Customized media based on miniaturized screening improve growth rate and cell yield of methane-oxidizing bacteria of the genus Methylomonas

The growth of twelve methanotrophic strains within the genus Methylomonas, including the type strains of Methylomonas methanica and Methylomonas koyamae, was evaluated with 40 different variations of standard diluted nitrate mineral salts medium in 96-well microtiter plates. Unique profiles of growth preference were observed for each strain, showing a strong strain dependency for optimal growth conditions, especially with regards to the preferred concentration and nature of the nitrogen source. Based on the miniaturized screening results, a customized medium was designed for each strain, allowing the improvement of the growth of several strains in a batch setup, either by a reduction of the lag phase or by faster biomass accumulation. As such, the maintenance of fastidious strains could be facilitated while the growth of fast-growing Methylomonas strains could be further improved. Methylomonas sp. R-45378 displayed a 50 % increase in cell dry weight when grown in its customized medium and showed the lowest observed nitrogen and oxygen requirement of all tested strains. We demonstrate that the presented miniaturized approach for medium optimization is a simple tool allowing the quick generation of strain-specific growth preference data that can be applied downstream of an isolation campaign. This approach can also be applied as a first step in the search for strains with biotechnological potential, to facilitate cultivation of fastidious strains or to steer future isolation campaigns.     

Cathepsin B-mediated Autophagy Flux Facilitates the Anthrax Toxin Receptor 2-mediated Delivery of Anthrax Lethal Factor into the Cytoplasm

Anthrax lethal toxin (LeTx) is a virulence factor secreted by Bacillus anthracis and has direct cytotoxic effects on most cells once released into the cytoplasm. The cytoplasmic delivery of the proteolytically active component of LeTx, lethal factor (LF), is carried out by the transporter component, protective antigen, which interacts with either of two known surface receptors known as anthrax toxin receptor (ANTXR) 1 and 2. We found that the cytoplasmic delivery of LF by ANTXR2 was mediated by cathepsin B (CTSB) and required lysosomal fusion with LeTx-containing endosomes. Also, binding of protective antigen to ANXTR1 or -2 triggered autophagy, which facilitated the cytoplasmic delivery of ANTXR2-associated LF. We found that whereas cells treated with the membrane-permeable CTSB inhibitor CA074-Me- or CTSB-deficient cells had no defect in fusion of LC3-containing autophagic vacuoles with lysosomes, autophagic flux was significantly delayed. These results suggested that the ANTXR2-mediated cytoplasmic delivery of LF was enhanced by CTSB-dependent autophagic flux.     

Physiological characterization of starch-utilizing <Emphasis Type="Italic">Saccharomyces</Emphasis> sp. SK0704 isolated from kimchi

Saccharomyces sp. SK0704 (further defined as SK0704) isolated from long-term-ripening kimchi was identified by a biochemical method with an API kit; its physiology was found to be very similar to that of S. cerevisiae ATCC 26603 (further defined as ATCC 26603), except in terms of starch utilization. SK0704 did not excrete extracellular glucoamylase, but utilized starch as a sole carbon source under only aerobic conditions. Crude enzyme excreted from SK0704 catalyzed the saccharification of starch to glucose, but ATCC 26603 did not. The PCR product obtained using the chromosomal DNA of SK0704 and the primers designed on the basis of the extracellular glucoamylase-coding gene of S. diastaticus was homologous with the intracellular sporulation-specific glucoamylase of S. cerevisiae. SDS-PAGE pattern of soluble protein extracted from yeast cells grown on glucose was greatly different from that on starch. From these results, we proposed that the SK0704 may have a specific physiological function for starch catabolism such as membrane transport system and intracellular sac-charification of starch.     

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.     

The essential DNA polymerases delta and epsilon are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae.

We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases delta and epsilon showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases delta and epsilon are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair.