腸内細菌のClostridium difficile胞子の発芽に及ぼす影響

金沢大学医学部研究課題/領域番号:61770290, 研究期間(年度):1986出典：研究課題「腸内細菌のClostridium difficile胞子の発芽に及ぼす影響」課題番号61770290（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-61770290/）を加工して作

C,difficileが産生する赤血球凝集活性を欠くトキシンAの性状解析

金沢大学医学部Clostridium difficile培養上清をモノQカラムを用いたFPLCにより分析した結果、トキシンA(エンテロトキシン)画分の細胞毒性ピ-クと赤血球凝集(HA)活性ピ-クとが解離することがわかった。StepーwiseのNaCl溶出実験により、この解離は溶出時のNaCl濃度差に基ずくものではないことが明らかにされ、C.Difficileの培養上清中にはHA活性を欠く変異性トキシンAが存在することが示唆された。培養上清のサイログロブリンアフィニティクマトグラフィ-素通り画分(HA活性陰性)を用いてQセフアロ-スFF及びモノQーFPLCを行ない変異型トキシンAを精製した。未変性及びSDSーPAGEの結果、変異型トキシンAの分子量はHA活性をもつトキシンAと同じく未変性状態で540kDa、変性状態で240kDa(主バンド)であった。トキシンAに対するモノクロ-ナル抗体を用いたSDSーPAGE後のイムノブロッティングにより、変異型トキシンAの240kDaの主バンドはトキシンAと同様に抗体と強く反応することが明らかにされた。また還元時、160kDaのマイナ-バンドが変異型トキシンAに比べトキシンAで強く検出された。変異型トキシンAの生物活性をトキシンAのそれと比較した。変異型トキシンAはトキシンAと同程度の細胞毒性、マウス致死毒性、腸管毒性(ウサギ結紮腸管試験による)をもつことが明らかにされた。キモトリプシンを含まないトリプシン(Type XIII)を用てい酵素処理をしても、変異型トキシンAのHA活性が回復されたり、トキシンAのHA活性が消失するようなことはなかった。トキシンAのHA活性の生物学的意義は未だ明らかにされていないが、本トキシンの腸管上皮細胞への結合の際に重要な役割をなすものと考えられている。しかし今回得られた結果は、トキシンAのHA活性は腸管毒性の発現に必要でないことを示唆している。研究課題/領域番号:02670178, 研究期間(年度):1990出典：研究課題「C,difficileが産生する赤血球凝集活性を欠くトキシンAの性状解析」課題番号02670178（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-02670178/）を加工して作

Temperature elevation enhances cell surface expression of measles virus fusion protein in infected cells

Cell fusion proceeded gradually in measles virus-infected cells incubated at 35°C. Shift-up of incubation temperature to 39°C induced rapidly increased cell fusion in spite of the cessation of de novo synthesis of the fusion (F) protein. Pulse-chase experiments showed that there was little difference in the acquisition of immunoreactivity by haemagglutinin (H) and F proteins between the two temperatures. H protein was detected on the cell surface 60 min after the chase at either temperature. However, appearance of F protein on the cell surface took less than 3 h at 39°C whereas it took 5 h at 35°C. These data indicate that temperature elevation induces more efficient expression of F protein on the cell surface accompanied by marked syncytium formation in measles virus-infected cells

Glycosylation of measles virus haemagglutinin protein in infected cells

Processing of the measles virus haemagglutinin (H) protein was analysed by the pulse-chase method, immunoprecipitation with an anti-H monoclonal antibody and SDS-polyacrylamide gel electrophoresis, combined with the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or monensin (inhibitors of intracellular processing of secretory proteins) to cultures and digestion of the protein with endoglycosidase H or neuraminidase. The apparent M(r) of the H protein was increased from 74K to 78K during the chase period. Addition of either CCCP or monensin to the chase medium inhibited the appearance of the 78K H protein, but not the immunoreactivity of the H protein or dimer formation, suggesting that these two events occur in the rough endoplasmic reticulum. The 74K H protein processed in the presence of CCCP was fully sensitive to endoglycosidase H digestion, whereas the 74K H protein processed in the presence of monensin was partially resistant to endoglycosidase H. In experiments using 3H-labelled sugars, [3H]galactose was incorporated into the 74K H protein in the presence of monensin. Neuraminidase treatment increased the electrophoretic mobility of the 78K H protein to 74K. Only the 78K H protein was detected on the surface of untreated cells, and it was resistant to endoglycosidase H digestion. These data suggest that after galactose addition sialic acid is added to the H protein in the trans-Golgi complex and then the mature 78K H protein is transported to the cell surface

Symbiotic Growth of a Thermophilic Sulfide-Oxidizing Photoautotroph and an Elemental Sulfur-Disproportionating Chemolithoautotroph and Cooperative Dissimilatory Oxidation of Sulfide to Sulfate

A thermophilic filamentous anoxygenic photosynthetic bacterium, Chloroflexus aggregans, is widely distributed in neutral to slightly alkaline hot springs. Sulfide has been suggested as an electron donor for autotrophic growth in microbial mats dominated with C. aggregans, but remarkable photoautotrophic growth of isolated C. aggregans has not been observed with sulfide as the sole electron source. From the idea that sulfide is oxidized to elemental sulfur by C. aggregans and the accumulation of elemental sulfur may have an inhibitory effect for the growth, the effects of an elemental sulfur-disproportionating bacterium that consumes elemental sulfur was examined on the autotrophic growth of C. aggregans, strain NA9-6, isolated from Nakabusa hot spring. A sulfur-disproportionating bacterium, Caldimicrobium thiodismutans strain TF1, also isolated from Nakabusa hot spring was co-cultured with C. aggregans. C. aggregans and C. thiodismutans were successfully co-cultured in a medium containing thiosulfate as the sole electron source and bicarbonate as the sole carbon source. Quantitative conversion of thiosulfate to sulfate and a small transient accumulation of sulfide was observed in the co-culture. Then the electron source of the established co-culture was changed from thiosulfate to sulfide, and the growth of C. aggregans and C. thiodismutans was successfully observed with sulfide as the sole electron donor for the autotrophic growth of the co-culture. During the cultivation in the light, simultaneous consumption and accumulation of sulfide and sulfate, respectively, were observed, accompanied with the increase of cellular DNAs of both species. C. thiodismutans likely works as an elemental sulfur scavenger for C. aggregans, and C. aggregans seems to work as a sulfide scavenger for C. thiodismutans. These results suggest that C. aggregans grows autotrophically with sulfide as the electron donor in the co-culture with C. thiodismutans, and the consumption of elemental sulfur by C. thiodismutans enabled the continuous growth of the C. aggregans in the symbiotic system. This study shows a novel symbiotic relationship between a sulfide-oxidizing photoautotroph and an elemental sulfur-disproportionating chemolithoautotroph via cooperative dissimilatory sulfide oxidation to sulfate

Outer Membrane Vesicles of Helicobacter pylori TK1402 are Involved in Biofilm Formation

<p>Abstract</p> <p>Background</p> <p><it>Helicobacter pylori </it>forms biofilms on glass surfaces at the air-liquid interface in <it>in vitro </it>batch cultures; however, biofilms of <it>H. pylori </it>have not been well characterized. In the present study, we analyzed the ability of <it>H. pylori </it>strains to form biofilms and characterized the underlying mechanisms of <it>H. pylori </it>biofilm formation.</p> <p>Results</p> <p>Strain TK1402 showed strong biofilm forming ability relative to the other strains in Brucella broth supplemented with 7% FCS. The strong biofilm forming ability of TK1402 is reflected the relative thickness of the biofilms. In addition, outer membrane vesicles (OMV) were detected within the matrix of only the TK1402 biofilms. Biofilm formation was strongly correlated with the production of OMV in this strain. We further observed that strain TK1402 did not form thick biofilms in Brucella broth supplemented with 0.2% β-cyclodextrin. However, the addition of the OMV-fraction collected from TK1402 could enhance biofilm formation.</p> <p>Conclusion</p> <p>The results suggested that OMV produced from TK1402 play an important role in biofilm formation in strain TK1402.</p

Human cytomegalovirus persistent infection in a human central nervous system cell line: Production of a variant virus with different growth characteristics

The susceptibility of human central nervous system cell lines to human cytomegalovirus (HCMV) and the fate of infected cultures were studied. Significant amounts of infectious progeny virus were produced in 118MGC glioma and IMR-32 neuroblastoma, but not in KGC oligodendroglioma cells when the cultures were infected with wild-type virus (HCMVwt) at an m.o.i. of 10 p.f.u. per cell. Further passage of infected 118MGC cells resulted in the establishment of a long-term persistent infection. This infection, designated 118MGC/Towne, continuously produced infectious virus (HCMVpi) with titres ranging from 102 to 105 p.f.u./106 cells up to 360 days post-infection (corresponding to 50 subcultures). Since no temperature-sensitive mutants, defective interfering particles or interferon-like activity were found in the 118MGC/Towne cultures, maintenance of the persistent infection seemed to be due to a balance between the release of infectious virus and the growth of uninfected cells. The HCMVpi produced in long-term persistently infected cultures was shown to be different from the HCMVwt originally used to infect by the following characteristics: (i) HCMVpi replicated slowly and yielded lower amounts of progeny virus than HCMVwt; (ii) HCMVpi induced a 73000 mol. wt. immediate early protein that was not synthesized in HCMVwt-infected cells; (iii) HCMVpi had a different DNA structure from that of HCMVwt. These results suggest that HCMVpi is a slower growing variant of HCMVwt and probably plays an important role in the maintenance of the persistent infection

The protective effect of CD40 ligand–CD40 signalling is limited during the early phase of Plasmodium infection

Abstractγδ T cells are essential for eliminating Plasmodium berghei XAT. Because administration of the agonistic anti-CD40 antibody can induce elimination of P. berghei XAT parasites in γδ T cell-deficient mice, we considered that γδ T cells might activate dendritic cells via CD40 signalling during infection. Here we report that administration of the anti-CD40 antibody to γδ T cell-deficient mice 3–10days post-P. berghei XAT infection could eliminate the parasites. Our data suggest that dendritic cell activation via γδ T cells expressing CD40 ligand is critical during the early phase of infection

Synthesis of M protein of HVJ (Sendai virus) in rat glial cells is selectively restricted at a non-permissive temperature

Production of HVJ (Sendai virus) wild-type in rat glial cells was characteristically restricted at high temperature. The synthesis of the M protein was selectively decreased in infected cells at 39° C. This temperature-sensitive (ts) character of HVJ was not observed in infection of LLCMK2 cells or chick embryo fibroblast cells. Newcastle disease virus, mumps virus and vesicular stomatitis virus did not show ts growth in glial cells

Enhanced replication of human cytomegalovirus in human fibroblasts treated with dexamethasone

The effect of glucocorticoid hormones on the replication of human cytomegalovirus (HCMV) was studied in human embryonic lung (HEL) cells. Treatment of cells with pharmacological concentrations of adrenal glucocorticoids such as dexamethasone enhanced HCMV replication; treatment with oestrogenic or androgenic hormones did not do so. In dexamethasone-treated HEL cells there was an approximately tenfold increase in virus yield, with the virus eclipse period shortened by 1 day compared to control cultures. Treatment of cells with the hormone also enhanced plaquing efficiency of the virus by approximately tenfold. As the synthesis of virus-specific immediate early proteins and antigens was notably enhanced together with an increase of HCMV DNA synthesis, it appeared that the early stages of the HCMV replication cycle might be under hormonal control. Moreover, the data presented suggest that the hormonal enhancement of HCMV replication involves specific receptor proteins and requires the synthesis of a specific cellular mRNA(s)