Purification and Properties of Lytic Enzymes from Streptomyces globisporus 1829

Two lytic enzymes capable of lysing Streptococcus mutans have been purified to give a single band on disc-gel electrophoresis, respectively. The M–1 and M–2 enzymes were both proved to be N-acetylmuramidases. However, these enzymes were entirely different on their enzymatic properties. The molecular weights were about 20,000 and 11,000 for M–1 and M–2 enzymes, respectively, The maximal lytic activity of M–1 enzyme was obtained at ionic strength 0.05, while lytic activity of M–2 enzyme did not change within the ionic strength range of 0 to 0.05. The M–1 enzyme constituted the majority of the total lytic activity against the cell walls of Streptococcus mutans BHT of cultured filtrate. The M–2 enzyme showed less specific lytic activity on the cell walls of Streptococcus mutans BHT than M–1 enzyme.     

Refeeding with a standard diet after a 48-h fast elicits an inflammatory response in the mouse liver

Unhealthy eating behaviors increase the risk of metabolic diseases, but the underlying mechanisms are not fully elucidated. Because inflammation contributes to the pathogenesis of metabolic diseases, it is important to understand the effects of unhealthy eating on the inflammatory state. The objective of our present study was to address the effects of a fasting–refeeding regime, a model of irregular eating, on the hepatic inflammatory responses in mouse. The animals were fasted for 48 h and then refed either a standard or low-carbohydrate/high-fat diet. Inflammatory gene expression in the liver was then sequentially measured for the first 17 h after initiation of refeeding. To assess the roles of dietary carbohydrates and toll-like receptor 2 (TLR2) in the refeeding-induced inflammatory changes, gene expression levels in mice refed only carbohydrates (α-corn starch and sucrose) at different doses and in TLR2-deficient mice refed a standard diet were also analyzed. Refeeding with a standard diet increased the liver expression of Tlr2, proinflammatory mediators (Cxcl10, Cxcl1, Cxcl2, Icam-1) and negative regulators of TLR-signaling (A20 and Atf3). These increases were attenuated in mice refed a low-carbohydrate/high-fat diet. Refeeding only α-corn starch and sucrose also increased the expression of these inflammatory pathway genes depending on the doses. TLR2 deficiency significantly attenuated the refeeding-induced increase in the liver expression of Cxcl10, Cxcl1, Icam-1 and A20. These findings suggest that an irregular eating behavior can elicit a liver inflammatory response, which is at least partly mediated by TLR2, and that dietary carbohydrates play critical roles in this process.     

Characterization of d-Alanyl-(d)-meso-2,6-diaminopimelic Acid Endopeptidase from Streptomyces globisporus 1829

d-Alanyl-(d)-meso-2,6-diaminopimelic acid endopeptidase was purified 47.4-fold with a yield of 40.5% from mutanolysin, which was partially purified from the cultural supernatant of Streptomyces globisporus 1829, by using ion exchange column chromatographies and a molecular sieve column. The purified enzyme was electrophoretically homogeneous. This enzyme had a molecular weight of 13,500 and an isoelectric point of pI 9.0. This enzyme was most active at pH 8.5 and stable between pHs 8.0 and 9.0. The hydrolyzing activity of this enzyme was enhanced by Co^{+ +} and Ca^{+ +} but inhibited appreciably by Zn^{+ +}, Cu^{+ +} and EDTA. The enzyme activity was not affected by β-lactam antibiotics and vancomycin. The Km values for bisdisaccharide heptapeptide and its derivative modified chemically by BOC-S were calculated to be 5.7 × 10^-4 and 4.0 × 10^-4 m, respectively.     

Protein extracts from cultured cells contain nonspecific serum albumin

Serum is an important component of cell culture media. The present study demonstrates contamination of intracellular protein extract by bovine serum albumin from the culture media and illustrates how this contamination can cause the misinterpretation of western blot results. Preliminary experiments can prevent the misinterpretation of some experimental results, and optimization of the washing process may enable specific protein detection.     

Cortisol and stress responses during a game and practice in female collegiate soccer players

The purpose of this study was to compare the cortisol responses from a regular season game and a typical practice session in female National Collegiate Athletic Association Division I collegiate soccer players. Eighteen players were assigned to 2 groups, 10 starters and 8 nonstarters, depending on their playing time. Salivary cortisol concentration, as well as competitive sport anxiety (somatic and cognitive anxiety, self-confidence), was monitored before and after 1 regular season game and 1 typical practice session. Although salivary cortisol levels increased postgame for both starters (+250%) and nonstarters (+140%), they increased to a greater extent for the starters. Practice salivary cortisol did not significantly change (p > 0.05). Cognitive and somatic anxiety was greater pre- and postgame when compared with the pre- and postpractice scores, respectively. These data clearly demonstrate the psychological and physiological differences between soccer competition and practice in collegiate women. It appears that both physiological and psychological variables combine to contribute to the large stress hormone response to an actual competitive game.     

Classification of genus Pseudomonas by MALDI-TOF MS based on ribosomal protein coding in S10-spc-alpha operon at strain level

We have proposed a rapid phylogenetic classification at the strain level by MALDI-TOF MS using ribosomal protein matching profiling. In this study, the S10-spc-alpha operon, encoding half of the ribosomal subunit proteins and highly conserved in eubacterial genomes, was selected for construction of the ribosomal protein database as biomarkers for bacterial identification by MALDI-TOF MS analysis to establish a more reliable phylogenetic classification. Our method revealed that the 14 reliable and reproducible ribosomal subunit proteins with less than m/z 15,000, except for L14, coded in the S10-spc-alpha operon were significantly useful biomarkers for bacterial classification at species and strain levels by MALDI-TOF MS analysis of genus Pseudomonas strains. The obtained phylogenetic tree was consisted with that based on genetic sequence (gyrB). Since S10-spc-alpha operons of genus Pseudomonas strains were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains, the ribosomal subunit proteins encoded in S10-spc-alpha operon were suitable biomarkers for construction and correction of the database. MALDI-TOF MS analysis using these 14 selected ribosomal proteins is a rapid, efficient, and versatile bacterial identification method with the validation procedure for the obtained results.     

Human Pluripotent Stem Cell-Derived Tumor Model Uncovers the Embryonic Stem Cell Signature as a Key Driver in Atypical Teratoid/Rhabdoid Tumor

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Substrate specificity,plasma membrane localization,and lipid modification of the aldehyde dehydrogenase ALDH3B1

The accumulation of reactive aldehydes is implicated in the development of several disorders. Aldehyde dehydrogenases (ALDHs) detoxify aldehydes by oxidizing them to the corresponding carboxylic acids. Among the 19 human ALDHs, ALDH3A2 is the only known ALDH that catalyzes the oxidation of long-chain fatty aldehydes including C16 aldehydes (hexadecanal and trans-2-hexadecenal) generated through sphingolipid metabolism. In the present study, we have identified that ALDH3B1 is also active in vitro toward C16 aldehydes and demonstrated that overexpression of ALDH3B1 restores the sphingolipid metabolism in the ALDH3A2-deficient cells. In addition, we have determined that ALDH3B1 is localized in the plasma membrane through its C-terminal dual lipidation (palmitoylation and prenylation) and shown that the prenylation is required particularly for the activity toward hexadecanal. Since knockdown of ALDH3B1 does not cause further impairment of the sphingolipid metabolism in the ALDH3A2-deficient cells, the likely physiological function of ALDH3B1 is to oxidize lipid-derived aldehydes generated in the plasma membrane and not to be involved in the sphingolipid metabolism in the endoplasmic reticulum.     

SRPX2 is a novel chondroitin sulfate proteoglycan that is overexpressed in gastrointestinal cancer

SRPX2 (Sushi repeat-containing protein, X-linked 2) has recently emerged as a multifunctional protein that is involved in seizure disorders, angiogenesis and cellular adhesion. Here, we analyzed this protein biochemically. SRPX2 protein was secreted with a highly posttranslational modification. Chondroitinase ABC treatment completely decreased the molecular mass of purified SRPX2 protein to its predicted size, whereas heparitinase, keratanase and hyaluroinidase did not. Secreted SRPX2 protein was also detected using an anti-chondroitin sulfate antibody. These results indicate that SRPX2 is a novel chondroitin sulfate proteoglycan (CSPG). Furthermore, a binding assay revealed that hepatocyte growth factor dose-dependently binds to SRPX2 protein, and a ligand-glycosaminoglycans interaction was speculated to be likely in proteoglycans. Regarding its molecular architecture, SRPX2 has sushi repeat modules similar to four other CSPGs/lecticans; however, the molecular architecture of SRPX2 seems to be quite different from that of the lecticans. Taken together, we found that SRPX2 is a novel CSPG that is overexpressed in gastrointestinal cancer cells. Our findings provide key glycobiological insight into SRPX2 in cancer cells and demonstrate that SRPX2 is a new member of the cancer-related proteoglycan family.