Molecular cloning and characterization of an alpha-amylase from Pichia burtonii 15-1

An alpha-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii alpha-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis alpha-amylase, 58% with Saccharomycopsis sp. alpha-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the K(m) values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the k(cat) values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the k(cat)/K(m) values of PBA were higher.     

High-throughput comprehensive analysis of D- and L-amino acids using ultra-high performance liquid chromatography with a circular dichroism (CD) detector and its application to food samples

A rapid and comprehensive analytical method for D- and L-enantiomers of proteinogenic amino acids was developed using ultra-high performance liquid chromatography (UHPLC) equipped with a circular dichroism (CD) detector. Pre-column derivatization reagents were examined for enhanced sensitivity and selectivity for UV and CD detection: 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was selected. The method, using a CD detector, does not require separation of optical isomers on a column to calculate the enantio ratio (%D) using the g-factor value and produces a simple chromatogram in comparison to other reported methods. Using this advantage, combined with UHPLC technology, analysis time for the derivatized proteinogenic amino acids was within 5.5 min. The UV detection limit was 4.9-23 pmol/injection and the CD detection limit was 11-64 pmol/injection. The method was applied to the analysis of D- and L-amino acids in food samples. D-Ala, D-Asp, D-Glu and D-Ser were detected at high concentrations in some Japanese black vinegars, fermented milks and yogurts. The results were identical to the results determined by the OPA method. We suggest the UHPLC-CD method would be useful in screening the D-amino acid content of foods and in helping to clarify the importance and reason for the presence of D-amino acids in foods.     

Comparative analysis of enzyme activities and mRNA levels of peptidyl prolyl cis/trans isomerases in various organs of wild type and Pin1-/- mice

We investigated the enzyme activity of peptidyl prolyl cis/trans isomerases (PPIases) in brain, testis, lung, liver, and mouse embryonic fibroblasts (MEF) of Pin1+/+ and Pin1-/- mice. The aim of this study is to determine if other PPIases can substitute for the loss of Pin1 activity in Pin1-/- mice and what influence Pin1 depletion has on the activities of other PPIases members. The results show that high PPIase activities of Pin1 are found in organs that have the tendency to develop Pin1 knockout phenotypes and, therefore, provide for the first time an enzymological basis for these observations. Furthermore we determined the specific activity (k(cat)/K(M)) of endogenous Pin1 and found that it is strongly reduced as compared with the recombinant protein in all investigated organs. These results suggest that posttranslational modifications may influence the PPIase activity in vivo. The activities originating from cyclophilin and FKBP are not influenced by the Pin1 knockout, but a basal enzymatic activity towards phosphorylated substrates could be found in Pin1-/- lysates. Real time PCR experiments of all PPIases in different mouse organs and MEF of Pin1+/+ and Pin1-/- mice support the finding and reveal the specific expression profiles of PPIases in mice.     

A novel type of transposon generated by insertion element Is102 present in a psc101 derivative

We describe a novel type of transposon in the tetracycline resistance plasmid pYM103, a derivative of pSC101 carrying a single copy of an insertion element IS102. The new transposons we found were identified as DNA segments, approximately 6 kb (Tn1021) and 10 kb (Tn1022) in length, able to mediate the cointegration of pYM1O3 with plasmid Col E1. The resulting cointegrate contains either of these pYM1O3 segments duplicated in a direct orientation at the junctions of the parent plasmids. A direct duplication of a 9 bp sequence at the target site in Col E1 is found at the junctions for cointegration. Both transposons have IS1O2 at one end and also contain different lengths of the pYM103 DNA adjacent to IS102, including the tetracycline resistance gene. Each transposon contains terminal inverted repeats of a short nucleotide sequence. These results and the fact that IS102 can itself mediate plasmid cointegration, giving rise to a duplication of a 9 bp target sequence, indicate that IS102 is responsible for generation of Tn1021 and Tn1022. They are quite different from the common IS-associated transposons, which are always flanked by two copies of an IS element, and may be similar to transposons such as those of the Tn3 family and phage Mu.     

Expression of Notch receptors and ligands on immature and mature T cells

Notch plays multiple roles in T cell development in the thymus and T cell differentiation in the periphery. In order to systematically examine the role of Notch in T cell biology, we determined the cell surface expression of all Notch receptors and ligands on various populations of T cells by using a panel of specific monoclonal antibodies we recently established. Notch1 and Notch3 were upregulated at double-negative (DN) 2-DN4 stages of immature thymocytes, then downregulated on mature single-positive thymocytes and peripheral T cells, but were rapidly upregulated again upon activation. Notch2 was consistently expressed on T cells while Notch4 was not. Jagged1 and Jagged2 were expressed at double-positive stage of immature T cells. Jagged2 was also inducible on mature T cells upon activation. In contrast, no Delta-like (Dll) 1 or Dll4 expression was observed on T cells. These comprehensive profiling of the expression of Notch receptors and ligands would be informative to fully understand the role of individual Notch receptors and ligands in T cell development and differentiation.     

Effect of alendronate on osteoclast differentiation and bone volume in transplanted bone.

Alendronate, one of the bisphosphonates, is known to have an inhibitory effect on bone resorption. The purpose of this study was to investigate the effects of alendronate on ectopic bone graft resorption and to determine the optimal dose in the mouse. The grafted bone in the control group disappeared due to resorption by osteoclasts within 5 weeks. In the experimental groups, the area of bone tissue decreased by only 20-40% at 5 weeks post-operatively. At 8 and 9 weeks after surgery, the decreased area of bone structure was significantly less in all the 10(-4) M injected alendronate-immersed groups than in the 10(-4) M non-injected alendronate-immersed. At 9 weeks after surgery, the number of osteoclasts were significantly less in the 10(-4) M injected alendronate-treated groups than in the 10(-4) M non-injected alendronate-treated groups. These results suggest that alendronate inhibits resorption of ectopic bone graft at concentrations of 10(-4) and 10(-6) M.     

The metabolism of 17beta-estradiol-3-glucosiduronate and estrone-3-glucosiduronate in the rabbit.

[6, 7-3H]-17beta-Estradiol-3-glucosiduronate, [6, 7-3H]-estrone-3-glucosiduronate or [6, 7-3H]-estrone was administered intravenously into the rabbit, and analysis and identification of the urinary metabolites were carried out. In either case, the major urinary metabolite was found to be a diconjugate. The sequential enzymic hydrolysis indicated that this diconjugate was glucosiduronate-N-acetyglucosaminide of 17alpha-estradiol. From these results, the conversion of the estrogen glucosiduronate into a diconjugate was thought a rather universal phenomenon in the rabbit.     

Effect of seaweed preparations on murine immunocytes

During a screening programme for new medical agents, many aqueous extracts from 59 species of seaweed were found to possess bioactivity against murine immunocytes. Thirty-eight extracts (8 green, 12 brown, 18 red algae) showed suppressive effects on the mitogenic response. Furthermore, 16 extracts (2 green, 6 brown, 8 red algae) suppressed the production of Interleukin 1 (1L-1) from murine macrophage. Using the murine mixed lymphocyte reaction assay, suppressive effects were observed in 4 red algae, but none in green or brown algae. Nine seaweed extracts suppressed the production of secondary antibody (IgG, IgM). Extracts of 3 red algae suppressed strongly the proliferation of bone marrow cells, but 2 other red algae caused stimulation above 200%. This is apparently the first report showing immunosuppressive activity from marine algae.