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Journal of Cell and
Molecular Biology
Volume 9 · No 2 · December 2011
http://jcmb.halic.edu.tr
•Circadian rhythm genes in cancer
•Tunneling nanotubes
•Genetic screening of Turkish barley genotypes
•Strontium ranelate induces genotoxicity
Journal of Cell and
Molecular Biology
Volume 9 · Number 2
December 2011
İstanbul-TURKEY
Editor-in-Chief
Nagehan ERSOY TUNALI
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:
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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Editorial Board
M. Baki YOKEŞ
Kürşat ÖZDİLLİ
Nural BEKİROĞLU
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Aslı BAŞAR
Editorial Assistance
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Özlem KURNAZ
Advisory Board
A.Meriç ALTINÖZ, Istanbul, Turkey
Tuncay ALTUĞ, İstanbul, Turkey
Canan ARKAN, Munich, Germany
Aglaia ATHANASSIADOU, Patras, Greece
E. Zerrin BAĞCI, Tekirdağ, Turkey
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
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
1
Tunneling nanotubes – Crossing the bridge
M. McGOWAN
11
Research Articles
Genetic screening of Turkish barley genotypes using simple sequence
repeat markers
H. SİPAHİ
19
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
27
Cloning, expression, purification, and quantification of the 17% Nterminal domain of apolipoprotein b-100
H. M. KHACHFE and D. ATKINSON
37
Cysteine protease from the malaria parasite, Plasmodium bergheipurification and biochemical characterization
E. AMLABU, A. J. NOK, H. M. INUWA, B. C. AKIN-OSANAIYE and E.
HARUNA
43
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
51
57
Journal of Cell and Molecular Biology 9(2):1-10, 2011
Haliç University, Printed in Turkey.
Review Article 1
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 correspondence͖selim@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 bir ağ 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
Yaklaşık Süre
Temel Fonksiyon
Araştırma Alanı
Evrimsel süreçlerde aktif olan
genler
Jeolojik devirler
Çeşitliliğin kökeni
Evrimsel biyoloji
Kalıtım Esnasında aktif olan
genler
Nesiller arası
Replikasyon ve
rekombinasyon
Genetik
Gelişim sırasında aktif olan
genler
Bir ömür boyu
Büyüme
Embriyoloji
Günlük- Haftalık
Metabolizma
Fonksiyonel genom
bilimi
Sirkadiyan ritime göre aktifleşen
genler
Sirkadiyan
Sistem fonksiyonlarının
Senkronizasyonu
Kronobiyoloji
Geç cevapta aktifleşen genler
4-8 saat
İmmunite
İmmunoloji
Erken cevabın ortalarında aktive
olan genler
1-2 saat
Çevresel cevap
Fiziko-nöroimmunoloji
Davranışlara cevap olarak aktive
olan genler
Değişken saatlik
dilimler
Uyanıklık, uyku, ruh
hali
Psikoloji
Fizyolojik değişikliklere bağlı
olarak aktive olan genler
Dakikalar-Saatler
Hafıza, öğrenme
Nöroloji
Erken cevabın başında hızlıca
aktive olan genler
Saniyeler- Dakikalar
Uyarılma, stres
Psikobiyoloji
Canlılığın devamı için
gerekli/sürekli aktif genler
Sirkadiyan ritim genleri ve kanser 3
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 bir ağ 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
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
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 mutasyona uğramış 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) anlamlı azalış 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 kat artış
bulunmuştur. Epiteliyal meme hücrelerinin
çoğalmasını indükleyen BDKRB2 geninin
transkripsiyonunda
2.1
kat,
metastazın
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
(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 genleri susturulmuş 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 geni susturulmuş meme kanseri hücre
kültürleri mutajenlerle muamele edildiğinde,
DNA hasarında kontrol hücrelerine oranla
anlamlı artış gözlenmiştir (Antoch ve
Kondratov, 2009). Bu bulgu CRY genlerinin
DNA tamirinde önemli rol oynadığını,
dolayısıyla
hücrenin
genotoksik
strese
duyarlılığını
etkilediğini
göstermektedir.
Tablo 2. Kanserde rol alan sirkadiyan ritim genleri (Ohdo ve ark., 2010).
Gen
PER2
(Fare)
Kanser Tipi
Lenfoma
Genotip/Gen ifadesi
Kanser Prognozuna
Etkisi
Tümör büyümesinde
artış
Eksik
Apoptozda azalma
artış
PER1,2,3
(İnsan)
Meme kanseri
PER2
(İnsan)
Akut myeloid lösemi
İfadesinde
azalma
Başlama ve/veya
ilerlemesi
PER2
(İnsan)
Kolorektal kanser
İfadesinde
azalma
Tümör oluşumunda
artış
PER1
(İnsan)
Prostat kanseri
İfadesinde
azalma
artış
p53 eksik farede
kanserin ortaya
çıkışında azalma ve
ömür uzaması
CRY1,2
(Fare)
P53 mutant farede
timik lenfoma
Eksik
Kanserin ortaya
çıkışında gecikme
CRY1/PER1 ifade
oranında değişim
Kronik lenfoid
löseminin olası
sonuçlarının
öngörülebilir hale
gelmesi
BMAL1
(İnsan)
B hücreli lenfoma,
akut lenfositik ve
myeloid lösemi
CK1
Npas2
(İnsan)
(Wu ve
ark., 2011)
Beta kateninde azalma
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
artış
Kanserde azalış
azalma
Kanserde artış
Nörodejeneratif
hastalıklar ve kanser
Düzenlenmesinde
bozukluk
Kanserde artış
Sinyal iletim yolaklarında
etkileşim
Non-Hodgkin
lenfoma ve meme
kanseri
Tek nükleotid
polimorfizmi
Kanserde artış
Bazı hücre döngüsü ve DNA
tamir genlerinin ifadelerinin
baskılanması
Apoptozda artış
Apoptozda rol oynayan
proteinlerde [Fas, Bax,c-Myc,
kaspaz-8, poli (ADP-riboz)
polimeraz (PARP)] değişimler
Meme kanseri
İfadesinde
CCAATT/artırıcı dizisine
bağlanan proteinlerdeki azalış
İmmun cevabın değişimi
Hepatik sistem gelişimindeki
değişiklikler
Tek nükleotid
polimorfizmi
Kronik lenfoid
lösemi
c-erbB2 ifadesinde düzensizlikler
Apoptozda azalma
Non-Hodgkin
lenfoma
CRY1
(İnsan)
Bmal1 ifadesinde azalma
c-myc represyonunun
engellenmesi
Tümör süpresör ve hücre
döngüsünde rol oynayan genlerin
düzenlenmesindeki bozukluklar
Androjen reseptör transkripsiyon
aktivitesinin düzenlenmesinde
bozukluklar
CRY2
(İnsan)
DEC2
(İnsan)
PER1 ve 2
promotorlarının
hipermetilasyonu
/İfadesinde azalma
Mekanizma
İfadesinde
İfadesinde
azalma
Baskılanmış
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
madde varmış gibi tümör oluşumu
gözlenecektir.
Sirkadiyan sistem, gerek kanserin
oluşum ve gelişim mekanizmalarını
çalışmada, gerekse kanserin tedavisini
yeni
kronoterapötik
kolaylaştırmada
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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Review Article 11
Journal of Cell and Molecular Biology 9(2): 11-18, 2011
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) and both F-actin and microtubules in thicker (>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 olarak keşfedilmiş 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) F aktin
bulunduran ve daha kalın nanotüplerde (>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,
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
hücreler
arası
transfer,
p53,
MAPK,
kanser.
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 and those >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 Ca2+ 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
Tunneling nanotubes 13
argued that Ca2+ transfer was more spontaneous
than provoked as TNTs were observed to house ER
and extend it through the TNT, which would allow
Ca2+ 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.
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
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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Research Article 19
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-98Başgül, 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 ayrımı yapılmış
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 ith pattern revealed by the jth
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
Turkish barley cultivar screening with SSR 21
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,
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/Obruk86/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
1-Tokak 157/37
Selection from Turkish land races
Row
2
End Use
Feed
Growth habit
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)
Repeat
PCRa
1H
(AC)23
3
1H
(AC)13
3
1H
(AC)24
2
1H
(GA)19
1
2H
(GA)13
1
3H
(CT)21
3
3H
(AC)13
3
3H
(AG)26
3
4H
(GA)22
3
4H
(AG)21
1
Hearnden PR et al. (2007)
Hayden MJ et al. (2008)
4H
(CT)11(
AC)20
2
4H
(AC)22
2
Ramsay et al. (2000)
Varshney RK et al. (2007)
5H
(AG)22
2
5H
(AG)16
3
5H
(AT)7(
AC)18
3
6H
(AG)29
3
7H
(AG)17(
AC)16
3
Sequence
Reference
Bmac0213
F:5’-ATGGATGCAAGACCAAAC-3’
R: 5’-CTATGAGAGGTAGAGCAGCC-3’
F:5’-ACTTAAGTGCCATGCAAAG-3’
R:5’- AGGGACAAAAATGGCTAAG-3’
F:5’- TCATTCGTTGCAGATACACCAC-3’
R:5’- TCAATGCCCTTGTTTCTGACCT-3’
Ramsay et al. (2000),
Liu et al. (1996)
EBmac0501
WMC1E8
HVM20
HVM36
F:5’- CTCCACGAATCTCTGCACAA-3’
R:5’- CACCGCCTCCTCTTTCAC-3’
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’
F:5’- CTACCTCTGAGATATCATGCC-3’
R:5’ -ATCTAGTGTGTGTTGCTTCCT-3’
Bmac0310
EBmac0679
Bmag0337
F:5’-ATTGGAGCGGATTAGGAT-3’
R:5’-CCCTATGTCATGTAGGAGATG- 3’
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’
F:5’-ATTATCTCCTGCAACAACCTA-3’
R:5’-CTCCGGAACTACGACAAG -3’
Bmag0217
a
Location
Primer
Liu et al. (1996)
Liu et al. (1996)
The numbers represent one of the three PCR conditions described in the materials and methods section.
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. Karatay94, 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
EBmac501
WMC1E8
HVM36
Bmac209
Bmag225
Bmag353
HVM68
Bmac310
EBmac679
Bmag337
Bmag387
Bmac113
Bmag500
HVM20
Bmag013
Bmag217
Mean
5
4
2
3
2
4
2
5
4
5
3
5
3
6
4
5
5
3.94±1.25
5
3
2
3
2
4
2
4
3
5
3
4
2
4
4
3
3
3.29±0.99
3.90
2.12
1.49
1.61
1.78
2.64
1.19
2.20
2.50
3.52
1.53
2.65
2.10
2.96
2.52
2.70
1.76
2.30±0.73
0.15, 0.06, 0.15, 0.32, 0.32
0.03, 0.15, 0.64, 0.18
0.21, 0.79
0.76, 0.06, 0.18
0.68, 0.32
0.12, 0.15, 0.55, 0.18
0.09, 0.91
0.09, 0.12, 0.64, 0.12, 0.03
0.03, 0.09, 0.41, 0.47
0.43, 0.15, 0.15, 0.21, 0.06
0.06, 0.79, 0.15
0.55, 0.03, 0.09, 0.12, 0.21
0.44, 0.03, 0.53
0.03, 0.53, 0.15, 0.15, 0.06, 0.08
0.44, 0.06, 0.44, 0.06
0.03, 0.03, 0.50, 0.32, 0.12
0.06, 0.15, 0.74, 0.03, 0.03
0.747
0.522
0.332
0.371
0.435
0.617
0,164
0.540
0.602
0.714
0.350
0.619
0.525
0.663
0.606
0.631
0,425
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 Karatay94/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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Sipahi H, Akar T, Yıldız M. A, Sayım I.
Determination of genetic variation and
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26 Hülya SİPAHİ
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Thesis. Çukurova University, 2005.
Research Article 27
Strontium ranelate induces genotoxicity in bone marrow and
peripheral blood upon acute and chronic treatment
Ayla ÇELİK*1, Serap YALIN2, Özgün SAĞIR2, Ülkü ÇÖMELEKOĞLU3, Dilek
EKE1
1
Department of Biology, Faculty of Science and Letters, Mersin University, Mersin, Turkey
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)
2
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<0.001).
Keywords: Strontium ranelate, polychromatic erythrocytes, genotoxicity, micronucleus, cytotoxicity
Stronsiyum ranelat akut ve kronik uygulama sonrası kemik iliği ve periferal kanda
genotoksisiteyi tetikliyor
Özet
Stronsiyum doğal bir element olup çevrede serbest metal ya da oksidasyon (II) halinde var olur. Bu
çalışmada, sıçanlar stronsiyumun tuzlu suda çözünmüş 500 mg/kg’lık dozu ile haftada 3 kez olmak
üzere 12 hafta için (kronik muamele) ve 24 saat için (akut muamele) gavaj yöntemi ile muamele
edildiler. Stronsiyum ranelatın genotoksik potansiyeli mikronukleus test sistemi kullanılarak Wistar
sıçanlarının periferik kanında ve kemik iliğinde araştırıldı. Bu test sistemine ilaveten sitotoksisite
belirteci olarak polikromatik eritrositlerin normokromatik eritrositlere oranı da araştırıldı. Stronsiyum
ranelat periferik kanda ve kemik iliğinde mikronükleus oluşumunu indüklemektedir. Stronsiyum
ranelatın periferik kan hücre populasyonu üzerine hem akut uygulamada hem de kronik uygulamada
sitotoksik etkiye sahip olduğu belirlenmiştir (p<0.001).
Anahtar Sözcükler: Stronsiyum ranelat, polikromatik eritrosit, genotoksisite, mikronukleus,
sitotoksisite
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.
Strontium ranelate genotoxicity 29
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.
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).
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-cyano3- 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
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.
difference (LSD) test. P≤ 0.05 was
considered as the level of significance.
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
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
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
Chronic treatment
(peripheral blood)
MN/2000
PCEs
PCE/2000
erythrocytes
MN/2000
PCEs
PCE/200
erythrocytes
1
2
3
4
5
1
2
2
2
1
1.60±0.24
2.1
2.2
3
3.4
2.1
2.60±0.25
2
2
3
2
2
2.20±0.20
108
105
100
102
102
103±1.40
1
2
3
4
5
6
4
6
4
4
4.80±0.48***
1
1
1
2
1
1.2±0.20**
75
75
82
80
72
76.8±1.82***
1
2
3
4
5
41
42
40
42
40
41.0±0.44***
1.2
1.3
1
1
1
1.10±0.06***
4
5
5
5
6
5.00±0.31*
**
20
22
21
20
21
20.8±0.37*
**
(n)
Isotonic saline
Mean ±SE
SR(500mg/kg
b.w.)
Mean ±SE
MMC(2
b.w.)
Mean±SE
g/kg
Acute treatment
(Bone marrow)
45
44
42
41
40
42.4±0.92***
***p<0.001, **p<0.01 MMC: Mitomycin C; SR: Strontium Ranelate; MN: Micronucleus; n: rat
number in study group. PCE: Polychromatic erytrocytes; SE: Standard error.
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<0.05). SR was shown to inhibit H2O2induced apoptosis of CRL–11372 cells in a
dose-dependent manner. Enhancement of
osteoblastic cell replication and activity by
SR, a stable salt of strontium, has been
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
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.
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59.pdf
Research Article 37
Journal of Cell and Molecular Biology 9(2):37-42, 2011
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
komponentidir. Artmış LDL düzeyleri ateroskleroz ve diğer koroner arter hastalıklarla ilişkilendirilmiştir.
Apo B’nin büyüklüğü (4536 aa) yapısal olarak organize olmuş 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 (AlAli 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
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
Expression, purification and quantification of B17 39
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
106 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
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.
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 MassSpectrometry (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.
Expression, purification and quantification of B17 41
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
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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caused by a mutation in the apolipoprotein B
gene that results in a truncated species of
apolipoprotein B (B-31): a unique mutation that
helps to define the portion of the apolipoprotein
B molecule required for the formation of
buoyant, triglyceride-rich lipoproteins. J Clin
Invest. 75:933-942, 1990.
Research Article 43
Cysteine protease from the malaria parasite, Plasmodium bergheipurification and biochemical characterization
Emmanuel AMLABU*1, Andrew Jonathan NOK2, Hajiya Mairo INUWA2, Bukola
Catherine AKIN-OSANAIYE3, Emmanuel HARUNA4
1
Department of Biochemistry, Kogi State University, Anyigba,Nigeria
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)
2
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 40OC 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 sıcaklıkla uyarılmış 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
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 4oC for
90 min. The supernatant was collected after an
initial centrifugation at 10,000 x g for 30 min at
4oC 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 37oC 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
Characterization of Plasmodium berghei cysteine protease 45
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
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 37OC 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 37oC 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
The activity of the enzyme was determined over a
temperature range of 4-60OC 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.
Figure 2. Compound PP-56
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.
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)
Table
Purification
Steps
Protein
(mg/ml)
Total activity
(µmol/min)
Crude
DE-52
Sephadex
G-50
10.06
4.50
9.0
6.0
Specific
activity
(µmol/min)
0.895
1.330
1.30
5.2
4.770
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).
Yield
(%)
Purification
Fold
100
67
1.00
1.19
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 23: 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
Initial velocity studies
Temperature dependent studies showed that the
enzyme was optimally active at 40oC (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).
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).
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
10
0
20
40
60
0
Temperature C
Log of activity
(µmol/min)
-
Activity (µmol/min )
pH and temperature studies
80
y = -0.1373x + 3.683
1
1
2
3
4
-3
1/T (x10 )
5
6
Figure 9. Arrhenius plot for the determination of
the Ea for Plasmodium berghei cysteine protease
activity.
1.4
1.2
1
0.8
0.6
0.4
0.2
0
12
y = 4.9261x + 2.3458
10
0
5
10
1/V (µmol/min)
Activity ( µmol/min )
Figure 6. Optimum temperature determination for
the activity of Plasmodium berghei cysteine
protease.
15
pH
Figure 7. Optimum pH determination for the
activity of Plasmodium berghei cysteine protease.
8
6
y = 1.9315x + 2.1625
4
2
0
-1.5
60
No Inhibitor
10
0
-2
0.5
1
1.5
2
1/S (mg/ml)
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.
20
-4
0
40
30
-6
-0.5
y = 10.305x + 4.1076
50
1/V(µmol/
min)
-1
0
2
4
6
1/S(mg/ml)
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.
10
y = 3.7648x + 3.4458
9
8
1/V (µmol/ml)
7
6
y = 1.9315x + 2.1625
5
4
3
2
1
0
-2
-1
No Inhibitor
0
1
2
1/S (mg/ml)
Com pd 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 40oC 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
Tropical Diseases 10: e849, 2010.
Miller LH, Good MF and Million G. Malaria
pathogenesis. Science. 264:1878-1883, 1994.
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.
More CM. Reaching maturity 25 years of TDR.
Parasitology Today. 16: 522-528, 2002.
Dominguez F and Cejudo FJ. Characterization of
the endoproteases appearing during wheat grain
development. Plant Physiology. 112: 12111217, 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
Tropical Medicine and Hygiene. Suppl2: 268278, 2004.
McKerrow JH and Doenhoff MJ. Schistosome
proteases. Parasitol. Today 4: 334-340, 1988.
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
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temperature dependent phase separation using
Triton X-114: Isolation of integral membrane
proteins of pathogenic parasites. J. Methods in
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Research Article 51
Optimization of cellulase enzyme production from corn cobs using
Alternaria alternata by solid state fermentation
Amir IJAZ1*, Zahid ANWAR2, Yusuf ZAFAR3, Iqbal HUSSAIN1, Aish MUHAMMAD1,
Muhammad IRSHAD2 and Sajid MEHMOOD2
1
National Agriculture Research Center (NARC), Islamabad, Pakistan
Nawaz Sharif Medical College (NSMC), University of Gujrat, Pakistan
3
Biological Division PAEC Islamabad, Pakistan
(*
author for correspondence; amirijaz79@yahoo.com)
2
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 (250C, 300C, 350C and 400C) and
pHs (3.0-9.0) were experimented for the production of cellulase. The optimum culture conditions were 96 hrs
of incubation at 350C 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 (250C, 300C, 350C ve 400C) ve pH’lar (3.0-9.0)
denenmiştir. Optimum kültür şartları 350C ve pH 6.0’da 96 saat inkübasyon olarak belirlenmiş 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 1010 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.
Substrate selection
Agricultural waste/samples of corn cobs were
collected from local industry of Gujranwala district,
Pakistan, the substrate was dried in oven at 700C
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.
Production of cellulase from corn cobs 53
Maintenance of Alternaria alternata
Strains of Alternaria alternata maintained on PDA
medium slants under sterilized conditions of LFH
and incubated at 300C for 72 hrs (Asgher et al.,
1999). T he p H o f me d i u m wa s ad j us ted to
4 . 8 wi t h 1 M H Cl /1 M N a O H a nd wa s
st eri liz ed a t 121 o C fo r 1 5 mi n ute s i n
au to c la ve. 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
370C and 150 rpm for 72 hrs and p H wa s
ad j u sted a t 5 .6 a nd wa s a uto cl a ved f o r 1 5
mi n u te s a t 1 5 lb / i n 2 i n a u to c la ve . After
specific incubation period inoculum of Alternaria
alternata was prepared. (Smith et al., 1996).
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 1210C 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).
Cellulose determination
Optimization of incubation period
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.
Duplicate Erlenmeyer flasks using corncobs
cultured with A. alternata were incubated at 300C
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.
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
Temperature optimization
Duplicate flasks inoculated with A. alternata were
kept at 250C, 300C, 350C and 400C, respectively to
determine the optimum temperature at which said
strain would express high cellulase activity was to
select.
pH optimization
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
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 500C for 30 min. Then, 500 µl enzyme
sample was boiled with 2.5 ml DNS 3, 5Dinitrosalicylic 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
A. alternata under SSF are described in Figure 3.
The A. alternata accounted maximum cellulase
activity 31.24 ± 0.16 µg/ml at 350C, so, its
optimum temperature was 350C.
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 (3.09.0) 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.
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 3rd (72 hrs) with maximum cellulase
activity 15.06 ± 0.17ug/ml.
Figure 4. Optimum pH for cellulase production on
corn cobs by Alternaria alternata
Discussion
Figure 2. Incubation period
production by Alternaria alternata
for
cellulose
Temperature is also an important factor to affect
cellulase yield. Different temperatures (25-400C)
for the production of cellulase using corn cobs by
The cellulase activity trend concerning corn cobs
was gradually ascended from 1st day to 3rd day and
descended from 4th day to 7th 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 25-350C
and then fell at 400C. The mentioned strain
Production of cellulase from corn cobs 55
exhibited minimum cellulase activity (21.34 ± 0.06
µg/ml) at 250C. 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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57
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Redford IR. Evidence for a general relationship
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Tccioli CE, Cottlieb TM, Blund T. Product of the
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REVISED
December 2011
Journal of Cell and
Molecular Biology
Volume 9 · No 2 · December 2011
Review Articles
The role of circadian rhythm genes in cancer / Kanserde sirkadiyan ritim genlerinin rolü
1
H. ATMACA and S. UZUNO)LU
11
M. McGOWAN
Research Articles
Genetic screening of Turkish barley genotypes using simple sequence repeat markers
19
H. SİPAHİ
Strontium ranelate induces genotoxicity in bone marrow and peripheral blood upon acute
and chronic treatment
27
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
37
H. M. KHACHFE and D. ATKINSON
Cysteine protease from the malaria parasite, Plasmodium bergheipurification and
biochemical characterization
43
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
51
A. IJAZ, Z. ANWAR , Y. ZAFAR , I. HUSSAIN, A. MUHAMMAD, M. IRSHAD and S.
MEHMOOD
Guidelines for Authors
57

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