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Biomedical subjects

M Hidaka

Publications and source records attributed to M Hidaka.

At least 91 records · Page 5Linked to original sources
PubMed1993

Phospholipase C-gamma 1 associates with viral and cellular src kinases.

A tyrosine kinase inhibitor, genistein, caused the subcellular translocation of phosphoinositide-specific phospholipase C (PLC) activity from membrane fractions to cytosolic fractions in rat 3Y1 fibroblasts and their transformants by Rous sarcoma virus, SR-3Y1. The ratio of PLC activities associated with the membrane fractions to those of the homogenate fractions was greater in SR-3Y1 (32.6%) than in 3Y1 (20.8%) whereas membrane-associated PLC activities were strikingly reduced to the same levels in both cells by treatment with genistein. Moreover, it was found by immunoblotting analyses of membrane fractions that the amounts of PLC-gamma 1 isozyme were reduced to 20.4% of initial level in SR-3Y1 and to 30.2% of that in 3Y1 cells. While the levels of PLC-delta, another detectable PLC isozyme, were not altered by genistein suggesting that tyrosine kinase plays an important role in the association of PLC-gamma 1 with membranes. PLC-gamma 1 molecules were detected in anti-p60arc antibody immunoprecipitates of both 3Y1 and SR-3Y1 cells. The amounts of PLC-gamma 1 co-immunoprecipitating with src kinases were higher in SR-3Y1 than 3Y1 cells and were reduced in both cell types by treatment with genistein. In addition, it was confirmed that PLC-gamma 1 purified from rat liver was phosphorylated at a tyrosine residue and associated with viral src kinase and that src kinases associated with the recombinant SH2 region of PLC-gamma 1, expressed in Escherichia coli, depending upon phosphorylation of tyrosine residues. These findings suggest that both viral and cellular src kinases associate with PLC-gamma 1 and may mediate cellular signaling in normal and transformant cell growth.

Animals↗
PubMed1993

Impaired glycosylation of glycosylphosphatidylinositol-anchor synthesis in paroxysmal nocturnal hemoglobinuria leucocytes.

Metabolic labeling with [3H]sugars in vivo or [3H]sugar nucleotides in vitro of glycosylphosphatidylinositol (GPI)-anchor precursors in peripheral blood granulocytes and cultured T lymphocytes of paroxysmal nocturnal hemoglobinuria (PNH) patients showed a synthetic defect in the GPI-anchor. Among the GPI-anchor precursors, phosphatidylinositol (PI) was normally synthesized, while the synthesis of glucosaminylphosphatidylinositol (GlcN-PI) and subsequent mannosylation of GlcN-PI were inhibited in affected cells. The defect in the GPI-anchor synthesis in PNH is thus attributed to interrupted glycosylation at plural sites in the synthesis of the common carbohydrate structure of the anchor.

Carbohydrate Sequence↗
PubMed1993

An inhibitor of inositol-phospholipid-specific phospholipase C.

Q12713 substance, a cyclic peptide from Actinomadura spp., strongly inhibited the enzyme activity of phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) but scarcely inhibited other phospholipases. Kinetic analysis demonstrated that the inhibition of PIP2-PLC activity was competitive with respect to phosphatidylinositol 4,5-bisphosphate. Calcium and magnesium ions had no significant effect on the inhibitory activity. On the contrary, potassium or rubidium ion was essential for the inhibitory activity. Furthermore, NaF and AlCl3-stimulated increase of phosphoinositides was decreased by Q12713 in the cultured 3T3 cells.

3T3 Cells↗
PubMed1993

Expression of decay-accelerating factor and CD59 in lymphocyte subsets of healthy individuals and paroxysmal nocturnal hemoglobinuria patients.

The expression of phosphatidylinositol (PI)-anchored complement-regulatory membrane proteins on circulating blood cells has been well clarified; however, the PI proteins on lymphocyte subsets have not been fully analyzed yet. We examined the expression of decay-accelerating factor (DAF) and CD59 on the T lymphocytes (CD2+, CD3+, CD4+, and CD8+) and CD20+ B lymphocytes in ten healthy volunteers and 12 paroxysmal nocturnal hemoglobinuria (PNH) patients by cytofluorometry. In healthy controls, each subset of lymphocytes showed a small population of cells weakly positive and a large population of cells strongly positive for DAF and CD59, while erythrocytes showed a single population of cells positive for the PI proteins. The two-population expression of DAF was most distinctive in CD8+ T cells among the subsets. In PNH, each subset of lymphocytes showed a moderately higher population of cells weakly positive and a smaller population of cells strongly positive for the membrane proteins compared with those in the healthy controls. Moreover, in some PNH cases, a negative population for the proteins was found in all subsets. Thus the analysis of PI-anchored proteins on lymphocytes subsets (especially CD8+ T cells) was considered to be of diagnostic value in PNH patients who receive blood transfusion after hemolytic attack of affected erythrocytes. Furthermore, the two-population expression of PI proteins in normal lymphocytes suggests that membrane PI protein would be a new subset marker of lymphocytes.

Adult↗
PubMed1993

Specific chromosomal sites enhancing homologous recombination in Escherichia coli mutants defective in RNase H.

To clone new replication origin(s) activated under RNase H-defective (rnh-) conditions in Escherichia coli cells, whole chromosomal DNA digested with EcoRI was to with a Kmr DNA fragment and transformed into an rnh- derivative host. From the Kmr transformants, we obtained eight kinds of plasmid-like DNA, each of which contained a specific DNA fragment, termed "Hot", derived from the E. coli genome. Seven of the Hot DNAs (HotA-G) mapped to various sites within a narrow DNA replication termination region (about 280 kb), without any particular selection. Because Hot DNA could not be transformed into a mutant strain in which the corresponding Hot region had been deleted from the chromosome, the Hot DNA, though obtained as covalently closed circular (ccc) DNA, must have arisen by excision from the host chromosome into which it had initially integrated, rather than by autonomous replication of the transformed species. While Hot DNA does not have a weak replication origin it does have a strong recombinational hotspot active in the absence of RNase H. This notion is supported by the finding that Chi activity was present on all Hot DNAs tested and no Hot-positive clone without Chi activity was obtained, with the exception of a DNA clone carrying the dif site.

Chromosomes, Bacterial↗
PubMed1993

A flavoprotein functional as NADH oxidase from Amphibacillus xylanus Ep01: purification and characterization of the enzyme and structural analysis of its gene.

Amphibacillus xylanus Ep01, a facultative anaerobe we recently isolated, shows rapid aerobic growth even though it lacks a respiratory pathway. Thus, the oxidative consumption of NADH, produced during glycolysis and pyruvate oxidation, should be especially important for maintenance of intracellular redox balance in this bacterium. We purified a flavoprotein functional as NADH oxidase from aerobically growing A. xylanus Ep01. The A. xylanus enzyme is a homotetramer composed of a subunit (M(r) 56,000) containing 1 mol of flavin adenine dinucleotide. This enzyme catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH as the preferred electron donor and exhibits no activity with NADPH. The flavoprotein gene of A. xylanus Ep01 was cloned by using a specific antibody. The amino acid sequence of 509 residues, deduced from the nucleotide sequence, showed 51.2 and 72.5% identities to the amino acid sequences of alkyl hydroperoxide reductase from Salmonella typhimurium and NADH dehydrogenase from alkalophilic Bacillus sp. strain YN-1, respectively. Bacillus spp. have a respiratory chain and grow well under aerobic conditions. In contrast, Amphibacillus spp., having no respiratory chain, grow equally well under both aerobic and anaerobic conditions, which distinguishes these two genera. Salmonella spp., which are gram-negative bacteria, are taxonomically distant from gram-positive bacteria such as Bacillus spp. and Amphibacillus spp. The above findings, however, suggest that the flavoprotein functional as NADH oxidase, the alkyl hydroperoxide reductase, and the NADH dehydrogenase diverged recently, with only small changes leading to their functional differences.

Amino Acid Sequence↗
PubMed1993

Mutational analysis of the amino acid residues essential for the cis and trans cleavage activity of the potato virus Y 50-kDa protease.

To examine the proteolytic activities of various truncated derivatives of the potato virus Y (PVY) 50-kDa protease, the derivatives were expressed in Escherichia coli in polyprotein forms fused with coat protein (CP). For the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-CP junction. The activity was evaluated by the amount of the mature CP released from the precursor by the intra- and intermolecular cleavage occurring in E. coli. By this experiment, we identified the moiety responsible for the proteolytic activity of the 50-kDa protease to be a 26-kDa polypeptide mapped to the C-terminal half of the protease. Introduction of His234-->Tyr, Asp269-->Asn, or Cys339-->Gly substitution in the putative catalytic triad of the protease abolished its activity. However, the mutated protease with Cys339-->Ser replacement retained a reduced proteolytic activity.

Amino Acid Sequence↗
PubMed1993

Cleavage of potato virus Y polyprotein in Escherichia coli depending on the proteolytic activity of viral protease.

We have cloned a 5-kb cDNA corresponding to the 3'-half of genomic RNA of potato virus Y (PVY), the type member of plant potyvirus. Based on its nucleotide sequence, the cDNA was presumed to encode PVY protease responsible for the self-processing of polyprotein precursor expressed from PVY genome. To test its proteolytic activity, the cDNA was modified so as to express the protease-polymerase-coat protein (CP) portion of PVY polyprotein in Escherichia coli. By Western blotting analysis of the cell extract of E. coli expressing the polyprotein, mature CP was detected. A large deletion in the polymerase-coding region did not affect the emergence of mature CP, but a small deletion in the putative protease region resulted in disappearance of mature CP and appearance of the intact polyprotein. These results indicated that the polyprotein expressed in E. coli was cleaved depending on the proteolytic activity of PVY protease.

Amino Acid Sequence↗
PubMed1992

Inversion of chromosome 16 and bone marrow eosinophilia in a myelomonocytic transformation of chronic myeloid leukemia.

We report a case of chronic myeloid leukemia (CML) in myelomonocytic transformation associated with bone marrow (BM) eosinophilia. At diagnosis, all BM cells showed a Ph chromosome. At the time of blastic phase, more than 50% of Ph+ cells had a pericentric inversion of chromosome 16, inv(16)(p13q22). This case confirms that blastic transformation of CML can involve any committed progenitor, and myelomonocytic leukemia with BM eosinophilia is specifically associated with rearrangement of chromosome 16 at band p13 and q22.

Blast Crisis↗
PubMed1992

Termination complex in Escherichia coli inhibits SV40 DNA replication in vitro by impeding the action of T antigen helicase.

DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases.

Antigens, Polyomavirus Transforming↗
PubMed1992

Identification of a site required for DNA replication fork blocking activity in the rRNA gene cluster in Saccharomyces cerevisiae.

The yeast genome has DNA replication fork blocking sites, that we have named sog sites, in the ribosomal RNA gene (rDNA) cluster. These are located at the 3' end of the 35S rRNA transcription unit and they block replication fork movement in a direction opposite to that of RNA polymerase I. We cloned this replication blocking site into a YEp-type plasmid and analyzed DNA replication intermediates, using two-dimensional (2D) agarose gel electrophoresis. The blocking activity remained even on a plasmid not involved in 35S rRNA transcription and inhibited fork movement in the same polar fashion as on the yeast chromosome. To define the site further, smaller fragments were subcloned into the YEp-type plasmid. A small 109 bp region exhibited sog activity and was located near the enhancer region for 35S rRNA transcription. It overlaps an essential element of the recombinational hot spot HOT1.

Base Sequence↗
PubMed1992

Nucleotide sequence of a carboxyphosphonoenolpyruvate phosphonomutase gene isolated from a bialaphos-producing organism, Streptomyces hygroscopicus, and its expression in Streptomyces lividans.

The carboxyphosphonoenolpyruvate (CPEP) phosphonomutase gene of bialaphos-producing Streptomyces hygroscopicus, which encodes a C-P bond forming enzyme was cloned into Streptomyces lividans and sequenced. The amino acid composition of the protein coded in an open reading frame of 295 codons and its calculated molecular mass, 32,800 Da, coincided well with those of the purified enzyme. Introduction of the CPEP phosphonomutase gene, the expression of which is controlled by the promoter of the aph gene, into S. lividans resulted in the production of this enzyme at a level almost equivalent to that in the parent strain.

Amino Acid Sequence↗
PubMed1992

Altered expression of protein kinase C in adult T-cell leukemia cells.

Protein kinase C (PKC) has been shown to be involved in the mitogenic response and in oncogenic cell transformation in many experimental models. We analyzed the expression of PKC in both highly purified leukemic T cells freshly isolated from adult T-cell leukemia (ATL) patients and control T lymphocytes obtained from healthy volunteers. PKC activity was decreased in the ATL cells as compared with the control T cells. Cytosolic PKC activity in the ATL cells was remarkably decreased, whereas particulate membrane PKC activity was similar to the control level. The percentage of PKC activity in the particulate fraction was 34% in the ATL cells and 19% in the control cells. Regarding the altered subcellular localization of PKC activity, phorbol ester-induced translocation of cytosolic PKC was inhibited in some ATL cases. Similarly to the decrease in PKC activity, there was a decrease in the expression of the major PKC isozymes II(beta) and III(alpha) in ATL cells. These results suggest impaired regulation of PKC expression in ATL as well as in many experimental cancers.

Adult↗