RESUMEN
Congenital heart defects comprise the most common form of major birth defects, affecting 0.7% of all newborn infants. Jacobsen syndrome (11q-) is a rare chromosomal disorder caused by deletions in distal 11q. We have previously determined that a wide spectrum of the most common congenital heart defects occur in 11q-, including an unprecedented high frequency of hypoplastic left heart syndrome (HLHS). We identified an approximately 7 Mb 'cardiac critical region' in distal 11q that contains a putative causative gene(s) for congenital heart disease. In this study, we utilized chromosomal microarray mapping to characterize three patients with 11q- and congenital heart defects that carry interstitial deletions overlapping the 7 Mb cardiac critical region. We propose that this 1.2 Mb region of overlap harbors a gene(s) that causes at least a subset of the congenital heart defects that occur in 11q-. We demonstrate that one gene in this region, ETS-1 (a member of the ETS family of transcription factors), is expressed in the endocardium and neural crest during early mouse heart development. Gene-targeted deletion of ETS-1 in mice in a C57/B6 background causes, with high penetrance, large membranous ventricular septal defects and a bifid cardiac apex, and less frequently a non-apex-forming left ventricle (one of the hallmarks of HLHS). Our results implicate an important role for the ETS-1 transcription factor in mammalian heart development and should provide important insights into some of the most common forms of congenital heart disease.
Asunto(s)
Eliminación de Gen , Defectos del Tabique Interventricular/genética , Ventrículos Cardíacos/anomalías , Síndrome de Deleción Distal 11q de Jacobsen/genética , Proteína Proto-Oncogénica c-ets-1/genética , Animales , Deleción Cromosómica , Mapeo Cromosómico , Cromosomas Humanos Par 11/genética , Defectos del Tabique Interventricular/embriología , Defectos del Tabique Interventricular/metabolismo , Ventrículos Cardíacos/embriología , Ventrículos Cardíacos/metabolismo , Humanos , Síndrome de Deleción Distal 11q de Jacobsen/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Proto-Oncogénica c-ets-1/metabolismoRESUMEN
Human myotonic dystrophy protein kinase (DMPK) is a member of a novel class of multidomain protein kinases that regulate cell size and shape in a variety of organisms. However, little is currently known about the general properties of DMPK including domain function, substrate specificity, and potential mechanisms of regulation. Two forms of the kinase are expressed in muscle, DMPK-1 and DMPK-2. We demonstrate that the larger DMPK-1 form (the primary translation product) is proteolytically cleaved near the carboxy terminus to generate the smaller DMPK-2 form. We further demonstrate that the coiled-coil domain is required for DMPK oligomerization; coiled-coil mediated oligomerization also correlated with enhanced catalytic activity. DMPK was found to exhibit a novel catalytic activity similar to, but distinct from, related protein kinases such as protein kinase C and A, and the Rho kinases. We observed that recombinant DMPK-1 exhibits low activity, whereas the activity of carboxy-terminally truncated DMPK is increased approximately 3-fold. The inhibitory activity of the full-length kinase was mapped to what appears to be a pseudosubstrate autoinhibitory domain at the extreme carboxy terminus of DMPK. To date, endogenous activators of DMPK are unknown; however, we observed that DMPK purified from cells exposed to the G protein activator GTP-gamma-S exhibited an approximately 2-fold increase in activity. These results suggest a general model of DMPK regulation with two main regulatory branches: short-term activation of the kinase in response to G protein second messengers and long-term activation as a result of proteolysis.
Asunto(s)
Distrofia Miotónica/enzimología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica , Péptidos/química , Péptidos/farmacología , Proteínas Serina-Treonina Quinasas/genética , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The genetic abnormality in myotonic muscular dystrophy, multiple CTG repeats lie upstream of a gene that encodes a novel protein kinase, myotonic dystrophy protein kinase (DMPK). Phospholemman (PLM), a major membrane substrate for phosphorylation by protein kinases A and C, induces Cl currents (I(Cl(PLM))) when expressed in Xenopus oocytes. To test the idea that PLM is a substrate for DMPK, we measured in vitro phosphorylation of purified PLM by DMPK. To assess the functional effects of PLM phosphorylation we compared I(Cl(PLM)) in Xenopus oocytes expressing PLM alone to currents in oocytes co-expressing DMPK, and examined the effect of DMPK on oocyte membrane PLM expression. We found that PLM is indeed a good substrate for DMPK in vitro. Co-expression of DMPK with PLM in oocytes resulted in a reduction in I(Cl(PLM)). This was most likely a specific effect of phosphorylation of PLM by DMPK, as the effect was not present in oocytes expressing a phos(-) PLM mutant in which all potential phosphorylation had been disabled by Ser --> Ala substitution. The biophysical characteristics of I(Cl(PLM)) were not changed by DMPK or by the phos(-) mutation. Co-expression of DMPK reduced the expression of PLM in oocyte membranes, suggesting a possible mechanism for the observed reduction in I(Cl(PLM)) amplitude. These data show that PLM is a substrate for phosphorylation by DMPK and provide functional evidence for modulation of PLM function by phosphorylation.
Asunto(s)
Proteínas de la Membrana/metabolismo , Distrofia Miotónica/enzimología , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Potenciales de la Membrana , Proteína Quinasa de Distrofia Miotónica , Oocitos/metabolismo , Oocitos/fisiología , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por Sustrato , XenopusAsunto(s)
Cromosomas Humanos Par 6/genética , Dedos de Zinc/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cartilla de ADN/genética , ADN Complementario/genética , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: The regulation and interaction of ACE and the angiotensin II (Ang II) type I (AT1) receptor in the failing human heart are not understood. METHODS AND RESULTS: Radioligand binding with 3H-ramiprilat was used to measure ACE protein in membrane preparations of hearts obtained from 36 subjects with idiopathic dilated cardiomyopathy (IDC), 8 subjects with primary pulmonary hypertension (PPH), and 32 organ donors with normal cardiac function (NF hearts). 125I-Ang II formation was measured in a subset of hearts. Saralasin (125I-(Sar1,Ile8)-Ang II) was used to measure total Ang II receptor density. AT1 and AT2 receptor binding were determined with the AT1 receptor antagonist losartan. Maximal ACE binding (Bmax) was 578+/-47 fmol/mg in IDC left ventricle (LV), 713+/-97 fmol/mg in PPH LV, and 325+/-27 fmol/mg in NF LV (P<0.001, IDC or PPH versus NF). In IDC, PPH, and NF right ventricles (RV), ACE Bmax was 737+/-78, 638+/-137, and 422+/-49 fmol/mg, respectively (P=0.02, IDC versus NF; P=0.08, PPH versus NF). 125I-Ang II formation correlated with ACE binding sites (r=0.60, P=0.00005). There was selective downregulation of the AT1 receptor subtype in failing PPH ventricles: 6.41+/-1.23 fmol/mg in PPH LV, 2.37+/-0.50 fmol/mg in PPH RV, 5.38+/-0.53 fmol/mg in NF LV, and 7.30+/-1.10 fmol/mg in NF RV (P=0.01, PPH RV versus PPH LV; P=0.0006, PPH RV versus NF RV). CONCLUSIONS: ACE binding sites are increased in both failing IDC and nonfailing PPH ventricles. In PPH hearts, the AT1 receptor is downregulated only in the failing RV.
Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Dominio Catalítico , Miocardio/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Receptores de Angiotensina/metabolismo , Adenilil Ciclasas/metabolismo , Adulto , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Ensayo de Unión Radioligante , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: The regulation of angiotensin II receptors and the two major subtypes (AT1 and AT2) in chronically failing human ventricular myocardium has not been previously examined. METHODS AND RESULTS: Angiotensin II receptors were measured by saturation binding of 125I-[Sar1,Ile8]angiotensin II in crude membranes from nonfailing (n = 19) and failing human left ventricles with idiopathic dilated cardiomyopathy (IDC; n = 31) or ischemic cardiomyopathy (ISC; n = 21) and membranes from a limited number of right ventricles in each category. The AT1 and AT2 fractions were determined by use of an AT1-selective antagonist, losartan. beta-Adrenergic receptors were also measured by binding of 125I-iodocyanopindolol with the beta 1 and beta 2 fractions determined by use of a beta 1-selective antagonist, CGP20712A, AT1 but not AT2 density was significantly decreased in the combined (IDC + ISC) failing left ventricles (nonfailing: AT1 4.66 +/- 0.48, AT2 2.73 +/- 0.39; failing: AT1 3.20 +/- 0.29, AT2 2.70 +/- 0.33 fmol/mg protein; mean +/- SE). The decrease in AT1 density was greater in the IDC than in the ISC left ventricles (IDC: 2.73 +/- 0.40, P < .01; ISC: 3.89 +/- 0.39 fmol/mg protein, P = NS versus nonfailing). beta 1 but not beta 2 density was decreased in the failing left ventricles. AT1 density was correlated with beta 1 density in all left ventricles (r = .43). AT1 density was also decreased in IDC right ventricles. In situ reverse transcription-polymerase chain reaction in sections of nonfailing and failing ventricles indicated that AT1 mRNA was present in both myocytes and nonmyocytes. CONCLUSIONS: AT1 receptors are selectively downregulated in failing human ventricles, similar to the selective downregulation of beta 1 receptors. The relative lack of AT1 downregulation in ISC hearts may be related to differences in the degree of ventricular dysfunction.
Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismo , Receptores de Angiotensina/biosíntesis , Adulto , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Membrana Celular/metabolismo , Regulación hacia Abajo , Femenino , Insuficiencia Cardíaca/patología , Ventrículos Cardíacos , Humanos , Cinética , Masculino , Miocardio/patología , Reacción en Cadena de la Polimerasa , Ensayo de Unión Radioligante , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Valores de ReferenciaRESUMEN
Myotonic muscular dystrophy is an autosomal dominant defect that produces muscle wasting, myotonia, and cardiac conduction abnormalities. The myotonic dystrophy locus codes for a putative serine-threonine protein kinase of unknown function. We report that overexpression of human myotonic dystrophy protein kinase induces the expression of skeletal muscle-specific genes in undifferentiated BC3H1 muscle cells. BC3H1 clones expressing myotonic dystrophy kinase appear equivalent to differentiated cells with respect to expression of myogenin, retinoblastoma tumor supressor gene, M creatine kinase, beta-tropomyosin, and vimentin. In addition, differential display analysis demonstrates that the pattern of gene expression exhibited by myotonic dystrophy kinase-expressing cells is essentially identical to that of differentiated BC3H1 muscle cells. These observations suggest that myotonic dystrophy kinase may function in the myogenic pathway.
Asunto(s)
Músculo Esquelético/enzimología , Distrofia Miotónica/enzimología , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Bases , Línea Celular , Creatina Quinasa/genética , Creatina Quinasa/metabolismo , ADN Complementario , Regulación Enzimológica de la Expresión Génica , Humanos , Isoenzimas , Datos de Secuencia Molecular , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The cardiac vasculature and myocardium contain components of the renin-angiotensin system (RAS), which may regulate local growth and cellular function. Alterations in the expression or action of these components, which include angiotensin converting enzyme (ACE), angiotensinogen, and angiotensin II type-1 receptors, may contribute to the development of disease, such as hypertension, left ventricular hypertrophy, myocardial infarction, and end-stage heart failure. ACE is one RAS component found to have genetic variants associated with cardiovascular disease. Molecular variants in any of the RAS components may affect signalling pathways, possibly increasing the risk of heart failure. In addition, variants may exacerbate the deleterious effects of altered RAS expression on cardiac function. Genetic variation in RAS components may affect therapy with ACE inhibitors and receptor-blocking agents. Although at present there is no compelling reason to target molecular variations for treatment, a new era in selective pharmacological therapy for cardiovascular disease may be imminent.
Asunto(s)
Angiotensinógeno/genética , Variación Genética , Cardiopatías/etiología , Peptidil-Dipeptidasa A/genética , Receptores de Angiotensina/genética , Angiotensina II/fisiología , Bradiquinina/fisiología , Gasto Cardíaco Bajo/fisiopatología , Cardiomegalia/fisiopatología , Humanos , Peptidil-Dipeptidasa A/fisiología , Receptores de Angiotensina/fisiología , Sistema Renina-Angiotensina/fisiologíaRESUMEN
The protein product of the myotonic dystrophy (DM) gene is a putative serine-threonine protein kinase (DM kinase). Previous reports have characterized the DM gene product as various 50-62-kDa proteins. The predicted protein size from DM cDNA sequence is 69 kDa. We therefore expressed a full-length recombinant human DM kinase protein and compared its size and expression to heart, cardiac Purkinje fibers, and skeletal muscle from normal and DM subjects. Recombinantly expressed DM kinase and endogenous DM kinase in human heart, displayed two immunoreactive DM kinase proteins with apparent molecular sizes of 71 and 80 kDa, suggesting that these prior reports are incorrect. In cardiac Purkinje fibers the 71-kDa protein was the major form, and in skeletal muscle the 80-kDa protein was the major form. Immunostaining showed DM kinase localized to neuromuscular junctions in skeletal muscle and intercalated discs in heart and Purkinje fibers. DM subjects showed low abundance of DM kinase in heart and skeletal muscle, suggesting haplotype insufficiency as a potential mechanism for disease expression. These studies describe differential expression of two protein forms of DM kinase, which are localized to specialized cellular structures associated with impulse transmission.
Asunto(s)
Expresión Génica , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/biosíntesis , Animales , Secuencia de Bases , Línea Celular , Membrana Celular/enzimología , Chlorocebus aethiops , Clonación Molecular , Cartilla de ADN , ADN Complementario , Humanos , Immunoblotting , Inmunohistoquímica , Riñón , Datos de Secuencia Molecular , Peso Molecular , Músculo Esquelético/enzimología , Miocardio/enzimología , Distrofia Miotónica/enzimología , Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Ramos Subendocárdicos/enzimología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Fracciones Subcelulares/enzimología , TransfecciónRESUMEN
Angiotensin II causes pulmonary vasoconstriction in man and in animals, and angiotensin-converting enzyme (ACE) inhibitors have prevented the development of chronic pulmonary hypertension in animals models. Angiotensin II may contribute to lung vascular remodeling in pulmonary hypertensive disease, since cilazapril, an inhibitor of ACE, reduces pulmonary vascular medical thickening in chronically hypoxic rats with established pulmonary hypertension. Furthermore, the ACE DD genotype, which has been associated with increased circulating and tissue ACE activity, has been associated with left ventricular hypertrophy in human hypertensive disorders. The ACE DD genotype may also 'permit' a greater hypertrophic adaptation of the pressure-over-loaded right ventricle. In fact, we have shown that pulmonary hypertension patients with maintained cardiac output and less right-heart failure fall into the group with the DD genotype and that patients with a low cardiac output and more severe right-heart failure fall into the group with the non-DD genotype, supporting the hypothesis. We assessed cardiopulmonary hemodynamics in patients with primary (unexplained) pulmonary hypertension and segregated the patients based on their ACE genotype. For similar mean pulmonary artery pressures in the DD and non-DD groups, the cardiac output was substantially lower in the patients with the non-DD genotype, whereas the values for mean right atrial pressure and pulmonary vascular resistance were double when compared with the DD group. Our data show that the ACE DD genotype is prevalent in patients with severe pulmonary hypertension and is a marker of maintained right ventricular function.
Asunto(s)
Hipertensión Pulmonar/fisiopatología , Peptidil-Dipeptidasa A/fisiología , Resistencia Vascular/efectos de los fármacos , Disfunción Ventricular Derecha/fisiopatología , Animales , Cilazapril/uso terapéutico , Modelos Animales de Enfermedad , Electroforesis en Gel de Agar , Genotipo , Hemodinámica/efectos de los fármacos , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Masculino , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Disfunción Ventricular Derecha/tratamiento farmacológicoRESUMEN
Plasma CK concentrations have been widely used as the primary muscle enzyme marker for diagnosis and progression of myositis. Recently, total CK and CK-MB serum concentrations have been compared to, and used in conjunction with, serum concentrations of aspartate aminotransferase in diagnosis of myositis. The algorithmic use of CK, AST, and aldolase plasma concentrations to diagnose and categorize patients with myopathy may be a useful method of diagnosing specific muscle disease without invasive procedures. CAIII, as a specific marker for skeletal muscle damage, may replace CK as the enzyme of choice in diagnosis and progression of myositis and other muscle disease. Additional studies are required to determine the usefulness of carbonic anhydrase for the diagnosis and assessment of myositis.
Asunto(s)
Miositis/diagnóstico , Biomarcadores/sangre , Humanos , Miositis/terapia , Resultado del TratamientoRESUMEN
Site-directed mutagenesis was used to alter the amino-acid residues at the presumed catalytic site Cys-283 and ATP binding site Asp-340 of human creatine kinase B cDNA. In addition, a highly conserved arginine residue, Arg-292, was also mutated. Transfection of 0.1 to 1 microgram of recombinant plasmid into COS cells produced increasing creatine kinase activity in the cell lysate. The expression of mutant Cys283-Tyr and Cys283-Ser resulted in complete abolition of homodimer BB isoform enzymatic activity without alteration of the capacity for dimerization. Expression of mutants Arg292-His, Arg292-Leu, and Arg292-Gln produced non-functional homodimers, whereas expression of mutant Arg292-Lys produced a homodimer with enzymatic activity that was 42% of the enzymatic activity of the wild type. Expression of the Asp340-Glu mutant creatine kinase did not alter enzyme activity as compared to the wild type. Following heterodimerization, there was inhibition of the normal subunit by the mutant subunit, for both the BB and the MB dimer. The results showed residues Cys-283 and Arg-292 are essential for enzyme catalysis. The best fit model for the dimer is one in which there is close apposition of the two catalytic sites. The interaction of the individual subunits during dimerization provides a molecular approach for dominant negative modulation of the creatine kinase isozyme system in future genetic manipulative experiments.
Asunto(s)
Creatina Quinasa/genética , Secuencia de Bases , Sitios de Unión , Creatina Quinasa/química , ADN Complementario/análisis , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-DirigidaRESUMEN
BACKGROUND: Familial hypertrophic cardiomyopathy (FHCM) is an autosomal dominant disease with protean clinical manifestations, ranging from asymptomatic to that of severe heart failure or sudden death. There is no known parameter in individuals with hypertrophic cardiomyopathy (HCM) that predicts a specific clinical event. This is particularly troublesome for premature sudden death that frequently occurs in young athletes without prior symptoms. Recent identification of mutations in the beta myosin heavy chain (beta MHC) gene that co-segregate with the inheritance of the disease provides an opportunity to determine whether certain mutations are more likely to induce a particular clinical event. In this study we analyzed the genotype and phenotype of individuals from two unrelated families with HCM in which the affected individuals have the same missense mutation in exon 13 (G1208A) of the coding sequence for beta MHC. METHODS: Individuals from two unrelated families with the diagnosis of FHCM were screened by history, physical examination, electrocardiography, and two dimensional echocardiography. After extraction of DNA from the blood of these individuals, the exon 13 of the beta MHC gene was amplified by polymerase chain reaction (PCR), and the PCR product was digested with Ddel restriction endonuclease. The digestion products were separated by gel electrophoresis and identified by ethidium bromide staining. RESULTS: We studied 54 individuals from the two families, 21 were affected with HCM of which eleven died prematurely, eight from sudden cardiac death. While most of the nine affected individuals studied had septal hypertrophy, three had concentric hypertrophy and six, left ventricular outflow tract obstruction. Onset of symptoms was in the second decade of life. Electrophoretic separation of the digested DNA (exon 13) from unaffected individuals provided two fragments of 84 and 70 bp in size, as expected. In contrast, DNA from individuals affected with HCM showed four fragments of 84 bp, 70 bp, 52 bp and 32 bp indicating they inherited the mutation. In only one 10 year old male was the mutation present without evidence of HCM which gives an overall penetrance of 86%. CONCLUSIONS: The missense mutation in exon 13 of the beta MHC gene in individuals with FHCM is associated with high penetrance, highly variable expressivity, severe disease, early in onset and a high incidence of premature sudden death. Based on these results we recommend individuals from families with HCM be screened for this missense mutation and if positive, be counselled to avoid combative sports, as it is these activities that often precipitate sudden death.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Muerte Súbita Cardíaca/prevención & control , Mutación , Miosinas/genética , Adulto , Secuencia de Bases , Cardiomiopatía Hipertrófica/fisiopatología , ADN/análisis , Análisis Mutacional de ADN , Muerte Súbita Cardíaca/etiología , Femenino , Pruebas Genéticas , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , FenotipoRESUMEN
The clinical manifestation of myotonic dystrophy (DM) is correlated to the extent of expansion of an unstable [CTG]n DNA motif. Recent studies have demonstrated that this trinucleotide motif forms part of the last, 3' untranslated exon of a gene which potentially encodes multiple protein isoforms of a serine/threonine protein kinase (myotonic dystrophy protein kinase, DM-PK). We report here on the development of antisera against synthetic DM-PK peptide antigens and their use in biochemical and histochemical studies. Immunoreactive DM-kinase protein of 53 kD is present at low levels in skeletal and cardiac muscle extracts of DM patients and normal controls. Immunohistochemical staining revealed that DM-PK is localised prominently at sites of neuromuscular and myotendinous junctions (NMJs and MTJs) of human and rodent skeletal muscles. Furthermore, very low levels of immunoreactive DM-PK protein are present in the sarcoplasm of predominantly type I fibres in various muscles. Strikingly, presence of the protein can also be demonstrated for NMJs of muscular tissues of adult and congenital cases of DM, with no gross changes in structural organisation. Our findings provide a basis for further characterisation of the role of the kinase in protein assembly processes or signal mediation at synaptic sites and ultimately for the understanding of the complex pathophysiology of DM.
Asunto(s)
Músculos/enzimología , Distrofia Miotónica/enzimología , Unión Neuromuscular/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos , Western Blotting , Encéfalo/enzimología , Exones , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica , Péptidos/síntesis química , Péptidos/inmunología , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/genética , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Creatine kinase (CK) isoenzymes play a central role in energy transfer. Expression of CK isoenzymes in rat uterus and placenta was analyzed to evaluate their contribution to energy metabolism during pregnancy and delivery. Tissue from the uterine horns and placentas of pregnant rats from day 14 of gestation to 17 days postpartum was analyzed for expression of "brain" CK (BCK) and ubiquitous mitochondrial CK (uMtCK) mRNA, protein, and enzyme activity. uMtCK mRNA expression is high in prepartum uterus, but rapidly falls (> 10-fold) after delivery to a nadir at 7 days postpartum. Prepartum BCK mRNA expression is coordinate with uMtCK but has a 15-fold greater expression than uMtCK. Both CK mRNAs rise by 17 days postpartum. Both BCK and uMtCK mRNA expressions are strongly induced in placenta at 20 days gestation with a rapid fall (> 6-fold) immediately before delivery. Protein expression of BCK and uMtCK is also coordinate. However, analysis of mRNA and protein expression indicates that significant posttranscriptional regulation of both kinds of CK also occurs. CK activity in uterus and placenta reflects BCK expression. By immunohistochemistry, BCK and uMtCK protein expression is highly localized in the placenta and endometrium of prepartum uterus. This expression shifts entirely to the uterine smooth muscle by 17 days postpartum. uMtCK mRNA expression is rapidly induced by beta-estradiol in vitro (> 6-fold), demonstrating estrogen-responsive elements in the uMtCK nuclear gene. Thus a second isoenzyme of CK, uMtCK, is expressed in rat uterus and placenta and is highly regulated with BCK. These results suggest an important role for CK in the maintenance and termination of pregnancy.
Asunto(s)
Creatina Quinasa/metabolismo , Placenta/enzimología , Preñez/metabolismo , Útero/enzimología , Animales , Secuencia de Bases , Northern Blotting , Células Cultivadas , Creatina Quinasa/genética , Femenino , Immunoblotting , Inmunohistoquímica , Isoenzimas , Sondas Moleculares/genética , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Placenta/citología , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Útero/citologíaRESUMEN
Polymorphism in the angiotensin-converting enzyme (ACE) gene has been shown to correlate with circulating ACE concentrations, and also to be an independent risk factor for the development of myocardial infarction, particularly in men thought to be at low risk by standard criteria. We determined the genotypes of individuals with end-stage heart failure due to either ischaemic dilated cardiomyopathy (102) or idiopathic dilated cardiomyopathy (112) and compared these to organ donors with normally functioning hearts (79). Genotypes were determined by the polymerase chain reaction with oligonucleotide primers flanking the polymorphic region in intron 16 of the ACE gene to amplify template DNA isolated from patients. Compared with the DD frequency in the control population, the frequency of the ACE DD genotype was 48% higher in individuals with idiopathic dilated cardiomyopathy (p = 0.008) and 63% higher in subjects with ischaemic cardiomyopathy (p = 0.008), suggesting that an ACE gene variant may contribute to the pathogenesis of both types of cardiomyopathy.
Asunto(s)
Cardiomiopatías/genética , Peptidil-Dipeptidasa A/genética , Adolescente , Adulto , Anciano , Cardiomiopatías/patología , Niño , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Factores de RiesgoRESUMEN
We developed standards for creatine kinase (CK; EC 2.7.3.2) assays by expressing human CK cDNAs in COS cells. Cells were transiently transfected with full-length cDNAs for CK subunits M and B, individually and in combination; and subsequently, high concentrations of CK activity were present in the cell lysate (1.2 U/mg protein). These proteins exhibited the characteristic isoenzyme-specific electrophoretic mobilities for CK MM and BB isoenzymes. We also produced subforms of CK MM and MB, identical to the modified CK variants produced in plasma after muscle or myocardial injury, by mutating the cDNA for the CK M subunit to delete the carboxy-terminal lysine residue. When this construct was cotransfected with the normal cDNAs for CK M and B, five electrophoretically distinct CK isoenzymes were detected by nondenaturing electrophoresis: MM3, MM2, MM1, MB2, and MB1. These proteins retained 100% of their activity after storage of the cell lysates -20 or 4 degrees C for 3 months.
Asunto(s)
Creatina Quinasa/sangre , Proteínas Recombinantes , Secuencia de Bases , Línea Celular , Creatina Quinasa/química , Creatina Quinasa/genética , Electroforesis en Gel de Agar , Estabilidad de Enzimas , Humanos , Isoenzimas , Datos de Secuencia Molecular , Infarto del Miocardio/enzimología , Reacción en Cadena de la Polimerasa , Control de Calidad , Proteínas Recombinantes/química , TransfecciónRESUMEN
The myotonic dystrophy mutation has recently been identified; however, the molecular mechanism of the disease is still unknown. The sequence of the myotonin-protein kinase gene was determined, and messenger RNA spliced forms were identified in various tissues. Antisera were developed for analytical studies. Quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to demonstrate that decreased levels of the messenger RNA and protein expression are associated with the adult form of myotonic dystrophy.
Asunto(s)
Músculos/metabolismo , Distrofia Miotónica/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , ARN Mensajero/genética , Adulto , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Expresión Génica , Humanos , Datos de Secuencia Molecular , Peso Molecular , Músculos/química , Distrofia Miotónica/metabolismo , Proteína Quinasa de Distrofia Miotónica , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/químicaRESUMEN
BACKGROUND: The beta-myosin heavy chain (beta-MHC) gene has been identified as a major locus for familial hypertrophic cardiomyopathy (FHCM). We recently showed that one of the common mutations associated with FHCM is expressed in the cardiac muscle messenger RNA (mRNA) of an affected individual. Since beta-MHC is a major sarcomeric protein of cardiac and skeletal muscle, studies were performed to determine whether the mutation is also expressed in skeletal muscle. METHODS AND RESULTS: Biopsies were obtained of skeletal muscle (biceps brachii) from a proband with FHCM known to have the missense mutation in exon 13 of the beta-MHC gene. RNA was extracted from skeletal muscle and lymphocytes by the RNAzol method. First-strand complementary DNA was synthesized by reverse transcription using an antisense primer to exon 16. Polymerase chain reaction (PCR) was performed using primers to exons 12 and 14 to amplify the segment encompassing exon 13. The PCR products were digested with Ddel restriction endonuclease. Undigested PCR product in the control and the proband was 321 base-pairs (bp). Ddel digestion of the PCR product from normal skeletal and lymphocytes showed two DNA fragments of 181 and 140 bp as expected, whereas digestion of the PCR product from the proband's skeletal muscle and lymphocytes showed four DNA fragments of 181, 149, 140, and 32 bp due to the mutation in exon 13. This indicates that the mutation in affected individuals is also expressed in the mRNA of skeletal muscle and lymphocytes. CONCLUSIONS: To our knowledge, this is the first documentation of a beta-MHC gene mutation expressed in skeletal muscle. This finding is provocative. Does it impair skeletal muscle function? If so, how? If not, why not? Is the impairment, or lack of it, a clue to the molecular defect of cardiac muscle? Furthermore, skeletal muscle provides a readily accessible source of mRNA for expression studies and for purification of the beta-MHC protein, which is probably essential to future investigation designed to unravel the molecular basis of this disorder.