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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↗
PubMed1995

Mutations in the v-mos gene abolish its ability to induce differentiation but not transformation.

The v-mos oncogene product has the ability to induce differentiation in human monocytic leukemia U937 cells, thereby arresting cell proliferation, and also exhibits transforming activity in mouse NIH3T3 cells. Mutation in the v-mos gene consisting of one or two amino acid substitutions in the putative ATP-binding domain impaired its differentiation-inducing activity although mutant proteins showed rather higher levels of autophosphorylation in vitro. Macrophage-specific characteristics such as their morphology, expression of C3b receptor and Fc receptor, and production of interleukin-1 beta and tumor necrosis factor alpha, were equally diminished in cells transfected with mutant mos genes when compared to those with intact v-mos. The ability of the gene to arrest the proliferation of U937 cells was likewise diminished, while the transforming efficiency of the intact and mutant mos genes were essentially the same. These results suggest that the mos product functions differently in cell differentiation and transformation.

3T3 Cells↗
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

Localization of caldesmon and its dephosphorylation during cell division.

Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle caldesmon to dissociate from microfilaments during prometaphase. (Yamashiro, S., Y. Yamakita, R. Ishikawa, and F. Matsumura. 1990. Nature (Lond.). 344:675-678; Yamashiro, S., Y. Yamakita, H. Hosoya, and F. Matsumura. 1991. Nature (Lond.) 349:169-172). To explore the functions of caldesmon phosphorylation during cytokinesis, we have examined the relationship between the phosphorylation level, actin-binding, and in vivo localization of caldesmon in cultured cells after their release of metaphase arrest. Immunofluorescence studies have revealed that caldesmon is localized diffusely throughout cytoplasm in metaphase. During early stages of cytokinesis, caldesmon is still diffusely present and not concentrated in contractile rings, in contrast to the accumulation of actin in cleavage furrows during cytokinesis. In later stages of cytokinesis, most caldesmon is observed to be yet diffusely localized although some concentration of caldesmon is observed in cortexes as well as in cleavage furrows. When daughter cells begin to spread, caldesmon shows complete colocalization with F-actin-containing structures. These observations are consistent with changes in the levels of microfilament-associated caldesmon during synchronized cell division. Caldesmon is missing from microfilaments in prometaphase cells arrested by nocodazole treatment, as shown previously (Yamashiro, S., Y. Yamakita, R. Iskikawa, and F. Matsumura. 1990. Nature (Lond.). 344:675-678). The level of microfilament-associated caldesmon stays low (12% of that of interphase cells) when some cells start cytokinesis at 40 min after the release of metaphase arrest. When 60% of cells finish cytokinesis at 60 min, the level of microfilament-associated caldesmon is recovered to 50% of that of interphase cells. The level of microfilament-associated caldesmon is then gradually increased to 80% when cells show spreading at 120 min. Dephosphorylation appears to occur during cytokinesis. It starts when cells begin to show cytokinesis at 40 min and completes when most cells finish cytokinesis at 60 min. These results suggest that caldesmon is not associated with microfilaments of cleavage furrows at least in initial stages of cytokinesis and that dephosphorylation of caldesmon appears to couple with its reassociation with microfilaments. Because caldesmon is known to inhibit actomyosin ATPase and/or regulate actin assembly, its continued dissociation from microfilaments may be required for the assembly and/or activation of contractile rings.

Actin Cytoskeleton↗
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↗