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Biomedical subjects

K Takahashi

Publications and source records attributed to K Takahashi.

At least 19 recordsLinked to original sources
PubMed1992

Acquisition of interleukin-3 independence in FDC-P2 cells after transfection with the activated c-H-ras gene using a bovine papillomavirus-based plasmid vector.

Since the ras family of proto-oncogenes is supposed to be involved in leukemogenesis by point-mutational activation, we studied the effect of the activated ras gene on the growth of a murine interleukin-3 (IL-3)-dependent cell line, FDC-P2. The human activated c-H-ras gene was transfected into FDC-P2 cells by electroporation using a high-level expression vector, BMGhph, which contains a partial DNA sequence from bovine papillomavirus (BPV) and a hygromycin B (hmB)-resistant gene as a selectable marker. The transformed FDC-P2 cells showed a high incidence of IL-3-independent growth and tumorigenicity in nude mice. These clones did not express or secrete IL-3, suggesting the acquisition of IL-3 independence by a nonautocrine mechanism. The high incidence of autonomous growth may be due to the use of the BMG vector, because (1) the activated ras gene in pBR322 vector (pHs-49) was not so efficient in the induction of IL-3 independence, (2) the c-H-ras genome copies per cell increased in number up to about 50 copies by using the BMG vector, and (3) cotransfection with the activated ras gene and the BPV gene in separate plasmids partly enhanced the incidence of autonomous growth without increasing the copy number of the ras gene compared with transfection with the activated ras gene alone. The present study supports the idea that the activation of ras gene is an important step in malignant transformation of hematopoietic cells and suggests that the BPV gene products may cooperate with ras gene activation probably by affecting the cellular genes that may be involved in multistep leukemogenesis. The BMG vector may be useful to test the transforming ability of oncogenes whose oncogenic potential is relatively low.

Animals↗
PubMed1992

Effects of Ca2+ antagonists and antiepileptics on tetrodotoxin-sensitive Ca(2+)-conducting channels in isolated rat hippocampal CA1 neurons.

All Ca2+ antagonists blocked tetrodotoxin-sensitive Ca2+ current (TTX-ICa) more potently than Na+ current (INa). Phenytoin and MK-801, at concentrations which had no effect on INa, could block TTX-ICa concentration-dependently. Valproic acid and phenobarbital had no effect on both TTX-ICa and INa. In particular, flunarizine and phenytoin have more potent inhibitory effects on TTX-ICa than other test drugs. These results suggest that the abnormal excess-excitation of TTX-sensitive Ca(2+)-conducting channels may be one of the trigger factors generating epilepsy.

Animals↗
PubMed1992

A protein kinase C inhibitor, staurosporine, activates phospholipase D via a pertussis toxin-sensitive GTP-binding protein in rabbit peritoneal neutrophils.

In rabbit peritoneal neutrophils prelabeled with [3H] lyso platelet-activating factor, a protein kinase C inhibitor, staurosporine (> 1 microM), increased [3H]phosphatidylethanol ([3H]PEt) level in the presence of ethanol in a concentration- and time-dependent manner, providing evidence for staurosporine activation of phospholipase D (PLD). The staurosporine activation of the enzyme absolutely required both extracellular calcium and cytochalasin B, and was almost completely inhibited by pretreatment of the cells with pertussis toxin (IAP). In a reconstituted system where the purified Gi1 had been incorporated into phospholipid vesicles, staurosporine activated GTPase activity of Gi1 in a concentration-dependent fashion, with a maximal 4-5-fold effect. ADP-ribosylation by IAP of Gi1 in vesicles significantly suppressed the staurosporine activation. As with the GTPase activity of Gi1, GTPase activities of other purified IAP-sensitive G proteins, such as Gi2 and G(o), were significantly stimulated by staurosporine, but the cholera toxin substrate Gs was appreciably less sensitive to the staurosporine stimulation. The staurosporine activation of GTPase was also observed in rabbit neutrophil membranes from control cells, but not in membranes from IAP-treated neutrophils. From these results, we conclude that the staurosporine activation of PLD in rabbit neutrophils is attributed to the direct activation of an IAP-sensitive G protein in a similar manner to receptors occupied by agonists. By contrast, staurosporine failed to activate phosphoinositide-specific phospholipase C (PI-PLC) under the conditions in which it activated PLD, indicating that there exists a PLD activation pathway independent of PI-PLC. Furthermore, it was found that N-acetyl-beta-glucosaminidase release from the granules of intact neutrophils was evoked by staurosporine to almost the same extent as by fMLP (100 nM), but O2- generation was not affected. These results suggest a possibility that PLD pathway plays an important role in enzyme release, but is not sufficient for O2- generation, in rabbit peritoneal neutrophils.

Acetylglucosaminidase↗
PubMed1992

Woodfruticosin (woodfordin C), a new inhibitor of DNA topoisomerase II. Experimental antitumor activity.

Woodfruticosin (woodfordin C) (WFC), a new inhibitor of DNA topoisomerase II (topo-II), was isolated from methanol extract of Woodfordia fruticosa Kurz (Lythraceae) and studied for in vitro and in vivo antitumor activities in comparison with Adriamycin (ADR) and etoposide (ETP), well known inhibitors of topo-II. The inhibitory activity against DNA topo-II shown by WFC was much stronger than that shown by ETP or ADR. WFC inhibited strongly intracellular DNA synthesis but not RNA and protein synthesis. On the other hand, WFC had a weaker growth inhibitory activity against various human tumor cells than ETP or ADR, but it showed remarkable activity against PC-1 cells and moderate activity against MKN45 and KB cells. Furthermore, WFC had in vivo growth inhibitory activity against s.c. inoculated colon38. These results indicate that the mechanism by which WFC exhibits antitumor activity may be through inhibition of topo-II.

Animals↗
PubMed1992

The active site titration of proteinases by using alpha 2-macroglobulin and high-performance liquid chromatography.

The active site titration for various proteinases relies on the development of optimal enzyme titrants for each proteinase, but these titrants are only available for a limited number of proteinases. We have described a new active site titration method applicable to various kinds of endoproteinases using small quantities of the enzymes. This method was carried out by using alpha 2-macroglobulin (alpha 2M) as a titrant and a high-performance liquid chromatography (HPLC) system. When the proteinase solution was treated with alpha 2M, the active proteinase was trapped by alpha 2M. In this reaction alpha 2M does not usually complex with inactive proteinase. After the reaction of proteinase with an excess of alpha 2M, the reaction mixture is applied to an HPLC gel column to separate the uncomplexed enzyme from the one complexed with alpha 2M. The active proteinase is complexed and eluted with alpha 2M, but the inactive proteinase is eluted at the original elution volume. The same amount of the enzyme was also applied to the column. From the decrease of the peak height at the elution position of the uncomplexed proteinase, we can estimate the ratio between enzymatically active proteinases and total proteinases. To test the usefulness of this method, we applied this method to chymotrypsin and trypsin whose activities were predetermined by conventional active site titration, and there was good agreement between both results. With this new method, we can estimate a proteinase activity with as little as 200 ng of the enzyme, a very small amount compared with those required in conventional methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1992

Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid chromatography after derivatization with N-tert.-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde.

The concurrent determination of free amino acid enantiomers and non-chiral amino acids in rat brain and serum was accomplished by high-performance liquid chromatography with fluorimetric detection after derivatization with N-tert.-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde. The method revealed the presence of a large amount of free D-serine (0.22 mumol/g of tissue; D/D + L ratio = 0.25) in the brain whereas D-aspartate and D-alanine were established to be at trace levels. These results further support the presence of D-serine in adult brain tissues as demonstrated by recent work using gas chromatography.

Amino Acids↗
PubMed1992

Insulin inhibits norepinephrine overflow from peripheral sympathetic nerve ending.

The effects of insulin on peripheral nervous system are unknown. We therefore studied the effects of insulin on sympathetic nerve activity in isolated mesenteric arteries of Sprague-Dawley rats. The overflow of norepinephrine (NE) by electrical stimulation was used as the index of sympathetic nervous system activity. Insulin (0.5 to 1U/l) decreased the NE release in a dose-dependent fashion. This inhibitory effect was, however, reversed by either 5 x 10(-5)M cocaine or 5 x 10(-4)M ouabain treatment. Thus, we postulate that insulin attenuates NE overflow from peripheral sympathetic nerve endings, probably due to enhanced NE reuptake.

Animals↗
PubMed1992

Detection of bcl-2 protein and bcl-2 messenger RNA in normal and neoplastic lymphoid tissues by immunohistochemistry and in situ hybridization.

bcl-2 protein has been detected in surgical specimens and cultured permanent cell lines of non-Hodgkin's lymphomas and leukemias using enzyme immunohistochemistry and immunofluorescence with anti-bcl-2 monoclonal antibodies. Of 40 surgical specimens, bcl-2 protein was expressed in 50% of B-cell and 41% of T-cell lymphomas, both with and without the bcl-2 gene rearrangement. In investigations of 38 hematopoietic cell lines, bcl-2 protein was detected not only in lymphoid cell lines but also in myeloid cell lines. In situ hybridization and immunohistochemical analysis of reactive lymph nodes showed that lymphocytes in mantle zones and paracortical areas expressed bcl-2 protein consistent with the messenger RNA distribution and that germinal center cells showed abundant bcl-2 transcript, despite the absence of detectable bcl-2 protein. These results suggest that bcl-2 protein is broadly expressed in various hematopoietic neoplasms not restricted in t(14; 18) lymphomas and that germinal center cells may be involved in some arrest of bcl-2 protein expression at the posttranscriptional level.

Blotting, Northern↗
PubMed1992

MY-1250, a major metabolite of the anti-allergic drug repirinast, induces phosphorylation of a 78-kDa protein in rat mast cells.

Repirinast (MY-5116; isoamyl 5,6-dihydro-7,8-dimethyl-4,5-dioxo-4H-pyrano [3,2-c]quinoline-2-carboxylate) is an anti-allergic drug of demonstrated effectiveness for treating bronchial asthma in humans. MY-1250 (5,6-dihydro-7,8-dimethyl-4,5-dioxo-4H-pyrano [3,2-c]quinoline-2-carboxylic acid), the major active metabolite of repirinast, inhibits antigen-induced histamine release from sensitized rat peritoneal exudate cells (PEC). When purified rat mast cells were treated with MY-1250 (2.5 x 10(-5) M) for 1 min, phosphorylation of a specific mast cell protein of apparent molecular mass of 78 kDa was observed as previously reported for sodium cromoglycate (SCG). Phosphorylation of this protein induced by MY-1250 and SCG occurred in a concentration-dependent manner with IC50 values of 2.0 x 10(-7) and 1.4 x 10(-5) M, respectively. MY-1250 did not inhibit calcium ionophore A23187 (1 microgram/mL)-induced histamine release from rat PEC. In the presence of calcium ionophore A23187 (1 microgram/mL), phosphorylation of this protein induced by MY-1250 was not evident. In conclusion, MY-1250 induced phosphorylation of a 78-kDa protein in rat mast cells and MY-1250 may inhibit histamine release by regulating phosphorylation of this protein in rat mast cells.

Animals↗
PubMed1992

Molecular cloning of a cDNA coding for neurofibromatosis type 1 protein isoform lacking the domain related to ras GTPase-activating protein.

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder, and a gene linked to NF1 was recently identified. Its gene product (NF1 protein) contains a domain functionally related to mammalian ras GTPase-activating protein (GAP). Here, we cloned a cDNA coding for NF1 protein isoform lacking the region related to GAP from a oligo(dT)-primed cDNA library of human placenta. This cDNA carries the insert of about 2.4 kb, coding for a protein of 551 amino acid residues, which shares the same aminoterminal 547 residues with authentic NF1 protein. We show that NF1 mRNAs of about 2.9, 11, and 13 kb are expressed in human tissues, and that the isolated cDNA may represent the 2.9-kb transcript.

Amino Acid Sequence↗
PubMed1992

Inhibitory effect of activated protein C on platelet aggregation induced by the prothrombin-converting reaction.

The present study was undertaken to elucidate the effect on platelet aggregation of the prothrombin-converting reaction on platelets with or without activated protein C (APC). A reaction mixture of washed platelets from human individuals, Factor Xa and prothrombin markedly induced platelet aggregation; maximum aggregation rates, 31.3-92.5%, and times to reach to maximum aggregation, 11.6 to 20.1 min. This aggregation was inhibited by the addition of APC with 50% inhibition concentration (IC50) value of 14.4 U/ml. APC also inhibited thrombin generation in the reaction mixture in a dose-dependent manner with IC50 value of 0.96 U/ml. However, APC did not inhibit the thrombin (0.1 CU/ml)-induced platelet aggregation at concentrations of up to 30 U/ml. These findings suggest that APC has no direct inhibitory effect on platelet aggregation and that APC inhibits platelet aggregation through inhibition of thrombin generation.

Blood Platelets↗
PubMed1992

[A descriptive epidemiology on sudden death among workers].

A survey on sudden unexpected death, that is, death within 24 hours from acute onset, among Japanese workers was carried out. Questionnaires were sent to full-time occupational health physicians who were active members of the Japan Association of Industrial Health at the time of the survey. Though 241 doctors answered (response rate: 61.5%), only 53 of them who had had both sudden death cases and the desire to join in this research co-operated in this survey. During the 5 years of observation (from Jan. 1, 1986 to Dec. 31, 1990), 143 cases of sudden death, 141 males and 2 females, were reported. The mean age was 46.5 (S. D. 10.5) for males. The ages of the 2 females were 31 and 44. Onset took place most frequently at home (55%) and most often during sleep (26%). This may be due to the higher rate not only of onset but of death resulting from acute onset at home and during sleep. Similar to the previous findings, a small peak of incidence was found early in the morning (4:00 AM--9:00 AM), although this was not statistically significant. However, the weekly and seasonal variations were a little different from previous reports. In this study, sudden death tended to occur more on Monday, Thursday, Friday, and Saturday (not statistically significant), and as for the seasonal variation, more cases occurred in April, November, and December which were statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Detection of lipopolysaccharide (LPS) and identification of its serotype by an enzyme-linked immunosorbent assay (ELISA) using poly-L-lysine.

A new solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for detection of LPS and identification of its serotype with antisera. Since LPS binds poorly to polystyrene microplates, precoating with poly-L-lysine was used before coating LPS on the surface of microplates. The small amount of LPS in complex mixtures (i.e., less than 1 microgram/ml) could be detectable in ELISA. Use of poly-L-lysine with high molecular weight (MW) provided a higher sensitivity than poly-L-lysine with low MW. Precoating with polymyxin B, or poly-L-histidine was less effective in the sensitivity than precoating with poly-L-lysine, but it was still better than no precoating. The newly developed ELISA technique could be also applied for detection of anti-LPS antibodies in sera or for screening of monoclonal anti-LPS antibody.

Animals↗
PubMed1992

Gastric procathepsin E and progastricsin from guinea pig. Purification, molecular cloning of cDNAs, and characterization of enzymatic properties, with special reference to procathepsin E.

Procathepsin E and progastricsin were purified from the gastric mucosa of the guinea pig. They were converted to the active form autocatalytically under acidic conditions. Each active form hydrolyzed protein substrates maximally at around pH 2.5. Pepstatin inhibited cathepsin E very strongly at an equimolar concentration, whereas the inhibition was much weaker for gastricsin. Molecular cloning of the respective cDNAs permitted us to deduce the complete amino acid sequences of their pre-proforms; preprocathepsin E and preprogastricsin consisted of 391 and 394 residues, respectively. Procathepsin E has unique structural and enzymatic features among the aspartic proteinases. Lys at position 37, which is common to various aspartic proteinases and is thought to be important for stabilizing the activation segment, was absent at the corresponding position, as in human procathepsin E. The rate of activation of procathepsin E to cathepsin E is maximal at around pH 4.0. It is very different from the pepsinogens and may be correlated with the absence of Lys37. Native procathepsin E is a dimer, consisting of two monomers covalently bound by a disulfide bridge between 2 Cys37. Interconversion between the dimer and the monomer was reversible and regulated by low concentrations of a reducing reagent. Although the properties of the dimeric and monomeric cathepsins E are quite similar, a marked difference was found between them in terms of their stability in weakly alkaline solution: monomeric cathepsin E was unstable at weakly alkaline pH whereas the dimeric form was stable. The generation of the monomer was thought to be the process leading to inactivation, hence degradation of cathepsin E in vivo.

Amino Acid Sequence↗
PubMed1992

The primary structure of the alpha subunit of a starfish guanosine-nucleotide-binding regulatory protein involved in 1-methyladenine-induced oocyte maturation.

Starfish-oocyte maturation induced by 1-methyladenine (MeAde) was inhibited by microinjection of pertussis toxin (PTX). The inhibition appeared to result from PTX-catalyzed ADP-ribosylation of a 39-kDa guanosine-nucleotide-binding regulatory protein (G protein) in the oocyte. These results strongly support the hypothesis that the MeAde-induced signals operate via a membrane receptor and are carried by the PTX-sensitive G protein. When PTX-injected oocytes were treated with dithiothreitol, 85% of them reinitiated meiosis, suggesting that dithiothreitol did not act on the MeAde receptor. We constructed a cDNA library from the immature ovary of starfish, Asterina pectinifera, and screened it with the cDNA of the alpha subunit of an inhibitory rat G protein (Gi-2). A positive cDNA clone contained an open reading frame of 1062 bases which had 74% identity with the rat Gi-2 cDNA. The deduced amino acid sequence was 85% and 89% identical to rat Gi-2 and rat Gi-1, respectively. The alpha subunit of the G protein purified from cortices of starfish oocytes was digested by trypsin and the resulting four peptides were microsequenced. Comparison of these amino acid sequences with the predicted one indicated that the isolated cDNA clone encoded the alpha subunit of the PTX-sensitive G protein in oocytes. The C-terminal sequence, KNNLKDCGLF, was identical to that of Gi, suggesting that the cysteine residue is the site of ADP-ribosylation.

Adenine↗
PubMed1992

[Analysis on combined effect of X-rays with 5-FU on rat subcutaneous gliomas].

In a series of experiments on the combined use of radio- and chemotherapy for malignant glioma, X-rays combined with ACNU or 5-FU treatment caused a supra-additive effect on multicellular spheroids in vitro. In the present experiment, the effect of X-rays combined with 5-FU treatment on subcutaneously transplanted rat gliomas of RGC-6 cells was analyzed. The dose-survival curve for X-rays given to tumors in air-breathing rats was biphasic with a terminal slope (D0 = 4.3 Gy) that was parallel to that for tumors in previously killed rats. The hypoxic cell fraction thus obtained from the ratio of the surviving fraction in two parallel curves at 20 Gy was about 7% in the subcutaneous tumors in air-breathing rats. X-ray-induced, potentially lethal cellular damage recovered within 8 hours in these tumors. The surviving fraction of cells in the tumors decreased to a minimum at 4-6 hours after a 5-FU injection, but increased thereafter. A biphasic dose-response curve for 5-FU was also obtained for cells in these tumors, indicating the presence of 5-FU resistant cells. The effect of X-irradiation given at about 8 hours after a 5-FU injection was greater than the additive effect of both agents acting independently. This was true when an X-ray dose of more than 5 Gy was given.

Animals↗
PubMed1992

Properties of 1-methyladenine receptors in starfish oocyte membranes: involvement of pertussis toxin-sensitive GTP-binding protein in the receptor-mediated signal transduction.

In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown. The 1-MA-initiated signal is, however, inhibited by prior microinjection of pertussis toxin into the oocytes, suggesting that a guanine nucleotide-binding protein (G protein) serving as the substrate of pertussis toxin is involved in the 1-MA receptor-mediated signal. We thus investigated properties of 1-MA receptors by means of binding of the radiolabeled ligand to the oocyte membranes. There were apparently two forms of 1-MA receptors with high and low affinities in the membranes. The high-affinity form was converted into the low-affinity one in the presence of a non-hydrolyzable analogue of GTP. A 39-kDa protein, which had been identified as the alpha-subunit of the major substrate G protein for pertussis toxin, was also ADP-ribosylated by cholera toxin only when 1-MA was added to the membranes. The ADP-ribosylated 39-kDa alpha-subunit could be immunoprecipitated with antibodies raised against the carboxy-terminal site of mammalian inhibitory G-alpha. These results indicate that 1-MA receptors are functionally coupled with the 39-kDa pertussis toxin-substrate G protein in starfish oocyte membranes.

Adenine↗