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Biomedical subjects

M Kuro-o

Publications and source records attributed to M Kuro-o.

At least 55 records · Page 3Linked to original sources
PubMed1993

Human smooth muscle myosin heavy chain isoforms as molecular markers for vascular development and atherosclerosis.

Smooth muscle myosin heavy chains (MHCs) exist in multiple isoforms. Rabbit smooth muscles contain at least three types of MHC isoforms: SM1 (204 kD), SM2 (200 kD), and SMemb (200 kD). SM1 and SM2 are specific to smooth muscles, but SMemb is a nonmuscle-type MHC abundantly expressed in the embryonic aorta. We recently reported that these three MHC isoforms are differentially expressed in rabbit during normal vascular development and in experimental arteriosclerosis and atherosclerosis. The purpose of this study was to clarify whether expression of human smooth muscle MHC isoforms is regulated in developing arteries and in atherosclerotic lesions. To accomplish this, we have isolated and characterized three cDNA clones from human smooth muscle: SMHC94 (SM1), SMHC93 (SM2), and HSME6 (SMemb). The expression of SM2 mRNA in the fetal aorta was significantly lower as compared with SM1 mRNA, but the ratio of SM2 to SM1 mRNA was increased after birth. SMemb mRNA in the aorta was decreased after birth but appeared to be increased in the aged. To further examine the MHC expression at the histological level, we have developed three antibodies against human SM1, SM2, and SMemb using the isoform-specific sequences of the carboxyl terminal end. Immunohistologically, SM1 was constitutively positive from the fetal stage to adulthood in the apparently normal media of the aorta and coronary arteries, whereas SM2 was negative in fetal arteries of the early gestational stage. In human, unlike rabbit, aorta or coronary arteries, SMemb was detected even in the adult. However, smaller-sized arteries, like the vasa vasorum of the aorta or intramyocardial coronary arterioles, were negative for SMemb. Diffuse intimal thickening in the major coronary arteries was found to be composed of smooth muscles, reacting equally to three antibodies for MHC isoforms, but reactivities with anti-SM2 antibody were reduced with aging. With progression of atherosclerosis, intimal smooth muscles diminished the expression of not only SM2 but also SM1, whereas alpha-smooth muscle actin was well preserved. We conclude from these results that smooth muscle MHC isoforms are important molecular markers for studying human vascular smooth muscle cell differentiation as well as the cellular mechanisms of atherosclerosis.

Aging↗
PubMed1993

[Production of transgenic mice overexpressing rabbit Na+/H+ antiporter].

Recent investigations have raised a possibility that abnormal ion transportation through cell membranes may be involved in the pathogenesis of essential hypertension. In order to test the hypothesis that increased activity of Na+/H+ antiporter may cause hypertension, we developed transgenic mice overexpressing the Na+/H+ antiporter. We isolated a full-length cDNA clone encoding the rabbit Na+/H+ antiporter and constructed the transgene by ligating it with the human elongation factor 1 alpha promoter. We obtained three transgenic strains which express the transgene in various tissues such as kidney, heart and aorta. These transgenic mice may be useful for the analysis of pathogenesis of essential hypertension.

Animals↗
PubMed1992

Heterogeneity in smooth muscle cell population accumulating in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery.

Rabbit smooth muscles contain at least three types of myosin heavy chain (MHC) isoforms; SM1, SM2 and SMemb (NMHC-B), the expression of which is developmentally regulated. We have recently reported that smooth muscles with the embryonic phenotype accumulate in the neointimas produced by endothelial denudation or high-cholesterol feeding. In this study, we examined MHC isoform expression in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery, and determined the phenotype of the smooth muscle cell in the dilated segment. We report here that neointimal cells in the dilated segment are smooth muscle cells with the embryonic phenotype as previously reported in our ballooning-injury study. The medial smooth muscles, however, are composed of heterogeneous population of smooth muscles which differ in stage of differentiation as determined by the MHC isoform expression. These results indicate that MHC isoforms are useful molecular markers to identify abnormally proliferating smooth muscles in diseased arteries and to understand the process of atherogenesis occurring following vascular injury.

Angioplasty, Balloon↗
PubMed1992

Identification of three types of PDGF-A chain gene transcripts in rabbit vascular smooth muscle and their regulated expression during development and by angiotensin II.

PDGF-like peptides secreted from smooth muscles have been suggested to be responsible for the smooth muscle growth. In order to elucidate the nature of PDGF-like molecules expressed in vascular smooth muscles, we have isolated and characterized cDNA clones for PDGF-A chain from a rabbit embryonic aorta cDNA library. One of the cDNA clones was found to encode a novel PDGF-A chain, named PDGF-A3 in this report. PDGF-A3 arises from a single PDGF-A chain gene by alternative RNA splicing and differs from the sequences of previously reported endothelial- or the glioma-type transcripts by a 110 bp insertion. Expression of PDGF-A3 mRNA was selectively induced by Angiotensin II in the smooth muscle cell in vitro. Total PDGF-A mRNA is most enriched in embryonic aortas, but its expression is down-regulated with vascular development. PDGF-A mRNA is markedly increased in primary-cultured smooth muscle cells during the log-phase growth. Our results suggest that autocrine production of PDGF-A chains from the smooth muscle cell may play a role in early vascular development and in Angiotensin II-induced smooth muscle cell proliferation.

Aging↗
PubMed1992

Germ line-restricted supernumerary (B) chromosomes in Eptatretus okinoseanus.

Cytogenetic studies were performed on two types of Japanese hagfish (Eptatretus okinoseanus) that eliminate about 45% (type A) and 55% (type B) of their DNA from presumptive somatic cells during the differentiation of somatic cells. The observations revealed inter- and intraindividual variations in the number of chromosomes in germ cells of both types of hagfishes. Although the modal number of chromosomes in the germ cells was 54 in both types, the percentage of cells with the modal number was rather low (38.6% [51/132] in five specimens of type A and 22.7% [25/110] in eight specimens of type B). In addition, one of seven type B specimens clearly had a modal number of 62 chromosomes. Another specimen of type B had a bimodal distribution of chromosome numbers, with peaks of 54 and 59 chromosomes. The observation of interindividual variations was supported by data on the amount of DNA in germ cells of type B specimens. However, these variations were rarely observed in somatic cells. These results suggest that supernumerary (B) chromosomes are maintained in germ cells and are eliminated together with some other chromosomes and/or chromatin from somatic cells.

Aneuploidy↗
PubMed1991

cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis.

Adult rabbit smooth muscles contain two types of myosin heavy chain (MHC) isoforms, SM1 and SM2 which are generated through alternative RNA splicing from a single gene (Nagai, R., Kuro-o, M., Babij, P. & Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). We previously reported that the expression of SM1 and SM2 during vascular development is differentially regulated at the level of RNA splicing, whereby SM1 is constitutively expressed from early development but SM2 appear after birth (Kuro-o, M., Nagai, R., Tsuchimochi, H., Katoh, H., Yazaki, Y., Ohkubo, A. & Takaku, F. (1989) J. Biol. Chem. 264, 18272-18275). We also demonstrated that embryonic vascular smooth muscles contain a third type of MHC isoform, referred to as SMemb in this report, which comigrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SM2. In the present study we have isolated and characterized a cDNA clone (FSMHC34) for SMemb. FSMHC34 encodes the light meromyosin region including the carboxyl terminus and showed 70% amino acid sequence identity with SM1 or SM2. SMemb is a nonmuscle-type MHC and identical with brain MHC, but clearly distinct from 196-kDa nonmuscle MHC in cultured smooth muscle cells. The expression of SMemb was predominant in embryonic and perinatal aortas, but down-regulated with vascular development. Interestingly SMemb was reexpressed in proliferating smooth muscle cells of arteriosclerotic neointimas. These results suggest that smooth muscle proliferation is coupled to the expression of SMemb and that dedifferentiation of smooth muscles toward the embryonic phenotype is involved in the mechanisms underlying atherosclerosis.

Aging↗
PubMed1990

Cytogenetic studies of Hynobiidae (Urodela). XI. Banding karyotype of Salamandrella keyserlingii Dybowski and a comparison with those of Hynobius species.

C- and R-banding analyses were performed on chromosomes of Salamandrella keyserlingii, in which 20 of 31 chromosome pairs could be identified. Banding patterns were compared between S. keyserlingii and specimens of Hynobius species of the family Hynobiidae. Between S. keyserlingii and six Hynobius species (2n = 56), there were four homoelogous and two partially homoeologous chromosome pairs. Between S. keyserlingii and H. retardatus (2n = 40), there were two homoeologous and two partially homoeologous chromosome pairs.

Animals↗
PubMed1989

Developmentally regulated expression of vascular smooth muscle myosin heavy chain isoforms.

Two types of smooth muscle myosin heavy chain (MHC) isoforms, SM1 and SM2, were recently identified to have different carboxyl termini (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). SM1 and SM2 are considered to be generated from a single gene through alternative RNA splicing. In this study we investigated expression of vascular MHC isoforms during development in rabbits at the mRNA, protein, and histological levels. In adults, all smooth muscle cells reacted with both anti-SM1 and anti-SM2 antibodies on immunofluorescence, suggesting the coexpression of SM1 and SM2 in a single cell. In fetal and perinatal rabbits, however, only anti-SM1 antibody consistently reacted with smooth muscles. Reactivity with anti-SM2 antibody was negative in the fetal and neonatal blood vessels and gradually increased during 30 days after birth. These developmental changes in SM1 and SM2 expression at the histological level coincided with mRNA expression of each MHC isoform as determined by S1 nuclease mapping, indicating that expression of SM1 and SM2 is controlled at the level of RNA splicing. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin from fetal and perinatal aortas revealed the presence of large amount of SM2. Interestingly, fetal SM2 did not cross-react with our anti-SM2 antibody on immunoblotting. We conclude that expression of SM1 and SM2 are differentially regulated during development and that a third type of MHC isoform may exist in embryonic and perinatal vascular smooth muscles.

Animals↗
PubMed1989

Identification of two types of smooth muscle myosin heavy chain isoforms by cDNA cloning and immunoblot analysis.

We previously reported the characterization of a rabbit uterus cDNA clone (SMHC29) which encoded part of the light meromyosin of smooth muscle myosin heavy chain (Nagai, R., Larson, D.M., and Periasamy, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1047-1051). We have now characterized a second cDNA clone (SMHC40) which also encodes part of the light meromyosin but differs from SMHC29 in the following respects. Nucleotide sequence analysis demonstrates that the two myosin heavy chain mRNAs are identical over 1424 nucleotides but differ in part of the 3'-carboxyl coding region and a portion of the 3'-nontranslated sequence. Specifically, SMHC40 cDNA encodes a unique stretch of 43 amino acids at the carboxyl terminus, whereas SMHC29 cDNA contains a shorter carboxyl terminus of 9 unique amino acids which is the result of a 39-nucleotide insertion. Recent peptide mapping of smooth muscle myosin heavy chain identified two isotypes with differences in the light meromyosin fragment that were designated as SM1 (204 kDa) and SM2 (200 kDa) type myosin (Eddinger, T. J., and Murphy, R.A. (1988) Biochemistry 27, 3807-3811). In this study we present direct evidence that SMHC40 and SMHC29 mRNA encode the two smooth muscle myosin heavy chain isoforms, SM1 and SM2, respectively, by immunoblot analysis using antibodies against specific carboxyl terminus sequences deduced from SMHC40 and SMHC29 cDNA clones.

Amino Acid Sequence↗
PubMed1988

Differences in response of myosin isozyme transition of ordinary and specialized myocardium to overload.

To investigate the response of myosin isozyme transition in specialized myocardium to cardiac overload, we examined immunohistochemically the distribution of myosin isozymes in sinus node cells of overloaded canine atria, using the monoclonal antibodies CMA19 and HMC14, which are specific for atrial myosin heavy chain (alpha-HC) and ventricular myosin heavy chain (beta-HC), respectively. Overloading in canine right atria was induced by artificial tricuspid valve regurgitation and pulmonary stenosis. Right atrial mean pressure rose to 15-20 mm Hg (n = 4) 2 months after surgery. In the working myocardium, cardiac overload caused redistribution of myosin isozymes, alpha-HC to beta-HC. Compared with the normal right atria, fewer myocytes were labeled with CMA19, but more were labeled with HMC14. However, the reactivity of sinus node cells with CMA19 and HMC14 was not changed between normal and overloaded right atria, indicating no redistribution of myosin heavy chain isozymes, alpha-HC to beta-HC. These results suggest that isozymes in myosin heavy chains in the specialized myocardium are protected from overload effects by their firm cytoskeletal framework or other mechanisms.

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↗
PubMed1986

Cytogenetic studies of Hynobiidae (Urodela). IV. DNA replication bands (R-banding) in the genus Hynobius and the banding karyotype of Hynobius nigrescens Stejneger.

The R-banding technique of Dutrillaux et al. (1973) was modified in order to analyze the chromosomes of salamanders in the genus Hynobius. Embryonic cells of Hynobius nigrescens Stejneger from Nakakubiki County, Niigata Prefecture, and Hakui County, Ishikawa Prefecture, were cultured in medium containing 5-bromodeoxyuridine (BrdU). Banded metaphases were obtained by the FPG (fluorescent-plus-Giemsa) technique (Perry and Wolff, 1974), with slight modifications. With this modified R-banding technique, multiple, clear DNA replication bands were obtained on the chromosomes, and 18 of 28 chromosome pairs could be identified easily by their replication patterns in embryos collected from Nakakubiki County. A distinct heteromorphism in banding pattern was detected on the long arm of chromosome 9 in these embryos, but the frequency of this variant was too low for chromosome 9 to be a sex chromosome. Chromosomes 1-14, except for 6, 12 (for which the data were not satisfactory), and 9 (the variant type from Nakakubiki County), had the same replication patterns in embryos obtained from Nakakubiki and Hakui Counties.

Animals↗
PubMed1986

Distribution of cardiac myosin isozymes in human conduction system. Immunohistochemical study using monoclonal antibodies.

To determine the presence and distribution of cardiac myosin isozymes in the human conduction system, we performed an immunohistochemical study using monoclonal antibodies CMA19 and HMC14, which are specific for myosin heavy chains of human atrial type (alpha-type) and ventricular type (beta-type), respectively. Serial frozen sections of human hearts were obtained from autopsy samples and examined by indirect immunofluorescence. Alpha-type was found in all myofibers of sinus node and atrio-ventricular node, and in 55.2 +/- 10.2% (mean +/- SD, n = 5) of the myofibers of ventricular conduction tissue, which consists of the bundle of His, bundle branches, and the Purkinje network. In contrast, beta-type was found in all myofibers of the atrio-ventricular node and ventricular conduction tissue, whereas almost all myofibers of the sinus node were unlabeled by HMC14. Although the number of ventricular myofibers labeled by CMA19 was small, the labeled myofibers were more numerous in the subepicardial region than in the subendocardial region. These findings show that the gene coding for alpha-type is expressed predominantly in specialized myocardium compared with the adjacent ordinary working myocardium.

Antibodies, Monoclonal↗
PubMed1986

Expression of myosin isozymes during the developmental stage and their redistribution induced by pressure overload.

Cardiac muscles contain at least two isozymes--referred to as alpha(HC alpha) and beta(HC beta)--of the myosin heavy chain. The proportional ratio of these isozymes varies depending upon the developmental stage and the physiological and/or the hormonal milieu of the cell. Using monoclonal antibodies (MoAb) specific for human cardiac HC alpha and HC beta, we have examined the expression of these isozymes in fetal through adult cardiac tissues and investigated whether isozymic redistribution occurs in pressure overloaded human ventricles. We found that although HC alpha was expressed in the atrium from the early embryonic stage, in embryonic ventricular myofibers, only HC beta was expressed without expression of HC alpha, but some myofibers replace HC beta by HC alpha after birth, and these HC alpha containing ventricular myofibers were found to be decreased by pressure overload, which suggested that isozymic redistribution from HC alpha to HC beta also occurred in the ventricles, as well as the atrium. In addition, we also found two subtypes of HC beta (beta 1, beta 2) in the human heart. In the ventricle, both beta 1 and beta 2 was present in all myofibers; in contrast, some myofibers contained beta 1 or beta 2 or both with or without expression of HC alpha in the atrium. beta 1 and beta 2 were distinctive in their expression during the developmental stage, since beta 1 was present in the embryonic heart from the early developmental stage, whereas beta 2 was not present in the early embryonic heart, but began to be expressed in the late embryonic stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Distribution of myosin isozymes in human atrial and ventricular myocardium: comparison in normal and overloaded heart.

We have prepared monoclonal antibodies specific for either atrial or ventricular myosin and defined the isomyosin composition of myocardium in normal and overloaded human hearts. In the atrial myocardium, normal isozymic pattern was V1 dominant which converted to being V3 dominant in an overloaded condition. In contrast, normal isomyosin pattern of the ventricular myocardium was exclusively V3 dominant, and only a small change in the proportion of isomyosin was observed in an overloaded condition. From this, we conclude that isozymic changes in cardiac myosin could occur in the human heart to meet increased work induced by cardiac overload. However, the physiological importance of these isomyosin redistributions in human myocardium seems to be much greater in the atrium than in the ventricle, since larger amounts of V1 isomyosin which could be transformed to V3 isomyosin were present in the atrial myocardium.

Adenosine Triphosphatases↗
PubMed1984

Isozymic changes in myosin of human atrial myocardium induced by overload. Immunohistochemical study using monoclonal antibodies.

An immunohistochemical study using monoclonal antibodies specific for the heavy chains of either human atrial (HC alpha) or ventricular (HC beta) myosin was performed to clarify the distribution of each isozyme in normal as well as pressure-overloaded human hearts. In normal human ventricles, all muscle fibers were stained by a monoclonal antibody (HMC14) specific for HC beta, whereas a small number of fibers reacted with a monoclonal antibody (CMA19) specific for HC alpha. In contrast, in normal human atria, almost all muscle fibers were stained by CMA19, and a relatively larger number of muscle fibers also reacted with HMC14. Furthermore, in pressure-overloaded atria, muscle fibers reactive with HMC14 were strikingly increased while those reactive with CMA19 showed a corresponding decrease. The extent of this isozymic redistribution was in good correlation with atrial pressure. These results not only confirmed the existence of isoforms of myosin heavy chain in human hearts, but also demonstrated that redistribution of iso-myosins could occur as an adaptation to pressure overload.

Animals↗