to the

author’s knowledge, no definite mechanism has

been found that can explain how TNTs form and

extend to a neighbouring cell.


Tunneling nanotubes 13

Figure 1. TNT formation in differentiated cells

signalling for passive transport from an injured to a

healthy cell. A) Left, a cell subjected to stress from

either serum depletion or ROS induced damage (Zhu et

al., 2005). Intracellular mechanisms are in place to begin

the activation of p53 signalling the cell for apoptosis or

senescence. B) Cell begins to form TNTs and extends

them to a nearby healthy cell (Wang et al., 2011). C) The

red dot represents a molecule being transported from the

stressed cell to the healthy cell as a means of salvage.

Figure 2. Potential mechanism of action of stem

cell-injured cell interaction and repair. A) Initial

stress to a cell. B) Stem cells are added into culture. C) A

TNT forms and extends to the stem cell (Yasuda et al.,

2011; Cselenyak et al., 2010,). D) Once coupled, the

stem cell then moves molecules and organelles in a

unidirectional manner towards the injured cell. Once

molecules have entered the injured cell repair

mechanisms begin rescuing it from cell death.


14 Marc McGOWAN

TNTs as a mechanism in disease

TNTs have now been demonstrated to be functional

in cellular communication and have the ability to

transport molecules to other cells, but what about

infected cells, incorrectly functioning cellular

organelles and drug resistance in cancers? Research

has now shown that viruses, prion exchange and

possible mechanisms of disease spreading amongst

cells without triggering an immune reaction have

all taken advantage of TNTs as a way of moving

without being detected.

Mitochondrial-related disease

Mitochondria are paramount for cellular ATP

synthesis. Diseases associated with the

mitochondria may have an additional migratory

bridge using TNTs to move mtDNA-damaged

mitochondria to healthy cells and increase mutated

mtDNA amongst cell populations. TNT formation

has been documented in astrocytes and glial cells

which have been found to passively transport

mitochondria (Agnati et al., 2010; Pontes et al.,

2008; Watts et al., 2005). The potential ability to

transport damaged mitochondria through TNTs may

reveal a possible mechanism of spreading diseases

such as Parkinson disease (PD) and Alzheimer’s

(AD). Mitochondria can be subjected to stress from

ROS leading to mutations in the mtDNA promoting

disease states (Rego and Oliveira, 2003)

PD is characterized by the death of dopamine

neurons in the substantia nigra of the brain. The

clinical characteristics of the disease are tremors,

slowness of movement and dementia. The cause of

the disease are still not clear but recent research has

highlighted a potential mechanism of cell death

being an over-expression of the protein α-synuclein

(α-syn) in the mitochondria of olfactory bulb,

hippocampus, striatum, and thalamus (Liu et al.,

2009). It was hypothesized that α-syn may have a

role in the degradation of mitochondria by

fragmentation (Nakamura et al., 2011). AD is

characterized as very similar to PD but does not

induce tremors. Instead, AD is characterized

clinically by impairment of judgement, language

skills and orientation to name a few. Pathological

characteristics are degeneration of neurons and

synapses. AD is induced by ROS damage to

mitochondria which can lead to mutations and

apoptosis of the cell (Su et al., 2008). Considering

that TNT formation occurs due to cellular stress,

this process may help explain rapid cellular

deterioration and onset of both PD and AD with the


potential passage of damaged mitochondria through

TNTs.

A part of the cancer puzzle

Amongst the intracellular molecules, surface

proteins can also be transferred as TNTs are an

elongated part of the cellular membrane.

Farnesylated endothelial growth factor proteins

(Farnesylated-EGFP) were observed to be

transported from one cell to another demonstrating

this type of trafficking (Rustom et al., 2004). Pglycoproteins

(P-gp) have also been found to

migrate from one cell to another via TNTs. P-gps

are transmembrane proteins found in many cancers

that can regulate and pump out cytotoxic drugs

(Gottesman and Pastan, 1993). Expression of P-gp

has also been found in many chemotherapyresistant

cancers that are able to efflux drugs before

they become active within the cell. Experiments

with breast cancers expressing P-gp were

conducted to determine if the protein could be

transferred from cells expressing P-gp(+) to those

not expressing P-gp(-) neighbouring cells. By coculturing

these two cell populations it was found

that the protein was able to be transferred from one

cell to another and also be functional (Pasquier et

al., 2011). In multidrug resistant cancers it has been

found that an overexpression of P-gp and other

multidrug resistant associated proteins enable a

cancerous cell to become resistant to chemotherapy

drugs (Gong et al., 2011). It has been shown that

TNTs can form in DU 145prostate cells (Vidulescu

et al., 2004) and in breast cancers (Pasquier et al.,

2011) that can become multidrug resistant by way

of multidrug resistant protein overexpression

(Sullivan et al., 1998). Could the transfer of

multidrug resistant protein via TNTs aid

neighbouring cells to become multidrug resistant?

With more research and in vivo assessments

TNTs may have a place in further describing cancer

mechanisms. Cytotoxic drugs are able to affect a

cancerous cell in many ways and all have different

mechanisms of action. Could the damage from

chemotherapic drugs be enough to promote the

formation of TNTs due to cellular stress?

The role of p53 in possible TNT formation is of

interest to many oncology research scientists.

During de novo oncogenesis a cancer cell may have

mutated p53 which has been found to prevent

expression of p21 and beginning of the subsequent

senescence and/or apoptosis cascade (Vousden and

Prives, 2009). p53 activation stems from cellular

stress including genetic damage like that of


chemotherapy which has many different

mechanisms of action within the cell preventing

proliferation. However, p53, being in high

concentration in cancers, may have the ability to

form TNTs. The questions that need to be asked

are: 1) Do cancers cells with high concentrations of

p53 also produce TNTs? 2) Does the treatment of

chemotherapy drugs increase TNT formation? 3)

Do resistant cancers have the ability to produce

TNTs when treated with chemotherapy drugs?

Virus and prion exchange

TNTs also provide a way for pathogens to migrate

from one cell to another and proliferate. HIV was

discovered to use TNTs to migrate from one cell to

another, evading the extracellular environment in

human monocyte-derived macrophages (MDM)

and avoiding the host’s immune cells (Kadiu and

Gendelman, 2011; Eugenin et al., 2009). It was

found that HIV depended upon entering a MDM

via clathrin-mediated endocytosis. This process

encapsulates the virus and allows it to pass through

F-actin and microtubule derived TNTs to a

neighbouring cell (Kadiu and Gendelman, 2011).

Again with T-cells, HIV was found to use TNTs as

a way of infecting neighbouring cells and also

increase the numbers of TNT formations without

having to spread via the extracellular fluid

(Sowinski et al., 2008). These data demonstrate

how viruses have the ability to use host immune

cells to migrate and proliferate in vitro without

contacting the extracellular fluid. Prions have also

been identified to migrate between cells using

TNTs (Gousset et al., 2009). Prions are misfolded

proteins that are capable of entering a cell and

altering wild-type proteins leading to diseases like

Creutzfeldt-Jakob disease (CJD) and can cause

necrosis (Brundin et al., 2010). It was identified

that TNTs can aid the spreading of prions in

cultured cells from Cath. a-differentiated (CAD)

cell line (Gousset et al., 2009).

The use and passage of mitochondria from

damaged to healthy cells in the brain may be a

cause of spreading neural diseases, e.g. AD and PD.

Three research questions arise in this area, which

are 1) Does a damaged cell have the ability to both

form and pass ROS-induced damaged mtDNA to a

healthy neighbouring cell? 2) Do the damaged

mitochondria have the ability to begin apoptosis in

the neighbouring cell and again signal for TNT

formation? 3) Can this process be reversed using

stem cells as previously described?

The process of viral entry and migration

through TNTs has now been documented and


Tunneling nanotubes 15

accepted. The ability to move from cell to cell

using TNTs without having to exit and migrate

through the extracellular fluid have provided a new

mechanism of infection for viruses and prions. This

process of “hijacking” TNTs will no doubt be of

interest to virology and more so for the spread of

HIV amongst immune cells. We now know that

viruses promote TNT formation in the infected cell

and allow safe passage to a recipient, can the same

process be blocked and prevent these “hijackers”

from migrating between cells using TNTs?

Stem cells and their ability to repair damage

via TNTs

Stem cells are very much at the forefront of medical

sciences and the hope of curing many diseases rests

upon these progenitor cells. It is surprising, though,

to discover their additional abilities and possible

mechanisms of aiding cells in distress in vitro. It

was discovered that endothelial progenitor cells

(EPC) – a precursor cell to endothelial cells – could

couple with both types of TNT sizes from HUVEC

(Yasuda et al., 2011). It was observed that EPC cocultured

with stressed HUVEC could produce

TNTs and traffic cellular components both ways,

but mainly observed to pass from EPC to HUVEC

(Yasuda et al., 2011). This promoted HUVEC to

recover from stress and to proliferate. Additionally,

mesenchymal stem cells (MSC) were also found to

traffic cellular components via TNTs to damaged

cardiomyoblasts and promote recovery (Cselenyak

et al., 2010). From the research it seems that stem

cells have the ability to repair cells which have

sustained stress. This process of repair rather than

salvaging (as found in non-stem cells in previous

sections of this review) may demonstrate how stem

cells can rescue damaged cells.

Discussion

With the discovery of TNTs and the subsequent

abilities they have in trafficking the molecules,

disease-spreading prions and viruses, it is clear that

these TNTs have a valid place in cellular biology. It

can be agreed that TNTs do provide a function in

cellular communication. There is still a mystery to

TNT-genesis in that it is not fully understood what

mechanisms are in place that signal aid via TNTs.

We do know that stress is a key factor and that

repair/apoptosis mechanisms are in place prior to

TNT development. With further research TNTgenesis

and key communication signals for

coupling may become better understood.

This review paper ventured into diseases

associated with intracellular molecules and viruses


16 Marc McGOWAN

with the notion of finding ways in which TNTs

move (and in some cases potentially) them from

one cell to another. This will require further

investigation as to the spreading of disease amongst

cells via TNTs as suggested in this review. This,

along with cancer research, provide great

opportunity to study mechanisms of TNT formation

within cell lines resistant to chemotherapy drugs by

the movement of surface proteins associated with

multidrug resistance. This would be very

interesting to see if cancer cells can help each other

when subjected to cytotoxic drugs.

Since the initial discovery by Gerdes’ team

(Rustom et al., 2004), TNTs have added another

level to our understanding of biological processes

for molecular and cell biologists. With each year

since their discovery, more and more cells are being

characterized as forming TNTs with their own

unique way of using them. It is now accepted that

TNTs provide direct cell-cell communication along

with gap junctions and synaptic signalling. It is

useful to find new and potential mechanisms of

disease spreading and observe how these pathogens

and mutated genes can migrate from one cell to

another without being targeted by the host’s

immune system. They also demonstrate a plausible

method of cellular repair such as the way stem cells

can meet the needs of a damaged cell. It thus can be

concluded here that TNTs are becoming very

important in direct cell-cell communication, repair

and disease transfer. There will no doubt be more

research papers coming to light after this review is

published and thus would not do justice to the new

work currently being conducted.

Acknowledgements

The author thanks to both Dr Stephen Merry and

Renate Simonsen for their time and support in

evaluating this paper.

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Journal of Cell and Molecular Biology 9(2): 19-26, 2011 Research Article 19

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Genetic screening of Turkish barley genotypes using simple

sequence repeat markers

Hülya SİPAHİ*

Biology Department, Faculty of Arts and Sciences, Sinop University, Turkey

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

Received: 29 April 2011; Accepted: 7 October 2011

Abstract

Thirty-four Turkish barley genotypes were differentiated and identified using barley simple sequence repeat

(SSR) markers. Amplification of SSR loci were generated using 17 SSR primers. These SSR primers totally

produced 67 alleles ranging from two to six alleles per locus with a mean value of 3.94 alleles per locus.

Genetic similarity ranged from 0.507 to 1.000. Maximum genetic similarity was found between Efes-98-

Başgül, and among Anadolu-86, Obruk-86, Anadolu-98, Tokak157/37 and Orza-96. Minimum genetic

similarity was between Bolayır and Angora. Although SSR markers cannot classify 34 Turkish barley

cultivars based on end use, growth habits and row groups, 27 Turkish barley genotypes could be identified

uniquely using 17 SSR primers. These results will are useful proves for protecting breeder’s rights and

designing new crossings.

Keywords: Barley (Hordeum vulgare L.), genetic discrimination, simple sequence repeats, molecular

markers, genetic similarity

Türk arpa genotiplerinin basit dizilim tekrarları işaretleyicileri ile genetik taranması

Özet

Basit Dizilim Tekrarları (BDT) işaretleyicileri kullanılarak, otuz dört Türk arpa genotipinin yapılmış

ayrımı

ve tanımlanmıştır. BDT lokuslarının çoğaltımı 17 BDT primeri kullanılarak yapılmıştır. Bu BDT primerleri

lokus başına ortalama 3.94 olmak üzere 2 ila 6 arasında değişen toplam 67 allel üretmiştir. Genetik benzerlik

0.507 ila 1.000 arasında değişim göstermektedir. En yüksek genetik benzerlik Efes-98 ve Başgül ile Anadolu

86, Obruk-86, Anadolu-98, Tokak157/37 ve Orza-96 arasında bulunmuştur. En düşük genetik benzerlik ise

Bolayır ve Angora arasındadır. BDT işaretleyicileri, 34 Türk arpa çeşidini, kullanım amaçlarına, yetişme

koşullarına ve başak sıralarına göre sınıflandıramamasına rağmen, 27 Türk arpa çeşidi 17 BDT primeri

kullanılarak tanımlanabilmiştir. Bu sonuçlar ıslahçı haklarını korunmasında ve yeni melezlerin

tasarlanmasında faydalı olabilecek kanıtlardır.

Anahtar Sözcükler: Arpa (Hordeum vulgare L.), basit dizilim tekrarları, genetik ayırım, moleküler

markörler, genetik benzerlik

Introduction

Barley (Hordeum vulgare L.) genotypes are

traditionally distinguished by morphological traits,

such as hairiness of leaf sheaths, intensity of

anthocyanin, number of rows, rachilla hair types,

plant length. In most cases, genotypes are obtained

from very similar parents. This makes the

morphological differentiation rather difficult. Seed

storage protein markers and molecular markers

have been used as tools to enhance barley cultivar

identification capabilities for several years. Among

different classes of molecular markers, SSR

markers have proved as markers of choice for

several applications in breeding because of their

multi-allelic nature, codominant inheritance,

reproducibility, abundance and wide genomic

distribution (Gupta and Varshney, 2000). SSRs are

particularly attractive for distinguishing between

cultivars because the level of polymorphism


20 Hülya SİPAHİ

detected at SSR loci is higher than that detected

with any other molecular assay (Saghai Maroof et

al., 1994; Powell et al., 1996).

So far, several investigations on the

discrimination between barley genotypes using

SSR markers have been carried out by Russell et al.

(1997), Pillen et al. (2000), Turuspekov et al.

(2001), and Chaabane et al. (2009). Limited

information is available on genetic discrimination

of Turkish barley cultivars. These research based

on analysis of Inter Simple Sequence Repeats

(ISSR) (Yalım, 2005), storage protein (hordein) and

Random Amplified Polymorphic DNA (RAPD)

(Sipahi et al., 2010). The purpose of the present

research was to distinguish 34 Turkish cultivars and

estimate the genetic relations among these cultivars

using SSR markers.

Materials and methods

Plant material

Thirty-four barley genotypes from Turkey used in

the present study are listed in Table 1. Seed

samples have been kindly provided by Central

Research Institute for Field Crops (CRIFC) Ankara,

Turkey. Barley seeds were germinated and grown

under standard conditions (25±1°C, 16 hours of

photoperiod for 14 days).

DNA extraction and SSR analysis

Total genomic DNA was isolated from seedlings of

each cultivar according to Anderson et al. (1992).

Seventeen microsatellite primer pairs were selected

based on their chromosomal positions (Table 2).

Polymerase chain reaction (PCR) reactions were

performed in 25 µL of a mixture containing 20 ng

DNA, 1X Taq Reaction Buffer, 5 units of Taq

DNA Polymerase, 0.2 mM dNTPs and 0.25 µM of

each primer. Depending on the primer used (Table

2), DNA amplifications were performed using one

of the following amplification parameters: (1)

Eighteen cycles of 1 min at 94°C for denaturation,

30 s at 64°C (decrease 1°C per 2 cycles until 55°C)

for annealing, 1 min extension at 72 °C, followed

by 30 cycles of 1 min at 94°C, 1 min at 55°C, 1 min

at 72° C and 5 mins final extension at 72°C. (2) 3

min denaturation at 94°C, 1 min annealing at 55°C,

1 min extension at 72°C, followed by 30 cycles of 1

min denaturation at 94°C, 1 min annealing at 55°C,

1 min extension at 72°C, and 5 mins final extension

at 72°C. (3) 1 cycle of 3 min denaturation at 94°C,

1 min annealing at 58°C, 1 min extension at 72°C,

followed by 30 cycles of 30 s denaturation at 94°C,

30 s annealing at 58°C, 30 s extension at 72°C,

followed by a single extension at 72°C for 5 mins.

PCR products were separated by electrophoresis

using 3% agarose gel and 6% non-denaturating

polyacrylamide gel in 1xTBE buffer, then stained

with ethidium bromide and visualized under UV

light. A 100 bp DNA ladder was used as a

molecular size standard.

Data analysis

SSR data were scored for the presence (1) or

absence (0) of clear bands. Only intense bands were

scored visually. The genetic similarities (GS)

among cultivars were calculated according to Nei

and Li (1979). Based on the similarity matrix, a

dendogram showing the genetic relationships

between genotypes was constructed using

unweighted pair group method with arithmetic

mean (UPGMA) (Sneath and Sokal, 1973) by using

the software NTSYS-pc version1.80 (Rohlf, 1993).

Polymorphic information content (PIC) values

were calculated for each primer according to the

formula: PIC = l - ∑(Pij) 2 , where Pij is the

frequency of the i th pattern revealed by the j th

primer summed across all patterns revealed by the

primers (Anderson et al., 1993).

Results

Seventeen SSR primers were used for cultivar

identification and estimation of the genetic relations

among 34 Turkish barley genotypes. Table 1 lists

the detail of the genotypes along with their

breeding parents. All 17 SSR primers generated

clear banding patterns with high polymorphism.

The Figure 1 shows an example of two

polymorphic bands between 150 and 200 bp

generated by Bmag0500 primer. Seventeen SSR

primers revealed a total of 67 alleles ranging from

two to six alleles per locus with a mean value of

3.94 alleles per locus (Table 3). The effective

number of alleles was less than observed alleles in

all loci, with an average of 2.30. The PIC values

ranged from 0.164 (Bmag353) to 0.747 (Bmac213)

with an average value of 0.523 (Table3). Bmac213

and EBmac679 revealed the highest PIC values

(0.747 and 0.714, respectively), which coincided

with their highest number of polymorphic bands

(5). The frequency of sixty percent of the 67 alleles

was lower than 0.20 (Table 3). Five alleles showed

frequencies higher than 0.70 and ten alleles had

frequency of 0.03. These results revealed the

distribution and representative aspect of the alleles

in Turkish barley cultivars. The number of rare

alleles, i.e. alleles found only in one genotype, was


determined. The frequency of rare alleles was 0.03.

Two alleles (~130 bp) at the locus Bmag387 and

Bmag500 and two alleles (~140 bp, 240 bp) at the

locus Bmag013 and Bmag217 was fixed in

Sladoran. The alleles (~140 bp, 220 bp, 200 bp, 150

bp, 230 bp, 130 bp) at locus EBmac501, HVM68,

Bmac113, Bmag013, Bmag217, Bmag310 were

fixed with only Kıral 97, Barbaros, Kalaycı 97,

Turkish barley cultivar screening with SSR 21

Angora, Bilgi 91 and Erginel genotypes,

respectively.

The genetic similarity matrix was established

using data generated by the seventeen SSR primers.

Genetic similarity ranged from 0.507 to 1.000.

Maximum and minimum similarities were found

for Efes-98/Başgül, Anadolu-86/Obruk-

86/Anadolu-98/Tokak157/37/Orza-96 and

Bolayır/Angora, respectively.

Table 1. Turkish barley (Hordeum vulgare L.) genotypes used in this study along with their pedigrees.

Name of cultivars Pedigrees Row End Use Growth habit

1-Tokak 157/37 Selection from Turkish land races 2 Feed Winter/Spring

2-Zafer 160 Selection from Turkish land races 6 Feed Spring

3-Yeşilköy 387 Zafer160 / land race from Kırklareli (gene bank no 3351) 6 Feed Spring

4-Yerçil 147 Strengs Frankengerste from Germany 2 Feed Spring

5- Obruk 86 Selection from Tokak 2 Feed Winter/Spring

6-Anadolu 86 Luther / BK 259-149/3 gün-82 2 Feed Winter

7-Bülbül 89 13GTH / land race ( Gene bank number 657) 2 Feed Winter

8-Erginel 90 Escourgeon / Hop2171 (France) 6 Feed Winter

9-Bilgi 91 Introduction from Mexico 2 Feed Spring

10-Şahin 91 Unknown 2 Malting Winter

11-Tarm 92 Tokak / land races no 4875 2 Feed Winter/Spring

12-Efes 3 Unknown 2 Malting Winter

13-Yesevi 93 Tokak / land race no 4857 2 Feed Winter/Spring

14-Karatay 94 3896/I-3/Toplani/3/Rekal/1128/90 Manhaists 2 Feed Winter

15-Orza 96 Tokak / land race no 4857 2 Feed Winter/Spring

16-Balkan 96 Unknown 2 Malting Winter

17-Kalaycı 97 Erginel 9 / Tokak 2 Feed Winter/Spring

18-Kıral 97 Unknown 6 Feed Winter

19-Sladoran Introduction from Yugoslavia 2 Malt Winter/Spring

20-Anadolu 98 Susuz selection / Berac (Turkey-Holland) 2 Malting Winter

21-Efes 98 Tercan selection / Tipper (Turkey-England) 2 Malting Winter

22-Angora (Triax / line 818 no ) / ( Malta X Ungar) /2/ (lineno 818/Sultan) 2 Malting Winter

23-Çetin 2000 Star (İran) / 4875 no line 6 Feed Winter

24-Aydanhanım GK Omega / Tarm 92 2 Malting Winter/Spring

25-Avcı 2002 Sci/3/Gi-72AB58,F1//WA1245141 6 Feed Winter/Spring

26-Çumra 2001 Tokak selection / Beka 2 Malting Winter/Spring

27-Çatalhöyük 2001 S 8602 / Kaya 2 Malting Winter

28-Zeynelağa (Anteres x KY63-1249) x Lignee 2 Malting Winter/Spring

29-Barbaros Introduction from France 6 Feed Winter

30-Larende ALM (4652)/Tokak//342TP/P-12-119/3/W.BELT22 2 Feed Winter/Spring

31-Çıldır 3896/28//284/28/CMM/14/624/682/5/WBQT12 2 Malting Winter/Spring

32- Başgül Severa/Tokak//Ad.Gerste/Clipper 2 Malting Winter/Spring

33- İnce Arpa 4671/Tokak/4648/P12-119/3/WBCB-4 2 Malting Winter/Spring

34- Bolayır OSK4.197/12-84//HB854/Astix/3/Alpha/Durna 2 Feed Winter


22 Hülya SİPAHİ

Table 2. Barley SSR primers, their sequences, the chromosomal location and repeat (F: Forward, R:Reverse)

Primer Sequence Reference Location Repeat PCR a

Bmac0213 F:5’-ATGGATGCAAGACCAAAC-3’

R: 5’-CTATGAGAGGTAGAGCAGCC-3’

EBmac0501 F:5’-ACTTAAGTGCCATGCAAAG-3’

R:5’- AGGGACAAAAATGGCTAAG-3’

WMC1E8 F:5’- TCATTCGTTGCAGATACACCAC-3’

R:5’- TCAATGCCCTTGTTTCTGACCT-3’

HVM20 F:5’- CTCCACGAATCTCTGCACAA-3’

R:5’- CACCGCCTCCTCTTTCAC-3’

HVM36 F:5’-TCCAGCCGACAATTTCTTG-3’

R:5’-AGTACTCCGACACCACGTCC-3’

Bmag0013 F:5’-AAGGGGAATCAAAATGGGAG-3’

R:5’-TCGAATAGGTCTCCGAAGAAA-3’

Bmac0209 F:5’-CTAGCAACTTCCCAACCGAC-3’

R:5’-ATGCCTGTGTGTGGACCAT-3’

Bmag0225 F:5’-AACACACCAAAAATATTACATCA-3’

R:5’-CGAGTAGTTCCCATGTGAC-3’

Bmag0353 F:5’-ACTAGTACCCACTATGCACGA-3’

R:5’ -ACGTTCATTAAAATCACAACTG-3’

HVM68 F:5’-AGGACCGGATGTTCATAACG-3’

R:5’-CAAATCTTCCAGCGAGGCT-3’

Bmac0310 F:5’- CTACCTCTGAGATATCATGCC-3’

R:5’ -ATCTAGTGTGTGTTGCTTCCT-3’

EBmac0679 F:5’-ATTGGAGCGGATTAGGAT-3’

R:5’-CCCTATGTCATGTAGGAGATG- 3’

Bmag0337 F:5’-ACAAAGAGGGAGTAGTACGC-3’

R:5’-GACCCATGATATATGAAGATCA-3’

Bmag0387 F:5’-CGATGACCATTGTATTGAAG-3’

R:5’-CTCATGTTGATGTGTGGTTAG-3’

Bmac0113 F:5’-TCAAAAGCCGGTCTAATGCT-3’

R:5’-GTGCAAAGAAAATGCACAGATAG-3’

Bmag0500 F:5’-GGGAACTTGCTAATGAAGAG-3’

R:5’-AATGTAAGGGAGTGTCCATAG-3’

Bmag0217 F:5’-ATTATCTCCTGCAACAACCTA-3’

R:5’-CTCCGGAACTACGACAAG -3’

Ramsay et al. (2000)

Hearnden PR et al. (2007)

1H (AC)23 3

Ramsay et al. (2000)

Varshney RK et al. (2007)

1H (AC)13 3

Ramsay et al. (2000),

Varshney RK et al. (2007)

Hearnden PR et al. (2007)

1H (AC)24 2

Liu et al. (1996) 1H (GA)19 1

Ramsay et al. (2000)

Varshney RK et al. (2000)

Liu et al. (1996)

Ramsay et al. (2000)

Varshney RK et al. (2007)

Hearnden PR et al. (2007)

Ramsay et al. (2000)

Varshney RK et al. (2007)

Hearnden PR et al. (2007)

Ramsay et al. (2000)

Varshney RK et al. (2007)

Hearnden PR et al. (2007)

2H

(GA)13

3H (CT)21

3H (AC)13 3

3H (AG)26 3

Ramsay et al. (2000)

Varshney RK et al. (2007)

Hearnden PR et al. (2007)

4H (GA)22 3

Liu et al. (1996) 4H (AG)21 1

Hearnden PR et al. (2007)

Hayden MJ et al. (2008)

4H (CT)11(

AC)20

Hearnden PR et al. (2007) 4H (AC)22 2

Ramsay et al. (2000)

Varshney RK et al. (2007)

Hearnden PR et al. (2007)

Ramsay et al. (2000)

Varshney RK et al. (2007)

Hearnden PR et al. (2007)

Ramsay et al. (2000)

Varshney RK et al. (2007)

Hearnden PR et al. (2007)

5H (AG)22

5H (AG)16 3

5H (AT)7(

AC)18

Hearnden PR et al. (2007) 6H (AG)29 3

Ramsay et al. (2000)

Varshney RK et al. (2007)

7H (AG)17(

AC)16

a The numbers represent one of the three PCR conditions described in the materials and methods section.

1

3

2

2

3

3


Turkish barley cultivar screening with SSR 23

Figure 1. Agarose gel showing the alleles of the Bmag0500 SSR marker in Turkish barley cultivars.

Tarm-92, 2. Yesevi-93, 3. Çetin-2000, 4. Yerçil, 5. Zeynelağa, 6. Çatalhöyük, 7.Kral-97, 8. Karatay-

94, 9. Anadolu-86, 10. Çumra-2001, 11. Anadolu-98, 12. Tokak157/37, 13.Orza-96, 14. Erginel,

15.Yeşilköy, 16. Sladoran, 17. Bülbül-89, 18. Balkan-96. M:Molecular size standard 50bp DNA

ladder.

A dendogram of the 34 barley cultivars was constructed by the UPGMA method (Figure 2).

According to this dendogram, genotypes were divided in five different groups and two of them were

also divided in two subgroups (Figure 2).

Figure 2. Dendogram constructed by the UPGMA method


24 Hülya SİPAHİ

The first group included two and six-row

genotypes and genotypes of diverse end use and

growth habit. The second group contained nine

genotypes. These genotypes were divided two subgroups.

While the sub-group A comprised only

feeding genotypes, the sub-group B was dominated

by malting and two-row genotypes. The

third group contained only two genotypes.

The fourth group comprised majority of

malting genotypes. The largest group was

group five. This group was two-row type,

except for Avcı, 2002.

Table 3. Number of observed, effective and polymorphic allele, frequencies of alleles and PIC values

of 17 SSR loci in 34 Turkish barley genotypes.

Locus Observed

number of

alleles

Number of

polymorphic

alleles

Effective

number of

alleles

Frequencies of alleles Polymorphic

information

content (PIC)

Bmac213 5 5 3.90 0.15, 0.06, 0.15, 0.32, 0.32 0.747

EBmac501 4 3 2.12 0.03, 0.15, 0.64, 0.18 0.522

WMC1E8 2 2 1.49 0.21, 0.79 0.332

HVM36 3 3 1.61 0.76, 0.06, 0.18 0.371

Bmac209 2 2 1.78 0.68, 0.32 0.435

Bmag225 4 4 2.64 0.12, 0.15, 0.55, 0.18 0.617

Bmag353 2 2 1.19 0.09, 0.91 0,164

HVM68 5 4 2.20 0.09, 0.12, 0.64, 0.12, 0.03 0.540

Bmac310 4 3 2.50 0.03, 0.09, 0.41, 0.47 0.602

EBmac679 5 5 3.52 0.43, 0.15, 0.15, 0.21, 0.06 0.714

Bmag337 3 3 1.53 0.06, 0.79, 0.15 0.350

Bmag387 5 4 2.65 0.55, 0.03, 0.09, 0.12, 0.21 0.619

Bmac113 3 2 2.10 0.44, 0.03, 0.53 0.525

Bmag500 6 4 2.96 0.03, 0.53, 0.15, 0.15, 0.06, 0.08 0.663

HVM20 4 4 2.52 0.44, 0.06, 0.44, 0.06 0.606

Bmag013 5 3 2.70 0.03, 0.03, 0.50, 0.32, 0.12 0.631

Bmag217 5 3 1.76 0.06, 0.15, 0.74, 0.03, 0.03 0,425

Mean 3.94±1.25 3.29±0.99 2.30±0.73 0.523±0,56

Discussion

The average PIC value in this study was lower than

what was reported in a previous study by Yalım

(2005) who discriminated 28 Turkish barley

genotypes using 10 ISSR primers. Ten ISSR

primers produced an average PIC value of 0.611.

The average PIC value of 0.523 detected in 34

Turkish cultivars is in accordance with Russell et

al. (1997) who found an average PIC value of 0.50

using eleven microsatellite loci in 24 barley

genotypes. The lower average PIC value was

reported by Pillen et al. (2000). They detected

average PIC value of 0.38 for 22 microsatellites in

25 German, 3 North American barley cultivars and

2 H. vulgare ssp. spontaneum accessions. Based on

the genetic similarity dendogram of seventeen SSR

primers, 27 Turkish cultivars could be

distinguished uniquely. On the other hand, more

SSR primers need to be used for reliable

discriminating of seven Turkish cultivars (Efes-98,

Başgül, Anadolu-86, Obruk-86, Anadolu-98,

Tokak157/37, Orza-96). In general, the

UPGMA cluster did not classify 34 Turkish

barley cultivars corresponding to their

pedigrees, the number of rows, end use and

growth habits.

Yalım (2005) noticed that 10 ISSR

primers were sufficient for separating 28

Turkish barley cultivars in which minimum

and maximum genetic distances were

between Efes-2/Yesevi-93 and Karatay-

94/Aday-4 cultivars, respectively. In order

to determine genetic variation and

relationships among barley genotypes

improved in Turkey using hordein and

RAPD, Sipahi et al. (2010) screened 34

barley cultivars and observed 15 different

hordein banding patterns twelve of which

were cultivar specific. RAPD variation

observed among cultivars higher than that of

hordein and cluster analyses based on

hordein data showed that most of the

cultivars are genetically closely related.


Moreover, correspondence analysis by using these

two marker systems showed that RAPD data could

distinguish almost all barley cultivars except Tokak

157/37 and Bülbül 89, whereas hordein data were

not able to discriminate the barley cultivars like

RAPDs.

Our SSR analysis showed that this technique

was time and labor saving, and effective approach

for barley cultivar identification. Seven barley

cultivars used in this study, which were not

identified by seventeen SSR primers, should also be

identified by combining different DNA based

techniques such as RAPD, ISSR, STS, SNP or

protein electrophoresis. Result of this investigation

will benefit barley breeders when selecting

potential parents to be used in crossing programs

and will also facilitate the germplasm management.

Acknowledgements

I am grateful to İsmail Sayım and Namuk Ergun for

providing Turkish barley genotypes.

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Tuberosa R. Genetic diversity of Japanese

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Röder MS, Stein N, Waugh, R, Langridge P,

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Yalım D. Türkiye’de yetişen arpa

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Journal of Cell and Molecular Biology 9(2): 27-35, 2011 Research Article 27

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Strontium ranelate induces genotoxicity in bone marrow and

peripheral blood upon acute and chronic treatment

Ayla ÇELİK *1 , Serap YALIN 2 , Özgün SAĞIR 2 , Ülkü ÇÖMELEKOĞLU 3 , Dilek

EKE 1

1 Department of Biology, Faculty of Science and Letters, Mersin University, Mersin, Turkey

2 Department of Biochemistry, Faculty of Pharmacy, Mersin University, Mersin, Turkey

3 Department of Biophysics, Faculty of Medical Science, Mersin University Mersin, Turkey

(* author for correspondence; a.celik@mersin.edu.tr)

Received: 19 September 2011; Accepted: 20 November 2011

Abstract

Strontium is a naturally occurring element that exists in the environment mainly as a free metal or in

the (II) oxidation state. In this study, rats were treated by gavage with 500 mg/kg of strontium ranelate

dissolved in saline three times per week for 12 weeks (chronic treatment) and 24 hours (acute

treatment). The genotoxic potential of strontium ranelate was investigated in Wistar rat peripheral

blood, using the micronucleus (MN) test systems. In addition to this test system, we also investigated

the ratio of polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs) as a

cytotoxicity marker. Strontium ranelate induced micronucleus formation in peripheral blood and bone

marrow of rats. It is determined that strontium ranelate has cytotoxic effect on peripheral blood cell

population upon both acute and chronic treatment (p


28 Ayla ÇELİK et al.

Introduction

Strontium is a naturally occurring element that

exists in the environment mainly as a free metal or

in the (II) oxidation state. Cyto-genotoxicity of

metals is important because some metals are

potential mutagens, which are able to induce

tumors in humans and experimental animals.

Strontium is fairly reactive and therefore is rarely

found in its pure form in the earth’s crust.

Examples of common strontium compounds

include strontium carbonate, strontium chloride,

strontium hydroxide, strontium nitrate, strontium

oxide, and strontium titanate. The most toxic

strontium compound is strontium chromate, which

is used in the production of pigments and can cause

cancer via inhalation route (Toxicological Profile

for Strontium U.S. Department of Health and

Human Services Health Service Agency for Toxic

Substances and Disease Registry, 2004).

The terminal elimination half-life for strontium

in humans has been estimated to be approximately

25 years. Estimates of the terminal elimination halflives

of strontium reflect primarily the storage and

release of strontium from bone. Over shorter time

periods after exposure, faster elimination rates are

observed, which reflect soft-tissue elimination as

well as elimination from a more rapidly

exchangeable pool of strontium in bone. Strontium

ranelate (SR), newly developed drug, was first

listed on the Pharmaceutical Benefits Scheme

(PBS) on April 1st, 2007 for the treatment of

established osteoporosis in postmenopausal

women. On November 1st, 2007 the listing of SR

was extended to the treatment of osteoporosis in

some postmenopausal women without fracture.

Cellular and subcellular functions of strontium

metal are not described in any detail (Meunier et al.

2004; Reginster et al. 2005). There is little evidence

for genotoxicity of stable strontium. However,

radioactive strontium isotopes release ionizing

radiation that, within an effective radius, is known

to damage DNA. No studies were located regarding

genotoxic effects in humans following exposure to

stable strontium. The only in vivo genotoxicity

study for stable strontium in animals involved acute

oral exposure (U.S. Department of Health and

Human Services, 2004). Genotoxicity testing of

pharmaceuticals prior to commercialization is

mandated by regulatory agencies worldwide. For

the most part, a three or four-test battery including

bacterial mutagenesis, in vitro mammalian

mutagenesis, in vitro chromosome

aberration analysis and an in vivo

chromosome stability assay are required.

These assays have not been modified

substantially since the initiation of their use

and they remain the best approach to

genotoxicity hazard identification (Snyder

and Green, 2001).

In recent years, the in vivo micronucleus

assay has become increasingly accepted as

the model of choice for evaluation of

chemically induced cytogenetic damage in

animals. The earliest applications of this

model focused on the frequency of

micronucleus in polychromatic (immature)

erythrocytes (MN-PCE) in rodent bone

marrow (Heddle, 1973). Reports were

eventually developed indicating that the

peripheral blood of treated rodent is an

acceptable cell population for this kind of

study as long as sampling schedule was

modified to account for the release of newly

formed micronucleated erythrocytes from

bone marrow to the blood (MacGregor et al.

1980; Schlegel and MacGregor 1983 ).

This approach opened the way for

incorporation of micronucleus assessments

into on-going repeat dose conventional

toxicology studies in mice (MacGregor et al.

1980; Ammann et al. 2007; Jauhar et al.,

1988). However, rat is the most frequently

used rodent species in repeat dose

toxicology studies. Several recent studies

have demonstrated the feasibility of

measuring MN-PCE in bone marrow at the

termination of repeat dose rat toxicology

studies (MacGregor et al., 1995; Albanese

and Middleton 1987; Garriot et al., 1995;

Çelik et al., 2003; Çelik et al., 2005) thus

taking advantage of the opportunity to

correlate genetic with conventional toxicity

data in this species. The circulating blood of

the mouse has been accepted as an

appropriate target for micronucleus

assessment for both acute and cumulative

damage. Very recently, studies conducted in

Japan have addressed the issue of the

suitability of rat blood for micronucleus

assessment. These studies support the use of

rat peripheral blood for evaluation of

micronucleus induction in PCE.


No studies on the genotoxic effect of SR on any

cell type could be found in the literature in vivo

and/or in vitro test systems. The aim of present

study is to provide new data on genotoxic potential

risks of strontium ranelate on the rat peripheral

blood using acridine orange staining- micronucleus

test in acute and chronic treatment.

Materials and methods

Animal treatment

The Institutional Animal Care and Use Committee

at Mersin University Medical Faculty approved the

experiments described in this study. Thirty, twelveweek-old

Sprague-Dawley female rats each

weighing 200–250 g were used. The animals were

acclimatized for 1 week to our laboratory

conditions before experimental manipulation. They

had free access to standard laboratory chow and

water ad libitum was maintained on 12 h/12 h light

dark cycle throughout the experiment. This study

utilizes two treatments, acute and chronic. Rats

were assigned randomly to a negative control group

(n=5), a positive control group (n= 5) and chronic

strontium group (n = 5). The rats were treated by

gavage with 500 mg/kg of SR (Figure 1) dissolved

in saline three times per week for 12 weeks for

chronic treatment and once for 24 hours. Each

treatment includes negative and positive control

groups. Since positive controls can be administered

by a different route and treatment schedule than the

test agent, a single dose of MMC (2 mg/kg, i.p.)

was administered at the 12th week dosing time.

Dose selection

Strontium ranelate [PROTOS® (strontium ranelate

2g)] was obtained as a characterized drug from

Servier Pharmaceuticals.

Description

Description of substance and solubility: Strontium

ranelate (SR) is a yellowish-white non-hygroscopic

powder. It crystallises as a nonahydrate form but

one water molecule is particularly labile and this

leads to a compound containing either 8 or 9 water

molecules per strontium ranelate molecule.

Strontium ranelate is slightly soluble in purified

water (3.7 mg/mL at saturation point) and

practically insoluble in organic solvents (eg,

methanol).

Strontium ranelate genotoxicity 29

Excipients

Aspartame (E951, a source of

phenylalanine), maltodextrin, mannitol.

Chemical name: Strontium ranelate. CAS

Registry Number: 135459-90-4 Molecular

formula: C12H6N2O8S, Sr2 (Figure 1). The

chemical name applied to SR is 5-[bis

(carboxymethyl)amino]-2-carboxy4-cyano-

3- thiophenacetic acid distrontium salt. The

Sr content of SR is 34.1% for a relative

molecular weight (anhydrous) of 513.49.

Figure 1. Chemical structure of strontium

ranelate

Presentation

Granules for oral suspension. PROTOS 2g

sachets contain 2g strontium ranelate as a

yellow powder. The dose selection of SR

was based on human exposures. The 500

mg/kg dose was an approximate

environmental daily level. In literature, there

are toxicity studies conducted on adult rats

with 225–900 mg/kg per day dose (Marie

2005; Ammann et al. 2007).

MMC (2 mg/kg) was used as a positive

control. The positive control and the

untreated control rats were identically

treated with equal volumes of normal saline

only via intraperitoneal (i.p.) injection. It is

acceptable that a positive control is

administered by a different route from or the

same as the test agent and that it is given

only a single time (Hayashi et al. 1994).

MMC was given as a single dose.

Tissue preparation

All the animals used for experiments were


30 Ayla ÇELİK et al.

anesthetized by ketamine hydrochloride (Ketalar,

Eczacibasi Ilac Sanayi ve Ticaret A.S., Istanbul,

Turkey). Blood samples were taken from their

hearts into tubes. Then the both femora bone were

removed by dissection.

MN assay in peripheral blood and bone marrow

smears

Whole blood smears were collected on the day

following the last strontium administration or 1st

day after chronic and MMC treatment. Whole

blood smears were prepared on clean microscope

slides, air dried, fixed in methanol and stained with

acridine orange (125 mg/ml in pH 6.8 phosphate

buffer) for 1 min just before the evaluation with a

fluoresence microscope using a 40X objective

(Hayashi et al., 1994). The frequency of PCEs per

total erythrocytes was determined using a sample

size of 2000 erythrocytes per animal. The number

of MNPCEs was determined using 2000 PCE per

animal.

The frequency of micronucleated erythrocytes

in femoral bone marrow was evaluated according to

the procedure of Schmid (1976), as performed in

femoral bone marrow, with slight modifications.

The bone marrow was flushed out from both

femora using 1 mL fetal bovine serum and

centrifuged at 2000 rpm for 10 min. The

supernatant was discarded. Bone marrow smears

were prepared on clean microscope slides, airdried,

fixed in methanol, and stained with acridine

orange (125 mg/ml in pH 6.8 phosphate buffer) for

1 min just before the evaluation with a fluorescence

microscope. In order to determine the frequency of

micronucleus, 2000 PCEs per animals were scored

to calculate the MN frequencies, and 200

erythrocytes (immature and mature cells) were

examined to determine the ratio of PCE to

normochromatic erythrocytes (NCEs) for bone

marrow analysis.

Briefly, immature erythrocytes, i.e. PCEs, were

identified by their orange–red color, mature

erythrocytes by their green color and micronuclei

by their yellowish color.

Statistical analysis

Data were compared by one-way variance analysis.

Statistical analysis was performed using the SPSS

for Windows 9.05 package program. Multiple

comparisons were carried out by least significant

difference (LSD) test. P≤ 0.05 was

considered as the level of significance.

Figure 2. Arrow indicates acridin-orange

stained micronucleus in immature

(polychromatic) erythrocyte of rat treated

with SR (500 mg/kg).

Results

A representative fluorescence

photomicrograph of MNPCE from a SRtreated

rat is shown in Figure 2. SR (500

mg/kg b.w) treatment induced the frequency

of MN in both rat bone marrow and

peripheral blood. There is a significant

difference between SR-treated rats and

negative control rats for micronucleus

induction. In peripheral blood and bone

marrow tissue, although the MNPCE

frequencies (4.80±0.48 and 5.00±0.31,

respectively) in rats treated with SR were

significantly higher than the frequency in

negative control (1.60±0.24 and 2.20±0.20,

respectively), they were much less than the

MNPCE frequency induced by the positive

control, 2 mg/kg MMC (41.0 ±0.44,

42.4±0.92, respectively). Table 1 represents

micronucleus induction and the PCEs/NCEs

ratios in bone marrow and peripheral blood.

SR treatment significantly decreased the

PCE number when compared to controls in

both bone marrow and peripheral blood (p <

0.001). SR is a toxic substance in both bone

marrow at acute treatment and peripheral

blood at chronic treatment. While PCE

number was 2.60±0.25 in the control group

of chronic treatment, this value reached

1.2±0.20 at chronic treatment of SR. While

PCE number was 103±1.40 in the control


group of acute treatment, this value reached

76.8±1.82 at acute treatment of SR.

Discussion

From a drug development standpoint, it is

important to have a thorough understanding of the

mechanism of any positive genetic toxicology

findings, so that informed decisions can be made

with respect to risk. This is particularly important

because of an increasing experience suggesting that

many “positive” gene-tox results may arise

artifactually as a consequence of cytotoxicity rather

than from true drug/DNA interactions. For

example, cytotoxicity may be due to lysosomal

damage and release of DNA endonucleases, ATP

Strontium ranelate genotoxicity 31

depletion or impairment of mitochondrial

function (Galloway, 2000). The field of

toxicology, especially toxicology practices

for regulatory purposes, has not changed in

several decades. Preclinical safety testing is

centered on in vivo laboratory animal

studies. These in vivo studies have been

valuable in the prevention of some toxic

drug candidates from further development,

as they are effective in the detection of

toxicity that are common to both humans

and non-human animals.

Table 1. Micronucleus induction and the PCEs/NCEs ratios in bone marrow and peripheral blood of

female Wistar rat induced by SR (500 mg/kg) treatment.

Groups

Isotonic saline

Mean ±SE

SR(500mg/kg

b.w.)

Mean ±SE

MMC(2 g/kg

b.w.)

Mean±SE

(n)

1

2

3

4

5

1

2

3

4

5

1

2

3

4

5

Chronic treatment

(peripheral blood)

MN/2000 PCE/2000

PCEs

erythrocytes

1

2

2

2

1

1.60±0.24

6

4

6

4

4

4.80±0.48***

41

42

40

42

40

41.0±0.44***

2.1

2.2

3

3.4

2.1

2.60±0.25

1

1

1

2

1

1.2±0.20**

1.2

1.3

1

1

1

1.10±0.06***

Acute treatment

(Bone marrow)

MN/2000 PCE/200

PCEs erythrocytes

2

2

3

2

2

2.20±0.20

4

5

5

5

6

5.00±0.31*

**

20

22

21

20

21

20.8±0.37*

**

108

105

100

102

102

103±1.40

75

75

82

80

72

76.8±1.82***

45

44

42

41

40

42.4±0.92***

***p


32 Ayla ÇELİK et al.

An advantage of animal studies is that they

provide a complete biological system, which can

evaluate the overall effect of subtle changes

observed in cell systems. Carefully controlled

animal studies are essential steps in the

extrapolation of biological effects to human health

safety. The fundamental similarities in cell

structure and biochemistry between animals and

humans provide a general valid basis for prediction

of likely effects of chemicals on human populations

(Garriot et al., 1995; Çelik et al., 2003). In this

study, SR induced micronucleus formation in both

peripheral blood and bone marrow and lead to

decreasing of the PCE number at chronic and acute

treatment in rats.

Important contribution to the knowledge of

strontium was obtained in the 1950s and 1960s. A

comprehensive review on strontium was published

in 1964. Strontium in human biology and pathology

has attracted less attention than the other divalent

metals such as magnesium and calcium and over

the years been an object of academic rather than

clinical interest. Strontium is not metabolized in the

body. However, strontium does bind with proteins

and, based on its similarity to calcium, probably

forms complex formation with various inorganic

anions such as carbonate and phosphate, and

carboxylic acids such as citrate and lactate.

Strontium is also found in the soft tissues, although

at much lower concentrations than in bone.

Strontium toxicity was studied by many

investigators. Intravenous administration of high

doses of strontium induces hypocalcaemia due to

increased renal excretion of calcium. Stable

strontium containing chemicals is considered as

harmful to humans (Meunier et al. 2004, U.S.

Department of Health and Human Services, 2004).

In this study, SR (new pharmaceutical) induced the

micronucleus frequency and decreased the PCE

ratio in peripheral blood and bone marrow chronic

and acute treatment, respectively.

Genotoxicity activity is normally indicated by a

statistically significant increase in the incidence of

micronucleated immature erythrocytes for the

treatment groups compared with the control group;

historical vehicle/negative control results are also

taken into account. Bone marrow cell toxicity (or

depression) is normally indicated by a substantial

and statistically significant decrease in the

proportion of immature erythrocytes; a very large

decrease in the proportion would be indicative of a

cytostatic or cytotoxic effect. Pollution by

heavy metals is an important problem due to

their stable and persistent existence in the

environment. The in vivo micronucleus test

used in this study was a very sensitive

method to evaluate the chromosomal

damage in mammalian cells exposed to

chemical substances. Micronuclei are

cytoplasmic chromatin masses with the

appearance of small nuclei that arise from

chromosome fragments or intact whole

chromosomes lagging behind in the

anaphase stage of cell division. Their

presence in cells is a reflection of structural

and/or numerical chromosomal aberrations

arising during mitosis (Holden et al., 1997,

Heddle et al., 1991). In general, genome

damage caused by accidental over exposure

may result from interactions such as the

formation of DNA damage directly or via

free radicals, but also from damage to the

nuclear membrane, lipid peroxidation,

methylation disturbances, activation of a

chain of signal molecules influencing the

expression of apoptosis, and other

mechanisms including hormonal, age related

bioaccumulation of pollutants, metabolism

and clearance (Giles, 2005)

Recently, strontium has been studied for

bone tissue engineering in osteoblastic

ROS17/2.8 cell culture. Osteoblastic cells

were seeded on strontium-doped calcium

polyphosphate scaffolds. This novel

strontium-releasing scaffold system was

found to be a promising material for bone

tissue engineering (Qiu et al., 2006).

Senkoylu et al. (2008) evaluated the effect

of SR on H2O2-induced apoptosis of CRL–

11372 cells. They assessed quantitatively

with a fluorescent dye and qualitatively with

agarose gel electrophoresis the apoptotic

index and viability of cells. Concentrations

of 1–100 µM of SR with a 6 h treatment and

only 1 µM concentration with a 12-h

treatment inhibited the apoptotic effect of

H2O2 on cultured osteoblasts significantly

(P


indicated in in vitro studies. Furthermore, SR

decreases preosteoclast differentiation and

osteoclastic activity dose dependently (Canalis et

al., 1996; Baron and Tsouderos, 2002).

The absorption of strontium and calcium from

the gastrointestinal tract is carried out by the same

mechanisms. It has long been suggested that

excessive doses of strontium could disturb the

calcium metabolism (Takahashi et al., 2003). In the

study performed to assess the toxic dose levels by

Morohashi et al. (1994), rats received daily

strontium doses ranging from 77–770 mg/kg per

day for 1 month. Net intestinal calcium absorption,

fractional calcium absorption (relative to intake)

and calcium retention in the body were all

markedly reduced in the group that received 770

mg/kg per day, but none of these parameters were

significantly affected in the groups receiving less

than 153 mg/kg per day. Morohashi et al. (1994)

determined that the toxic effect of strontium is

dependent on doses. Some drugs such as

alenderonate and tibolone, is advised in order to

therapy the osteoporosis. In another study

performed in postmenopausal women with

osteoporosis, Bayram et al. (2006) investigated the

genotoxic effects of the alendronate treatment with

or without tibolone using comet assay. They

reported that the Comet assay revealed that tibolone

did not cause any DNA damage, but alendronate

did at the end of the 1-year administration of these

drugs. In other studies performed in relation to

drugs used in osteoporesis treatment, conclusive

results were obtained for genotoxic damage. Şahin

et al. (2000) reported that alendronate did not show

any signs of genotoxic effects according to the

sister chromatid exchange (SCE) assay. However,

some of the bisphosphonates like pamidronate and

zoledronate have been reported to cause DNA

fragmentation (Şahin et al., 2000). Taking into

consideration the long years of accumulation of

these drugs in the bone, DNA damage may be

important. Considering that there is still a lack of

information regarding the essentiality and toxicity

of SR, plasma data showed large individual

variation, resulting in uncertain pharmacokinetic

profiles. No studies on the genotoxic effect of SR

on cells could be found in the literature. Oral

administration of 130 mg strontium/kg body weight

as strontium chloride to Swiss albino female mice

increased the incidence of chromosomal aberrations

(gaps, breaks, nondisjunction, polyploidy) in bone

Strontium ranelate genotoxicity 33

marrow cells 5-fold after 6 hours (Ghosh et

al., 1990). Genotoxicity in male mice

administered a similar dose (140 mg/kg) was

only doubled, and therefore, less severe than

in females. At higher dose (1,400 mg/kg),

the incidence of chromosomal aberrations

was similar in both sexes after 6, 12, or 24

hours. In study performed by Berköz et al.

(2008) it is shown that SR decreased the

paraoxonase level in rats receiving SR only

one time, underwent ovariectomy operation

and did not receive any drug and treated

with strontium ranelate for three months

after three months from the ovariectomy

operation. Paraoxonase protects from

oxidation the lipoproteins, Therefore in our

opinion, this issue is very important in

explaining for its use in treatment of

established osteoporosis in postmenopausal

women.

In conclusion, although the studies

regarding the geno-cytotoxic effects of

drugs used in osteoporosis therapy are

contradictory, our results clearly

demonstrated that chronically and acutely

administration of SR (500 mg/kg)

significantly increased the frequency of

MNPCEs and decreased the % PCEs in

peripheral blood of rats. Evaluation of the

role of drug metabolism and toxicity is

arguably a necessary activity for the

evaluation of human drug toxicity. It allows

a rationale design of a safer molecule (e.g.

by blocking sites critical for toxic metabolite

formation), assessment of sensitive human

population (e.g. populations with high level

of the drug metabolizing enzyme pathway

for the formation of toxic metabolites;

populations with low detoxifying activities;

environmental factors leading to high levels

of “activating” activities or low levels of

“detoxifying” activities). Future studies will

be necessary on experimental animal models

using different doses-period and test

methods.

Acknowledgements

Authors are grateful to Dr. Gökhan Coral

(Ph.D.) for the assistance in preparation of

the schematic figure of micronucleus and for

laboratory availability.


34 Ayla ÇELİK et al.

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59.pdf


Journal of Cell and Molecular Biology 9(2):37-42, 2011 Research Article 37

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Cloning, expression, purification, and quantification of the 17% Nterminal

domain of apolipoprotein b-100

Hassan M. KHACHFE * 1, 2, 3 and David ATKINSON 3

1 Faculty of Sciences-V, Lebanese University, Nabatieh, Lebanon

2 Departments of Biological and Biomedical Sciences, Lebanese International University, Beirut, Lebanon

3 Department of Physiology and Biophysics and Center For Advanced Biomedical Research, Boston

University School of Medicine, 715 Albany Street, Boston MA 02118, USA

(*author for correspondence; hassan.khachfe@liu.edu.lb )

Received: 22 August 2011; Accepted: 9 December 2011

Abstract

Apolipoprotein B-100 (apo B) is the sole protein component of normal human low density lipoprotein (LDL).

Elevated levels of LDL have been correlated with atherosclerosis and other coronary artery diseases. The

large size of apo B (4536 aa) necessitates that it be studied in pieces corresponding to its structurally

organized domains. The 17% N-terminal domain of apo B, simply B17, poses as one of these domains,

having very specific structural characteristics. The current report describes a set of protocols for the cloning,

expression, purification, and quantification of this important part of the protein.

Keywords: Apolipoprotein (Apo B), C127 cells, cloning, low-density lipoprotein, Sf9 cells

Apolipoprotein B-100’ün %17 N-Terminal bölgesinin klonlanması, anlatımı, saflaştırılması ve

kantifikasyonu

Özet

Apolipoprotein B-100 (apo B) normal insan düşük yoğunluklu lipoprotein (LDL)’in yegane protein

Artmış komponentidir. LDL düzeyleri ateroskleroz ve diğer koroner arter hastalıklarla ilişkilendirilmiştir.

Apo B’nin büyüklüğü (4536 aa) yapısal olarak olmuş organize domeynlere karşılık gelen parçalar halinde

çalışılmasını gerektirmektedir. ApoB’nin N-terminal bölgesinin %17’si olan B17, çok özel yapısal özellikleri

olan bu domeynlerden biridir. Bu makale proteinin bu önemli parçasının klonlaması, anlatımı, saflaştırılması

ve kantifikasyonu için protokolleri açıklamaktadır.

Anahtar Sözcükler: Apolipoprotein (Apo B), C127 hücreleri, klonlama, düşük yoğunluklu lipoprotein

(LDL), Sf9 hücreleri.

Introduction

Standing as one of the largest known proteins

known, apolipoprotein B100 is the sole protein

constituent of LDL (Mahley et al., 1984). The

entire protein is a single peptide chain composed of

4536 amino acid residues, plus a 27 amino acid

signal sequence. When glycosylated, the protein

has a molecular mass of ~550 kDa, but the mature

de-glycosylated chain is 512,937 Da. (Chen et al.,

1986; Cladaras et al., 1986; Knott et al., 1986; Law

et al., 1986; Yang et al., 1986). Apo B100 is

synthesized in the liver and packaged into VLDL

within the inner leaflet of the endoplasmic

reticulum (Olofsson et al., 1987; Pease et al.,

1991).

Apo B100 has 25 cysteine residues of which

sixteen form disulfide bonds (Yang et al., 1990;

Shelness and Thornburg, 1996; Huang and

Shelness, 1997). Except for the Cys-1/Cys-3 and

Cys-2/Cys-4 bridges, all the other disulfide bonds

occur between neighboring cysteines. All of the

disulfide bonds occur in regions that are releasable


38 Hassan M. KHACHFE and David ATKINSON

from the particle by trypsin digestion (Yang et al.,

1989).

Because of the size and insolubility of apo B,

determination of its structural motifs responsible

for the lipid association has been so difficult that

only indirect probing has been done on this

nonexchangeable protein. Biochemical and

biophysical techniques, as well as computer

algorithms have been used to study the domain

structure and rearrangements of apo B. These

studies have deepened our understanding of the

domain arrangement of this huge protein.

Therefore, it is necessary to study the structure

of the protein in pieces, perhaps corresponding to

structural or functional domains. For this reason,

genetically engineered truncated forms have been

obtained to study the domain organization in the

protein. The N-terminal portion of the protein

posed as an interesting candidate for structural

studies for several reasons:

1) The striking homology it shows with other lipid

transporting proteins, e.g., lipovitellin, whose

structure was solved and studied vis-à-vis its

function, and therefore, opened the door for

computer modeling of the structure of apo B (Al-

Ali and Khachfe, 2007).

2) This portion of the protein shows an optimal

interaction with the microsomal triglyceride

transfer protein (MTP). The presence of MTP

complexed to the protein disulfide isomerase (PDI)

found in the endoplasmic reticulum is an absolute

requirement for the assembly of neutral lipids and

phospholipids into chylomicrons and VLDL

particles (Hussain et al., 1997).

3) Although truncated, this part of the protein is

readily associated with a variety of phospholipids

to from large discoidal particles (Herscovitz et al.,

2001), and has interesting metabolic behaviors

based on its glycosylation state, such that the

Q158N mutation of the single glycosylation site in

this domain decreases the secretion of the protein,

but has little effect on its synthesis or its

intracellular distribution (Vukmirica et al., 2002).

4) As mentioned above, the fact that seven out of

the eight disulfide bonds found in apo B100 are

located in the N-terminal domain suggest that this

portion is compact, highly organized, and most

likely globular (Prassl and Laggner, 2009).

Hence, the 17% N-terminal domain of apo B100 was

expressed with an aim to later characterize its

structure and eventually relate to its function in the

full-length protein. One of the restriction enzymes

used to cut the apo B100 gene yielded a portion that

corresponds to the 17% N-terminal part of the

protein (Herscovitz et al., 1991). Following the

same nomenclature process that described the

different truncated forms of apo B100, this portion of

the protein that corresponded to the N-terminal

17% of the full-length protein was then called apo

B17, or simply B17.

Materials and methods

Materials

Murine mammary carcinoma cells (C127)

overexpressing B17 were obtained from Dr. V.

Zannis (BUMC, Medicine) (Cladaras et al., 1987).

Sf9 cells were from Life Technologies

(Gaithersburg, MD). Baculovirus particles cloned

with the B17 gene were a kind gift from Dr. G.

Carraway. Dulbecco’s modified Eagle’s medium

(DMEM), Sf-900 II serum-free medium (SFM),

bovine fetal serum (BFS), penicillin / streptomycin

(PS), and trypsin-EDTA were from Life

Technologies (Gaithersburg, MD). N-Acetyl-Lleucinyl-L-leucinyl-L-norleucine

(ALLN),

aprotinin, leupeptin, phenyl-methyl-sulfonylfluoride

(PMSF), sodium azide, and ethylenediamine

tetraacetate (EDTA) were from Sigma (St.

Louis, MO). Broad range protein marker (6,500 –

200,000 MW) was from BioRad (Hercules, CA).

Gelatin Sepharose and Protein-G Sepharose 4

Fast Flow were from Pharmacia Amersham

(Piscataway, NJ). Polyclonal goat anti-human apo

B IgG, alkaline phosphatase-conjugated rabbit antigoat

IgG, and horse-radish peroxidase (HRP)conjugated

rabbit anti-goat IgG were from

BioDesign (Saco, ME). Polyclonal sheep antihuman

apo B IgG was from Roche Molecular

Biochemicals (Indianapolis, IN).

C-127 cells were permanently transfected with

the gene coding for B17 as previously described

(Claderas et al., 1987; Herscovitz et al., 1991).

Mass expression of the protein was achieved using

roller bottles (Claderas et al., 1987) or a Verax

System-1 Bioreactor (Verax, Lebanon, NH) that

was modified – in-house – and coupled with a

ceramic core reactor, which eventually increased

the number of cells to near tissue density while

automating the media feed and harvest processes.

The harvested or stored media were then

vacuum-filtered through 0.45 µm pore size filter

paper, and then concentrated 25-fold using an

Amicon stirred-cell with a 30,000 MWCO

membrane. The concentrate was processed for

protein purification.

Sf9 – Spodoptera frugiperda – insect cells

adapted to serum-free suspension culture in Sf-900


II SFM were grown in 100 – 500 ml Erlenmeyer

flasks on an orbital shaker at 29ºC. The suspension

culture was infected with the virus carrying the B17

gene when the cells were in mid-exponential

growth and the density of cells is between 1 – 3 x

10 6 cells/ml. 2 plaque-forming units (pfu) were

added to each cell in suspension, a parameter that is

usually called multiplicity of infection, MOI.

Harvesting is done 24 to 48 hours later. The media

is then centrifuged at 250xg for 5 minutes, and the

supernatant is collected and stored in 2 μg/ml

PMSF or aprotinin, 2 mM EDTA, and 0.05% NaN3

at 4 ºC.

The transfection with the B17 gene was done

with a pDLST8 plasmid containing the B17 cDNA

sequence into a recombinant donor plasmid. The

donor plasmid was then hosted for one day in

competent DH10Bac E. coli cells, and

subsequently transposed for antibiotic selection for

2 days in E. coli (Lac7) containing a recombinant

bacmid. In day 4, the recombinant bacmid DNA

was introduced into the Sf-9 cells for recombinant

virus particles to be produced the next two days. A

viral titer was done by plaque assay to determine

the number of pfus in the stock and to concentrate,

if necessary. The viral stock was then used to infect

other suspension cultures.

Aliquots of the stored media stock were

incubated for two hours in glass tubes containing

protein-G Sepharose in the ratio 4:1 to remove

media IgGs prior to the last incubation for two

hours in an immuno-adsorbant column. This

immobilization column contained a similar bed

volume of protein-G sepharose coupled with antiapo

B IgG. The anti-B IgG was crosslinked to the

protein-G Sepharose with DMP in TEA or a basic

Na-phosphate buffer, and the blocking was

achieved with ethanolamine. The immobilized B17

was then eluted with an acidic glycine buffer (pH

2.5), and the eluate was brought a neutral pH by

adding a volume of tris (pH 8) amounting to 10%

of the total elution volume. The eluate was then

tested for purity, dialyzed against a K-phosphate

buffer (pH 7.4) and stored at 4ºC.

The concentration of B17 in solution was

initially determined by the Lowry method using

bovine serum albumin as the standard (Lowry et

al., 1951). Alternatively, a multiplicity factor was

determined so that the concentration can be

approximated from the direct measurement of the

protein UV absorbance at 280 nm, A280. This was

achieved by preparing two sets of samples with the

same amount of protein in each sample. One set

was diluted with differing volumes of chemical

Expression, purification and quantification of B17 39

denaturant (e.g. urea), while the other was diluted

with the same volumes of the sample buffer. The

absorbance A280 was then measured for both sets

and the one with denaturant was compared with the

reported A280 of tryptophan and tyrosine. A

multiplicity factor of 2 was then determined such

that the concentration of B17 in that solution is

equal to A280 x 2 (mg/ml).

Results

Figure 1 shows the initial protein production check

that was done in the early stages of cell growth –

before introduction to roller bottles or bioreactor.

The Western blot shows a single band

corresponding to B17 – probed by the apoB

polyclonal antibody.

Figure 1. B17 production from C-127 cells. The

band corresponding to B17 on (A) Coomassie

stained SDS-PAGE and (B) the Western blot of the

identical gel, aligned with Bio-Rad broad range

protein marker.

The large-scale mass expression in the roller

bottle or the bioreactor, however, showed that, upon

prolonged incubation necessitated by these

techniques, degradation products begin to form

(Figure 2), a problem that was solved by the

addition of a protease inhibitor, PMSF, to the

conditioned media (Figure 3).

Figure 2. Degradation problem in the mammalian

system. Western blot of B17 from the bioreactor

media samples taken at different incubation times

showing the appearance of the degradation product

as a function of time.


40 Hassan M. KHACHFE and David ATKINSON

The Sf-9 cell system was analyzed for protein

production as early as the first virus infections took

place (Figure 4). The degradation problem was also

present in this system. Although the incubation time

between infection and harvesting in the Sf-9 cells

was less than half of that in the C-127 cells (Figure

4), the relative intensity of both the B17 band and

the degradation product band were comparable to

those of the C-127 cell system. The problem was

solved by the addition of EDTA to the suspension

media (Figure 5).

Figure 6 shows a comparison between the two

expression systems in terms of their protein

production and purity. While both the mammalian

and insect systems produced identical products in

terms of their PAGE behavior, the yield from the

Sf-9 cells was 15-fold higher than that of the C-127

cells. Upon confirming that both products were

identical in terms of their secondary structural

contents (CD data not shown), we decided to abort

the protein production from the C-127 cells, and

continue with the Sf-9 cells. The purity of the

protein was further assessed using Mass-

Spectrometry (data not presented), which showed a

single peak at around 88 kDa proving that the

expressed protein is pure and has a molecular mass

of 88 kDa (in agreement with the calculated

molecular mass of 87.7936 kDa).

Figure 3. Analyzing expression by C-127 cells.

The PMSF protease inhibitor treatment. (A) shows

the Western blot bands corresponding to B17 and

the degradation product, while (B) shows the single

band corresponding to B17 following treatment

with PMSF as described in the methods. (C)

represents the Coomassie stained gel band for B17

after immuno-affinity purification. (D) is the

accompanying BioRad broad range protein marker

lane.

Figure 4. The Western blot of B17 overexpressed

in Sf-9 cells and harvested after 30 hours (A),

compared to B17 overexpressed in C-127 cells and

harvested after 72 hours (B).

Figure 5. Analyzing expression by the insect

system. Western blot demonstrating protection by

different protease inhibitors. The control lane (A)

corresponds to untreated media; individual lanes to

the right correspond to samples from media treated

by adding: (B) 0.5% FBS; (C) 0.05 mM EDTA; (D)

40 µg/ml ALLN; (E) 40 µg/ml Aprotinin; (F) 100

µg/ml PMSF. Addition of PMSF killed the cells

immediately and no detectable amount of protein

was expressed. (G) SDS-PAGE of purified B17

from EDTA-treated media.

Figure 6. Product comparison. Coomassie-stained

SDS-PAGE gels showing B17 purified from the

insect (A) and mammalian (B) (bioreactor)

systems.


Several truncated forms of apo B100 have been

identified in the plasma of human subjects, the

shortest of which was denoted apo B31,

corresponding to 31% portion of the full length

protein (Havel, 1989; Young et al., 1990). Although

shorter forms are indeed synthesized, they either

don't find their way to the plasma because they are

not secreted or, once in the plasma, they are rapidly

degraded (Collins et al., 1988). The present study

reaffirms previously reported results showing that

shorter forms of apo B100, namely B17, can be

secreted by C127 cells (Herscovitz et al., 1991) and

Sf-9 cells (Choi et al., 1995).

However, this study shows that the mass

expression of B17 results in degradation products

that are clearly correlated with the larger number of

cells used in the expression system. The use of a

protease inhibitor, PMSF, in the C127 cell system

prevented the degradation, indicating that proteases

in the media and/or inside the cells were

responsible for this effect. On the other hand,

addition of EDTA to the insect cell media to a final

concentration of 20µM EDTA also prevented the

degradation in that system.

SDS-PAGE analysis of the expressed and

purified proteins from both expression systems

showed that the two products are similar in size.

Further assessment using CD spectroscopy

confirmed that the products expressed and purified

from the two systems are identical in terms of their

structural contents.

Discussion

Apolipoprotein B100 (apo B) is the only protein

found on human low density lipoprotein (LDL)

particles. LDL is the agent provocateur for

atherosclerosis and other coronary heart diseases.

Apo B is a large (4536 amino acids, 550 kDa)

secretory glycoprotein that has unique structural

properties. The large size of apo B necessitated that

it be studied in pieces corresponding to its

structurally organized domains. In the present

work, we studied the conformational and stability

properties of the 17% N-terminal domain of apo B,

B17. This portion of the protein is secreted

predominantly lipid-free, and plays an important

role in the initiation and assembly of the LDL

particle (Herscovitz et al., 2001).

Mass expression of B17 was achieved via two

different cell lines: Mammalian and insect. The

mammalian-derived murine C127 cells were

transfected with a bovine papilloma virus-based

expression vector, while the insect-derived Sf-9

Expression, purification and quantification of B17 41

cells were transfected with a baculovirus-based

expression vector. Previously reported methods and

protocols were enhanced and fine-tuned to

overcome a degradation problem associated with

the mass expression of the protein. The protein

yields from both systems were compared for purity

and homogeneity, and were found to be identical.

Acknowledgement

The initial C127 cell batch and the initial

baculovirus particles were kind gifts from Drs. V.

Zannis and G. Carraway, respectively. This project

was partially supported by an award from the

National Health Institutes (NIH).

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Journal of Cell and Molecular Biology 9(2): 43-49, 2011 Research Article 43

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Cysteine protease from the malaria parasite, Plasmodium berghei-

purification and biochemical characterization

Emmanuel AMLABU* 1 , Andrew Jonathan NOK 2 , Hajiya Mairo INUWA 2 , Bukola

Catherine AKIN-OSANAIYE 3 , Emmanuel HARUNA 4

1 Department of Biochemistry, Kogi State University, Anyigba,Nigeria

2 Department of Biochemistry, Ahmadu Bello University, Zaria,Nigeria

3 Department of Chemistry, University of Abuja, Gwagwalada,Nigeria

4Department of Biochemistry, Kaduna State University, Nigeria

(* author for correspondence; ninmac2000@yahoo.com)

Rceived: 6 August 2011; Accepted: 22 December 2011

Abstract

Plasmodium berghei was isolated from mice red blood cells and phase-separated by Triton X-100

temperature-induced phase separation procedures. The enzyme cysteine protease was purified 5.33 fold with

a recovery of 58 %. SDS-PAGE analysis of the enzyme revealed two protein bands with molecular weights

corresponding to 18 and 40 kDa, respectively. The enzyme was optimally active at temperature of 40 O C and

at a pH of 5.0 (50 mM acetate buffer). Activation energy (6.27 kJ/mole) of the enzyme was determined from

Arrhenius plots and initial velocity studies revealed KM and Vmax values of 2.5 mg/ml and 0.2 µmol/min,

respectively. The enzyme was inactive on the substrates, albumin and myoglobin. The enzyme was

exclusively sensitive to the cysteine protease specific inhibitor iodoacetate (IAA), but was insensitive to

Phenylmethylsulphonyl chloride (PMSF), 1, 10 phenanthroline, soybean trypsin inhibitor (SBTI), pepstatin A

and EDTA. The synthetic compounds PP-54 and PP-56, currently being evaluated for their anti-malaria

potential, competitively inhibited the enzyme activity with corresponding Ki values of 48.88 µg/ml and 0.14

µg/ml, respectively.

Keywords: Cysteine protease, Plasmodium berghei, malaria parasite, iodoacetate, enzyme activity

Malarya paraziti Plasmodium berghei’den sistein proteaz- saflaştırılması ve karakterizasyonu

Özet

Fare kırmızı kan hücrelerinden Plasmodium berghei izole edilmiştir ve Triton X-100 uyarılmış sıcaklıkla faz

ayırım yöntemleri ile faz ayırımı yapılmıştır. Sistein proteaz enzimi %58 geri kazanımla 5.33 kat

saflaştırılmıştır. Enzimin SDS-PAGE analizi sırasıyla 18 ve 40kDa moleküler ağırlıklarına karşılık gelen iki

protein bantı ortaya çıkarmıştır. Enzim pH 5.0’te (50 mM asetat tamponu) ve 40ºC sıcaklıkta optimal olarak

aktiftir. Enzimin aktivasyon enerjisi (6.27 KJ/mol) Arrhenius grafiğinden belirlenmiştir ve Km ve Vmax

değerleri başlangıç hız çalışmaları ile sırasıyla 2.5 mg/ml ve 0.2 µmol/dk olarak belirlemiştir. Enzim albumin

ve miyoglobin substratlarında inaktiftir. Enzim özellikle sistein protez spesifik inhibitör iyodoasetata

duyarlıdır; fakat PMSF, 1,10 fenantrolin, SBTI, pepstatin A ve EDTA’ya duyarlı değildir. PP-54 ve PP-56

sentetik bileşiklerinin malaryaya karşı potansiyel yarışmalı olarak inhibe edilen enzim aktivitesine karşılık

gelen Ki değerleri sırasıyla 48.88 µg/ml ve 0.14 µg/ml olarak ölçülmüştür.

Anahtar Sözcükler: Sistein proteaz, Plasmodium berghei, malarya paraziti, iyodoasetat, enzim aktivitesi


44 Emmanuel AMLABU et al.

Introduction

Malaria remains a tremendous public health burden

especially for people living in the tropics,

particularly in Africa. About 300-500 million

people are infected with the malaria parasite, with

up to 1-3 million deaths per year due to the disease

(Miller et al., 1994; More, 2002; Martin et al.,

2004). The global resistance of malaria parasites to

mainstay anti-malarial drugs has intensified the

need for the identification of novel

chemotherapeutic targets and the development of

an effective malaria vaccine.

Malaria proteases play distinct roles in the

modification of parasite proteins involved in host

cell recognition and invasion of red blood cells.

Cysteine proteases of parasites have been suggested

to have an extracorporeal function in the digestion

of host tissues (Rhoads and Fetterer, 1997).

Maturing schistosomula and adult schistosomes

degrade hemoglobin using cysteine protease for

viability maintenance and egg production in the

host (McKerrow and Doenhoff, 1988). Plasmodium

cysteine protease has been reported to have a

critical role in hemoglobin degradation within the

food vacuole of Plasmodium falciparum (Rosenthal

et al., 1988).

Despite the availability of literature on cysteine

proteases from malaria parasites, the properties of

the enzyme from the aqueous and/or detergent

phase(s) of the parasite have not been described. In

the present work, we report some properties of the

enzyme isolated from the detergent-treated phase of

the malaria parasite which can be exploited for

precise drug targeting.

Materials and methods

Materials

The compounds (PP-54 and PP-56) were

synthesized in India and obtained by Professor A.J

Nok and are presently undergoing trials as potential

anti-malarials. Other chemicals used in this study

were obtained from Sigma, USA. The malaria

parasite Plasmodium berghei was obtained from the

Kuvin Medical Centre, Hebrew University of

Jerusalem Ein keren, Israel. The strain was

maintained in our laboratory by serial blood

passage from mouse to mouse.

Experimental infection

A donor mouse with rising parasitemia of 20 % was

sacrificed and blood was drawn in heparinized

syringe and diluted in phosphate buffered saline.

Infection was initiated by needle passage of the

parasite preparation from a donor mouse to healthy

mice via intraperitoneal route (Peter and Anatoli,

1998; Klemba and Golberg, 2002). Each mouse

received 0.2 ml of the diluted infected blood.

Course of infection

Parasitemia was monitored by microscopic

Giemsa-stained thin blood smears. The number of

parasitized erythrocytes in about 10-50 fields were

counted twice and the average was computed to

give the parasitemia of each mouse.

Separation of parasite proteins

The malaria parasites were phase-separated by

Triton X-100 temperature-induced phase separation

procedures, using the previously described protocol

by Smythe et al. (1990) with slightly modifications.

Briefly, 0.5 % Triton X-100 in Tris-buffered saline

was added to the parasites and incubated at 4 o C for

90 min. The supernatant was collected after an

initial centrifugation at 10,000 x g for 30 min at

4 o C and was layered on 6 % sucrose containing

0.06 % Triton X-100 followed by incubation at

37˚C for 5 min. The aqueous and detergent phases

were collected after an initial centrifugation at

900xg for 5 min at 37 o C and were precipitated with

cold acetone. The resulting precipitates were

referred as the aqueous and detergent phase

proteins, respectively. The pellets from each

preparation were suspended to 6 ml in 50 mM

phosphate buffered saline, pH 7.2

Enzyme activity assays

The aqueous and detergent phases of the malaria

parasite were used for activity assays by incubating

50 µl of the sample with 500 µl of 100 mM sodium

acetate buffer, pH 4.5, and 100 µl of 3 % gelatin.

The reaction volume was adjusted to 1 ml with

distilled water. Assays were carried out at 37 ºC for

an hour and were stopped by the addition of 200 µl

of 20 % (v/v) trichloroacetic acid. The precipitated

protein was removed by centrifugation (10,000 x g

for 5 min at room temperature) and absorbance of

the supernatant was read at 366 nm (Dominguez

and Cejudo, 1996). One unit of proteolytic activity

was defined as 1µmole of tyrosine hydrolyzed per

hour under standard assay conditions.

Enzyme purification

The crude proteins from the detergent-treated phase

of the parasite was applied onto a DEAE-cellulose

column (1 cm X 12 cm) pre-equilibrated with 50


mM of phosphate buffer (pH 7.2) containing 10

mM cysteine), after repeated washing with the

operating buffer which removed any unbound

material, the protein was eluted in a step-gradient

of NaCl (0.0-0.3 M) prepared in 50 mM phosphate

buffer and twenty fractions were collected. The

collected fractions were analyzed for proteolytic

activity and total protein content.

Fractions with high specific activity were

pooled and purified by gel permeation

chromatography (GPC) on Sephadex G-50

chromatography column (1cmX12 cm) preequilibrated

with 50 mM acetate buffer (pH 5.0)

Proteins were eluted isocratically from the column

with the operating buffer and thirty two fractions

were collected and analyzed for proteolytic activity

and total protein content. The active fraction

(protein peak B) which was exclusively sensitive to

the cysteine protease (CP) specific inhibitor was

characterized.

SDS-PAGE

Electrophoresis was conducted under denaturing

conditions in 12 % polyacrylamide gel as described

previously by Laemmli (1970). Protein bands were

located by staining with Coomassie Brilliant Blue

R-250.

pH activity profile

The activity profile of the purified enzyme was

determined as a function of pH using 3 % gelatin as

substrate. The buffers, 10 mM sodium acetate (pH

2-5), 10 mM Tris-HCl (pH 6-8), 20 mM

bicarbonate-carbonate (pH 9-10) were prepared at

different pH values in the range of pH 2.0-10 and

the activity of the enzyme was determined. A plot

of enzyme activity against pH was prepared to

determine the optimum pH.

Temperature activity profile

The activity of the enzyme was determined over a

temperature range of 4-60 O C and Arrhenius plot

was used to determine the activation energy (Ea) of

the enzyme.

Initial velocity studies

The substrate gelatin was prepared at a

concentration range of 0.2– 1.5 mg/ml in acetate

buffer (pH 5.0). The activity of the enzyme was

determined as described. Lineweaver-Burk plots of

the reciprocal initial velocities were plotted against

the inverse of substrate concentrations. The KM and

Vmax of the enzyme were determined from the plot.

Characterization of Plasmodium berghei cysteine protease 45

Inhibition studies

The substrate (gelatin) was prepared at a

concentration range of 3 - 0.075 gml -1 by serial

dilutions in 100 µl of 100 mM acetate buffer (pH

4.5). 50 µl of the each of the synthetic compounds

were added to the reaction mixture and was made

to a final concentration of 5 µgml -1, which is preincubated

with 50 µl of the enzyme at 37 O C for an

hour. The reaction was stopped with 200 µl of 20 %

trichloroacetic acid and absorbance was read at 366

nm.

Effects of some compounds on the enzyme

activity

The evaluation of the class of protease was based

on the pre-incubation of the purified enzyme with

0.05 mM 1,10 phenanthroline, 0.05 mM soybean

trypsin inhibitor (SBTI), 0.05 mM iodoacetate

(IAA), 0.05 mM phenylmethylsulphonyl chloride

(PMSF) and 0.05 mM pepstatin A at 37 o C for 2 hrs.

The residual enzyme activity was determined as

previously described.

Results

Synthetic compounds

The molecular weights of the compounds PP-54

(Figure 1) and PP-56 (Figure 2) are 249.31 and

336.34, and the crystallization solvents are

methanol and methanol+acetone, respectively.

Figure 1. Compound PP-54

Figure 2. Compound PP-56


46 Emmanuel AMLABU et al.

Purification of cysteine protease from Plasmodium

berghei

Initially, three protein peaks that possessed

enzymatic activity were eluted from the DE-52

Cellulose column (Figure 3). However, only the

peak with the highest specific activity was

submitted for subsequent purification steps (Table

1). The active fraction which had the highest

specific activity and was exclusively sensitive to

the cysteine protease specific inhibitor was applied

onto a Sephadex G-50 column and two protein

peaks (Peaks A and B) emerged with proteolytic

activities (Figure 4).

Figure 3. Typical elution profile for the chromatography of

Plasmodium berghei cysteine protease on DE-52 Cellulose

column.

Table 1. Purification scheme for Plasmodium berghei cysteine protease. (1U of proteolytic activity was

defined as the amount of enzyme that hydrolyzes 1 µmole of tyrosine per hour under standard assay

conditions)

Purification

Steps

Protein

(mg/ml)

Total activity

(µmol/min)

Figure 4. Gel filtration of Plasmodium berghei cysteine protease

DE-52 cellulose fraction on Sephadex G-50 Column.

SDS PAGE analysis revealed that the enzyme had

molecular weights corresponding to 18 and 40 kDa

(Figure 5). The protease was sensitive to a typical

cysteine protease inhibitor, IAA, and was

insensitive to soya bean trypsin inhibitors PMSF,

pepstatin A and 1, 10 phenanthroline. This

observation indicates the absence of other forms of

proteases (Table 2).

Specific

activity

(µmol/min)

Yield

(%)

Purification

Fold

Crude

DE-52

10.06

4.50

9.0

6.0

0.895

1.330

100

67

1.00

1.19

Sephadex

G-50

1.30 5.2 4.770 58 5.33

Figure 5. SDS-PAGE analysis of partially purified Plasmodium

berghei cysteine protease on 12 % polyacrylamide gels. Lane 1:

Molecular weight standards (Fermentas) (14-116 kDa). Lane 2-

3: GPC Purified cysteine protease.

Table 2. Effect of specific inhibitors on P. berghei cysteine

protease activity

Inhibitor Relative activity (%)

Control 100 ± 0.5

1,10 phenanthroline 93±1.5

IAA 116 ± 1.

PMSF 91 ± 1.7

SBTI 98 ± 0.3

Pepstatin A 15 ± 0.9

EDTA 105 ± 1.4


pH and temperature studies

Temperature dependent studies showed that the

enzyme was optimally active at 40 o C (Figure 6).

Arrhenius plot of the log of initial velocity as a

function of the reciprocal of absolute temperature

gave an Ea of 6.27 kJ/mol (Figure 7). pH dependent

studies revealed that the enzyme was optimally

active at pH 5.0 (Figure 8).

Activity (µmol/min - )

0.16

0.14

0.12

0.1

0.08

0.06

0.04

0.02

0

0 20 40 60 80

Temperature 0 C

Figure 6. Optimum temperature determination for

the activity of Plasmodium berghei cysteine

protease.

Activity ( µmol/min)

1.4

1.2

1

0.8

0.6

0.4

0.2

0

0 5 10 15

pH

Figure 7. Optimum pH determination for the

activity of Plasmodium berghei cysteine protease.

1/V(µmol/

min)

60

50

40

30

20

10

y = 10.305x + 4.1076

0

-6 -4 -2 0 2 4

1/S(mg/ml)

6

Figure 8. Lineweaver-Burk plot relating

Plasmodium berghei Cysteine protease reaction

velocity to gelatin concentration. KM was calculated

as milligram gelatin /ml. Each point represents the

average of three experiments.

Characterization of Plasmodium berghei cysteine protease 47

Initial velocity studies

Lineweaver Burk plots of initial velocity studies of

the enzyme gave the KM and Vmax values of 2.5

mg/ml and 0.2 µmol/min, respectively (Figure 9).

Inhibitory studies conducted with the synthetic

compounds PP-56 and PP-54, currently being

validated for their anti-malarial activity, revealed

competitive inhibition patterns with Ki values of

48.88 µg/ml (Figure 10) and 0.14 µg/ml (Figure

11).

10

Log of activity

(µmol/min)

1

y = -0.1373x + 3.683

1 2 3 4 5 6

1/T (x10 -3 )

Figure 9. Arrhenius plot for the determination of

the Ea for Plasmodium berghei cysteine protease

activity.

1/V (µmol/min)

12

10

8

6

4

2

y = 4.9261x + 2.3458

y = 1.9315x + 2.1625

0

-1.5 -1 -0.5 0 0.5 1 1.5 2

1/S (mg/ml)

No Inhibitor Compd P56

Figure 10. Lineweaver-Burk plots of initial

velocity data for the determination of inhibition

pattern on Plasmodium berghei Cysteine protease

by compound PP-56 using gelatin as substrate. Data

from three experiments were used to plot the graph

using MS Excel program.


48 Emmanuel AMLABU et al.

1/V (µmol/ml)

10

9

8

7

6

5

4

3

2

1

0

y = 3.7648x + 3.4458

y = 1.9315x + 2.1625

-2 -1 0 1 2

1/S (mg/ml)

No Inhibitor Compd P54

Figure 11. Lineweaver-Burk plots of initial

velocity data for the determination of inhibition

pattern on Plasmodium berghei Cysteine protease

by compound PP-54 using gelatin as substrate. Data

from three experiments were used to plot the graph

using MS Excel program.

Discussion

Herein, we have characterized cysteine Protease

from Plasmodium berghei and SDS-PAGE analysis

revealed that the enzyme migrated at sizes

corresponding to 18 and 40 kDa, respectively. We

have reported previously that the molecular weight

of this enzyme ranges between 20-47 kDa based on

our analysis on disc gel electrophoresis which

revealed a possible existence of variant forms of

parasitic enzyme (Emmanuel et al., 2011).

Several genes that encode potential cysteine

proteases have been identified and characterized in

Plasmodium (Shenai et al., 2000; Rosenthal, 2004).

However, refolded berghepain-2 has been reported

to be processed from 36 kDa to an enzymatically

active protein of 30 kDa upon exposure to an acidic

buffer and a purified recombinant vivapain from

Plasmodium vivax has also been reported to be

37kDa in size (Byoung-Kuk Na et al., 2010). These

reports further support the existence of cysteine

protease as a low molecular weight protein.

The protease lost a significant level of activity

in the presence of IAA. However the same

preparation was unaffected by EDTA, 1,10

phenanthroline, SBTI and PMSF. These

observations further confirm that the enzyme is

indeed a cysteine protease and excludes other forms

of proteases.

Moreover, the enzyme was activated in the

presence of cysteine and dithiothrietol (data not

shown), both compounds are thiol (-SH) containing

ingredients required for the activity of the enzyme.

This observation is supported by a previous work

on cysteine protease from T. aestivum, which is

reported to be activated by β-mercaptoethanol and

dithiothreitol (Afaf et al., 2004).

Also the pH optima of 5.0 suggest a preference

for acidic environments by the P.berghei cysteine

protease. Indeed the acidic microenvironment such

as the food vacuole (Choi et al., 1999) is an

indication that this environment will contribute to

the enhancement of the enzymatic activity as such

the pathology of malaria.

The enzyme was optimally active at 40 o C with

Ea of 6.27 kJ/mol. Such low Ea is

thermodynamically favorable, implying less

frequency of collision required to surmount the

activated complex and form the products. The KM

and Vmax values are clear indications on the

physiological efficiency of the enzyme because a

Vmax of 0.2 µmol/min presupposes that at least 12

mmol of the product will be released within an

hour. Such a level of released metabolite could be

significant in the infection mediated by the parasite.

The pattern of inhibition shown by these

compounds PP-54 and PP-56 was competitive and

mix competitive inhibition and the kinetics of

inhibition of the enzyme cysteine protease revealed

that compounds PP-56 and PP-54 inhibited the

enzyme activity with Ki values of 48.88 µg/ml and

0.14 µg/ml, respectively.

Basically, a competitive pattern of inhibition

implies that the inhibitor acts as a substrate

analogue of the enzyme by competing efficiently

with the substrate at the active site of the enzyme.

We have evaluated the anti-malaria potential of

both compounds in rodent models and both

compounds have demonstrated tremendous effect at

diminishing parasitaemia in infected mice with a

concomitant curative effect.

Also, our opinion at this time is that the antimalarial

potential of these compounds could in part

be linked to the inhibition of Plasmodium

proteases.

References

Afaf SF, Ahmed AA and Saleh AM. 2004.

Characterization of a cysteine protease from

wheat Triticum aestivum (cv.Giza 164).

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Byoung-Kuk N, Young-An B, Young-Gun Z,

Youngchoo , Seon-Hee K, Prashant VD,

Mitchell AA, Charles SC, Tong-Soo K,

Rosenthal PJ and Yoon K. Biochemical

Properties of a Novel Cysteine Protease of


Plasmodium vivax, Vivapain-4 PLoS Neglected

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Choi MH, Choe SE and Lee SH. A 54 kDa cysteine

protease purified from the crude extract of

Neodiplostomum seoulense adult worms.

Korean Journal of Parasitology. 37:39-46,

1999.

Dominguez F and Cejudo FJ. Characterization of

the endoproteases appearing during wheat grain

development. Plant Physiology. 112: 1211-

1217, 1996.

Emmanuel A, Nok AJ, Mairo IH, Catherine AB

and Haruna E. Effect of Immunization with

Cysteine Protease from Phase-Separated

Parasite Proteins on the Erythrocytic Stage

Development of the Chloroquine-Resistant,

Plasmodium berghei in BALB/C Mice. J

Bacteriol Parasitol. 2:119, 2011.

Klemba M and Goldberg DE. Biological roles of

proteases in parasitic protozoa. Annual Review

in Biochemistry. 71: 275-305, 2002.

Laemmli UK. Cleavage of structural proteins

during the assembly of the head of

bacteriophage T4. Nature. 227(5259):680–685,

1970.

Martin SA, Bygbjerg IC and Joil GB. Are

multilateral malaria researches and control

programs the most successful? Lesson from the

past 100 years in Africa. American journal of

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278, 2004.

McKerrow JH and Doenhoff MJ. Schistosome

proteases. Parasitol. Today 4: 334-340, 1988.

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Miller LH, Good MF and Million G. Malaria

pathogenesis. Science. 264:1878-1883, 1994.

More CM. Reaching maturity 25 years of TDR.

Parasitology Today. 16: 522-528, 2002.

Peter IT and Anatoli VK. The current global

malaria situation. Malaria parasite biology and

protection. ASM Press. WDC. 11-22, 1998.

Rhoads ML and Fetterer RH. Extracellular matrix:

A tool for defining the extracorporeal functions

of parasite proteases. Parasitology Today 13:

119-122, 1997.

Rosenthal PJ. Cysteine proteases of malaria

parasites. International Journal of Parasitology.

3: 1489 -1499, 2004.

Rosenthal PJ, Mckerrow JH, Aikawa M, Nagasawa

H and Leech JHA. Malarial cysteine protease is

necessary for hemoglobin degradation by

Plasmodium falciparum. Journal of Clinical

Investigation 82: 1560-1565, 1988.

Shenai BR, Sijwali PS, Singh A and Rosenthal PJ.

Characterization of native and recombinant

falcipain-2, a principal trophozoite cysteine

protease and essential hemoglobinase of

Plasmodium falciparum. Journal of Biological

Chemistry. 275: 29000–29010, 2000.

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Journal of Cell and Molecular Biology 9(2): 51-56, 2011 Research Article 51

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

Optimization of cellulase enzyme production from corn cobs using

Alternaria alternata by solid state fermentation

Amir IJAZ 1* , Zahid ANWAR 2 , Yusuf ZAFAR 3 , Iqbal HUSSAIN 1 , Aish MUHAMMAD 1 ,

Muhammad IRSHAD 2 and Sajid MEHMOOD 2

1 National Agriculture Research Center (NARC), Islamabad, Pakistan

2 Nawaz Sharif Medical College (NSMC), University of Gujrat, Pakistan

3 Biological Division PAEC Islamabad, Pakistan

(* author for correspondence; amirijaz79@yahoo.com)

Received: 3 August 2011; Accepted: 23 December 2011

Abstract

Cellulase is an important industrial enzyme which can be obtained from cheap agrowastes. Pakistan is an

agriculture country, producing tons of waste in the form of wheat straw, rice bran, sugarcane bagasee, corn

cobs, corn stover etc. The aim of the present study was to produce cellulase by using abundant agrowastes

like corn cobs. The conditions were optimized by using corn cobs and culturing Alternaria alternata with

solid state fermentation. Different incubation times (1-7days), temperatures (25 0 C, 30 0 C, 35 0 C and 40 0 C) and

pHs (3.0-9.0) were experimented for the production of cellulase. The optimum culture conditions were 96 hrs

of incubation at 35 0 C and pH 6.0, giving enzyme activities of 15.06 µg/ml, 31.2406 µg/ml, 26.4106 µg/ml,

respectively.

Keywords: Cellulase, corn cobs, agrowaste, solid state fermentation, Alternaria alternata.

Katı hal fermentasyonu ile mısır koçanlarından Alternaria alternata kullanılarak selülaz enzimi

üretiminin optimizasyonu

Özet

Selülaz ucuz zirai atıktan elde edilen önemli bir endüstriyel enzimdir. Pakistan buğday samanı, pirinç kepeği,

şeker kamışı posası, mısır koçanı vb. şekillerde tonlarca atık üreten bir tarım ülkesidir. Bu çalışmanın amacı

mısır koçanı gibi bol tarım atıklarını kullanarak selülaz üretmektir. Bu nedenle koşullar mısır koçanı

kullanılarak ve Alternaria alternata katı hal fermentasyonu ile kültür edilerek optimize edilmiştir. Selülaz

üretimi çin farklı inkübasyon süreleri (1-7 gün), sıcaklıklar (25 0 C, 30 0 C, 35 0 C ve 40 0 C) ve pH’lar (3.0-9.0)

denenmiştir. Optimum kültür şartları 35 0 C ve pH 6.0’da 96 saat inkübasyon belirlenmiş olarak ve bu

şartlarda enzim aktiviteleri sırasıyla 31.2406 µg/ml, 26.4106 µg/ml ve 15.06 µg/ml olarak tespit edilmiştir.

Anahtar Sözcükler: Selülaz, mısır koçanı, zirai atık, katı hal fermentasyonu, Alternaria alternata

Introduction

Agricultural waste is one of the major

environmental pollutants, their biotechnological

conversion is not only a remedy for environmental

problems but also the source of suitable microbial

byproducts like food, fuel and chemicals (Milala et

al., 2005). Agro-industrial wastes, e.g. wheat and

rice bran, sugar cane bagasse, corn cobs, citrus and

mango peel, are one of important wastes of food

industries of Pakistan. Their unchecked

accumulation on land serves as a source of

environmental pollution (Government of Pakistan,

2001). The most abundant renewable organic

compound in the biosphere is cellulose, which

accounts for 40-50% of plant composition and its

production is expected to be 10 10 tones from cell

wall of plants per year (Thu et al., 2008). Pakistan

contributes about 50 to 60 agro-waste million tons

per year. An agricultural waste is a cheap source of

cellulose for the production of different useful

products all over the world (Ali and Saad, 2008).

Cellulase production from agrowastes is


52 Amir IJAZ et al.

economical as compared to production from pure

cellulose (Chahal, 1985). Three major structural

polymers combined to make up lignocellulose are

called cellulose (a homopolymer of ß-D-glucosyl

units), hemicellulose (a cluster of heteropolymers

which contain xylans, arabinans, mannans,

galactans), and lignin (an intricate polyphenolic

polymer) (Rajoka, 2005).

Cellulases are a group of enzymes that break

down cellulose into glucose monomers (Yi et al.,

1999). Bacterial and fungal cellulases are

traditionally separated into three classes:

Endoglucanases (EGs) (EC 3.2.1.4), exoglucanases

(EC 3.2.1.91), and ß-glucosidases (EC 3.2.1.21)

(Kim, 2008) based on the ability to degrade

carboxymethylated cellulose (CMC), whereas EGs

being the most efficient (Henriksson et al., 1999).

The endo-ß-glucanase is responsible for the scission

of the inner bonds in the cellulose chains yielding

glucose and cell-oligosaccharides. Exo-ß-glucanase

(cellobiohydrolases) cleaves non-reducing end of

cellulose with cellobiose as the main structure

(Be´guin, 1990; Tomme et al., 1995). The ßglucosidase

(cellobiase) hydrolyses cellobiose to

glucose (Eveleigh, 1987).

Cellulase enzyme, having its importance due to

major role in industrial applications (Bhat, 2000). It

is used for bioremediation, waste water treatment

and also for single cell protein (Alam, 2005). It has

also importance in food sciences like food

processing in coffee, drying of beans by for

efficient purification of juices when used mixed

with pectinases, paper and pulp industry and as a

supplement in animal feed industry. This enzyme

helpful for plant protoplast isolation, plant viruses

investigations, metabolic and genetic modification

studies (Bhat, 2000; Chandara et al., 2005; Shah,

2007). This enzyme have also pharmaceutical

importance, treatment of phytobezons (a type of

bezoar cellulose existing in humans stomach) and a

key role in textile industry especially as its

detergent applications to recover properties of

cellulose related textiles and biofuels production

from cellulosic biomass(Ali and Saad, 2008).

Cellulases producing fungi include genra Aspergilli

(Ali and Saad, 2008) Aspergillus niger and

Aspergillus terreus, Rhizopus stolonifer (Pothiraj,

2006) Trichoderma, Penicillium, Botrytis

Neurospora etc. (Pandey et al., 1999). Fungi are

capable of decomposing cellulose, hemicellulose

and lignin in plants by secreting multifarious set of

hydrolytic and oxidative enzymes (Abd Elzaher and

Fadel, 2010).

Solid State Fermentation (SSF) is a way of

fermenting substrate in the presence of excessive

moisture in growth medium in spite of large

amount of water being provided. SSF is an

environmental friendly (less waste water

production), low energy required and economical

technology in synthesizing cellulase enzyme in

response to submerged fermentation (Pandey,

2003). SSF from last decade has made its

importance in the production of value added

products i.e., secondary metabolites, alkaloids,

enzymes, organic acids, bio-pesticides

(mycopesticides and bio-herbicides), biosurfactants,

biofuels, aroma compounds, biopulping,

degradation of toxic compounds,

biotransformation, nutritional improvement of

crops, biopharmaceuticals and bioconversion of

agricultural waste (Pandey et al., 2000).

Pakistan has to spend about 106, 986.45 million

rupees per month to import organic chemicals

(Monthly Review of Foreign Trade, 2010). A huge

quantity agricultural waste is produced from agroindustries

of Pakistan can be advantageous in

making useful by-products. A large amount of

money of our country is consumed in importing

various types of enzymes including cellulases for

local industries and research activities. The aim of

this study was to obtain a high yield of cheap

cellulase by using a local novel strain Alternaria

alternata through solid state fermentation and also

exploiting local agro-waste like corn cobs. This

study will help in proper disposal of agro-waste

resulting in resolution of the environmental

problems.

Materials and methods

Substrate selection

Agricultural waste/samples of corn cobs were

collected from local industry of Gujranwala district,

Pakistan, the substrate was dried in oven at 70 0 C

and grinded mechanically with electric grinder to

make it in powdered form and sieve to 40 meshes.

Microorganism selection

Fungal strain of Alternaria alternata was selected

for production of cellulase enzyme. The strain was

obtained from fungal bank’s stock cultures of

Institute of Plant Pathology and Mycology, Punjab

University, Lahore.


Maintenance of Alternaria alternata

Strains of Alternaria alternata maintained on PDA

medium slants under sterilized conditions of LFH

and incubated at 30 0 C for 72 hrs (Asgher et al.,

1999). The pH of medium was adjusted to

4.8 with 1M HCl/1M NaOH and was

sterilized at 121 o C for 15 minutes in

autoclave. The spores of cultured Alternaria

alternata on PDA medium were isolated aseptically

using sterilized water with 0.1% Tween 80

followed by inoculation in PDA broth. Then

inoculated flasks were placed in shaker incubator at

37 0 C and 150 rpm for 72 hrs and pH was

adjusted at 5.6 and was autoclaved for15

minutes at 15 lb/in 2 in autoclave. After

specific incubation period inoculum of Alternaria

alternata was prepared. (Smith et al., 1996).

Cellulose determination

Raw cellulose contents of corn cobs were

determined by using Weendize method as described

previously (Henneberg, 1975) and were shown as a

schematic diagram Figure 1.

1g of sample in 200mL flask

Add 1.25 of 200mL of sulphuric acid

(Remove all glucid)

Boil for 30 minutes

Filter and wash several time with hot water

Add 200 ml sodium hydroxide 1,25%

(Remove proteins by hydrolysis and fats by saponification)

Boil for 30 minutes

Filter and wash several time with hot water

the assay is treated with ethyl alcohol

(remove dyes, tannins, fats marks, the raw ash complex).

Residue is dried at 105°C, cooled and weighed residue

Figure 1. Cellulose determination procedure

Production of cellulase from corn cobs 53

Composition of culture medium

Solid state fermentation was carried out in

Erlenmeyer duplicate flasks containing 5g of corn

cobs, moistened with 10 ml distilled water,

autoclaved at 121 0 C followed by inoculation with 3

ml sporulation medium of Alternaria alternata.

Substrate (5g), moisture level (10 ml), and fungal

inoculum (3ml) were kept constant for all

optimizing steps.

Selection of optimum conditions for cellulase

production under SSF

The strategy was adopted for optimizing the

engaged parameters enhancing cellulase yield was

to optimize one specific parameter and process it at

the optimized level in the next experiment

(Sandhya and Lonsane, 1994).

Optimization of incubation period

Duplicate Erlenmeyer flasks using corncobs

cultured with A. alternata were incubated at 30 0 C

temperature for a period of 1-7 days to select the

optimum incubation period of A. alternata for the

production of cellulases. The growth was assessed

every 24 hrs and the best incubation period at

which employed strain would give maximum

cellulase activity was selected.

Temperature optimization

Duplicate flasks inoculated with A. alternata were

kept at 25 0 C, 30 0 C, 35 0 C and 40 0 C, respectively to

determine the optimum temperature at which said

strain would express high cellulase activity was to

select.

pH optimization

pH was optimized from 3.0-9.0 (50 mM) to select

optimum pH at which A. alternata would exhibit

hyper cellulase activity was selected.

Culture harvesting/ Isolation of crude cellulase

enzyme

The product of fermented cultures (cellulases) was

collected by simple contact method (Krishna and

Chandrasekaran, 1996) followed by addition of 100

ml distilled water due to neutral pH (except in case

of pH optimization where used 100 ml pH solutions

ranging 3.0-9.0 for each duplicate flask) shaking at

180 rpm in orbital shaker incubator for 45 min.

The shaked flasks were filtered and centrifuged

at 4000 rpm for 10 minutes to eliminate impurities

and insoluble materials. The supernatants were


54 Amir IJAZ et al.

carefully collected with the help of auto-pipette and

filtered through Millipore filter to make it spore

free.

Bioassay of cellulase (FPase)

Bioassay of cellulase (FPase) was performed by

taking 1ml of crude enzyme and 1ml of sodium

citrate buffer (pH 4.8) which were added in each

test tube containing 50 mg filter paper No. 1,

incubated at 50 0 C for 30 min. Then, 500 µl enzyme

sample was boiled with 2.5 ml DNS 3, 5-

Dinitrosalicylic acid for 15 minutes, following

cooling, absorbance of sample was taken at 540 nm

(Mandel et al., 1976). The absorbance was

translated by plotting against regression equation to

get µg/ml/min of glucose by inserting into the

following formula to calculate units of enzyme

activity.

Enzyme activity = Absorbance of enzyme solution x Regression equation

(µg/ml/min) Time of incubation

One unit of enzyme activity was defined as the

amount of glucose (μg) released per ml of enzyme

solution per minute.

Results

The cellulose contents in corn cobs were

determined to be 24.54 %. The incubation period is

directly associated with the production of enzyme

and other physiological functions up to a certain

extent. Incubation period for cellulase production

by Alternaria alternata under SSF is represented in

Figure 2, corn cobs and sugarcane bagasse showed

optimum day 3 rd (72 hrs) with maximum cellulase

activity 15.06 ± 0.17ug/ml.

Figure 2. Incubation period for cellulose

production by Alternaria alternata

Temperature is also an important factor to affect

cellulase yield. Different temperatures (25-40 0 C)

for the production of cellulase using corn cobs by

A. alternata under SSF are described in Figure 3.

The A. alternata accounted maximum cellulase

activity 31.24 ± 0.16 µg/ml at 35 0 C, so, its

optimum temperature was 35 0 C.

Figure 3. Optimum temperature for cellulase

cellulase production by Alternaria alternata

pH is also one of the main factors having direct

impact on cellulase production. Different pH (3.0-

9.0) for cellulase production using corn cobs by A.

alternata is represented in Figure 4, the cellulase

activity was highest at an acidic pH 6.0 (26.41 ±

0.08 ug/ml) & lowest at pH 9.0 (11.84 ±

0.07ug/ml), indicating its optimum pH 6.0.

Figure 4. Optimum pH for cellulase production on

corn cobs by Alternaria alternata

Discussion

The cellulase activity trend concerning corn cobs

was gradually ascended from 1 st day to 3 rd day and

descended from 4 th day to 7 th day. The falling of

cellulase activity might be due to loss of moisture

and inactivation of enzyme resulting from

fluctuation in pH during fermentation (Melo et al.,

2007). Using banana waste culturing Bacillus

subtilis gave maximum cellulase activity after

72hrs of incubation (Krishna, 1999). Our results

can be correlated with the said results. The

cellulase activity increased gradually from 25-35 0 C

and then fell at 40 0 C. The mentioned strain


exhibited minimum cellulase activity (21.34 ± 0.06

µg/ml) at 25 0 C. Using Trichoderma harzianum

T2008 grown on empty fruit bunches under SSF

exhibited maximum FPase activity (8.2 IU/g) at

32°C after 4 days of incubation in Erlenmeyer flask

(Alam et al., 2009). Our findings are in agreement

with the mentioned results. The cellulase activity

trend was increased gradually from pH 3.0-5.0 and

then settled down from pH 6.0-9.0 (showing acidic

nature of enzyme). The highest cellulase activity of

48.70 U/ mL was obtained by using bacillus strain

of BOrMGS-3 at an acidic pH or pH 5.0 (Tabao

and Monsalud, 2010). Our highest activity attained

at pH of 6.0 by showing that results were in

accordance with the mentioned results. Thus, the

maximum cellulase activity could be achieved in a

range of pH 5-6 culturing Trichoderma viride

strains; as pH increased up to 5.5, the hyper

activities of exoglucanase (2.16 U/ml),

endoglucanase (1.94 U/ml) and β-glucosidase (1.71

U/ml) were observed (Gautam et al., 2010).

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Journal of Cell and Molecular Biology

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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

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Tccioli CE, Cottlieb TM, Blund T. Product of the

XRCCS gene and its role in DNA repair and

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Ohlrogge JB. Biochemistry of plant acyl carrier

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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

November 13, 2002, from http:// www.

windspeed.net.au/jenny/seadragons/

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Deubert K. Referencing, not plagiarism.

Retrieved October 31, 2002 from http:

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Journal of Cell and

Volume 9 · No 2 · December 2011

Review Articles

Molecular Biology

The role of circadian rhythm genes in cancer / Kanserde sirkadiyan ritim genlerinin rolü

H. ATMACA and S. UZUNOLU

Tunneling nanotubes – Crossing the bridge

M. McGOWAN

Research Articles

Genetic screening of Turkish barley genotypes using simple sequence repeat markers

SİPAHİ H.

Strontium ranelate induces genotoxicity in bone marrow and peripheral blood upon acute

and chronic treatment

A. ÇELİK, S. YALIN, Ö. SAĞIR, Ü. ÇÖMELEKOĞLU and D. EKE

Cloning, expression, purification, and quantification of the 17% N-terminal domain of

apolipoprotein b-100

H. M. KHACHFE and D. ATKINSON

Cysteine protease from the malaria parasite, Plasmodium berghei- purification and

biochemical characterization

E. AMLABU, A. J. NOK, H. M. INUWA, B. C. AKIN-OSANAIYE and E. HARUNA

Optimization of cellulase enzyme production from corn cobs using Alternaria alternata by

solid state fermentation

A. IJAZ, Z. ANWAR , Y. ZAFAR , I. HUSSAIN, A. MUHAMMAD, M. IRSHAD and S.

MEHMOOD

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