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S Inui

Publications and source records attributed to S Inui.

At least 55 records · Page 3Linked to original sources
PubMed1996

Characterization of a toxin-deficient Clostridium perfringens strain, KZ1340.

Clostridium perfringens KZ1340, previously classified as Clostridium plagarum, is an isolate from Antarctic soil, and was identified as an alpha-, theta-, and kappa-toxin non-producing variant. On Southern hybridization, the variant was found to be defective in the pfoA (theta-toxin) gene, but the plc (alpha-toxin) and colA (kappa-toxin) genes were present on the same EcoRI fragment as in the standard strain, NCTC8237. Northern analysis revealed that mature plc mRNA was transcribed in KZ1340 though less efficiently than in NCTC8237, while no mature colA mRNA was present in KZ1340. After transformation of the pfoA and plc genes into the KZ1340 via shuttle vector, pJIR418, the pfoA gene was successfully expressed but the plc gene was not efficiently expressed, suggesting that in KZ1340 there is negative regulation of plc gene expression. Toxin-deficient C. perfringens KZ1340 might be a suitable host for expression analysis of the pfoA gene and other clostridial virulence genes, if expressed efficiently, because it produces a small amount of extracellular toxins.

Bacterial Toxins↗
PubMed1996

Diagnosis of lymphoma in paraffin wax sections by nested PCR and immunohistochemistry.

AIMS: To investigate whether nested polymerase chain reaction (PCR) and immunohistochemistry can be used to diagnose malignant lymphoma. METHODS: Paraffin wax embedded tissue sections from 31 patients with malignant lymphoma were analysed by nested PCR and immunohistochemistry using standard protocols. RESULTS: Nested PCR amplification of 1 pg DNA confirmed monoclonality in B cell lymphoma; PCR amplification of 10 pg DNA confirmed monoclonality in T cell lymphoma. Twenty seven (87%) samples were diagnosed as malignant lymphoma by nested PCR, and 24 (77%) by immunohistochemistry. Seven samples were diagnosed as malignant lymphoma by nested PCR, but not by immunohistochemistry, whereas the use of both procedures gave a diagnosis of malignant lymphoma in all 31 samples. CONCLUSIONS: A combination of immunohistochemistry and nested PCR can be used to diagnose malignant lymphoma in routine paraffin wax embedded sections.

Adult↗
PubMed1996

Demonstration of infectious bovine rhinotracheitis virus antigen by immunoperoxidase method in tissues of aborted bovine fetuses preserved for 25 years in paraffin blocks.

Infectious bovine rhinotracheitis (IBR) virus antigen was demonstrated by the immunoperoxidase method in tissues of two aborted bovine fetuses, which had been stored for 25 years after fixing in formalin and embedding in paraffin. Necrotic foci were detected in the liver, kidney, adrenal gland, and thymus of the fetuses. Coincidenting with the distribution of the necrotic foci, IBR virus antigen was demonstrated by immunostaining. The present study proved the immunoperoxidase method in one of useful techniques to demonstrate IBR virus antigen in tissue sections from preserved paraffin blocks.

Abortion, Veterinary↗
PubMed1995

Dose-dependent suppression of toluene metabolism by isopropyl alcohol and methyl ethyl ketone after experimental exposure of rats.

In order to examine possible suppression of toluene metabolism due to coexposure to other solvents, female Wistar rats were exposed for 8 h to toluene alone (at 50 or 100 ppm), or in combination with either methyl ethyl ketone (at 50, 100, 200 or 400 ppm) or isopropyl alcohol (at 50, 100, 200, 400, 800 or 1600 ppm). Urine samples were collected for 24 h after initiation of each exposure, and subjected to analysis for two toluene metabolites, hippuric acid and o-cresol, both by HPLC. The excretion of hippuric acid, a major metabolite, was not modified when the concentrations of methyl ethyl ketone or isopropyl alcohol were low, i.e. 100 ppm or below, whereas it was reduced when methyl ethyl ketone or isopropyl alcohol concentrations were twice or more times higher than that of toluene. There were no changes in any cases in excretion of o-cresol, a minor metabolite. The observation after coexposure to methyl ethyl ketone or isopropyl alcohol at low concentration is in line with the negative interaction between toluene and methyl ethyl ketone as well as between toluene and isopropyl alcohol after occupational exposures at low concentrations. Metabolic interaction may take place when the exposure intensity is high, as observed in the present study and also after experimental exposure of volunteers to toluene and m-xylene, or occupational exposure to benzene and toluene.

1-Propanol↗
PubMed1995

Molecular cloning of a cDNA clone encoding a phosphoprotein component related to the Ig receptor-mediated signal transduction.

We have shown previously that a 52-kDa phosphoprotein (p52) co-precipitated with Ig receptor (IgR)-associated MB-1 protein was inducibly phosphorylated by the stimulation with 12-O-tetradecanoyl-phorbol-13-acetate. By immunizing the 52-kDa protein co-precipitated with MB-1, we prepared a mAb (19-14), which can immunoprecipitate the p52 and the associated kinase molecule. Immune complex kinase assay with the 19-14 mAb showed that the p52 is associated with a novel kinase molecule and is involved in IgR-mediated signal transduction. Here we isolated a cDNA clone (alpha 4) from a murine bone marrow cDNA library in lambda gt-11 vector by the 19-14 mAb. The alpha 4 cDNA encodes a novel protein of 340 amino acids with multiple potential phosphorylation sites. The 1.4-kb alpha 4 mRNA is expressed in various cells including cell lines of B lineage, T lineage, monocytic, fibroblast, and normal organs such as liver, spleen, thymus, and brain. To study the molecule encoded by the alpha 4 cDNA, we produced a chimeric protein of the glutathione S transferase (GST)-alpha 4 in pGEX-3X expression vector. The 19-14 mAb binds to the GST-alpha 4 fusion protein. Rabbit anti-alpha 4 Ab prepared by immunizing the GST-alpha 4 fusion protein immunoprecipitated a 52-kDa phosphoprotein from WEHI-231 cells. The 52-kDa phosphoprotein immunoprecipitated by the anti-alpha 4 Ab is tightly associated with kinase activity that is similarly observed with the p52 protein reported previously. Moreover, the 52-kDa phosphoprotein immunoprecipitated with the anti-alpha 4 Ab is identical to the p52 by the protein comparison on non-equilibrium pH gradient gel electrophoresis/SDS-PAGE, isoelectric focusing/SDS-PAGE, and V8 protease mapping. The 52-kDa phosphoprotein is associated with functional components that are inducibly phosphorylated by anti-IgM stimulation. These results suggested that the alpha 4 gene-encoded molecule is functionally involved in the IgR-mediated signal transduction in B cells.

Amino Acid Sequence↗
PubMed1995

Anti-CD40 monoclonal antibody induces the proliferation of murine B cells as a B-cell mitogen through a distinct pathway from receptors for antigens or lipopolysaccharide.

To study the activation and differentiation of murine B cells, we prepared a hybridoma secreting monoclonal antibody, LB429, which can directly induce the proliferation of murine B cells in vitro. LB429 recognizes a B cell specific surface molecule of 45 kDa. It recognizes an epitope of murine CD40 produced as a soluble fusion protein with glutathione S-transferase. LB429 stains COS-7 transfectant with murine CD40 cDNA and mature B-cell lines but does not stain pre-B cell lines. Two color staining demonstrated that the epitope recognized with LB429 appears on the surface of B220+ cells of spleen and bone marrow. LB429 can induce a strong proliferation of murine B cells from spleen in the absence of initial triggering with anti-IgM antibody or with anti-IgM antibody + IL-4. LB429 induced the cell size enlargement and the cell cycle transition of resting B cells as well as lipopolysaccharide (LPS). LB429 and LPS stimulate B cells synergistically in vitro by accumulating 44.7% of cells in S/G2/M phases of cell cycle. However, stimulation of spleen B cells with LB429 resulted in the increase of sIgM high+ sIgD(high)+ B cells, in contrast LPS showed the proliferation of both sIgM(high)+ sIgD(high)+ B cells and sIgM(low)+ sIgD(high)+ B cells. These results suggested that LB429 and LPS cause the proliferation of B cells through different stimulatory pathways. This anti-mouse CD40 antibody (LB429) is a very useful reagent to study the activation and differentiation of B cells in vitro.

Animals↗
PubMed1994

B cell Ag receptor mediates different types of signals in the protein kinase activity between immature B cell and mature B cell.

Ig receptor (IgR) on the surface of B cells mediates the Ag-specific stimulatory signal for B cell proliferation and differentiation. In immature B cells, the stimulatory signal causes an inhibitory effect which is believed to be a key phenomenon in B cell tolerance or B cell anergy. Here, we studied the molecular mechanism of the inhibitory response of the IgR-mediated signal transduction that results in the programmed cell death of immature B cells. To analyze the downstream molecules of the IgR-mediated signal transduction, we prepared a mAb against a 160-kDa membrane protein (p160) that can coprecipitate the kinase molecule(s) acting on serine, threonine, and tyrosine residues. Anti-IgR stimulation induces the increase of the kinase activity coprecipitated with the p160 protein in mature B cell BAL17 and normal adult spleen B cells. This result suggest that the p160-associated kinase activity is one of the downstream events of the IgR-mediated signal transduction cascade. Interestingly, immature B cell lymphoma WEHI-231 and the neonatal spleen B cells showed the adverse reaction of the p160-associated kinase which results in the transient loss of the kinase activity. Moreover, the transient decrease of the p160-associated kinase was caused by the tyrosine phosphatase activity induced by the stimulation of IgR in WEHI-231. The results suggest that this molecular difference in the downstream events of the IgR-mediated signal transduction between immature B cells and mature B cells already begins at the transmembrane level in the IgR-mediated signal transduction pathway.

Animals↗
PubMed1994

Identification of a 52-kDa molecule (p52) coprecipitated with the Ig receptor-related MB-1 protein that is inducibly phosphorylated by the stimulation with phorbol myristate acetate.

Triggering of the Ig receptor (IgR) induces the activation in multiple intracellular signal transduction reactions including protein tyrosine phosphorylation, activation of phospholipase C, increased inositoltriphosphate, increased diacylglycerol, intracellular Ca2+ mobilization, and activation of protein kinase C. The IgR-complex, composed of mu-chain, L chain, Ig-alpha (MB-1), and Ig-beta (B29) proteins, is a functional unit both for expression of IgR and for signal transduction into cells, possibly by physical association with the down-stream functional molecules. An important functional motif ((D or E)-X7-(D or E)-Y-X3-L-X7-Y-X2-(L or I)) in the cytoplasmic domain of MB-1 molecule was shown to bind with several phosphoprotein components including src-type tyrosine kinases and phosphatidylinositol-3 kinase. To further study the functional components, we analyzed the phosphoprotein molecules coprecipitated with MB-1 protein. We found that a 52-kDa protein is coprecipitated with MB-1 protein and is inducibly phosphorylated by the stimulation with PMA. A rat mAb, prepared by immunizing the 52-kDa protein purified from SDS-PAGE, could detect the similar 52-kDa phosphoprotein (p52) expressed on the cell surface. In comparison with the 52-kDa protein in the immunoprecipitate of MB-1, the p52 migrated to the same position on 2-D gel electrophoresis (nonequilibrium pH gradient gel electrophoresis/SDS-PAGE). An in vitro kinase reaction analysis demonstrated that the p52 is tightly associated with the tyrosine kinase molecule(s), one of which is an 80-kDa protein containing an apparent autophosphorylation activity. These molecules would provide the informations of the down-stream molecules in the cascade reactions of the IgR-mediated signal transduction.

Amino Acid Sequence↗
PubMed1994

Occupational exposure to solvent mixtures: effects on health and metabolism.

Exposure monitoring by personal diffusive samplers, biological monitoring of toluene exposure by urinary hippuric acid determination, haematology, serum biochemistry for liver function, and a subjective symptom survey by questionnaire were conducted on 303 male solvent workers. They were exposed to a mixture of solvents including toluene (geometric mean 18 ppm), methyl ethyl ketone (MEK; 16 ppm), isopropyl alcohol (IPA; 7 ppm), and ethyl acetate (9 ppm). The intensity was mostly below unity using the additiveness formula based on current Japanese occupational exposure limits, but more than eight times unity at the maximum. The results were compared with the findings in 135 non-exposed male workers of similar ages. Haematology and liver function tests did not show any exposure related abnormality, and subjective symptoms were mostly related to central nervous system depression and local irritation. Further analysis suggested that the irritation effects were not related to exposure to MEK. Analysis of the relation between toluene exposure and hippuric acid excretion in urine showed that there was no metabolic interaction between MEK and toluene, or between IPA and toluene. Overall, therefore, it is concluded that there was no sign or symptom detected to suggest anything other than toluene toxicity, that there was no evidence to indicate any modification of toluene toxicity or metabolism due to coexposure, and that the additiveness assumption is reasonable for risk assessment for the combination of solvents under these exposure conditions.

1-Propanol↗
PubMed1994

Down regulation of Ia expression in macrophages following incubation with mycobacteria.

Macrophages are known to release cytokines in response to various kinds of stimulators. In the present study, peritoneal macrophages from C3H/He or C3H/HeJ mice were incubated in vitro with heat-killed M. lepraemurium, M. intracellulare or M. gordonare for 3 days followed by harvest culture supernatant to analyze cytokine activities. It, therefore, seems that macrophages phagocytizing these mycobacteria, released interleukin-1 (IL-1) and tumor necrosis factor (TNF) in culture media. The amount of release was dose dependent on mycobacteria employed. In addition, macrophages, as already have reported elsewhere, treated with IFN for 2 to 3 days showed enhanced expression of surface Ia; although the expression was inhibited if the cells phagocytized mycobacteria. Similarly, the reduced expression of Ia was observed in peritoneal macrophages from MRL/lpr mice after 3 day-culture with mycobacteria in vitro. More importantly, in the presence of the supernatant obtained from macrophages incubated with mycobacteria, IFN gamma-treated normal macrophages exhibited suppressed expression of Ia. These results demonstrate that cytokine release and reduced expression of surface Ia in macrophages are simultaneous phenomena after phagocytosis of mycobacteria. Suppression of Ia may be in part induced by Ia suppressive factor(s) released from mycobacterium-phagocytized macrophages.

Animals↗
PubMed1993

Induction of tyrosine phosphorylation in human B lineage cells by crosslinking MB-1 molecule of B cell receptor-related heterodimer complex.

B cell antigen receptor is composed of immunoglobulin and associated MB-1 and B29. Here, we found that anti-human MB-1 stimulation induced tyrosine phosphorylation in immature B cells (FL4.4 and Nalm-6) but not in mature B cells (Daudi). Coprecipitated complex with the heterodimer component in Daudi and Nalm-6 contained the kinase molecule(s) which act on the heterodimer protein, while the complex in early lymphoid cell with germ line antigen receptor genes (FL4.4) did not. Candidate Fyn and Lyn are expressed in Nalm-6 and Daudi but are not expressed in FL4.4. These results suggested that src-type tyrosine kinases as Fyn and Lyn are responsible for the phosphorylation of MB-1 and B29 heterodimer, but anti-MB-1 stimulation can induce tyrosine phosphorylation reaction mediated by other kinase molecule(s) in the progenitor type cells.

Amino Acid Sequence↗
PubMed1993

Cross-linking of B cell receptor-related MB-1 molecule induces protein tyrosine phosphorylation in early B lineage cells.

Cross-linking of surface B cell Ag receptor (BCR) induces tyrosine phosphorylation of BCR-associated components through a receptor-mediated signal transmission pathway. B cell-specific mb-1 and B29 genes encode the alpha/beta components of the BCR-associated complex in mature sIgM+ B cells. Here, we studied the involvement of the mb-1 gene product, MB-1, in the BCR-related structure of immature B cells. Affinity-purified anti-MB-1 antibody coprecipitated mu chain/20-kDa/15-kDa proteins together with monomer MB-1 and Ig-alpha/Ig-beta heterodimer components from digitonin lysates of the pre-B cell line 18.81. The monomer MB-1 and Ig-alpha in the pre-B cell line were shown to migrate with identical patterns in nonequilibrium pH gradient gel electrophoresis/SDS-PAGE. Western blot analysis showed that MB-1 protein is coprecipitated with mu chain from the pre-B cell line. We studied the tyrosine phosphorylation response of bone marrow B lineage cells as well as spleen B cells after cross-linking of BCR-related components with anti-mu, anti-kappa, and anti-MB-1 antibodies. We identified the activation of tyrosine kinase by direct cross-linking of MB-1 expressed on the surface of early B lineage cells. Anti-mu antibody stimulation induced the activation of tyrosine kinase in early (5- to 10-min) and late (30- to 120-min) responses in bone marrow early B lineage cells. Anti-MB-1 mAb (11-18-5) induced the late response exclusively but anti-kappa antibody induced only the early response. These results clearly indicate that MB-1 acts in the BCR-mediated signal transmission in early B lineage cells. To explore the molecular mechanism of protein tyrosine phosphorylation in bone marrow B lineage cells, we studied associated components of the BCR complex by using an in vitro kinase reaction and observed the phosphorylation of a 60-kDa protein in pre-B cell lines. The 60-kDa phosphoprotein coprecipitated with MB-1 and the BCR-related complex is very similar to the Src-type Fyn tyrosine kinase or a Fyn-related protein.

Animals↗
PubMed1992

Activated human T cells express a ligand for the human B cell-associated antigen CD40 which participates in T cell-dependent activation of B lymphocytes.

To identify the ligand for the B cell-associated antigen CD40, we constructed a chimeric immunoglobulin molecule where the extracellular portion of the CD40 protein replaced the normal immunoglobulin variable region. No binding was detected on resting peripheral blood T cells. However, following T cell activation with phorbol esters and ionomycin, the chimeric protein bound specifically to activated human T cells and precipitated a 35-kDa protein from such cells. The induction of the CD40 ligand was detectable on the cell surface after 1 h, with maximal expression after 8 h of stimulation. The T cells expressing CD40 ligand were predominantly CD4 positive, although a proportion of CD8-positive cells also expressed the protein. There was no particular correlation with CD45 phenotype. Finally, we found that soluble CD40 inhibited T-dependent B cell proliferation. The results are discussed in the context of cognate interactions between B and T cells.

Adult↗
PubMed1992

Establishment of a murine pre-B cell clone dependent on interleukin-7 and stem cell factor.

To identify cytokines required for proliferation of murine pre-B cells, we established a pre-B cell clone MH11 (B220+ MB-1+ sIgM-) on a stromal cell line ST2 from day 13 fetal liver. The growth of MH11 is dependent on ST2. Another stromal cell line PA6, non-secretor of IL-7, could not support MH11 unless IL-7 was added. We investigated the effect of cytokines on proliferation of MH11 with or without stromal cells. IL-7 had a stimulatory effect on proliferation of MH11, but IL-7 alone could not support MH11 growth without ST2. Recombinant stem cell factor (rSCF) also had a positive effect on MH11. rSCF and rIL-7, when added together, could maintain the growth of MH11 in the absence of stromal cells. Moreover, the growth of MH11 on ST2 was inhibited almost completely by anti-c-kit monoclonal antibody (mAb). These results demonstrate that direct SCF/c-kit interaction is involved in the stimulation of pre-B cells.

Animals↗
PubMed1992

Cyclic AMP mimics IL-1 action in augmenting the differentiation of a mouse myeloid leukemic cell line (M1).

We have shown previously that recombinant human interleukin 1(IL-1) and interleukin 6 (IL-6) inhibited the proliferation of a mouse myeloid leukemic cell line (M1), and that IL-6 induced differentiation of the cells into macrophage-like cells and that IL-1 augmented this differentiation. Using this model we investigated the action mechanisms of IL-1 and IL-6. IL-6, but not IL-1, stimulated prostaglandin E2 (PGE2) production. The differentiative effect of IL-6 however, was not suppressed by indomethacin, although PGE2 induction by IL-6 was completely inhibited. Exogenously added PGE2 neither augmented the differentiative effect of IL-6 nor induced differentiation in combination with IL-1. Therefore, stimulation of PGE2 production did not appear to be essential for differentiative effects of these cytokines. Dibutyryl cAMP, 8-Br-cAMP and two adenylate cyclase-activating reagents, cholera toxin (CT) and forskolin (FK), all exhibited the similar augmenting effects as IL-1. These reagents augmented M1 cell differentiation by IL-6, and they did not induce differentiation in combination with IL-1. cAMP derivatives, CT, FK, IL-1 and IL-6 all inhibited the proliferation of M1 cells. CT and FK increased the intracellular cAMP levels. However, neither IL-1 nor IL-6 increased the cAMP levels. In contrast to the cAMP derivatives and reagents that activate adenylate cyclase activity, phorbol 12-myristate 13-acetate (PMA) and calcium ionophore neither induced nor augmented the differentiation in combination with either IL-1 or IL-6. Intracellular Ca2+ concentration was not altered by IL-1 or IL-6 suggesting that Ca2+/Calmodulin kinase and protein kinase C activation are not involved in this signal transduction pathway. Therefore, the present study suggests that IL-1 exhibits an effect similar to that of cAMP without affecting intracellular cAMP level.

Adenylyl Cyclases↗
PubMed1992

[Monokine production by mouse peritoneal macrophages after phagocytosis of mycobacteria].

It is well known that monokines, IL-1 (Interleukin-1) and TNF (Tumor Necrosis Factor), are produced by macrophages after stimulated with various agents. These cytokines are involved in various aspects of the inflammatory process and immunological response in addition to their original activities to proliferate T lymphocytes and causing tumor necrosis, respectively. Recently, there have been reported that IL-1 and TNF also play an important role in mycobacterial infections such as granuloma formation. In the present study, IL-1 and TNF productions were observed by mouse peritoneal exudate and resident macrophages after incubation with heat-killed M. lepraemurium and M. avium in vitro. The production was enhanced by phagocytosis of these mycobacteria in a dose dependent manner, and the time course of the production was maximum within 24 hr after phagocytosis of these mycobacteria. It was also shown of morphological changes and enhanced glucose consumption in media by these macrophages. Above results suggest that phagocytosis of mycobacteria by macrophages leads to monokine production, which would not only causes well known immunological reactions but also makes characteristic phenomena to be observed in mycobacterial infections.

Animals↗
PubMed1991

Inactivation of exogenous surfactant by pulmonary edema fluid.

Modified natural porcine surfactant was mixed with edema fluid sampled from the airways of hyperoxia-exposed adult rabbits. By varying the concentration of surfactant lipids (10, 25, and 50 mg/mL) and edema fluid proteins (0-280 mg/mL), we obtained a series of preparations with protein to surfactant lipid weight ratios ranging from 0 to 11.2. The surfactant activity of these various mixtures was analyzed with a pulsating bubble (at a lipid concentration of 10 mg/mL) or in experiments on immature newborn rabbits (at lipid concentrations of 25 or 50 mg/mL). For the latter purpose, animals were delivered at a gestational age of 27 d and ventilated with a standardized sequence of insufflation pressures after receiving 0.1 mL of the surfactant-edema sample into the airways at birth. Nearly complete in vitro inhibition of surfactant (markedly delayed film adsorption and a minimum surface tension of 23 mN/m during pulsation) was observed at a protein to surfactant lipid ratio of 4.5. Under in vivo conditions, nearly complete surfactant inhibition (tidal volumes reduced to less than 20% of the values for littermates ventilated with the same pressure after receiving surfactant without admixture of edema fluid) was documented at a protein to surfactant lipid ratio of 11.2. Our data suggest that the functional properties of an immature neonatal lung, in which serum proteins tend to leak into the airspaces after the onset of ventilation, depend on the stoichiometric relation between surfactant lipids and inhibitory proteins in the lung liquid.

Animals↗