Factors influencing potent antagonistic effects of Escherichia coli and Bacteroides ovatus on Staphylococcus aureus in anaerobic continuous flow cultures

We examined factors related to the potent antagonistic effect of Escherichia coli and Bacteroides ovatus on Staphylococcus aureus in anaerobic continuous flow cultures. In the presence of sugars fermentable by E. coli alone or both E. coli and S. aureus, motile E. coli strains exerted a potent antagonistic effect and S. aureus was expelled from the culture vessel within a few days. Conversely, in the presence of a sugar fermentable by S. aureus alone, the antagonistic effect of E. coli was diminished and S. aureus persisted at ca. 5 x 10(5) cfu/mL. B. ovatus alone exerted only a weak antagonistic effect on S. aureus in any culture conditions; however, when B. ovatus was cocultivated with E. coli and S. aureus, even in the presence of a sugar fermentable by S. aureus but not by E. coli, the potent antagonistic effect was restored. Escherichia coli showed the same level of antagonistic effect either in the presence of acetic acid (ca. 32 mM), propionic acid (4 mM), butyric acid (17 mM) and hydrogen sulfide (5 x 10(-1) mM) or when these metabolic products, except for a small amount of acetic acid (1.2 mM) were not present. In these culture conditions, S. aureus populations were lost at rates much higher than theoretical wash out rates of resting cells. These results indicate the presence of some bactericidal factors other than the volatile fatty acids and hydrogen sulfide. The bactericidal factors were not found in cultures of E. coli heated in boiling water for 10 min and in cell-free culture filtrates. Thus, the bactericidal factors seem to be associated with live E. coli cells. The nature of the bactericidal factors is not clear at present.     

Molecular genotyping of N-acetylation polymorphism to predict phenotype

Summary N-acetylation polymorphism is one of the representative pharmacogenetic traits that underlie interindividual and interethnic differences in response to xenobiotics. To develop a practical genotyping method to predict acetylator phenotype, we studied the conditions for accurate phenotyping, and identified the phenotype in 51 Japanese. Then we performed Southern blot analysis of genomic DNA from these subjects using ³²P-labeled cDNA for polymorphic N-acetyltransferase in the liver, and found that four N-acetyltransferase alleles generated six genotypes. The present genotyping method predicted the rapid, intermediate, and slow acetylators correctly in 48 of 51 overall subjects (96%) and in all of 4 slow acetylators.     

Excitation of indole analogs by phagocytosing leukocytes

The system suspended with phagocytosing leukocytes and related system produce weak light which could be greatly amplified by indole analogs with plain fatty acids at 3 position. Main emitting species in indole-3-acetic acid or indole-3-propionic acid-sensitized system was analyzed spectrometrically in the dark and ascribed to the transition of an excited indole compound in triplet state to its ground state. Such an excited species would be generated by the oxidative way of the indole analogs but not through the dioxetane structure of 2 and 3 positions on indole ring.     

Vibrio tetraodonis subsp. pristinus subsp. nov., isolated from the coral Acropora cytherea at Palmyra Atoll,and creation and emended description of Vibrio tetraodonis subsp. tetraodonis subsp. nov

Strain OCN044^T was isolated from the homogenised tissue and mucus of an apparently healthy Acropora cytherea coral fragment collected from the western reef terrace of Palmyra Atoll in the Northern Line Islands and was taxonomically evaluated with a polyphasic approach. The morphological and chemotaxonomic properties are consistent with characteristics of the genus Vibrio: Gram-stain-negative rods, oxidase- and catalase-positive, and motile by means of a polar flagellum. Strain OCN044^T can be differentiated as a novel subspecies based on 21 differences among chemotaxonomic features (e.g., fatty acids percentages for C_12:0 and C_18:1 ω7c), enzymatic activities (e.g., DNase and cystine arylamidase), and carbon sources utilized (e.g., L-xylose and D-melezitose) from its nearest genetic relative. Phylogenetic analysis and genomic comparisons show close evolutionary relatedness to Vibrio tetraodonis A511^T but the overall genomic relatedness indices identify strain OCN044^T as a distinct subspecies. Based on a polyphasic characterisation, differences in genomic and taxonomic data, strain OCN044^T represents a novel subspecies of V. tetraodonis A511^T, for which the name Vibrio tetraodonis subsp. pristinus subsp. nov. is proposed. The type strain is OCN044^T (=?LMG 31895^T?=?DSM 111778^T).

     

Editor's choice: Correction of Down syndrome and Edwards syndrome aneuploidies in human cell cultures

Aneuploidy, an abnormal number of chromosomes, has previously been considered irremediable. Here, we report findings that euploid cells increased among cultured aneuploid cells after exposure to the protein ZSCAN4, encoded by a mammalian-specific gene that is ordinarily expressed in preimplantation embryos and occasionally in stem cells. For footprint-free delivery of ZSCAN4 to cells, we developed ZSCAN4 synthetic mRNAs and Sendai virus vectors that encode human ZSCAN4. Applying the ZSCAN4 biologics to established cultures of mouse embryonic stem cells, most of which had become aneuploid and polyploid, dramatically increased the number of euploid cells within a few days. We then tested the biologics on non-immortalized primary human fibroblast cells derived from four individuals with Down syndrome—the most frequent autosomal trisomy of chromosome 21. Within weeks after ZSCAN4 application to the cells in culture, fluorescent in situ hybridization with a chromosome 21-specific probe detected the emergence of up to 24% of cells with only two rather than three copies. High-resolution G-banded chromosomes further showed up to 40% of cells with a normal karyotype. These findings were confirmed by whole-exome sequencing. Similar results were obtained for cells with the trisomy 18 of Edwards syndrome. Thus a direct, efficient correction of aneuploidy in human fibroblast cells seems possible in vitro using human ZSCAN4.     

Action of multiple endoplasmic reticulum chaperon-like proteins is required for proper folding and polarized localization of Kre6 protein essential in yeast cell wall β-1,6-glucan synthesis

Saccharomyces cerevisiae Kre6 is a type II membrane protein essential for cell wall β-1,6-glucan synthesis. Recently we reported that the majority of Kre6 is in the endoplasmic reticulum (ER), but a significant portion of Kre6 is found in the plasma membrane of buds, and this polarized appearance of Kre6 is required for β-1,6-glucan synthesis. An essential membrane protein, Keg1, and ER chaperon Rot1 bind to Kre6. In this study we found that in mutant keg1-1 cells, accumulation of Kre6 at the buds is diminished, binding of Kre6 to Keg1 is decreased, and Kre6 becomes susceptible to ER-associated degradation (ERAD), which suggests Keg1 participates in folding and transport of Kre6. All mutants of the calnexin cycle member homologues (cwh41, rot2, kre5, and cne1) showed defects in β-1,6-glucan synthesis, although the calnexin chaperon system is considered not functional in yeast. We found synthetic defects between them and keg1-1, and Cne1 co-immunoprecipitated with Keg1 and Kre6. A stronger binding of Cne1 to Kre6 was detected when two glucosidases (Cwh41 and Rot2) that remove glucose on N-glycan were functional. Skn1, a Kre6 homologue, was not detected by immunofluorescence in the wild type yeast, but in kre6Δ cells it became detectable and behaved like Kre6. In conclusion, the action of multiple ER chaperon-like proteins is required for proper folding and localization of Kre6 and probably Skn1 to function in β-1,6-glucan synthesis.     

Eudesmane-type sesquiterpene lactones inhibit multiple steps in the NF-κB signaling pathway induced by inflammatory cytokines

Inflammatory cytokines, such as interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), induce the intracellular signaling pathway leading to the activation of nuclear factor κB (NF-κB). A series of eudesmane-type sesquiterpene lactones possessing an α-methylene γ-lactone group and/or an α-bromo ketone group were synthesized and evaluated for their inhibitory effects on the NF-κB-dependent gene expression and signaling pathway. Our present study reveals that eudesmane-type α-methylene γ-lactones and α-bromo ketones inhibit multiple steps in the NF-κB signaling pathway induced by IL-1α and TNF-α.     

Virtual ligand screening of α-glucosidase: Identification of a novel potent noncarbohydrate mimetic inhibitor

5-Thiazoleacetamide derivatives of AR122 and AR125 were screened as α-glucosidase inhibitors by in silico high-throughput screening from commercial drug-like small compound libraries. Inhibition of α-glucosidase with AR122 and AR125 is time dependent: with no preincubation, AR122 and AR125 are relatively moderate inhibitors, but interestingly, after a 120 min incubation, they were 50-fold more potent (AR122: IC(50)=2.47 μM and AR125: IC(50)=27.1 μM). Plots of ln [residual α-glucosidase activity %] versus preincubation time show a pseudo-first order kinetics for both inhibitors. Through dialysis of enzyme-inhibitor complexes, no activity recovery was shown. These results suggest that AR122 and AR125 constitute a new class of noncarbohydrate mimetic inhibitor with an irreversible mechanism.     

Mature hepatocyte growth factor/scatter factor on the surface of human granulocytes is released by a mechanism involving activated factor Xa

Serum hepatocyte growth factor (HGF) is rapidly increased in patients suffering from various tissue injuries including arterial occlusive diseases. However, the cellular sources of the HGF increase remain largely unknown. In the present study, we showed that bioactive mature HGF is constitutively present on the surface of granulocytes in human peripheral blood. Exogenously added 125I-labeled iodo-HGF efficiently bound to granulocyte surface, whereas only a scarce amount of HGF mRNA was detected in granulocytes, indicating that the mature HGF on granulocytes is likely to be derived from other cell types. Interestingly, treatment of granulocytes with human serum rapidly induced the release of the cell surface-associated HGF. In vivo, thromboplastin injection into mice increased HGF release from transplanted human granulocytes, which was inhibited by the pretreatment with DX9065a, a specific inhibitor of factor Xa. Furthermore, DX9065a also inhibited the serum-induced HGF release from human granulocytes in vitro, suggesting that the HGF-releasing factor(s) in serum is associated with factor Xa activation. Thus, human granulocytes may function as a transporter of HGF in the peripheral blood, releasing HGF at the injured sites caused by blood coagulation, where HGF may promote tissue repair.     

Identification of human UDP-glucuronosyltransferase isoform(s) responsible for the C-glucuronidation of phenylbutazone

Glucuronidation is a major metabolic pathway in the biotransformation of many xenobiotics and endogeneous compounds. There have been many studies on the formation of O-, N- or S-glucuronides and identification of the UDP-glucuronosyltransferase (UGT) isoforms responsible for the formation of these glucuronides. However, there is no information available on which UGT isoform(s) catalyzes C-glucuronidation. In the present study, 16 human UGTs (UGTs 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B11, 2B15, 2B17 and 2B28) were cloned and expressed in baculovirus-infected insect cells and investigated to determine their C-glucuronidating activity toward phenylbutazone (PB). Among the UGT isoforms investigated, only UGT1A9 catalyzed PB C-glucuronidation. Human liver and kidney microsomes, which are well known to express UGT1A9, had C-glucuronidating activity toward PB. However, the jejunum, which did not express UGT1A9, had no C-glucuronidating activity. These results demonstrate for the first time that PB C-glucuronidation is catalyzed by only UGT1A9.