Biomedical subjects

T Yokochi

Publications and source records attributed to T Yokochi.

At least 91 records · Page 5Linked to original sources

PubMed1996

Characterization of lipopolysaccharides of polymyxin-resistant and polymyxin-sensitive Klebsiella pneumoniae O3.

Lipopolysaccharides isolated from the polymyxin-resistant Klebsiella pneumoniae O3 mutant OM-5 and its polymyxin-sensitive parent LEN-1 were analyzed for chemical composition, and their lipid A portions were structurally characterized. The lipopolysaccharide of OM-5 contained approximately five times more 4-amino-4-deoxy-L-arabinopyranose than that of LEN-1. Other saccharide and phosphate components exhibited no significant differences. Structural characterization, including analyses by phosphorus magnetic resonance spectroscopy and by fast atom bombardment mass spectrometry, revealed a novel type of lipid A. In the OM-5 lipopolysaccharide, both phosphates of lipid A were almost totally present as phosphodiesters with 4-amino-4-deoxy-L-arabinopyranose. In the sensitive-type LEN-1 lipid A, the extent of this substitution was much lower, especially in the glycosidically linked phosphate. Phosphate in these K. pneumoniae lipopolysaccharides was almost exclusively found in lipid A. These results show that cationic substituents of phosphates of lipid A play a decisive role in determining polymyxin reactivity. OM-5 was also found to contain a large proportion of heptaacyl lipid A, which represented only a small fraction of lipid A in LEN-1.

Carbohydrate Sequence↗

PubMed1996

Regulation of IFN-alpha/beta genes: evidence for a dual function of the transcription factor complex ISGF3 in the production and action of IFN-alpha/beta.

BACKGROUND: Efficient production of interferons (IFNs) in virally infected cells is an essential aspect of the host defence. The transcription factor complex ISGF3 (IFN-stimulated gene factor 3) was originally identified as a critical mediator of the IFN signal; it is formed upon IFN receptor (IFNR) stimulation and binds to ISREs (IFN-stimulated response elements) to activate IFN-inducible genes. It has recently been shown that the DNA binding component of ISGF3, p48 (ISGF3gamma) also binds to virus-inducible elements in the IFN-alpha/beta genes, suggesting a potential new role of p48 in IFN production. RESULTS: Primary cells from mice with a targeted disruption of the p48 gene show severe defects in virus-induced IFN-alpha/beta gene expression. A similar defect was also observed in cells lacking type I IFNR or Stat1, further demonstrating the role of IFN signalling in the induction of these IFN genes. ISGF3 in fact binds to the virus-inducible elements within the IFN-alpha/beta promoters. We also provide evidence showing that these elements are additionally controlled by an unidentified factor(s) which presumably triggers the primary phase of IFN gene induction. CONCLUSIONS: Our results demonstrate that the IFN signal transducing complex ISGF3 plays a crucial role in IFN production and suggest that ISGF3 may participate directly in the activation of IFN-alpha/beta promoters. This dual function of ISGF3 may insure the efficient operation of this cytokine system in the host defence.

Animals↗

PubMed1996

DNA nicking by Escherichia coli topoisomerase IV with a substitution mutation from tyrosine to histidine at the active site.

BACKGROUND: Escherichia coli topoisomerase IV is a type II topoisomerase composed of ParC and ParE subunits and plays a major role in the decatenation of replicated molecules. The reaction with type II topoisomerases involves the cutting through transesterification between an active-site tyrosine and a DNA phosphodiester bond and a rejoining of cleaved DNA. RESULTS: To genetically analyse the mechanism of this reaction, we isolated dominant-negative topoisomerase IV mutants. In one of them, the parC10 mutant, there was a substitution by histidine of the active-site tyrosine. Purified mutant topoisomerase IV did not show normal DNA cutting activity but retained DNA nicking activity, even in the absence of ATP. The DNA ends of the product were not covalently bound to the ParC subunits, suggesting that the DNA is not cut via transesterification but by hydrolysis. CONCLUSIONS: We have shown genetically that the 120th tyrosine residue is important for the DNA cutting step in the topoisomerase reaction. The 120th amino acid residue, tyrosine or histidine, appears to be activated and the histidine residue forces the hydrogen-bonded water to attack the phosphoryl group of the DNA in hydrolysis, while the tyrosine residue directly attacks the phosphoryl group during transesterification.

Base Sequence↗

PubMed1996

Lipopolysaccharide induces apoptotic cell death of B memory cells and regulates B cell memory in antigen-nonspecific manner.

Lipopolysaccharide (LPS) was administered into sheep red blood cells (SRBC)-primed mice, and the effect of LPS on SRBC-specific memory cells was investigated. Spleen cells from SRBC-primed mice which were injected with LPS exhibited much lower in vitro secondary plaque-forming cells (PFC) responses to SRBC than those from untreated SRBC-primed mice. The in vitro anti-SRBC response of the spleen cells to LPS was also reduced. The combination experiments of B cells and T cells from SRBC-primed mice which were injected with or without LPS demonstrated that the reduction of immune responses to SRBC after administration of LPS was caused by the defect of SRBC-specific B memory cells, but not T memory cells. B cell type rosette-forming cells (RFC) for SRBC markedly decreased after injection of LPS, while PFC as antibody-forming cells did not increase subsequently. Therefore, the reduction of RFC was not due to their differentiation into PFC. The lymphoid follicles in the spleens from mice injected with LPS were stained positively by in situ nick end labeling specific for fragmented DNA. A large percentage of Ig+ spleen cells from SRBC-primed mice which were injected with LPS was also stained positively. The injection of glucocorticoids into SRBC-primed mice induced similar reduction of B memory cells. It was suggested that LPS might induce apoptosis of B memory cells and regulate B cell memory in antigen-nonspecific manner.

Animals↗

PubMed1996

Differential release of smooth-type lipopolysaccharide from Pseudomonas aeruginosa treated with carbapenem antibiotics and its relation to production of tumor necrosis factor alpha and nitric oxide.

Endotoxin release from Pseudomonas aeruginosa treated with cell wall-active carbapenem antibiotics and its effect on the production of tumor necrosis factor alpha and nitric oxide were examined. Treatment of bacteria with imipenem induced much lower levels of endotoxin release than treatment with meropenem. The endotoxin released was demonstrated to be of the smooth type and O-specific polysaccharide-rich. The exposure of the filtrates of P. aeruginosa treated with imipenem to physiologically relevant cells caused low-level production of tumor necrosis factor alpha and nitric oxide, while similar treatment with meropenem induced high levels of production.

Carbapenems↗

PubMed1996

Augmented immunological activities of recombinant lipopolysaccharide possessing the mannose homopolymer as the O-specific polysaccharide.

Recombinant lipopolysaccharide possessing the mannose homopolymer as the O-specific polysaccharide was manufactured genetically by transforming Escherichia coli K-12 with various rfb genes capable of synthesizing the mannose homopolymer. Recombinant lipopolysaccharide exhibited levels of anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity much higher than those exhibited by the original rough-type lipopolysaccharide from E. coli K-12 or lipopolysaccharide possessing the heteropolysaccharide from E. coli O111. Immunological activities of recombinant lipopolysaccharide were as strong as those of wild-type lipopolysaccharide possessing the mannose homopolymer. Characteristic activities of wild-type lipolysaccharide possessing the mannose homopolymer were exhibited by recombinant lipopolysaccharide. The abilities of lipopolysaccharide to activate B cells polyclonally and to produce cytokines did not seem to be related to the presence of the mannose homopolymer. Therefore, it was suggested that the mannose homopolymer in the O-specific polysaccharide might exclusively enhance anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity among various lipid A activities.

Adjuvants, Immunologic↗

PubMed1996

Apoptotic cell death in the response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model.

The apoptotic cell death induced in D-galactosamine-sensitized mice by administration of lipopolysaccharide was characterized. Administration of lipopolysaccharide caused apoptotic cell death in livers of D-galactosamine-sensitized mice. Apoptotic cells were also detected in the kidney, thymus, spleen, and lymph node. Severe hepatic apoptosis in D-galactosamine-sensitized mice was reproduced by transfer of the sera from mice injected with D-galactosamine and lipopolysaccharide. The hepatocyte apoptosis induced by lipopolysaccharide was completely prevented by an anti-tumor necrosis factor alpha antibody but not by an anti-gamma interferon antibody. Administration of recombinant tumor necrosis factor into D-galactosamine-sensitized mice also caused hepatocyte apoptosis. Lipopolysaccharide-induced hepatocyte apoptosis in D-galactosamine-sensitized mice did not seem to be mediated by Fas antigen. It was suggested that lipopolysaccharide- induced hepatic injury and failure in D-galactosamine-sensitized mice was due to the apoptotic cell death of hepatocytes caused by tumor necrosis factor alpha released in the circulation.

Animals↗

PubMed1996

Expression of heat shock protein 72 on peripheral blood mononuclear cells from patients with pustulosis palmaris et plantaris.

The expression of heat shock protein (HSP) 72 on peripheral blood mononuclear cells (PBMC) from patients with pustulosis palmaris et plantaris (PPP) was studied. PBMC isolated freshly from patients with PPP expressed HSP72, while those from psoriasis patients did not. PBMC from patients with PPP continued to express it in in vitro cultures at 37 degrees C. This expression was further augmented by in vitro heat stimulation at 43 degrees C. Immunofluorescence studies showed that approximately 20% of PBMC from patients with PPP were stained positively with anti-HSP72 antibody. HSP72 was expressed on both nonadherent and adherent cells of PBMC. These findings suggest that PBMC from patients with PPP may produce HSP72 spontaneously through their in vivo exposure to stressful agents.

Adult↗

PubMed1996

A screening method for antimitotic and antifungal substances using conidia of Pyricularia oryzae, modification and application to tropical marine fungi.

A bioassay method detecting deformations of mycelia germinated from conidia of Pyricularia oryzae P-2b, has been modified to give quantitative estimations. The method was first developed using antimitotic agents which showed characteristic curling effect. Morphological deformations include curling, swelling, hyper-divergency, beads shape and so on, and inhibition of the germination was also observed. For quantitative estimations, indices were introduced for the hyphal growth inhibition and a quantity of conidia in each assay cell and concentration of test solutions were adjusted. Details of the modified method and the application to screening assay of marine fungi isolated in Yap Islands are described. Eight strains of 109 tested showed morphological deformations, and chaetoglobosin A was isolated from the broth filtrate of a strain assigned to Chaetomium sp. This bioassay is a cheap, quick and easy method to be applied to the primary screening for antimitotic and antifungal substances from natural sources.

Antifungal Agents↗

PubMed1995

Dissociated control by glycyrrhizin of proliferation and IL-2 production of murine thymocytes.

The action of glycyrrhizin (GL) modulating the proliferation and IL-2 production of murine thymocytes in response to anti-CD3 and concanavalin A was studied. Different from the previously reported GL effect of accelerating both IL-2 production and proliferation of mature T lymphocytes, GL displayed a dissociated action on immature thymocytes promoting IL-2 production/IL-2 receptor expression but inhibiting cell growth. Hydrocortisone-resistant mature thymocytes behaved like peripheral T lymphocytes, demonstrating the dependency of the GL action on cell maturation stage. GL-mediated growth inhibition of thymocytes was not due the cytotoxic action of GL that induces cell death or DNA fragmentation. In parallel to these dissociated actions, GL promoted the tyrosine phosphorylation of p56 but suppressed the phosphorylation of p40 induced by anti-CD3. Moreover, GL and anti-CD3 showed a combination effect suppressing the transcription of c-fos, which was promoted by anti-CD3 alone or GL alone. It is suggested that whereas mature and immature T cells share a common signal pathway for IL-2 production augmented by the action of GL, they have signaling steps for DNA synthesis which are under different mechanisms receiving the modulation effects of GL in opposite directions.

Animals↗

PubMed1995

T cell receptor-mediated signaling events in CD4+CD8+ thymocytes undergoing thymic selection: requirement of calcineurin activation for thymic positive selection but not negative selection.

The goal of this study was to identify the differences of intracellular signals between the processes of thymic positive and negative selection. The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocked the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression were inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation did not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) was not inhibited by in vivo treatment with FK506 and that there was no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. DNA fragmentation induced by TCR activation of DP thymocytes in vitro was not affected by FK506. In addition, different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes.

Animals↗

PubMed1995

Molecular epidemiology of Salmonella enteritidis. An outbreak and sporadic cases studied by means of pulsed-field gel electrophoresis.

Large outbreaks of diarrhoea due to Salmonella enteritidis in Aichi-ken, Japan, provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. This was performed by comparing the plasmid profile types, phage types, antimicrobial resistance, and the restriction fragment length polymorphisms (RFLPs) by pulsed-field gel electrophoresis (PFGE) of S. enteritidis from outbreaks and sporadic cases. Among the isolates studied, 10 distinctive RFLP types were found with XbaI and four with NotI, while 12 combination types were identified among the 68 isolates from 16 Health Centres in Aichi-ken, Japan. A total of 22 isolates from four outbreaks, however, had the same RFLP and phage types. The RFLP type was subdivided by means of the plasmid profile and phage type. Conversely, plasmid profile and phage type were separated by means of RFLP. This PFGE method may prove useful for subclassifying S. enteritidis and differentiating isolates of the same plasmid profile and phage type.

Bacteriophage Typing↗

PubMed1995

Evidence for conservative (two-progeny) DNA double-strand break repair.

The double-strand break repair models for homologous recombination propose that a double-strand break in a duplex DNA segment is repaired by gene conversion copying a homologous DNA segment. This is a type of conservative recombination, or two-progeny recombination, which generates two duplex DNA segments from two duplex DNA segments. Transformation with a plasmid carrying a double-strand gap and an intact homologous DNA segment resulted in products expected from such conservative (two-progeny) repair in Escherichia coli cells with active E. coli RecE pathway (recBC sbcA) or with active bacteriophage lambda Red pathway. Apparently conservative double-strand break repair, however, might result from successive events of nonconservative recombination, or one-progeny recombination, which generates only one recombinant duplex DNA segment from two segments, involving multiple plasmid molecules. Contribution of such intermolecular recombination was evaluated by transformation with a mixture of two isogenic parental plasmids marked with a restriction site polymorphism. Most of the gap repair products were from intramolecular and, therefore, conservative (two-progeny) reaction under the conditions chosen. Most were conservative even in the absence of RecA protein. The double-strand gap repair reaction was not affected by inversion of the unidirectional replication origin on the plasmid. These results demonstrate the presence of the conservative (two-progeny) double-strand break repair mechanism. These experiments do not rule out the occurrence of nonconservative (one-progeny) recombination since we set up experimental conditions that should favor detection of conservative (two-progeny) recombination.

Bacteriophage lambda↗

PubMed1995

Expression of Lewis Y (Le(y)) antigen in human anagen hair follicles.

Lewis Y (Le(y)) antigen, a difucosylated tetrasaccharide found on type 2 blood group oligosaccharides of glycolipids and glycoproteins, is thought to be a phenotypic marker predictive of cell differentiation. The distribution of this antigen in human anagen hair follicles was examined by immunohistochemical staining using a monoclonal antibody (AH-6) to Le(y). In the bulbar and suprabulbar portion of anagen hair follicles, Le(y) antigen was detected in the three layers of the inner root sheath. Subsequently, the positive staining became translocated to the innermost layer of the outer root sheath in the middle part of the hair follicles. In the upper portion of the hair follicles, Le(y) antigen was found in the outer cells of the outer root sheath. These findings suggested that the expression of Le(y) antigen in the anagen hair follicles was correlated with the processes of keratinization or terminal differentiation.

Antibodies, Monoclonal↗

PubMed1995

Natural killer (NK)-like cytotoxicity of murine intraepithelial lymphocytes in the small intestine (iIEL) and the effect of the serine proteases.

The natural killer (NK)-like cytotoxicity of murine intraepithelial lymphocytes in the small intestine (iIEL) and the participation of serine proteases in it were investigated. We monitored the cytotoxicity of iIEL with a sensitive cytotoxic assay using laser flow cytometry. iIEL exhibited NK-like cytotoxicity on YAC-1 target cells. Benzamidine, a serine protease inhibitor, inhibited significantly both Na-CBZ-L-lysine thiobenzyl ester (BLT)-specific serine protease activity and iIEL-mediated NK-like cytotoxicity. These results suggest that BLT-specific serine proteases may participate in NK-like cytotoxicity of murine iIEL.

Amino Acid Sequence↗

PubMed1995

Strong adjuvant action of Klebsiella O3 lipopolysaccharide and its inhibition of systemic anaphylaxis.

Immunization with lipopolysaccharide from Klebsiella O3 as an immunological adjuvant did not cause the death of mice in systemic anaphylaxis to bovine serum albumin. On the other hand, most mice immunized with lipopolysaccharide from Escherichia coli O111, Klebsiella O4 and Salmonella minnesota did die. Klebsiella O3 lipopolysaccharide enhanced IgM and IgG antibody response to BSA more markedly than Escherichia coli O111 lipopolysaccharide, while it affected the production of IgE antibody only slightly. therefore, it is suggested that the inhibition of systemic anaphylaxis by Klebsiella O3 lipopolysaccharide adjuvant might be related to its strong adjuvant action on IgM and IgG class antibody production, and that high levels of circulating IgM and IgG antibodies might act as blocking antibodies in the development of IgE-mediated systemic anaphylaxis.

Adjuvants, Immunologic↗

PubMed1995

Role of tumor necrosis factor-alpha and glucocorticoid on lipopolysaccharide (LPS)-induced apoptosis of thymocytes.

Administration of bacterial lipopolysaccharide (LPS) into mice markedly induced the apoptosis of CD4+8+ thymocytes. The injection of anti-tumor necrosis factor (TNF)-alpha antibody or RU38486, a glucocorticoid receptor antagonist, into mice definitely inhibited LPS-induced apoptosis of thymocytes. Addition of the sera 1 h after injection of LPS into in vitro cultures of thymocytes caused thymocyte apoptosis. It was also prevented by either anti-TNF-alpha antibody or RU38486. Further, recombinant TNF-alpha and hydrocortisone collaborated in induction of the thymocyte apoptosis in vitro. The in vivo phenomenon of LPS-induced apoptosis of thymocytes was reproducible by the in vitro experimental system. It was therefore suggested that both TNF-alpha and glucocorticoid participate and collaborate as effector molecules in LPS-induced apoptosis of thymocytes.

Animals↗

PubMed1995

Binding of mannose-binding protein to Klebsiella O3 lipopolysaccharide possessing the mannose homopolysaccharide as the O-specific polysaccharide and its relation to complement activation.

Lipopolysaccharide from Klebsiella pneumoniae O3, which possesses the mannose homopolysaccharide as the O-specific polysaccharide, exhibits an extraordinarily high ability to activate the human complement system. We isolated the mannose-binding protein with a Klebsiella O3 lipopolysaccharide affinity column. The protein isolated had a molecular mass of much higher than 200 kDa, and it consisted of subunits with an apparent molecular mass of 32 kDa. The NH2-terminal sequence of the 32-kDa subunits was completely consistent with a part of the amino acid sequence of human serum mannose-binding protein. In immunoblotting, an anti-mannose-binding protein monoclonal antibody was definitely reactive with the isolated protein with the higher molecular mass. The protein isolated was bound exclusively to lipopolysaccharides possessing the mannose homopolysaccharide, not to lipopolysaccharide possessing the heteropolysaccharides. Klebsiella O3 lipopolysaccharide did not exhibit a high anticomplement activity in the serum from which the mannose-binding protein was depleted. It was concluded that the serum factor that bound to Klebsiella O3 lipopolysaccharide may be mannose-binding protein and that it may play a crucial role in the strong complement activation by Klebsiella O3 lipopolysaccharide.

Amino Acid Sequence↗