Geomicrobiological exploration and characterization of a novel deep-sea hydrothermal system at the TOTO caldera in the Mariana Volcanic Arc

Novel hydrothermal activities accompanying effluent white smokers and elemental sulfur chimney structures at the north-east lava dome of the TOTO caldera depression in the Mariana Volcanic Arc have been explored and characterized by geochemical and microbiological surveys. White smoker hydrothermal fluids were observed in the potential hydrothermal activity centre of the field and represented the maximal temperature of 170 degrees C and the lowest pH of 1.6. The chimney structures, all consisting of elemental sulfur (sulfur chimney), were also unique to the TOTO caldera hydrothermal field. Microbial community structures in a sulfur chimney and its formation hydrothermal fluid with a high concentration of hydrogen sulfide (15 mM) have been investigated by culture-dependent and -independent analyses. 16S rRNA gene clone analysis and fluorescence in situ hybridization (FISH) analysis revealed that epsilon-Proteobacteria dominated the microbial communities in the sulfur chimney structure and formed a dense microbial mat covering the sulfur chimney surface. Archaeal phylotypes were consistently minor components in the communities and related to the genera Thermococcus, Pyrodictium, Aeropyrum, and the uncultivated archaeal group of 'deep-sea hydrothermal vent euryarchaeotal group'. Cultivation analysis suggested that the chemolithoautotrophs might play a significant ecological role as primary producers utilizing gas and sulfur compounds provided from hydrothermal fluids.     

Detection of urovirulence factors in Escherichia coli by multiplex polymerase chain reaction

Abstract Primers to amplify the genes encoding the virulence factors of uropathogenic Escherichia coli , such as pilus associated with pyelonephritis ( pap ), haemolysin ( hly ), aerobactin ( aer ) and cytotoxic necrotizing factor 1 ( cnf 1) genes, were designed. The above primers along with previously reported primers for S fimbriae ( sfa ) and afimbrial adhesin I ( afaI ) genes were combined to develop a multiplex polymerase chain reaction (PCR) for detection of the respective virulence factors and for the identification of uropathogenic E. coli . The multiplex PCR to detect pap, sfa, afa I, hly, aer and cnf 1 genes was highly specific and the sensitivity was found to be about 5 × 10³ colony forming units of E. coli per ml. A total of 194 E. coli strains isolated from patients with simple acute cystitis were examined by the multiplex PCR and the results were in complete agreement with that obtained by DNA colony hybridization test. The multiplex PCR developed was, therefore, concluded to be a useful, sensitive and rapid assay system to identify uropathogenic E. coli .     

Enzymatic and genetic characterization of carbon and energy metabolisms by deep-sea hydrothermal chemolithoautotrophic isolates of Epsilonproteobacteria

The carbon and energy metabolisms of a variety of cultured chemolithoautotrophic Epsilonproteobacteria from deep-sea hydrothermal environments were characterized by both enzymatic and genetic analyses. All the Epsilonproteobacteria tested had all three key reductive tricarboxylic acid (rTCA) cycle enzymatic activities--ATP-dependent citrate lyase, pyruvate:ferredoxin oxidoreductase, and 2-oxoglutarate:ferredoxin oxidoreductase--while they had no ribulose 1,5-bisphosphate carboxylase (RubisCO) activity, the key enzyme in the Calvin-Benson cycle. These results paralleled the successful amplification of the key rTCA cycle genes aclB, porAB, and oorAB and the lack of success at amplifying the form I and II RubisCO genes, cbbL and cbbM. The combination of enzymatic and genetic analyses demonstrates that the Epsilonproteobacteria tested use the rTCA cycle for carbon assimilation. The energy metabolisms of deep-sea Epsilonproteobacteria were also well specified by the enzymatic and genetic characterization: hydrogen-oxidizing strains had evident soluble acceptor:methyl viologen hydrogenase activity and hydrogen uptake hydrogenase genes (hyn operon), while sulfur-oxidizing strains lacked both the enzyme activity and the genes. Although the energy metabolism of reduced sulfur compounds was not genetically analyzed and was not fully clarified, sulfur-oxidizing Epsilonproteobacteria showed enzyme activity of a potential sulfite:acceptor oxidoreductase for a direct oxidation pathway to sulfate but no activity of AMP-dependent adenosine 5'-phosphate sulfate reductase for a indirect oxidation pathway. No activity of thiosulfate-oxidizing enzymes was detected. The enzymatic and genetic characteristics described here were consistent with cellular carbon and energy metabolisms and suggest that molecular tools may have great potential for in situ elucidation of the ecophysiological roles of deep-sea Epsilonproteobacteria.     

Herbal medicine Sho-saiko-to (TJ-9) increases expression matrix metalloproteinases (MMPs) with reduced expression of tissue inhibitor of metalloproteinases (TIMPs) in rat stellate cell

We have reported that Sho-saiko-to (TJ-9) prevents liver fibrosis in vivo. To gain further insights into the effect of TJ-9, the matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) balance was examined. Hepatic stellate cells (HSCs) were isolated from male Wistar rats and cultured with TJ-9 (0-1000 microg/ml) on uncoated plastic dishes for 4 days. To elucidate the effects on the MMPs/TIMPs balance by TJ-9, quantitative analysis of type IV collagen-degrading activity, gelatin zymography and reverse zymography were carried out. Northern blot analysis was performed to determine the expression of MMP-2, 13 and TIMP-1 mRNAs. TJ-9 treatment resulted in dose-dependent upregulation of MMP-2, 13 mRNA and downregulation of TIMP-1 mRNA up to 500 microg/ml. Gelatin zymography, reverse zymography and quantitative analysis of type IV collagen-degrading activity confirmed that TJ-9 increased MMP-2 activity and prevented TIMP-1, 2 activities in a dose-dependent manner. SB203580 diminished the reduction of mRNA as well as the activity of TIMP-1 by TJ-9 and induction of mRNA as well as the activity of MMP-2. These results show that TJ-9 increased MMP-2, 13 activity with reduced TIMP-1, 2 activities on HSCs possibly via P38 pathway.     

Molecular signature associated with plasticity of bone marrow cell under persistent liver damage by self-organizing-map-based gene expression

The mechanism that regulates the plasticity of bone marrow cells (BMCs) into hepatocytes is poorly understood. We developed a green fluorescent protein/carbon tetrachloride model to find that BMC transplantation recovered liver damage. Serum albumin level and liver fibrosis were recovered by BMC transplantation. To understand the mechanism, we used DNA-chip technology to profile the change of transient gene expression before and after BMC transplantation. On the basis of gene expression with self-organizing map using specific equation, genes were classified into 153 clusters. The information is useful to understand the dramatic gene activation during the process of the plasticity of BMC.     

Spatial distribution of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems

Distribution profiles of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems were mapped with culture-independent molecular techniques. Planktonic samples were obtained from the waters surrounding two geographically and geologically distinct hydrothermal systems, and the abundance of marine crenarchaeota group I was examined by 16S ribosomal DNA clone analysis, quantitative PCR, and whole-cell fluorescence in situ hybridization. A much higher proportion of marine crenarchaeota group I within the microbial community was detected in deep-sea hydrothermal environments than in normal deep and surface seawaters. The highest proportion was always obtained from the ambient seawater adjacent to hydrothermal emissions and chimneys but not from the hydrothermal plumes. These profiles were markedly different from the profiles of epsilon-Proteobacteria, which are abundant in the low temperatures of deep-sea hydrothermal environments.     

Combination of monocyte-derived dendritic cells and activated T cells which express CD40 ligand: a new approach to cancer immunotherapy

Interactions between dendritic cells (DCs) and activated T cells are critically important for the establishment of an effective immune response. To develop the basis for a new DC-based cancer vaccine, we investigated cell-to-cell interactions between human monocyte-derived DCs and autologous T cells that are activated to express the CD40 ligand (CD40L). Peripheral blood monocytes were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) to induce differentiation of DCs. Activated T cells (ATs) consisted of autologous peripheral blood lymphocytes that had been activated with phytohemagglutinin (PHA) and then stimulated with calcium ionophore to up-regulate expression of CD40L. Coculture of these DCs and ATs induced significant production of interleukin 12 (IL-12) and also enhanced the production of interferon (IFN-). The production of IL-12 was blocked by an anti-CD40L antibody or by separation of the DC and AT fractions by a permeable membrane. Furthermore, coculture of DCs and ATs induced DCs to upregulate CD83 expression and stimulated migration of DCs toward the macrophage inflammatory protein 3- (MIP-3). ATs also migrated toward the MIP-3. These results suggest a combination of DCs and ATs as a potentially effective therapeutic strategy.     

Diversifying selection in human papillomavirus type 16 lineages based on complete genome analyses

Human papillomavirus type 16 (HPV16) is the primary etiological agent of cervical cancer, the second most common cancer in women worldwide. Complete genomes of 12 isolates representing the major lineages of HPV16 were cloned and sequenced from cervicovaginal cells. The sequence variations within the open reading frames (ORFs) and noncoding regions were identified and compared with the HPV16R reference sequence. This whole-genome approach gives us unprecedented precision in detailing sequence-level changes that are under selection on a whole-viral-genome scale. Of 7,908 base pair nucleotide positions, 313 (4.0%) were variable. Within the 2,452 amino acids (aa) comprising 8 ORFs, 243 (9.9%) amino acid positions were variable. In order to investigate the molecular evolution of HPV16 variants, maximum likelihood models of codon substitution were used to identify lineages and amino acid sites under selective pressure. Five codon sites in the E5 (aa 48, 65) and E6 (aa 10, 14, 83) ORFs were demonstrated to be under diversifying selective pressure. The E5 ORF had the overall highest nonsynonymous/synonymous substitution rate (omega) ratio (M3 = 0.7965). The E2 gene had the next-highest omega ratio (M3 = 0.5611); however, no specific codons were under positive selection. These data indicate that the E6 and E5 ORFs are evolving under positive Darwinian selection and have done so in a relatively short time period. Whether response to selective pressure upon the E5 and E6 ORFs contributes to the biological success of HPV16, its specific biological niche, and/or its oncogenic potential remains to be established.