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Biomedical subjects

S Muramatsu

Publications and source records attributed to S Muramatsu.

At least 19 recordsLinked to original sources
PubMed1992

Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.

Antigen-presenting, major histocompatibility complex (MHC) class II-rich dendritic cells are known to arise from bone marrow. However, marrow lacks mature dendritic cells, and substantial numbers of proliferating less-mature cells have yet to be identified. The methodology for inducing dendritic cell growth that was recently described for mouse blood now has been modified to MHC class II-negative precursors in marrow. A key step is to remove the majority of nonadherent, newly formed granulocytes by gentle washes during the first 2-4 d of culture. This leaves behind proliferating clusters that are loosely attached to a more firmly adherent "stroma." At days 4-6 the clusters can be dislodged, isolated by 1-g sedimentation, and upon reculture, large numbers of dendritic cells are released. The latter are readily identified on the basis of their distinct cell shape, ultrastructure, and repertoire of antigens, as detected with a panel of monoclonal antibodies. The dendritic cells express high levels of MHC class II products and act as powerful accessory cells for initiating the mixed leukocyte reaction. Neither the clusters nor mature dendritic cells are generated if macrophage colony-stimulating factor rather than granulocyte/macrophage colony-stimulating factor (GM-CSF) is applied. Therefore, GM-CSF generates all three lineages of myeloid cells (granulocytes, macrophages, and dendritic cells). Since > 5 x 10(6) dendritic cells develop in 1 wk from precursors within the large hind limb bones of a single animal, marrow progenitors can act as a major source of dendritic cells. This feature should prove useful for future molecular and clinical studies of this otherwise trace cell type.

Animals↗
PubMed1992

Inhibition of Ca(2+)-dependent catecholamine release by myosin light chain kinase inhibitor, wortmannin, in adrenal chromaffin cells.

To elucidate the possible involvement of myosin light chain kinase (MLCK) in the mechanism of exocytosis, we studied effects of MLCK inhibitor, wortmannin, on the secretory function of bovine adrenal chromaffin cells. Preincubation of chromaffin cells with wortmannin inhibited both acetylcholine- and high K(+)-evoked catecholamine (CA) release. The IC50 for high K(+)-evoked CA release was 1 microM. When the cells were permeabilized with digitonin after wortmannin preincubation, Ca(2+)-dependent exocytosis was inhibited in a dose-dependent manner (IC50, 1 microM). These findings suggest the implication of MLCK in the Ca(2+)-triggered process in the machinery of exocytosis.

Acetylcholine↗
PubMed1992

Preparation of immunoaffinity mini-columns for the analysis of platelet activating factor (PAF) in biological samples.

Using an antibody to BN 52719, an analogue of platelet activating factor (PAF), immunoaffinity mini-columns for the separation of PAF from biological samples were prepared. Rabbits were immunized with BN 52719 and immunoglobulin G (IgG) from the antiserum was coupled with Sepharose 4B. The resulting suspension of the IgG-coated Sepharose 4B in 25 mM phosphate buffer (pH 6.9) was poured into a plastic mini-column (bed volume 2.0 x 0.8 cm). Stepwise elution of the column with methanol revealed that lyso-PAF is eluted with 20-30% methanol in water whereas PAF is eluted with 50-80% methanol. For the determination of PAF in biological samples, it is recommended that lipids are extracted from the samples and the extract, reconstituted in 20% methanol, is loaded on the column. The column is then washed with 50% methanol followed by elution of PAF with 80% methanol. A small amount of [3H]PAF is added to the samples for measurement of the recoveries of PAF during the procedures of extraction and elution. The PAF is then quantified by radioimmunoassay or bioassay. Employing the immunoaffinity mini-column and radioimmunoassay, the contents of PAF in macrophages and conditioned medium after stimulation with calcium ionophore A23187, or tumor promoters such as TPA and thapsigargin, were measured.

Animals↗
PubMed1992

Suppression of macrophage Ia antigen expression by endogenous interferon-alpha/beta.

Exogenous interferon-alpha (IFN-alpha) and interferon-beta (IFN-beta) (type I IFNs) are known to suppress the IFN-gamma-dependent expression of class II MHC (Ia) antigens on macrophages (M phi). We report here that the endogenous type I IFNs produced by M phi in response to IFN inducers regulate Ia expression of the M phi themselves. Coculture of M phi with IFN-gamma and polyinosinic-polycytidylic acid [poly(I):poly(C)] resulted in the reduction of Ia expression in comparison with those cultured without poly(I):poly(C). Pretreatment of M phi with poly(I):poly(C) or a bacterial lipopolysaccharide (LPS), which is also a potent IFN inducer, in vitro or in vivo, before being exposed to IFN-gamma was also effective in suppressing the Ia expression. Such suppression was abolished by the addition of anti-IFN-alpha/beta antibodies to the M phi culture along with IFN-gamma. M phi cultured with L-cell conditioned medium (LCM) containing M-CSF were less capable of expressing Ia antigens than those cultured without LCM. The Ia-expressing ability of LCM-treated M phi was also restored by the addition of anti-IFN-alpha/beta antibodies. M phi in the early stage of sterile inflammation were less responsive to IFN-gamma than those in the late stage. These results suggest that endogenous type I IFNs, which are produced in response to natural or synthetic IFN-inducers, regulate M phi Ia expression in an autocrinal manner.

Animals↗
PubMed1992

Regulatory role of the GTP-binding protein, G(o), in the mechanism of exocytosis in adrenal chromaffin cells.

To elucidate the possible involvement of GTP-binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and antibodies against the G proteins (Gi and G(o)) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine-evoked catecholamine release. High K(+)-, histamine-, or gamma-aminobutyric acid-evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10(-4) M acetylcholine was decreased significantly in PTX-treated cells. In digitonin-permeabilized cells, PTX pretreatment resulted in a decrease of the half-maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP-gamma-S (a nonhydrolyzable GTP analogue) inhibited Ca(2+)-dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the alpha subunit of the PTX-sensitive G protein, G(o). Treatment with similar antibodies against the alpha subunit of Gi had no effect. These findings suggest that G(o) directly controls the Ca(2+)-triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.

Animals↗
PubMed1992

Fibrinogen Lima: a homozygous dysfibrinogen with an A alpha-arginine-141 to serine substitution associated with extra N-glycosylation at A alpha-asparagine-139. Impaired fibrin gel formation but normal fibrin-facilitated plasminogen activation catalyzed by tissue-type plasminogen activator.

An A alpha-arginine-141 to serine substitution has been identified in a homozygous dysfibrinogen, fibrinogen Lima, associated with impaired fibrin polymerization. The point mutation created an asparagine-X-serine-type glycosylation sequence, and indeed, extra, mainly disialylated biantennary oligosaccharides have been isolated from A alpha asparagine-139 of the patient's fibrinogen. This type of glycosylation sequence is unique for human fibrinogen, because the sequences shown for normal and abnormal fibrinogens are all asparagine-X-threonine types. The terminal sialic acids of the extra oligosaccharides seem to have largely contributed to the impaired fibrin gel formation, as evidenced by its correction to a near normal level by desialylation. Nevertheless, the polymerizing fibrin facilitated tissue-type plasminogen activator-catalyzed plasmin formation in a normal fashion, indicating that the initial two-stranded fibrin protofibrils had been constructed normally. Thus the impaired fibrin gel formation could be attributed to the delay in their subsequent lateral association, most probably because of the repulsive forces generated by the negative electric charge of the extra sialic acids. The substitution of a basic residue arginine to a noncharged residue serine may also have contributed to the impaired function in a similar manner or by steric hindrance in association with bulky extra oligosaccharide chains.

Amino Acids↗
PubMed1992

Metabolism of pravastatin sodium in isolated rat hepatocytes. I. Glutathione conjugate formation reaction.

1. The metabolic fate of pravastatin sodium (sodium (+)-(3R,5R)-3,5-dihydroxy-7-((1'S,2'S,6'S,8'S,8'aR)-6'-hydroxy-2'methyl- 8'-[(S)-2"-methylbutyryloxy]-1',2',6',7',8', 8'a-hexahydro-1'-naphthyl) heptanoate) was studied in isolated rat hepatocytes. 2. Two polar metabolites were isolated and identified as a glutathione conjugate and a dihydrodiol. 3. Both metabolites were formed via an epoxide which has been identified as the 4'a beta,5' beta-epoxide on the decalin moiety. 4. Formation of the glutathione conjugate was enzymic, while the dihydrodiol was formed by non-enzymic hydrolysis of the epoxide accompanied by the intramolecular migration of the double bond.

Animals↗
PubMed1992

Gene analyses of abnormal fibrinogens with a mutation in the gamma chain.

In four abnormal fibrinogens with a point mutation in the gamma chain, all characterized by impaired fibrin polymerization, we identified single base exchanges in the respective mutant gamma chain genes by polymerase chain reaction followed by DNA sequence analysis. These base exchanges accounted for the amino acid substitutions previously reported from our laboratory. They were exchanges of C to T (CGC for gamma Arg-275 to TGC for Cys) in fibrinogen Osaka II, T to G (AAT for gamma Asn-308 to AAG for Lys) in fibrinogen Kyoto I, T to C (ATG for gamma Met-310 to ACG for Thr) in fibrinogen Asahi, and G to T (GAT for gamma Asp-330 to TAT for Tyr) in fibrinogen Kyoto III. These base exchanges were found to reside in exon VIII of the gamma chain gene. Since many abnormal molecules are associated with polymerization defects, unless associated with the impaired release of fibrinopeptides A and/or B, exon VIII of the gamma chain gene may deserve careful study to define the structural alterations.

DNA Mutational Analysis↗
PubMed1991

Effects of recombinant alpha-interferon-gelatin conjugate on in vivo murine tumor cell growth.

A recombinant human alpha-interferon A/D (IFN), also known to be effective in mice, was conjugated to gelatin with a water-soluble carbodiimide. The IFN-gelatin conjugate was much more efficient than free IFN in activating mouse peritoneal macrophages (Mø) in an in vitro experiment to inhibit the growth of IFN-resistant subline cells (RR1) of murine fibrosarcoma. A single i.p. injection of the conjugate administered to normal mice was also more effective than one of free IFN in activating peritoneal Mø and natural killer cells in peritoneal exudate cell and spleen cell populations. In the investigation on body distribution of the IFN-gelatin conjugate, an enhanced affinity to Mø as well as a prolonged retention were observed in comparison with free IFN. An injection of the IFN-gelatin conjugate i.p. was more effective than one of free IFN in suppressing the in vivo growth of not only IFN-sensitive SS2 cells but also RR1 cells in the peritoneal cavity of mice, although RR1 cells were only susceptible to the indirect effect of IFN via host cells, in contrast to SS2 cells. In addition to an increased recruitment of Mø to the peritoneal cavity in RR1-bearing mice receiving i.p. injection of the IFN-gelatin conjugate, these Mø were activated to inhibit the in vitro growth of RR1 cells. These results indicate that the IFN-gelatin conjugate is a promising antitumor agent that is much more effective than free IFN. The dose of IFN in the conjugate required for exerting the antitumor effects is much lower than that of free IFN, which leads to a reduction of adverse effects of IFN.

Animals↗
PubMed1991

A sensitive nonisotopic method for the determination of intracellular azidothymidine 5'-mono-, 5'-di-, and 5'-triphosphate.

In order to analyze the efficacy of azidothymidine (AZT), it is important to know intracellular concentrations of AZT metabolites. However, it has been impossible to measure intracellular AZT 5'-monophosphate (AZT-MP), AZT 5'-diphosphate (AZT-DP), and AZT 5'-triphosphate (AZT-TP) without using isotopes. In the present study, we developed a new method to measure intracellular AZT metabolites without radiolabeled compounds. The method employed was a high-performance liquid chromatography (HPLC) system programmed for column switching technique, in which two columns were used: column 1 (TSK-G2000-SW, 300 x 7.5 mm) to preseparate AZT metabolites from major cell components, and column 2 (YMC-A-312-ODS, 150 x 6 mm) to determine the metabolites. The limit of detectability of this system was 3.3 pmol/injection. When MT-4 cells were incubated with various concentrations of AZT, intracellular concentrations of AZT-MP increased in parallel with extracellular AZT. Those of AZT-DP and AZT-TP, however, reached plateaus at 5 and 2 microM of AZT, respectively. In MT-4 and Molt-4 cells incubated with 5 microM AZT, concentrations of AZT-MP increased time dependently, while the AZT-DP/AZT-MP ratios decreased with time. These data suggest that high dose of AZT may not necessarily increase intracellular concentration of AZT-TP. The concentrations of AZT metabolites in peripheral blood mononuclear cells in a patient with AIDS and an asymptomatic carrier were measured; the concentrations were comparable to those in cultured cells. Quantitative analysis of intracellular AZT metabolites without the use of isotopes will increase safety and convenience of measurement, and take an effective step in studying pharmacokinetics of AZT in clinical materials.

Acquired Immunodeficiency Syndrome↗
PubMed1991

An A alpha Ser-434 to N-glycosylated Asn substitution in a dysfibrinogen, fibrinogen Caracas II, characterized by impaired fibrin gel formation.

We have identified a unique N-glycosylated Asn substitution for a Ser at position 434 of the A alpha chain of an abnormal fibrinogen designated fibrinogen Caracas II. This dysfibrinogen was characterized by impaired fibrin monomer aggregation. Since there were 4 Thr residues immediately following the mutation, a new Asn-X-Thr/Ser-type consensus sequence, Asn-Thr-Thr arose for N-glycosylation of the Asn. The extra oligosaccharide was found to consist mainly of a disialylated biantennary structure comprising 81.9%, while a neutral and a monosialylated biantennary oligosaccharide represented only 3.6% and 14.5%, respectively. The mutation resides in the carboxyl-terminal region of the A alpha chain, which could fold back to form an extra small globular region located near the central region of the molecule (Erickson, H.P., and Fowler, W.E. (1983) Ann. N. Y. Acad. Sci. 408, 146-163; Weisel, H.P., Stauffacher, C.V., Bullitt, E., and Cohen, C. (1985) Science 230, 3124-3133). Therefore, the participation of this region, referred to as an additional central domain or an alpha domain, in fibrin gel formation is strongly implicated.

Amino Acids↗
PubMed1991

Effect of L-arginine on the retention of macrophage tumoricidal activity.

It has been reported that the tumoricidal activity of macrophages (M phi) depends on L-arginine and that L-arginine metabolites such as reactive nitrogen intermediates alter M phi physical capacities. The aim of this report is to investigate the dose-related effect of L-arginine on the expression and retention of M phi tumoricidal activity. Cytotoxicity of M phi activated by IFN-gamma plus LPS was detected in the presence of about 0.1 mM or more of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. On the other hand, activated M phi were destined to die and lost their tumoricidal activity with time in the presence of 0.3 mM or more L-arginine. They retained, however, considerable activity in the absence or presence of 0.15 mM L-arginine. This retention of M phi cytotoxicity was longer when M phi were preactivated by 100 ng/ml than 10 ng/ml of LPS in combination with IFN-gamma. Addition of indomethacin, an inhibitor of prostaglandin production, did not prevent the decay of M phi cytotoxicity but rather facilitated it even in the absence of L-arginine. Regardless of indomethacin, consecutive stimulation with LPS or LPS plus IFN-gamma during culture was effective in maintaining the tumoricidal activity at a high level. In addition, we found that M phi which had lost tumoricidal activity during culture in L-arginine deficient medium could be reactivated by LPS to attack tumor target cells.

Animals↗
PubMed1991

Distinct mechanisms of neonatal tolerance induced by dendritic cells and thymic B cells.

To assess the role of different types of antigen-presenting cells (APC) in the induction of tolerance, we isolated B cells, macrophages, and dendritic cells from thymus and spleen, and injected these into neonatal BALB/c mice across an Mls-1 antigenic barrier. One week after injection of APC from Mls-1-incompatible mice or from control syngeneic mice, we measured the number of thymic, Mls-1a-reactive, V beta 6+ T cells and the capacity of thymocytes to induce a graft-vs.-host (GVH) reaction in popliteal lymph nodes of Mls-1a mice. Injection of thymic but not spleen B cells deleted thymic, Mls-1a-reactive V beta 6+ T cells and induced tolerance in the GVH assay. The thymic B cells were primarily of the CD5+ type, and fluorescence-activated cell sorter-purified CD5+ thymic B cells were active. Injection of dendritic cells from spleen or thymus also induced tolerance, but the V beta 6 cells were anergized rather than deleted. Macrophages from thymus did not induce tolerance. Dendritic cells and thymic B cells were also effective in inducing tolerance even when injected into Mls-, major histocompatibility complex-incompatible, I-E- mice, but only thymic B cells depleted V beta 6-expressing T cells. Therefore, different types of bone marrow-derived APC have different capacities for inducing tolerance, and the active cell types (dendritic cells and CD5+ thymic B cells) can act by distinct mechanisms.

Animals↗
PubMed1991

Systematic characterization of curved DNA segments randomly cloned from Escherichia coli and their functional significance.

In addition to the set of curved DNA segments isolated previously from Escherichia coli, another set of curved DNA segments has now been isolated. To gain an insight into the functional significance of these curved DNA sequences, systematic analyses were carried out, which included not only mapping of the precise locations of the segments on the E. coli chromosome but also clarification of the gene organization in the chromosomal regions surrounding the curved DNA sequences. It was demonstrated that most of the curved DNA sequences, which have been characterized so far, appear to be located immediately upstream of the coding sequences of adjacent genes. It was also demonstrated that an E. coli histone-like protein, named H-NS (or H1a), exhibits a strong affinity for naturally occurring curved DNA sequences in regions upstream promoters.

Bacterial Proteins↗
PubMed1991

Macrophage-activating factor extracted from mycoplasmas.

Mycoplasmas (M. gallisepticum, chicken mycoplasmas), in concert with interferon gamma (IFN gamma), were effective in activating macrophages (M theta) to be tumoricidal. The M theta-activating capacity of mycoplasmas was maintained after treatment with heat. 0.1 M NaOH, 1 M HCl, or trypsin. M theta-activating factor was extracted from mycoplasmas with chloroform/methanol and water (Mf-B). Mf-B was also effective in activating M theta in the presence of IFN gamma. The threshold dose of Mf-B for M theta of ordinary C3H/He mice and that for those of C3H/HeJ mice, the latter being known to be low responders to bacterial lipopolysaccharide, were actually the same. This seems to indicate that the effectiveness of Mf-B was not attributable to possibly contaminating lipopolysaccharides, and that the pathway of activity of Mf-B is different from that of lipopolysaccharides. Since the M theta-activating principle was only a very small part of Mf-B, we have not yet succeeded in identifying it, but there was no evidence that it was protein, nucleic acid, sugar, or lipid. The cytotoxicity of M theta activated by Mf-B plus IFN gamma was dependent on L-arginine in the culture, suggesting that arginine metabolites are involved in M theta cytotoxicity. Mf-B induced a small amount of tumor necrosis factor in M theta, and this induction was markedly enhanced by IFN gamma.

Animals↗
PubMed1991

Differential roles of the caudate nucleus and putamen in motor behavior of the cat as investigated by local injection of GABA antagonists.

In order to reveal a functional difference between the two distinctly separate nuclei of the striatum, i.e. the caudate nucleus (Cd) and the putamen (Put), we studied the effects of local bicuculline (BIC) and picrotoxin (PTX) injection into these nuclei on the motor behavior in the cat. The extent of diffusion of the injected BIC could be approximated by determining the extent of spreading of the dye fast green FCF (FCF) mixed with BIC solution since the extent of diffusion of radioactive [3H]BIC mixed with BIC and FCF solution was almost the same as that of FCF.BIC and PTX are GABA antagonists and are assumed to activate efferent neurons of the Cd and Put by removing the action of GABA-ergic inhibitory synapses on them. A total of 28 BIC and 22 PTX injections was made in 20 adult cats, 22 aimed at the Cd and 28 at the Put. Injection of BIC or PTX to either the head or body of the Cd unilaterally induced locomotor hyperactivity without any postural asymmetry or circling tendency. When BIC or PTX was injected into the Put, dystonic movements (dystonia) towards the contralateral side appeared frequently in the neck and trunk. Even though the injected BIC or PTX often spread to the external segment of the globus pallidus, claustrum, or anterior sylvian gyrus, none of these areas was consistently associated with dystonia. These results demonstrate that the Cd and Put are differentially associated with locomotor and postural functions.

Animals↗