PubMed Health⌕ Search

Biomedical subjects

Y Hiroi

Publications and source records attributed to Y Hiroi.

At least 37 records · Page 2Linked to original sources
PubMed1999

Familial atrial septal defect and atrioventricular conduction disturbance associated with a point mutation in the cardiac homeobox gene CSX/NKX2-5 in a Japanese patient.

Atrial septal defect (ASD) is the most common form of congenital cardiac defect in humans. Recently, point mutations in the cardiac homeobox gene CSX/NKX2-5 have been reported to cause the autosomal dominant form of familial ASD. Notably, all the affected patients exhibit atrioventricular conduction disturbance and some of them died suddenly. The first case of familial ASD with a mutation of the CSX/NKX2-5 gene in a Japanese patient is reported here. Identification of CSX/NKX2-5 mutations in ASD patients would be very important because the existence of such mutations may predict sudden cardiac death.

Heart Block↗
PubMed1998

Purification by column chromatographies of beta-amyloid precursor proteins and their association with other 95 kDa protein in rat brain.

Beta-amyloid precursor proteins (APPs) in the subcellular fractions of the homogenate of rat brain were detected immunologically. They were found to be localized in both the cytosol and microsome fractions in generally equal amounts. APPs were purified from the cytosol fraction of rat brain by column chromatography in a DEAE-anion-exchanger, Blue-Sepharose, Ni-charged chelating Sepharose, and Sephacryl S-300 columns. They migrated at about 400 kDa or above in a final gel filtration column with trypsin inhibitor activity. They gave two broad protein bands of 80 and 100 kDa and several other protein bands in sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE). The 80 and 100 kDa bands were highly concentrated during purification. They gave the same amino terminal sequence and were identified as rat APPs without an amino terminal signal sequence. These results suggest that rat brain APPs form a complex with themselves or with other proteins and contain APP isoforms including a serine protease inhibitor domain, APP770 or APP751, or both. An antibody produced by a rabbit immunized with the final preparation of APPs reacted with a 95 kDa protein band which migrated between the 80 and 100 kDa bands of APPs in SDS-PAGE, but it did not react with the bands of APPs. The 80 and 100 kDa APP bands were coprecipitated with a 95 kDa antigen protein band by reacting this antibody with the partially purified APPs. We conclude that APPs in the rat brain are associated directly or indirectly with another protein to yield the 95 kDa band demonstrated by SDS-PAGE.

Amino Acid Sequence↗
PubMed1998

Mechanical stretch induces hypertrophic responses in cardiac myocytes of angiotensin II type 1a receptor knockout mice.

Many lines of evidence have suggested that angiotensin II (AngII) plays an important role in the development of cardiac hypertrophy through AngII type 1 receptor (AT1). To determine whether AngII is indispensable for the development of mechanical stress-induced cardiac hypertrophy, we examined the activity of mitogen-activated protein kinase (MAPK) family and the expression of the c-fos gene as hypertrophic responses after stretching cultured cardiac myocytes of AT1a knockout (KO) mice. When cardiac myocytes were stretched by 20% for 10 min, extracellular signal-regulated protein kinases (ERKs) were strongly activated in KO cardiomyocytes as well as wild type (WT) myocytes. Both basal and stimulated levels of ERKs were higher in cardiomyocytes of KO mice than in those of WT mice. Activation of another member of the MAPK family, p38(MAPK), and expression of the c-fos gene were also induced by stretching cardiac myocytes of both types of mice. An AT1 antagonist attenuated stretch-induced activation of ERKs in WT cardiomyocytes but not in KO cardiomyocytes. Down-regulation of protein kinase C inhibited stretch-induced ERK activation in WT cardiomyocytes, whereas a broad spectrum tyrosine kinase inhibitor (genistein) and selective inhibitors of epidermal growth factor receptor (tyrphostin, AG1478, and B42) suppressed stretch-induced activation of ERKs in KO cardiac myocytes. Epidermal growth factor receptor was phosphorylated at tyrosine residues by stretching cardiac myocytes of KO mice. These results suggest that mechanical stretch could evoke hypertrophic responses in cardiac myocytes that lack the AT1 signaling pathway possibly through tyrosine kinase activation.

Animals↗
PubMed1998

Cell type-specific angiotensin II-evoked signal transduction pathways: critical roles of Gbetagamma subunit, Src family, and Ras in cardiac fibroblasts.

Angiotensin II (Ang II) induces hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. To determine the molecular mechanism by which Ang II displayed different effects on cardiac myocytes and fibroblasts, we examined signal transduction pathways leading to activation of extracellular signal-regulated kinases (ERKs). Ang II-induced ERK activation was abolished by pretreatment with pertussis toxin and by overexpression of the Gbetagamma subunit-binding domain of the beta-adrenergic receptor kinase 1 in cardiac fibroblasts but not in cardiac myocytes. Inhibition of protein kinase C strongly inhibited activation of ERKs by Ang II in cardiac myocytes, whereas inhibitors of tyrosine kinases but not of protein kinase C abolished Ang II-induced ERK activation in cardiac fibroblasts. Overexpression of C-terminal Src kinase (Csk), which inactivates Src family tyrosine kinases, suppressed the activation of transfected ERK in cardiac fibroblasts. Ang II rapidly induced phosphorylation of Shc and association of Shc with Grb2. Cotransfection of the dominant-negative mutant of Ras or Raf-1 kinase abolished Ang II-induced ERK activation in cardiac fibroblasts. Overexpression of Csk or the dominant-negative mutant of Ras had no effects on Ang II-induced ERK activation in cardiac myocytes. These findings suggest that Ang II-evoked signal transduction pathways differ among cell types. In cardiac fibroblasts, Ang II activates ERKs through a pathway including the Gbetagamma subunit of Gi protein, tyrosine kinases including Src family tyrosine kinases, Shc, Grb2, Ras, and Raf-1 kinase, whereas Gq and protein kinase C are important in cardiac myocytes.

Angiotensin II↗
PubMed1998

Molecular cloning of human homolog of yeast GAA1 which is required for attachment of glycosylphosphatidylinositols to proteins.

Anchoring proteins to cell surface membranes by glycosylphosphatidylinositols (GPIs) is important. We have isolated a component of the putative transamidase machinery, hGaa1p (human GPI anchor attachment protein). hGAA1 cDNA is approximately 2 kb in length and codes 621 amino acids. The amino acid sequence of hGaa1p is 25%, identical and 57% homologous to that of yeast Gaa1p. Moreover, Kite-Dolittle hydrophobicity plots of both proteins show marked similarity. hGAA1 gene is expressed ubiquitously and mRNA levels are higher in the undifferentiated state. Overexpression of antisense hGAA1 in human K562 cells significantly reduced the production of a reporter GPI-anchored protein.

Amino Acid Sequence↗
PubMed1998

The Rho family G proteins play a critical role in muscle differentiation.

The Rho family GTP-binding proteins play a critical role in a variety of cytoskeleton-dependent cell functions. In this study, we examined the role of Rho family G proteins in muscle differentiation. Dominant negative forms of Rho family proteins and RhoGDI, a GDP dissociation inhibitor, suppressed transcription of muscle-specific genes, while mutationally activated forms of Rho family proteins strongly activated their transcription. C2C12 cells overexpressing RhoGDI (C2C12RhoGDI cells) did not differentiate into myotubes, and expression levels of myogenin, MRF4, and contractile protein genes but not MyoD and myf5 genes were markedly reduced in C2C12RhoGDI cells. The promoter activity of the myogenin gene was suppressed by dominant negative mutants of Rho family proteins and was reduced in C2C12RhoGDI cells. Expression of myocyte enhancer binding factor 2 (MEF2), which has been reported to be required for the expression of the myogenin gene, was reduced at the mRNA and protein levels in C2C12RhoGDI cells. These results suggest that the Rho family proteins play a critical role in muscle differentiation, possibly by regulating the expression of the myogenin and MEF2 genes.

Actins↗
PubMed1998

Efficient inhibition of the development of cardiac remodeling by a long-acting calcium antagonist amlodipine.

The purpose of the present study was to examine the effects of a long-acting calcium antagonist, amlodipine, on the development of cardiac remodeling. Dihydropyridine calcium antagonists have been used widely for many years in the treatment of hypertension and angina pectoris. It has been reported, however, that a prototype of dihydropyridines, nifedipine, does not reduce mortality of patients with ischemic heart disease, possibly because of reflex stimulation of the sympathetic nervous system. A calcium antagonist, amlodipine, has been reported to have potential benefits by virtue of a gradual onset of action and a long duration of effects. Amlodipine (8 mg/kg per day, once a day) or nifedipine (24 mg/kg per day, three times a day) was administered to spontaneously hypertensive 12-week-old rats for 12 weeks. Left ventricular wall thickness was measured by echocardiography, and relative amounts of myosin heavy chain isoforms were assessed by pyrophosphate gels. Expressions of "fetal type" genes and type 1 collagen gene were examined by Northern blot analysis. Amlodipine and nifedipine both markedly reduced systolic blood pressure. However, the decrease in systolic blood pressure caused by nifedipine continued for no more than 8 hours, whereas the blood pressure-lowering effect of amlodipine continued for more than 16 hours post dose. Amlodipine markedly reduced left ventricular wall thickness, whereas nifedipine only weakly attenuated an increase in the wall thickness. Amlodipine, but not nifedipine, prevented an increase in the relative amount of V3 myosin heavy chain isoform and suppressed an increase in mRNA levels of beta-myosin heavy chain, skeletal alpha-actin, and type 1 collagen. Unlike nifedipine, amlodipine effectively prevented cardiac remodeling secondary to high blood pressure at biochemical levels and morphological levels. These results suggest that a long-acting calcium antagonist is more effective than a short-acting one in preventing organ injury in hypertensive subjects.

Amlodipine↗
PubMed1998

Degradation of serum albumin by rat liver and kidney lysosomes.

Lysosomes were isolated from the livers and from the kidneys of rats treated or not treated with the cysteine proteinase inhibitor leupeptin, and the levels of the intralysosomal serum albumin of the leupeptin-treated rats were compared with those of the saline-treated control rats. Leupeptin caused an intralysosomal accumulation of albumin in vivo because of its potent inhibition of lysosomal protein degradation. In fact, the lysosomes isolated from the livers and kidneys of leupeptin-treated rats almost completely lost their ability to degrade rat albumin in vitro. These findings show that the lysosomes are subcellular sites of the degradation of unlabeled serum albumin in these tissues. They also suggest that cysteine proteinases sensitive to leupeptin are involved in the lysosomal degradation of albumin. Albumin was degraded by total lysosomal enzymes in vitro. It was also degraded by the lysosomal extract being devoid of cathepsins H and J, prepared from rat kidney. The degradation of albumin by total lysosomal enzymes in vitro was greatly suppressed by a cysteine proteinase inhibitor, cystatin alpha, with no inhibition of cathepsins B and L. It was slightly suppressed by N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prol ine (CA-074), a selective inhibitor of cathepsin B, and by pepstatin, an inhibitor of cathepsin D, whereas it was markedly suppressed by a combination of cystatin alpha and either CA-074 or pepstatin. These and associated findings show that cystatin alpha-sensitive cysteine proteinase(s), which is distinct from cathepsins B, H, L, and J, and cathepsins B and D are involved in the lysosomal degradation of albumin.

Animals↗
PubMed1997

Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats.

A growing body of evidence has suggested that oxidative stress causes cardiac injuries during ischemia/reperfusion. Extracellular signal-regulated kinases (ERKs) have been reported to play pivotal roles in many aspects of cell functions and to be activated by oxidative stress in some types of cells. In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of ERKs in cultured cardiomyocytes of neonatal rats, and determined their role in oxidative stress-induced cardiomyocyte injuries. ERKs were transiently and concentration-dependently activated by hydrogen peroxide (H2O2) in cardiac myocytes. A specific tyrosine kinase inhibitor, genistein, suppressed H2O2-induced ERK activation, while inhibitors of protein kinase A and C or Ca2+ chelators had no effects on the activation. When CSK, a negative regulator of Src family tyrosine kinases, or dominant-negative mutant of Ras or of Raf-1 kinase was overexpressed, activation of transfected ERK2 by H2O2 was abolished. The treatment with H2O2 increased the number of cells stained positive by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and induced formation of DNA ladder and activation of CPP32, suggesting that H2O2 induced apoptosis of cardiac myocytes. When H2O2-induced activation of ERKs was selectively inhibited by PD98059, the number of cardiac myocytes which showed apoptotic death was increased. These results suggest that Src family tyrosine kinases, Ras and Raf-1 are critical for ERK activation by hydroxyl radicals and that activation of ERKs may play an important role in protecting cardiac myocytes from apoptotic death following oxidative stress.

Animals↗
PubMed1997

Norepinephrine induces the raf-1 kinase/mitogen-activated protein kinase cascade through both alpha 1- and beta-adrenoceptors.

BACKGROUND: Although norepinephrine induces cardiac hypertrophy by activating protein kinase A and C through beta- and alpha 1-adrenoceptors, respectively, protein kinase A has been reported to inhibit cell growth in many other cell types. METHODS AND RESULTS: To elucidate the molecular mechanism of norepinephrine-induced hypertrophic responses, we examined the effects of protein kinase A and protein kinase C on the activities of raf-1 kinase and mitogen-activated protein (MAP) kinases and on protein synthesis rates using cultured cardiomyocytes of neonatal rats. Norepinephrine-induced activation of MAP kinases was partially inhibited by either an alpha 1-adrenoceptor blocker (prazosin) or a beta-adrenoceptor blocker (propranolol) and was completely abolished by both blockers. Both a beta-adrenoceptor agonist, isoproterenol, and an alpha 1-adrenoceptor agonist, phenylephrine, increased the activities of raf-1 kinase and MAP kinases and phenylalanine incorporation into proteins. Furthermore, isoproterenol and phenylephrine synergistically activated these kinases and protein synthesis. Similar synergistic activation of MAP kinases was observed when other protein kinase A-activating agents such as forskolin, dibutyryl cAMP, and isobutyl-methylxanthine were used with a protein kinase C-activating agent at the same time. Chelation of extracellular Ca2+ completely abolished isoproterenol- and phenylephrine-evoked MAP kinase activation. CONCLUSIONS: Norepinephrine activates the raf-1 kinase/MAP kinase cascade through both alpha 1- and beta-adrenergic stimulation, and signaling pathways from the two receptors synergistically induce cardiomyocyte hypertrophy.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Downregulation of polo-like kinase correlates with loss of proliferative ability of cardiac myocytes.

Cardiac myocytes rapidly increase the cell number during the fetal and early neonatal period, but they lose their proliferative ability soon after birth. To understand the mechanism of how cardiac myocytes exit from the cell cycle, we examined the role of a newly identified serine/threonine kinase, polo-like kinase (Plk), in the process of proliferation of cardiac myocytes. Northern blot analysis revealed that Plk gene was abundantly expressed in cardiac myocytes and non-myocytes of fetal and neonatal rats but not in cardiocytes of adult rats. Western blot analysis showed that Plk protein was also detected only in fetal and neonatal hearts. During the early stage of cardiac differentiation. Plk expression was well correlated with the proliferative ability of cardiocytes. Plk mRNA was most abundant in undifferentiated embryonic stem (ES) cells and the mRNA levels decreased along with cardiac differentiation in the developing ES cell system. Once serum was deprived from the culture media, expression levels of Plk were markedly decreased and DNA was not synthesized in both cardiac myocytes and non-myocytes of neonatal rats. Re-addition of serum stimulated Plk gene expression and DNA synthesis in non-myocytes but not in cardiomyocytes. All these results taken together with the critical role of Plk in DNA synthesis in many cell types suggest that downregulation of Plk is important for the permanent withdrawal of cardiomyocytes from the cell cycle.

Animals↗
PubMed1997

Protein kinase A and protein kinase C synergistically activate the Raf-1 kinase/mitogen-activated protein kinase cascade in neonatal rat cardiomyocytes.

Adrenoceptor agonists play an important role in cardiac hypertrophy. In cardiomyocytes, activation of alpha- and beta-adrenoceptors induces a variety of hypertrophic responses via activation of protein kinase C (PKC) and protein kinase A (PKA), respectively. Although PKC evokes activation of the Raf-1 kinase (Raf-1)/mitogen-activated protein (MAP) kinase cascade, PKA has been shown to inhibit the activation of Raf-1 and MAP kinases induced by growth factors in various cell types. The present study was performed to elucidate the role of PKA and PKC in cardiomyocyte hypertrophy. PKA activators such as forskolin (FSK), isobutylmethylxanthine, dibutyryl cAMP and isoproterenol, significantly activated Raf-1 and MAP kinases with a peak at 2 and 8 min, respectively, followed by an increase in protein synthesis in cardiac myocytes. Similar responses were observed when cardiomyocytes were stimulated with PKC activators such as 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II, phenylephrine and mechanical stretch. After depleting extracellular Ca2+ with EGTA, FSK did not activate MAP kinases, while down-regulation of PKC by long exposure with TPA did not influence FSK-induced MAP kinase activation. Furthermore, FSK and TPA synergistically activated Raf-1. Similar synergistic activation of MAP kinases was observed when other PKC activators were added to cardiac myocytes with FSK at the same time. In conclusion, unlike other cell types, PKA activates Raf-1 and MAP kinases followed by an increase in protein synthesis in cardiac myocytes.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Autoregulation of human cardiac homeobox gene CSX1: mediation by the enhancer element in the first intron.

Csx/Nkx-2.5 is a murine homeobox gene expressed predominantly in cardiocytes and their progenitor cells. The highly lineage-restricted expression pattern of Csx/Nkx-2.5 gene suggests the existence of a positive autoregulatory loop in the transcriptional regulation of Csx/Nkx-2.5. The first intron of CSX1, a human homolog of Csx/Nkx-2.5 gene, had two potential CSX1-binding sequences. Activity of the CSX1 minimal promoter in cultured cardiac myocytes was significantly increased by placing the 3' half of the CSX1 first intron downstream of the reporter gene, suggesting that this region functions as a positive enhancer element. Transient transfection experiments in nonmuscle cells demonstrated that the reporter construct containing the CSX1 minimal promoter and the 3' half of the CSX1 first intron was strongly transactivated by overexpression of CSX1, whereas the CSX1 minimal promoter alone was not. Together these results suggest that the highly lineage-restricted expression of CSX1 is accomplished by autoactivation, which may be mediated by the enhancer element in the first intron.

Animals↗
PubMed1996

Protein kinase C, but not tyrosine kinases or Ras, plays a critical role in angiotensin II-induced activation of Raf-1 kinase and extracellular signal-regulated protein kinases in cardiac myocytes.

Angiotensin II (AngII) induces cardiac hypertrophy through activating a variety of protein kinases. In this study, to understand how cardiac hypertrophy develops, we examined AngII-evoked signal transduction pathways leading to the activation of extracellular signal-regulated protein kinases (ERKs), which are reportedly critical for the development of cardiac hypertrophy, in cultured cardiac myocytes isolated from neonatal rats. Inhibition of protein kinase C (PKC) with calphostin C or down-regulation of PKC by pretreatment with a phorbol ester for 24 h abolished AngII-induced activation of Raf-1 and ERKs, and addition of a phorbol ester conversely induced a marked increase in the activities of Raf-1 and ERKs. Pretreatment with two chemically and mechanistically dissimilar tyrosine kinase inhibitors, genistein and tyrphostin, did not attenuate AngII-induced activation of ERKs. In contrast, genistein strongly blocked insulin-induced ERK activation in cardiac myocytes. Although pretreatment with manumycin, a Ras farnesyltransferase inhibitor, or overexpression of a dominant-negative mutant of Ras inhibited insulin-induced ERK activation, neither affected AngII-induced activation of ERKs. Overexpression of a dominant-negative mutant of Raf-1 completely suppressed ERK2 activation by AngII, endothelin-1, and insulin. These results suggest that PKC and Raf-1, but not tyrosine kinases or Ras, are critical for AngII-induced activation of ERKs in cardiac myocytes.

Angiotensin II↗
PubMed1996

Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy.

We have recently shown that mechanical stress induces cardiomyocyte hypertrophy partly through the enhanced secretion of angiotensin II (ATII). Endothelin-1 (ET-1) has been reported to be a potent growth factor for a variety of cells, including cardiomyocytes. In this study, we examined the role of ET-1 in mechanical stress-induced cardiac hypertrophy by using cultured cardiomyocytes of neonatal rats. ET-1 (10(-8) approximately 10(-7) M) maximally induced the activation of both Raf-1 kinase and mitogen-activated protein (MAP) kinases at 4 and 8 min, respectively, followed by an increase in protein synthesis at 24 h. All of these hypertrophic responses were completely blocked by pretreatment with BQ123, an antagonist selective for the ET-1 type A receptor subtype, but not by BQ788, an ET-1 type B receptor-specific antagonist. BQ123 also suppressed stretch-induced activation of MAP kinases and an increase in phenylalanine uptake by approximately 60 and 50%, respectively, but BQ788 did not. ET-1 was constitutively secreted from cultured cardiomyocytes, and a significant increase in ET-1 concentration was observed in the culture medium of cardiomyocytes after stretching for 10 min. After 24 h, an approximately 3-fold increase in ET-1 concentration was observed in the conditioned medium of stretched cardiomyocytes compared with that of unstretched cardiomyocytes. ET-1 mRNA levels were also increased at 30 min after stretching. Moreover, ET-1 and ATII synergistically activated Raf-1 kinase and MAP kinases in cultured cardiomyocytes. In conclusion, mechanical stretching stimulates secretion and production of ET-1 in cultured cardiomyocytes, and vasoconstrictive peptides such as ATII and ET-1 may play an important role in mechanical stress-induced cardiac hypertrophy.

Animals↗
PubMed1995

Activation of Na(+)-H+ antiporter (NHE-1) gene expression during growth, hypertrophy and proliferation of the rabbit cardiovascular system.

The Na(+)-H+ antiporter is a unique transmembrane protein with multiple roles in cellular functions through intracellular alkalization. It participates in the regulation of intracellular pH, cell volume and intracellular signalling in response to various mitogenic stimuli. To clarify its role as a subcellular signal in cardiovascular remodeling like vascular hyperplasia or cardiac hypertrophy, we determined mRNA levels of the Na(+)-H+ antiporter isoform, NHE-1, in vascular smooth muscles and pressure-overloaded hearts in rabbits. The NHE-1 mRNA levels in rabbit aortas and hearts were developmentally regulated with high levels at embryonic and neonatal stages than in adults. In primary-cultured smooth muscle cells (SMC), the mRNA levels were increased during exponential growth, but decreased to initial levels at confluency. Growth of a mutant SMC line, C5, which is deficient in Na(+)-H+ antiporter activity, was markedly reduced in bicarbonate-free medium. However, when the activity was restored by transfecting cells with a full-length NHE-1 cDNA in an expression vector, the growth rate of C5 was accelerated again. After balloon injury to the vascular wall, the NHE-1 mRNA levels of the injured arteries were also increased, suggesting that Na(+)-H+ antiporter contributes to the network of the growth promoting systems in smooth muscle cells in vivo. Pressure-overload on the ventricle increased the NHE-1 mRNA levels in hearts approximately two-fold of sham-operated rabbits after 3 days and remained for at least two weeks (P < 0.05). We further demonstrated that 3-methylsulfonyl-4-piperidino-benzoyl guanidine mesylate (Hoe 694), a potent antagonist of Na(+)-H+ antiporter, partially inhibited stretch-induced activation of mitogen-activated kinase (MAP kinase) in the cultured cardiomyocytes. From these results, we conclude that activation of the Na(+)-H+ antiporter and its gene expression is involved in molecular mechanisms of both cardiac hypertrophy and vascular smooth muscle cell proliferation, indicating a potential target in developing new therapeutics for cardiovascular diseases.

Aging↗