Chicken- and duck-associated Bacteroides–Prevotella genetic markers for detecting fecal contamination in environmental water

Bacteroides–Prevotella group is one of the most promising targets for detecting fecal contamination in water environments, principally due to its host-specific distributions and high concentrations in feces of warm-blooded animals. We developed real-time PCR assays for quantifying chicken/duck-, chicken-, and duck-associated Bacteroides–Prevotella 16S rRNA genetic markers (Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac). A reference collection of DNA extracts from 143 individual fecal samples and wastewater treatment plant influent was tested by the newly established markers. The quantification limits of Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac markers in environmental water were 54, 57, and 12 copies/reaction, respectively. It was possible to detect possible fecal contaminations from wild ducks in environmental water with the constructed genetic marker assays, even though the density of total coliforms in the identical water samples was below the detection limit. Chicken/Duck-Bac marker was amplified from feces of wild duck and chicken with the positive ratio of 96 and 61 %, respectively, and no cross-reaction was observed for the other animal feces. Chicken-Bac marker was detected from 70 % of chicken feces, while detected from 39 % of cow feces, 8.3 % of pig feces, and 12 % of swan feces. Duck-Bac marker was detected from 85 % of wild duck feces and cross-reacted with 31 % of cow feces. These levels of detection specificity are common in avian-associated genetic markers previously proposed, which implies that there is a practical limitation in the independent application of avian-associated Bacteroides–Prevotella 16S rRNA genetic markers and a combination with other fecal contamination markers is preferable for detecting fecal contamination in water environments.     

Ras GTPase Activating Protein CoIra1 Is Involved in Infection-Related Morphogenesis by Regulating cAMP and MAPK Signaling Pathways through CoRas2 in Colletotrichum orbiculare

Colletotrichum orbiculare is the causative agent of anthracnose disease on cucurbitaceous plants. Several signaling pathways, including cAMP–PKA and mitogen-activating protein kinase (MAPK) pathways are involved in the infection-related morphogenesis and pathogenicity of C. orbiculare. However, upstream regulators of these pathways for this species remain unidentified. In this study, CoIRA1, encoding RAS GTPase activating protein, was identified by screening the Agrobacterium tumefaciens-mediated transformation (AtMT) mutant, which was defective in the pathogenesis of C. orbiculare. The coira1 disrupted mutant showed an abnormal infection-related morphogenesis and attenuated pathogenesis. In Saccharomyces cerevisiae, Ira1/2 inactivates Ras1/2, which activates adenylate cyclase, leading to the synthesis of cAMP. Increase in the intracellular cAMP levels in coira1 mutants and dominant active forms of CoRAS2 introduced transformants indicated that CoIra1 regulates intracellular cAMP levels through CoRas2. Moreover, the phenotypic analysis of transformants that express dominant active form CoRAS2 in the comekk1 mutant or a dominant active form CoMEKK1 in the coras2 mutant indicated that CoRas2 regulates the MAPK CoMekk1–Cmk1 signaling pathway. The CoRas2 localization pattern in vegetative hyphae of the coira1 mutant was similar to that of the wild-type, expressing a dominant active form of RFP–CoRAS2. Moreover, we demonstrated that bimolecular fluorescence complementation (BiFC) signals between CoIra1 and CoRas2 were detected in the plasma membrane of vegetative hyphae. Therefore, it is likely that CoIra1 negatively regulates CoRas2 in vegetative hyphae. Furthermore, cytological analysis of the localization of CoIraI and CoRas2 revealed the dynamic cellular localization of the proteins that leads to proper assembly of F-actin at appressorial pore required for successful penetration peg formation through the pore. Thus, our results indicated that CoIra1 is involved in infection-related morphogenesis and pathogenicity by proper regulation of cAMP and MAPK signaling pathways through CoRas2.     

Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities

Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain-truncated form (HBdeltatm) of the molecule. HB(uc/uc) mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBdeltatm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control.     

W-heterochromatin of chicken; its unusual DNA components,late replication,and chromatin structure

About 65% of DNA in the chicken W chromosome has been shown to consist ofXhoI andEcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kbXhoI and the 1.2-kbEcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescencein situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed alongXhoI andEcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the β-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of theHaeIII-digested MSB-1 nuclei yielded a chromatin fraction in whichXhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for theXhoI family sequence were present in this fraction.     

Phosphorylation of p66shc mediates 6-hydroxydopamine cytotoxicity

6-Hydroxydopamine (6-OHDA) is a neurotoxin that has been widely used to generate Parkinson's disease (PD) models. Increased oxidative stress is suggested to play an important role in 6-OHDA-induced cell death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66shc, this study investigated the role of p66shc in the cytotoxicity of 6-OHDA. 6-OHDA induced cell death and p66shc phosphorylation at Ser36 in SH-SY5Y cells. Pre-treatment with the protein kinase C β (PKCβ) inhibitor hispidin suppressed 6-OHDA-induced p66shc phosphorylation. Elimination of H(2)O(2) by catalase reduced cell death and p66shc phosphorylation induced by 6-OHDA. Cells deficient in p66shc were more resistant to 6-OHDA-induced cell death than wild-type cells. Furthermore, reconstitution of wild-type p66shc, but not the S36A mutant, in p66shc-deficient cells increased susceptibility to 6-OHDA. These results indicate that H(2)O(2) derived from 6-OHDA is an important mediator of cell death and p66shc phosphorylation induced by 6-OHDA and that p66shc phosphorylation at Ser36 is indispensable for the cytotoxicity of 6-OHDA.     

Synthesis,structural characterization and antimicrobial activities of 12 zinc(II) complexes with four thiosemicarbazone and two semicarbazone ligands

Twelve zinc(II) complexes with thiosemicarbazone and semicarbazone ligands were prepared and characterized by elemental analysis, thermogravimetric and differential thermal analysis (TG/DTA), FT-IR and 1H and 13C NMR spectroscopy. Seven three-dimensional structures of zinc(II) complexes were determined by single-crystal X-ray analysis. Their antimicrobial activities were evaluated by MIC against four bacteria (B. subtilis, S. aureus, E. coli and P. aeruginosa), two yeasts (C. albicans and S. cerevisiae) and two molds (A. niger and P. citrinum). The 5- and 6-coordinate zinc(II) complexes with a tridentate thiosemicarbazone ligand (Hatsc), ([Zn(atsc)(OAc)](n) 1, [Zn(Hatsc)(2)](NO(3))(2).0.3H(2)O 2, [ZnCl(2)(Hatsc)] 3 and [Zn(SO(4))(Hatsc)(H(2)O)].H(2)O 4 [Hatsc=2-acetylpyridine(thiosemicarbazone)]), showed antimicrobial activities against test organisms, which were different from those of free ligands or the starting zinc(II) compounds. Especially, complex 2 showed effective activities against P. aeruginosa, C. albicans and moderate activities against S. cerevisiae and two molds. These facts are in contrast to the results that the 5- or 6-coordinate zinc(II) complexes with a tridentate 2-acetylpyridine-4N-morpholinethiosemicarbazone, ([Zn(mtsc)(2)].0.2EtOH 5, the previously reported catena-poly [Zn(mtsc)-mu-(OAc-O,O')](n) and [Zn(NO(3))(2)(Hmtsc)] [Hmtsc=2-acetylpyridine (4N-morpholyl thiosemicarbazone)]), showed no activities against the test microorganisms. The 5- and 6-coordinate zinc(II) complexes with a tridentate 2-acetylpyridinesemicarbazone, ([Zn(OAc)(2)(Hasc)] 6 and [Zn(Hasc)(2)](NO(3))(2) 7 [Hasc=2-acetylpyridine(semicarbazone)]), showed no antimicrobial activities against bacteria, yeasts and molds. Complex [ZnCl(2)(Hasc)] 8, which was isostructural to complex 3, showed modest activity against Gram-positive bacterium, B. subtilis. The 1:1 complexes of zinc(II) with pentadentate thiosemicarbazone ligands, ([Zn(dmtsc)](n) 9 and [Zn(datsc)](n) 10 [H(2)dmtsc=2,6-diacetylpyridine bis(4N-morpholyl thiosemicarbazone) and H(2)datsc=2,6-diacetylpyridine bis(thiosemicarbazone)]), did not inhibit the growth of the test organisms. On the contrary, 7-coordinate zinc(II) complexes with one pentadentate semicarbazone ligand and two water molecules, ([Zn(H(2)dasc)(H(2)O)(2)](OAc)(2).5.3H(2)O 11 and [Zn(H(2)dasc)(H(2)O)(2)](NO(3))(2).H(2)O 12 [H(2)dasc=2,6-diacetylpyridine bis(semicarbazone)]), showed modest to moderate activities against bacteria. Based on the X-ray structures, the structure-activity correlation for the antimicrobial activities was elucidated. The zinc(II) complexes with 4N-substituted ligands showed no antimicrobial activities. In contrast to the previously reported nickel(II) complexes, properties of the ligands such as the ability to form hydrogen bonding with a counter anion or hydrated water molecules or the less bulkiness of the 4N moiety would be a more important factor for antimicrobial activities than the coordination number of the metal ion for the zinc(II) complexes.     

Golgin-84 is a rab1 binding partner involved in Golgi structure

Members of the golgin family of coiled-coil proteins have been implicated in the tethering of vesicles to Golgi membranes and cisternal membranes to each other. Many also bind to rab GTPases. Golgin-84 is a membrane-anchored golgin that we now show binds preferentially to the GTP form of the rab1 GTPase. It is also present throughout the Golgi stack by immuno-EM. Antibodies to golgin-84 inhibit stacking of cisternal membranes in a cell-free assay for Golgi reassembly, whereas the cytoplasmic domain of golgin-84 stimulates stacking and increases the length of re-assembled stacks. Transient expression of golgin-84 in NRK cells helps prevent the disassembly of the Golgi apparatus normally triggered by treatment with brefeldin A. Together these data suggest that golgin-84 is involved in generating and maintaining the architecture of the Golgi apparatus.     

Construction and expression of anti-Tn-antigen-specific single-chain antibody genes from hybridoma producing MLS128 monoclonal antibody

Anti-Tn-antigen monoclonal antibody MLS128 has affinity for three consecutive Tn-antigens (Tn3) more than Tn2. The major aim of this study was to isolate genes encoding MLS128 variable domains to produce a large quantity of recombinant MLS128 antibodies, in turn, allowing the conduct of studies on precise interactions between Tn3- or Tn2-epitopes and MLS128. This study describes cloning of the variable region genes of MLS128, construction of the variable region genes in single-chain variable fragments (scFv) and two scFvs conjugated with human IgG(1) hinge and Fc regions (scFv-Fc) types, and their respective expression in bacterial and mammalian cell. MLS128 scFv protein with the expected specificity and affinity was successfully prepared from inclusion bodies accumulating in Escherichia coli. Construction, expression and purification of two types of MLS128-scFv-Fc proteins with differing linker lengths in Chinese hamster ovary cells demonstrated that the purified scFv-Fc proteins had binding activity specific to the glycoprotein-expressing Tn-antigen clusters. These results revealed that VL and VH genes cloned from the hybridoma represent those of MLS128 and that recombinant antibodies produced from these genes should provide sufficient amounts of binding domains for use in 3D structural studies such as NMR and X-ray analysis.