Biomedical subjects

M Kurabayashi

Publications and source records attributed to M Kurabayashi.

At least 127 records · Page 7Linked to original sources

PubMed1993

[Regulation of the myosin heavy chain gene expression during cardiac hypertrophy induced by pressure-overload].

In response to pressure-overload, the myocardium exhibits a complex molecular changes, characterized by the reexpression of a fetal phenotypes. Although signaling pathways involved in these changes are still largely unknown, studies on the differential expression of the myosin heavy chain (MHC) genes will provided a framework for understanding the molecular mechanisms underlying the reprogramming of cardiac gene expression in response to mechanical load. Recently, in vitro medel of myocardial stretch on the silicone sheet has demonstrated the up regulation of the "cardiac fetal genes", including beta-MHC, skeletal alpha-actin, and atrial natriuretic factor (ANF), which is known to be a hallmark of the altered gene expression in the hypertrophic heart. Transient transfection experiments using this model will be useful for identifying the "stretch-response element" within the promoters of these genes. Furthermore, this approach will eventually identify the stretch-induced intracellular pathways.

Animals↗

PubMed1991

Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes. Possible role of protein kinase C activation.

To examine the molecular mechanisms by which mechanical stimuli induce cardiac hypertrophy and specific gene expression, we cultured rat neonatal cardiocytes in deformable dishes and imposed an in vitro mechanical load by stretching the adherent cells. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos and skeletal alpha-actin. Nuclear run-off transcription assay revealed that this increase in c-fos mRNA level by stretching at least partially reflects changes in the transcriptional status. The transfected chloramphenicol acetyltransferase gene linked to upstream sequences of the fos gene indicated that sequences containing a serum response element were required for efficient transcription by stretching and that sequences containing a cAMP/calcium response element might not be involved in the c-fos response to myocyte stretching. The accumulation of c-fos mRNA by stretching was suppressed by protein kinase C inhibitors at the transcriptional level and inhibited markedly by down-regulation of protein kinase C. Moreover, myocyte stretching increased inositol phosphate levels, and activation of protein kinase C by phorbol esters stimulated the expression of c-fos and skeletal alpha-actin genes. These findings suggest that mechanical stimuli (myocyte stretching) might directly induce cardiac hypertrophy and specific gene expression possibly via protein kinase C activation.

Actins↗

PubMed1990

Functional identification of the transcriptional regulatory elements within the promoter region of the human ventricular myosin alkali light chain gene.

We have identified and functionally characterized DNA sequences that regulate the expression of the human ventricular/slow twitch isoform of myosin alkali light chain (VLC1) gene. By using primer extension and S1 nuclease mapping techniques, we have shown that the VLC1 gene is transcribed from the identical site in the ventricular and slow twitch skeletal muscles. Comparison of the VLC1 sequences from +1 to -1296 in the genes for human and mouse showed that the 5'-proximal flanking region, up to about 220 nucleotides, was highly conserved (83% homology). To determine the location of sites that may be important for the function of the VLC1 promoter, a series of transient expression vectors containing progressive deletions of the VLC1 gene 5'-flanking sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) gene was introduced into myogenic and nonmyogenic cells. Deletion mutagenesis of sequences between -357 and +40 revealed the presence of positive and negative activity in all the cells tested. We demonstrated that the minimal promoter sequence required to generate muscle cell-specific expression is the region between -94 to -64 upstream from the cap site and a sequence element located between -107 and -94 was found to have a positive effect in both myogenic cells and nonmyogenic cells. These two proximal regions located between -107 and -64 appear to act together to determine the cell type-specific high level expression of the VLC1 gene in muscle cells. Competition gel retardation assays revealed that the CArG sequence located between -96 and -87 interacts specifically with nuclear extracts from myogenic and nonmyogenic cells and compete for binding with the CArG sequence present in the human cardiac alpha-actin gene and with the serum response element of the c-fos gene. These results strongly suggested that similar, if not identical, the CArG box binding proteins interact with the functionally different promoter element in the VLC1, cardiac alpha-actin, and c-fos genes.

Animals↗

PubMed1990

Stretching cardiac myocytes stimulates protooncogene expression.

Recently cellular protooncogenes have been found to be induced as an early response to pressure overload in cardiac hypertrophy. To examine whether mechanical stimuli directly induce specific gene expression in the heart, we cultured rat neonatal cardiocytes in elastic silicone dishes and stretched these adherent cells. Myocyte stretching stimulated expression of the protooncogene, c-fos, in a stretch length-dependent manner, followed by an increase in amino acid incorporation into proteins. c-fos mRNA levels were enhanced within 15 min by cardiocyte stretching, peaked at 30 min, and declined to undetectable levels by 240 min. In the presence of cycloheximide, a greater increase in c-fos mRNA was seen by stretching. The transfected chloramphenicol acetyltransferase gene linked to upstream sequences of the fos gene including its promoter was also activated by stretching cardiac myocytes. These results suggest that mechanical loading directly regulates gene transcription without the participation of humoral factors in cardiocytes.

Animals↗

PubMed1990

Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates.

Applicability of negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) was examined in trace mixture analyses and structural assignments of some isoprenoid diphosphates. Negative ion FAB-MS spectra using a glycerol matrix of these isoprenoid diphosphates showed predominantly molecular ions (M-H)- together with fragment ions at m/z 177 (H3P2O7)-, 176 (H2P2O7)-, 159 (HP2O6)-, and 79 (PO3)- which were characteristic of the diphosphate ester moiety. The molecular ions did not overlap with peaks arising from any impurities even when crude sample such as butanol extracts from enzymatic reaction mixtures were directly analyzed without any purification. Moreover, collisionally activated dissociation spectra of the molecular ion showed many structurally significant fragment ions which enabled us to elucidate the structures of such irregular alkyl chain moieties as those having a homoisoprenoid skeleton or substituted structures. These studies indicate that negative ion FAB-MS/MS is a simple and useful technique for trace mixture analysis and structure elucidation of isoprenoid diphosphates.

Esters↗

PubMed1990

Molecular cloning of gene sequences from rat heart rapidly responsive to pressure overload.

Although pressure overload induces cardiac hypertrophy and reexpression of contractile protein isogenes, the molecular mechanism of both is not well understood. We demonstrated the early change in the translational activity of specific cardiac messenger RNA by two-dimensional gel electrophoresis of in vitro translational products. A total of over 400 translational products were detected by fluorography, and the relative predominance of eight species was increased by pressure overload whereas that of two translational products was decreased. We cloned four pressure-overload-responsive complementary DNA clones, pPO-1, -4, -5, and -6, by differential dot blot hybridization. Two clones were increased from the early period after the imposition of pressure overload; the other two were decreased. The expression pattern of each complementary DNA clone in the pressure-overloaded hearts was similar to that in fetal hearts. Pressure overload also induced the additional messenger RNA, which hybridized with pPO-4. This mRNA was also recognized in fetal, neonatal, and adult spontaneously hypertensive rat hearts. The induction of this transcript by pressure overload was not suppressed but, rather, stimulated by cycloheximide. These results suggest that there are some genes that rapidly respond to pressure overload and that may play some role in the development of cardiac hypertrophy.

Acute Disease↗

PubMed1990

The myosin gene switching in human cardiac hypertrophy.

Cardiac hypertrophy is associated with qualitative as well as quantitative changes in myocardial cells. To analyze the molecular basis of isozymic transitions of cardiac myosins in response to pressure overload, we have constructed and characterized two types of myosin heavy chain (MHC) cDNA clones, specifying alpha- and beta-MHCs, and two types of myosin alkali light chain cDNA clones, complementary to atrial type (ALC1) and ventricular type (VLC1) mRNAs from a human fetal heart cDNA library. Using the S1 nuclease mapping procedure, we showed that the MCH isozymic transitions from alpha- to beta-MHC in the pressure overloaded atria are produced by changes in the relative level of alpha- and beta-MHC gene expression. In addition, we observed that the expression of VLC1 gene is also induced in the atria subjected to severe pressure overload. Thus, it appears that the increased expression of VLC1 gene, together with the isogene switch from alpha- to beta-MHC gene, may participate in the adaptation of myocardium to new functional requirement. Then, to get a better understanding of the genetic mechanisms involved in the regulation of isogene expression, we have isolated and sequenced genomic clone for VLC1 isoform. Sequence analysis has identified multiple potential cis regulatory elements within a 686-bp upstream region. This region includes 28-bp alternating purine/pyrimidine sequences and two segment exhibiting homology to consensus sequence proposed for viral and cellular enhancer elements. In particular, a comparison of the VLC1 upstream gene sequence with those available for several muscle-specific genes revealed that CC(A + T-rich)6GG elements and CATTCCT sequence are conserved. These results suggested that CArG box (-96 to -87) has an important role in the positive regulation of the VLC1 gene and this element may be involved in the co-regulation of VLC1 and cardiac alpha-actin genes.

Adaptation, Physiological↗

PubMed1990

Molecular mechanism of cardiac hypertrophy.

Pressure overload induces cardiac hypertrophy and reexpression of contractile protein isogenes. To ascertain the molecular mechanism of these events, we examined the expression of cellular oncogenes and the early change in the translational activity of specific cardiac mRNA by two-dimensional gel electrophoresis of in vitro translational products. Pressure overload increased the expression levels of c-fos, c-myc, and c-Ha-ras genes. The relative predominance of 8 species out of over 400 translational products was increased by pressure overload while that of 2 translational products was decreased. We cloned four pressure-overload-responsive cDNA clones by differential dot blot hybridization. The expression pattern of each cDNA clone in the pressure-overloaded hearts was similar to that in fetal hearts. To examine whether mechanical stimuli directly induce specific gene expression in the heart, we cultured rat neonatal cardiocytes in elastic silicone dishes and stretched these adherent cells. Myocytes stretching stimulated amino acid uptake and expression of the c-fos gene, which was blocked by protein kinase C inhibitors. These results suggest that there are some early responsive genes in cardiac hypertrophy and that mechanical loading directly stimulates gene expression possibly via protein kinase C activation.

Amino Acids↗

PubMed1989

Molecular adaptation to pressure overload in human and rat hearts.

The process of enlargement of the heart due to overload involves a significant reconstitution of the organ including myocytes and intracellular constituents. We demonstrated the distribution of two types of cardiac myosin heavy chains (HC alpha and HC beta) in the human heart using monoclonal antibodies. The ventricle comprised mainly HC beta which has low ATPase activity, whereas the atrium was predominantly composed of HC alpha which has high ATPase activity. We also demonstrated isozymic transition of HC alpha to HC beta in the human atrium and ventricle by hemodynamic overload, regarded as a compensatory mechanism to meet an increased demand in work. To examine the molecular mechanism for the expression of these HCs, we have isolated human HC alpha and HC beta cDNA clones from a fetal heart cDNA library. Comparison of the nucleotide and amino acid sequences deduced from the DNA between these cDNA clones showed 91 and 96% homology, respectively. Using HC alpha and HC beta gene-specific sequences, we demonstrated that the transition of HC alpha to HC beta in the overloaded human heart was induced by the expression of HC beta-gene. To determine the role of cellular oncogenes in the process of cardiac growth and hypertrophy, we examined the expression pattern of eight cellular oncogenes during the developmental stage and pressure-overloaded hypertrophy of the rat heart by Northern blot analysis. c-fos, c-myc and c-Ha-ras were expressed in the heart in response to pressure overload and in a stage-specific manner, suggesting that these cellular oncogenes participate in the normal developmental process and hypertrophy of the heart. We also cloned the genes of which expression level was rapidly changed by pressure overload by differential hybridization technique. Our results suggest that clone 4 may be involved in the molecular mechanism for the development of cardiac hypertrophy due to overload.

Adaptation, Physiological↗

PubMed1989

Molecular cloning and characterization of a Ca2+ + Mg2+-dependent adenosine triphosphatase from rat cardiac sarcoplasmic reticulum. Regulation of its expression by pressure overload and developmental stage.

To investigate the regulation of expression of cardiac Ca2+ + Mg2+-dependent ATPase (Ca2+-ATPase) in sarcoplasmic reticulum (SR), we isolated cDNA (pHA6) encoding a Ca2+-ATPase of rat cardiac SR. The clone consisted of 2,311 mRNA-derived nucleotides, which covered half the coding region and the entire 3'-untranslated regions. The nucleotides and deduced amino acid sequences of pHA6 showed striking homology, 89 and 98%, respectively, to those of rabbit Ca2+-ATPase of the slow-twitch form. Northern blot analyses revealed that the mRNA levels of Ca2+-ATPase were decreased by pressure overload and became 32% of sham in 1 mo. During the developmental stage the mRNA levels were very low in the fetal period and steeply increased around birth. These changes in mRNA levels were correlated with the corresponding protein levels. These results suggest that the expression of cardiac Ca2+-ATPase in SR is regulated by pressure overload and the developmental stage, at least in part, at the pretranslational level.

Aging↗

PubMed1988

Molecular cloning and characterization of human atrial and ventricular myosin alkali light chain cDNA clones.

We have isolated essentially full-length cDNA clones for atrial (ALC1) and ventricular (VLC1) myosin alkali light chains from a human fetal heart cDNA library. Comparison of overall nucleotide sequences of ALC1 and VLC1 cDNA clones has revealed that, while these two inserts show significant DNA sequence homology (78.4%) with respect to their coding regions, the 5'- and 3'-untranslated regions are highly divergent. Our statistical analysis suggests that human ALC1 and VLC1 diverged approximately 300 million years ago, during the time of separation of birds and mammals. RNA blot analysis shows that ALC1 mRNA is expressed in fetal ventricular and fetal skeletal muscles as well as fetal and adult atrial muscles and VLC1 mRNA is expressed in adult slow skeletal muscle as well as fetal and adult ventricular muscles. Southern blot analysis indicates that each protein is encoded by a single gene. Finally, we show that VLC1 mRNA is induced in pressure-overloaded human atrium.

Adult↗

PubMed1988

Expression of cellular oncogenes in the myocardium during the developmental stage and pressure-overloaded hypertrophy of the rat heart.

Proto-oncogenes have been revealed to participate in normal cell proliferation as well as in cell transformation. Since cardiac myocytes are terminally differentiated, they cannot divide except in the fetal period. To determine the role of cellular oncogenes in the growth of the heart, the expression pattern of eight cellular oncogenes during the developmental stage and pressure-overloaded hypertrophy of the rat hearts were examined in vivo. Northern blot analysis was performed with eight oncogene probes (myc, fos, Ha-ras, src, erbA, erbB, sis, myb). Pressure overload increased the levels of cellular (c-) fos, c-myc, and c-Ha-ras. An increase of c-fos and c-myc was detected at 30 minutes and 2 hours, respectively; the levels peaked at 8 hours, and they returned to baseline by 48 hours after aortic constriction. However, the level of c-Ha-ras showed a gradual increase. During the course of development, the expression of c-myc was detectable only in the embryonic stage, whereas the expression of c-fos was not detected in the fetal period, was increased after birth, and peaked in 200-day-old adults. The expression of c-Ha-ras was almost the same throughout the development. Cellular oncogenes were expressed in the heart in response to pressure overload and in a stage-specific manner. These results suggest that cellular oncogenes may participate in the normal developmental process and hypertrophy of hearts and that the cellular hypertrophy induced by pressure overload may share a similar mechanistic pathway with cell proliferation.

Animals↗

PubMed1988

Heterogeneity of beta-type myosin isozymes in the human heart and regulational mechanisms in their expression. Immunohistochemical study using monoclonal antibodies.

To investigate the existence of heterogeneity of beta-type myosin isozymes (HC beta) in human hearts, immunohistochemical studies using monoclonal antibodies (MoAbs) raised against human ventricular myosin heavy chains were performed. Two types of MoAbs recognized some muscle fibers in the atrium, whereas both reacted with all ventricular muscle fibers. Since atrial muscle fibers reactive with each MoAb were found to be clearly different, the existence of two immunologically distinct HC beta (beta 1, and beta 2) was suggested in the atrium. By using affinity chromatography, two molecular variants of HC beta were isolated from the bovine atrium, and differences in the primary structure of beta 1 and beta 2 were confirmed by analysis of peptides produced by chymotryptic digestion. In pressure-overloaded human atria, myofibers containing beta 1 and/or beta 2 increased in accordance with decrement of myofibers containing alpha-type myosin isozyme (P less than 0.01). But they differed in expression during the developmental stage, since beta 2 did not exist in the early embryonic bovine heart, but beta 1 did. Thus, there are two distinct HC beta whose expression is regulated by at least two factors: pressure overload and developmental stage.

Adult↗

PubMed1988

Molecular cloning and characterization of human cardiac alpha- and beta-form myosin heavy chain complementary DNA clones. Regulation of expression during development and pressure overload in human atrium.

We have constructed and characterized two types of myosin heavy chain (MHC) cDNA clones (pHMHC2, pHMHC5) from a fetal human heart cDNA library. Comparison of the nucleotide and deduced amino acid sequences between pHMHC2 and pHMHC5 shows 95.1 and 96.2% homology, respectively. The carboxyl-terminal peptide and 3'-untranslated (3'-UT) regions are highly divergent and specific for these cDNA clones. By using the synthetic oligonucleotide probes that are complementary to the unique 3'-UT regions of these cDNA clones, we demonstrate that pHMHC2 is exclusively transcribed in the atrium, whereas the mRNA for pHMHC5 is predominantly expressed in the ventricle. This result indicates that pHMHC2 and pHMHC5 code for alpha- and beta-form MHCs, respectively. Furthermore, we show that beta-form MHC mRNA is expressed in adult atrium at a low level but scarcely expressed in fetal atrium. Finally, we demonstrate that MHC isozymic transition in pressure-overloaded atrium is, at least in part, regulated at a pretranslational level.

Adult↗

PubMed1987

Isolation and characterization of myosin heavy chain isozymes of the bovine conduction system.

To determine the characteristics of cardiac myosin in the conduction system, a pure Purkinje fiber preparation, consisting of atrioventricular nodes and the ventricular conduction system, was obtained from bovine hearts. Two types of myosin heavy chain isozymes, alpha-type and beta-type, were fractionated by affinity chromotography using monoclonal antibodies CMA19 and HMC50, which are specific for the alpha-type heavy chain and beta-type heavy chain, respectively. Competitive enzyme-linked immunosorbent assay demonstrated that the content of beta-type in the atrioventricular node (30-40%) was higher than that in atrial ordinary myocardium (10-20%) and that of the alpha-type was 30-40% in the ventricular conduction system, which was much higher than that in the ventricular ordinary myocardium (less than 10%). By one- and two-dimensional electrophoresis of the peptides produced by partial and complete digestion, the peptide compositions of alpha-type and beta-type in the conduction system were shown to be very similar to those of alpha-type and beta-type in ordinary myocardium, respectively. The CA2+-activated ATPase activity of myosin of the atrioventricular nodes was lower than that of ordinary atrial myosin (0.46 +/- 0.03 versus 0.58 +/- 0.02 mumol Pi/mg/min, mean +/- SEM, p less than 0.05) and in contrast, that of ventricular specialized myocardium was higher than that of myosin in the ventricular ordinary working myocardium (0.32 +/- 0.03 versus 0.22 +/- 0.01 mumol Pi/mg/min, p less than 0.05). This was in good agreement with the relative proportion of myosin isozymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Distribution of cardiac myosin isozymes in cardiomyopathy: immunohistochemical and gene analysis.

We characterized cardiac myosin isoforms by immunohistochemical approaches using monoclonal antibodies and demonstrated the existence of a distinctive type of cardiac myosin heavy chain which predominates in the fetal stage but is depressed during postnatal development. Furthermore, we showed that this type of cardiac myosin heavy chain was markedly expressed in patients with dilated cardiomyopathy. The results suggested that the pathologic process involved in dilated cardiomyopathy affects myocardial differentiation by the inhibition of the myosin gene switching that normally occurs during muscle maturation. However, we could not find any polymorphism in myosin gene in our population sample of dilated cardiomyopathy. The pathophysiological role of fetal type cardiac myosin expression in dilated cardiomyopathy will be clarified by the characterization of the myosin near future.

Adult↗

PubMed1986

Isolation and characterization of two isozymes of myosin heavy chain from canine atrium.

To clarify the characteristics of myosin isozymes in the atrium, we fractionated two isoforms of myosin heavy chain (HC), atrial HC alpha (A-HC alpha) and HC beta (A-HC beta), from the canine heart by affinity chromatography, using monoclonal antibodies specific for HC alpha (CMA19) and HC beta (HMC50), respectively, and then compared their peptide composition and enzymatic properties with those of ventricular HC alpha (V-HC alpha) and HC beta (V-HC beta). The reactivity of these isozymes with three monoclonal antibodies revealed that there are at least three different epitopes between A-HC alpha and A-HC beta. Differences in the primary structure of A-HC alpha and A-HC beta were confirmed by one- and two-dimensional gel electrophoretic analyses of these peptides, produced by digestion with alpha-chymotrypsin and cyanogen bromide (CNBr). A-HC alpha and V-HC alpha were indistinguishable proteins, and A-HC beta was also very similar to V-HC beta. Furthermore, there were differences between A-HC alpha and A-HC beta in their Ca2+-activated ATPase activities. The ATPase activity of A-HC beta was lower than that of A-HC alpha and was similar to that of V-HC beta. We concluded that there are two different isozymes of myosin heavy chain in the atrium (A-HC alpha and A-HC beta), as well as in the ventricle (V-HC alpha and V-HC beta), and that A-HC beta is very similar to V-HC beta, the predominant form of ventricular myosin, in its molecular structure and enzymatic activity.

Adenosine Triphosphatases↗

PubMed1986

A case of Takayasu's disease with ruptured carotid aneurysm.

Recently, a number of cases of Takayasu's disease having dilatative or aneurysmal lesions have been reported. Such lesions have come to be considered important manifestations of Takayasu's disease. A case, whose right common carotid artery perforated spontaneously and became a pseudoaneurysm, without any other stenotic lesion is presented with a review of the literature. Surgical treatment was performed successfully.

Adult↗