Molecular survey of Babesia microti, Ehrlichia species and Candidatus neoehrlichia mikurensis in wild rodents from Shimane Prefecture, Japan

A significant number of patients are diagnosed with "fevers of unknown origin" (FUO) in Shimane Prefecture in Japan where tick-borne diseases are endemic. We conducted molecular surveys for Babesia microti, Ehrlichia species, and Candidatus Neoehrlichia mikurensis in 62 FUO cases and 62 wild rodents from Shimane Prefecture, Japan. PCR using primers specific for the Babesia 18S small-subunit rRNA (rDNA) gene and Anaplasmataceae groESL amplified products from 45% (28/62) and 25.8% (16/62) of captured mice, respectively. Of the 28 18S rDNA PCR positives, 23 and five samples were positive for Hobetsu- and Kobe-type B. microti, respectively. In contrast, of the 16 groESL PCR positives, eight, one and seven samples were positive for Ehrlichia muris, Ehrlichia sp. HF565 and Candidatus N. mikurensis, respectively. Inoculation of selected blood samples into Golden Syrian hamsters indicated the presence of Hobetsu- and Kobe-type B. microti in four and one sample, respectively. Isolation of the latter strain was considered important as previous studies suggested that the distribution of this type was so far confined to Awaji Island in Hyogo Prefecture, where the first case of transfusion-associated human babesiosis originated. DNA samples from 62 FUO human cases tested negative for B. microti 18S rDNA gene, Anaplasmataceae groESL gene, Rickettsia japonica 17K genus-common antigen gene and Orientia tsutsugamushi 56K antigen gene by PCRs. We also conducted seroepidemiological surveys on 62 human sera collected in Shimane Prefecture from the FUO patients who were suspected of carrying tick-borne diseases. However, indirect immunofluorescent antibody tests using B. microti- and E. muris-infected cells detected IgG against E. muris in only a single positive sample. This study demonstrates the presence of several potentially important tick-borne pathogens in Shimane Prefecture and suggests the need for further study on the causative agents of FUOs.     

Novel reporter gene expression systems for monitoring activation of the Aspergillus nidulans HOG pathway

The Aspergillus nidulans high-osmolarity glycerol response (AnHOG) pathway is involved in osmoadaptation. We found that fludioxonil, a fungicide, causes improper activation of HogA mitogen-activated protein kinase (MAPK) in A. nidulans. Here we present novel reporter systems for monitoring activation of the AnHOG pathway. The promoter region of gfdB (glycerol-3-phosphate dehydrogenase), whose expression depends on the presence of HogA, was fused to a beta-glucuronidase uidA gene (GUS) to construct the reporter, which was introduced into A. nidulans wild type and hogADelta. Increased GUS activity was detected in the wild type only when it was treated with high osmolarity or fludioxonil, while reporter activity was scarcely stimulated in the hogADelta mutant. These results indicate that the reporter activity is controlled via HogA activation. Furthermore, we present possible applications of the reporter systems in screening new antifungal compounds.     

Streptococcus panodentis sp. nov. from the oral cavities of chimpanzees

Three strains TKU9, TKU49 and TKU50^T, were isolated from the oral cavities of chimpanzees (Pan troglodytes). The isolates were all gram‐positive, facultative anaerobic cocci that lacked catalase activity. Analysis of partial 16S rRNA gene sequences showed that the most closely related species was Streptococcus infantis (96.7%). The next most closely related species to the isolates were S. rubneri, S. mitis, S. peroris and S. australis (96.6 to 96.4%). Based on the rpoB and gyrB gene sequences, TKU50^T was clustered with other member of the mitis group. Enzyme activity and sugar fermentation patterns differentiated this novel bacterium from other members of the mitis group streptococci. The DNA G + C content of strain TKU50^T was 46.7 mol%, which is the highest reported value for members of the mitis group (40–46 mol%). On the basis of the phenotypic characterization, partial 16S rRNA gene and sequences data for two housekeeping gene (gyrB and rpoB), we propose a novel taxa, S. panodentis for TKU 50^T (type strain = CM 30579^T = DSM 29921^T), for these newly described isolates.     

Expression and Localization of Type II Diacylglycerol Kinase Isozymes δ and η in the Developing Mouse Brain

The functions of type II diacylglycerol kinase (DGK) δ and -η in the brain are still unclear. As a first step, we investigated the spatial and temporal expression of DGKδ and -η in the brains of mice. DGKδ2, but not DGKδ1, was highly expressed in layers II–VI of the cerebral cortex; CA–CA3 regions and dentate gyrus of hippocampus; mitral cell, glomerular and granule cell layers of the olfactory bulb; and the granule cell layer in the cerebellum in 1- to 32-week-old mice. DGKδ2 was expressed just after birth, and its expression levels dramatically increased from weeks 1 to 4. A substantial amount of DGKη (η1/η2) was detected in layers II–VI of the cerebral cortex, CA1 and CA2 regions and dentate gyrus of the hippocampus, mitral cell and glomerular layers of the olfactory bulb, and Purkinje cells in the cerebellum of 1- to 32-week-old mice. DGKη2 expression reached maximum levels at P5 and decreased by 4 weeks, whereas DGKη1 increased over the same time frame. These results indicate that the expression patterns of DGK isozymes differ from each other and also from other isozymes, and this suggests that DGKδ and -η play distinct and specific roles in the brain.     

The nucleoporin Seh1 forms a complex with Mio and serves an essential tissue-specific function in Drosophila oogenesis

The nuclear pore complex (NPC) mediates the transport of macromolecules between the nucleus and cytoplasm. Recent evidence indicates that structural nucleoporins, the building blocks of the NPC, have a variety of unanticipated cellular functions. Here, we report an unexpected tissue-specific requirement for the structural nucleoporin Seh1 during Drosophila oogenesis. Seh1 is a component of the Nup107-160 complex, the major structural subcomplex of the NPC. We demonstrate that Seh1 associates with the product of the missing oocyte (mio) gene. In Drosophila, mio regulates nuclear architecture and meiotic progression in early ovarian cysts. Like mio, seh1 has a crucial germline function during oogenesis. In both mio and seh1 mutant ovaries, a fraction of oocytes fail to maintain the meiotic cycle and develop as pseudo-nurse cells. Moreover, the accumulation of Mio protein is greatly diminished in the seh1 mutant background. Surprisingly, our characterization of a seh1 null allele indicates that, although required in the female germline, seh1 is dispensable for the development of somatic tissues. Our work represents the first examination of seh1 function within the context of a multicellular organism. In summary, our studies demonstrate that Mio is a novel interacting partner of the conserved nucleoporin Seh1 and add to the growing body of evidence that structural nucleoporins can have novel tissue-specific roles.     

Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori

Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air–liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2–3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402.     

Daily expression of clock genes in whole blood cells in healthy subjects and a patient with circadian rhythm sleep disorder

In recent years, circadian rhythm sleep disorders in humans have been increasing. Clinical features characteristic of this disorder are well known, but the specific causes remain unknown. However, various derangements of circadian expression of the clock gene are a probable cause of this disease. We have attempted to elucidate the relationship between the expression of the clock genes in whole blood cells and the clinical features characteristic of this disorder. In this study, we indicate the daily expression of clock genes period (Per) 1, 2, 3, Bmal1, and Clock in whole blood cells in 12 healthy male subjects. The peak phase of Per1, Per2, and Per3 appeared in the early morning, whereas that of Bmal1 and Clock appeared in the midnight hours. Furthermore, in one patient case with circadian rhythm sleep disorder, we observed variations of the peak phase in clock genes by treatments such as light therapy, exercise therapy, and medicinal therapy. This study suggested that the monitoring of human clock genes in whole blood cells, which may be functionally important for the molecular control of the circadian pacemaker as well as in suprachiasmatic nucleus, might be useful to evaluate internal synchronization.