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I Yahara

Publications and source records attributed to I Yahara.

At least 19 recordsLinked to original sources
PubMed2001

Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse.

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.

Actin Depolymerizing Factors↗
PubMed2001

Cofilin-2, a novel type of cofilin, is expressed specifically at aggregation stage of Dictyostelium discoideum development.

BACKGROUND: A conventional cofilin, cofilin-1 in Dictyostelium discoideum plays significant roles in cell proliferation, phagocytosis, chemotactic movement and macropinocytosis. RESULTS: We identified a new member of the cofilin family, named cofilin-2 in D. discoideum. Cofilin-2 shows significant homology to a conventional Dictyostelium cofilin, cofilin-1, through its entire sequence, and contains residues conserved among the cofilin family that are responsible for actin-binding. On the other hand, several residues that are conserved among the cofilin family are missing from cofilin-2. Purified cofilin-2 depolymerized actin filaments in a dose- and pH-dependent manner and reduced the apparent viscosity of an actin solution, although they did not co-sediment with actin filaments at all. Cofilin-2 was not expressed in vegetative cells, but was transiently induced during the aggregation stage of development, whereas cofilin-1 was predominantly expressed in vegetative cells. Immunocytochemistry revealed that cofilin-2 localizes at substrate adhesion sites, where cofilin-1 is almost completely excluded. Disruption of the cofilin-2 gene caused an increase in actin accumulation at the substrate adhesion sites. We also found that cofilin-2 did not rescue Deltacof1 yeast cells, whereas cofilin-1 did. CONCLUSIONS: Cofilin-2 may play a distinct role from that of cofilin-1 in destabilization of the actin cytoskeleton during Dictyostelium development.

Actin Depolymerizing Factors↗
PubMed2001

[Toxicity study of cefmatilen hydrochloride hydrate (S-1090) (1)--Single oral and intravenous dose toxicity studies in rats].

A single oral dose toxicity study of Cefmatilen hydrochloride hydrate (S-1090) and a single intravenous dose toxicity study of its sodium salt (S-1090-Na) were conducted in rats. One dose level of 2000 mg potency/kg was set in both studies. Single oral dose toxicity study of S-1090 No deaths occurred. Diarrhea occurred on the dosing day and slightly soft feces lasted until 6 days after administration. These changes were considered to result from changes of intestinal flora induced by the antibiotic activity of S-1090. Reddish-brown feces (due to chelated products of S-1090 or its decomposition products with Fe3+ in the diet) were also observed until the next day after administration. Body weights increased favorably, and no S-1090-related pathological changes were observed. The oral lethal dose of S-1090 was estimated to be more than 2000 mg potency/kg. Single intravenous dose toxicity study of S-1090-Na No deaths occurred. The rats showed characteristic clinical signs such as hypoactivity, abnormal gait and hypopnea immediately after dosing, and some rats showed prone position or paleness of eyeballs and ear auricles in due course. These signs disappeared by 4 hr after administration. Slightly soft feces and reddish-brown feces were observed much the same as in the orally-treated rats. Body weights increased favorably. In the pathological examinations, slight cecal enlargement and increased basophilia, dilatation and calcification of the renal tubules in the kidney were observed. The intravenous lethal dose of S-1090-Na was estimated to be more than 2000 mg potency/kg.

Administration, Oral↗
PubMed2001

[Toxicity study of cefmatilen hydrochloride hydrate (S-1090) (4)--One- and three-month repeated oral dose toxicity studies in dogs].

One- or three-month repeated oral dose toxicity studies of Cefmatilen hydrochloride hydrate (S-1090) were conducted in beagle dogs. Doses were set at 25, 100 and 400 mg potency/kg/day in both studies. In both studies, no deaths occurred, and reddish-brown feces (due to chelated products of S-1090 and its decomposition products with Fe3+ in the diet) were observed in all treated groups. A transient excretion of reddish urine was observed in the 400 mg potency/kg group and a slight increase in plasma irons was also observed in the 100 and 400 mg potency/kg groups of both studies. However, as no changes suggesting anemia or hepatic injury were noted in these groups, the change of plasma irons was considered to have no toxicological significance. Plasma S-1090 concentrations increased in a manner less than dose-proportional in both studies. In the one-month toxicity study, no toxicologically significant changes, including the above findings, were noted, so the NOAEL was assessed to be 400 mg potency/kg/day. In the three-month toxicity study, urinalysis in the 400 mg potency/kg group revealed a positive reaction to occult blood and erythrocytes in sediments. In the pathological examinations, submucosal edema, hemorrhage, inflammatory cell infiltration and occasionally focal mucosal thickening were observed in the urinary bladder of the 400 mg potency/kg group. The cystisis was considered to result from chronic stimulation with the metabolite(s) of S-1090 in urine, and the reversibility was demonstrable upon one-month drug withdrawal. From these results, the NOAEL of S-1090 in the three-month toxicity study was assessed to be 100 mg potency/kg/day.

Administration, Oral↗
PubMed2001

[Toxicity study of cefmatilen hydrochloride hydrate (S-1090) (5)--Six-month repeated oral dose toxicity study and supplement study in rats].

Cefmatilen hydrochloride hydrate (S-1090) was orally administered to rats at dose levels of 100, 300 and 1000 mg potency/kg once daily for 6 months. All the S-1090 treated groups showed soft feces, reddish-brown feces (due to chelated products of S-1090 or its decomposition products with Fe3+ in the diet), abdominal distention, increased food and water consumption, lower urine pH, and a decrease of white blood cells counts (except for males of the 100 mg potency/kg group). One male in the 300 mg potency/kg group showed mucous feces and marked decrease in body weight, and diet in the middle stage of the administration period. In necropsy of the survivors of all treated groups, marked cecal enlargement was noted. No remarkable changes were observed in the other examination items. From the early stage of the withdrawal period, animals in the 1000 mg potency/kg group showed again soft or mucous feces and a marked decrease in body weight. Of these animals, one male died and another male was sacrificed in a moribund state at about 2 weeks of the withdrawal period. Enterocolitis was observed in these cases. Almost all animals recovered within 3 weeks of withdrawal. A supplemental study of the 6-month toxicity study was conducted to examine the mechanisms of enterocolitis and the changes observable in the 100 or 300 mg potency/kg groups after drug withdrawal. As a reference, cefdinir (CFDN), an oral cephem antibiotic the same as S-1090, was added in the 1000 mg potency/kg group. No deaths occurred in any groups. Decreased intestinal flora were noted in all the groups treated with S-1090 or CFDN at the end of the dosing period. At 2 weeks of the withdrawal period, C. difficile and its D-1 toxin in the cecal contents were highly detected in the S-1090 300 and 1000 mg potency/kg groups and CFDN group. Inflammatory changes in the cecum and colon were observed in these groups. At 4 weeks of the withdrawal period, intestinal flora in the S-1090 groups almost returned to the condition before dosing, but those in the CFDN group were retained highly. Cecal D-1 toxin in the CFDN group was positive and higher than in the S-1090 groups. It was thus considered that the critical condition with enterocolitis resulted from C. difficile, which proliferated more rapidly than the other bacteria and D-1 toxin produced by this bacteria in the withdrawal period. Above changes were commonly observed in the CFDN group. The NOAEL of S-1090 was assessed to be 100 mg potency/kg/day which induced no enteritis.

Administration, Oral↗
PubMed2000

Interaction of the human DnaJ homologue, HSJ1b with the 90 kDa heat shock protein, Hsp90.

The 90 kDa heat shock protein (Hsp90) is a major cytoplasmic molecular chaperone associating with numerous other proteins. Both genetic and in vitro refolding experiments using reticulocyte lysate have suggested a functional interaction of Hsp90 with yeast human homologues of E. coli DnaJ. Here we present direct evidence using surface plasmon resonance that Hsp90 and the human DnaJ homologue, HSJ1b, bind to each other. We also show that Hsp90 and HSJ1b transfer alpha-lactalbumin to each other in an ATP-dependent manner. The two chaperones have additive effects in preventing rhodanese aggregation.

HSP40 Heat-Shock Proteins↗
PubMed2000

A critical role for the proteasome activator PA28 in the Hsp90-dependent protein refolding.

The 90-kDa heat shock protein, Hsp90, was previously shown to capture firefly luciferase during thermal inactivation and prevent it from undergoing an irreversible off-pathway aggregation, thereby maintaining it in a folding-competent state. While Hsp90 by itself was not sufficient to refold the denatured luciferase, addition of rabbit reticulocyte lysate remarkably restored the luciferase activity. Here we demonstrate that Hsc70, Hsp40, and the 20 S proteasome activator PA28 are the effective components in reticulocyte lysate. Purified Hsc70, Hsp40, and PA28 were necessary and sufficient to fully reconstitute Hsp90-initiated refolding. Kinetics of substrate binding support the idea that PA28 acts as the molecular link between the Hsp90-dependent capture of unfolded proteins and the Hsc70- and ATP-dependent refolding process.

Amino Acid Sequence↗
PubMed2000

p53-independent association between SV40 large T antigen and the major cytosolic heat shock protein, HSP90.

The simian double strand DNA tumor virus SV40 encodes the 90-kDa multi-functional protein, large T antigen (LT). LT functions by binding to DNA, as well as to many cellular target proteins such as p53 and retinoblastoma protein (pRB). We report here the identification of a cellular heat shock protein, HSP90, as a previously undescribed LT-associated protein. Immunoprecipitates by anti-HSP90 antibodies from LT-expressing cell lysates contained LT protein, as revealed by Western blotting. Conversely, anti-LT antibody co-immunoprecipitated HSP90. Co-immunoprecipitation of HSP90 and LT was observed even after complete immuno-depletion of p53, indicating that the association of LT with HSP90 is p53-independent. LT-HSP90 complexes can be reconstituted from purified HSP90 and unfolded-LT in vitro in an ATP-independent manner but not from HSP90 and native LT, suggesting that non-mature conformation of LT is required for the efficient association with HSP90. Moreover, geldanamycin, an anti-tumor drug that specifically binds and inhibits HSP90, reduced the intracellular concentration of LT by destabilizing newly synthesized LT. The above results suggest that HSP90 associates with immature forms of LT both in vivo and in vitro, and thus might assist LT in the formation of a functional, mature structure.

Animals↗
PubMed2000

Role of HSP90 in salt stress tolerance via stabilization and regulation of calcineurin.

The role of HSP90 in stress tolerance was investigated in Saccharomyces cerevisiae. Cells showing 20-fold overexpression of Hsc82, an HSP90 homologue in yeast, were hypersensitive to high-NaCl or H-LiCl stresses. Hsc82-overexpressing cells appeared similar to calcineurin-defective cells in salt sensitivity and showed reduced levels of calcineurin-dependent gene expression. Co-overexpression of Cna2, the catalytic subunit of calcineurin, suppressed the hypersensitivity. Cna2 and Hsc82 coimmunoprecipitated from control cells grown under normal conditions but not from stressed cells. In contrast, coimmunoprecipitation was detected with Hsc82-overexpressing cells even after exposure to stresses. Cna2 immune complexes from stressed control cells showed a significant level of calcineurin activity, whereas those from stressed Hsc82-overexpressing cells did not. Treatment of extracts from Hsc82-overexpressing cells with Ca(2+)-calmodulin increased the calcineurin activity associated with Cna2 immune complexes. Geldanamycin, an inhibitor of HSP90 abolished the coimmunoprecipitation but did not activate calcineurin. When the expression level of Hsc82 decreased to below 30% of the normal level, cells also became hypersensitive to salt stress. In these cells, the amount of Cna2 was reduced, likely as a result of degradation. The present results showed that Hsc82 binds to and stabilizes Cna2 and that dissociation of Cna2 from Hsc82 is necessary for its activation.

Blotting, Western↗
PubMed1999

Two activities of cofilin, severing and accelerating directional depolymerization of actin filaments, are affected differentially by mutations around the actin-binding helix.

The biochemical activities of cofilin are controversial. We demonstrated that porcine cofilin severs actin filaments and accelerates monomer release at the pointed ends. At pH 7.1, 0.8 microM cofilin cut filaments (2.2 microM actin) about every 290 subunits and increased the depolymerization rate 6.4-fold. A kink in the major alpha-helix of cofilin is thought to constitute a contact site for actin. Side chain hydroxyl groups of Ser119, Ser120 and Tyr82 in cofilin form hydrogen bonds with main chain carbonyl moieties from the helix, causing the kink. We eliminated side chain hydroxyls by Ser-->Ala and/or Tyr-->Phe mutagenesis. Severing and depolymerization-enhancing activities were reduced dramatically in an Ala120 mutant, whereas the latter was decreased in a Phe82 mutant with a relatively small effect on severing, suggesting different structural bases for the two activities of cofilin. The Ala120-equivalent mutation in yeast cofilin affected cell growth, whereas that of the Phe82-equivalent had no effect in yeast. These results indicate the physiological significance of the severing activity of cofilin that is brought about by the kink in the helix.

Actin Depolymerizing Factors↗
PubMed1999

Monomer arrangement in HSP90 dimer as determined by decoration with N and C-terminal region specific antibodies.

Electron microscopy using the low-angle rotary shadowing replica method showed that the HSP90 dimer consists of four globular domains aligning in a tandem fashion. When decorated with two monoclonal antibodies against epitopes mapped on the N-terminal region of HSP90, these antibodies bound to both ends of the HSP90 dimer. A C-terminal region specific antibody was shown to bind to the side of HSP90. These results support a model for HSP90 dimer whereby two HSP90 monomers are arranged in an antiparallel fashion and dimerize through the C-terminal domain. Treatment of HSP90 at elevated temperatures or with ATP at room temperature, though not with ADP, induces molecular transformation of the linear HSP90 dimer into an O-ring-shaped structure.

Adenosine Triphosphate↗
PubMed1999

Interactions of Hsp90 with histones and related peptides.

The 90 kDa heat shock protein (Hsp90) induces the condensation of the chromatin structure [Csermely, P., Kajtár, J., Hollósi, M., Oikarinen, J., and Somogyi, J. (1994) Biochem. Biophys. Res. Commun. 202, 1657-1663]. In our present studies we used surface plasmon resonance measurements to demonstrate that Hsp90 binds histones H1, H2A, H2B, H3 and H4 with high affinity having dissociation constants in the submicromolar range. Strong binding of the C-terminal peptide of histone H1 containing the SPKK-motif and a pentaeicosa-peptide including the Hsp90 bipartite nuclear localization signal sequence was also observed. However, a lysine/arginine-rich peptide of casein, and the lysine-rich platelet factor 4 did not display a significant interaction with Hsp90. Histones and positively charged peptides modulated the Hsp90-associated kinase activity. Interactions between Hsp90, histones, and high mobility group (HMG) protein-derived peptides raise the possibility of the involvement of Hsp90 in chromatin reorganization during steroid action, mitosis, or after cellular stress.

Amino Acid Sequence↗
PubMed1999

Cooperation of two actin-binding proteins, cofilin and Aip1, in Saccharomyces cerevisiae.

BACKGROUND: Cofilin is a low-molecular weight actin-modulating protein, and is structurally and functionally conserved among eukaryotes. Cofilin is encoded by COF1 in Saccharomyces cerevisiae, and is essential for cell viability. Cofilin binds to and severs actin filaments in vitro, and also enhances their depolymerization. A partner protein that cooperates with cofilin in vivo has not been identified. RESULTS: When COF1 was over-expressed in yeast cells under the GAL1 promoter in a medium containing galactose as a sole carbon source, the cells did not survive. These results indicate that cells can grow only when the expression of cofilin is appropriately regulated. Several temperature sensitive (ts-) mutants were independently created by the random mutagenesis of COF1 with hydroxylamine. Mutated amino acids in ts-mutants were mapped in the sequences that were presumed to be involved in actin binding. A gene on a multicopy plasmid which suppresses the ts-phenotype of cof1-101, a typical ts-cofilin mutant, was isolated. The suppressor gene, SCF1, was found to be identical to AIP1, a gene encoding an actin-interacting protein. Although SCF1/AIP1 is not essential for cell viability, a combination of cof1-101 and Deltascf1/aip1 is synthetic lethal. Immunofluorescence staining of a wild-type strain using anti-Aip1 antibodies revealed that Aip1 was distributed in cortical actin patches where cofilin was also co-localized. Thick and long fibres stained with anti-cofilin antibody were detected in Deltascf1/aip1 cells, but not in SCF1/AIP1 cells. CONCLUSIONS: These results suggest the cooperative modulation of the actin cytoskeleton by cofilin and Aip1.

Actin Depolymerizing Factors↗
PubMed1999

Hyperosmotic stress-induced reorganization of actin bundles in Dictyostelium cells over-expressing cofilin.

BACKGROUND: Cofilin is a low-molecular weight actin-modulating protein, which binds to, severs, and depolymerizes actin filaments in vitro. Aip1, an actin-interacting protein, was recently identified as a product of a gene on a multicopy plasmid which suppresses the temperature-sensitive phenotype of a cofilin mutant in Saccharomyces cerevisiae. Actin cytoskeleton plays an essential role in resistance to hyperosmotic stress in Dictyostelium discoideum. The roles of cofilin and Aip1 in this resistance are not known. RESULTS: In response to hyperosmotic stress, D. discoideum cells round up. This stress-induced morphological change involves the redistribution of cofilin, together with actin filaments, into cortical contractile portions of the cells, followed by their contraction. Over-expression of cofilin increases and thickens cortical actin bundles in cells. The bundles become tight and are reorganized into a ring-shaped structure in response to hyperosmotic stress. The ring structure of actin bundles had two characteristic bands across them; bright and dark bands, heavily stained and not stained with phalloidin. In the bundles, straight filaments with a diameter of 5.3-nm were aligned parallel by cross-bridge structures. In cells lacking the myosin-II heavy chain, the bundles, which were induced by an over-expression of cofilin, shortened and became straight following hyperosmotic stress, forming a polygonal structure. D. discoideum Aip1/Wrp2 enhanced the severing of actin filaments by cofilin in vitro and colocalized with cofilin in cells, including those that were over-expressing cofilin before and after exposure to hyperosmotic stress. CONCLUSIONS: Cofilin plays a pivotal role in concert with Aip1/Wrp2 in the reorganization of actin architectures into bundles that contract in a myosin-II-independent manner, in response to hyperosmotic stress.

Actin Depolymerizing Factors↗
PubMed1999

The role of HSP90 in evolution.

A mechanism by which morphological mutations are stored without expressing phenotypes was unraveled by Rutherford & Lindquist (1998) through genetic studies of Hsp83 (HSP90) in Drosophila. Cryptic mutations are essentially neutral and therefore evolve in the absence of selective constraint. A shift from neutral mutations to selective mutations is induced when flies are exposed to environmental stress. This is a step toward understanding macroevolution in molecular terms.

Animals↗
PubMed1998

Interaction of vanadate oligomers and permolybdate with the 90-kDa heat-shock protein, Hsp90.

The 90-kDa heat-shock protein (Hsp90) is a molecular chaperone that aids the folding of nuclear hormone receptors and protein kinases. Hsp90 x protein complexes can be stabilized by molybdate and by other transition metal oxyanions such as vanadate. Our earlier findings [Csermely, P., Kajtár, J., Hollósi, M., Jalsovszky, G., Holly, S., Kahn, C. R., Gergely, P. Jr, Söti, C., Mihály, K. & Somogyi, J. (1993) J. Biol. Chem. 268, 1901-1907] showed that vanadate and molybdate can induce a large conformational change of Hsp90. Here we provide direct evidence for the binding of vanadate and molybdate to Hsp90 by demonstrating that surface-plasmon-resonance measurements indicate binding of various vanadate oligomers to Hsp90. 51V-NMR measurements show an extensive interaction of decavanadate with the chaperone, and permolybdate treatment of Hsp90 induces a marked mobility shift of the protein and its tryptic fragments. Our results indicate the flexibility of molybdate/vanadate-binding sites of Hsp90, which are able to accommodate various species of these transition metal anions.

Amino Acid Sequence↗