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Biomedical subjects

M Kimoto

Publications and source records attributed to M Kimoto.

At least 109 records · Page 6Linked to original sources
PubMed1994

Parotin subunit as a potent polyclonal B cell activator binds to newly found glycosylphosphatidylinositol (GPI)-anchored proteins on human B cell surfaces.

Parotin subunit (PS) is a unique glycoprotein, isolated from bovine parotid glands, which possesses the ability to induce polyclonal antibody production and IL-1-like activity. The present studies investigated the existence of receptors for PS on B cell surfaces using PS-affinity chromatography. No PS-binding proteins (PSR) solubilized from human B cell surfaces with Triton X-100 were detected, whereas the PSR released from human B cell membranes with phosphatidylinositol-specific phospholipase C (PI-PLC) treatment were composed of 75- and 40-kDa proteins. PI-PLC treatment markedly reduced polyclonal antibody responses to PS but weakly inhibited the responses to PWM, xanthan gum, and LPS in human and mouse lymphocytes. Addition of PSR caused a dose-related reduction in polyclonal IgM and IgG antibody responses to PS. These results suggest that PSR can act as glycosylphosphatidylinositol-anchored receptors for PS.

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

Antibody production system modulated by oral administration of human milk and TGF-beta.

Human milk contains large amounts of transforming growth factor-beta (TGF-beta), which exerts powerful suppressive effects on many immune functions. Oral administration of human milk and TGF-beta in mice strongly inhibited anti-sheep red blood cell (SRBC) responses obtained following oral immunization with SRBC, but enhanced the responses elicited by intraperitoneal, intrabronchial or intravenous immunization with SRBC. Anti-SRBC antibody responses stimulated by intraperitoneal or intravenous immunization with SRBC failed to augment when TGF-beta-depleted human milk was given orally. These findings suggest that TGF-beta in human milk functions as an adjuvant for protective antibody responses against parenteral infections.

Adjuvants, Immunologic↗
PubMed1994

A calcium- or manganese-dependent epitope on the integrin beta 1 chain recognized by a unique mAb.

A calcium- or manganese-dependent epitope was demonstrated on the beta 1 integrin chain by a newly established mAb. In immunoprecipitation, the mAb was able to bind to the solubilized beta 1 integrin chain in the presence of calcium but not magnesium. Cation chelating reagents completely affected the binding of the antibody to the intact beta 1 integrin chain on a cell surface. Calcium but not magnesium restored the binding of the antibody. Quantitative analyses revealed that chelating reagents reduced the affinity of the antibody. Moreover, manganese also rescued epitope expression. These results suggested that divalent cations, particularly calcium or manganese, had a crucial role in the conformation of the beta 1 integrin chain recognized by the SG/7 antibody. This antibody would be of great use for studying the divalent cation-dependent modulation of the integrin molecules and bring another aspect to the understanding of the regulatory mechanisms of ligand binding of the integrin molecules.

Antibodies, Monoclonal↗
PubMed1994

Signal transmission through MHC class II molecules in a human B lymphoid progenitor cell line: different signaling pathways depending on the maturational stages of B cells.

The function of MHC class II HLA-DR molecules expressed on a human B lymphoid progenitor cell line FL8.2.4.4 (abbreviated as FL4.4) was examined. FL4.4 cells expressed HLA-DR molecules and stimulation of the DR molecules by anti-DR mAb or by superantigen TSST-1 induced strong augmentation of homocytic aggregation and protein tyrosine phosphorylation in FL4.4 cells. Induced homocytic aggregation in FL4.4 consists both of LFA-1/ICAM-1-dependent and -independent pathways as revealed by mAb blocking experiments. Metabolic inhibitors, NaN3 and cytochalasin B, blocked the induced homocytic aggregation of FL4.4. Early mature Daudi B cell lines also showed a similar type of homocytic aggregation by stimulation with anti-DR mAb. Daudi cells are more sensitive to protein kinase inhibitors herbimycin A and H7 than FL4.4 cells in their blocking of induced homocytic aggregation, while W7 showed stronger inhibitory effects on FL4.4 cells than on Daudi cells. Western blotting analysis revealed that the stimulation of DR molecules induced protein tyrosine phosphorylation of 100-kDa, 90-kDa, 60-kDa and 55-kDa proteins in FL4.4 cells, while, in Daudi cells 110-kDa, 100-kDa and 80-kDa proteins were phosphorylated. These results suggest that different signaling pathways through class II molecules are employed depending on the maturational stage of B-cell differentiation.

Antibodies, Monoclonal↗
PubMed1994

In vivo induction of IgG anti-DNA antibody by autoreactive mixed haplotype A beta z/A alpha d MHC class II molecule-specific CD4+ T-cell clones.

We characterized autoreactive T-cell clones derived from (NZB x NZW)F1 (B/WF1) mice. These autoreactive T-cell clones are shown to be CD4+ by immunofluorescence staining and to belong to Th2 type by cytokine release assay. Specificity analysis revealed the existence of mixed haplotype A beta z/A alpha d major histocompatibility complex (MHC) class II molecule-specific T-cell clones as well as A beta z/A alpha z- or A beta d/A alpha d-specific T-cell clones. Some but not all of the mixed haplotype A beta z/A alpha d-specific autoreactive T-cell clones showed strong activity to induce IgG anti-DNA antibody production upon transfer to young (4-month-old) B/WF1 mice, indicating that T cells with these specificities might be involved in B/WF1 autoimmunity.

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

Mixed haplotype A beta Z/A alpha d class II molecule in (NZB x NZW)F1 mice detected by T cell clones.

We have tried to demonstrate the existence of a mixed haplotype MHC class II molecule in (NZB x NZW)F1 (B/WF1) mice. When a large panel of keyhole limpet hemocyanin-specific T cell clones derived from B/WF1 mice was analyzed, several clones were shown to be restricted by a F1-specific A beta Z/A alpha d class II molecule. Autoreactive A beta Z/A alpha d-specific T cell clones were also obtained. The ability of the association and expression of A beta Z with A alpha d was confirmed by hybridoma and transfection experiments. Hybridoma cell lines created by fusion of NZW (H-2z) spleen cells with M12.C3 (a A beta d- variant cell line derived from M12.4.1 (H-2d) B lymphoma) cells expressed A beta Z determinants. Transfection of A beta Z genomic DNA to M12.C3 cells resulted in the expression of A beta Z determinants. These hybridoma cell lines and transfectants were able to stimulate A beta Z/A alpha d-specific T cell clones, suggesting the expression of A beta Z/A alpha d molecules on the cell surface. However, attempts to demonstrate the existence of mixed haplotype MHC class II molecules in B/WF1 mice by two-dimensional (nonequilibrium pH gradient gel electrophoresis/SDS-PAGE) gel electrophoresis analysis with the use of anti-class II mAb failed to demonstrate the existence of mixed haplotype A beta Z/A alpha d or A beta d/A alpha z class II molecules in B/WF1 mice. Analysis of mixture of TA beta Z cell and B/WF1 spleen cell lysates immunoprecipitated by anti-A beta Z mAb suggested that the amount of haplotype mixed A beta Z/A alpha d molecules in B/WF1 spleen cells is less than 1/10 that of haplotype matched A beta/A alpha pairs. Our results suggest that, although undetectable by biochemical analysis, small amounts of mixed haplotype A beta Z/A alpha d molecules exist in B/WF1 spleen cells. Also, T cell clones which recognize them exists in B/WF1 mice. Because autoimmune symptoms of B/WF1 mice are shown to be related to heterozygosity at the H-2 region, autoreactive T cell clones which recognize the mixed haplotype A beta Z/A alpha d class II molecule might be involved for the induction of autoimmunity in B/WF1 mice.

Animals↗
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↗
PubMed1993

Detection of NG,NG-dimethylarginine dimethylaminohydrolase in the nitric oxide-generating systems of rats using monoclonal antibody.

In order to elucidate the biological role of NG,NG-dimethylarginine dimethylaminohydrolase (EC 3.5.3.18), we prepared monoclonal antibodies (mAbs) against the enzyme from rat kidney and examined the distribution of the enzyme in rats. Four mAbs have been obtained by the fusion of the spleen cells from BALB/c mouse immunized with the sodium dodecyl sulfate-denatured or native enzyme and P3X63Ag8U1 myeloma cells. All the mAbs were shown to bind to the denatured enzyme, but none of them could recognize the native enzyme. The occurrence of the enzyme protein in various rat tissues and cell systems such as peritoneal neutrophils and macrophages was examined using an immunoblotting technique with one of the mAbs. The immunoblotting analyses showed that the enzyme protein is widely distributed in rats, particularly, in kidney, pancreas, liver, brain, and aorta at high concentrations. Furthermore, the enzyme protein was clearly shown to exist in peritoneal neutrophils and macrophages. Since NG-monomethylarginine and NG,NG-dimethylarginine have been suggested to be specific blockers of the systems generating nitric oxide (NO), the above findings are of great interest in connection with the regulation of the NO production in such tissues and cell systems as aorta, brain, peritoneal neutrophils, and macrophages.

Amidohydrolases↗
PubMed1993

Generation of cells with morphological and antigenic properties of microglia from cloned EBV-transformed lymphoid progenitor cells derived from human fetal liver.

Single-cell clones from the Epstein Barr virus transformed lymphoid progenitor-like cell line established from human fetal liver at 8-week gestation, have been derived and characterized. These clones retained immunoglobulin (Ig) and T cell receptor (TCR) genes in their germ line configuration. They expressed HLA-DR and some B lymphoid markers such as CD19, CD20, and in some, the T lymphoid marker, CD2. They did not express surface Igs, CD3, CD4, CD8 or TCRs (alpha/beta, gamma/delta). A sensitive RT-PCR assay revealed that they did not express mRNA for a recombination activating gene-1, which is expressed after commitment to lymphoid cells. These results suggest that the established cloned lines are very early lymphoid progenitors that have not yet been committed to lymphoid cell lineage. In one of the lines, FL8.2.1.4, a marked morphological change that resembled microglia was induced when the cells were cultured in the presence of phorbol myristate acetate (PMA). After 72 hr of culture, 5-10% of FL8.2.1.4 cells developed a microglial morphology when stimulated with 10 to 100 ng/ml PMA. The newly generated cells with microglial morphology expressed HLA-DR and stained with Recinus communis agglutinin-1, which has been reported to bind specifically to brain microglia. In contrast, expression of lymphoid markers on cells with microglia-shaped morphology was remarkably diminished by PMA stimulation. Thus, the early lymphoid progenitor cells have the capacity to differentiate into cells with the morphological and antigenic properties of microglia cells. This system might be useful for further understanding of the characteristics and functions of microglia cells distributed in the central nerve system.

Antigens, CD↗
PubMed1993

Antimetastatic activity of lipopolysaccharide against a NK-resistant murine fibrosarcoma.

An intravenous injection of bacterial lipopolysaccharide (LPS) into mice exerted prominent antimetastatic activities against a NK-resistant weakly immunogenic NFSa fibrosarcoma. The number of visible metastases in the lung was increased by a pretreatment of anti-asialoGM1 (asGM1) antibody or silica, but pretreatment of asGM1 antibody or silica scarcely affected the antimetastatic activities of LPS. The pulmonary retention of radiolabeled tumor cells showed that LPS accelerated the detachment of the tumor cells from the lung, and that this acceleration was not suppressed by anti-asGM1 antibody. Sialic acids of lung endothelial cell surface, essential components of various receptors, was diminished by the i.v. injection of LPS. These results suggested that the antimetastatic effect of LPS against NK-resistant NFSa cells was partly the result of modulations of lung endothelial cell surface.

Animals↗
PubMed1993

Significance of platelets in an antimetastatic activity of bacterial lipopolysaccharide.

Recently we reported an antimetastatic activity of bacterial lipopolysaccharide (LPS) on a NK-cell-resistant murine fibrosarcoma (NFSa). Here we investigate and report the mechanistic significance of platelets in this activity. The number of circulating platelets was reduced to 63% of the control 3 days after an i.v. injection of 1.0 micrograms LPS, and then recovered to the level of control at day 10. Aggregation efficiency of platelets was impaired by LPS. The number of metastatic lung colonies after an i.v. injection of tumor cells was maximally reduced to 2.2% of the control at day 3 and increased in proportion to the recovery of platelet number. Neuraminidase (Ndase), which caused a non-immunological thrombocytopenia, also inhibited lung metastasis when injected prior to an i.v. tumor cell challenge. LPS and Ndase showed an identical pattern against five other syngeneic tumors; these agents inhibited lung metastases of the FSa fibrosarcoma and the SCC VII squamous cell carcinoma but failed to inhibit those of the NR-S1 squamous cell carcinoma, the MMCa#4 mammary adenocarcinoma and the NR-PG parotid gland tumor. All the three cells which were not responsive to any agents possessed a high aggregating activity of platelets while the other three tumors responsive to both agents did not show a detectable level of this activity. Platelet transfusion failed to modify the antimetastatic activity of LPS. These results suggest that platelets play an important role in the antimetastatic activity of LPS, though whether the role is principal or assistant remains to be seen.

Animals↗
PubMed1993

Preparation and application of monoclonal antibodies for a sandwich enzyme-linked immunosorbent assay of the major soybean allergen, Gly m Bd 30K.

In order to obtain probes suitable for the determination of the major soybean allergen, Gly m Bd 30 K, in soybean-related processed foods, we have prepared two monoclonal antibodies, F5 of IgG2a and H6 of IgM, by the fusion of P3U1 myeloma cells with the spleen cells of BALB/c mice immunized with the reductively carboxymethylated allergen (RCM-allergen). The two monoclonal antibodies were shown to be specific to the intact allergen as well as the RCM-allergen and to recognize distinct epitopes on the allergen. Of the monoclonal antibodies, F5 was conjugated with peroxidase. H6 and the labeled F5 were applied as the fixing antibody and the labeled first antibody, respectively, for a direct sandwich enzyme-linked immunosorbent assay of the allergen. When the allergen was extracted from the soybean-related foods with Tris-HCl buffer containing sodium dodecylsulfate and mercaptoethanol, the allergen, Gly m Bd 30 K, was shown to be measured in a range of 5-500 ng in this assay.

Allergens↗
PubMed1992

Dual effects of oleic acid on Ca2+ mobilization and protein phosphorylation in human platelets in presence or absence of platelet activating factor.

This laboratory demonstrated earlier that oleic acid inhibited platelet activating factor (PAF)-induced aggregation and serotonin release of rabbit platelets (M. Miwa, C. Hill, R. Kumar, J. Sugatani, M. S. Olson, and D. J. Hanahan, 1987, J. Biol. Chem. 262, 527-530). More recently, we reported that oleic acid caused a decrease in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2), but did not affect the level of inositol-1,4,5-trisphosphate (IP3), in rabbit platelets (D. Nunez, J. Randon, C. Gandhi, A. Siafaka-Kapadai, M. S. Olson, and D. J. Hanahan, 1990, J. Biol. Chem. 265, 18330-18838). These results suggested that oleic acid did not stimulate phospholipase C. In contrast, PAF induced a decrease in PIP2 and an increase in PIP level and IP3. These effects were shown to be attenuated by oleic acid. In this current study, our experiments show that (a) oleic acid blocked PAF-induced rise in intracellular [Ca2+] (to provide a mechanism in agreement with our previous experiments which showed that oleic acid inhibited PAF-induced IP3 rise in platelets) and (b) oleic acid itself induced a gradual rise in [Ca2+]i, which would provide a mechanism for oleic acid-induced aggregation despite the fact that oleic acid did not cause the production of IP3 (Nunez et al., 1990). Oleic acid, in a dose-dependent manner, was shown to inhibit PAF-induced Ca2+ mobilization from intra- and extracellular sources. The inhibition was closely related to the suppressive effect of oleic acid on PAF-induced aggregation. Furthermore, oleic acid inhibited the PAF-stimulated phosphorylation of the 20- and 40-kDa proteins. At concentrations above 20 microM, oleic acid itself could induce platelet aggregation and Ca2+ mobilization, but the time sequence of these two responses in human platelets was significantly different from those obtained with PAF. Oleic acid alone, at 20 microM, caused a 1.4-fold increase in the cAMP level in platelets which was followed by a decline to a basal value at higher concentrations of this fatty acid. It seemed clear that elevation of adenylate cyclase activity was not associated with free fatty acid inhibition of platelet activation. Interestingly, both PAF and oleic acid added separately to human platelets induced protein-tyrosine phosphorylation, but oleic acid did not cause any inhibition of PAF-induced protein-tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Platelets↗
PubMed1992

[The effects of centrifugal pump on liver support system with porcine liver perfusion].

The effects of vaneless centrifugal pump (Bio-pump P-80) on energy metabolism were studied, as compared with the roller pump, in an isolated porcine liver perfused with human blood for 6 hours. According to preliminary examination, flow rates were decided to 0.4, 0.8 ml/min/g in roller pump group, in Bio-pump pump group, respectively, not to increase more than 15 mmHg in initial portal pressure. During the perfusion, actual portal pressure was measured and circulatory resistance (portal pressure/flow rate) was calculated before perfusion and hourly after perfusion. Acetoacetate, beta-hydroxybutyrate, lactate and pyruvate, were measured at same times, from which values the ketone body ratio (acetoacetate/beta-hydroxybutyrate, KBR), reflecting the redox state of liver mitochondria, was calculated. In the Bio-pump group, in spite of its high flow, circulatory resistance was low at every hour during the perfusion. KBR was increased rapidly from 0.40 to 1.39, 2.59, 2.75, 2.38, 2.41, and 1.82 and lactate was decreased rapidly from 7.96 to 3.90, 1.77, 1.29, 1.33, 1.34, and 1.25 mmol/L at the respective hours after perfusion. There were significant differences at 2 and 4 hours after perfusion in KBR and after 2 hours of perfusion in lactate as compared with the roller pump group (p < 0.05). These results suggested that the Bio-pump is available for constant and high flow to the liver and helpful to elevate the mitochondrial NAD/NADH ratio (oxidized and reduced forms of free nicotinamide-adenine dinucleotides), leading to and enhancement of metabolic capacity of the perfused liver.

Adult↗