RESUMEN

Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

Asunto(s)

Baculoviridae/enzimología , Flavina-Adenina Dinucleótido/metabolismo , Oxidorreductasas/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Baculoviridae/genética , Baculoviridae/metabolismo , Secuencia Conservada , Flavina-Adenina Dinucleótido/genética , Sistemas de Lectura Abierta/genética , Alineación de Secuencia , Análisis Espectral , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación

RESUMEN

Reporter gene transactivation by human p53 is compromised in S. cerevisiae lacking the TRR1 gene encoding thioredoxin reductase. The basis for p53 inhibition was investigated by measuring the redox state of thioredoxin and glutathione in wild-type and Deltatrr1 yeast. The Deltatrr1 mutation affected the redox state of both molecules. About 34% of thioredoxin was in the disulfide form in wild-type yeast and increased to 70% in Deltatrr1 yeast. About 18% of glutathione was in the GSSG form in wild-type yeast and increased to 32% in Deltatrr1 yeast. The Deltatrr1 mutation also resulted in a 2.9-fold increase in total glutathione per mg extract protein. Highcopy expression of the GLR1 gene encoding glutathione reductase in Deltatrr1 yeast restored the GSSG:GSH ratio to wild-type levels, but did not restore p53 activity. Also, p53 activity was shown to be unaffected by a Deltaglr1 mutation, even though the mutation was known to result in glutathione oxidation. In summary, the results show that, although glutathione becomes more oxidized in Deltatrr1 cells, glutathione oxidation is neither sufficient nor necessary for p53 inhibition. The results indicate that p53 activity has a specific requirement for an intact thioredoxin system, rather than a general dependence on the intracellular reducing environment.

Asunto(s)

Genes p53 , Glutatión/metabolismo , Saccharomyces cerevisiae/genética , Reductasa de Tiorredoxina-Disulfuro/genética , Tiorredoxinas/metabolismo , Activación Transcripcional/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Disulfuros/metabolismo , Eliminación de Gen , Disulfuro de Glutatión/metabolismo , Humanos , Oxidación-Reducción , Compuestos de Sulfhidrilo/metabolismo

RESUMEN

The acute administration of acetaminophen to isolated, perfused guinea pig hearts appears to have cardioprotective effects against the injury/mechanical dysfunction caused by global, low-flow, myocardial ischemia and reperfusion. In the current study we selected ischemia/reperfusion and administration of sodium pentobarbital as perturbations of the electrical stability of the myocardium. We investigated their ability to induce ventricular arrhythmias and changes in the characteristics of monophasic action potentials in the absence and presence of acetaminophen (0.35 mmol/l). The numbers of ventricular premature beats and ventricular salvos encountered in the presence of pentobarbital were significantly (P < 0.05) reduced by acetaminophen. The combined frequency of these arrhythmias was 0.14+/-0.06/min vs 0.03+/-0.01/min (P < 0.05) in the absence and presence of acetaminophen, respectively. The incidence of ventricular salvos increased steadily in vehicle-treated hearts after administration of pentobarbital. No such trend was seen with acetaminophen. After 10 min of global, low-flow myocardial ischemia, MAP50 and MAP90 (monophasic action potentials at 50 and 90% repolarization, respectively) decreased without acetaminophen (e.g. MAP50, 31+/-4 ms) but did not change during the same time interval with acetaminophen (e.g. MAP50, 57+/-6 ms)(P < 0.05). During ischemia and reperfusion, acetaminophen attenuated the release of hydroxyl radicals and peroxynitrite. Collectively these data reveal cardioprotective, antioxidant behavior of acetaminophen. Under selected conditions (e.g. those causing release of free radicals and other oxidants) such behavior might also prevent ventricular arrhythmias.

Asunto(s)

Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Antioxidantes/farmacología , Cardiotónicos/farmacología , Isquemia Miocárdica/tratamiento farmacológico , Potenciales de Acción/efectos de los fármacos , Animales , Femenino , Moduladores del GABA , Cobayas , Sistema de Conducción Cardíaco/efectos de los fármacos , Radical Hidroxilo/análisis , Técnicas In Vitro , Masculino , Miocardio/química , Pentobarbital , Ácido Peroxinitroso/análisis , Complejos Prematuros Ventriculares/inducido químicamente , Complejos Prematuros Ventriculares/tratamiento farmacológico

RESUMEN

Stimulation of target gene transcription by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. LexA/p53 fusion proteins were used to study the basis for thioredoxin reductase dependence. A fusion protein containing all 393 of the residues of p53 efficiently and specifically stimulated transcription of a LexOP-LacZ reporter gene in wild-type yeast but was several-fold less effective in delta trr1 yeast lacking the thioredoxin reductase gene. Thus, even when p53 was tethered to a reporter gene by a heterologous DNA-binding domain, reporter gene transactivation remained dependent on thioredoxin reductase. A fusion protein containing only the activation domain of p53 stimulated reporter gene transcription equally in wild-type and delta trr1 cells, suggesting that p53 residues downstream from the activation domain created the requirement for thioredoxin reductase. Experiments using additional LexA/p53 truncation mutations indicated that the p53 negative regulatory domain, rather than the DNA-binding or oligomerization domains, created the requirement for thioredoxin reductase. The fusion protein results suggested that, under oxidative conditions, the negative regulatory domain inhibited the ability of DNA-bound p53 to stimulate transcription. However, deletion of the negative regulatory domain did not alleviate the requirement of non-LexA-containing p53 for thioredoxin reductase. The results, thus, suggest that oxidative conditions inhibit both DNA binding and transactivation by p53, and that inhibition of the latter requires the negative regulatory domain.

Asunto(s)

Genes p53 , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Activación Transcripcional , Genes Reporteros , Humanos , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Reductasa de Tiorredoxina-Disulfuro/genética , Transcripción Genética , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genética

RESUMEN

5-Aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR) is taken up by perfused skeletal muscle and phosphorylated to form 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuraosyl-5'-monopho sph ate (analog of 5'-AMP) with consequent activation of AMP-activated protein kinase, phosphorylation of acetyl-CoA carboxylase, decrease in malonyl-CoA, and increase in fatty acid oxidation. This study was designed to determine the effect of increasing levels of palmitate on the rate of fatty acid oxidation. Malonyl-CoA concentration was manipulated with AICAR at different palmitate concentrations. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine red cells, 200 microU/ml insulin, 10 mM glucose, and different concentrations of palmitate (0. 1-1.0 mM) without or with AICAR (2.0 mM). Perfusion with medium containing AICAR was found to activate AMP-activated protein kinase in skeletal muscle, inactivate acetyl-CoA carboxylase, and decrease malonyl-CoA at all concentrations of palmitate. The rate of palmitate oxidation increased as a function of palmitate concentration in both the presence and absence of AICAR but was always higher in the presence of AICAR. These results provide additional evidence that malonyl-CoA is an important regulator of the rate of fatty acid oxidation at palmitate concentrations in the physiological range.

Asunto(s)

Malonil Coenzima A/metabolismo , Músculo Esquelético/metabolismo , Palmitatos/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Quinasas Activadas por AMP , Acetil-CoA Carboxilasa/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Ácidos Grasos/metabolismo , Miembro Posterior/irrigación sanguínea , Miembro Posterior/fisiología , Hipoglucemiantes/farmacología , Masculino , Complejos Multienzimáticos/metabolismo , Oxidación-Reducción , Consumo de Oxígeno/fisiología , Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/fisiología , Ribonucleótidos/farmacología

RESUMEN

This chapter describes methods for analyzing the cell cycle kinetics of asynchronous mammalian cells and for preparing synchronous cultures. The described asynchronous cell methods include determination of mean generation time and proliferative fraction, determination of mitotic and [3H]thymidine-labeling indices, and estimation of cell cycle phase durations by the labeled mitoses procedure and by flow cytometry. The described synchronization methods include release from G0 arrest, release from M-phase and S-phase blocking agents, mitotic detachment, and centrifugal elutriation. Caveats in interpreting cell synchrony experiments are discussed.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Animales , Ciclo Celular , Humanos

RESUMEN

Reperfusion of blood flow to an ischemic myocardium is imperative to survival; ironically, it may also manifest several pathophysiological conditions. The most important of these are reperfusion arrhythmias and tissue injury and/or death. The mechanisms involved in reperfusion arrhythmias remain to be fully elucidated; however, increasing evidence indicates that reperfusion-induced arrhythmias are a free radical-mediated phenomenon. Acute administration of conjugated equine estrogen to dogs attenuates ischemia- and reperfusion-induced arrhythmias. The cardioprotective effect of estrogens in postmenopausal women is well documented, and recent studies suggest that estrogens possess strong antioxidant properties, with equine estrogens most potent. In this study we show that administration of conjugated equine estrogen to fully anesthetized dogs abolishes the burst of .OH radicals typically produced on reperfusion of the myocardium. This indicates that estrogen might attenuate reperfusion-induced ventricular arrhythmias by virtue of its antioxidant properties, suggesting a novel cardioprotective effect of the hormone.

Asunto(s)

Estrógenos Conjugados (USP)/farmacología , Gentisatos , Radical Hidroxilo/metabolismo , Isquemia Miocárdica/metabolismo , Reperfusión Miocárdica , Animales , Perros , Estrógenos Conjugados (USP)/sangre , Femenino , Hidroxibenzoatos/sangre , Masculino , Aturdimiento Miocárdico/metabolismo , Factores Sexuales

RESUMEN

The acute administration of conjugated equine estrogen (CEE) to dogs significantly attenuated the severity and incidence of ventricular arrhythmias during ischemia and reperfusion. We hypothesized that one of the cardioprotective mechanisms of estrogen might be the ability to maintain electrical stability of the heart during ischemia. The current study was conducted to determine the effect of chronic administration of estrogen, simulating hormone replacement therapy, on the ventricular arrhythmias of ischemia and reperfusion. Chronically-treated (100 micrograms/kg/week CEE, or vehicle) male beagles were anesthetized and subjected to regional ischemia (20 min) and reperfusion. Although there was a trend towards a lower incidence of arrhythmias during ischemia in estrogen-treated dogs, values did not achieve significance at P < 0.05. Baseline coronary vascular resistance was significantly higher in estrogen-treated dogs (2.3 vs 1.5 mmHg/ml/min/100 g, P < 0.05) indicating an increase in vasomotor tone. There was also an increase in the time it took hyperemic coronary blood flow to reach a peak value upon reperfusion (71 sec in estrogen-treated dogs vs 12 sec in vehicle-treated dogs, P < 0.05). This slower reflow is consistent with increased coronary vascular resistance upon reflow in estrogen-treated dogs. We conclude that the chronic administration of CEE to male dogs increased coronary vascular tone, and impaired the rate of reperfusion, but did not decrease the incidence of ventricular arrhythmias caused by ischemia.

Asunto(s)

Arritmias Cardíacas/prevención & control , Vasos Coronarios/efectos de los fármacos , Estrógenos Conjugados (USP)/administración & dosificación , Corazón/efectos de los fármacos , Daño por Reperfusión/prevención & control , Animales , Perros , Esquema de Medicación , Ventrículos Cardíacos , Hemodinámica/efectos de los fármacos , Inyecciones Intramusculares , Masculino , Isquemia Miocárdica/fisiopatología , Resistencia Vascular/efectos de los fármacos

RESUMEN

The prevalence of p53 gene mutations in many human tumors implies that p53 protein plays an important role in preventing cancers. Central among the activities ascribed to p53 is its ability to stimulate transcription of other genes that inhibit cells from entering S phase with damaged DNA. Human p53 can be studied in yeast where genetic tools can be used to identify proteins that affect its ability to stimulate transcription. Although p53 strongly stimulated reporter gene expression in wild type yeast, it only weakly stimulated reporter gene expression in Deltatrr1 yeast that lacked the gene encoding thioredoxin reductase. Furthermore, ectoptic expression of TRR1 in Deltatrr1 yeast restored p53-dependent reporter gene activity to high levels. Immunoblot assays established that the Deltatrr1 mutation affected the activity and not the level of p53 protein. The results suggest that p53 can form disulfides and that these disulfides must be reduced in order for the protein to function as a transcription factor.

Asunto(s)

Proteínas de Unión al ADN/química , Eliminación de Gen , Regulación Fúngica de la Expresión Génica/genética , Saccharomyces cerevisiae/enzimología , Reductasa de Tiorredoxina-Disulfuro/genética , Proteína p53 Supresora de Tumor/fisiología , Disulfuros/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Genes Reporteros/genética , Humanos , Modelos Moleculares , Saccharomyces cerevisiae/genética , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/química

RESUMEN

Women generally exhibit angina rather than myocardial infarction as the first manifestation of heart disease. Postmenopausal use of hormone replacement therapy, specifically estrogens, is associated with reduced incidence of major cardiac events suggesting estrogen may protect the heart during ischemia. We recently showed that acute administration of conjugated equine estrogens prior to ischemia attenuated the ventricular arrhythmias of ischemia as well as those of reperfusion. This study looks at basal effects of estrogen on coronary blood flow and the effects of estrogen on regional blood flow during ischemia to determine if estrogen exerts its antiarrhythmic effects during ischemia by altering blood flow. Under conditions of natural blood flow, estrogen caused cyclic changes in blood flow. When coronary blood flow was controlled and limited, estrogen increased coronary perfusion pressure (118 +/- 8 mmHg vs. 85 +/- 10 mmHg in non-treated dogs, P < 0.05) demonstrating an overall vasoconstrictor effect. Coronary blood flow and regional myocardial perfusion were determined before and during ischemia in anesthetized dogs with and without acutely-administered estrogen. Colored microspheres were injected at steady state prior to ischemia, and during steady state myocardial ischemia. Conjugated equine estrogen (10 micrograms/kg), administered about 6 min before ischemia, had no effect on regional perfusion under steady state conditions, nor in the non-ischemic zone during ischemia. Perfusion in the subepicardial and subendocardial ischemic zones in estrogen-treated dogs was significantly lower than in non-treated dogs [0.14 +/- 0.01 ml/min/g vs. 0.23 +/- 0.02 ml/min/g (P < 0.05) in the epicardial ischemic zone; and, 0.15 +/- 0.02 ml/min/g vs. 0.22 +/- 0.03 ml/min/g (P < 0.05) in the endocardial ischemic zone]. We conclude that the acute, systemic administration of estrogen in the anesthetized dog decreases regional perfusion in the ischemic myocardium and causes significant coronary vaso-constriction when flow is controlled and limited.

Asunto(s)

Circulación Coronaria/efectos de los fármacos , Estrógenos Conjugados (USP)/farmacología , Anestesia , Animales , Perros , Femenino , Masculino , Resistencia Vascular/efectos de los fármacos

RESUMEN

Mlu1 cell cycle box (MCB) elements are found near the start site of yeast genes expressed at G1/S. Basal promoters dependent on the elements for upstream activating sequence activity are inactive in Deltaswi6 yeast. Yeast were screened for mutations that activated MCB reporter genes in the absence of Swi6. The mutations identified a single complementation group. Functional cloning revealed the mutations were alleles of the TRR1 gene encoding thioredoxin reductase. Although deletion of TRR1 activated MCB reporter genes, high copy expression did not suppress reporter gene activity. The trr1 mutations strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle box)-containing reporter genes, but also weakly (3-fold) stimulated reporter genes that lacked these elements. The trr1 mutations did not affect the level or periodicity of three endogenous MCB gene mRNAs (TMP1, RNR1, and SWI4). Deletion of thioredoxin genes TRX1 and TRX2 recapitulated the stimulatory effect of trr1 mutations on MCB reporter gene activity. Conditions expected to oxidize thioredoxin (exposure to H2O2) induced MCB gene expression, whereas conditions expected to conserve thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression. The results suggest that thioredoxin oxidation contributes to MCB element activation and suggest a link between thioredoxin-oxidizing processes such as ribonucleotide reduction and cell cycle-specific gene transcription.

Asunto(s)

Proteínas Fúngicas/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Ciclo Celular , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Genes Reporteros , Peróxido de Hidrógeno/metabolismo , Mutación , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Oxidación-Reducción , Periodicidad , Unión Proteica , ARN Mensajero/metabolismo , Saccharomyces cerevisiae , Reductasa de Tiorredoxina-Disulfuro/genética , Tiorredoxinas/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) has previously been reported to be taken up into cells and phosphorylated to form ZMP, an analog of 5'-AMP. This study was designed to determine whether AICAR can activate AMP-activated protein kinase (AMPK) in skeletal muscle with consequent phosphorylation of acetyl-CoA carboxylase (ACC), decrease in malonyl-CoA, and increase in fatty acid oxidation. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine red blood cells, 200 microU/ml insulin, and 10 mM glucose with or without AICAR (0.5-2.0 mM). Perfusion with medium containing AICAR was found to activate AMPK in skeletal muscle, inactivate ACC, and decrease malonyl-CoA. Hindlimbs perfused with 2 mM AICAR for 45 min exhibited a 2.8-fold increase in fatty acid oxidation and a significant increase in glucose uptake. No difference was observed in oxygen uptake in AICAR vs. control hindlimb. These results provide evidence that decreases in muscle content of malonyl-CoA can increase the rate of fatty acid oxidation.

Asunto(s)

Acetil-CoA Carboxilasa/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Eritrocitos/fisiología , Glucosa/metabolismo , Malonil Coenzima A/metabolismo , Complejos Multienzimáticos/metabolismo , Músculo Esquelético/fisiología , Ácido Palmítico/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Ribonucleósidos/farmacología , Proteínas Quinasas Activadas por AMP , Nucleótidos de Adenina/farmacología , Aminoimidazol Carboxamida/farmacología , Animales , Bovinos , Activación Enzimática , Miembro Posterior , Insulina/farmacología , Cinética , Masculino , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Fosforilación , Ratas , Ratas Sprague-Dawley , Ribonucleótidos/farmacología , Albúmina Sérica Bovina/farmacología

RESUMEN

The transcription factors SBF and DSC1/MBF bind SCB and MCB promoter elements, respectively, and are essential for the cell cycle progression of Saccharomyces cerevisiae through the control of G1 cyclin gene expression. We isolated a gene (BRY1; Bacterial Response regulator in Yeast) able to activate either MCB or SCB promoter elements on a reporter plasmid which, when overexpressed, can bypass the normally essential requirement for SBF and DSC1/MBF by the stimulation of CLN1 and CLN2 expression. In the case of CLN2 at least, this expression depends upon the MCB and SCB promoter elements. In wild-type yeast, the disruption of BRY1 has no apparent phenotype, but under conditions where the activities of SBF and DSC1/MBF are reduced, BRY1 becomes essential. Our data imply the existence of a third pathway affecting cyclin expression. BRY1 is the same gene as SKN7 which has significant sequence homology to the receiver domains found in response regulator proteins from the bacterial two-component signal transduction pathways. SKN7 is thought to affect cell wall structure, and when highly overexpressed we find that BRY1/SKN7 is lethal perhaps because of perturbations in cell wall biosynthesis. The lethality is partially rescued by genes from the protein kinase C pathway, but genetic data imply that BRY1/SKN7 and protein kinase C are not in the same pathway. Our results suggest that Bry1/Skn7 can influence the expression of MCB- and SCB-driven gene expression in budding yeast, perhaps including genes involved in cell wall metabolism, via a two-component signal transduction pathway which activates Bry1/Skn7 in response to an unidentified signal.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Fase G1 , Genes Fúngicos , Factores de Transcripción del Choque Térmico , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de Transcripción/genética

RESUMEN

The purpose of this investigation was to determine if exogenous estrogen could attenuate the ventricular arrhythmias caused by myocardial ischemia and reperfusion. Conjugated equine estrogen, administered as an intravenous bolus injection (100 micrograms) to anesthetized, instrumented beagles of both genders, significantly attenuated the incidence of ventricular arrhythmias during a 20-min period of ischemia (2 +/- 1 vs. 19 +/- 16% ectopy) and in the first 5 min of reperfusion (15 +/- 9 vs. 69 +/- 20% ectopy). By 15-20 min of ischemia, ventricular salvos and nonsustained ventricular tachycardia were frequently observed in nontreated dogs. One dog in this group fibrillated during ischemia. In contrast, estrogen-treated dogs exhibited only an occasional ventricular premature beat during the same period of ischemia. When compared with baseline values, the percent ectopy during ischemia in estrogen-treated dogs was insignificant. During reperfusion, nontreated dogs displayed severe, life-threatening arrhythmias such as sustained ventricular tachycardia. In two of these dogs ventricular tachycardia deteriorated to ventricular fibrillation. In comparison, estrogen-treated dogs displayed only innocuous ventricular arrhythmias during reperfusion, i.e., ventricular premature beats, ventricular salvos, and ventricular bigeminy. In addition to the effect of estrogen on arrhythmias, there was a gradual increase in coronary blood flow on reperfusion in estrogen-treated dogs. This effect of estrogen was preceded by a significantly higher coronary perfusion pressure during ischemia (31 +/- 2 vs. 18 +/- 4 mmHg, P < 0.05). In conclusion, our findings suggest that antiarrhythmic effects of estrogen treatment might stabilize ventricular rhythmicity during ischemia and reperfusion.

Asunto(s)

Arritmias Cardíacas/fisiopatología , Estrógenos Conjugados (USP)/farmacología , Hemodinámica/efectos de los fármacos , Isquemia Miocárdica/fisiopatología , Daño por Reperfusión Miocárdica/fisiopatología , Disfunción Ventricular/fisiopatología , Fibrilación Ventricular/fisiopatología , Animales , Arritmias Cardíacas/etiología , Presión Sanguínea/efectos de los fármacos , Circulación Coronaria/efectos de los fármacos , Perros , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Hemodinámica/fisiología , Masculino , Análisis Multivariante , Taquicardia/etiología , Taquicardia/fisiopatología , Disfunción Ventricular/etiología , Fibrilación Ventricular/etiología

RESUMEN

Eighteen anesthetized, instrumented beagles (both genders, 10.4 +/- 0.5 kg) were used to investigate the effects of administered adenosine (n = 6), erythro-9-(2-hydroxy, 3-nonyl)adenine (EHNA), a potent inhibitor of endogenous adenosine deaminase (n = 6), and saline (n = 6), on the incidence of ventricular arrhythmias caused by systemic hypoxia (5% O2, 95% N2, PaO2 = 21 +/- 3 mmHg). After dogs were instrumented and monitored variables were in the steady-state, the above compounds were infused continuously into the cannulated left anterior descending (LAD) coronary artery for three minutes before, and throughout a four-minute period of hypoxia. After approximately 4 min of hypoxia the rates of ventricular ectopy [(total beats-normal beats)/total beats x 100 = % ectopy] were 73 +/- 9%, 73 +/- 11%, and 35 +/- 8% for the three groups, respectively. The percent ectopy of the adenosine- and EHNA-treated dogs was significantly greater (p < 0.05) than that for the saline-treated controls. These findings suggest that adenosine contributes to the ventricular arrhythmias of experimental systemic hypoxia.

Asunto(s)

Inhibidores de la Adenosina Desaminasa , Adenosina/farmacología , Arritmias Cardíacas/fisiopatología , Ventrículos Cardíacos/fisiopatología , Hipoxia/fisiopatología , Adenina/análogos & derivados , Adenina/farmacología , Adenosina/administración & dosificación , Animales , Arritmias Cardíacas/etiología , Perros , Electrocardiografía , Inhibidores Enzimáticos/farmacología , Femenino , Ventrículos Cardíacos/enzimología , Hemodinámica , Masculino , Miocardio/enzimología

RESUMEN

The three objectives of this study were: 1) to determine whether an initial exposure to systemic hypoxia could affect the ventricular ectopy caused by a second period of hypoxia, 2) to see whether the duration of the intervening period of reoxygenation was important, and 3) to determine whether propranolol could produce results similar to those caused by an inadequate period of reoxygenation. Anesthetized instrumented beagles of either sex weighing 10.2 +/- 0.4 kg (n = 25) were exposed to dual periods of hypoxia (4 min each, PO2 approximately 15 +/- 3 mmHg). Dogs were divided into four groups according to the duration of reoxygenation and propranolol treatment: group 1 (n = 7), 20 min of reoxygenation; group 2 (n = 7), 40 min of reoxygenation; group 3 (n = 6), 60 min of reoxygenation; group 4 (n = 5), 50 min of reoxygenation plus propranolol. Dogs in groups 1 and 4 experienced a significant reduction in percent ectopy during their second exposure to hypoxia [group 1; 47 +/- 9% vs. 11 +/- 6%, group 4; 50 +/- 1% vs. 1 +/- 2%, (P < 0.05)]. There were no significant differences in percent ectopy between the two periods of hypoxia in either group 2 or group 3 dogs (e.g., group 2; 50 +/- 1% vs. 51 +/- 11%). The results show that a first exposure to hypoxia can precondition the myocardium against arrhythmogenic effects of a second period of hypoxia.

Asunto(s)

Arritmias Cardíacas/etiología , Hipoxia/complicaciones , Oxígeno/farmacología , Animales , Arritmias Cardíacas/tratamiento farmacológico , Perros , Femenino , Hipoxia/terapia , Masculino , Propranolol/uso terapéutico

RESUMEN

Twenty-six beagles of either sex, weighing 10.4 +/- 0.3 kg, were used to investigate the role of adenosine in the genesis of ventricular arrhythmias during systemic hypoxia. After instrumentation dogs were randomly assigned to one of four treatment groups: 14 dogs were pretreated before hypoxia with adenosine deaminase (n = 7, group I) or its vehicle (n = 7, group II) while 12 other dogs were pretreated with the A1 selective adenosine receptor antagonist BW A1433U (n = 6, group III) or its vehicle (n = 6, group IV). Each dog was exposed to a 3-min period of hypoxic ventilation [3% O2-5% CO2-92% N2; PO2 in arterial blood 96 +/- 3 Torr (before hypoxia), 21 +/- 1 Torr (during hypoxia)]. The percentages of ventricular ectopic beats (19) experienced in the four groups after 3 min of hypoxia were 21 +/- 10% (group I, P < 0.05 relative to group II), 50 +/- 2% (group II), 15 +/- 8% (group III, P < 0.05 relative to group IV), and 42 +/- 7% (group IV). Ventricular bigeminy, the most prominent arrhythmia seen in this study, was significantly reduced by adenosine deaminase and BW A1433U. No significant differences in other monitored cardiovascular variables were seen between adenosine deaminase and BW A1433U treatment groups and their corresponding vehicles. These findings implicate endogenous adenosine as an arrhythmogenic mediator during hypoxia and point to a mechanism involving the A1 adenosine receptor.

Asunto(s)

Adenosina Desaminasa/farmacología , Arritmias Cardíacas/prevención & control , Xantinas/farmacología , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/fisiopatología , Circulación Coronaria/efectos de los fármacos , Perros , Femenino , Ventrículos Cardíacos , Hipoxia/complicaciones , Masculino , Antagonistas Purinérgicos , Receptores Purinérgicos/fisiología

RESUMEN

After yeast cells commit to the cell cycle in a process called START, genes required for DNA synthesis are expressed in late G1. Periodicity is mediated by a hexameric sequence, known as a MCB element, present in all DNA synthesis gene promoters. A complex that specifically binds MCBs has been identified. One polypeptide in the MCB complex is Swi6, a transcription factor that together with Swi4 also binds G1 cyclin promoters and participates in a positive feedback loop at START. The finding that Swi6 is directly involved in both START and DNA synthesis gene control suggest a model in which Swi6, activated through its participation in START, serves as the central transcription factor in coordinating late G1 gene expression. The mechanism may be conserved in all eukaryotic cells.

Asunto(s)

Replicación del ADN , ADN de Hongos/biosíntesis , Genes Fúngicos , Saccharomyces cerevisiae/genética , Animales , Secuencia de Bases , Ciclo Celular/genética , Replicación del ADN/genética , ADN de Hongos/genética , Retroalimentación , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Origen de Réplica , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Vertebrados/genética

RESUMEN

Fifteen mongrel dogs weighing 22-34 kg were instrumented to investigate the antiarrhythmic effects of ammonia (0.1-0.2 mmol/min ammonium hydroxide), adenosine (1.87 mumol/min), and saline (0.9% NaCl) during norepinephrine-driven ventricular tachycardia, under conditions of controlled and natural coronary blood flow. Under natural flow conditions, the severe ectopy caused by norepinephrine (100-800 ng.kg-1.min-1) was reduced by 42 +/- 4% after 30 s of ammonia infusion. Adenosine infusion reduced percent ectopy by 97 +/- 2% at 30 s. Ammonia also significantly increased coronary blood flow by 26 +/- 4%, while adenosine increased blood flow by 72 +/- 14%. Saline infusion had no significant effect on either the severity of ventricular tachycardia or coronary blood flow. Norepinephrine consistently caused coronary functional hyperemia as previously reported. When coronary blood flow was controlled by a peristaltic pump to match natural coronary blood flow and to prevent norepinephrine-induced coronary functional hyperemia, the antiarrhythmic effects of ammonia were lost while those of adenosine were unaffected. Additionally, increasing coronary blood flow manually during norepinephrine-induced ventricular tachycardia, to a level seen with combined norepinephrine and ammonia under natural flow conditions, appeared to worsen the ventricular arrhythmias. We conclude that the antiarrhythmic properties of ammonia against norepinephrine-driven ventricular tachycardia might be dependent on coronary blood flow, while those of adenosine are independent of coronary blood flow.

Asunto(s)

Amoníaco/farmacología , Antiarrítmicos/farmacología , Taquicardia/tratamiento farmacológico , Adenosina/farmacología , Animales , Circulación Coronaria/fisiología , Perros , Femenino , Masculino , Norepinefrina , Taquicardia/inducido químicamente , Taquicardia/fisiopatología

RESUMEN

In this investigation, an isolated, perfused rat stomach system was used to elucidate the roles of histamine, serotonin, and the action of cimetidine, methysergide, and propranolol in relation to the in vivo and in vitro administration of compound 48/80. While histamine administered both in vivo and in vitro stimulated acid secretion in the perfused rat stomach, serotonin, added in vitro, inhibited histamine-induced gastric acid secretion. Cimetidine, given either in vivo or in vitro, blocked histamine-induced acid secretion, and methysergide, but not propranolol, reversed the serotonin-induced inhibition of histamine-stimulated acid secretion. Compound 48/80, given in vitro, caused gastric acid secretion that was blocked by pretreatment with cimetidine. Administered in vivo, however, compound 48/80 inhibited both basal and histamine-stimulated acid secretion. This inhibition was partially reversed by pretreatment with methysergide. The absence of inhibition of acid secretion by in vitro-administered compound 48/80 may be related to the timing of the serotonin effect. When serotonin was given prior to histamine, it blocked acid secretion, whereas no inhibition occurred when serotonin was administered together with histamine. None of the other agents investigated affected basal acid secretion.

Asunto(s)

Ácido Gástrico/metabolismo , Histamina/farmacología , Serotonina/farmacología , p-Metoxi-N-metilfenetilamina/farmacología , Animales , Cimetidina/farmacología , Femenino , Técnicas In Vitro , Metisergida/farmacología , Propranolol/farmacología , Ratas , Ratas Endogámicas