Comparison of two recombinant systems for expression of cholera toxin B subunit from Vibrio cholerae.

PURPOSE: The aim of this study was to assess the production of recombinant cholera toxin B subunit (rCTB) protein in two different expression systems (pAE_ctxB and pQE_ctxB constructs) in Escherichia coli BL21 (DE3). MATERIALS AND METHODS: The ctxB fragment was amplified from Vibrio cholerae O 1 ATCC14035 and cloned in pGETM-T easy vector after which it was transformed to E. coli Top 10F' and grown on LB-ampicillin agar medium. Sequence analysis confirmed the complete ctxB gene sequence in the construct which was further subcloned to pQE-30 vector. The construct was subsequently transformed to E. coli M15 (pREP4). The recombinant pAE_ctxB and pQE_ctxB were transformed to competent E. coli BL21 (DE3) cells to express CTB protein. RESULT: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the maximum expression of rCTB in both systems at 5 h after induction and western blot analysis confirmed the presence of recombinant CTB in blotting membranes. CONCLUSION: Expression of rCTB in pAE_ctxB construct was more efficient (15-fold) than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3) is more compatible with the former construct. This expression system can be used to produce recombinant CTB in high yield which may enable us to study the oral tolerance or mucosal adjuvant properties of rCTB using animal models.

Paclitaxel loaded niosome nanoparticle formulation prepared via reverse phase evaporation method: an in vitro evaluation.

Niosoms are nanoparticles used in drug delivery systems. Niosomes are prepared by various methods. In this research niosoms were prepared by reverse phase evaporation and the factors affecting the niosomes formation were studied. Percent of paclitaxel pegylated and non-pegylated prepared with Span 60 were 95 and 92, respectively while for those of pegylated and non-pegylated niosomes with Span 20, 94 and 90, respectively. In addition, the average diameters of pegylated and no-pegylated prepared with Span 60 and 20 were determined to be 191, 214, 244 and 284 nm, respectively. The amount of released drug (48 h) from pegylated and non pegylated formulations in the presence of Spans 60 and 20 were 8, 10, 6, 7%, respectively. Cytotoxicities ofpaclitaxel niosom polyethyleneglycol, paclitaxel niosome and free paclitaxel on MCF-7 cell line after 48 hours were studied by MTT assay. The results showed the formulation prepared with Span 60 is more effective than that of Span 20 and the IC50 of the former was decreased twice while IC50 of the later decreased 1.5 times.

Study of nano-fiber cellulose production by Glucanacetobacter xylinum ATCC 10245.

Bacterial Celluloses (BC) are gaining importance in research and commerce due to numerous factors affecting the bacterial cellulose characteristics and application in different industries. The aim of the present study was to produce bacterial cellulose in different media using different cultivation vessels. Bacterial cellulose was produced by static cultivation of Glucanacetobacter xylinum ATCC 10245 in different culture media such as Brain Heart Agar, Luria Bertani Agar /Broth, Brain Heart Infusion, Hestrin-Schramm and medium no. 125. Cultivation of bacterium was conducted in various culture vessels with different surface area. The cellulose membrane was treated and purified with a 0.1 M NaOH solution at 90 degreesC for 30 min and dried by a freeze- drier at -40 degreesC to obtain BC. The prepared bacterial cellulose was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy and X-ray diffraction (XRD). The amount of produced BC was related directly to the surface area of culture vessels.

Immobilization of Pichia pastoris cells containing alcohol oxidase activity.

BACKGROUND AND OBJECTIVES: The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. MATERIALS AND METHODS: Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. RESULTS: At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD (420.) There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. CONCLUSION: Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media.

Serotyping and Cross-Reactivity's Between Different Pseudomonas aeruginosa Isolates Prevalent in Iran.

BACKGROUND AND OBJECTIVES: 300 Pseudomonas aeruginosa strains were isolated from hospitalized patients in Iran. Using international antigenic typing system (IATS) antibodies, all strains were classified into 16 serotypes while serotype 14 was not identified among the 17 known serotypes. To evaluate the rate of cross-reactivity between O- antigenic determinants, monospecific polyclonal antibodies were made against whole-killed-cells and live cells of each serotype. MATERIALS AND METHODS: Each antiserum was challenged against homologous and heterologous antigens using slide agglutination test. The degree of agglutination reaction is shown by -ve, 1+ve, 2+ve, 3+ve and 4+ve for 0, 25%, 50%, 75% and 100% agglutination respectively. Then, the results were tabulated for further study. RESULTS: The rate of cross-reactivity between O-antigenic determinants demonstrated that strains 10.55 and 15.14 had the highest agglutination reaction with serum of all the homologous and heterologous serotypes. CONCLUSION: Evaluation of the results obtained from the present study can be applied in production of reliable vaccines and antisera as therapeutic agents or as diagnostic kits.

A flow cytometric opsonophagocytic assay for measurement of functional antibodies elicited after immunization with outer membrane vesicle of Neisseria meningitidis serogroup B.

Opsonophagocytosis mediated by antibody and complement is the major defense mechanism for clearing Neisseria meningitidis from the host. Therefore, a newly developed phagocytosis assay based on flow cytometry (flow assay) was using sera obtained from rabbit postvaccination with outer membrane vesicle of N. meningitidis serogroup B, was done in order to evaluation of the potential efficacy of (experimental) meningococcal vaccines. The Outer Membrane Vesicles (OMVs) and control were injected intramuscularly into groups of five rabbit with boosters on 14, 28 and 42 days after the primary immunization. The serum on 0, 14, 28, 42 and 56 days were collected and stored at -20 degrees C for next analysis. Phagocytic function of and intracellular oxidative burst generation by rabbit polymorphonuclear (PMN), against N. meningitidis serogroup B, was measured with flow cytometer, using dihydrorhodamine-123 as probes, respectively. We use a Coulter Epics XL-profile (USA) with an argon laser operating at 488 nm. The results of quantitative flow cytometric analysis of rabbit PMN function in hyperimmun sera with OMVs revealed a highly significant increase in opsonophagocytic responses against serogroup B meningococci after 56 day in comparison with the control group (p < 0.05). Present results indicated that OMVs could be as a candidate for vaccine toward serogroup B meningococci and a new standard flow cytometric method to measure the opsonophagocytosis activity by rabbit PMNs was shown by this study.

Mutation of Streptomyces griseoflavus in order to obtain high yield desferrioxamine producing fused cells.

Streptomyces griseoflavus PTCC 1130 was mutated by UV irradiation. Two mutants were obtained (C7031 and S7011). These two mutants were able to produce desferioxamine. Desferrioxamine was extracted from the culture broth of the two mutated strains and the thin layer chromatogram of the products showed the R(F) values of 0.461, 0.463 and 0.456 for S7011, C7031 and the standard, respectively. The protoplasts of mutated Streptomyces griseoflavus were isolated and fused together. Total numbers of 58 fusions were obtained and only 17 fusions showed significant resistance to sodium azide and crystal violet. In terms of production of desferrioxamine only fusion PF9 and PF10 increased 68.3 and 81.8% desferrioxamine production as compared to parent strain (PTCC 1130), respectively.

Induction of diabetes by Streptozotocin in rats.

The objective of this study is to induce experimental diabetes mellitus by Streptozotocin in normal adult Wistar rats via comparison of changes in body weight, consumption of food and water, volume of urine and levels of glucose, insulin and C-peptide in serum, between normal and diabetic rats. Intra-venous injection of 60mg/kg dose of Streptozotocin in adult wistar rats, makes pancreas swell and at last causes degeneration in Langerhans islet beta cells and induces experimental diabetes mellitus in the 2-4 days. Induction of experimental diabetes mellitus is indeed the first step in the plan of purification of pancreatic Langerhans islet cells of normal rats for transplanting under the testis subcutaneous of experimentally induced diabetic rats. Streptozotocin induces one type of diabetes which is similar to diabetes mellitus with non-ketosis hyperglycemia in some animal species. For induction of experimental diabetes in male adult rats weighted 250-300 grams (75-90 days), 60mg/kg of Streptozotocin was injected intravenously. Three days after degeneration of beta cells, diabetes was induced in all animals. The diabetic and normal animals were kept in the metabolic cages separately and their body weight, consumption of food and water, urine volume, the levels of serum glucose, insulin and C-peptide quantities in all animals were measured and then these quantities were compared. For a microscopic study of degeneration of Langerhans islet beta cells of diabetic rats, sampling from pancreas tissue of diabetic and normal rats, staining and comparison between them, were done. Induction of diabetes with Streptozotocin decreases Nicotinamide-adenine dinucleotide (NAD) in pancreas islet beta cells and causes histopathological effects in beta cells which probably intermediates induction of diabetes. In this study, we used Streptozotocin for our experiments in induction of experimental diabetes mellitus. After Induction of diabetes, consumption of food and water, volume of urine and glucose increased in the diabetic animals in comparison with normal animals, but the weight of body and the volume of insulin and C-peptide decreased in the diabetic animals. Sampling and staining of pancreas tissue of diabetic and normal rats showed that the Langerhans islet beta cells of diabetic rats have been clearly degenerated. In three days, Streptozotocin makes pancreas swell and at last causes degeneration in Langerhans islet beta cells and induces experimental diabetes. It also changes normal metabolism in diabetic rats in comparison with normal rats. Consumption of water and food, volume of urine, serum glucose increases in diabetic animals in comparison with normal rats but the levels of serum insulin, C-peptide and body weight decreases.