Simple Detection of Xylosidase Activity in Single Colonies,Using 1-Naphthyl-β-d-xylopyranoside

Since the xylosidase of Bacillus pumilus hydrolyzed 1-naphthyl-β-d-xylopyranoside (naphthyl-X) to produce xylose and 1-naphthol and a chromogenic azo compound is produced by coupling 1-naphthol and Fast Blue Salt B, a simple method for detection of xylosidase activity in single colonies was studied. Escherichia coli JM109 carrying the xylosidase gene of B. pumilus was cultivated at 37°C for 18 h on an LB plate containing 0.5 mg/ml naphthyl-X, and then the plate was overlaid with 3 ml of a top layer containing 24 mg of agar and 6 mg of Fast Blue Salt B. After incubation of the plate at 37°C for 1 h, each colony became reddish-brown. Even a small colony with the xylosidase on the plate was easily distinguished from colonies without the enzyme.     

A Simple and Efficient Method for High Fidelity PCR Cloning Using Antibody-neutralizing Technology

We introduce the TA cloning antibody method for the high-fidelity PCR product amplified by family B DNA polymerase without purification. This method uses antibodies and Thermus aquaticus (Taq) DNA polymerase. The antibodies can inhibit only the activity of family B DNA polymerase, and Taq can co-work for A-tailing. This method has nearly cloning efficiency to that of the PCR product of Taq.     

The Purification and the Properties of Three Kinds of Lipases from Rhizopus delemer

The study was undertaken to clarify whether three kinds of lipases (EC 3.1.1.3) secreted from Rhizopus delemar are originally different or identical with each other. First of all, the purification of those lipases was carried out and their enzymatic properties were examined. Their properties including the stability on heat and pH, precipitabilities at a certain pH, the behaviours on a SE-Sephadex C50 column and on a Sephadex G200 column and so on were compared.

From the results, A-lipase is clearly different from the other two lipases. On the other hand, it seems that B- and C-lipases are originally identical.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.     

Grape seed proanthocyanidin extracts prevent high glucose-induced endothelia dysfunction via PKC and NF-κB inhibition

In our study, it has been detected in vivo and in vitro that GSPE reversed high glucose-induced the increase of ICAM-1 and VCAM-1. It is shown that by western blotting detection, GSPE significantly inhibited the activation of NF-κB induced by high glucose while there was significant decrease of the expression of PKC with GSPE intervention. By adding the NF-κB blocker PDTC and the PKC inhibitor peptide 19–31(10^?6 M), no significant difference was found in the levels of VCAM-1 and ICAM-1 among GSPE group, the PKC inhibitor peptide 19–31-added GSPE group and the PDTC-added GSPE group. So the conclusion could be drawn that PKC inhibition must be involved in GSPE decreasing the level of ICAM-1 and VCAM-1.We proved for the first time that GSPE prevented high glucose-induced the increase of ICAM-1 and VCAM-1 by PKC and NF-κB inhibition. These findings show a novel mechanism of the action GSPE preventing endothelial dysfunction, which may have clinical application values.     

Neutral Aroma Constituents in Burley Tobacco

The constituents of the neutral volatiles from air-cured Burley tobacco were studied using distillation, silicic acid column chromatography, preparative gas chromatography and GC–MS. The isolation and identification of 84 compounds are reported of which 27 are newly identified as tobacco constituents and 4 are new natural products.     

Palmitate Contributes to Insulin Resistance through Downregulation of the Src-Mediated Phosphorylation of Akt in C2C12 Myotubes

The mechanisms of free fatty acid (FFA)-induced peripheral insulin resistance remain elusive. This study aimed to investigate the effect of palmitate, a saturated fatty acid, on glucose metabolism in C2C12 myotubes, and to explore the underlying mechanisms. In it, palmitate decreased insulin-stimulated glucose uptake and consumption in a dose-dependent manner, and it reduced the insulin-stimulated phosphorylation of Akt at Thr308 and Ser473, but had no effect on the protein expression of PI3K-p85 or the activity of PI3K. Additionally, it inhibited the insulin-stimulated phosphorylation of Src at Tyr416, causing a reduction in the Src-mediated phosphorylation of Akt. Inhibition of Src by PP2 resulted in decreases in insulin-stimulated glucose uptake and phosphorylation of Src at Tyr416 and Akt at Thr308 and Ser473. The findings indicate that palmitate contributes to insulin resistance by inhibiting the Src-mediated phosphorylation of Akt in C2C12 myotubes, and this provides insight into the molecular mechanisms of FFA-induced insulin resistance.     

Functional Expression and Characterization of Dipeptidyl Peptidase IV from the Black-Bellied Hornet Vespa basalis in Sf21 Insect Cells

The maturation of mastoparan B, the major toxin peptide in the venom of Vespa basalis, requires enzymatic cleavage of its prosequence presumably via sequential liberation of dipeptides. The putative processing enzyme, dipeptidyl peptidase IV, was expressed as a glycosylated His-tag fusion protein (rDPP-IV) via the baculovirus expression system. rDPP-IV purified by one-step nickel-affinity chromatography was verified by Western blot and LC-MS/MS analysis. The k_cat/K_m of rDPP-IV was determined to be in the range of 10–500 mM^?1·S^?1 for five synthetic substrates. The optimal temperature and pH for rDPP-IV were determined to be 50 °C and pH 9. Enzymatic activity of rDPP-IV was significantly reduced by 80 and 60% in the presence of sitagliptin and phenylmethylsulfonyl fluoride respectively.     

Overexpression of mutant cell division cycle 25 homolog B (CDC25B) enhances the efficiency of selection in Chinese hamster ovary cells

The effects of mutant cell division cycle 25 homolog B (CDC25B) overexpression on the generation of cells producing a monoclonal antibody were investigated in Chinese hamster ovary (CHO) cells. Mutant CDC25B (m-CDC25B) expression plasmids were transfected into CHO DG44-derived cells producing a monoclonal antibody, and the frequency of highly producing cells was assessed following gene amplification in the presence of 250 nM methotrexate. Most of the clones obtained from the m-CDC25B-overexpressing cells had higher antibody titers than did mock-transfected control cells. This arose from either higher transgene copy numbers or higher mRNA expression levels for the antibody. However, the high mRNA expression levels were not always accompanied by increases in transgene copy numbers. Our results suggest that cells producing high levels of a monoclonal antibody can be selected efficiently using m-CDC25B overexpression.