2-Pyrrolidone synthesis from γ-aminobutyric acid produced by Lactobacillus brevis under solid-state fermentation utilizing toxic deoiled cottonseed cake.

There is an increasing demand of γ-aminobutyric acid (GABA) as drug and food additive, as well as feedstock to produce 2-pyrrolidone, a precursor for the synthesis of nylon 4. 2-Pyrrolidone is a petrochemical and depleting reserve which raises concern for its bio-based production. The study herein describes bio-based economical GABA production from Lactobacillus brevis by solid-state fermentation (SSF) using toxic deoiled cottonseed cake (CSC) as substrate. In general, the use of cottonseed cake remains restricted due to the presence of toxic gossypols. Thus, simultaneous detoxification observed during fermentation also widens the scope of utilization of this residual seedcake for feed use vis-a-vis production of other value added chemicals. The SSF conditions were optimized for maximum GABA production, viz., 19.7 mg/g, CSC of GABA was obtained at 6th day of fermentation with 70 % degradation of gossypols simultaneously. The potential of this bio-based GABA as a platform chemical is demonstrated in the synthesis of 2-pyrrolidone. Thus, a simple and cost-effective strategy for utilizing toxic biomass has been developed as an alternate to chemical synthetic route.

Production of Sporotrichum thermophile xylanase by solid state fermentation utilizing deoiled Jatropha curcas seed cake and its application in xylooligosachharide synthesis.

De-oiled Jatropha curcas seed cake, a plentiful by-product of biodiesel industry was used as substrate for the production of a useful xylanase from Sporotrichum thermophile in solid state fermentation. Under the optimized conditions, 1025U xylanase/g (deoiled seed cake) was produced. The xylanase exhibited half life of 4h at 45°C and 71.44min at 50°C respectively. It was stable in a broad pH range of 7.0-11.0. Km and Vmax were 12.54mg/ml and 454.5U/ml/min respectively. S. thermophile xylanase is an endoxylanase free of exoxylanase activity, hence advantageous for xylan hydrolysis to produce xylooligosachharides. Hydrolysis of oat spelt xylan by S. thermophile xylanase yielded 73% xylotetraose, 15.4% xylotriose and 10% xylobiose. The S. thermophile endoxylanase thus seem potentially useful in the food industries.

Utilization of deoiled Jatropha curcas seed cake for production of xylanase from thermophilic Scytalidium thermophilum.

Jatropha curcas is a major biodiesel crop. Large amount of deoiled cake is generated as by-product during biodiesel production from its seeds. Deoiled J. curcas seed cake was assessed as substrate for the production of xylanase from thermophilic fungus Scytalidium thermophilum by solid-state fermentation. The seed cake was efficiently utilized by S. thermophilum for its growth during which it produced good amount of heat stable extracellular xylanase. The solid-state fermentation conditions were optimized for maximum xylanase production. Under the optimized conditions viz. deoiled seed cake supplemented with 1% oat-spelt xylan, adjusted to pH 9.0, moisture content 1:3 w/v, inoculated with 1×10(6) spores per 5 g cake and incubated at 45 °C, 1455 U xylanase/g deoiled seed cake was obtained. The xylanase was useful in biobleaching of paper pulp. Solid-state fermentation of deoiled cake appears a potentially viable approach for its effective utilization.

A novel process for extraction of edible oils: Enzyme assisted three phase partitioning (EATPP).

Three phase partitioning (TPP), a technique used in protein purification has been evaluated, for extraction of oil from three different plant sources viz: mango kernel, soybean and rice bran. The process consists of simultaneous addition of t-butanol (1:1,v/v) and ammonium sulphate (w/v) to a crude preparation/slurry. Under optimized condition, the protein appears as an interfacial precipitate between upper t-butanol containing oil and lower aqueous phase. Pretreatment of the slurries with a commercial enzyme preparation of proteases, Protizyme, followed by three phase partitioning resulted in 98%, 86% and 79% (w/w) oil yields in case of soybean, rice bran and mango kernel, respectively. The efficiency of the present technique is comparable to solvent extraction with an added advantage of being less time consuming and using t-butanol which is a safer solvent as compared to n-hexane used in conventional oil extraction process.

A polyherbal vaginal pessary with spermicidal and antimicrobial action: evaluation of its safety.

A polyherbal vaginal pessary (Praneem) has been formulated that has antimicrobial properties against genital pathogens in addition to spermicidal action. Thus, it has dual potential as a barrier method for contraception and for providing protection against some sexually transmitted infections. The present study reports the findings of a multicentre trial that was conducted to evaluate the safety of this product. Trials were carried out in 23 women in three centres in India: the Postgraduate Institute of Medical Education and Research, Chandigarh; Safdarjang Hospital, New Delhi; and Kamla Nehru Memorial Hospital, Allahabad. Thorough clinical and pelvic examinations were carried out as well as cervical cytology, blood biochemistry and haematology before and after use of the polyherbal pessary intravaginally once daily for 7 consecutive days. No toxicity was observed on clinical examination or by laboratory investigations. Daily intravaginal use of this pessary for 7 days had no adverse effects on cervical cytology or on metabolic and organ functions.

Three phase partitioning for extraction of oil from soybean.

Three phase partitioning, a method generally used for protein separation, has been evaluated for extraction of oil from soybean. 82% oil was extracted within 1 h using this process which required simultaneous addition of t-butanol (1:1, v/v) and 30% ammonium sulphate to the soybean slurry.

PKC-delta inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells.

BACKGROUND & AIMS: Previous studies showed decreased protein kinase C (PKC)-delta expression in azoxymethane-induced rat and sporadic human colonic tumors. To elucidate the role of PKC-delta on the neoplastic phenotype of human colon cancer cells, we established stable transfectants of this isoenzyme in CaCo-2 cells. METHODS: Human PKC-delta complementary DNA was subcloned into 2 distinct metallothionein-regulated expression vectors. Polyclonal populations of PKC-delta transfectants were characterized by Western blotting. PKC-delta activity was measured in situ using a PKC-delta-specific substrate. Proliferation was determined by Coulter counter, and cell cycle distribution was analyzed by flow cytometry. In vitro transformation was assessed by growth in soft agar and differentiation by changes in alkaline phosphatase and sucrase isomaltase. Apoptosis was evaluated by 4',6-diamidino-2-phenylindole dihydrochloride and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining. RESULTS: In the presence of Zn(2+), PKC-delta transfectants expressed a 4-fold increase in the protein and a 2-fold increase in activity of PKC-delta. PKC-delta transfectants exhibited a 30% decrease (P < 0.05) in cell growth and an enhanced differentiation phenotype. Increased PKC-delta expression induced a significant G0/G1 arrest, inhibited anchorage-independent growth (50%, P < 0.05), and caused a 2-fold increase in apoptosis (P < 0.05). CONCLUSIONS: Our studies show that increased expression of PKC-delta inhibits anchorage-dependent and -independent growth, while inducing cellular differentiation and limiting survival of this human colon cancer cell line.

Increase in the immunogenicity of HIV peptide antigens by chemical linkage to polytuftsin (TKPR40).

The use of synthetic peptide antigens in human prophylaxis still suffers from the very important problem of finding suitable carriers devoid of side effects. A desirable carrier for use in humans would be poorly immunogenic by itself, yet it would enhance the immune response to the peptide antigen. In the study reported herein, we examined the role of polytuftsin (TKPR40), a synthetic polymer of the natural immunomodulator tuftsin, as a carrier for synthetic peptides of HIV derived from the gp41 and gp120 proteins. Chimeric immunogens were constructed by chemical linkage between synthetic peptides of HIV and polytuftsin. These were employed for immunization of mice of different MHC haplotypes, and the humoral and cellular immune responses developed against the peptides were assessed by measuring total IgG, IgG, subclasses, T-cell proliferation, and in vitro cytokine release. A significantly stronger immune response was observed in mice immunized with the peptide-polytuftsin conjugates than in mice receiving the peptide dimers (peptide-peptide). Peptide-polytuftsin conjugates induced IgG2a and IgG2b isotype switching after both primary and secondary immunization. In addition, there was a positive correlation between the amounts of cytokines and the shift in the IgG isotypes. These data suggest that the use of polytuftsin as a carrier may increase the immune response against poorly immunogenic synthetic peptides.

HLA-B27 heavy chains contribute to spontaneous inflammatory disease in B27/human beta2-microglobulin (beta2m) double transgenic mice with disrupted mouse beta2m.

MHC class I allele, HLA-B27, is strongly associated with a group of human diseases called spondyloarthropathies. Some of these diseases have an onset after an enteric or genitourinary infection. In the present study, we describe spontaneous disease in HLA-B27 transgenic mice where endogenous beta2-microglobulin (beta2m) gene was replaced with transgenic human beta2m gene. These mice showed cell surface expression of HLA-B27 similar to that of human peripheral blood mononuclear cells. In addition, free heavy chains (HCs) of HLA-B27 were also expressed on thymic epithelium and on a subpopulation of B27-expressing PBLs. These mice developed spontaneous arthritis and nail changes in the rear paws. Arthritis occurred primarily in male animals and only when mice were transferred from the pathogen-free barrier facility to the conventional area. Transgenic mice expressing HLA-B27 with mouse beta2m have undetectable levels of free HCs on the cell surface and do not develop arthritis. In vivo treatment with anti-HC-specific antibody delayed the onset of disease. Our data demonstrate specific involvement of HLA-B27 'free' HCs in the disease process.

Protein kinase C isoforms in the chemopreventive effects of a novel vitamin D3 analogue in rat colonic tumorigenesis.

BACKGROUND & AIMS: We recently showed that dietary supplementation with an analogue of 1alpha,25-dihydroxy-vitamin D3, 1alpha,25-dihydroxy-16-ene-23-yne-26,27 F6-vitamin D3 (RO24-5531), reduced the incidence of colonic tumors in rats treated with azoxymethane (AOM). The aim of this study was to determine whether alterations in specific isoforms of protein kinase C (PKC) are involved in this phenomenon. METHODS: Protein abundance and subcellular distribution of several PKC isoforms were examined and compared in AOM-induced tumors of rats fed control and RO24-5531-supplemented diets. RESULTS: In both AOM-induced colonic adenomas and carcinomas, a significant down-regulation of PKC-alpha, -delta, and -zeta and an up-regulation of PKC-beta11 were found compared with control colonocytes. Dietary RO24-5531 preserved the expression of PKC-zeta and increased the abundance of PKC-epsilon in carcinogen-induced adenomas. CONCLUSIONS: Because identical changes in specific isoforms of PKC were found in AOM-induced adenomas and carcinomas, these alterations may be involved in the early stage(s) of colonic malignant transformation. Moreover, the ability of RO24-5531 to block the changes in PKC-zeta induced by AOM, as well as to up-regulate PKC-epsilon, may underlie its ability to prevent adenomas from progressing to carcinomas.

Mechanism of action of chemoprotective ursodeoxycholate in the azoxymethane model of rat colonic carcinogenesis: potential roles of protein kinase C-alpha, -beta II, and -zeta.

Several lines of evidence from our laboratory and others indicate that epigenetic alterations in protein kinase C (PKC) are involved in colonic carcinogenesis in both man and experimental animals. Furthermore, bile salts, known activators of PKC, have also been implicated in colonic tumor development. Recently, however, our laboratory has demonstrated that, whereas dietary cholic acid increased the occurrence of azoxymethane (AOM)-induced rat colonic tumors, ursodeoxycholic acid was associated with a significant protective effect. In the present studies, we therefore examined changes in PKC isoforms that accompanied AOM-induced tumor formation and investigated whether the chemopromotional and/or chemopreventional actions of these supplemental dietary bile salts involved changes in specific isoforms of PKC. Rats treated with vehicle (saline) or AOM and maintained on bile salt unsupplemented or supplemented diets were used to isolate control colonocytes and carcinogen-induced tumors, which were then subjected to subcellular fractionation. The homogenates and subcellular fractions were then probed for individual PKC isoforms by quantitative Western blotting using isoform-specific antibodies. Normal rat colonocytes expressed PKC-alpha, -beta II, -delta, -epilson, and -zeta. AOM, in unsupplemented or cholate-supplemented groups, caused significant down-regulation of PKC-alpha, -delta and -zeta and up-regulation of PKC-beta II, while increasing particulate PKC-alpha, -beta II, and -zeta in carcinogen-induced tumors compared to normal colonocytes. Dietary supplementation with ursodeoxycholic acid, in marked contrast to these groups, prevented the changes in the subcellular distributions of PKC-alpha, -beta II, and -zeta, and preserved the expression of PKC-zeta in AOM-induced tumors. These studies suggest that changes in specific isoforms of PKC (particularly, PKC-alpha, -beta II, -delta, and/or -zeta) are involved in colonic malignant transformation in the AOM model but do not account for the chemopromotional actions of cholic acid in this model. Furthermore, the ability of ursodeoxycholic acid to block AOM-induced increases in particulate PKC-alpha, -beta II, and -zeta, and/or inhibit down-regulation of PKC-zeta, may contribute to the chemopreventive effects of this bile acid.

1 alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol, a noncalcemic analogue of 1 alpha,25-dihydroxyvitamin D3, inhibits azoxymethane-induced colonic tumorigenesis.

Vitamin D3 and its metabolites, particularly 1 alpha,25-dihydroxyvitamin D3 (1 alpha, 25(OH)2D3), have received increasing attention as potential anticarcinogens in the prevention of cancers in a number of organs, including the colon. These agents, however, have the potential to induce hypercalcemia, thus limiting their practical use for these purposes. In the present studies it was, therefore, of interest to determine whether dietary supplementation with 1 alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalcifero l (RO24-5531), a recently synthesized apparently noncalcemic analogue of 1 alpha,25(OH)2D3, inhibited colon cancer induced by azoxymethane (AOM). Rats were placed on a standard diet or fed this diet with supplemental RO24-5531 (2.5 nmol/kg feed) before and during (initiation arm), or after AOM or vehicle administration (postinitiation arm). After 34 weeks of study, animals in each group were sacrificed, and their colons were removed and examined macroscopically and microscopically for the presence of tumors. At the time of sacrifice, the animals' serum calcium, phosphorus, 25-hydroxyvitamin D3 and 1 alpha,25(OH)2D3 levels were also analyzed. The results of these studies demonstrated that dietary RO24-5531 supplementation during the initiation arm of these experiments significantly reduced (by 70%) the incidence of AOM-induced colonic tumors compared to rats on the standard diet without RO24-5531. Moreover, this dietary regimen abolished the development of adenocarcinomas in this model. Although there was also a trend for dietary RO24-5531 supplementation during the postinitiation arm of this study to reduce the incidence of colon tumors, this did not reach statistical significance (P > 0.05). In addition, neither dietary RO24-5531 supplementation regimen significantly influenced the animals' rates of growth or their serum levels of calcium, phosphorus, or 25-hydroxyvitamin D3. These studies, therefore, demonstrate for the first time that supplemental dietary RO24-5531 is a chemopreventive agent in the AOM model of experimental colonic carcinogenesis. They also suggest that this agent may ultimately prove useful in clinical colon cancer chemopreventive trials.

Trace element imbalances in amyotrophic lateral sclerosis.

Concentrations of 15 elements were determined by instrumental neutron activation analysis in brain, spinal cord, blood cells, serum and nails of Amyotrophic Lateral Sclerosis (ALS) patients and appropriately matched control subjects. Several significant imbalances were detected in trace element levels in ALS samples compared to control samples. Some of these changes are probably secondary to the loss of tissue mass, especially in spinal cord. However the widespread changes observed in Hg and Se levels in ALS tissues deserve special attention. The significance of these alterations in trace element levels in relation to the pathogenesis of ALS is discussed.

Effect of phophatidylcholine and phosphatidylethanolamine enrichment on the structure and function of yeast membrane.

The phospholipid composition of yeast plasma membrane was manipulated by two different methods: (i) by using two auxotrophic strains KA101 (cho1) and MC13 (Cho+) which required phospholipid bases for growth and (ii) by supplementing Saccharomyces cerevisiae (3059) cells with high concentration of choline or ethanolamine. It was possible to enrich the plasma membrane with phosphatidylcholine (PC) or phosphatidylethanolamine (PE) by both methods. The uptake of amino acids, e.g., glycine, glutamic acid, leucine, lysine methionine, phenylalanine, proline and serine, was significantly reduced in PC- or PE-enriched cells. However, the extent of reduction in transport was variable among different strains. A fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS), was used to monitor the structural changes induced by altered phospholipid composition. It was observed that the relative fluorescence intensity of bound ANS was decreased as a consequence of PC or PE enrichment. The decrease in fluorescence was probably associated with reduced number of available binding sites (n) and increased apparent dissociation constant (Kd). Furthermore, our results also suggest that a critical level of PE or PC is required for proper functioning of yeast membrane.