R.L. Deopurkar

Comparison of Montanide adjuvants, IMS 3012 (Nanoparticle), ISA 206 and ISA 35 (Emulsion based) alongwith incomplete Freund's adjuvant for hyperimmunization of equines used for production of polyvalent snake antivenom.

The use of adjuvant is of fundamental importance in vaccines formulations and antisera production. Currently selection and use of adjuvant systems in snake antivenom preparation has become a major issue in terms of animal welfare as well as economics. In order to minimize disadvantages associated with traditionally used Freunds adjuvant (FA) in equines and to produce potent polyvalent antivenom against four Indian snake venoms in minimum possible period, a comparison was made between various commercially available non-emulsion/emulsion based adjuvants like IMS 3012, ISA 206 and ISA 35 with Incomplete Freunds adjuvant (IFA) for their immunopotentiation capacity and safety in donor animals. The present study was conducted in 33 new horses, randomly divided into four groups and hyperimmunized using crude mixture of snake venoms, viz.; Cobra venom (CV), Russells viper venom (RV), Krait venom (KV) and Saw-scaled viper (EV) along with four above mentioned adjuvants through subcutaneous (s.c.) route at intervals of two weeks. Periodic standard safety assessments were done. Immunopotentiation ability of each adjuvant group in terms of percent responders were estimated at 14th, 21st, 30th and 43rd week. The neutralization activity (ED(50)) of pooled sera samples by 43(rd) week, obtained with IMS 3012 group for CV, RV, KV and EV venoms were 0.133, 0.143, 0.070 and 0.270 mg venom/ml of serum respectively. The antivenom potency with IMS 3012 and overall responding horses (100%) even against weak immunogen like CV was significantly higher (p<0.05) than other three adjuvants studied. The horses of IMS 3012 group showed minimum local reactions at injection site, while horses from other three groups exhibited moderate (++) reactions; 66.7% in ISA 206, 12.5% in ISA 35 and 14.3% in IFA respectively, however these were transient and reabsorbed or healed subsequently. Finally, we conclude that, nanoparticle adjuvant IMS 3012 could be a possible alternative to the emulsion adjuvants for primary phase of immunization in antivenom preparation considering its better immunopotentiation capacity and safety in donor animals.

Association of small Rho GTPases and actin ring formation in epithelial cells during the invasion by Candida albicans.

Invasion of epithelial cells is a major virulence determinant of Candida albicans; however, the molecular events that occur during invasion are not discerned. This study is aimed to elucidate the role of the hosts actin remodeling and involvement of small GTPases during invasion. Actin filaments formed a rigid ring-like structure in the rabbit corneal epithelial cell line SIRC after C. albicans invasion. During invasion, an increase in the mRNA content of Cdc42, Rac1 and RhoA GTPase was observed in SIRC cells. Immunochemical staining and expression of chimeric green fluorescent protein (GFP)-GTPases showed that all three GTPases colocalize at invasion and actin polymerization sites. This colocalization was not seen in SIRC cells expressing a GFP-tagged dominant-negative mutant of GTPases. Inhibition of invasion was observed in SIRC cells expressing dominant-negative mutants of Rac1 and RhoA GTPases. Involvement of zonula occludens-1 (ZO-1) was observed in the process of actin-mediated endocytosis of C. albicans. Actin, GTPases and ZO-1 were colocalized in epithelial cells during uptake of polymethylmethacrylate beads coated with spent medium from a C. albicans culture. The results indicate that host actin remodeling and recruitment of small GTPases occur during invasion and molecules that are shed or secreted by C. albicans are probably responsible for cytoskeletal reorganization.

β-Xylanase production by Aureobasidium pullulans grown on sugars agricultural residues

Aureobasidium pullulans grew well in media containing glucose, fructose, xylan or xylose but β-xylanase was only produced with xylan or xylose. Lactose and maltose were poor substrates for growth. β-Xylanase production was repressed in media containing glucose or fructose along with xylose. Agricultural residues, such as wheat bran, paddy husk and rice straw, could be used as carbon sources for growth and β-xylanase production of Aureobasidium pullulans. Tween 80 at 0.5% (v/v) increased the yield of β-xylanase by up to 20%.

Agro-Industrial Wastes for Production of Biosurfactant by Bacillus subtilis ANR 88 and Its Application in Synthesis of Silver and Gold Nanoparticles

Biosurfactants, surface-active amphiphilic compounds, despite having a wide range of applications, have a high cost of production, which severely restricts their use. For cheaper production of biosurfactant, we investigated the potential of the indigenously isolated biosurfactant producing organism, Bacillus subtilis ANR 88, to grow on different cheap carbon sources (molasses, whey, and extracts of potato peels, orange peels, banana peels, and bagasse). We found that, B. subtilis ANR 88 used significant amounts of total sugar to produce cell biomass and biosurfactant. The biosurfactant production in minimal medium containing glucose as sole source of carbon was 0.207 g/l and the same with molasses as carbon source was 0.241 g/l. With whey as carbon source, isolate failed to produce biosurfactant. Amongst the extracts of the agro-wastes, the extracts of bagasse and orange peels gave 0.127 and 0.089 g/l of biosurfactant respectively. One-variable-at-a-time (OVAT) studies carried out to optimize the production of biosurfactant by B. subtilis ANR 88 resulted into maximum biosurfactant yield of 0.513 g/l in medium: molasses 4%, ammonium ferric citrate 0.25%, pH 7. Plackett–Burman design based statistical method for optimization increased the production of biosurfactant to 0.746 g/l, which is 3.6-fold of that produced on glucose. The biosurfactant produced by B. subtilis ANR 88 was analyzed by Fourier Transform Infrared Spectroscopy (FT-IR); it showed that the biosurfactant contained alkyl as well as peptide groups. The biosurfactant of B. subtilis ANR 88 was found effective in the synthesis of silver as well as gold nanoparticles in the total absence of conventional chemical reducing agents. Interestingly, nanoparticles produced were almost uniform in their size and shapes i.e., spherical silver (4–18 nm) and hexagonal gold nanoparticles (40–60 nm), as evident in TEM images.

Evaluation of health status of horses immunized with snake venom and montanide adjuvants, IMS 3012 (nanoparticle), ISA 206 and ISA 35 (emulsion based) during polyvalent snake antivenom production: hematological and biochemical assessment.

Several biochemical and hematological changes in horses are observed during production of snake antivenom. Although conventional adjuvants like Freunds (Complete and Incomplete) are good immunopotentiators, they produce considerable local reactions in animals. Variety of commercial adjuvants, like montanide adjuvants, having high immunopotentiation and showing lesser side effects are available. The prime objective during antivenom production is to strike a balance between safety of immunized horses and efficacy of the product. In our earlier work, efficacy of montanide group of adjuvants in antivenom production has already been established. The aim of the present work was to assess the safety parameters in horses, viz.: biochemical and hematological, during production of snake antivenom. In the present study, 33 new horses were randomly divided into four groups and hyperimmunized using mixture of snake venoms, viz.: Cobra venom, Russells viper venom, Krait venom and Echis venom along with montanide adjuvants, IMS 3012, ISA 206, ISA 35 and Incomplete Freunds adjuvant as a control adjuvant; through subcutaneous route at intervals of two weeks. During the immunization period, biochemical and hematological parameters were monitored at 0th, 14th, 21st, 30th and 42nd weeks. The mean hemoglobin values dropped slightly during initial immunization but subsequently regained to normal levels. The mean serum total protein values and globulin levels showed an increment in all the four groups, compared to day zero, vice-versa a slight drop was observed in albumin levels. No significant changes were observed in serum creatinine, bilirubin, alkaline phosphatase and blood urea nitrogen values. Finally, we conclude that montanide adjuvants could be a safer alternative to the conventional adjuvants for primary phase of immunization in antivenom production.

Beta‐galactosidase from Aureobasidium pullulans

Aureobasidium pullulans is a new source of enzyme beta‐galactosidase with optimum activity at pH 6·8 and a temperature of 45°C. Lactose induced enzyme synthesis, but low constitutive levels of enzyme were seen with other sugars. Addition of Mn2+ and Ca2+ increased enzyme production and activity. Co2+ was a strong activator of enzyme activity. The enzyme is stable over a pH range of 5–10 and temperatures up to 45°C. These studies are significant for studying production of pullulan by using lactose or cheese whey.

Saponified starch-g-polyacrylonitrile gels as carbon source in bacterial culturing

Water-absorbing gels are being increasingly used in our daily lives. Some of these gels are synthesized by modification of starch. They are of the superabsorbing type. The chemical modifications to starch, involved in the synthesis of gels, are likely to present an environmental threat on account of the loss of its inherent biodegradability. Alkali hydrolysed starch-graft-polyacrylonitrile (HSPAN), a starch-based gel, was studied for biodegradability using indigenously isolated bacterial culture. Interestingly, the bacterial culture isolated in the laboratory used HSPAN as the sole source of carbon for its growth and progenesis. The microbial characteristics of the culture were Gram-positive rods with centrally located spores.

Distribution of molybdenum in yeasts grown in the presence of sodium molybdate

The distribution of Mo99 in Saccharomyces cerevisiae and Candida krusei grown in the presence of sodium molybdate has been examined by physical fractionation methods. The major fraction of molybdenum was bound to the cell wall; however, 48% of bound molybdenum penetrated the cells of Saccharomyces cerevisiae. In Candida krusei cells, no intra‐cytoplasmic molybdenum was detected.