Characterization of toxigenic vibrios isolated from the freshwater environment of Hiroshima, Japan.

The occurrence and characterization of toxigenic vibrios in surface water and sediment samples of the fresh water environment of the Ohta River were studied. The membrane filter, pad preenrichment technique, followed by the placement of membranes onto thiosulfate citrate-bile salt-sucrose agar, was used for the enumeration of total vibrios. Qualitative examination of pathogenic vibrios was also attempted. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples of the Ohta River and the Hiroshima coast. In the identification of 361 strains, 12 species of Vibrio and two species of Listonella were observed. Non-01 Vibrio cholerae was prevalent among the members of the genus Vibrio. Vibrio parahaemolyticus (serotype 04:K34), isolated in fresh water, is significant and suggests that some still unknown conditions promote the survival of these organisms in fresh water. An estimated 132 strains were hemolytic by a simple agar method, and further characterization revealed that 82% of the hemolytic vibrios (107 strains) produced various toxins. About 71% (93 strains) elaborated cytotoxin, 55% (72 strains) produced hemolysin, and 44% (58 strains) responded for both cytotoxin and hemolysin in the crude toxin extracts. All the non-01 V. cholerae showed cytotoxic activity, and the virulent strains of Vibrio fluvialis and Vibrio spp. showed cytotonic responses in RK-13 cells. Of 36 sediment samples tested, 10 harbored C. botulinum spores (28%) and were isolated invariably in all the regions of the Hiroshima coast and in the Ohta River, except the upper region of the Ohta River.     

Molecular evidence of digestion and absorption of epibiotic bacterial community by deep-sea crab Shinkaia crosnieri

The hydrothermal vent crab Shinkaia crosnieri is considered to obtain nutrition from the epibiotic bacteria found on the setae, but previous studies have not shown how nutrients can be transferred from the epibionts to the host. In this study, microscopic observations of S. crosnieri intestinal components detected autofluorescent setae fragments and pigmentation derived from the digestion of epibionts in a dye-stained epibiont tracer experiment. An in vitro digestion experiment with epibiotic populations using an intestinal extract demonstrated the degradation of epibiotic cells by digestive enzymes. A phylogenetic analysis showed that many of the bacterial 16S ribosomal RNA gene sequences obtained from the intestine were closely related to the sequences of the epibionts, thus they were probably derived from the epibionts. A stable isotope tracer experiment also indicated that ¹³C assimilated by the epibionts provided a carbon (nutrition) source for the host. Both activity measurements and isotope studies showed that chemosynthetic metabolism by the gut microbial components were inactive. Together with the feeding behaviour of living S. crosnieri, these results indicate that S. crosnieri ingests the epibionts using maxillipeds and assimilates them via its digestive organs as a nutrient source. The results of this study elucidate the mechanism of nutritional transfer in ectosymbiosis between chemosynthetic bacteria and deep-sea invertebrates.     

Genomics insights into ecotype formation of ammonia-oxidizing archaea in the deep ocean

Various lineages of ammonia-oxidizing archaea (AOA) are present in deep waters, but the mechanisms that determine ecotype formation are obscure. We studied 18 high-quality genomes of the marine group I AOA lineages (alpha, gamma and delta) from the Mariana and Ogasawara trenches. The genomes of alpha AOA resembled each other, while those of gamma and delta lineages were more divergent and had even undergone insertion of some phage genes. The instability of the gamma and delta AOA genomes could be partially due to the loss of DNA polymerase B (polB) and methyladenine DNA glycosylase (tag) genes responsible for the repair of point mutations. The alpha AOA genomes harbour genes encoding a thrombospondin-like outer membrane structure that probably serves as a barrier to gene flow. Moreover, the gamma and alpha AOA lineages rely on vitamin B₁₂-independent MetE and B₁₂-dependent MetH, respectively, for methionine synthesis. The delta AOA genome contains genes involved in uptake of sugar and peptide perhaps for heterotrophic lifestyle. Our study provides insights into co-occurrence of cladogenesis and anagenesis in the formation of AOA ecotypes that perform differently in nitrogen and carbon cycling in dark oceans.     

Vitamin K therapy for cortical bone fragility caused by reduced mechanical loading in a child with hemiplegia

Fractures frequently occur at cortical bone sites in children with cerebral palsy, but there is no established therapy. We previously found that treatment with vitamins D and K increased cortical bone mass in children with severe physical disability, and have hypothesized that vitamin K could play a significant role in pediatric cortical bones under conditions with reduced mechanical loading. In the present case report, we treated a right hemiplegic ambulant eight-year-old boy with oral vitamin K (15 mg per day) for eight months. Cortical bone geometries at mid-diaphyseal sites in bilateral tibiae were evaluated before and after the treatment. The cross-sectional total, bone and marrow areas of non-hemiplegic tibia increased by 8.8%, 7.4% and 12.0%, respectively, while those of hemiplegic tibia changed by 9.0%, 14.9% and -3.4%, respectively. As a result, the polar moment of inertia, an indicator of the resistance to torsion forces, increased by 13.0% in the non-hemiplegic tibia and by 63.7% in the hemiplegic tibia. Vitamin K may restrict cortical bone fragility, caused by reduced mechanical loading, through its actions at the endosteal bone marrow interface. Further studies are needed to confirm these findings and to clarify the mechanisms involved.     

Structural basis of functional cooperation of Tim15/Zim17 with yeast mitochondrial Hsp70

Mitochondrial heat-shock protein 70 (mtHsp70) and its partner proteins drive protein import into the matrix. Tim15/Zim17/Hep1 is a mtHsp70 partner protein on the matrix side of the inner mitochondrial membrane. We determined the nuclear magnetic resonance (NMR) structure of the core domain of Tim15. On the basis of the NMR structure, we created Tim15 mutants and tested their ability to complement the functional defects of Tim15 depletion and to suppress self-aggregation of mtHsp70 in vivo. A pair of basic residues, Arg 106 and His 107, conserved Asp 111 and flexible loop 133-137, and were important (Arg 106-His 107 pair and Asp 111) or partly important (the loop 133-137) for yeast cell growth, mitochondrial protein import and the suppression of mtHsp70 aggregation. Therefore, the function of Tim15 in yeast cell growth is well correlated with its ability to suppress mtHsp70 aggregation, although it is still unknown whether inhibition of mtHsp70 aggregation is the primary function of Tim15.     

Identification of innate IL-5-producing cells and their role in lung eosinophil regulation and antitumor immunity

IL-5 is involved in a number of immune responses such as helminth infection and allergy. IL-5 also plays roles in innate immunity by maintaining B-1 B cells and mucosal IgA production. However, the identity of IL-5-producing cells has not been unambiguously characterized. In this report, we describe the generation of an IL-5 reporter mouse and identify IL-5-producing non-T lymphoid cells that reside in the intestine, peritoneal cavity, and lungs in naive mice. They share many characteristics with natural helper cells, nuocytes, and Ih2 cells, including surface Ags and responsiveness to cytokines. However, these phenotypes do not completely overlap with any particular one of these cell types. Innate non-T IL-5-producing cells localized most abundantly in the lung and proliferated and upregulated IL-5 production in response to IL-25 and IL-33. IL-33 was more effective than IL-25. These cells contribute to maintaining sufficient numbers of lung eosinophils and are important for eosinophil recruitment mediated by IL-25 and IL-33. Given that eosinophils are shown to possess antitumor activity, we studied lung tumor metastasis and showed that innate IL-5-producing cells were increased in response to tumor invasion, and their regulation of eosinophils is critical to suppress tumor metastasis. Genetic blockade or neutralization of IL-5 impaired eosinophil recruitment into the lung and resulted in increased tumor metastasis. Conversely, exogenous IL-5 treatment resulted in suppressed tumor metastasis and augmented eosinophil infiltration. These newly identified innate IL-5-producing cells thus play a role in tumor surveillance through lung eosinophils and may contribute to development of novel immunotherapies for cancer.     

Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.