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H Hosoya

Publications and source records attributed to H Hosoya.

At least 19 recordsLinked to original sources
PubMed1995

Cloning of the cDNA encoding neural adhesion molecule F3 from bovine brain.

We cloned a bovine cDNA encoding the neural adhesion molecule F3 and analyzed its nucleotide sequence. The coding region consisted of 3054 bp encoding 1018 amino acid (aa) residues. The M(r) calculated from the deduced aa sequence was 113,383. Bovine F3 had 93, 94 and 77% aa identity with the mouse, human and chicken homologs, respectively. Bovine F3, similar to those of chicken and human, was devoid of two aa residues (Ile-Thr) in the sixth immunoglobulin type C2-like domain, as compared with the mouse homolog. Parts of bovine F3 protein were overproduced in Escherichia coli. The antibodies raised against the recombinant proteins in rabbits reacted specifically with F3. F3 protein was detected in cerebellum, cerebrum and spinal cord in Western blot analysis.

Amino Acid Sequence↗
PubMed1995

Splicing isoforms of rat Ash/Grb2. Isolation and characterization of the cDNA and genomic DNA clones and implications for the physiological roles of the isoforms.

We obtained three types of cDNA clones homologous to Ash/Grb2(Ash-l) cDNA from rats. One of these clones, Ash-psi, was an unusual transcribed gene having 93% identity in the nucleotide sequence to Ash-l. The other two clones, Ash-m and -s, had nucleotide sequences identical with Ash-l cDNA in the amino-terminal region. The coding sequence of Ash-m cDNA is 42 nucleotides shorter than that of Ash-l cDNA. The defective region of Ash-m cDNA encodes 14 amino acid residues (157 to 170 of Ash-l), which comprise the most conserved region of the second SH3 domain. On the other hand, the coding sequence of Ash-s terminated at the end of the first SH3 domain due to a stop codon at the boundary of the sequence, thereby differing from Ash-l cDNA. Cloning of the genomic DNA of the Ash-l-encoding gene, determination of the gene organization, and nucleotide sequencing revealed that the two isoforms, as well as Ash-l, are generated from a single gene by unusual alternative splicings. The gene spans more than 16 kilobases and contains 6 exons and 5 introns. Ash-m and Ash-s mRNAs were detected in various tissues by reverse-transcribed polymerase chain reaction. Ash-m physically associated with dynamin, but the association with Sos was less effective than that of Ash-l in rat pheochromocytoma PC12 cell lysates, irrespective of treatment with nerve growth factor. In contrast, Ash-s formed a complex with dynamin and Sos in cell lysates. Moreover, the newly formed carboxyl-terminal SH3 of Ash-m by splicing bound different proteins from those bound to the carboxyl-terminal SH3 domain of Ash-l, suggesting that Ash-m generates different signals. Microinjection of Ash-m or Ash-s into Balb/c 3T3 cells inhibited DNA synthesis induced by platelet-derived growth factor. These results show that these isoforms act as dominant negative regulators of mitogenic signals by Ash-l.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Developmental expression of the neural adhesion molecule F3 in the rat brain.

We cloned a cDNA encoding rat F3 and analyzed the nucleotide sequences. The results have shown that rat F3 is comprised of 1021 amino acid residues. It shared 99% and 76% identities with mouse and chicken homologs, respectively, at the amino acid sequence level. During postnatal development of the rat brain, cells expressing F3 mRNA appeared in the cortex, hippocampus, superior and inferior colliculi, anterior olfactory nucleus, olfactory bulb and cerebellum, whereas little was observed at postnatal day 1 (P1). Extraordinarily high expression of F3 mRNA was observed in the cerebral cortical neurons of layer 5 at P7. The number of cells with high expression of F3 mRNA expanded to the entire region of the cerebral cortex at P14. The whole cerebrum displayed expression at P90 in which the cortex still showed the highest expression level, although the overall signals were weak in comparison with those at P14. In the hippocampal formation, F3-expressing granule cells of the dentate gyrus were restricted to the outer aspect, then expanded to the inner aspect during development. Finally the granule cells in the entire region of the dentate gyrus transcribed F3 mRNA. We discuss the significance of the expression pattern of F3 mRNA during development.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of dynamin by cdc2 kinase.

A 100kD microtubule-bundling protein dynamin was phosphorylated in vitro by cdc2 kinase to approximately 1 mol of phosphate/mol of dynamin at a serine residue. These phosphorylations of dynamin greatly reduced its binding ability to microtubules.

Animals↗
PubMed1994

Evidence for direct binding of intracellularly distributed ganglioside GM2 to isolated vimentin intermediate filaments in normal and Tay-Sachs disease human fibroblasts.

Although some intracellularly distributed glycosphingolipids are reported to be associated with vimentin intermediate filaments or colchicine sensitive cytoskeleton, no direct evidence for such an association has yet been shown. In this report we demonstrated that the intracellularly distributed ganglioside GM2 directly binds to isolated vimentin intermediate filaments in normal and Tay-Sachs disease human fibroblasts. Indirect immunofluorescence microscopy using a GM2-specific monoclonal antibody demonstrated filamentously distributed GM2 in the cytoplasm. A double staining of Tay-Sachs fibroblasts with anti-GM2 and anti-vimentin monoclonal antibodies strongly suggested that the GM2 positive filaments are vimentin intermediate filaments. We then isolated vimentin, in the presence of a detergent and urea, from the normal human skin fibroblasts and murine mastocytoma cells. In a solid phase enzyme-linked immunosorbent assay, the isolated vimentin dose-dependently reacted with both anti-vimentin and anti-GM2 monoclonal antibodies but not with anti-GM3 or anti-GM1 monoclonal antibody. The molar ratio of GM2 to vimentin was approximately 20:1. The lipid fraction extracted from the purified vimentin preparation was immunostained with anti-GM2 on a thin-layer chromatography plate. Furthermore, only one band was detected at the molecular weight of 57 kDa, after electroblotting and simultaneous immunostaining with anti-GM2 and anti-vimentin monoclonal antibodies. These results clearly indicated that ganglioside GM2 directly binds to vimentin.

Animals↗
PubMed1994

Promoter screening from Pseudomonas species by a promoter probe transposon and its structure.

By use of the promoter probe transposon Tn5-B21, two promoter fragments were isolated. One promoter (822 bp) (GC = 63.5%) isolated from P. putida loses all of its promoter activity by exonuclease or restriction enzyme deletion. Another promoter (264 bp) (GC = 49.2%) isolated from P. fluorescens could be shortened to 154 bp by exonuclease deletion without any effect on its promoter activity in several Pseudomonas species.

DNA Transposable Elements↗
PubMed1994

DNA sequence of proline permease gene from Pseudomonas fluorescens and predicted structure of proline permease.

The proline permease gene of Pseudomonas fluorescens has been isolated from the promoter region isolated by using a promoter probe (transposon Tn5-B21). By DNA sequencing of 2222 bp the primary structure of permease (494 aa) was deduced. The DNA sequence is 71.0% and 70.7% identical and the amino acid sequence is 75.8% and 76.0% similar to those of Escherichia coli and Salmonella typhimurium, respectively.

Amino Acid Sequence↗
PubMed1993

Regulation of hsc70 expression in the human histiocytic lymphoma cell line, U937.

The transcription of hsc70, a cognate member of the hsp70 gene family, is suppressed in the terminally differentiated human histiocytic lymphoma cell line, U937, and appears to be regulated in the absence of heat shock. We have examined the 5' upstream regulatory region of 450 bp which contains a putative regulatory sequence of 18 bp in addition to known cis-elements such as HSEs, CCAAT boxes, Sp1 binding sites and AP2 binding sites. The 18 bp cis-element formed larger complexes with nuclear localized transfactors when extracts were prepared from differentiated rather than from proliferating cells. These larger complexes were also detected in nuclear extracts of serum-deprived cells, suggestng that such complexes may be related to the suppression of hsc70 gene transcription in Go arrested cells. The level of HSC70 protein as determined using monoclonal antibodies, increased threefold in differentiated cells. The apparent discrepancy between the levels of hsc70 mRNA and HSC70 protein can be explained by a rapid turnover of HSC70 in proliferating cells.

Base Sequence↗
PubMed1992

Ca(2+)-regulated actin and phospholipid binding protein (68 kD-protein) from bovine liver: identification as a homologue for annexin VI and intracellular localization.

An F-actin binding protein was purified from bovine liver by means of DNase I affinity, hydroxylapatite and DEAE-cellulose column chromatographies. It consisted of a single polypeptide chain having an apparent molecular weight of 68,000 with a Stokes radius of 35 A. Electron microscopy of rotary shadowed specimens showed that the 68 kD protein is a globular protein. This protein showed a higher affinity for F-actin in the presence of Ca2+ than in its absence, which is opposite to the actin-binding property shown by nonmuscle alpha-actinin or fimbrin. The 68 kD protein had no F-actin severing and capping activity. Interestingly, the 68 kD protein was found to aggregate liposomes at micromolar Ca2+ concentrations. Immunoblot analysis and partial protein sequence data identified the 68 kD protein as an annexin VI (p68) homologue. Immunocytochemical studies showed that the 68 kD protein was localized along stress fibers as well as membrane ruffles, microspikes and focal contacts, raising the possibility that annexin VI may contribute to control membrane-microfilament interaction in the cell.

Actins↗
PubMed1992

Differentiation and dedifferentiation of the human monocytic leukemia cell line, U937.

U937 cells were differentiated into macrophages after being treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA) for the first two days and dedifferentiated with daily medium renewal for 10 days. Cell proliferation slowed down and the number of cells reached the maximum level on day 2. By day 4, all of the cells had spread and attached firmly to the culture dish, and more than 90% of the cells expressed the Fc-receptor and produced superoxide anion. From there on, the number of adherent, living cells decreased gradually to about half the initial count. Most of the cells eliminated from the culture by cell death were in the S phase at the time of TPA treatment. After day 8, the number of cells expressing macrophage-specific phenotypes gradually decreased, cell adhesion was weakened, and at the same time, DNA synthesis was initiated anew. The cells became round and began to proliferate as floating cells on days 9 to 10, and thereafter they became sensitive to the second round of TPA treatment. On the basis of all the results taken together, it is suggested that fully differentiated U937 cells were dedifferentiated after being cultured with frequent medium renewal.

Autoradiography↗
PubMed1991

Mitosis-specific phosphorylation of myosin light chain kinase.

Cell cytosol preparations from mitotic HeLa cells exhibit a kinase activity that phosphorylates myosin light chain kinase (MLCK). This MLCK kinase activity is apparently distinct from the known MLCK kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, Ca(2+)-activated phospholipid-dependent protein kinase, or Ca(2+)-calmodulin-dependent protein kinase II, based on the following criteria. First, the MLCK kinase activity of mitotic cells does not respond to a variety of characteristic activators or inhibitors of these known kinases. Second, one- and two-dimensional peptide maps have revealed that the site of phosphorylation by the MLCK kinase of mitotic cells differs from those by these known kinases. The mitotic MLCK kinase phosphorylates MLCK at a threonine residue at a ratio of up to 1 mol of phosphate/mol of chicken gizzard MLCK. The MLCK kinase is mitosis-specific because mitotic cell extracts show much higher phosphorylation activity than nonmitotic cell extracts.

Animals↗
PubMed1991

Phosphorylation of non-muscle caldesmon by p34cdc2 kinase during mitosis.

One of the profound changes in cellular morphology which occurs during mitosis is a massive alteration in the organization of the microfilament cytoskeleton. This change, together with other mitotic events including nuclear membrane breakdown, chromosome condensation and formation of mitotic spindles, is induced by a molecular complex called maturation promoting factor. This consists of at least two subunits, a polypeptide of relative molecular mass 45,000-62,000 (Mr 45-62K) known as cyclin, and a 34K catalytic subunit which has serine/threonine kinase activity and is known as cdc2 kinase. Non-muscle caldesmon, an 83K actin- and calmodulin-binding protein, is dissociated from microfilaments during mitosis, apparently as a consequence of mitosis-specific phosphorylation. We now report that cdc2 kinase phosphorylates caldesmon in vitro principally at the same sites as those phosphorylated in vivo during mitosis, and that phosphorylation reduces the binding affinity of caldesmon for both actin and calmodulin. Because caldesmon inhibits actomyosin ATPase, our results suggest that cdc2 kinase directly causes microfilament reorganization during mitosis.

Actin Cytoskeleton↗
PubMed1990

Primary structure of profilins from two species of Echinoidea and Physarum polycephalum.

Profilin is a small G-actin-binding protein, the amino acid sequence of which was previously reported for calf, human, Acanthamoeba and yeast. Here the amino acid sequences of three profilins obtained from eggs of two species of Echinoidea, Clypeaster japonicus (order, Clypeasteroida) and Anthocidaris crassispina (order, Echinoida), and plasmodium of Physarum polycephalum were determined. Two echinoid profilins were composed of 139 amino acid residues, N-termini were acylated and the molecular mass was calculated to be 14.6 kDa, slightly larger than that of 13 kDa estimated by SDS/PAGE [Mabuchi, I. & Hosoya, H. (1982) Biomed. Res. 3, 465-476]. On the other hand, Physarum profilin was composed of 124 amino acid residues, the N-terminus was acylated, and the calculated molecular mass was 13132 Da. The sequences of C. japonicus and A. crassispina profilins were homologous (84% identical). However, the similarity of these profilins with those form other organisms was low. The sequence of Physarum profilin was homologous with Acanthamoeba profilin isoforms (51% identical) and with yeast profilin (42% identical), but not with other profilins. The relatively conservative sequence of profilins from yeast, Physarum, Acanthamoeba, echinoid eggs and mammalian cells was found in the N-terminal region, which was suggested to be a common actin-binding region. The C-terminal region was also conserved, although to a lesser extent than the N-terminal region.

Amino Acid Sequence↗
PubMed1988

Preparation of tubulin from Caulerpa, a marine green alga, using casein as a protective agent against proteolytic degradation.

Bidirectional organelle movements taking place in the cytoplasm of the rhizomes of Caulerpa, a coenocytic marine green alga, have been indicated to be dependent on microtubules (Kuroda, K. & Manabe, E. (1983) Proc. Jpn. Acad. 59B, 131-134; Manabe, E. & Kuroda, K. (1984) Proc. Jpn. Acad. 60B, 118-121). However, when a crude extract of Caulerpa rhizomes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting with monoclonal anti-tubulin antibody, no reacting band could be detected. This apparent absence of tubulin in the extract was found to be a result of the complete degradation of tubulin by potent intrinsic proteolytic activity. All of the commercially available protease inhibitors so far tested (p-chloromercuriphenylsulfonic acid, phenyl methylsulfonyl fluoride, 1-chloro-4-phenyl-3-tosylamido-2-butanone, 7-amino-1-chloro-3-tosylamido-2-heptanone, p-tosyl-L-arginine methyl ester, soybean trypsin inhibitor, antipain, chymostatin, leupeptin, and pepstatin) failed to inhibit the activity completely. But addition of casein at the concentration of 1% (weight per volume) to the solutions used for preparation was effective in protecting tubulin from proteolytic degradation, thus making it possible to prepare tubulin from the crude extract of Caulerpa. On SDS-PAGE, the Caulerpa alpha-tubulin thus prepared was a little smaller in molecular weight than that of rabbit brain.

Animals↗
PubMed1988

The amino acid sequence, immunofluorescence and microinjection studies on the 15 kDa calcium-binding protein from sea urchin egg.

The 15 kDa protein is the most abundant low molecular weight Ca2+-binding protein, different from calmodulin, in eggs of sea urchin, Hemicentrotus pulcherrimus. The data from the amino acid sequence demonstrated that the 15 kDa protein belonged to the troponin C superfamily. Based on immunofluorescent and immunomicroscopic observations, we showed that the 15 kDa protein localized in the nuclei of fertilized eggs and mitotic apparatus of dividing eggs. Microinjection of the antibody against 15 kDa protein into sea urchin blastomeres resulted in the arresting of cell division. These results suggest that the 15 kDa protein plays an important role in mitosis of sea urchin egg.

Amino Acid Sequence↗
PubMed1987

Melanoma dynein: evidence that dynein is a general "motor" for microtubule-associated cell motilities.

Platyfish-swordtail hybrid melanoma cells exhibit pigment aggregation in response to adrenergic stimulation or melanophore-concentrating hormone. This translocation of pigment granules is thought to be related to radially arrayed microtubules. Very little is known about the molecular "motor" that powers the translocation. We present evidence that dynein is located on these microtubules and is a candidate for the "motor". Vanadate and erythro-9-[3-(2-hydroxynonyl)]adenine, which are potent inhibitors of dynein ATPase, prevent the transport of melanosome granules in Brij-treated melanoma cells. Direct identification of dynein in melanoma cells and tissues is demonstrated by immunofluorescence microscopy and immunoblotting using anti-fragment A (tryptic fragment of sea urchin sperm dynein) serum. The cytoplasm of melanoma cells is stained with the antiserum and gives rise to a pattern similar to the distribution of microtubules. Western blotting shows that the molecular weight of an immunoreactive polypeptide in melanoma tissues coincides with that of the heavy chain of sea urchin sperm dynein.

Adenosine Triphosphatases↗