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Y Inaguma

Publications and source records attributed to Y Inaguma.

At least 19 recordsLinked to original sources
PubMed1999

AlphaB-crystallin in the rat lens is phosphorylated at an early post-natal age.

We determined the developmental changes in the phosphorylation state of alphaB-crystallin in lenses from rats at various post-natal ages by isoelectric focusing gel electrophoresis or sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of lenses using antibodies that recognized the carboxy-terminal sequence or each of the three phosphorylated serine residues (Ser-19, Ser-45 and Ser-59) in alphaB-crystallin. Phosphorylated forms of alphaB-crystallin were barely detected at birth but they became detectable at 3 weeks of age and reached plateau levels at 8 weeks of age. The phosphorylation of alphaB-crystallin at Ser-45 was observed preferentially. The active form of p44/42 MAP kinase, which is responsible for the phosphorylation of Ser-45 in alphaB-crystallin, also increased in a development-dependent manner. Thus we found that the developmental increase of the phosphorylation at Ser-45 of alphaB-crystallin in the rat lens was due to the developmental activation of p44/42 MAP kinase.

Animals↗
PubMed1998

Phosphorylation of alphaB-crystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation.

The immunofluorescence localization of alphaB-crystallin in U373 MG human glioma cells with an antibody specific for alphaB-crystallin that had been phosphorylated at Ser-45 revealed an intense staining of cells in the mitotic phase of the cell cycle. Phosphorylated forms of alphaB-crystallin in mitotic cells were detected in all cell lines examined and in tissue sections of mouse embryos. Increases in the levels of alphaB-crystallin that had been phosphorylated at Ser-45 and Ser-19, but not at Ser-59, were detected biochemically by isoelectric focusing or SDS-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of cells collected at the mitotic phase. When we estimated the phosphorylation activity specific for alphaB-crystallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated alphaB2-crystallin as the substrate, we found that the activities responsible for the phosphorylation of Ser-45 and Ser-19 were markedly enhanced but that the activity responsible for the phosphorylation of Ser-59 was suppressed. The protein kinases responsible for the phosphorylation of Ser-45 and Ser-59 in the amino-terminal 72-amino acid peptide were partially purified from extracts of cells that had been stimulated by exposure to H2O2 in the presence of calyculin A. The activities responsible for the phosphorylation of Ser-45 and Ser-59 were eluted separately from a column of Superdex 200 at fractions corresponding to about 40 and 60 kDa, respectively, while the kinase for Ser-19 was unstable. p44/42 mitogen-activated protein (MAP) kinase and MAP kinase-activated protein (MAPKAP) kinase-2 were concentrated in the Ser-45 kinase fraction and Ser-59 kinase fraction, respectively. Recombinant human p44 MAP kinase and MAPKAP kinase-2 purified from rabbit muscle selectively phosphorylated Ser-45 and -59, respectively. The Ser-45 kinase fraction and Ser-59 kinase fraction phosphorylated myelin basic protein and hsp27, respectively. These results suggest that the phosphorylations of Ser-45 and Ser-59 in alphaB-crystallin are catalyzed by p44/42 MAP kinase and MAPKAP kinase-2, respectively, in cells and that the phosphorylation of Ser-45 by p44/42 MAP kinase is enhanced while the phosphorylation of Ser-59 by MAPKAP kinase-2 is suppressed during cell division.

Animals↗
PubMed1998

Cloning and developmental expression of a nuclear ubiquitin-conjugating enzyme (DmUbc9) that interacts with small heat shock proteins in Drosophila melanogaster.

In a two hybrid screen designed to identify proteins that interact with small heat shock proteins (sHsps), a Drosophila melanogaster homologue of yeast and human ubc9 (Dmubc9) was found to interact with Drosophila Hsp23. Further, two-hybrid system analysis reveals DmUbc9 interaction with Drosophila and mammalian Hsp27. In situ hybridization localizes Dmubc9 as a doublet at locus 21D on chromosome 2L, and genomic cloning of the gene reveals a single open reading frame without introns. The predicted Dmubc9 protein sequence shares a very high level of homology with mouse (85.4%) and human (> or = 82.9%) Ubc9. Genetic complementation analysis show that Dmubc9 functionally rescues a temperature-sensitive S. cerevisiae ubc9ts mutant. Co-immunoprecipitation with antibody raised against DmUbc9 confirms the interaction with Drosophila Hsp23 and Hsp26 and preferentially with Hsp27. The DmUbc9 protein, which localizes primarily to the nucleus in Drosophila S2 cells, is found at high levels in embryos but is also present at lower levels throughout development. The significance of the sHsp-Ubc9 interaction is discussed.

Amino Acid Sequence↗
PubMed1996

cDNA cloning of a 20-kDa protein (p20) highly homologous to small heat shock proteins: developmental and physiological changes in rat hindlimb muscles.

A cDNA clone encoding p20, a novel member of the small heat-shock protein family in mammals, was isolated from a rat soleus cDNA library. The clone contained an insert of 1.3 kb with an open reading frame specifying a polypeptide of 162 amino-acid residues. Southern blot analysis suggested that the p20 gene is a single gene in rat genome. Developmental changes and a sciatic nerve denervation experiment suggested that the expression of p20 in rat hindlimb muscle is related to muscle contraction, and specifically in slow-twitch muscles.

Animals↗
PubMed1996

Synthesis and accumulation of alphaB crystallin in C6 glioma cells is induced by agents that promote the disassembly of microtubules.

When C6 cells in culture were exposed at 37 degrees C to 1 microM colchicine or to 1 microM colcemid, a tubulin-binding antimitotic alkaloid, levels of alphaB crystallin in cells began to increase after about 10 h, reaching a maximum of more than 1 microg/mg protein after 24 h. The level of alphaB crystallin returned to near the control level within two subsequent days of culture in the normal medium. Northern blot analysis showed that the accumulation of alphaB crystallin was preceded by an increase in the level of the mRNA for alphaB crystallin. Nuclear run-off transcription assays showed that colchicine induced new synthesis of mRNA for alphaB crystallin. Immunofluorescence staining revealed that alphaB crystallin accumulated in the peripheral areas of cells, as did the depolymerized tubulin, after several hours of treatment with colcemid, and then it gradually became more conspicuous in the cytoplasm. Vinblastine and nocodazole, which also promote the disassembly of microtubules by binding to tubulins, also induced the synthesis of alphaB crystallin. Furthermore, induction of alphaB crystallin by these drugs was observed in quiescent cells that had been cultured in serum-free medium. However, taxol, a microtubule-stabilizing antimitotic agent, did not stimulate the synthesis of alphaB crystallin, but rather, it suppressed the induction of synthesis of alphaB crystallin by the microtubule-disrupting drugs. Induction of alphaB crystallin by colchicine or by other drugs that promote the disassembly of microtubules was sensitive to staurosporine, an inhibitor of protein kinases, and the induction was completely suppressed in the presence of 10 nM staurosporine. These results suggest that the expression of alphaB crystallin is stimulated, via phosphorylation reactions that are sensitive to staurosporine, when the depolymerization of microtubules is enhanced.

Animals↗
PubMed1996

Enhancement of stress-induced synthesis of hsp27 and alpha B crystallin by modulators of the arachidonic acid cascade.

The regulation by intrinsic factors of responses to stress of two small stress proteins, hsp27 and alpha B crystallin, was examined in C6 rat glioma cells. Levels of hsp27 and alpha B crystallin were low in C6 glioma cells in confluent cultures. However, levels of the two proteins increased after exposure of cells to heat (42 degrees C for 30 min) or arsenite (50 microM for 1 h) stress. When cells were exposed to arsenite or hear in the presence of indomethacin (50 microM), an inhibitor of cyclooxygenase, or in the presence of nordihydroguaiaretic acid (NDGA; 50 microM), an inhibitor of lipoxygenase, induction of hsp27 and alpha B crystallin was markedly stimulated as detected by specific immunoassays, Western blot analysis, and Northern blot analysis. The presence of melittin (1 microM), an activator of phospholipase A2, during the stress period also stimulated the induction of the two proteins. The expression of hsp70 to each stress was also enhanced in the presence of indomethacin, NDGA, or melittin. The gel mobility shift assay revealed that these chemicals prolonged the arsenite-induced activation of heat shock element (HSE)-binding activity of heat shock transcriptional factor (HSF) in cells. Induction of hsp27 and alpha B crystallin in adrenal glands of heat-stressed (42 degrees C for 15 min) rats was also enhanced by prior injection of aspirin, another inhibitor of cyclooxygenase. These results indicate that the responses to stress of hsp27 and alpha B crystallin, as well as the response of hsp70, are coupled with the metabolic activity of the arachidonic acid cascade and the mechanism for regulation of stress responses observed in C6 cells is operative in tissues and organs in vivo.

Animals↗
PubMed1996

Elevated concentrations of gamma-enolase in renal cell tumors in rats: similarity to renal cell carcinoma in man.

Concentrations of enolase isozymes in normal kidney and renal cell tumors in rats were determined using a highly sensitive enzyme immunoassay, and the isozymes were immunohistochemically localized in tissue sections. Levels of alpha-enolase in renal cell tumors were significantly lower than in normal kidney, whereas those of gamma-enolase were significantly elevated (mean +/- SD: 211 +/- 129 ng/mg protein, n = 15, as compared to 27.1 +/- 2.9 ng/mg protein, n = 7). The proportion of gamma-enolase in the total enolases in the tumor tissues (1.6 +/- 0.5%) was significantly higher than in normal kidney (0.15 +/- 0.05%). Immunohistochemistry revealed epithelial cells of all nephron segments to be positive for the alpha-isozyme, whereas gamma-enolase staining was strongly positive only in the loops of Henle, being faint in the distal tubules and absent in the proximal tubules. Both alpha- and gamma-enolases demonstrated positive immunostaining in all of the seven renal cell tumors studied. These findings indicate that an isozyme switch from alpha- to gamma-enolase occurs during rat kidney carcinogenesis, taking into account the derivation from proximal tubules, consistent with the findings for renal cell carcinomas in man.

Animals↗
PubMed1996

Modulation of the stress-induced synthesis of hsp27 and alpha B-crystallin by cyclic AMP in C6 rat glioma cells.

The possible participation of cyclic AMP in the stress-induced synthesis of two small stress proteins, hsp27 and alpha B-crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50-100 microM for 1 h) or heat (42 degrees C for 30 min), expression of hsp27 and alpha B-crystallin was stimulated, with levels of the two proteins reaching a maximum after 10-16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 microM) or epinephrine (20 microM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of beta-adrenergic receptors. Cholera toxin (2 micrograms/ml), forskolin (20 microM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite- or heat-induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of alpha B-crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and alpha B-crystallin showed that the modulation of the stress-induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and alpha B-crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of alpha B-crystallin is suppressed.

Adrenergic beta-Agonists↗
PubMed1996

[Mammalian small stress proteins and responses to stress].

When cells are exposed to heat stress or chemical stress, expression of genes for heat shock proteins or stress proteins (HSPs) is enhanced and the proteins are accumulated in cells. The cells with increased HSPs exhibit tolerance against the additional stress. HSPs are expressed also in unstressed tissues or cells for essential biochemical cellular processes including growth and differentiation. Since the responses of HSPs in tissues to stress loaded to a whole living body are much more sensitive compared to those in cultured cells, it is suggested that endogenous factors modulate the stress-induced expression of HSPs. Here we summarize the responses of small HSPs (alpha crystallins, HSP27 and p20) to stress and their modifications by various factors.

Amino Acid Sequence↗
PubMed1995

Induction of the synthesis of hsp27 and alpha B crystallin in tissues of heat-stressed rats and its suppression by ethanol or an alpha 1-adrenergic antagonist.

The levels of hsp27 and alpha B crystallin in various tissues of rats that had been subjected to heat stress were determined by specific immunoassays. When rats were immersed in a water bath at 42 degrees C for 20 min, the levels of hsp27 in most tissues, including central nervous tissue, liver, lung, spleen, adrenal glands, and hypophysis, had increased dramatically 8 to 16 h after the treatment. alpha B crystallin was also induced at high levels in the liver and adrenals, but not much was induced in the central nervous tissue. The increases in the levels of both hsp27 and alpha B crystallin in response to heat stress were markedly inhibited when ethanol or an alpha 1-adrenergic antagonist, prazosin, was administered before, but not after, the stress period. The expression of mRNA for hsp27 was suppressed in the livers of rats that had received ethanol or prazosin. A beta-adrenergic antagonist, propranolol, and an alpha 2-adrenergic antagonist, yohimbine, did not inhibit induction of the synthesis of the two proteins. Accumulation of hsp70 after heat stress, at least in the liver, was inhibited by ethanol or prazosin in a similar manner to that of hsp27, as detected on Western blot analysis. These results suggest that the induction of hsp27 and alpha B crystallin, as well as of hsp70, in tissues of heat-stressed rats is controlled by a physiological process(es) that is sensitive to ethanol and prazosin, and is operative for a short time during the application of heat stress.

Adrenergic alpha-Antagonists↗
PubMed1995

Enhancement of stress-induced synthesis of stress proteins by mastoparan in C6 rat glioma cells.

The levels of two small stress proteins, hsp27 and alpha B crystallin, were low in C6 glioma cells confluency. However, the levels of the two proteins increased after exposure of cells to heat (42 degrees C for 30 min) or arsenite (50-100 microM for 1 h) stress. When cells were exposed to arsenite or heat in the presence of mastoparan, a peptide toxin from wasp venom, the induction of hsp27 and alpha B crystallin was markedly stimulated, as detected by means of specific immunoassays, Western blot analysis, and Northern blot analysis. The response of hsp70 to each stress was also enhanced in the presence of mastoparan. Treatment of cells with 40 microM mastoparan alone barely induced the accumulation of hsp27 and alpha B crystallin. The stimulatory effect of mastoparan was little affected in cells that had been treated with pertussis toxin, but it was strongly suppressed in the presence of quinacrine, an inhibitor of phospholipase A2. These results suggest that mastoparan, which is an activator of phospholipase A2, enhances the responses to stress of hsp27, alpha B crystallin and hsp70 by increasing the metabolic activity of the arachidonic acid cascade.

Animals↗
PubMed1995

Modulation of the stress-induced synthesis of stress proteins by a phorbol ester and okadaic acid.

The expression of alphaB crystallin, hsp27, and hsp70 in C6 cells increased when they were exposed to arsenite (50 microM for 1 h) or heat (42 degrees C for 30 min), as detected by specific immunoassays, Western blot analysis, and Northern blot analysis. When cells were exposed to arsenite in the presence of 0.1 microM phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, or 0.2 microM okadaic acid, an inhibitor of phosphoserine/phosphothreonine protein phosphatases, expression of alphaB crystallin was markedly enhanced. The induction of hsp27 and hosp70 expression was also stimulated to a considerable extent in the same cells. The stimulatory effect of PMA was further enhanced in the presence of okadaic acid, but it was strongly inhibited in the presence of 0.5 microM staurosporine, an inhibitor of protein kinase C. PMA and okadaic acid also stimulated the response to heat stress of the expression of alphaB crystallin, but they barely stimulated the response to heat stress of hsp27. The extent of stimulation of the arsenite-induced responses by PMA and okadaic acid was greater when the concentration of arsenite (i.e. the magnitude of the stress) was relatively low (25-50 microM). The arsenite-induced release of arachidonic acid from cells was also stimulated in the presence of PMA and/or akadaic acid, and the stimulatory effects of PMA and okadaic acid on the arsenite-induced accumulation of alphaB crystallin and hsp27 were strongly suppressed by quinacrine, an inhibitor of phospholipase A2. These results suggest that the stimulatory effects of PMA and okadaicacid on the stress responses are cuased, in part, by the increased metabolic activity of the arachidonic acid cascade, as a consequence of the activation of phospholipase A.

Alkaloids↗
PubMed1994

Purification and characterization of a 20-kDa protein that is highly homologous to alpha B crystallin.

A 20-kDa protein (p20) that had internal amino acid sequences highly similar to those of alpha B crystallin was purified from rat and human skeletal muscle. p20 co-eluted with alpha B crystallin and HSP27/28 during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. p20 was separated from alpha B crystallin and HSP27/28 and was resolved into two fractions, a minor first peak and a major second peak, by column chromatography on S-Sepharose in the presence of 7 M urea. During chromatography on a column of Superdex 75pg, even in the presence of 7 M urea, p20 in the second peak was eluted as aggregates near the exclusion volume of the column, whereas p20 in the first peak was eluted in fractions that corresponded to a lower molecular mass. Further chromatography on a TSK-SP-5PW column yielded pure preparations of each of the two forms of rat and human p20. The fragmentation patterns of the two forms of the respective p20 proteins generated by digestion with endoproteinase Asp-N were identical. The primary structures of rat and human p20, determined with an NH2-terminal sequenator, were highly homologous to those of alpha B crystallin and HSP27/28. p20 was present in all rat tissues examined and at high levels (> 1 micrograms/mg protein) in the soleus muscle, heart, and diaphragm, as are alpha B crystallin and HSP27. Centrifugation on sucrose density gradients allowed detection of the aggregated form and the small form of p20, as well as of HSP27, in extracts of rat muscle tissues. During heating at 45 degrees C of tissue in vitro, p20 in rat diaphragm was redistributed from the cytoplasm to the insoluble fraction, and dissociation of the aggregated p20 to the small form was enhanced. These results suggest that p20 is related to stress proteins.

Amino Acid Sequence↗
PubMed1994

Dissociation as a result of phosphorylation of an aggregated form of the small stress protein, hsp27.

We have found that the small stress protein, hsp27, exists in extracts of U251 MG human glioma cells in two forms: a large or aggregated form (L-hsp27, 300-400 kDa) and a small or dissociated form (S-hsp27, < 70 kDa), as indicated by centrifugation on sucrose density gradients. Dissociation of L-hsp27 to S-hsp27 was enhanced by incubation of cells with phorbol 12-myristate-13 acetate, interleukin-1 alpha, tumor necrosis factor alpha, or okadaic acid, all of which are known to enhance or mimic the effects of phosphorylation of hsp27 without stimulation of its synthesis. Exposure of cells to chemical stressors, namely, NaAsO2 and CdCl2, also enhanced the dissociation of L-hsp27. hsp27 that had been labeled with [32P]H3PO4 in U251 MG cells was detected mostly in fractions that contained S-hsp27, and the incorporation of radioactivity to S-hsp27 was enhanced under conditions that stimulated the dissociation of L-hsp27. L-hsp27 present in the (NH4)2SO4 fraction (0-50% saturation) of cell extracts were dissociated to 32P-labeled hsp27 when incubated in the presence of [gamma-32P]ATP and Mg2+. These results indicate that the molecular configuration of hsp27 in cells is determined in part by phosphorylation and dephosphorylation of this protein by protein kinase(s) and phosphatase(s) and, moreover, that the rapid dissociation of the aggregated form of hsp27 by phosphorylation might be involved in a cellular defense mechanism for protection against stress.

Arsenates↗