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Y Inaguma

Publications and source records attributed to Y Inaguma.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1993

Responses to heat shock of alpha B crystallin and HSP28 in U373 MG human glioma cells.

Responses to heat shock of alpha B crystallin and small heat-shock protein HSP28, which are expressed at considerable levels in human astroglioma U373 MG cells (2-4 micrograms of each per mg soluble protein in confluent cultures), were analysed quantitatively by specific immunoassays. Concentrations of alpha B crystallin and HSP28 in soluble extracts of U373 MG cells decreased to about 50% of original values, with an increase in the insoluble fraction, during heat treatment for 15 min at 45 degrees C. The concentrations of alpha B crystallin and HSP28 increased gradually upon return to 37 degrees C, reaching and then exceeding the control levels within 5 h and 10 h, respectively, after heat shock. During centrifugation on sucrose density gradients both alpha B crystallin and HSP28 in extracts from untreated and heat-treated cells sedimented at the same position, which corresponded to a molecular mass of > 540 kDa. This result suggests that the sizes of aggregates of the two proteins in the cytoplasm are not affected by heat shock. Both alpha B crystallin and HSP28 in an extract of U373 MG cells were trapped on and coeluted from an affinity column prepared with antibodies against alpha B crystallin. These results suggest that the two proteins are also associated in U373 MG cells.

Cell Division↗
PubMed1993

Alpha B crystallin and HSP28 are enhanced in the cerebral cortex of patients with Alzheimer's disease.

The localization of two small heat shock proteins, alpha B crystallin and 28-kDa heat shock protein (HSP28), was studied immunochemically and immunohistochemically in cerebral cortex of patients with Alzheimer's disease (AD) and control groups. By specific immunoassays for alpha B crystallin and HSP28, we found that the concentrations of alpha B crystallin are elevated in the temporal and frontal lobes, while those of HSP28 are elevated in the temporal, frontal, and parietal lobes in the AD brains. Immunohistochemically, alpha B crystallin was localized in astrocytes and oligodendrocytes, while HSP28 was present in degenerating neurons in the cerebral cortex of both AD and control brains. However, alpha B crystallin-immunoreactive astrocytes and HSP28-immunoreactive degenerating neurons were more frequently observed in AD brains. The immunoreactivity for HSP28 was also found in the senile plaques in the AD brains. These findings suggest that the increased accumulations of these small heat shock proteins appear to be part of reactive processes of glial cells and neurons under pathologic conditions.

Aged↗
PubMed1993

Physiological and pathological changes in levels of the two small stress proteins, HSP27 and alpha B crystallin, in rat hindlimb muscles.

The two small stress proteins, HSP27 and alpha B crystallin, are expressed widely in normal rat tissues and abundantly in skeletal muscle. In order to clarify the physiological significance of these stress proteins, the changes in their levels were determined immunochemically, in the slow-twitch soleus muscle and fast-twitch extensor digitorum longus muscle or rectus femoris muscle of growing rats, and in those of adult rats during denervation and tenotomy. HSP27 was quantitated by specific immunoassay, similar to that for alpha B crystallin, with antibodies raised in rabbits against purified rat HSP27. In adult rats, HSP27 was present at high levels in tissues composed of striated muscle, and it was present at much higher levels in the soleus muscle than in the rectus femoris or extensor digitorum longus muscle, as is alpha B crystallin. However, in rats of perinatal age (from prenatal day 2 to postnatal day 3), levels of HSP27 in the rectus femoris muscle were enhanced like those in the soleus muscle, reaching the maximum levels at postnatal day 3. Thereafter HSP27 in the fast-twitch muscle showed a steep decrease. The increase in alpha B crystallin in the hindlimb muscles was also observed in the perinatal period. However, alpha B crystallin concentrations in the soleus muscle of perinatal rats were as low as those in rectus femoris muscle. The transection of the sciatic nerve resulted in decreases in the levels of HSP27 and alpha B crystallin in the soleus muscle of adult rats, together with increases in the levels of the two proteins in the extensor digitorum longus muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Coinduction of two low-molecular-weight stress proteins, alpha B crystallin and HSP28, by heat or arsenite stress in human glioma cells.

The responses of two low-molecular-weight stress proteins, alpha B crystallin and HSP28, to various types of stress were determined quantitatively by specific immunoassays in a human glioblastoma cell line (U118 MG). Levels of alpha B crystallin (2-4 ng/mg protein) and HSP28 (1-1.5 micrograms/mg protein) in the soluble fraction from cells that had been cultured at 37 degrees C increased about 100-fold and 3-fold, respectively, within 24 h after heat treatment for 15 min at 45 degrees C, with a temporary decrease, due to redistribution to the insoluble fraction, during the heat treatment. Exposure of cells to arsenite (NaAsO2, 100 microM for 1 h) also induced the two proteins with a time course similar to that observed after heat stress, but without a decrease during the stress period. L-Azetidine-2-carboxylate (5 mM for 5 h) was also effective in inducing the two proteins, but to a lesser extent. Other chemicals, including CdCl2, ZnCl2, AlCl3, ethanol, caffeine, nicotine, NaN3, dibutyryl 3',5'-cyclic AMP, forskolin, and a phorbol ester, did not induce the two proteins. Expression of alpha B crystallin and HSP28 mRNAs in cells was enhanced after heat stress and after exposure to arsenite. When cells were challenged with heat stress in the presence of arsenite, the effect on the induction of the two proteins was synergistic. Ethanol (1-2%) enhanced the responses to heat stress or arsenite stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Arsenites↗
PubMed1992

Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.

Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a column of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.

Amino Acid Sequence↗