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Biomedical subjects

M Nakamura

Publications and source records attributed to M Nakamura.

At least 19 recordsLinked to original sources
PubMed1992

Three types of Gi alpha protein of the guinea-pig lung: cDNA cloning and analysis of their tissue distribution.

cDNA clones encoding three types of Gi alpha, the alpha subunit of GTP-binding protein (Gi1 alpha, Gi2 alpha, and Gi3 alpha), were isolated from a cDNA library of the guinea-pig lung. Nucleotide sequence analysis revealed a high degree of homology with other mammalian Gi alpha cDNAs. By RNA blot analysis, the expression pattern of Gi1 alpha was more tissue-specific than those of other types of Gi alphas in the guinea-pig tissues examined. While Gi2 alpha and Gi3 alpha mRNAs were ubiquitously expressed in all tissues examined, Gi1 alpha mRNA was mainly expressed in the brain, lung and kidney. These results suggest that each Gi alpha protein may have a different role.

Amino Acid Sequence↗
PubMed1992

Endotoxin transduces Ca2+ signaling via platelet-activating factor receptor.

Lipopolysaccharide (LPS) is a pathogenic substance causing severe multiple organ failures and high mortality. Although several LPS binding proteins have been identified, the molecular mechanism underlying the LPS signaling pathway still remains obscure. We have found that the LPS-induced Ca2+ increase in platelets and platelet aggregation is blocked by selective platelet-activating factor (PAF) receptor antagonists, thus suggesting a cross-talk between LPS and the PAF receptor. Next, we confirmed this hypothesis using the cloned PAF receptors [(1991) Nature 349, 342-346; (1991) J. Biol. Chem. 266, 20400-20405] expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells. In both systems, cells responded to LPS only when PAF receptors were expressed, and specific PAF binding was successfully displaced and reversibly dissociated by LPS. PAF receptor activation by LPS may represent a novel important pathway in the pathogenesis of circulatory collapse and systemic thrombosis caused by endotoxin.

Animals↗
PubMed1992

Inhibition by 5-fluorouracil of cis-diamminedichloroplatinum(II)-induced DNA interstrand cross-link removal in a HST-1 human squamous carcinoma cell line.

To elucidate the mechanism of the synergistic cytotoxicity of 5-fluorouracil (5-FU) and cis-diamminedichloroplatinum(II) (CDDP), we studied the interaction of these agents using a human squamous carcinoma cell line (HST-1). Exposure to 5-FU for 24 h and to CDDP for 1 h produced a 50% inhibitory concentration of 1.0 micrograms/ml (7.7 microM) and 2.5 micrograms/ml (8.3 microM), respectively. The cytotoxic action of CDDP was augmented, and a greater than additive effect was observed when the cells were exposed to 5-FU (1.0 micrograms/ml; 7.7 microM) for 24 h before the CDDP treatment. This synergistic activity was maximal when the interval between 5-FU and CDDP exceeded 24 h. In contrast, the cytotoxicity of CDDP was attenuated when it preceded the exposure to 5-FU. Thymidine did not alter the 5-FU-CDDP interaction. Evaluation of the kinetics of the removal of DNA interstrand cross-links, measured by alkaline elution, showed a significant reduction of this removal in the cells exposed to 5-FU followed by CDDP with a drug-free interval of 48 h, as compared with cells exposed to CDDP alone, or to 5-FU immediately followed by CDDP, although no differences were found in the formation of DNA interstrand cross-links by CDDP among these cells. No significant differences in the accumulation of intracellular platinum were detected by atomic absorption spectrophotometry. These findings suggest that 5-FU modulates the repair of platinum-DNA adducts, thereby potentiating the antitumor activity of CDDP.

Carcinoma, Squamous Cell↗
PubMed1992

Total metabolic flow of glycosphingolipid biosynthesis is regulated by UDP-GlcNAc:lactosylceramide beta 1-->3N-acetylglucosaminyltransferase and CMP-NeuAc:lactosylceramide alpha 2-->3 sialyltransferase in human hematopoietic cell line HL-60 during differentiation.

We have previously reported that ganglioside GM3 was remarkably increased during monocytoid differentiation of human myelogenous leukemia cell line HL-60 cells and that neolacto series gangliosides (NeuAc-nLc) were enriched during granulocytoid differentiation. In addition, HL-60 was differentiated into monocytic lineage by exogenous GM3 and into granulocytoid by NeuAc-nLc. In the present report, the enzymatic bases of glycosphingolipid biosynthesis in HL-60 during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid were investigated. The following results were of particular interest. (i) Lactosylceramide alpha 2-->3 sialyltransferase (GM3 synthase) was remarkably up-regulated during monocyte differentiation, while the GM3 synthase level did not change in granulocytic differentiation. (ii) By contrast, lactosylceramide beta 1-->3N-acetylglucosaminyltransferase (Lc3Cer synthase) was down-regulated during monocytic differentiation, while the activity of Lc3Cer synthase was found to increase in granulocytic differentiation. (iii) The activities of four downstream glycosyltransferases (for synthesis of NeuAc-nLc) were found to increase or to remain unchanged during monocytic and granulocytic differentiation. These results strongly suggested the following. The dramatic GM3 increase and the decrease of NeuAc-nLc during monocytic differentiation are the consequences of the up-regulation of GM3 synthase and the down-regulation of Lc3Cer synthase, although the downstream enzymes are ready to catalyze their enzyme reactions. The notable increase of NeuAc-nLc and the relative decrease of GM3 during granulocytic differentiation are the results of the unchanged level of GM3 synthase and the up-regulation of Lc3Cer synthase together with the activation of the downstream glycosyltransferases. These results suggest that these two key upstream glycosyltransferases, GM3 synthase and Lc3Cer synthase, play critical roles in regulating the glycosphingolipid biosynthesis in HL-60 cells during differentiation. This switching mechanism of these two glycosyltransferases, together with our previous findings, might be one of the most important parts of the determining system of differentiation direction in human myeloid cells into monocytic or granulocytic lineages.

Antibodies, Monoclonal↗
PubMed1992

Cloning and sequencing of a rat type II activin receptor.

A full-length cDNA for a rat type II activin receptor was cloned by hybridization from a rat ovary cDNA library. The deduced amino acid sequence (513 residues) containing a single membrane-spanning domain and an intracellular kinase domain with predicted serine/threonine specificity. The amino acid sequence is 99.8% and 99.4% identical in the coding region with the previously cloned mouse and human type II activin receptor, and only 66.7% identical in the coding region with the previously cloned rat type IIB activin receptor. We examined the effect of PMSG-hCG on the mRNA level of type II activin receptor in immature rat ovaries. Northern blot analysis of ovarian RNA revealed two mRNAs (3.0 kb and 6.0 kb).

Activin Receptors↗
PubMed1992

Dynamic analysis of efficient heme rotation in myoglobin by NMR spectroscopy.

Sperm whale myoglobin was reconstituted with 1,4,5,8-tetramethylhemin. The hyperfine-shifted proton NMR signals from the prosthetic group exhibit remarkable pattern changes around 15 degrees C, while the globin resonances are normal to obey the Curie law. The NMR anomaly specifically observed for the heme signals suggests a slow to rapid rotational transition of the hemin about the iron-histidine bond. The temperature-dependent pattern changes were quantitatively analyzed by a dynamic NMR method. Two sets of analyses with the heme-methyl and pyrrole-proton lines consistently afforded delta H not equal to = 16.3 kcal/mol, delta S not equal to = 14.0 e.u., delta G not equal to = 12.1 kcal/mol at 298 K, and a frequency of 90 degrees heme rotation 5600 s-1 at 20 degrees C. The relatively large activation entropy suggests that structural rearrangements at the direct heme vicinity are involved and that efficient heme rotation is accomplished by a number of fluctuative local heme-globin contacts within a conserved crevice structure.

Animals↗
PubMed1992

Cell type-specific tyrosine phosphorylation of IL-2 receptor beta chain in response to IL-2.

Functional activities of the IL-2 receptor (IL-2R) beta chain exogenously expressed on lymphoid and non-lymphoid cells were examined in terms of phosphorylation of IL-2R beta and cell growth. Lymphoid MOLT-4 and its transfectants expressing IL-2R beta either alone or with IL-2R alpha chain were found to be rapidly phosphorylated predominantly at tyrosine residues of IL-2R beta and to be affected in their growth in an IL-2-dependent manner. In contrast, IL-2 induced neither phosphorylation of IL-2R beta nor cell growth in non-lymphoid transfectants derived from COS7, HeLa and L929, even though they acquired the IL-2 binding ability when coexpressed as IL-2R beta and IL-2R alpha. These results suggest that IL-2 induces activation of a tyrosine kinase possibly associated with IL-2R beta in a cell type-specific manner.

Cell Division↗
PubMed1992

The role of the C-terminal region of rat brain natriuretic peptide in receptor selectivity.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have different C-terminal tail structures compared with the rather conservative ring structures which consist of 17 amino acid residues. To examine the different effects of the tail structures of ANP and BNP on their interaction with receptors, we synthesized several peptide analogs and measured their biological actions in three different assay systems. Deletion of the C-terminal tail from rat BNP did not effect the vasorelaxation activity against rat aorta, but it promoted cGMP production in cultured rat aortic smooth muscle cells (RASMC). Deletion of the C-terminal tail from rat ANP diminished both vasorelaxant and cGMP producing activities. In a binding competition assay with RASMC and [125I]rat ANP-(1-28), the competition activities of both ANP and BNP were greatly reduced by C-terminal deletion. In addition, we obtained agonists with novel receptor selectivity.

Animals↗
PubMed1992

Identification of an amino acid residue involved in the substrate-binding site of rat liver uricase by site-directed mutagenesis.

Computer analysis has shown that a conserved amino acid sequence (Leu 160 to Lys 164) of rat liver uricase is also present in other enzymes with purine substrates. The significances of the amino acids in this sequence were studied by site-directed mutagenesis. Replacement of Lys 164 by Glu or Ile resulted in loss of uricase activity and decrease in binding of the competitive inhibitor xanthine. The far ultraviolet circular dichroic spectra of the mutant uricases were identical to that of the wild type protein, indicating that the replacement of Lys 164 by other amino acids did not result in serious modification of the conformation of uricase. These findings suggest that this amino acid is involved in the substrate-binding site of the enzyme.

Amino Acid Sequence↗
PubMed1992

Novel variant transthyretin gene (Ser50 to Ile) in familial cardiac amyloidosis.

We detected a point mutation in the transthyretin (TTR) gene in a patient with familial cardiac amyloidosis by using PCR-DCP (DNA conformation polymorphism) analysis that is based on the diversity in electrophoretic mobility of single-stranded DNAs and/or heteroduplex DNAs in PCR products. The PCR products of the transthyretin gene were denatured in the presence of formamide and electrophoresed in a non-denaturing polyacrylamide gel to detect an electrophoretic change due to a sequence variation. An unusual DNA fragment was visualized by silver staining in the PCR products of the exon 3 from the patient. Subsequent sequencing analysis revealed a T to A transversion and led to a replacement of Ser by Ile at codon 50 of the TTR gene.

Amyloidosis↗
PubMed1992

Secretion of autocrine growth-promoting activity by renal-carcinoma cells treated with 5-fluorouracil.

A study was made of the auto-proliferative activity of human renal-carcinoma cells in the supernatant from a carcinoma-cell culture in serum-free medium to which an anticancer agent had been added (5-FU). The human renal-cancer cells used in this study were of 3 strains: ACHN, VMRC-RCW and NT. When each line was cultured in medium containing no 5-FU, the supernatant showed almost no activity for stimulating DNA synthesis. However, when the line was cultured in the presence of 5-FU, the supernatant showed autocrine growth-promoting activity which strongly stimulated DNA synthesis of 3 renal-cancer cell lines in a dose-dependent manner. Activity could be detected in a 4- to 6-kDa fraction by gel filtration. This fraction increased the DNA-synthesis-promoting activity of epidermal growth factor, transforming growth factor-beta, basic fibroblast growth factor and insulin, and was acid- and heat-stable. It was also stable against pepsin and dithiothreitol. DNA synthesis in BALB/c 3T3, adult rat hepatocytes and rabbit renal tubular cells was not affected by this fraction which was thus considered not to affect non-cancerous cells. Renal-cell carcinoma responds poorly to anticancer agents, and the autocrine activity of the fraction may possibly be a factor accounting for this resistance.

3T3 Cells↗
PubMed1992

Molecular cloning and characterization of the inositol 1,4,5-trisphosphate receptor in Drosophila melanogaster.

We isolated a cDNA encoding an inositol 1,4,5-trisphosphate receptor (InsP3R) of Drosophila melanogaster. The predicted Drosophila InsP3R (2,833 amino acids) has extensive sequence similarity to the mouse InsP3R. The polypeptide encoded by the cDNA was functionally expressed and showed characteristic InsP3-binding activity. The Drosophila InsP3R gene is located at the region 83A5-9 on the third chromosome and expresses throughout development but predominantly in the adult. Localization of the InsP3R mRNA in adult tissues suggests strong expression in the retina and antenna, indicating the involvement of the InsP3R in visual and olfactory transduction. In addition, the InsP3R mRNA is abundant in the legs and thorax, which are enriched with a muscular system. Such localization is apparently consistent with the quantitatively predominant sites for [3H]InsP3 binding in Drosophila and the fleshfly (Boettcherisca peregrina). The present study points to the likely functional importance of the InsP3/Ca2+ signaling system in Drosophila.

Amino Acid Sequence↗
PubMed1992

Reduction of alpha-tocopherolquinone to alpha-tocopherolhydroquinone in rat hepatocytes.

The contents of alpha-tocopherolhydroquinone (TQH2), alpha-tocopherolquinone (TQ) and alpha-tocopherol (Toc) in isolated rat hepatocytes and liver homogenates were determined by HPLC under anaerobic conditions, because TQH2 easily autoxidizes to TQ under aerobic conditions. The viable hepatocytes were used for the determination without homogenization. The hepatocytes contained 3.1, ND and 5.0, 3.1-9.0, and 31.3-63.2 nmol of TQH2, TQ and Toc/g liver, respectively. However, TQH2 was not detected in liver homogenates because endogeneous TQH2 autoxidizes to TQ during preparation of homogenates under aerobic conditions. The homogenates contained 2.0-23.5 and 36.5-54.9 nmol of TQ and Toc/g liver, respectively. Addition of TQ showed that TQ was reduced and converted into TQH2 in isolated hepatocytes. The TQH2 formation from TQ was also observed in liver homogenates in the presence of either NADPH or NADH. The formation was further analysed and confirmed by HPLC and mass spectrometry. The formation of TQH2 was also found to occur in mitochondria, microsomes and cytosol. The specific activity of NADPH-dependent TQ reductase activity was in the order of mitochondria greater than or equal to microsomes greater than cytosol. Furthermore, NADPH-cytochrome P450 reductase was found to catalyse TQH2 formation from TQ.

Animals↗
PubMed1992

Identification of new epitopes recognized by human monoclonal antibodies with neutralizing and antibody-dependent cellular cytotoxicity activities specific for human T cell leukemia virus type 1.

We have generated a number of EBV-transformed B cell lines producing human mAb against human T cell leukemia virus type 1 (HTLV-1) from the peripheral blood B lymphocytes obtained from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis. Various synthetic peptides corresponding to antigenic regions of HTLV-1 gag and env proteins were used for the screening of antibodies in ELISA. In our study, four IgG mAb to the gag p19 amino acids 100 to 130, and 5 IgG mAb to the env p46 amino acids 175 to 199 were characterized. An immunofluorescence assay showed that all of these mAb specifically bound to the surface of HTLV-1-bearing cell lines. Among these mAb, one anti-gp46 mAb, designated KE36-11, neutralized the infectivity of HTLV-1 as determined by both the inhibition of HTLV-1-induced syncytium formation and transformation assays in vitro. An antibody-binding assay using overlapping oligopeptides revealed that KE36-11 recognized a new epitope locating between the gp46 amino acid sequence 187-193 (Ala-Pro-Pro-Leu-Leu-Pro-His). Another anti-gp46 mAb, designated KE36-7, showed antibody-dependent cellular cytotoxicity against HTLV-1-bearing cell line. KE36-7 bound strongly to the 10-mer peptide-gp46 187-196, and weakly to peptides containing the gp46 amino acid sequence 191-196 (Leu-Pro-His-Ser-Asn-Leu). These two epitopes, which are associated with HTLV-1 neutralization and antibody-dependent cellular cytotoxicity, are thus the first epitopes identified in human HTLV-1 infection. It is possible that passive immunization of humans with these two human mAb are effective on the protection of HTLV-1 infection in vivo.

Amino Acid Sequence↗
PubMed1992

Calreticulin is present in the acrosome of spermatids of rat testis.

We have already succeeded in purifying a calcium-binding protein (CalBP) from rat spermatogenic cells [Nakamura et al., Biochem. Biophys. Res. Commun., 176 (1991) 1358]. In this study, the location of this protein within rat testis was examined, using a rabbit antisera for this protein. The antigen was localized on the developing acrosomes during spermiogenesis. The NH2-terminal amino acid sequence obtained for rat CalBP was identical to that of calreticulin obtained for the skeletal muscle of mice and closely resembled that for rabbit calreticulin. On the immunoblot analysis, the purified rat CalBP reacted with an antibody raised against rabbit skeletal muscle calreticulin. The results indicate that calreticulin is present in the acrosome of spermatids of rat testes.

Acrosome↗
PubMed1992

Antisera to calreticulin inhibits sperm motility in mice.

Mouse sperm were rapidly immobilized when exposed to rabbit antisera against rat calreticulin. The inhibition of sperm motility was concentration dependent at dilutions 1:50-350. The velocity of sperm did not change significantly as long as they were motile. Neither motility nor velocity of sperm was affected by adding sheep antisera to bovine calmodulin. The antisera to calreticulin also inhibited in vitro fertilization of mouse eggs. Using the avidin-biotin-peroxidase complex method, the antigen was found to be localized in the acrosome of sperm. The results indicate that calreticulin is present in the acrosome of mouse sperm and may play an important role in sperm motility.

Animals↗