Expression of biologically active measles virus hemagglutinin glycoprotein by a recombinant baculovirus

In this study, one of the measles virus membrane proteins, named hemagglutinin (H) which has a key role in tropism, receptor binding, hemagglutinating activity and also induction of protective immunity against viral infection, was expressed by the baculovirus expression system using specific plasmid (pDONR221) to produce entry clone. Measles Virus (AIK-C strain) genome was extracted from infected Vero cells. H gene was amplified by specific primers during RT-PCR reaction and inserted into the specific plasmid (pDONR221) using BP recombination reaction. Recombinant baculovirus harboring H gene was consequently constructed by LR reaction. Insect cells (Sf9) were infected with recombinant baculovirus. In order to increase viral titer, recombinant baculoviruses were passaged four times in Sf9 cells. Synthesis of H protein was verified by SDS-PAGE, western-blot and indirect immunoflourescene using goat polyclonal antibody against Measles Virus. The results showed that H protein was partially glycosylated but it appeared to be active in hemagglutination assay. © 2008 Asian Network for Scientific Information

Structural Basis for Certain Naturally Occurring Bioflavonoids to Function as Reducing Co-Substrates of Cyclooxygenase I and II

Recent studies showed that some of the dietary bioflavonoids can strongly stimulate the catalytic activity of cyclooxygenase (COX) I and II in vitro and in vivo, presumably by facilitating enzyme re-activation. In this study, we sought to understand the structural basis of COX activation by these dietary compounds.A combination of molecular modeling studies, biochemical analysis and site-directed mutagenesis assay was used as research tools. Three-dimensional quantitative structure-activity relationship analysis (QSAR/CoMFA) predicted that the ability of bioflavonoids to activate COX I and II depends heavily on their B-ring structure, a moiety known to be associated with strong antioxidant ability. Using the homology modeling and docking approaches, we identified the peroxidase active site of COX I and II as the binding site for bioflavonoids. Upon binding to this site, bioflavonoid can directly interact with hematin of the COX enzyme and facilitate the electron transfer from bioflavonoid to hematin. The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity. Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation

Decolorization of different azo dyes by Phanerochaete chrysosporium RP78 under optimal condition

Detoxification of synthetic dyes is one of the main challenges in clearing textile industry wastes. Biodegradation of azo-dyes using Phanerochaete chrysosporium is one the most environmentally friendly methods available. The main enzymes responsible for mycodecolorization process are lignin and manganese peroxidases. Here, optimization of expression conditions has been carried out with manipulating culture condition and nutrient sources. Therefore, the effects of buffer and temperature as well as nitrogen source on lignin peroxidase and manganese peroxidase production were investigated at two levels and four levels, respectively. For this purpose, P. chrysosporium RP78 based on Taguchi design of experiment has been applied. Maximum lignin and manganese peroxidase activities of 182 ± 2.5 U/L and 850 ± 41 U/L were obtained under predicted optimum conditions, respectively. Thereby, about 100 % decolorization was achieved after 24 h for two most widely used groups of azo dyes in textile industry consisting reactive and acidic. The physical adsorption of the azo dyes by mycelia was not significant which indicated that the enzymatic degradation of the dyes was occurred. Time profile of these enzymes showed that manganese peroxidase was peaked on 9th day while lignin peroxidase peaked on 13th. day and remained stable in the culture. The extracellular expression profiles of both were studied by 2 dimensional gel electrophoresis to partially characterize the enzymes

Enhanced Fe2+ oxidation by mixed culture originated from hot spring: Application of response surface method

For maximum oxidation efficiency and minimum amount of jarosite, a total of 30 experimental runs were conducted and the experimental data fitted to the experimental quadratic model. The analysis of variance (ANOVA) demonstrated that the model was highly significant. Three dimensional plots were illustrated to depict the interaction between all the factors. The experimental results indicated that, the optimal conditions for the desired objects were 1.3, 7 g/l, 141 rpm and 35°C for the media initial pH, Fe2+ ion concentration, agitation rate and temperature, respectively. Using the regression model, a maximal oxidation efficiency of 55% and minimum jarosite precipitation of 0.34 g/l were achieved with the consideration of the optimal condition for the parameters at the end of 26 days of operation. The experiment was carried out under optimal condition and resulted in efficient oxidation and jarosite amount of 52% and 0.32 g/l, respectively. Similar values were predicted by the model. According to these results, the response surface methodology was not only applied to determine the significance information on the interaction between the factors, but also was conducted at optimal conditions for the desired objectives. It was also concluded that, the mixed culture isolated from the hot spring had the potential to oxidize ferrous to ferric irons.Key words: Biooxidation, response surface method, jarosite,  biodesulfurization, process optimization

Optimization of probiotic lactobacillus casei ATCC 334 production using date powder as carbon source

This study was conducted to optimize culture conditions for economic production of a probiotic bacterium, Lactobacillus casei ATCC 334, in which palm date powder was applied for the first time as a low-cost main carbon source. The effect of eleven factors on bacterial growth was investigated using the Taguchi experimental design, and three factors including palm date powder, tryptone and agitation rate were found to be the most significant parameters. The optimum conditions including date powder concentration, 38 g/L; tryptone concentration, 30 g/L; and an agitation rate of 320 rpm were determined by response surface methodology of Box-Behnken. A third-order polynomial model was suggested to predict the design space following which the predicted values were validated experimentally. The maximum log value of the viable cells in the optimized alternative medium was 9.97 at 24 h of incubation which was comparable to that obtained in the complex and expensive MRS medium (10.06)