Biomedical subjects
R K Saxena
Publications and source records attributed to R K Saxena.
Interplay of matrices in cartilage synovial fluid combine under moderate & high loadings.
Interplay of the constituents of the articular cartilage synovial fluid combine and its role was examined through the biochemical and rheological studies on bovine joints. The results showed an inverse relationship between the changes in the hyaluronic acid of synovial fluid and the proteoglycans content in articular cartilage together with alterations in the rheological properties of synovial fluid. The study indicated that the inter-movement of fluid solutes across the cartilage and synovial fluid may have an important role in the pathophysiology of osteoarthritis.
Purification and characterization of a regiospecific lipase from Aspergillus terreus.
Aspergillus terreus lipase was purified to homogeneity with 18.0% yield. The specific activity of the enzyme increased from 20.80 to 250 U/mg of protein. Ion exchange on Q-Sepharose was highly effective in the purification process. The molecular mass of the purified enzyme was 41+/-1 kDa as determined by SDS/PAGE. The purified lipase showed excellent temperature tolerance (15-90 degreesC) and was highly thermostable, retaining 100% activity at 60 degreesC for 24 h. It showed good pH tolerance (3.0-12.0) and was stable over a pH range of 4.0-10.0 for 24 h. The activity of the enzyme was inhibited by ionic detergents, whereas non-ionic detergents stimulated enzyme activity. Mg2+ and Ca2+ ions stimulated lipase activity, whereas Co2+, Cu2+, Ni2+ and Fe3+ ions caused inhibition. The enzyme was unaffected by the metal chelator EDTA or by 2-mercaptoethanol and potassium ferrocyanide. At a concentration of 100 microM, 3,4-dichloroisocoumarin caused weak inhibition with 40% loss of activity, but diethyl p-nitrophenyl phosphate at the same concentration strongly inhibited enzyme activity (98.12% loss of activity), confirming that the A. terreus lipase is a serine hydrolase. The lipase was highly active on pig fat (151% relative activity) and groundnut oil (103% relative activity) and least active on kusum oil (18% relative activity). Extensive dialysis did not affect enzyme activity up to 168 h, suggesting the absence of any dialysable cofactor in the enzyme. The A. terreus lipase retained significant activity on freeze-drying and had a shelf-life of more than 6 months at room temperature. The A. terreus lipase exhibited 1,3-regiospecificity and was stable in various organic solvents.
Oscillatoria anguistissima: a promising Cu2+ biosorbent.
Oscillatoria anguistissima rapidly adsorbs Cu2+ from aqueous solution. The adsorption of Cu2+ followed Freundlich Isotherm, and the amount of Cu2+ removed from solution increased with increasing Cu2+ concentration. The adsorption is pH dependent, and maximum Cu2+ removal occurs at pH 5. Of the various pretreatments, HCl treatment of the biomass increased the capacity for Cu2+ removal. Presence of Mg2+ and Ca2+ resulted in decline in the Cu2+ adsorption capacity of Oscillatoria cells. This species could also effectively remove Cu2+ from mine water containing 68.4 microg/ml of Cu2+ at pH 3.45.
Mechanism of interaction of pH 3.0 treated tumor cells expressing lower levels of class I MHC antigens, with IL-2 activated NK cells.
Effect of lowering the levels of class I MHC antigens, on the ability of three murine (YAC, P815, SP2O) and three human (Molt4, Raji, HR7) tumor cell lines, to compete in cold target inhibition assays, was studied. Specific reduction in class I MHC levels (range 60-76%) was induced by brief exposure to citrate buffer (pH 3.0). Susceptibility of the acid pH treated tumor cells to lysis by IL-2 activated NK cells, was found to be significantly increased in all cases with the exception of YAC cells. Reduction in class I MHC antigen expression by acid pH treatment, had no significant effect on competing ability of the tumor cells. In contrast, tumor cells on which MHC class I antigens were upregulated by interferon treatment, had significantly lower ability to compete. These results suggest that the mechanisms of altered interactions between MHC I up or down regulated target cells may be different. A model compatible with increased NK susceptibility along with an unaltered ability to compete for acid pH treated tumor cells, has been proposed.
Ontogeny of inhibitory receptors for MHC molecules on NK cells.
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A rapid plate assay for screening L-asparaginase producing micro-organisms.
A pH and dye-based fast procedure for screening L-asparaginase producing micro-organisms is reported. The procedure is suitable for bacterial and fungal screening. The results are obtained within 24 and 48 h for bacteria and fungi, respectively. The results correlate with quantitative estimations in culture broths.
Microorganisms as an alternative source of protein.
Demand for human food and animal feed proteins from nonconventional sources has increased, particularly in developing countries. Microbial protein is one such source. It is desirable because it is amenable to controlled intensive cultivation and is less dependent on variations in climate, weather, and soil. Microbial proteins must be evaluated for nutritive value, safety, and economic considerations before mass production is undertaken.
Bcl-2 transfection protects Hut78 cell line from different types of cytotoxic effector cells.
The human T lymphoma cell line Hut78 was transfected with Bel-2 gene or with control G418 Neomycin resistance-conferring plasmid. Bcl-2 transfected and Neomycin selected Hut78 cells expressed about ten-fold greater Bcl-2 protein than the neo-transfected controls. Susceptibility of Bcl-2-transfected Hut78 cells to human NK cells, human mixed lymphocyte reaction-generated cytotoxic effector cells, and mouse cytotoxic spleen cells generated by immunization with Hut78 cells was examined. In all systems tested, Bcl-2-transfected Hut78 target cells were significantly less susceptible to lysis than the neo-transfected control target cells. These results suggest that in Hut78 cells, Bcl-2 gene product may confer partial resistance to different types of cytotoxic effector mechanisms.
Polyamines as modulators of microcycle conidiation in Aspergillus flavus.
Since polyamines (PAs) play a potential role in the regulation of growth and developmental processes in a wide variety of organisms, we have examined the influence of the PAs putrescine (Put) and spermidine (Spd) and the PA biosynthetic inhibitors alpha-difluoromethylornithine (DFMO), alpha-difluoromethylarginine (DFMA), methylglyoxal bis-(guanylhydrazone) (MGBG) and cyclohexylamine (CHA), singly and in combinations on microcycle conidiation (MC) in Aspergillus flavus. The exogenous application of the diamine Put (concentrations ranging from 0.1 to 5 mM) caused a sharp decline of MC in a dose-dependent fashion, but induced vegetative growth. However, the triamine Spd (0.1-5 mM) had a minimal effect on MC and induced a shift from MC to normal condition. PA inhibitors, especially DFMO, MGBG and CHA, produced greater inhibition of MC and complete inhibition of MC was observed at 5 mM of these inhibitors. DFMA even at 5 mM had only a weak inhibitory effect on MC. DFMO also inhibited conidial germination and germ tube growth. MGBG and CHA, while having an inhibitory effect on MC, induced vegetative growth. The inhibitory effect of PA inhibitors was partially reversed by exogenous Put or Spd, with Spd being more effective than Put. The analysis of free PA levels during various phases of MC revealed that undifferentiated spores contained a high Put/Spd ratio and there was a dramatic decrease in Put/Spd ratio before and during microcycle conidiophore maturity. The change in spermine titres could not be detected. These observations imply that Put is essential for vegetative growth, while Spd is involved in MC, and that a low Put/Spd ratio seems to be important for spore differentiation to MC.
Microbial lipases: production and applications.
Lipases occupy a prominent place among biocatalysts and have a wide spectrum of biotechnological applications. Lipases are unique as they hydrolyse fats into fatty acids and glycerol at the water-lipid interface and can reverse the reaction in non-aqueous media. The stability of these enzymes in organic solvents have pushed them into the frontier areas of organic synthesis leading to the designing of novel drugs, surfactants, bioactive compounds and oleochemicals. In addition, lipase-catalysed trans-esterification and inter-esterification reactions have been exploited in the fat industry. Looking into the wide scenario of lipase applications, commercialization of lipase production is a prime area of interest for microbiologists, process engineers and biochemists. Research carried out in this field has revealed that microbes, especially fungi and bacteria, are the tools of choice for commercial production. Recently, the structure determination of a few microbial lipases has widened our knowledge about the unique mechanism of catalysis of this enzyme.
Correlation of class I MHC antigen levels on some human tumor cell lines with susceptibility to LAK cells and performance in cold target inhibition assays.
Relationships between (a) levels of class I MHC antigens, (b) susceptibility to LAK cells, and (c) ability to perform in cold target competition assays were studied using K562, MOLT-4, Raji, Daudi, and HR7 human tumor cell lines. The hypothesis of inverse correlation between class I MHC antigen expression and susceptibility to LAK cells was concurrent with results using K562, HR7, and Daudi cell lines. For Raji cells, IFN-gamma had no effect on the levels of class I MHC antigen but induced resistance to LAK cells, whereas for MOLT-4 cells, enhanced MHC I expression was not accompanied with any change in susceptibility to LAK cells. In competition experiments, all five tumor cell lines were used as competitors as well as targets. Different specificities of competition were observed for different tumor cells. These results indicated that the different tumor cell lines could share one or more target structures recognized by LAK effector cells. IFN-gamma treatment lowered the ability of several tumor cell lines to inhibit target lysis in cold competition assays. Changes in the competition efficacy of tumor cells correlated better with their susceptibility to LAK cells rather than levels of class I MHC antigens.
Changes in LAK susceptibility of tumor cells as their MHC class I antigen expression levels regenerate after treatment at pH 3.0.
Changes in LAK susceptibility of tumor cells in which MHC class I antigen expression was undergoing regeneration following treatment at pH 3.0, were studied. P815 (murine mastocytoma) and RAJI (human B-cell leukemia) cell lines were briefly exposed to pH 3.0 buffer which brought down the MHC class I expression on these cells by 70-80% and resulted in a 3-fold increase in their susceptibility to murine and human LAK cells, respectively. During the MHC class I regeneration phase, LAK susceptibility fell and reached normal levels at a time point when MHC class I antigens had regenerated to a level which was 60-70% of the normal. Highly significant inverse correlations were obtained between LAK susceptibility and the log of MHC class I antigen expression, for both P815 and RAJI cell lines. These results indicate that during the MHC class I regeneration phase, there is an inverse correlation between MHC class I antigen levels and LAK susceptibility. Moreover, these results also suggest that the extent of change in LAK susceptibility may depend upon the levels of basal MHC class I expression on the tumor cells.
Chitinase production by Streptomyces viridificans: its potential in fungal cell wall lysis.
Streptomyces viridificans was found to be a good chitinase producer among nine species of Streptomyces screened. Minimum levels of constitutive enzyme were observed with both simple and complex carbon substrate. Arabinose doubled the enzyme production amongst the various pentoses and hexoses used with chitin. However, with glucose end-product inhibition and catabolite repression were observed. The enzyme tolerated a wide range of temperature (30-55 degrees C) and pH (3-7.5). Among various divalent cations Mn2+ and Hg2+ completely inhibited the purified enzyme while beta-mercaptoethanol stimulated its activity. Crude and purified enzyme had potential for cell wall lysis of many fungal pathogens tested.
Role of major histocompatibility complex class I antigens in modulating the performance of murine tumour cells in cold target competition assays.
The role of class I major histocompatibility complex (MHC) antigen levels on the ability of five murine tumour cell lines (YAC, P815, EL4, SP20 and L929) to competitively inhibit their own lysis, as well as the lysis of other targets by lymphokine-activated killer (LAK) effector cells was examined. Basal LAK susceptibilities of the cell lines were in the order P815 > YAC > SP20 > EL4 > L929, whereas the basal class I MHC antigen levels were in the order P815 > SP20 > L929 > YAC > EL4. Treatment with interferon-gamma (IFN-gamma) induced augmentation of class I MHC antigen levels on all cell lines. A concomitant decline in LAK susceptibility was seen for P815, YAC, SP20 and L929 cells, but not for EL4 target cells. On the basis of competition results, tumour cells appear to fall into two groups (group 1: P815, YAC and SP20; group 2: EL4 and L929). Members of each group could in general competitively inhibit the lysis of cell lines of their own group only. Treatment with IFN-gamma suppressed the ability of all tumour cell lines, except EL4, to cause competitive inhibition. These results support the proposition that class I MHC antigens may interfere with the recognition of target cells by effector LAK cells.
Regulation of NK susceptibility by target cell class I MHC molecules.
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Rock phosphate solubilization under alkaline conditions by Aspergillus japonicus and A. foetidus.
Aspergillus japonicus and A. foetidus were found to solubilize five types of Indian rock phosphates at pH 8 and 9. Solubilization was higher in the presence of pyrite than in controls lacking either pyrite or fungal inoculum. Both the aspergilli were found to be good pyrite solubilizers and could grow over a wide pH range. Solubilization of rock phosphates was the result of organic acid release and pyrite oxidation.
CPA and medical services: missing prerequisite.
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