Immobilization of amyloglucosidase on polystyrene anion exchangeresin II. Kinetics and stabilities

Summary Amyloglucosidase (exo-1, 4- -D-glucosidase, EC 3.2.1.3), was coupled to glutaraldehyde activated Indion 48-R (a cross-linked macroporous anion exchanger) by Schiff base reaction. Immobilization brought about a marginal increase in the apparent K_m. The bound enzyme exhibited increased stability towards urea and metal ions, but was less stable in the presence of guanidine hydrochloride. Immobilized amyloglucosidase could be stored at 4°C (in wet state) for 6–8 months without any apparent loss of activity.     

A lipase preparation with dual pH optima,wide temperature optima and broad substrate specificity for multiple applications

Summary Optimised procedures for the production and recovery of lipase at 25 litre scale by submerged fermentation using aRhizopus species isolated from petroleum-soaked soil are developed. These lipase preparations are characterized by dual pH optima (4.0 and 8.0), wide temperature optima (over 80% activity between 40–57°C), broad specificity for substrates, high degree of tolerance for common salts and excellent stability during usage as well as storage.     

An optimized protocol for the production of high purity maltose

An economical protocol, which is simple, rapid and reproducible for the production of maltose by enzymatic hydrolysis of tapioca starch, has been optimized. The protocol involves liquefaction of 35% (w/w) tapioca starch by bacterial -amylase at 78±2°C to 3 to 5% (w/w) reducing sugars, followed by maximal (85±3% w/w maltose equivalent) saccharification with barley -amylase and pullulanase at 50°C for 24 to 30 h. The post-saccharification recovery protocol comprised decolourization by charcoal, de-dextrinization by denatured spirit precipitation, de-ionization by passage through cation and anion exchangers and dehydration by vacuum drying. A white crystalline maltose powder was obtained with specifications comparable to commercial high purity maltose. The protocol yields at least 60% (w/w) recovery of maltose and is suitable for use by the pharmaceutical industry. The protocol is unique in that it utilizes cheap and easily hydrolysed tapioca starch, leaves no mother liquor, enabling higher recovery of maltose, and allows almost quantitative recovery of limit maltodextrins, a value-added marketable by-product.     

Isolation of Bacillus licheniformis mutants for stable production profiles of alkaline protease

Summary A phenotypic requirement for cysteine was introduced inBacillus licheniformis producing alkaline protease to facilitate its isolation from poor or nonproducerBacillus species. This facilitated purification of the strain in cases of cross-contamination, preparation of good inocula for commercial production and stabilization of alkaline protease harvest values, alleviating economic losses incurred through cross-contamination.     

Inhibition of human immunodeficiency virus type 1 infection and syncytium formation in human cells by V3 loop synthetic peptides from gp120.

Because V3 loop-specific antibodies have been shown to inhibit human immunodeficiency virus type 1 (HIV-1) infection of human cells and because specific mutations in the V3 loop render the virus ineffective for infection and syncytium formation, we tested the anti-HIV effects of V3 loop peptides from different HIV-1 strains. We obtained evidence that V3 loop synthetic peptides of 8 to 15 amino acids at nanogram concentrations efficiently blocked HIV-1 IIIB infection of several human T-cell lines and of freshly prepared normal human T cells. More importantly, syncytium formation by three different primary clinical HIV isolates was inhibited by the V3 loop peptide from HIV-1 IIIB at a concentration of 1 micrograms/ml. Concentrations of V3 peptides up to 50 micrograms/ml were not toxic to any of the human cells studied. Additionally, V3 peptides incubated in normal human serum or plasma exhibited biological and physical stability for up to 24 h. Taken together, these results suggest that the V3 loop peptides have medical utility as therapeutic reagents to either prevent HIV-1 infection in humans or reduce the spread of virus infection in HIV-infected individuals. These findings are especially significant because a number of reports in the literature indicate that the V3 loop region in gp120 plays an important role in the initial stages of HIV-1 infection of cells.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

A comparison of cell-mediated immune responses in rhesus macaques housed singly, in pairs, or in groups

A variety of psychosocial factors have been shown to influence immunological responses in laboratory primates. The present investigation examined the effects of social housing condition on cell-mediated immune responses, comparing rhesus macaques (Macaca mulatta) in three housing conditions (single, pair, and group). Subjects included 12 adults of both sexes in each housing condition (N=36). Multiple blood samples (0, 4, 8, and 12 months) were collected for immunological analyses, including lymphocyte subsets, lymphocyte proliferation to pathogens and nonspecific mitogens, natural killer cell activity, and cytokine production. CD4(+) to CD8(+) ratios differed significantly across housing conditions and singly caged subjects had significantly lower CD4(+)/CD8(+) after the 4-month timepoint than did socially housed (pair and group) subjects. CD4(+) to CD8(+) ratios were positively correlated within subjects, suggesting a trait-like aspect to this parameter. Lymphocyte proliferation responses to all four gastrointestinal pathogens differed across housing conditions (at least at the 0.08 level), as did proliferation responses to StaphA, and the production of cytokines (IFN-gamma, IL-2, and IL-10). Proliferation responses of singly caged monkeys did not differ from socially housed monkeys and the highest levels of both IFN-gamma and IL-10 were produced by group housed subjects. The data demonstrate that social housing condition affects immune responses. While not unidirectional, these effects generally suggest enhanced immune responses for socially housed animals. Since rhesus monkeys live socially in nature, and the immune responses of singly housed animals differed from those housed socially, there is considerable motivation and justification for suggesting that the use of singly housed rhesus macaques may complicate interpretations of normal immunological responses. This may have important implications for the management, treatment, and selection of primate subjects for immunological studies.     

Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC

Protein function prediction is very important in establishing the roles of various proteins in bacteria; however, some proteins in the E. coli genome have their function assigned based on low percent sequence homology that does not provide reliable assignments. We have made an attempt to verify the prediction that E. coli genes ygiC and yjfC encode proteins with the same function as glutathionylspermidine synthetase/amidase (GspSA). GspSA is a bifunctional enzyme that catalyzes the ATP-dependent formation and hydrolysis of glutathionylspermidine (G-Sp), a conjugate of glutathione (GSH) and spermidine. YgiC and YjfC proteins show 51% identity between themselves and 28% identity to the synthetase domain of the GspSA enzyme. YgiC and YjfC proteins were expressed and purified, and the properties of GspSA, YgiC, and YjfC were compared. In contrast to GspSA, proteins YgiC and YjfC did not bind to G-Sp immobilized on the affinity matrix. We demonstrated that all three proteins (GspSA, YgiC and YjfC) catalyze the hydrolysis of ATP; however, YgiC and YjfC cannot synthesize G-Sp, GSH, or GSH intermediates. gsp, ygiC, and yjfC genes were eliminated from the E. coli genome to test the ability of mutant strains to synthesize G-Sp conjugate. E. coli cells deficient in GspSA do not produce G-Sp while synthesis of the conjugate is not affected in ΔygiC and ΔyjfC mutants. All together our results indicate that YgiC and YjfC are not glutathionylspermidine synthetases as predicted from the amino acid sequence analysis.