Iranian Journal of Biotechnology
Volume:1 Issue: 2, Spring 2003

Vacuolating cytotoxin (VacA) is one of the most important virulence factors of H. pylori (Hp), which is the only toxic protein that is secreted from Hp cell into the culture supernatant. The effects of VacA on eukaryotic systems are the subjects of many previous and on going research studies. Intracellular targets for this toxin include: late endosomal and lysosomal compartments, mitochondria, cell-cell junctions and phospholipid bilayers. Its effects on these targets include vacuolation of late endosomal and lysosomal compartments, apoptosis and channel formation, which result in the increase of ion uptake specially anions. The aim of this review is to increase the perception on this toxin and its functions.

In order to determine the different genotypes of Toxoplasma gondii strains, genetic analysis of SAG2 locus by PCR-RFLP assay was performed for the first time in Iran. Twenty-one isolates obtained from Toxoplasma infected human and mice from Iran (Tehran and Lahijan) were used for molecular typing. None of the isolates had any passage in mice. Genotype determination was made directly on samples. Type II was the predominant lineage, accounting for 85.7% of the isolates. Type I was found in 14.3% of cases with more presentation in animal samples, whereas type III lineage was not seen. No correlation was found between genotype and source of sample (blood, CSF and amniotic fluid). This study indicates a higher prevalence of type II lineage of T. gondii in infected human and mice in the above mentioned regions of Iran.

Prior to the production of human gamma interferon using recombinant DNA technology, it had been produced mainly upon mitogenic induction of lymphocytes in very low amounts, which evidently hampered its characterization and its medical applications. The recombinant gamma interferons produced in larger quantities in prokaryotic systems retain their biological activities, and can be used clinically in the treatment of various viral, neoplastic and immunosuppressed conditions or diseases. In this study, a cDNA sequence coding for human gamma interferon was synthesized from mRNA template extracted from induced human T lymphocytes. The cDNA was then amplified by PCR, cloned in an expression vector, and transformed into Escherichia coli. The polypeptide produced through the expression of this DNA sequence in E. coli showed immunological and chemical properties resembling authentic human IFN

Sixty four Iranian patients with cystic fibrosis (CF) were studied for colonization with Pseudomonas aeruginosa. The patients age ranged between 2 months to 18 years old. Twenty one patients were colonized, 15 with non-mucoid and 6 with mucoid strains of P. aeruginosa. The colonization rate increased with age and the mucoid phenotype was only recovered from the older patients. All mucoid strains came from patients with respiratory disease whereas most of the non-mucoid isolates (n=13) were from patients with gastrointestinal disorder. The antibiograms of the isolates showed 100% sensitivity to Imipenem and Collistin followed by Ciprofloxacin (90.5%), Ceftazidime, and Tobramycin (85.7%), Amikacin, Piperacillin and Tazobactam-Piperacillin (81%), Ticarcillin (76%), Gentamycin (62%), Mezlocillin (52.4%) and Carbenicillin (43%). The MICs for Ceftazidime, Gentamycin and Tobramycin agreed with the disk test results. However, MIC determination for Amikacin showed a 100% sensitivity compared to the disk test where 81% sensitivity was observed. The discrepancy may be due to the fact that over 20% of the isolates had borderline MIC values for Amikacin. The genomic fingerprinting of the 21 isolates as well as the non-mucoid revertants of the mucoid strains was carried out by RAPD-PCR using primer 272 which was previously used for typing P. aeruginosa isolates from CF patients. Thirteen genotypes were found among the 21 isolates. One fingerprint (A) was found in 6 patients and another (B) was shared by 2 patients, all from the same health center. The idea of the hospital as environmental source or cross infection between patients cannot be ruled out.

With the aim of the production of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) in the periplasmic space of Escherichia coli, the expression of hGM-CSF cDNA was examined under the regulation of a T7-based as well as a lac-based expression systems. For the efficient expression of hGM-CSF cDNA, the first five codons at the N-terminal were altered based on the E.coli major codon usage. The hGM-CSF cDNA, fused to pelB signal sequence, was expressed using the two inducible promoters. The expression analysis of the 2 recombinant plasmids were performed in the BL21(DE3) and TG1 strains of E. coli, respectively. After induction with 1mM isopropyl--D-Thiogalactopyranoside (IPTG) the recombinant E. coli with T7 promoter produced hGM-CSF more efficiently than did the lac promoter. Under inducing conditions both of the recombinant bacteria allowed successful secretion of hGM-CSF into the periplasmic space. The optimal temperature for the over-expression of the recombinant protein under the T7-based system was 30C and that of the lac regulated system was 28C. The optimization of growth condition for the recombinant bacteria, produced in this work, provides mean for studying the function of environmental as well as genetic factors on the over-expression of recombinant proteins in the periplasmic space of E. coli.

3-beta hydroxysteroid dehydrogenase (3BHSD) is secreted by the cortex of adrenal gland functioning in stress conditions. The gene encoding the 3-beta hydroxysteroid dehydrogenase protein was PCR amplified from a lgt11 cDNA library using specific primers. The amplified PCR product was then cloned into pGEX-4T-1 expression vector under Ptac promoter and the expression of the enzyme was examined in E. coli (BL21). Upon optimization of the expression condition, the enzyme was produced as a glutathione S-transferase (GST) fusion protein, which was purified by affinity chromatography using glutathione sepharose column. The GST part was then removed by selective proteolytic digestion with thrombin. The purified recombinant enzyme could be used in construction of diagnostic kits for screening the patients with premature ovarian failure (POF) for the presence of autoantibodies against 3BHSD as an important molecular target.

Removing nitrogen, as one of the most common and abundant pollutant of ground and surface waters is very important. For this purpose, biological nitrification and denitrification as the most economical method should be investigated. Feasibility of high load (Chemical Oxygen Demand) COD (1000-2000 mg/l) and NH4 (1000-2000 mg/l) wastewater treatment, at different Hydraulic Retention Times (HRTs) was studied in two 9-lit anaerobic-aerobic system in pre-denitrification mode. Moving Bed Biofilm Reactor (MBBR) is a new system, having all the advantages of activated sludge, fluidized bed and fixed bed processes, without disadvantages of each system, that the biofilm production takes place on the packings, moving along the height of the reactor. From the experiments carried out in this system, result, showed higher ammonia removals take place at higher ammonia and lower organic loads. Denitrification increases at higher nitrification rates because of the increasing effect of NO3- entering the anaerobic reactor. In spite of the fact that nitrifying bacteria are more sensitive than COD and NO3- removing bacteria, after toxic shock by phenol as organic source, nitrification rate increases and COD removal decreases according to the damaging effect of phenol on COD removing bacteria. Total COD removal during the study varies between 80-100%, this value changes to 30-80% for ammonia and 30-80% for ammonia and 40-90% for nitrate.

A dibenzothiophene (DBT) degrading bacterium, which utilizes DBT as the sole source of sulfur, was isolated from soil contaminated with crud oil collected from oil refinery of Tabriz (Iran). A convenient spectrophotometric assay (Gibbs assay) was used to determine the quantity of desulfurized product (Hydroxybiphenyl). This isolate did not grow on DBT, dibenzothiophene sulfone (DBTO2), or 2-Hydroxybiphenyl (HBP) as sole carbon sources. Biodesulfurization activity was observed only in growing cultures and depressed by free sulfate. The desulfurization trait was expressed at increasing levels during the exponential phase of growth and then declined in stationary-phase cells. This gram-positive, non-spore-forming, partially acid fast, polymorphic bacterium with the ability to desulfurize DBT or DBTO2 was identified as Rhodococcus sp. and designated strain FMF