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Extraction, Purification and Characterization of Lipopolysaccharide from Escherichia coli and Salmonella typhi 2 2 Lipopolysaccharide (LPS) is an important structural component of the outer cell membrane complex of gram-negative microorganisms. Its causative role in gram-negative bacteria-induced diseases and broad applications in different kinds of cell stimulation experiments provided a conceptual basis for studies directed at the isolation, purification, and detailed chemical characterization of LPS. The main problem with LPS purification protocols is the contamination of the end product with nucleic acids and proteins in variable proportions which could potentially interfere with downstream applications. In this study, a simple procedure for purification of LPS from Escherichia coli (E. coli) and Salmonella typhi (S. typhi) with high purity and very low contaminating nucleic acids and proteins based on the hot phenol-water extraction protocol has been introduced. The purity of extracted LPS was evaluated by silver and coomassie blue staining of SDS-PAGE gels and HPLC analysis. Limulus Amebocyte Lysate (LAL) coagulation activity and rabbit pyrogen assay were exploited to monitor the functionality of purified LPS. The results showed that DNase and RNase treatment of the sample is essential after the sonication step to eliminate nucleic acid contamination in the LPS fraction. Silver staining demonstrated ladder pattern which is characteristic of LPS. No contaminating protein was found as assessed by Coomassie blue staining. HPLC fractionation revealed high degree of purity comparable with commercial LPS. Parenteral administration of purified LPS resulted in substantial increase of rabbits’ body temperature (mean: 1.450C). LAL coagulation assay confirmed the functional activity of the purified LPS. In conclusion, the protocol presented here could be employed for isolation of LPS with high purity and functional activity. 1 7 Simin Rezania Department of Microbiology, School of Medicine, Tehran University of Medical Sciences Department of Microbiology, School of Medicine, Tehran University of Medical Sciences Iran Noor Amirmozaffari Department of Microbiology, School of Medicine, Tehran University of Medical Sciences Department of Microbiology, School of Medicine, Tehran University of Medical Sciences Iran Bahman Tabarraei Department of Bacterial Vaccine and Antigen Production, Pasteur Institute of Iran Iran Mahmood Jeddi-Tehrani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECRReproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECRReproductive Biotechnology Research Center, Avicenna Research Institute, ACECR IranIran Omid Zarei Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Reza Alizadeh Nanobiotechnology Research Center, Avicenna Research Institute, ACECR Nanobiotechnology Research Center, Avicenna Research Institute, ACECR Iran Faramarz Masjedian Department of Microbiology, School of Medicine, Tehran University of Medical Sciences Department of Microbiology, School of Medicine, Tehran University of Medical Sciences Iran Amir-Hassan Zarnani Nanobiotechnology Research Center, Avicenna Research Institute, ACECRImmunology Research Center, Tehran University of Medical Sciences Nanobiotechnology Research Center, Avicenna Research Institute, ACECRImmunology Research Center, Tehran University of Medical Sciences IranIran <i>Escherichia coli</i>EndotoxinExtractionLipopolysaccharidePurificationS. typhi 53.pdf Rietschel ET, Brade H. Bacterial endotoxins. Sci Am 1992;267(2):54-61.##Shnyra A, Luchi M, Morrison DC. Preparation of endotoxin from pathogenic gram-negative bacteria. In: Evans TJ (eds). Methods in molecular medicine (Vol. 36 Septic Shock Methods and Protocols). Totowa: Humana press; 2000, 13-25.##Erridge C, Bennett-Guerrero E, Poxton IR. Structure and function of lipopolysaccharides. Microbes Infect 2002;4(8):837-851.##Luderitz O, Galanos Ch, Lehmann V, Nurminen M, Rietschel ET, Rosenfelder G, et al. Lipid A: Chemical structure and biological activity. J Infect Dis 1973; 128 (Suppl l):17-29. ##Luderitz O, Westphal O, Staub AM, Nikaido H. Isolation and chemical and immunological characterization of bacterial lipopolysaccharides. In: Weinbaum G, Kadis S, Ajl SJ (eds). Microbial toxins. New York: Academic press; 1972, 145-233.##Staub AM. Bacterial lipido-proteinopolysaccharides (“O” somatic antigens). Extraction with tricloroacetic acid. In: Whistler RL, Wolfan ML (eds). Methods in carbohydrate chemistry. New York: Academic press; 1965, 92-93.##Morrison DC, Leive L. Fractions of lipopolysaccharide from Escherichia coli O111:B4 prepared by two extraction procedures. J Biol Chem 1975;250(8): 2911-2919.##Delahooke DM, Barclay GR, Poxton IR. A re-appraisal of the biological activity of bacteroides LPS. J Med Microbiol 1995;42(2):102-112.##Sonesson A, Jantzen E, Bryn K, Larsson L, Eng J. Chemical Composition of Lipopolysaccharide from Legionella pnumophila. Arch Microbiol 1989;153 (1):72-78.##Eidhin DN, Mounton C. A rapid method for preparation of rough and smooth lipopolysaccharde from bacteroides, Porphyromonas and Prevotella. FEMS Microbiol Lett 1993;110(2):133-138.##Galanos C, Luderitz O, Westphal O. A new method for the extraction of R lipopolysaccharide. Eur J Biochem 1969;9(2):245-249.##Nurminen M, Vaara M. Methanol extracts LPS from deep rough bacteria. Biochem Biophys Res Commun 1996;219(2):441-444.##Perdomo R, Montero V. Purification of E.coli 055: B5 lipopolysaccharides by size exclusion chromatography. Biotecnología Aplicada 2006;23(2):124-129.##Westphal O, Jann K. Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. In: Whistler RL, Wolfan ML (eds). Methods in carbohydrate chemistry. New York: Academic press; 1965, 83-91.##Yin ET, Galanos C, Kinsky S, Bradshow RA, Wessler S, Luderitz O, et al. Picogram-sensitive assay for endotoxin: gelation of Limulus polyphemus blood cell lysate induced by purified lipopolysaccharides and lipid A from gram-negative bacteria. Biochem Biophys Acta 1972;261(1):284-289.##Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on toll-like receptors. Nat Immunol 2010;11(5):373-384.##De Smedt T, Pajak B, Muraille E, Lespagnard L, Heinen E, De Baetselier P, et al. Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo. J Exp Med 1996;184(4):1413-1424.##AL-Harbi AH, Austin B. The immune response of turbot, Scophthalmus maximus (L.), to lipopolysaccharide from a fish-pathogenic cytophaga- like bacterium. J Fish Disease 1992;15(5):449-452.##Baba T, Imamura J, Izawa K. Immune protection in carp, Cyprinus carpio L., after immunization with Aeromonas hydrophila crude lipopolysaccharide. J Fish Disease 1988;11(3):237-244.##Ingram GA, Alexander JB. The immune response of the brown trout Salmo trutta to lipopolysaccharide. J Fish Biol 1980;16(2):181-197.##Saeed MO, Plumb AJ. Immune response of channel catfish to lipopolysaccharides and whole cell Edwarshiella ictaluri vaccines. Dis Aquat Organ 1986;2:21-25.##Salati F, lkeda Y, Kusuda R. Effect of Edwarsiella tarda lipopolysaccharide immunization on phagocytosis in the eel. Nippon Suisan Gakkaishi 1987;53(2):201-204.##Hitchcock PJ, Morrison DC. The protein component of bacterial endotoxin. In: Rietschel E (eds). Handbook of endotoxin: Chemistry of endotoxin. Amsterdam: Elsevier Science Publishers; 1984, 339-375.##Apicella MA. Isolation and characterization of lipopolysaccharides. In: Evans TJ (eds). Methods in molecular medicine (Vol. 431 Bacterial pathogenesis). Totawa: Humana press; 2000, 13-25.##Hendrick CA, Sequeira L. Lipopolysaccharide defective mutants of the wilt pathogen Pseudomonas solanacearum. Appl Environ Microbiol 1984;48(1): 94-101.##Hitchcock PJ, Brown TM. Morphological heterogeneity among Salmonella lipopolysaccharides chemotypes in silver stained polyacrylamide gels. J Bacteriol 1983;154(1):269-277.##Kido N, Ohta M, Kato N. Detection of lip polysaccharide by ethidium bromide staining after sodium dodecylsulfate-polyacrylamide gel electrophoresis. J Bacteriol 1990;172(2):1145-1147. ##Sledjeski DD, Weiner RM. Hyphomonas Spp., Shewanella Spp., and other marine bacteria lack heterogeneous (ladder like) lipopolysaccharides. Appl Environ Microbiol 1991;57(7):2094-2096.##Chart H. Lipololysaccharide chemotyping. In: Howard J, Whitcombe DM (eds). Methods in molecular biology (Vol. 46 Diagnostic bacteriology protocols). Totowa: Humana press; 1995: 41-48.##

A Nonlinear Pattern Recognition of Pandemic H1N1 Using a State Space Based Methods 2 2 Genomic Signal Processing is a relatively new field in bioinformatics, in which signal processing algorithms and methods are used to study functional structures in the DNA. An appropriate mapping of the DNA sequence into one or more numerical sequences enables the use of many digital signal processing tools in the analysis of different genomic sequences. Also, a novel Influenza A (H1N1) virus of swine origin emerged in the spring of 2009 and spread very rapidly among people. The severity of the disease and the number of deaths caused by a pandemic virus varies greatly and can change over time. Throughout this work, Pandemic H1N1 genomic sequences were characterized according to nonlinear dynamical features such as moment invariants and largest Lyapunov exponents and then compared to those features that extracted from classical H1N1 genomic sequences. The proposed methods were applied to a number of sequences encoded into a time series using a coding measure scheme employing Electron-Ion Interaction Pseudopotential (EIIP). The aim of this work is to extract genomic features that can distinguish the new swine flu from the classical H1N1 existed before using sequences from segment 8 of the influenza genome that consists of 8 RNA segments which encodes two important proteins for immune system attack (NS1 and NS2). According to the obtained results it is evident that variability is present based on a significance test in both groups; pandemic and classical H1N1 sequences. 1 5 Mai S.Mabrouk Biomedical Engineering Department, Misr University for Science and Technology (MUST) Biomedical Engineering Department, Misr University for Science and Technology (MUST) Egypt GenomeH1N1 SubtypePandemicsSequence 54.pdf Cox NJ, Subbarao K. Influenza. Lancet 1999;354: 1277-1282.##Carrat F, Vergu E, Ferguson NM, Lemaitre M, Cauchemez S, Leach S, et al. Time lines of infection and disease in human influenza: a review of volunteer challenge studies. Am J Epidemiol 2008;167(7):775-785.##Shinde V, Bridges CB, Uyeki TM, Shu B, Balish A, Xu X, et al. Triple-reassortant swine influenza A (H1) in humans in the United States, 2005–2009. N Engl J Med 2009;360:2616-2625. ##Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, Balish A, et al. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 2009;325(5937):197-201.##Chen GW, Chang SC, Mok CK, Lo YL, Kung YN, Huang JH, et al. Genomic signatures of human versus avian influenza A viruses. Emerg Infect Dis 2006;12(9):1353-1360. ##Cristea P. Large scale features in DNA genomic signals. Signal Processing 2003;83(4):871-888.##Cristea P. Genomic signals of re-oriented ORFs. EURASIP JASP 2004;2004. ##Cristea P. Conversion of nitrogenous base sequences into genomic signals. J Cell Mol Med 2002;6(2):279-303.##Nair AS, Sreenadhan SP. A coding measure scheme employing electron-ion interaction pseudopotential (EIIP). Bioinformation 2006;1(6): 197-202.##Mabrouk MS, Solouma NH, Youssef AM, Kadah YM. Eukaryotic gene prediction by an investigation of nonlinear dynamical modeling techniques on EIIP coded sequences. Int J Biol Sci 2007;3(4):225-230. ##http://www.physik3.gwdg.de/tstool/.##Mamistvalov AG. n-dimensional moment invariants and conceptual mathematical theory of recognition n-dimensional solids. IEEE Trans Pattern Anal Mach Intell 1998;20(8):819-831.##Mamistvalov AG. On the construction of affine invariants of n-dimensional patterns. Bull Acad Science Georgian SSR 1974;76(1):61-64.##Owis MI, Abou-Zied AH, Youssef AM, Kadah YM. Study of features based on nonlinear dynamical modeling in ECG arrhythmia detection and classification. IEEE Trans Biomed Eng 2002;49(7):733-736.##

Protective Effects of Capparis zeylanica Linn. Leaf Extract on Gastric Lesions in Experimental Animals 2 2 The aim of the study was to study the anti-ulcer activity of the methanolic extract of the leaves of Capparis zeylanica Linn on experimental animal models. The methanol extract of Capparis zeylanica Linn. leaves was investigated for anti-ulcer activity against aspirin plus pylorus ligation induced gastric ulcer in rats. HCl-Ethanol induced ulcer in mice and Indomethacin induced ulcer in rats at 200 mg/kg body weight p.o. A significant (p<0.01, p<0.001) anti-ulcer activity was observed in all the models. Pylorus ligation showed significant (p<0.01) reduction in gastric volume, free acidity and ulcer index as compared to control. It also showed 88.5% ulcer inhibition in HCl-ethanol induced ulcer and 83.78% inhibition in Indomethacin induced ulcer. 1 5 Karanayil R.Sini Department of Pharmaceutical Chemistry, Grace College of Pharmacy India Barij N.Sinha Department of Pharmaceutical Sciences India Aiyolu Rajasekaran KMCH College of Pharmacy India AspirinIndomethacin RatsPlant extractsMiceGastric ulcer 56.pdf Ojewole EB. Peptic ulcer disease. In: Aguwa CN (eds). Therapeutic basis of clinical pharmacy in the tropics. Enugu: SNAAP Press; 2004,541-564.##Del Valle J, Chey W, Scheiman J. Acid peptic disorders. In: Yamada T, Aplers DH, Kaplowitz N (eds). Textbook of gastroenterology. Philadelphia: Lippincott Williams and Wilkins; 2003,1321-1376.##Berardi RR, Welage S. Peptic ulcer disease. In: Dipiro JT, Talbert RL, Yee GC, Matzke GR, Wells BG, Posey LM (eds). Pharmacotherapy: A pathophysiologic approach. New York: McGraw-Hill; 2005,629-648.##Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med 2002; 347:1175-1186.##Alkofahi A, Atta AH. Pharmacological screening of the anti-ulcerogenic effects of some Jordanian medicinal plants in rats. J Ethnopharmacol 1999; 67(3):341-345.##Peskar BM, Maricic N. Role of prostaglandins in gastroprotection. Dig Dis Sci 1998;43(9):23-29.##Toma W, Hiruma-Lima CA, Guerrer RO, Brito AR. Preliminary studies of Mammea americana L. (Guttiferae) bark/latex extract point to an effective anti-ulcer effect on gastric ulcer models in mice. Phytomedicine 2005;12(5):345-350.##Vela SM, Souccar C, Lima-Landman M, Lapa AJ. Inhibition of gastric acid secretion by the aqueous extract and purified extract of Stachytarpheta cayennensis. Planta Med 1997;63(1):36-39.##Goulart YCF, Sela V, Obici S, Martins JVC, Otobone F, Cortez DA, et al. Evaluation of gastric anti-ulcer activity in a hydro-ethanolic extract from Kielmeyera coriacea. Braz Arch Bio Tech 2005;48(2):211-216.##Aguwa CN, Ukwe C. Gastrointestinal activities of Sterculia tragacantha leaf extracts. Fitoterapia1997;68(2):127-131.##Guha Bakshi DN, Sensarma P, Pal DC. A lexicon of medicinal plant in India (Vol 2). Calcutta: Naya Prokash; 1999.##Asolkar LV, Kakkar KK, Chakre OJ, Chopra RN, Nayer SL, Chopra IC. Glossary of Indian medicinal plants. 2nd supplement. New Delhi: Publications & Information Directorate; 1992.##Kirtikar KR, Basu BD. Indian medicinal plants (Vol 1). 2nd ed. Dehradun: International Book Distributors; 1993.##Joshi SG. Medicinal plants (Vol 3). Calcutta: Oxford and IBH Publication; 1997.##Raghunathan K, Mitra R. Pharmacognosy of indigenous drugs (Vol 3). New Delhi: Central council for research in Ayurveda and Siddha; 1999.##Ghule BV, Murugananthan G, Nakhat PD, Yeole PG. Immunostimulant effect of Capparis zeylanica Linn. leaves. J Ethnopharmacol 2006;108(2):311-315.##Mali RG, Hundiwale JC, Sonawane RS, Patil RN, Hatapakki BC. Evaluation of Capparis deciduas for antihelmintic and antimicrobial activities. Indian J Nat Prod 2004;20(4):10-13.##Chaudhary SR, Chavan MJ, Gaud RS. Ant inflammatory and analgesic activity of Capparis zeylanica root extracts. Indian J Nat Prod 2004;20(1):36-39.##Blakelock RA, Townsend CC. Capparidaceae. In: Townsend CC, Guest E (eds), Flora of Iraq. Iraq: Ministry of Agriculture of the Republic of Iraq; 1980,139-145.##Mukaherjee PK. Quality control of herbal drugs: an approach to evaluation of botanicals. 1st ed. New Delhi: Business Horizons; 2002.##Ecobichon DJ. The basis of toxicity testing. New York: CRC Press; 1997.##Shay H, Komarov SA, Fele SS, MeranzeD, Gruenstein H, Siplet H. A simple method for uniform production of gastric ulceration in rat. Gastroenterology 1945;5:43-61.##Ganguly AK, Bhatnagar OP. Effect of bilateral adrenolectomy on production of restraint ulcers in the stomach of albino rats. Can J Physiol Pharmacol 1973;51(10):748-750.##Kulkarni SK. Handbook of experimental pharmacology. 3rd ed. New Delhi: Vallabh Prakashan; 1999.##Urushidani T, Kasuya Y, Okabe S. The mechanism of aggravation of indomethacin induced gastric ulcer by adrenolectomy in rats. Jpn J Pharmacol 1979;29(5):775-780.##Robert A, Nezamis JE, Lancaster C, Hanchar AJ. Cytoprotection by prostaglandins in rats: Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl and thermal injury. Gastroenterology 1979;77(3):433-443.##Pihan G, Regillo C, Szabo S. Free radicals and lipid peroxidation in ethanol- or aspirin-induced gastric mucosal injury. Dig Dis Sci 1987;32(12):1395-1401.##Lanza FL. A guideline for the treatment and prevention of NSAID induced ulcers. Am J Gastroenterol 1998;93(11):2037- 2046.##Parmar NS, Parmar S. Anti-ulcer potential of flavonoids. Indian J Physiol Pharmacol 1998;42(3):343-351.##Goel RK, Bhattacharya SK. Gastro duodenal mucosal defence and mucosal protective agents. Indian J Expl Biol 1991;29:701-714.##Carlo GD, Mascolo N, Izzo AA, Capasso F, Autore G. Effect of quercetin on gastrointestinal tract in rats and mice. Phytother Res 1994;8(1):42-45.##

Design and Construction of Two Yeast Shuttle Vectors Containing Human Procollagen Genes Expression Cassette for Expression in Yeast 2 2 Collagens are the most abundant proteins in the human body. Their main function is to provide structural and mechanical support for the tissues, but they are also involved in a number of other biological functions including cell attachment, migration and differentiation. Collagens and gelatins are widely used in pharmaceutical and medical applications. Every year, more than 50,000 tons of collagen and gelatin are used in medical applications. These materials may have some viral and prion impurity and/or stimulate allergic responses in human body. Therefore, scientists have produced human collagen in recombinant systems. In this study we have constructed two yeast shuttle vectors containing human procollagen genes expression cassette for expression in yeast. Total RNA was extracted from human skin fibroblast cell line, and cDNA synthesis was done by oligo dt. Then gene fragments were amplified from the cDNA with the necessary changes by Polymerase Chain Reaction (PCR). Finally they were cloned in yeast vector pPICZαA containing regulatory sequences for expressing and secreting the polypeptide product. Two yeast shuttle vectors containing human COL1A1 and COL1A2 expression cassettes were created. Final constructs were confirmed by enzymatic digestion, PCR of desired fragment and sequencing. The yeast shuttle vectors containing human COL1A1 and COL1A2 can be transferred into the yeast in the later stages to determine the scale of expression. 1 8 Baharak Abdemami National Cell Bank of Iran, Pasteur Institute of IranPasteur Research and Production Complex IranIran Mohammad Ali Shokrgozar National Cell Bank of Iran, Pasteur Institute of Iran Iran Hossein Khanahmad Pasteur Research and Production Complex Iran Mehdi Ghavami National Cell Bank of Iran, Pasteur Institute of IranScience and Research Branch, Islamic Azad University Science and Research Branch, Islamic Azad University IranIran CollagenFibroblastsYeasts 57.pdf Symowicz J, Adley BP, Gleason KJ, Johnson JJ, Ghosh S, Fishman DA, et al. Engagement of collagen-binding integrins promotes matrix metalloproteinase-9-dependent E-cadherin ectodomain shedding in ovarian carcinoma cells. Cancer Res 2007;67(5):2030-2039.##Olsen D, Yang C, Bodo M, Chang R, Leigh S, Baez J, et al. Recombinant collagen and gelatin for drug delivery. Adv Drug Deliv Rev 2003;55(12):1547-1567.##Koch FP, Yekta SS, Merkel C, Ziebart T, Smeets R. The impact of bisphosphonates on the osteoblast proliferation and collagen gene expression in vitro. Head Face Med 2010;6:12.##Akashi T, Minami J, Ishige Y, Eishi Y, Takizawa T, Koike M, et al. Basement membrane matrix modifies cytokine interactions between lung cancer cells and fibroblasts. Pathobiology 2005;72(5):250-259.##Alvares K, Siddiqui F, Malone J, Veis A. Assembly of the type 1 procollagen molecule: selectivity of the interactions between the alpha 1(I)- and alpha 2(I)-carboxyl propeptides. Biochemistry 1999;38(17):5401-5411.##Bernocco S, Finet S, Ebel C, Eichenberger D, Mazzorana M, Farjanel J, et al. Biophysical characterization of the C-propeptide trimer from human procollagen III reveals a tri-lobed structure. J Biol Chem 2001;276(52):48930-48936.##Koch M, Veit G, Stricker S, Bhatt P, Kutsch S, Zhou P, et al. Expression of type XXIII collagen mRNA and protein. J Biol Chem 2006;281(30):21546-21557.##Baez J, Olsen D, Polarek JW. Recombinant microbial systems for the production of human collagen and gelatin. Appl Microbiol Biotechnol. 2005;69(3):245-252.##Vuorela A, Myllyharju J, Nissi R, Pihlajaniemi T, Kivirikko KI. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J 1997;16(22):6702-6712.##Sutter M, Siepmann J, Hennink WE, Jiskoot W. Recombinant gelatin hydrogels for the sustained release of proteins. J Control Release 2007;119(3):301-312.##Schreiber RE, Blease K, Ambrosio A, Amburn E, Sosnowski B, Sampath TK. Bone induction by AdBMP-2/collagen implants. J Bone Joint Surg Am 2005;87(5):1059-1068.##Nokelainen M, Tu H, Vuorela A, Notbohm H, Kivirikko KI, Myllyharju J. High-level production of human type I collagen in the yeast Pichia pastoris. Yeast 2001;18(9):797-806.##Han S, Makareeva E, Kuznetsova NV, DeRidder AM, Sutter MB, Losert W, et al. Molecular mechanism of type I collagen homotrimer resistance to mammalian collagenases. J Biol Chem 2010;285:22276-22281.##Pabbruwe MB, Kafienah W, Tarlton JF, Mistry S, Fox DJ, Hollander AP. Repair of meniscal cartilage white zone tears using a stem cell/collagen-scaffold implant. Biomaterials 2010;31(9):2583-2591.##Keizer-Gunnink I, Vuorela A, Myllyharju J, Pihlajaniemi T, Kivirikko KI, Veenhuis M. Accumulation of properly folded human type III procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris. Matrix Biol 2000;19(1):29-36.##Myllyharju J. Recombinant collagen trimers from insect cells and yeast. Methods Mol Biol 2000;139:39-48.##Eskelin K, Ritala A, Suntio T, Blumer S, Holkeri H, Wahlstrom EH, et al. Production of a recombinant full-length collagen type I alpha-1 and of a 45-kDa collagen type I alpha-1 fragment in barley seeds. Plant Biotechnol J 2009;7(7):657-672.##Cereghino GP, Cereghino JL, Ilgen C, Cregg JM. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 2002;13(4):329-332.##Cregg JM, Cereghino JL, Shi J, Higgins DR. Recombinant protein expression in Pichia pastoris. Mol Biotechnol 2000;16(1):23-52.##Fichard A, Tillet E, Delacoux F, Garrone R, Ruggiero F. Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing. J Biol Chem 1997;272:30083-30087.##Toman PD, Pieper F, Sakai N, Karatzas C, Platenburg E, de Wit I, et al. Production of recombinant human type I procollagen homotrimer in the mammary gland of transgenic mice. Transgenic Res 1999;8(6):415-427.##Ruggiero F, Exposito JY, Bournat P, Gruber V, Perret S, Comte J, et al. Triple helix assembly and processing of human collagen produced in transgenic tobacco plants. FEBS Lett 2000;469(1):132-136.##Merle C, Perret S, Lacour T, Jonval V, Hudaverdian S, Garrone R, et al. Hydroxylated human homotrimeric collagen I in Agrobacterium tumefaciens-mediated transient expression and in transgenic tobacco plant. FEBS Lett 2002;515(1):114-118.##Tomita M, Munetsuna H, Sato T, Adachi T, Hino R, Hayashi M, et al. Transgenic silkworms produce recombinant human type III procollagen in cocoons. Nat Biotechnol 2003;21(1):52-56.##Ritala A, Wahlstrom EH, Holkeri H, Hafren A, Makelainen K, Baez J, et al. Production of a recombinant industrial protein using barley cell cultures. Protein Expr Purif 2008;59(2):274-281.##Goldberg I, Salerno AJ, Patterson T, Williams JI. Cloning and expression of a collagen-analog-encoding synthetic gene in Escherichia coli. Gene 1989;80(2):305-314.##Toman PD, Chisholm G, McMullin H, Giere LM, Olsen DR, Kovach RJ, et al. Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae. J Biol Chem 2000;275(30):23303-23309.##Hartner FS, Ruth C, Langenegger D, Johnson SN, Hyka P, Lin-Cereghino GP, et al. Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucl Acids Res 2008;36(12):e76.##Vuorela A, Myllyharju J, Pihlajaniemi T, Kari I. Coexpression with collagen markedly increases the half-life of the recombinant human prolyl 4-hydroxylase tetramer in the yeast Pichia pastoris. Matrix Biol 1999;18(5):519-522.##Cregg JM, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T. Expression in the yeast Pichia pastoris. Methods Enzymol 2009;463:169-189.##Olsen DR, Leigh SD, Chang R, McMullin H, Ong W, Tai E, et al. Production of human type I collagen in yeast reveals unexpected new insights into the molecular assembly of collagen trimers. J Biol Chem 2001;276(26):24038-24043.##

Editorial 2 2 We all know that cancer is a major medical condition that millions of individuals are afflicted by it throughout the world. No cure is found for it yet and the diagnostic tools currently available to detect cancer at an early stage are not considered to be as sensitive and specific to meet the requirement in the clinics. However, for some cancers there are treatments available in major industrial countries but not available in the developing countries with low to medium income range. Indeed why the developing countries are not investing more in prevention and early diagnosis methods? Obviously if health officials and the public are educated to focus on prevention of cancer and early detection of cancer, the burden of cancer would be much lower for any population in the near and long term. In fact for some countries (including Iran) it is predicted cancer incidence to be on the rise which could reach its climax by year 2025. The gap between the rich and poor countries is a well established fact and international organizations such as IMF, world bank,… have been established to help the economies of such developing countries which may take many decades depending on a given country. However, what is urgently needed for many of these low- to medium income level countries is an assistance in education and research programs, enabling these countries to defend their populations against cancer. In the most recent issue of Science (1), some numbers on the gap between the rich and poor countries are given that are shocking, for example: "A child suffering from leukemia in Western Europe has an 85% chance of survival; in the 25 poorest countries in the world, it's just over 10%. For a man with testicular cancer, the numbers are about 95% and just over 40%. Estimates suggest that less than 5% of the world's cancer resources are spent in the developing world". These data suggest that the developing world should pay more attention to the problem of cancer, especially in the areas of prevention and diagnosis and establish organizations that can effectively work with the developed countries in order to transfer their experiences in fighting cancer. Hopefully the Iranian ministry of health would spend more resources for fighting cancer in the coming years simply because all data suggest that cancer will be the number one killing disease in Iran in the near future if mechanisms are not found to stop its spread due to both life-style changes and environmental pollutants that are considered among the major contributors to cause cancer. 18 18 Ali M. Ardekani Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Editorial 161.pdf Enserink M. A push to fight cancer in the developing world. Science 2011;331(6024):1548-1550.##

Cloning and Expression of S1 Subunit of Pertussis Toxin in Escherichia coli 2 2 Bordetella pertussis is a gram negative bacterium that causes respiratory tract infection in human (whooping cough). Pertussis toxin (PT) is the main component of current acellular pertussis vaccine and the S1 (subunit1) is the main immunogenic part of it. Thus, S1 has been the target of many studies as a potent candidate of acellular vaccine against Bordetella pertussis, lacking the side effects of whole cell based ones. S1 gene was amplified and inserted in three expression vectors including pET-14, pET-22b(+) and pAED4. The possibility and level of expression of these constructs were investigated in BL21 (DE3) strain of Escherichia coli (E.coli) as expression host. The highest expression was in pET-22b(+)-S1. Best expression achieved 6 hr post induction with 0.2 mM IPTG in LB broth containing ampicillin, at 30°C with shaking (250 rpm). Recombinant S1 protein was observed in two distinct separated proteins with 28 and 31 kDa estimated molecular weight. In spite of toxicity of PT and S1 in the E.coli, considerable amount of S1 was expressed in E.coli. Two rS1 bands were detected on SDS-PAGE. Both were confirmed as S1 in western blot with specific monoclonal and polyclonal antibodies against pertussis toxin. Appearance of two distinct bands could be the result of leader peptidase activity or nonspecific peptidase from E.coli on recombinant S1. As the recombinant S1 is a suitable antigen for studies as a candidate acellular vaccine or development of ELISA for detection of Bordetella pertussis, further studies are underway. 19 24 Abolfazl Khafri Department of Bacteriology, Faculty of Medical Science, Tarbiat Modares University Department of Bacteriology, Faculty of Medical Science, Tarbiat Modares University Iran Khosrow Aghaiypour Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute (RVSRI) Iran Shahin Najar Peerayeh Department of Bacteriology, Faculty of Medical Science, Tarbiat Modares University Department of Bacteriology, Faculty of Medical Science, Tarbiat Modares University Iran Reihaneh Ghorbani Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute (RVSRI) Iran Bordetella pertussis<i>Escherichia coli</i>Pertussis toxin 51.pdf Hazes B, Boodhoo A, Cockle SA, Read RJ. Crystal structure of the pertussis toxin–ATP Complex: A molecular sensor. J Mol Biol 1996;258(4):661-671.##Locht C, Cieplak W, Marchitto KS, Sato H, Keith JM. Activities of complete and truncated forms of pertussis toxin subunits S1 and S2 synthesized by Escherichia coli. Infect Immun 1987;55(11):2546-2553.##Yuen CT, Canthaboo C, Menzies JA, Cyr T, Whitehouse LW, Jones C, et al. Detection of residual pertussis toxin in vaccines using a modified ribosylation assay. Vaccine 2002;21(1-2):44–52.##Williamson YM, Moura H, Schieltz D, Rees J, Woolfitt AR, Pirkle JL, et al. Mass spectrometric analysis of multiple pertussis toxins and toxoids. J Biomed Biotechnol 2010;942365. Epub 2010 May 23.##Askelöf P, Rodmalm K, Wrangsell G, Larsson U, Svenson SB, Cowell JL, et al. Protective immunogenicity of two synthetic peptides selected from the amino acid sequence of Bordetella pertussis toxin subunit S1. Proc Natl Acad Sci USA 1990;87(4):1347-1351. ##Andreasen C, Powell DA, Carbonetti NH. Pertussis toxin stimulates IL-17 production in response to Bordetella pertussis infection in mice. Plos One 2009;4(9):1-11.##Castro MG, McNamara U, Carbonetti NH. Expression, activity and cytotoxicity of pertussis toxin S1 subunit in transfected mammalian cells. Cell Microbiol 2001;3(1):45-54.##Barbieri JT, Pizza M, Cortina G, Rappoli R. Biochemical and biological activities of recombinant S1 subunit of pertussis toxin. Infect Immun 1990;58(4):999-1003.##Barbieri JT, Rappouli R, Collieri RJ. Expression of the S-1 catalytic subunit of pertussis toxin in Escherichia coli. Infect Immun 1987;55(5):1321-1323.##Antoines R, Lochto C. The NAD-Glycohydrolase activity of the pertussis toxin S1 subunit involvement of the catalytic HIS-35 residue. J Biol Chem 1994;269 (9):6450-6457. ##Cyr T, Menzies AJ, Calver J, Whitehouse LW. A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products. Biologicals 2001;29(2):81-95.##Smith AM, Yan H, Groves N, Pozza TD, Walker MJ. Co-expression of the Bordetella pertussis leader peptidase I results in enhanced processing and expression of the pertussis toxin S1 subunit in Escherichia coli. FEMS Microbiol Lett 2000;191(2):177-182.##Barbieri JT, Moloney BK, and Mende-mueller LM. Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli. J Bacteriol 1989;171(8):4362-4369.##Pande AH, Moe D, Jamnadas M, Tatulian SA, Teter K. The pertussis toxin S1 subunit is a thermally unstable protein susceptible to degradation by the 20S proteasome. Biochemistry 2006;45(46):13734-13740.##Carbonetti NH, Mays RM, Artamonova GV, Plaut RD, Worthington ZE. Proteolytic cleavage of pertussis toxin S1 subunit is not essential for its activity in mammalian cells. BMC Microbiol 2005;5(7)1-14.##

Antidiabetic Activity of Aqueous Leaves Extract of Sesbania sesban (L) Merr. in Streptozotocin Induced Diabetic Rats 2 2 The aqueous leaves extract of Sesbania sesban (L) Merr. (Family: Fabaceae) was evaluated for its antidiabetic potential on normal and streptozotocin (STZ)-induced diabetic rats. In the chronic model, the aqueous extract was administered to normal and STZ- induced diabetic rats at the doses of 250 and 500 mg/kg body weight (b.w.) p.o. per day for 30 days. The fasting Blood Glucose Levels (BGL), serum insulin level and biochemical data such as glycosylated hemoglobin, Total Cholesterol (TC), Triglycerides (TG), High Density Lipoproteins( HDL) and Low Density Lipoproteins (LDL) were evaluated and all were compared to that of the known anti-diabetic drug glibenclamide (0.25 mg/kg b.w.). The statistical data indicated significant increase in the body weight, liver glycogen, serum insulin and HDL levels and decrease in blood glucose, glycosylated hemoglobin, total cholesterol and serum triglycerides when compared with glibenclamide. Thus the aqueous leaves extract of Sesbania sesban had beneficial effects in reducing the elevated blood glucose level and lipid profile of STZ-induced diabetic rats. 37 43 Ramdas B.Pandhare MES College of Pharmacy, Sonai, Newasa, AhmednagarResearch Scholar Department of Pharmacy, Suresh Gyan Vihar University, Jaipur Research Scholar Department of Pharmacy, Suresh Gyan Vihar University, Jaipur IndiaIndia B. Sangameswaran Department of Pharmacognosy, TIT-Pharmacy India Popat B.Mohite MES College of Pharmacy, Sonai, Newasa, Ahmednagar India Shantaram G.Khanage MES College of Pharmacy, Sonai, Newasa, Ahmednagar India Diabetes mellitusHyperglycemiaSesbania sesbanStreptozotocin 55.pdf Aslan M, Orhan DD, Orhan N, Sezik E, Yesilada E. In vivo antidiabetic and antioxidant potential of Helichrysum plicatum ssp. plicatum capitulums in Streptozotocin-induced-diabetic rats. J Ethnopharmacol 2007;109(1):54-59. ##Khare CP. Indian Medicinal Plants - An illustrated dictionary. Berlin: Springer-Verlag; 2007.##Yusuf M, Chowdhury JU, Wahab MA, Begum J. Medicinal plants of Bangladesh, BCSIR, Dhaka, Bangladesh: Bangladesh Council of Scientific and Industrial Research; 1994. ##Chapman & Hall. Dictionary of Natural Products. London: Taylor and Francis. 2002. ##Gupta AK, Grasdalen H. Nmr studies of composition and side-chain arrangement in Sesbania aegyptiaca seed galactomannan. Carbohydrate Res 1989:81:239-244. ##Upadhyaya JS, Singh SP. Chromatographic studies of oxidation products of lignin from Sesbania sesban. Cellul Chem Technol 1991;25:219-226. ##El-Sayed NH. A rare kaempferol trisaccharide anti-tumor promoter from Sesbania sesban. Pharmazie 1991;46(9):679-680. ##Jain SR. Hypoglycemic principal in the Musa sapeintum and its isolation. Planta Med 1968; 16(1):43-47.##Latha M, Pari L. Effect of an aqueous extract of Scoparia dulcis on blood glucose, plasma insulin and some polyol pathway enzymes in experimental rat diabetes. Braz J Med Biol Res2004;37(4):577-586.##Kokate CK. Practical Pharmacognosy. 4th ed. New Delhi: Vallabh Prakashan; 1994.##Khandelwal KR. Practical Pharmacognosy Techniques and Experiments. 15th ed. Pune: Nirali Prakashan. 2000.##Organization for economic co-operation and development. OECD Guidelines. Guidance document on acute oral toxicity testing (2001) series on testing and assessment no. 24. OECD environment, health and safety publications. Paris, January 2007.##Chakrabarti S, Biswas TK, Seal T, Rokeya B, Ali L, Azad Khan AK, et al. Antidiabetic activity of Caesalpinia bonducella F. in chronic type 2 diabetic model in Long-Evans rats and evaluation of insulin secretagogue property of its fractions on isolated islets. 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New Delhi: New Age International Pvt. Ltd.; 1996.##Seifter S, Dayton S, Novic B, Muntwyler E. The estimation of glycogen with anthrone reagent. Arch Biochem 1950;25:191-200.##Swanston-Flat SK, Day C, Bailey CJ, Flatt PR, Traditional plant treatment for diabetes: studies in normal and streptozotocin diabetic mice. Diabetologia 1990;33(8):462-464.##Chatterjee MN, Shinde R. Textbook of Medical Biochemistry. 5th ed. New Delhi: Jaypee Brothers Medical Publishers; 2002.##Reshma SP, Sushma AM. Hypolipidemic activity of Acorus calamus L. in rat. Fitoterapia 2002; 73:451-455.##Hall PM, Cook JGH, Sheldon J, Rutherford SM, Gould BJ. Glycosylated hemoglobin and glycosylated plasma proteins in the diagnosis of diabetes mellitus and impaired glucose tolerance. Diabetes Care 1984;7(2):147-150.##

Lack of Association between Interleukin 12 C(-1188)A Polymorphism and Irritable Bowel Syndrome 2 2 Irritable Bowel Syndrome (IBS) is a functional gastrointestinal disorder, characterized by recurrent abdominal pain and altered bowel habits. This study was performed to investigate the important role of interleukin-12 (IL-12) in intestinal inflammation. For this study seventy one patients with IBS and 140 controls were investigated. The allele and genotype frequencies of IL-12 C(-1188)A were determined using polymerase chain reaction with sequence-specific primers. The allele A was more common that the allele C in both groups of patients and controls. There was not any significant difference on IL-12 alleles and genotypes between patients and controls. The AA genotype was the most common genotypes, which was seen in 57.4% of the patients and 51.4% of the controls (p=0.53). Although frequency of the CC genotype in the control group was lower than the patient group, this difference was not significant (5.7% vs. 11.5%, respectively, p=0.16). Considering the lack of association between IL-12 C(-1188)A polymorphism and IBS, this cytokine gene polymorphism may not have significant role in the pathophysiology of disease. 45 48 Elham Barkhordari Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Iran Ali Akbar Amirzargar Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Iran Naser Ebrahimi-Daryani Department of Gastroenterology and Hepatology, Imam Khomeini Hospital, Tehran University of Medical Sciences Department of Gastroenterology and Hepatology, Imam Khomeini Hospital, Tehran University of Medical Sciences Iran Mahdi Mahmoudi Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Iran Bita Ansaripour Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Iran Maryam Alighardashi Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Iran Hamid Reza Ahmadi-Ashtiani Biochemistry and Nutrition Department, Zanjan University of Medical Sciences, Zanjan; and Clinical Biochemistry Department, School of Medical Science, Tarbiat Modarres University Biochemistry and Nutrition Department, Zanjan University of Medical Sciences, Zanjan; and Clinical Biochemistry Department, School of Medical Science, Tarbiat Modarres University Iran Mohammad Bashashati Gastrointestinal Research Group, University of Calgary, Calgary Gastrointestinal Research Group, University of Calgary, Calgary Canada Nima Rezaei Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Research Center for Immunodeficiencies, Pediatrics Center of Excellence, Children’s Medical Center, Tehran University of Medical Sciences Molecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical Sciences Research Center for Immunodeficiencies, Pediatrics Center of Excellence, Children’s Medical Center, Tehran University of Medical Sciences IranIran Genetic polymorphismInterleukin-12Irritable bowel syndrome 52.pdf Jones R, Lydeard S. Irritable bowel syndrome in the general population. BMJ 1992;304:87-90.##Thompson WG, Longstreth GF, Drossman DA, Heaton KW, Irvine EJ, Müller-Lissner SA. Functional bowel disorders and functional abdominal pain. Gut 1999;45 (Suppl 2):1143-1147.##Chey WJ, Jin HO, Lee MH, Sun SW, Lee KY. Colonic motility abnormality in patients with irritable bowel syndrome exhibiting abdominal pain and diarrhea. Am Gastroenterol 2001;96(5): 1499-1506.##Azpiroz F, Bouin M, Cammileri M, Mayer EA, Poitras P, Serra J, et al. Mechanisms of hypersensitivity of IBS and functional disorder. Neurogastroenterol Motil 2007;19(Suppl 1):62-88. ##Barbara G, DE Giorgio R, Stanghellini V, Cremon C, Corinaldesi R. A role for inflammation in iritable bowel syndrome? Gut 2002;51(Suppl 1): 41-44.##Chadwick VS, Chen W, Shu D, Paulus B, Bethwaite P, Tie A, et al. Activation of the mucosal immune system in irritable bowel syndrome. Gastroenterology 2002;122(7):1778-1783.##Drossman DA, McKee DC, Sandler RS, Mitchell CM, Cramer E, Lowman BC, et al. Psychosocial factors in the irritable bowel syndrome. A multivariate study of patients and nonpatients with irritable bowel syndrome. Gastroenterology 1988; 95(3):701-708.##Whitehead WE, Palsson OS. Is rectal pain sensitivity a biological marker for irritable bowel syndrome: psychological influences on pain perception. Gastroenterology 1998;115(5):1263-1271.##Hotoleanu C, Popp R, Trifa AP, Nedelcu L, Dumitrascu DL. Genetic determination of irritable bowel syndrome. World J Gastroenterol 2008;14 (43):6636-6640.##Adam B, Liebregts T, Holtmann G. Mechanisms of disease: genetics of functional gastrointestinal disorders-searching the genes that matter. Nat Clin Pract Gastroenterol Hepatol 2007;4(2):102-110.##Talley NJ. Genes and environment in irritable bowel syndrome: one step forward. Gut 2006;55 (12):1694-1696.##Monteleone G, Biancone L, Marasco R, Morrone G, Marasco O, Luzza F, et al. Interleukin 12 is expressed and actively released by Crohn’s disease intestinal lamina propria mononuclear cells. Gastroenterology 1997;112(4):1169-78.##Parronchi P, Romagnani P, Annunziato F, Sampognaro S, Becchio A, Giannarini L, et al. Type 1 T-helper cell predominance and interleukin-12 expression in the gut of patients with Crohn’s disease. Am J Pathol 1997;150(3):823-832.##Chikano S, Sawada K, Shimoyama T, Kashiwamura SI , Sugihara A, Sekikawa K, et al. IL-18 and IL-12 induce intestinal inflammation and fatty liver in mice in an IFN-γ dependent manner. Gut 2000;47(6):779-786##Stanilova S, Miteva L. Taq-I polymorphism in 3′UTR of the IL12B and association with IL-12p40 production from human PBMC. Genes Immun 2005;6:364-366.##Seegers D, Zwiers A, Strober W, Peña AS, Bouma G. A TaqI polymorphism in the 3'UTR of the IL-12 p40 gene correlates with increased IL-12 secretion. Genes Immun 2002;3(7):419-423.##Shokrgozar MA, Sarial S, Amirzargar AA, Shokri F, Rezaei N, Arjang Z, et al. IL-2, IFN-γ, and IL-12 gene polymorphisms and susceptibility to multiple sclerosis. J Clin Immunol 2009;29(6):747-751.##Hall MA, McGlinn E, Coakley G, Fisher SA, Boki K, Middleton D, et al. Genetic polymorphism of IL-12 p40 gene in immune-mediated disease. Genes Immun 2000;1(3):219-224.##Barkhordari E, Rezaei N, Ansaripour B, Larki P, Alighardashi M, Ahmadi-Ashtiani HR, et al. Proinflammatory cytokine gene polymorphisms in irritable bowel syndrome. J Clin Immunol 2010;30 (1):74-79.##Barkhordari E, Rezaei N, Mahmoudi M, Larki P, Ahmadi-Ashtiani HR, Ansaripour B, et al. T-helper 1, T-helper 2, and T-regulatory cytokines gene polymorphisms in irritable bowel syndrome. Inflammation 2010;33(5):281-286.##Amirzargar AA, Naroueynejad M, Khosravi F, Dianat S, Rezaei N, Mytilineos J, et al. Cytokine single nucleotide polymorphisms in Iranian populations. European Cytokine Network 2008;19: 104-112.##Hotoleanu C, Popp R, Trifa AP, Nedelcu L, Dumitrascu DL. Genetic determination of irritable bowel syndrome. World J Gastroenterol 2008;14 (43):6636-6640.##Gately MK, Renzetti LM, Magram J, Stern AS, Adorini L, Gubler U, et al. The interleukin-12/interleukin-12 receptor system: role in normal and pathologic immune responses. Annu Rev Immunol 1998;16:495-521.##Huang D, Cancilla M, Morahan G. Complete primary structure, chromosomal localization, and definition of polymorphisms of the gene encoding the human interleukin-12 p40 subunit. Genes Immun 2000;1(8):515-520.##O’Mahony L, McCarthy J, Kelly P, Hurley G, Luo F, Chen K, et al. Lactobacillus and bifidobacterium in irritable bowel syndrome: Symptom responses and relationship to cytokine profiles. Gastroenterology 2005;128(3):541-551.##