Culture-Independent Evaluation of Nonenveloped-Virus Infectivity Reduced by Free-Chlorine Disinfection

The inability of molecular detection methods to distinguish disinfected virions from infectious ones has hampered the assessment of infectivity for enteric viruses caused by disinfection practices. In the present study, the reduction of infectivity of murine norovirus S7-PP3 and mengovirus vMC0, surrogates of human noroviruses and enteroviruses, respectively, caused by free-chlorine treatment was characterized culture independently by detecting carbonyl groups on viral capsid protein. The amount of carbonyls on viral capsid protein was evaluated by the proportion of biotinylated virions trapped by avidin-immobilized gel (percent adsorbed). This culture-independent approach demonstrated that the percent adsorbed was significantly correlated with the logarithm of the infectious titer of tested viruses. Taken together with the results of previous reports, the result obtained in this study indicates that the amount of carbonyls on viral capsid protein of four important families of waterborne pathogenic viruses, Astroviridae, Reoviridae, Caliciviridae, and Picornaviridae, is increased in proportion to the received oxidative stress of free chlorine. There was also a significant correlation between the percent adsorbed and the logarithm of the ratio of genome copy number to PFU, which enables estimation of the infectious titer of a subject virus by measuring values of the total genome copy number and the percent adsorbed. The proposed method is applicable when the validation of a 4-log reduction of viruses, a requirement in U.S. EPA guidelines for virus removal from water, is needed along with clear evidence of the oxidation of virus particles with chlorine-based disinfectants.     

N-terminal region of chitinase I of Bacillus circulans KA-304 contained new chitin-biding domain

Chitinase I (CHI1) of Bacillus circulans KA-304 forms protoplasts from Schizophyllum commune mycelia when the enzyme is combined with α-1,3-glucanase of B. circulans KA-304. CHI1 consists of an N-terminal unknown region and a C-terminal catalytic region classified into the glycoside hydrolase family-19 type. An N-terminal region-truncated mutant of CHI 1 (CatCHI1), which was expressed in Escherichia coli Rosetta-gami B (DE3), lost colloidal chitin- and powder chitin-binding activities. The colloidal chitin- and the powder chitin-hydrolyzing activities of CatCHI1 were lower than those of CHI1, and CatCHI1 was not effective in forming the protoplast. A fusion protein of the N-terminal region of CHI1 and green fluorescent protein (Nterm-GFP) was expressed in E. coli, and the fusion protein was adsorbed to colloidal chitin, powder chitin, and chitosan. Fluorescence microscopy analysis showed that Nterm-GFP bound to the S. commune cell-wall.     

Microbial communities associated with acetate-rich gas-petroleum reservoir surface facilities

We evaluated the microbial communities in acetate-rich production waters from separators of a high-temperature gas-petroleum reservoir in Higashi-Niigata, Japan. Bacterial and archaeal 16S rRNA gene libraries constructed from these waters were dominated by Acetobacterium-, Methanofollis-, and Methanosarcina-related sequences. The libraries constructed from enrichment cultures of the production waters were dominated by sequences related to the Acetobacterium- and Methanofollis-related sequences.     

Synthesis of microbial signaling molecules and their stereochemistry-activity relationships

Microbial signaling molecules such as autoinducers and microbial hormones play important roles in intercellular communication in microorganisms. Information transfer between the individual cells of a microorganism is one of the most important biological events among them. Researchers often suffer from extremely low levels of microbial signaling molecule contents, which prevent them from understanding chemistry and biology of intercellular communication in microorganisms. Chemical synthesis is a powerful tool to obtain sufficient amounts of sample and to clarify the structure of a molecule. This review focuses on the synthesis and stereochemistry-bioactivity relationships of five microbial signaling molecules, Vibrio cholerae autoinducer-1 (CAI-1), AI-2 precursor (DPD), an acylhomoserine lactone from Rhizobium leguminosarum (small bacteriocin), a diffusible extracellular factor of Xanthomondas campestris pv. campestris, and Phytophthora mating hormone α1.     

A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation

The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 μM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.     

The second Phytophthora mating hormone defines interspecies biosynthetic crosstalk

The heterothallic species of the agricultural pest Phytophthora use mating hormones α1 and α2 to regulate their sexual reproduction. Here we describe the absolute stereostructure of the second mating hormone α2 as defined by spectroscopic analysis and total synthesis. We have uncovered not only the interspecies universality of α hormones but also the pathway by which α2 is biosynthesized from phytol by A2-mating type strains and metabolized to α1 by A1 strains.     

β-Actin specifically controls cell growth, migration, and the G-actin pool

Ubiquitously expressed β-actin and γ-actin isoforms play critical roles in most cellular processes; however, their unique contributions are not well understood. We generated whole-body β-actin-knockout (Actb(-/-)) mice and demonstrated that β-actin is required for early embryonic development. Lethality of Actb(-/-) embryos correlated with severe growth impairment and migration defects in β-actin-knockout primary mouse embryonic fibroblasts (MEFs) that were not observed in γ-actin-null MEFs. Migration defects were associated with reduced membrane protrusion dynamics and increased focal adhesions. We also identified migration defects upon conditional ablation of β-actin in highly motile T cells. Of great interest, ablation of β-actin altered the ratio of globular actin (G-actin) to filamentous actin in MEFs, with corresponding changes in expression of genes that regulate the cell cycle and motility. These data support an essential role for β-actin in regulating cell migration and gene expression through control of the cellular G-actin pool.     

A Src family inhibitor (PP1) potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells

Connexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity. We have recently reported that Cx32 acts as a tumor suppressor gene in renal cancer cells due to the inhibition of Src-dependent signaling. In line with the previous study, here we examined if a Src family inhibitor (PP1) could potentiate tumor-suppressive effect of Cx32 in Caki-2 cell from human renal cell carcinoma. In order to clarify the potentialization of PP1, using Cx32-transfected Caki-2 cells and mock-transfected Caki-2 cells, we estimated difference in cytotoxic effect of PP1 on the two cell clones in vitro as well as in vivo. PP1 showed more cytotoxic effect on Caki-2 cells having Cx32 positive expression than that of Cx32 negative expression at lower doses. This potentialization was also observed in xenograft model of nude mice. The potentialization of the effect mainly depended on the induction of apoptosis but not the control of cell growth. In conjugation with this event, the reduction of anti-apoptotic molecules (Bcl-2 and Bcl-xL) was caused by the combination of Cx32 expression and PP1 treatment in Caki-2 cells. These results suggest that PP1 potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells through the reduction of anti-apoptotic molecules.     

Neurokinins and inflammatory cell iNOS expression in guinea pigs with chronic allergic airway inflammation

In the present study we evaluated the role of neurokinins in the modulation of inducible nitric oxide synthase (iNOS) inflammatory cell expression in guinea pigs with chronic allergic airway inflammation. In addition, we studied the acute effects of nitric oxide inhibition on this response. Animals were anesthetized and pretreated with capsaicin (50 mg/kg sc) or vehicle 10 days before receiving aerosolized ovalbumin or normal saline twice weekly for 4 wk. Animals were then anesthetized, mechanically ventilated, given normal saline or N(G)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg ic), and challenged with ovalbumin. Prechallenge exhaled NO increased in ovalbumin-exposed guinea pigs (P < 0.05 compared with controls), and capsaicin reduced this response (P < 0.001). Compared with animals inhaled with normal saline, ovalbumin-exposed animals presented increases in respiratory system resistance and elastance and numbers of total mononuclear cells and eosinophils, including those expressing iNOS (P < 0.001). Capsaicin reduced all these responses (P < 0.05) except for iNOS expression in eosinophils. Treatment with l-NAME increased postantigen challenge elastance and restored both resistance and elastance previously attenuated by capsaicin treatment. Isolated l-NAME administration also reduced total eosinophils and mononuclear cells, as well as those cells expressing iNOS (P < 0.05 compared with ovalbumin alone). Because l-NAME treatment restored lung mechanical alterations previously attenuated by capsaicin, NO and neurokinins may interact in controlling airway tone. In this experimental model, NO and neurokinins modulate eosinophil and lymphocyte infiltration in the airways.