Saccharomyces cerevisiae mutant displaying beta-glucans on cell surface

The deletion of MCD4 leads to an increase in beta-1,6-glucan level and a decrease in glycosylphosphatidylinositol-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae, suggesting that mcd4 deletion mutant (mcd4Delta) displays beta-glucans on the cell surface without a mannan cover. An observation of the cell surface of mcd4Delta cells and an examination of the effect of contact between mcd4Delta cells and mouse macrophages indicated that macrophages were activated by contact with mcd4Delta cells displaying beta-glucans on the cell surface. We further examined the effect of intraperitoneal ethanol-fixed mcd4Delta cells on the survival period of mice infected with Candida albicans. mcd4Delta cells prolonged the survival period, implying that mcd4Delta cells may enhance the immune function of mice via macrophage activation. Moreover, we examined the structures of beta-glucans (i.e., alkali- and acetic acid-insoluble beta-glucans) extracted from mcd4Delta with (13)C-NMR and the effect of extracted beta-glucans on TNF-alpha secretion from macrophages. The structures of the beta-glucans from mcd4Delta differed from those of wild type (WT); however, there was no difference in tumor necrosis factor-alpha (TNF-alpha) secretion level between beta-glucans from mcd4Delta and those from WT. The yield of purified beta-glucans obtained from dry cells of mcd4Delta was higher than that obtained from dry cells of WT. mcd4Delta may be a superior strain for the preparation of beta-glucans.     

Archaeal population on supporting material in methanogenic packed-bed reactor

The population of methanogenic archaea in a packed-bed reactor was determined. A difference between populations on the supporting material and in effluent was observed at operation under a high organic loading rate and a short hydraulic retention time. This difference was characterized by the predominance of Methanosarcina sp. on the supporting material.     

Purification and characterization of enzyme responsible for N-myristoylation of octapeptide in aqueous solution without ATP and coenzyme a from Pseudomonas aeruginosa

The enzyme that catalyzes N-acyl linkage between myristic acid and the NH(2)-terminal glycine residue of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-NH(2) in aqueous solution without ATP and coenzyme A was found in Pseudomonas aeruginosa. The enzyme was purified from cell-free crude extract using DEAE-Cellulose, Sephadex G-200, CM-Sephadex C-50, and hydroxyapatite column chromatographies, and then purified approximately 1900-fold with about 1.5% recovery of enzyme activity from the crude extract. Finally, the purified enzyme showed a main band on SDS polyacrylamide gel electrophoresis after staining with Coomassie Brilliant Blue. The band corresponded to a molecular mass of approximately 60 kDa. The K(m)s of the purified enzyme for the substrate myristic acid and the octapeptide were 0.36 and 2.6 mM, respectively. When myristoyl-CoA instead of myristic acid was used as the substrate for the enzyme reaction, myristoyl octapeptide could be synthesized as observed in the case of myristic acid. The K(m) of myristoyl-CoA was 0.17 mM.     

Evaluation of the Anti-Proliferative Activity of Rare Aldohexoses against MOLT-4F and DU-145 Human Cancer Cell Line and Structure-Activity Relationship of D-Idose

D-Allose (D-All), a C-3 epimer of D-glucose (D-Glc), is a naturally rare monosaccharide, which shows anti-proliferative activity against several human cancer cell lines. Unlike conventional anticancer drugs, D-All targets glucose metabolism and is non-toxic to normal cells. Therefore, it has attracted attention as a unique “seed” compound for anticancer agents. However, the anti-proliferative activities of the other rare aldohexoses have not been examined yet. In this study, we evaluated the anti-proliferative activity of rare aldohexoses against human leukemia MOLT-4F and human prostate cancer DU-145 cell lines. We found that D-All and D-idose (D-Ido) at 5 mM inhibited cell proliferation of MOLT-4F cells by 46 % and 60 %, respectively. On the other hand, the rare aldohexoses at 5 mM did not show specific anti-proliferative activity against DU-145 cells. To explore the structure–activity relationship of D-Ido, we evaluated the anti-proliferative activity of D-sorbose (D-Sor), 6-deoxy-D-Ido, and L-xylose (L-Xyl) against MOLT-4F cells and found that D-Sor, 6-deoxy-D-Ido, and L-Xyl showed no inhibitory activity at 5 mM, suggesting that the aldose structure and the C-6 hydroxy group of D-Ido are important for its activity. Cellular glucose uptake assay and western blotting analysis of thioredoxin-interacting protein (TXNIP) expression suggested that the anti-proliferative activity of D-Ido is induced by inhibition of glucose uptake via TXNIP-independent pathway.     

Construction of hydrogen fermentation from garbage slurry using the membrane free bioelectrochemical system

The aim of this study was to show the effectiveness of the membrane free bioelectrochemical system (BES) using three electrodes on inhibition of methanogenesis and construction of hydrogen fermentation from the artificial garbage slurry. The electrical redox-potential on the working electrode was adjusted to -1.0V (vs. Ag/AgCl) that has positive effect on methanogenesis. The redox-potential on the counter electrode was measured to be 1.6V. The pH in the effluents was 5.5-6.4. Hydrogen production rate at the cathode side was similar to that at the anode side and much higher than that calculated from current, and reached a maximum of 2445±815 (average±standard deviation) mL?L(-1)?d(-1) at an organic loading rate of 58.7g dichromate chemical oxygen demand per L d(-1). Methane production was negligible throughout the experiment. Acetate and butyrate were the main products of the fermentation using a BES; these offered favorable conditions for hydrogen production. The bacterial community in the bioelectrochemical hydrogen fermentor differed from that in the methanogenic seed sludge and included hitherto unknown species. These results show that high redox-potential on the anodic electrode and acidic pH in the membrane free BES can be utilized for hydrogen fermentation from the artificial garbage slurry by avoiding methanogenesis.     

Prevalence, populations and pheno- and genotypic characteristics of Listeria monocytogenes isolated from ready-to-eat vegetables marketed in São Paulo, Brazil

Detection of food-borne viruses such as noroviruses, rotaviruses and hepatitis A virus in food products differs from detection of most food-borne bacteria, as most of these viruses cannot be cultivated in cell culture to date. Therefore, detection of food-borne viruses in food products requires multiple steps: first, virus extraction; second, purification of the viral genomic material (RNA for the majority of food-borne viruses); and last, molecular detection. This review is focused on the first step, the virus extraction. All of the numerous published protocols for virus extraction from food samples are based on 3 main approaches: 1) (acid adsorption-) elution-concentration; 2) direct RNA extraction; and 3) proteinase K treatment. This review summarizes these virus extraction approaches and the results obtained from published protocols. The use of process controls is also briefly described.     

Magnesium interlayered diamond coating on silicon

Diamond thin films have been deposited using hot filament chemical vapour deposition technique on manually scratched p-Si(1 0 0) substrate, with and without magnesium interlayer. In spite of magnesium melting point being lower (T_m = 649 °C) than the growth temperature of the substrate (T_s  750 °C) used in these experiments, it was found that high quality diamond films could be grown on Mg covered substrate. A liquid substrate is probably generated during the diamond film growth. Raman spectroscopy analysis exhibited only the triply degenerate, zone centre optical phonon peak at 1333 cm⁻¹ indicating that nearly stress free crystallites were present. Broadening of the Raman peak (11.76 cm⁻¹) indicates that some small crystallites also are present. Scanning electron and atomic force microscopy accompanied by X-ray diffraction analysis where used to compare the details of diamond film growth directly on scratched Si(1 0 0) and Mg interlayered scratched Si(1 0 0) substrates.     

Molecular cloning of an isoform of Doc2 having two C2-like domains

We previously isolated a novel protein having two C2-like domains known to interact with Ca2+ and phospholipid, and named Doc2 (Double C2). Doc2 is predominantly expressed in brain and is implicated in Ca(2+)-dependent neurotransmitter release. We have isolated here an isoform of Doc2 and named the original one Doc2 alpha and the new one Doc2 beta. Doc2 beta alsp has two C2-like domains and is 61% identical to Doc2 alpha at the amino acid level. In contrast to Doc2 alpha, the Doc2 beta mRNA is expressed ubiquitously. These results indicate that there are at least two isoforms of Doc2, and suggest that Doc2 beta is involved in Ca(2+)-dependent intracellular vesicle trafficking in various types of cells.