RESUMEN
Endothelins, nitric oxide, and oxygen-derived free radicals decisively regulate vascular tone. An imbalance in the biosynthesis of these substances in pathophysiologic conditions may trigger vasospasm and promote the development of atherosclerosis. Previous studies have shown that oxygen-derived free radicals can increase the synthesis of endothelin-1 in cultured endothelial cells. Interestingly, conditions of increased oxidative stress within smooth muscle cells as induced by angiotensin II infusion or hypercholesterolemia have been shown to be associated with increased autocrine synthesis of endothelin-1. Because endothelin-1 formed in smooth muscle cells can trigger hypersensitivity to vasoconstrictors, we tested whether oxidative stress per se may affect endothelin expression in vascular smooth muscle cells. Cultured human coronary artery smooth muscle cells were exposed to oxidative stress generated by the xanthine/xanthine oxidase reaction or by hydrogen peroxide. Preproendothelin-1 mRNA content was quantitated by means of quantitative polymerase chain reaction and endothelin-1 protein was measured by radioimmunoassay. Incubation with xanthine/xanthine oxidase significantly increased preproendothelin-1 mRNA synthesis, whereas GAPDH remained unchanged. Likewise, xanthine/xanthine oxidase also led to a dose-dependent increase of intracellular endothelin-1. The increase in ET-1 expression induced by xanthine/xanthine oxidase was significantly inhibited by superoxide dismutase but not by catalase. We conclude that oxygen-derived free radicals can stimulate the synthesis of endothelin-1 in endothelial and vascular smooth muscle cells by increasing preproendothelin-1 mRNA content and that this effect is mediated predominantly by superoxide anions. We therefore have identified a new mechanism in the interaction of oxidative stress and endothelin-1 expression in smooth muscle cells that may have important implications in diseases such as atherosclerosis and hypertension.
Asunto(s)
Vasos Coronarios/metabolismo , Endotelina-1/biosíntesis , Músculo Liso Vascular/metabolismo , Estrés Oxidativo/fisiología , Células Cultivadas , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , ARN Mensajero/biosíntesis , Especies Reactivas de Oxígeno/farmacología , Xantina Oxidasa/farmacologíaRESUMEN
RGS proteins (regulators of G protein signalling) negatively regulate G protein function as GTPase-activating proteins (GAP) for G protein alpha-subunits. The existence of mRNAs of different size for some of the RGS proteins, e.g. RGS3, suggests that these proteins may exist in isoforms due to alternative splicing. We therefore investigated RGS3 mRNA and protein expression in different human tissues. Ribonuclease protection assays and Northern blot analysis showed two specific mRNAs for RGS3 (RGS3L, RGS3S) in human myocardium, suggesting an additional, N-terminally truncated form of approximately 168 aa. When expressed as a recombinant protein RGS3S was recognized at approximately 23 kDa by an antipeptide antiserum originally raised against an RGS2 sequence. In membranes of human tissues this antiserum detected specific signals for RGS3L (approximately 70 kDa), RGS2 (approximately 30 kDa) and a 25-kDa protein, most likely RGS3S. Both RGS3S mRNA and the 25 kDa protein were abundant in human heart, whereas expression in liver, brain and myometrium was much weaker. To characterize RGS3S functionally, single turnover GTPase, adenylyl cyclase (AC) and phospholipase C (PLC) activities were determined. Both recombinant RGS3S and RGS16 increased Pi release from Galphai1 by about 150% and increased GTP- and GTP plus isoprenaline-stimulated AC activity by 20-30% in human left ventricular myocardial membranes. Additionally, both RGS proteins reduced basal and endothelin-stimulated PLC activity in these membranes by about 40%. We conclude that an additional truncated form of RGS3 is expressed in the human heart. As described for the full-length protein, RGS3S negatively regulates the activity of Gi/o- and Gq-, but not Gs-subfamily members.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa , Hígado/metabolismo , Miocardio/metabolismo , Proteínas RGS/fisiología , Humanos , Isoformas de Proteínas , Proteínas RGS/genéticaRESUMEN
Modulation of the biosynthesis of the vasoconstrictor peptide endothelin-1 by oxygen-derived free radicals generated by xanthine oxidase or hydrogen peroxide was studied in cultured endothelial cells. Endothelin-1 metabolism was investigated at the level of endothelin-1 promoter, preproendothelin-1 mRNA and intracellular big endothelin-1. Endothelin-1 mRNA, as characterized by Northern blotting, was increased both time- and dose-dependently by xanthine oxidase to up to 500% above baseline. Analysis of endothelin-1 promoter activity using a construct containing 1329 bp of the endothelin-1 promoter revealed that promoter activity was increased up to eight-fold by incubation with xanthine oxidase. Specificity was ascertained by co-incubation with superoxide dismutase and catalase leading to inhibition of the effect of xanthine oxidase. A significant contribution of nitric oxide was ruled out, since NOS III-mRNA transcription remained unchanged and l -NAME did not significantly alter endothelin-1 promoter activity. Synthesis of intracellular big endothelin-1 protein was increased dose-dependently by xanthine oxidase. Our results indicate that oxidative stress leads to increased endothelial synthesis of big endothelin-1, which is a previously unknown mechanism and may help to understand the detrimental association of increased oxidative stress and elevated endothelin-1 levels in pathophysiological conditions promoting atherosclerosis.
Asunto(s)
Endotelina-1/genética , Endotelina-1/metabolismo , Endotelinas/biosíntesis , Estrés Oxidativo , Regiones Promotoras Genéticas , Precursores de Proteínas/biosíntesis , Animales , Aorta/citología , Aorta/metabolismo , Northern Blotting , Catalasa/metabolismo , Bovinos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Radicales Libres/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Luciferasas/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , ARN Mensajero/metabolismo , Radioinmunoensayo , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Transfección , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , Xantina Oxidasa/farmacologíaRESUMEN
The nucleotide sequence of an 11,142-bp region including the stx2 operon in the genome of the temperate bacteriophage 933W in the EDL933 strain of Escherichia coli O157 was determined and compared to the respective regions derived from other lambdoid bacteriophages. In phage 933W, a region of ORFs interlinked by overlapping start-stop codons (ATGA) was detected preceding the toxin gene. These ORFs show a high degree of sequence identity to genes of the nin region of phage lambda. Immediately downstream of these nin genes we identified an ORF that may code for an anti-terminator similar to the lambda Q protein. It is concluded that toxin expression is directly associated with the initiation of cell lysis. Downstream of the stx2 operon we identified an ORF that is homologous to the holin gene S of bacteriophage PA-2. PCR primers were designed, which, based on a comparison of the phage sequences, appeared to be common to both stx1- and stx2-harbouring phages. However, only seven of the 22 STEC strains investigated from serogroups O157, O26, O103 and O111 yielded the expected PCR amplification product. The data reported here may be useful in developing new strategies for inhibiting the expression of Stx and for developing universal diagnostic primers for use in tracking the origin and evolution of Shiga toxins and the phages that carry them.
Asunto(s)
Toxinas Bacterianas/genética , Bacteriófagos/genética , Genoma Viral , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Toxinas ShigaRESUMEN
Chronic treatment of rats with carbachol downregulates M-cholinoceptors and inhibitory, pertussis toxin (PTX)-sensitive G proteinalpha-subunits (Gialpha) and sensitizes the heart to arrhythmogenic effects of isoprenaline (ISO), suggesting a causal relationship. To test this hypothesis by a more direct and quantitative approach, nine groups of rats were treated for 24 h with increasing doses of PTX (1.25-200 microg/kg i.v.). Inactivation of cardiac Gialpha was determined biochemically by 32P-ADP-ribosylationin vitro and functionally by measuring contractile effects of carbachol. Effects of ISO were studied in spontaneously beating right atria (RA) and isolated papillary muscles (PM; paced at 1 Hz). PTX increased heart rate in conscious animals (ECG) with a bell-shaped dose-dependency (maximal increase 120 beats/min at 7.5 microg/kg). PTX dose-dependently inactivated 25-85% of total cardiac Gialpha, which linearly correlated with a loss of the direct negative chronotropic effect of carbachol in atria, but not with a loss of its indirect negative inotropic effect in PM. The latter was resistant up to PTX 20 microg/kg (=70% inactivation). The decrease in Gialpha closely correlated with an increased efficacy of ISO to induce spontaneous contractile activity (automaticity) in PM. At 3 micromol/l ISO, all PM from PTX 200 microg/kg beat spontaneously compared to 10% in control. In contrast, pretreatment with PTX only modestly and not clearly dose-dependently increased the inotropic potency of ISO (PTX 100 microg/kg: EC50 28v 81 nmol/l in control) and did not affect the chronotropic effect of ISO. The disparity of the functional consequences of PTX treatment suggest that under physiological conditions, Gialpha serve mainly to suppress arrhythmogenic, but not or to a minor extent, positive chronotropic or inotropic effects of beta-adrenoceptor activation.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Frecuencia Cardíaca/fisiología , Isoproterenol/farmacología , Contracción Miocárdica/fisiología , Músculos Papilares/fisiología , Receptores Adrenérgicos beta/fisiología , Adenosina Difosfato Ribosa/metabolismo , Animales , Carbacol/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Atrios Cardíacos , Frecuencia Cardíaca/efectos de los fármacos , Técnicas In Vitro , Masculino , Contracción Miocárdica/efectos de los fármacos , Músculos Papilares/efectos de los fármacos , Toxina del Pertussis , Ratas , Ratas Wistar , Receptores Adrenérgicos beta/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
A number of molecular and cellular alterations have been identified in the failing human heart that help to understand contraction and relaxation abnormalities. Cyclic AMP dependent pathways are desensitized due to quantitative changes in beta-adrenoceptors, beta-adrenoceptor kinase, and inhibitory G-proteins. Calcium homeostasis is impaired, characterized by a decreased calcium reuptake rate of the sarcoplasmic reticulum, an increased threshold of the calcium release channel, and an increased Na+/Ca2+ exchanger expression. Myofibrillar function may be affected by a decrease in Mg2(+)-ATPase activity and in troponin I phosphorylation, and by changes in TnT isoform expression. These alterations seem to occur independently of the underlying etiology of heart failure and are most likely consequences rather than primary causes of the disease. Most likely, chronic neurohumoral activation and abnormal mechanical load initiate the majority of the hitherto known changes in the myocardium and promote the further progression of cardiac failure as part of a vicious circle. Further extension of knowledge of pathophysiological mechanisms should improve therapeutical strategies which aim at slowing the progression of heart failure and at reversing secondary alterations by interrupting the deleterious influence of neurohumoral activation. Future progress will depend on answers to current gaps in our knowledge of heart failure, including the unknown primary cause of idiopathic dilated cardiomyopathy, factors underlying the greatly variable progression of pump failure, as well as the exact pathophysiological role of the molecular alterations as described in this review.
Asunto(s)
Proteínas Contráctiles/metabolismo , Insuficiencia Cardíaca/metabolismo , Contracción Miocárdica , Miocardio/metabolismo , Transducción de Señal , Calcio/metabolismo , Cardiotónicos/uso terapéutico , Diástole , Expresión Génica , Corazón/efectos de los fármacos , Corazón/fisiopatología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/fisiopatología , Homeostasis , Humanos , SístoleRESUMEN
The enkephalins are derived from a common precursor protein known as preproenkephalin (ppENK). Enkephalins appear to be one of the endogenous ligands for the opiate receptors. In the rat the ventricular myocardium contains more ppENK mRNA than any other tissue. To gain further insight into the role of cardiac enkephalins, the regional and developmental distribution of ppENK mRNA was studied by Northern blotting and in situ hybridization. In the early postnatal period, ppENK mRNA is low in atrial and ventricular myocardium. With maturation, ppENK expression increases threefold in left and right ventricular tissue, but not in the atria or cardiac conductive system. Interestingly, ppENK mRNA levels are four times higher in the left than in the right chamber. Thus, to our knowledge, ppENK is the only gene exhibiting marked differences in expression between the adult right and left ventricle. Given the left-side preference of ppENK expression, the possibility is raised that the left ventricle is an endocrine organ that supplies the body with enkephalins.
Asunto(s)
Encefalinas/genética , Regulación del Desarrollo de la Expresión Génica , Corazón/crecimiento & desarrollo , Miocardio/metabolismo , Precursores de Proteínas/genética , ARN Mensajero/biosíntesis , Transcripción Genética , Envejecimiento , Animales , Animales Recién Nacidos , Northern Blotting , Peso Corporal , Encefalinas/biosíntesis , Femenino , Ventrículos Cardíacos , Hibridación in Situ , Masculino , Tamaño de los Órganos , Precursores de Proteínas/biosíntesis , Ratas , Ratas WistarRESUMEN
OBJECTIVE: The aim of our study was to analyse the single-channel properties of L-type calcium channels from failing human heart and to compare them to the respective animal data. Furthermore, we intended to evaluate the feasibility of future single-channel studies on the role of calcium channels in the pathophysiology of heart failure. METHODS: Single L-type calcium channels were recorded in ventricular myocytes from explanted failing human heart, using the cell-attached configuration of the patch-clamp technique. RESULTS: One or more successful registrations of calcium channels could be obtained in 11 of 19 cell isolations. Determination of single-channel conductance yielded a mean value of 16.6 +/- 1.2 pS (70 mM Ba2+ as the charge carrier) under control conditions and 23.7 +/- 2.8 pS in presence of the calcium-channel agonist FPL 64176. The rapid gating process could be described by a C<-->C<-->O gating scheme. Slow gating analysis revealed a highly significant clustering of active and non-active sweeps. CONCLUSION: Single-channel measurements of L-type calcium channels in human failing ventricle are feasible and reproducible despite the varying patient characteristics. Their channel properties are qualitatively comparable to those found in other mammals. Whether there are quantitative differences due to the underlying heart failure can be elucidated in further studies.
Asunto(s)
Canales de Calcio/fisiología , Insuficiencia Cardíaca/fisiopatología , Corazón/fisiopatología , Adulto , Anciano , Calcio/metabolismo , Agonistas de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Femenino , Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos , Humanos , Activación del Canal Iónico/efectos de los fármacos , Masculino , Persona de Mediana Edad , Técnicas de Placa-Clamp , Pirroles/farmacologíaRESUMEN
OBJECTIVE: The goal of the present study was to examine the ability of failing myocardium to respond to enhanced preload with an increase in force development. METHODS: The effect of various preload conditions (2.5-15 mN) on force development was studied in right ventricular trabeculae carneae from explanted human failing hearts with ischemic cardiomyopathy (ICM, n = 5, 42 preparations) or idiopathic dilated cardiomyopathy (DCM, n = 9, 77 preparations). To determine the severity of cardiac impairment we measured the positive inotropic effect of beta-adrenoceptor stimulation and calcium (ISO/Ca2+ ratio) and the expression of atrial natriuretic peptide (ANP) mRNA in all hearts. RESULTS: (1) Force of contraction increased with stepwise augmentation of preload (length at 2.5 mN preload to length of maximal force development) from 3.7 +/- 0.5 (ICM) and 2.7 +/- 0.4 (DCM) to 8.3 +/- 0.9 and 6.5 +/- 0.8 mN/mm2, respectively (p < 0.05). (2) The ISO/Ca2+ ratio was 0.40 +/- 0.04 (ICM) and 0.35 +/- 0.03 (DCM), respectively. (3) ANP mRNA was expressed in all preparations, albeit at greatly varying levels (ICM 22.5 +/- 6.1 and DCM 18.7 +/- 4.7 normalized arbitrary units). (4) Contraction experiments performed in left ventricular tissue (n = 3, 32 preparations) essentially confirmed the results. CONCLUSION: The Frank-Starling mechanism is preserved in terminally failing human hearts irrespective of the underlying etiology. We found no relation between the severity of cardiac impairment as assessed by either ANP expression or the ISO/Ca2+ ratio and the ability of failing human myocardium to respond to enhanced preload with an increase in force development.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Factor Natriurético Atrial/genética , Calcio/farmacología , Insuficiencia Cardíaca/fisiopatología , Isoproterenol/farmacología , Contracción Miocárdica/efectos de los fármacos , Autorradiografía , Northern Blotting , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Expresión Génica , Insuficiencia Cardíaca/metabolismo , Ventrículos Cardíacos , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Miocardio/metabolismo , ARN Mensajero/análisis , Estimulación QuímicaRESUMEN
The objectives of the present study were to investigate the risk of B. burgdorferi s.1. (Bb)-transmission by I. ricinus-nymphs to a host (i) after different periods of feeding, and (ii) with regard to the particular method of tick removal. On each of 72 Mongolian gerbils 3 tick nymphs taken from a highly infected batch were allowed to feed in a small capsule. Feeding ticks were removed 16.7, 28.9, 47.0, and 65.2 hrs post-attachment. In each of these 4 groups 3 sub-groups with 6 gerbils each were deticked by (a) pulling ticks out with forceps without any pretreatment, (b) pulling ticks out after 3 min of intensive squeezing, and (c) applying nail polish to ticks 1.1 hrs before removal. The infection status in each gerbil was subsequently determined by larval xenodiagnosis. All gerbils with ticks removed > or = 47 hrs post-attachment were found to be infected. After 16.7 hrs as well as after 28.9 hrs of tick feeding, approximately 50% of the gerbils had acquired a transmissible infection, thus Bb-transmission to a host may even occur in the early phases of I. ricinus feeding. There is no evidence from this study that the tick removal method used has any significant influence on a host's Bb-infection risk.
Asunto(s)
Borrelia/patogenicidad , Enfermedad de Lyme/transmisión , Animales , Técnica del Anticuerpo Fluorescente Indirecta , Gerbillinae/parasitología , Interacciones Huésped-Parásitos , Ixodes/parasitología , Enfermedad de Lyme/parasitologíaRESUMEN
End-stage human heart failure is associated with changes in expression of steady-state messenger RNA (mRNA) levels. These changes correspond to alterations in protein levels and myocardial function and may have clinical implications regarding etiology, clinical state, or prognosis. However, analysis of mRNA levels in endomyocardial biopsies can be accomplished only by the quantitative polymerase chain reaction, which is difficult to standardize. The aim of the study was to evaluate whether the RNase protection assay is applicable to measure mRNAs of multiple genes simultaneously in small amounts of ventricular myocardium comparable to myocardial biopsies. Total RNA was prepared from left ventricular myocardium from terminally failing hearts with idiopathic (n=9) or ischemic cardiomyopathy (n=7) and from nonfailing control hearts (n=10). mRNA was measured by an optimized RNase protection assay for the beta1-adrenoceptor, the stimulatory G protein alpha-subunit (Gsalpha), phospholamban, the calcium ATPase of the sarcoplasmic reticulum (SERCA), beta-myosin heavy chain (beta-MHC), and the atrial natriuretic peptide (ANP). We extracted 10.7+/-2.1 microg total RNA from three myocardial biopsies taken in vitro. All of the six genes were measurable in duplicate in a total of 7 microg RNA. mRNAs of beta1-adrenoceptor, phospholamban, and SERCA were lower in failing than in nonfailing myocardium by 50%, 33%, and 42% respectively, whereas beta-MHC and Gsalpha mRNAs were unchanged. mRNA of ANP was expressed at high levels only in the failing myocardium, providing a highly specific and sensitive marker for discriminating nonfailing and failing hearts. A direct comparison with ANP and Gsalpha levels obtained by Northern blot analysis with 7.5 microg total RNA showed a good correlation between the two methods. The RNase protection assay is thus a suitable method for simultaneous measurements of multiple mRNA levels in human myocardial biopsies. Changes in mRNA levels closely reflected those identified by other methods using larger amounts of RNA. Increased myocardial ANP mRNA levels determined by the RNase protection assay may serve as a molecular marker of heart failure.
Asunto(s)
Expresión Génica , Ventrículos Cardíacos/metabolismo , Miocardio/metabolismo , ARN/biosíntesis , Adolescente , Adulto , Northern Blotting , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ribonucleasas/metabolismoRESUMEN
A cosmid library of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL 933 was constructed and clones carrying the stx2 gene were identified by colony blot hybridization with a stx2B specific probe. Nucleotide sequencing upstream of the stx2A gene revealed high sequence identities of 89.5% to the ileX tRNA gene found in E. coli. The ileX gene was located 260 bp from the translational start codon of stx2A. PCR analysis with primers specific for this analyzed region showed that in 11 Stx2-producing EHEC strains from patients with hemolytic uremic syndrome, all PCR-positive strains carried the ileX tRNA gene. However, PCR analysis of the respective region in 11 Stxl-producing EHEC strains detected no ileX genes. Although the role of ileX in Stx2-producing EHEC strains is not clear, its function in regard to the use of rare codons and as an integration site is discussed.
Asunto(s)
Toxinas Bacterianas/genética , Enterotoxinas/genética , Escherichia coli O157/genética , ARN de Transferencia de Isoleucina/genética , Clonación Molecular , Cósmidos , ADN Bacteriano/análisis , Regulación Bacteriana de la Expresión Génica/genética , Biblioteca de Genes , Genes Bacterianos/genética , Operón/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Toxinas ShigaRESUMEN
Myoglobin levels are decreased in various animal models of heart failure, a change that has been associated with compromised energy supply. The underlying mechanisms by which myoglobin content decreases in failing myocardium are unknown. Bovine hereditary cardiomyopathy (bCMP) displays several characteristics of human dilated cardiomyopathy with a marked desensitization of the beta-adrenoceptor signal cascade. The aim of the present study was to investigate whether a similar reduction of myoglobin can be seen in this animal model, and to elucidate the possible mechanism of this reduction. Myoglobin protein concentration was decreased by 46-47% (P < 0.05) in left and right ventricular myocardium of failing hearts (n = 9) compared to control hearts (n = 11). No difference was found between atria of diseased and control animals. Immunohistochemistry with a polyclonal antibody against myoglobin revealed a strong and uniform labeling in cardiomyocytes of non-failing hearts. Using microscopic densitometry, immunosignals were significantly decreased in ventricular myocytes of bCMP hearts (168 +/- 5.3 v 118 +/- 8.6 arbitrary units, P < 0.05). Moreover, myoglobin was heterogeneously distributed in bCMP hearts, with single myocytes showing no staining. Slot blot analysis of total RNA demonstrated a 40-50% reduction (P < 0.05) of myoglobin mRNA levels in ventricular but not in atrial myocardium of bCMP hearts. The results support the view that a decrease of myocardial myoglobin is a general phenomenon in end-stage heart failure. It appears to be primarily due to reduced gene expression but may be aggravated by leaking from single myocytes. The decrease of myoglobin may contribute to the imbalance between energy production and energy expenditure in heart failure.
Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Enfermedades de los Bovinos/metabolismo , Miocardio/metabolismo , Mioglobina/metabolismo , Animales , Northern Blotting , Bovinos , Femenino , Atrios Cardíacos/química , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/química , Ventrículos Cardíacos/metabolismo , Humanos , Inmunohistoquímica , Masculino , Miocardio/química , Mioglobina/química , Mioglobina/inmunología , Proteínas/química , Proteínas/metabolismo , ARN/química , ARN/metabolismoRESUMEN
In this study, we determined the nucleotide sequence of the p gene contained within a 5-kb EcoRI restriction fragment cloned from Shiga-like toxin II (SLT-II)-converting phage 933W of Escherichia coli O157:H7 strain EDL933. The p gene was 702 bp long and had 95.3% sequence similarity to the p gene of phage lambda. Multiple hybridization patterns were obtained when genomic DNA fragments were hybridized with both p and slt-I, slt-II, or slt-IIc sequences. All O157 isolates also possessed an analog of lambda gene p which was not linked with either slt-I or slt-II. Restriction fragment length polymorphism comparisons of clinical O157 isolates and derivates undergoing genotype turnover during infection were made, and loss of large DNA fragments that hybridized with slt-II and p sequences was observed. To further analyze the DNA region containing the p and slt genes, we amplified fragments by using a PCR with one primer complementary to p and the other complementary to either the slt-I or the slt-II gene. PCR analysis with enterohemorrhagic E. coli O157 and non-O157 strains yielded PCR products that varied in size between 5.1 and 7.8 kb. These results suggest that even within O157 isolates, the genomes of SLT-converting phages differ. The methods described here may assist in further investigation of SLT-encoding phages and their role in the epidemiology of infection with enterohemorrhagic E. coli.
Asunto(s)
Toxinas Bacterianas/genética , Bacteriófago lambda/genética , Enterotoxinas/genética , Escherichia coli O157/genética , Genes Bacterianos , Genes Virales , Southern Blotting , Sondas de ADN , ADN Bacteriano/genética , Infecciones por Escherichia coli/microbiología , Síndrome Hemolítico-Urémico/genética , Síndrome Hemolítico-Urémico/microbiología , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Toxina Shiga IIRESUMEN
We examined 30 children with classical hemolytic-uremic syndrome (HUS) for the presence of enterohemorrhagic Escherichia coli (EHEC) strains in stool samples and determined the specific immune response to O157 lipopolysaccharide in acute-phase serum samples from these patients. EHEC O157 strains were isolated from stool samples of 18 (60%) of the patients, and non-O157 EHEC strains were isolated from 5 (17%) of the patients. For O157 strain isolation from stools, we introduced a selective enrichment step using O157-specific antibodies attached to paramagnetic particles (immunomagnetic separation [IMS] method). This procedure allowed the detection of O157 strains at 10(2) CFU/g of stool in the presence of 10(7) coliform background flora organisms. By using IMS followed by plating on sorbitol MacConkey (SMAC) agar and cefixime-tellurite SMAC (CT-SMAC) agar, O157 strains were detected in 18 samples, whereas colony hybridization detected a subset of 12 positive samples and direct culture on CT-SMAC or SMAC agar detected only 7. Three of the 18 O157-positive stools were negative by cytotoxicity assay performed with stool filtrates and by direct PCR with DNA extracted from stools. The IMS technique allowed the isolation of O157 strains from 18 of 20 patients with serological evidence for O157 infection. Apart from the increase in sensitivity in O157 detection compared with that of direct culture, the IMS technique also has the advantage of being less labor-intensive and less time-consuming than the molecular methods. IMS can therefore be considered an efficient method for wide-spread use in the detection of O157 strains in clinical microbiology laboratories. However, because a significant number of HUS cases were attributable to non-O157 EHEC serogroups, the use of additional methods besides IMS in the bacteriological diagnosis of HUS is necessary.
Asunto(s)
ADN Bacteriano/análisis , Escherichia coli O157/aislamiento & purificación , Síndrome Hemolítico-Urémico/microbiología , Separación Inmunomagnética , Anticuerpos Antibacterianos/sangre , Niño , Preescolar , Escherichia coli O157/genética , Escherichia coli O157/inmunología , Heces/microbiología , Humanos , Lactante , Sensibilidad y EspecificidadRESUMEN
Increased expression of the inhibitory G protein Gi alpha-2 is assumed to contribute to desensitization of adenylyl cyclase in human heart failure. The mechanisms of upregulation involve increases in myocardial Gi alpha-2 protein, mRNA and gene transcriptional activity. To elucidate these mechanisms in more detail, the 5' flanking region of the human Gi alpha-2 gene (-1214/+115 bp) was cloned upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene and transfected in embryonic chick cardiomyocytes. CAT activity was measured 48 h after transfection. Unstimulated activity of the -1214/+115 bp construct was about 10-fold higher than activity of the basal CAT-construct (pGEMCAT). 5' deletion from -1214/+115 to -85/+115 bp upstream of the transcriptional start site increased, further stepwise deletions to 46/+115 gradually decreased promotor activity. Deletion from -46/+115 to -33/+115 bp completely abolished promotor activity. Stimulation of cardiomyocytes that had been transfected with the -1214/+115 CAT-construct with isoprenaline (10 microM), forskolin (10 microM), forskolin (10 microM) plus IBMX (10 microM) or dibutyryl-cAMP (1 mM) for 24 h induced an increase in CAT activity to 139 +/- 12% (n = 9), 211 +/- 18% (n = 12), 256 +/- 20% (n = 5) and 198 +/- 28% (n = 7) of unstimulated values, respectively. We conclude: 1) In chicken cardiomyocytes a sequence element of 52 bp between -85 and -33 bp is necessary to provide basal Gi alpha-2 promotor activity. 2) Elevation of cAMP has a stimulatory effect on the human Gi alpha-2 promotor, thereby offering a mechanism for beta-adrenoceptor-mediated increases in Gi alpha-2 in the heart.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Miocardio/metabolismo , Animales , Bucladesina/farmacología , Células Cultivadas , Embrión de Pollo , Cloranfenicol O-Acetiltransferasa/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Corazón/efectos de los fármacos , Corazón/embriología , Humanos , Mutación , Miocardio/citología , Plásmidos/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , Transfección , Regulación hacia Arriba/genéticaAsunto(s)
Calcinosis/diagnóstico por imagen , Mano/diagnóstico por imagen , Enfermedad de Raynaud/diagnóstico por imagen , Esclerodermia Sistémica/diagnóstico por imagen , Calcinosis/cirugía , Femenino , Mano/irrigación sanguínea , Mano/cirugía , Humanos , Isquemia/cirugía , Persona de Mediana Edad , Radiografía , Enfermedad de Raynaud/cirugía , Esclerodermia Sistémica/cirugíaRESUMEN
Elevation of cytosolic sodium is thought to be correlated with an increase in force of contraction due to an activation of sodium-calcium exchange. We investigated the inotropic response mediated by the new sodium-channel activator BDF 9148 (0.01-100 mumol/l) on failing human myocardium. Force of contraction was studied using electrically driven human papillary muscle strips from moderately (NYHA II-III, mitral valve replacement) and terminally (NYHA IV, heart transplantation) failing hearts. We also investigated the effects in auricular trabeculae from non-failing hearts (aortocoronary bypass operation). Results were compared with inotropic responses to DPI 201-106 (DPI, 0.1-3 mumol/l), Ca2+ (1.8-15 mmol/l) and isoprenaline (0.001-1 mumol/l). Carbachol (100 mumol/l) and adenosine (1000 mumol/l) were examined in the presence of BDF 9148 and isoprenaline. Both sodium-channel activators, BDF 9148 and DPI 201-106, increased force of contraction in a dose-dependent manner in papillary muscle strips as well as in auricular trabeculae. BDF 9148 and DPI 201-106 were more effective (max. PIE NYHA II-III 1.6 +/- 0.2 mN, NYHA IV 5.9 +/- 0.7 mN, P less than 0.05) and more potent (EC50 (in mumol/l): NYHA IV 0.35, 0.19-0.66; NYHA II-III 1.85, 1.37-2.41) in terminally failing as compared to moderately failing left ventricular myocardium. Moreover, the positive inotropic effects of BDF 9148 were greater than those of DPI 201-106 in NYHA IV (max. PIE 2.7 +/- 0.3 mN, P less than 0.05). In NYHA IV, BDF 9148 was as effective as CA2+ (max. PIE 5.1 +/- 0.4 mN). In the same hearts, the positive inotropic effects of isoprenaline were reduced in NYHA IV (max. PIE 2.1 +/- 0.3 mN) compared to NYHA II-III (max. PIE 3.4 +/- 0.4 mN, P less than 0.05). Adenosine as well as carbachol did not affect the positive inotropic response of BDF 9148 or DPI 201-106 but reduced the effectiveness of isoprenaline (P less than 0.05). In myocardial membranes, BDF 9148 was 1000-fold less effective in competition experiments with 3H-ouabain than ouabain. We conclude that (1) sodium-channel activators may produce a significant cAMP-independent positive inotropic effect in left ventricular myocardium from failing human hearts; (2) the inotropic effect of sodium-channel activators were more potent and more effective in NYHA IV as compared to NYHA II-III. The degree of myocardial failure does not reduce the effectiveness of the sodium-channel activator BDF 9148.
Asunto(s)
Azetidinas/farmacología , Cardiomiopatía Dilatada/tratamiento farmacológico , Cardiotónicos , Corazón/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Adulto , Azetidinas/química , Azetidinas/uso terapéutico , Cardiotónicos/química , Cardiotónicos/uso terapéutico , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas In Vitro , Isoproterenol/farmacología , Cinética , Masculino , Persona de Mediana Edad , Estructura Molecular , Contracción Miocárdica/efectos de los fármacos , Miocardio/metabolismo , Ouabaína/metabolismo , Piperazinas/farmacologíaRESUMEN
The purpose of the present study was to characterize the effects of xamoterol in the human myocardium. In the presence of forskolin or milrinone, xamoterol increased isometric force of contraction, contraction velocity, and relaxation velocity in isolated, electrically driven preparations from human myocardium, but had no effect alone. There was no difference in the effect of xamoterol between right atrial myocardium and left ventricular myocardium from nonfailing (NF), moderately failing (NYHA II-III), and severely failing (NYHA IV) human hearts. The positive inotropic and lusitropic effects of isoprenaline were reduced depending on the severity of heart failure in left ventricular myocardium (i.e., NF greater than NYHA II-III greater than NYHA IV). In the presence of norepinephrine, xamoterol produced negative inotropic effects similar to those of the beta-adrenoceptor antagonists pindolol and propranolol. Xamoterol alone had no effects on force of contraction, whereas pindolol and propranolol markedly reduced contractile force. In NYHA class IV, isoprenaline stimulated adenylate cyclase about twofold but xamoterol, like pindolol or propranolol, had no effect. Experiments with the beta 1- and beta 2-selective antagonists CGP 207.12A and ICI 118.551, respectively, showed that the positive inotropic and lusitropic effects of xamoterol were mediated by beta 1-adrenoceptors. Consistently, xamoterol had a selectivity of 13.8 at beta 1-adrenoceptors as measured in radioligand binding experiments. It is concluded that xamoterol acts as a beta 1-adrenoceptor antagonist with a selectivity of 13.8 in human ventricular myocardium. The compound has an intrinsic sympathomimetic activity, as it produces beta 1-adrenoceptor-mediated positive inotropic and lusitropic effects in the presence of forskolin. The beneficial effects of xamoterol in patients with heart failure could be due to prevention of the detrimental effects of norepinephrine such as beta 1-adrenoceptor downregulation of an increase of Gi (inhibitory guanine-nucleotide binding protein).