RESUMEN
We have generated a mouse IgG1 monoclonal antibody (mAb) that recognizes amino acids 1-58 of Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA 2) of type 1 EBV strain B95-8. mAb Y101 also reacted with EBNA 2 of EBV type 2 strains MISP and Jijoye in immunoblots, whereas Jijoye EBNA 2 was not detected by the widely used mAb PE2. mAb Y101, in contrast to mAb PE2, reacted with faster migrated, hypophosphorylated proteins of type 1 EBNA 2 as intensely as slower migrated, hyperphosphorylated ones. mAb Y101 did not react in fixed-cell immunostaining or cell extract immunoprecipitation. The results implicate that the amino-terminal epitope is not exposed in a native form, consistent with the previously reported idea of self-association of EBNA 2 through the amino-terminus. mAb Y101 is the first mAb to the EBNA 2 amino-terminus and will be useful for further analyses of the structure and function of EBNA 2.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular , Mapeo Epitopo , Antígenos Nucleares del Virus de Epstein-Barr/administración & dosificación , Herpesvirus Humano 4/inmunología , Herpesvirus Humano 4/metabolismo , Inmunización , Ratones , Ratones Endogámicos BALB C , Proteínas ViralesRESUMEN
The causes of reduced cardiac force development in congestive heart failure (CHF) are still uncertain. We explored the subcellular mechanisms leading to decreased force development in trabeculae from rats with a myocardial infarction. We defined CHF according to clinical and pathological criteria and compared properties of trabeculae from animals with CHF (cMI) to those of animals with a myocardial scar but without evidence of CHF (uMI), and sham-operated animals. The new findings of this study on properties of cMI trabeculae are that (1) maximal twitch force following post-extrasystolic potentiation is unchanged; (2) the sensitivity of cMI trabeculae to [Ca(2+)](o) is increased; (3) spontaneous diastolic sarcomere length (SL) fluctuations (SA) are increased in cMI at all levels of SR Ca(2+) loading; and (4) SA is accompanied by a proportional reduction of F(max). The results suggest that the probability of spontaneous diastolic opening of SR Ca(2+) channels is increased in CHF. These data provide the basis for a novel mechanism underlying systolic and diastolic dysfunction as well as arrhythmias in hearts in CHF. If SA proves to be a component of myocardial dysfunction in human CHF, our thinking about therapy of the patient with CHF may be profoundly changed.
Asunto(s)
Insuficiencia Cardíaca/fisiopatología , Infarto del Miocardio/fisiopatología , Sarcómeros/fisiología , Animales , Masculino , Contracción Miocárdica , Ratas , Ratas Endogámicas LewAsunto(s)
Ciclosporina/farmacocinética , Trasplante de Riñón/inmunología , Área Bajo la Curva , Ciclosporina/sangre , Ciclosporina/uso terapéutico , Monitoreo de Drogas/métodos , Estudios de Seguimiento , Prueba de Histocompatibilidad , Humanos , Inmunosupresores/sangre , Inmunosupresores/farmacocinética , Inmunosupresores/uso terapéutico , Japón , Donadores Vivos , Análisis de Regresión , Factores de TiempoRESUMEN
OBJECTIVES AND METHODS: Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1), which is essential for EBV latency, homodimerizes and binds to the EBV replication origin, oriP. We analyzed the dimerization/DNA-binding domain of EBNA-1 by random and site-directed amino acid substitution. RESULTS: Random point mutations that resulted in reduced DNA binding clustered in the DNA contact region (a.a. 461-473) and at or near the termini of alpha-helix II (514-527). Three substitutions of Gly in the DNA contact region each greatly reduced binding to a single binding site oligonucleotide. Substitutions at and near the termini of alpha-helix II diminished DNA binding. A helix-deforming substitution in alpha-helix I (477-489) blocked DNA binding. A helix-deforming substitution in alpha-helix III (568-582) abolished dimerization and DNA binding. Similarities in surface electrostatic properties and conserved amino acids were found between alpha-helix II and recognition helices of papillomavirus E2 proteins. CONCLUSIONS: The basic DNA contact region is crucial for the specific interaction of EBNA-1 with a single binding site. Alpha-helix I477 is indispensable for oriP binding, and alpha-helix III568 contributes to the homodimeric structure of EBNA-1. Alpha-helix II514 contributes to oriP binding, perhaps changing its alignment with DNA.
Asunto(s)
Sustitución de Aminoácidos/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4 , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía Líquida de Alta Presión , Secuencia Conservada/genética , ADN Viral/genética , Proteínas de Unión al ADN/genética , Dimerización , Antígenos Nucleares del Virus de Epstein-Barr/genética , Glicina/metabolismo , Herpesvirus Humano 4/química , Herpesvirus Humano 4/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Puntual/genética , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Origen de Réplica/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Branched-chain alpha-keto acid dehydrogenase kinase is responsible for the inactivation and phosphorylation of the branched-chain alpha-keto acid dehydrogenase complex, the enzyme that catalyses the committed step of branched-chain amino acid catabolism. The activity of the branched-chain alpha-keto acid dehydrogenase complex is inversely correlated with kinase activity, suggesting that the relative activity of the kinase is the primary regulator of the activity of the complex. It has been shown that kinase activity and expression are affected by nutritional states imposed by low-protein diet feeding, starvation, diabetes, and exercise. Evidence has also been presented that certain hormones, particularly insulin, glucocorticoid, thyroid hormone and female sex hormones, affect the activity and expression of the kinase. The findings indicate that nutritional and hormonal control of the activity and expression of branched-chain alpha-keto acid dehydrogenase kinase provides an important means of control of the activity of the branched-chain alpha-keto acid dehydrogenase complex, with inactivation serving to conserve branched-chain amino acids for protein synthesis in some situations and activation serving to provide carbon for gluconeogenesis in others.
Asunto(s)
Aminoácidos de Cadena Ramificada/metabolismo , Cetona Oxidorreductasas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Quinasas/metabolismo , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Dieta con Restricción de Proteínas , Regulación hacia Abajo , Ejercicio Físico/fisiología , Expresión Génica , Hormonas/fisiología , Humanos , Fosforilación , Inanición/enzimologíaRESUMEN
Branched-chain alpha-keto acid dehydrogenase (BCKDH) complex catalyzes the committed step of branched-chain amino acid catabolism, and its activity is regulated by the phosphorylation-dephosphorylation cycle. BCKDH kinase is responsible for inactivation of the complex by phosphorylation. In the present study, we examined acute exercise on the activity state of the complex as well as the amounts of bound and free forms of the kinase in rat liver and skeletal muscle. Acute exercise activated the complex in association with a decrease in the bound form of kinase in both liver and muscle. The free form of kinase in both tissues was slightly increased but the total amount of the kinase was not affected by acute exercise. The protein amount ratio of bound kinase to E1beta component of the complex was much higher in muscle than in the liver of rats, reflecting the low activity state of the complex in muscle. These results suggest that the amount of the bound kinase plays an important role in regulation of the activity state of the complex. We propose that the alteration in the amount of bound BCKDH kinase is a short-term regulatory mechanism for determining the activity of BCKDH complex.
Asunto(s)
Condicionamiento Físico Animal , Proteínas Quinasas/metabolismo , Animales , Glucemia/metabolismo , Activación Enzimática , Ácidos Grasos/metabolismo , Femenino , Immunoblotting , Hígado/enzimología , Músculo Esquelético/enzimología , Fosforilación , Pruebas de Precipitina , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
The influence of timing and magnitude of arterial wave reflection (WR) on afterload-dependent relaxation was evaluated in patients with a variety of heart diseases (group 1, age < 30 yr; group 2, age > 40 yr) and in dogs. While both femoral arteries were compressed (FC), WR returned just after the dicrotic notch (early diastole) in group 1 but before the dicrotic notch (late systole) in group 2. The time constant of the left ventricular pressure decay (tau) was shortened during FC in group 1, whereas it was prolonged in group 2. In dogs, a constriction of the thoracic aorta induced a late systolic augmentation of WR with a prolongation of tau (cf. group 2), whereas constriction of the lower abdominal aorta induced an early diastolic augmentation of WR with a shortening of tau (cf. group 1). With aortic constriction, coronary flow increased, and there was a close correlation between the peak change in backward aortic pressure and that in coronary flow regardless of the timing of WR. Thus the time at which WR returns during the cardiac cycle may have an important effect on left ventricular relaxation and coronary flow.
Asunto(s)
Enfermedad Coronaria/fisiopatología , Arteria Femoral/fisiopatología , Cardiopatías/fisiopatología , Hemodinámica/fisiología , Función Ventricular Izquierda/fisiología , Adulto , Anciano , Animales , Aorta Abdominal/fisiología , Aorta Torácica/fisiología , Presión Sanguínea , Circulación Coronaria/fisiología , Perros , Arteria Femoral/fisiología , Frecuencia Cardíaca , Humanos , Persona de Mediana Edad , Pulso Arterial , Análisis de Regresión , VasoconstricciónRESUMEN
Branched-chain 2-oxo acid dehydrogenase (BCODH) kinase is responsible for inactivation of BCODH complex by phosphorylation of the complex. Activity of the kinase towards its substrate, the E1 component of the BCODH complex, is known dependent upon binding of the kinase to the E2 component. The possible existence as well as importance of unbound mitochondrial BCODH kinase has been largely ignored in previous studies. Evidence is presented here for the existence of free and bound BCODH kinase in the matrix space of rat liver mitochondria. Furthermore, in female rats, in which diurnal variations in liver BCODH complex and kinase activities occur, the amount of the kinase bound to the complex changes between morning and evening without a change in total kinase protein. Activity of the kinase correlates with the amount of bound rather than total kinase protein, suggesting only the bound form is active. Changes in amount of kinase bound and therefore active appear responsible for diurnal variation in BCODH complex activity in the female rat. We propose that change in the amount of bound BCODH kinase is a key feature of a novel regulatory mechanism for determining the activity state of the BCODH complex.
Asunto(s)
Cetona Oxidorreductasas/metabolismo , Mitocondrias Hepáticas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Quinasas/metabolismo , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Aminoácidos de Cadena Ramificada/metabolismo , Animales , Ritmo Circadiano , Activación Enzimática , Femenino , Immunoblotting , Mitocondrias Hepáticas/enzimología , Fosforilación , Pruebas de Precipitina , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
The branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex is the rate-limiting enzyme in the catabolism of branched-chain amino acids. In the present study, we examined the effects of exercise training on the activity and enzyme expression of the hepatic BCKDH complex in diabetic rats. The rats were prepared by intravenous injections of streptozotocin (50 mg/kg BW), and exercise training was accomplished by treadmill running for 45 min/d for 4 wk. The total and actual activities of hepatic BCKDH complex were significantly increased to approximately 160% by 4 wk of diabetes. On the other hand, diabetic rats in the trained group had the same level of activities as those in the normal rats, indicating that exercise training inhibited the diabetes-induced increase in the enzyme activities. The activity state (% active form) of the enzyme complex was about 100% in all groups and was not affected by diabetes or training. The protein amounts of the enzyme subunits (E1alpha and E2) and the abundance of mRNA for the E2 subunit, but not for the other subunits, in the liver had the same trend as the activities. These results suggest that the capacity for branched-chain amino acid catabolism in streptozotocin-induced diabetic rats is reduced by exercise training and that this modification is associated with the suppression of diabetes-induced BCKDH complex expression in the liver.
Asunto(s)
Diabetes Mellitus Experimental/enzimología , Cetona Oxidorreductasas/metabolismo , Hígado/enzimología , Complejos Multienzimáticos/metabolismo , Condicionamiento Físico Animal/fisiología , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Aminoácidos de Cadena Ramificada/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal/fisiología , Diabetes Mellitus Experimental/metabolismo , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica , Cetona Oxidorreductasas/genética , Hígado/metabolismo , Masculino , Complejos Multienzimáticos/genética , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , EstreptozocinaRESUMEN
Catabolism of alpha-ketoisocaproate in liver is mediated by cytosolic alpha-ketoisocaproate dioxygenase (KICD) and mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). The latter is believed to be involved in the main pathway of the KIC catabolism. In the present study, we measured the activities of KICD and BCKDC in human and rat livers. The KICD activity in human liver was 0.9 mU/g tissue, which was 14.2% of the total activity of BCKDC, and that in rat liver was 4.2 mU/g tissue, which was only 1.0% of the total activity, suggesting that KICD in human liver plays a relatively important role in the alpha-ketoisocaproate catabolism. The KICD activity in human liver was significantly increased by cirrhosis. In rat liver, the enzyme activity was markedly increased by physical training and streptozotocin-induced diabetes, but not by feeding of a diet rich in branched-chain amino acids, although BCKDC activity was increased by feeding of the diet.
Asunto(s)
Dioxigenasas , Cetoácidos/metabolismo , Hígado/enzimología , Oxigenasas/metabolismo , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Aminoácidos de Cadena Ramificada/administración & dosificación , Aminoácidos de Cadena Ramificada/farmacología , Animales , Diabetes Mellitus/enzimología , Diabetes Mellitus/metabolismo , Femenino , Fibrosis/enzimología , Fibrosis/metabolismo , Humanos , Cetona Oxidorreductasas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Persona de Mediana Edad , Complejos Multienzimáticos/metabolismo , Condicionamiento Físico Animal/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
We have previously reported that exercise training prevents a maturation-induced decrease in insulin sensitivity and suggested that an improvement of insulin sensitivity by exercise training was attributable, in part, to an increase in insulin-sensitive GLUT-4 on the skeletal muscle plasma membrane. In this study, we examined the effects of maturation and exercise training on the gene expression and protein content of the components of post-insulin receptor signal transduction in rat skeletal muscle. Rats aged 3 weeks were sedentary or trained by voluntary running through 4 or 27 weeks of age, and then the rats in both the sedentary and trained groups were killed and the gastrocnemius muscle was immediately removed for analysis of mRNA and protein content. The concentration of mRNA and protein for insulin receptor substrate-1 (IRS-1) in sedentary rats significantly decreased with maturation (49% and 63%, respectively, at age 27 weeks v age 4 weeks), but in trained rats they did not decrease with maturation. Although the level of phosphatidylinositol 3-kinase (PI 3-kinase) mRNA in sedentary rats was not altered with maturation, PI 3-kinase protein in sedentary rats significantly decreased with maturation (73% at 27 weeks v 4 weeks). However, PI 3-kinase protein in trained rats did not decrease with maturation. These results suggest that the prevention of maturation-induced decreases in the protein content of IRS-1 and PI 3-kinase is involved in the mechanisms responsible for the improvement of insulin sensitivity by exercise training, and exercise training may affect transcriptional regulation of the IRS-1 gene and posttranscriptional regulation of PI 3-kinase expression.
Asunto(s)
Músculo Esquelético/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/análisis , Condicionamiento Físico Animal , Animales , Femenino , Insulina/sangre , Proteínas Sustrato del Receptor de Insulina , Fosfatidilinositol 3-Quinasas/genética , Fosfoproteínas/genética , ARN Mensajero/análisis , Ratas , Ratas WistarRESUMEN
The effects of a diet supplemented with branched-chain amino acids (BCAA; 4.8% or 6.2%) on BCAA catabolism and glycogen metabolism in rats were examined. Rats were fed a BCAA diet or control diet for 4 wk and part of the rats were subjected to exercise training during the experimental period. Feeding the BCAA diet increased serum BCAA concentrations and activity of the hepatic branched-chain alpha-keto acid dehydrogenase complex, the rate-limiting enzyme in the catabolism of BCAA, suggesting that dietary BCAA promotes BCAA catabolism. Although the serum glucose concentration and glycogen contents in the liver and gastrocnemius muscle of rested rats were not significantly affected by feeding of the BCAA diet, those in rats exhausted by acute exercise were 2-4-fold higher in rats fed the BCAA diet than in rats fed the control diet. The activity of pyruvate dehydrogenase complex in the liver and gastrocnemius muscle after acute exercise showed reverse trends; the complex activities (especially in liver) tended to be less in the BCAA diet group than in the control diet group. These results suggest that dietary BCAA spares glycogen stores in liver and skeletal muscle during exercise and that the decrease in pyruvate dehydrogenase complex activity in these tissues by dietary BCAA is involved in the mechanisms.
Asunto(s)
Aminoácidos de Cadena Ramificada/administración & dosificación , Proteínas en la Dieta/administración & dosificación , Glucógeno/metabolismo , Esfuerzo Físico/fisiología , Aminoácidos de Cadena Ramificada/metabolismo , Aminoácidos de Cadena Ramificada/farmacología , Animales , Proteínas en la Dieta/metabolismo , Proteínas en la Dieta/farmacología , Hígado/enzimología , Hígado/metabolismo , Masculino , Músculo Esquelético/enzimología , Músculo Esquelético/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The pathogenesis of haemophagocytic lymphohistiocytosis (HLH) in children without a known familial pattern of inheritance is often difficult to establish. Splenic enlargement, one of the main clinical findings in this disorder, has led to the use of splenectomy for uncontrollable coagulopathy, persistent cytopenia or both. This procedure is also thought to be a useful tool in making a differential diagnosis in cases of the immunochemotherapy-resistant HLH. We report here five cases of splenectomized childhood HLH, in which subsets of mononuclear spleen cells were analysed either by flow cytometry or immunohistochemistry, and the results were compared with those from cases of hereditary spherocytosis (controls). There was a statistically significant depletion of CD19+ B cells in the HLH cases (3.8 +/- 3.2% vs. 52.6 +/- 4.5%, P < 0. 0001) associated with an increase of T cells in three cases and of natural killer cells in another. The histopathological findings included atrophic white pulps, B-cell depletion with fibrosis and haemosiderosis in all five cases. Despite temporary therapeutic benefits, three of the HLH patients had a rapidly deteriorating post-splenectomy course and all three eventually died. These results demonstrate striking depletion of B cells in the enlarged spleens of children with HLH, which may be an intrinsic feature of HLH pathogenesis. Further study is needed to establish the therapeutic value of splenectomy in this disease.
Asunto(s)
Antígenos CD19/inmunología , Linfocitos B/inmunología , Histiocitosis de Células no Langerhans/inmunología , Histiocitosis de Células no Langerhans/cirugía , Esplenectomía , Linfocitos B/patología , Estudios de Casos y Controles , Preescolar , Resultado Fatal , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Lactante , Células Asesinas Naturales/patología , Recuento de Linfocitos , Masculino , Esferocitosis Hereditaria/inmunología , Linfocitos T/patologíaRESUMEN
The abundance of mRNAs for pyruvate dehydrogenase kinase (PDK) isoenzymes in four brain regions of young (10 wk) and aged (50 wk) rats was investigated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNAs for PDK1, 2, and 4 were detected in all the regions examined. The level of PDK2 mRNA was the most abundant among the isoenzymes in all the brain regions when judged from the PCR cycles. The level of PDK1 mRNA was relatively high in cerebellum and cerebral cortex compared to medulla oblongata and hippocampus. Aging decreased the levels of mRNAs for PDK1 and 2 in cerebellum and increased the PDK2 mRNA in hippocampus and cerebral cortex. The level of PDK4 mRNA was not affected by aging. These results provide the first evidence suggesting that there is the regional difference in the abundance of mRNAs for PDK isoenzymes in rat brain and that the levels of mRNAs for the isoenzymes were affected by aging.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/enzimología , Regulación del Desarrollo de la Expresión Génica , Proteínas Quinasas/genética , Transcripción Genética , Animales , Encéfalo/crecimiento & desarrollo , Cerebelo/enzimología , Corteza Cerebral/enzimología , Regulación Enzimológica de la Expresión Génica , Hipocampo/enzimología , Isoenzimas/genética , Masculino , Proteínas Serina-Treonina Quinasas , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , ARN Mensajero/análisis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Vascular hypertrophy is considered to be an adaptive response to increased arterial wall stress in hypertension. Although there are several reports concerning the effect of angiotensin II inhibition on the development of vascular hypertrophy, little information is available as to its effect on vascular hypertrophy in parallel with the evaluation of arterial wall characteristics. The goal of this study was to evaluate the effect of angiotensin II type 1 receptor antagonist TCV-116 on pressure overload-induced vascular hypertrophy in parallel with the assessment of aortic impedance. Low dose (LD; 0.3 mg/kg/day) or high dose (HD; 3.0 mg/kg/day) of TCV-116 was administered to abdominal aortic-banded rats over 4 weeks; then hemodynamics and morphology were evaluated. In both the LD and HD groups, blood pressures were decreased to a similar extent compared with those of the vehicle-treated group (P < .05). Left ventricular (LV) weight and LV weight/body weight ratio was inhibited in both TCV-116-treated groups (P < .05), whereas the media cross-sectional area of the aorta was inhibited only in the HD group (P < .05). After the treatment of TCV-116 (LD, HD), total systemic resistance was decreased compared with the vehicle-treated group (P < .05), but there was no significant difference between the TCV-116-treated groups. In contrast, the first harmonic of the impedance modulus revealed the decrease only in the HD group (P < .05). TCV-116 attenuated the development of pressure overload LV hypertrophy and vascular hypertrophy as well; however, the dose of TCV-116 required for the inhibition of vascular hypertrophy was significantly higher than that for LV hypertrophy. Vascular hypertrophy may be less pressure dependent than cardiac hypertrophy. On chronic addition of high dose of TCV-116, arterial wave reflection was decreased in association with the attenuation of vascular hypertrophy.
Asunto(s)
Antagonistas de Receptores de Angiotensina , Aorta Abdominal/fisiopatología , Aorta Abdominal/cirugía , Vasos Sanguíneos/patología , Tetrazoles , Animales , Antihipertensivos/farmacología , Aorta Abdominal/efectos de los fármacos , Bencimidazoles/farmacología , Compuestos de Bifenilo/farmacología , Presión Sanguínea/efectos de los fármacos , Vasos Sanguíneos/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Constricción , Relación Dosis-Respuesta a Droga , Hipertrofia/prevención & control , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Izquierda/prevención & control , Masculino , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
An optimized method for the determination of flecainide in serum is presented. Extraction using a solid-phase C18 column and chromatography on a stabilized fluorocarbon-bonded silica gel column effectively separate flecainide from an internal standard (a positional isomer of flecainide). The HPLC apparatus and conditions were as follows: analytical column, Fluofix 120N; sample solvent, 20 microl; column temperature, 40 degrees C; detector, Shimadzu RF-5000 fluorescence spectrophotometer (excitation wavelength = 300 nm, emission wavelength = 370 nm); mobile phase, 0.06% phosphoric acid containing 0.1% tetra-n-butyl ammonium bromide-acetonitrile (75:25, v/v); flow-rate, 1.0 ml/min. The standard curves for flecainide were linear in the concentration range examined (10-2000 ng/ml). The regression equation was y = 0.08+0.0078x (r = 0.9998). The minimum detectable amount of flecainide was approximately 5 ng/ml. In the within-day study, the precision coefficients of variation were 2.66, 2.18, 2.54, 2.72, 2.88, 2.24, and 3.29% for the 10, 50, 100, 200, 500, 1000, and 1500 ng/ml standards, respectively. The absolute recovery rates of flecainide at each concentrations were 94-100%. The method described provides analytical sensitivity, specificity and reproducibility suitable for both biomedical research and therapeutic drug monitoring.
Asunto(s)
Antiarrítmicos/sangre , Cromatografía Líquida de Alta Presión/métodos , Flecainida/sangre , Fluorocarburos/química , Humanos , Reproducibilidad de los Resultados , Gel de Sílice , Dióxido de Silicio/química , Espectrometría de FluorescenciaRESUMEN
By using primers (AL18F2 and AL18R2) designed from goose parvovirus (GPV) strain IHC, an 806-bp band was amplified by polymerase chain reaction (PCR) from all of 17 samples from Thailand. Specificity to GPV was confirmed by Southern hybridization. With restriction enzyme digestion of the PCR products, two isolates differed from the other 15 isolates by the absence of restriction sites for HincII and BglII and the presence of EcoR1 site. Nucleotide sequence analysis of the PCR products from the different groups revealed that one group is GPV and the other group is Muscovy duck parvovirus (MDPV). Thus restriction enzyme fragment length polymorphism analysis of the PCR products could be used to distinguish GPV and MDPV. The data showed that GPV and MDPV are present in Thailand.
Asunto(s)
Patos/virología , Gansos/virología , Infecciones por Parvoviridae/veterinaria , Parvovirus/genética , Parvovirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Enfermedades de las Aves de Corral , Animales , Secuencia de Bases , Brotes de Enfermedades/veterinaria , Datos de Secuencia Molecular , Infecciones por Parvoviridae/diagnóstico , Infecciones por Parvoviridae/epidemiología , Parvovirus/clasificación , Reacción en Cadena de la Polimerasa/veterinaria , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Tailandia/epidemiologíaRESUMEN
The goal of this study was to examine the effect of an angiotensin II type 1 (AT1)-receptor antagonist (TCV-116) on left ventricular (LV) geometry and function during the development of pressure-overload LV hypertrophy. A low (LD; 0.3 mg x kg(-1) x day(-1)) or a high (HD; 3.0 mg x kg(-1) x day(-1)) dose of TCV-116 was administered to abdominal aortic-banded rats over 4 wk, and hemodynamics and morphology were then evaluated. In both LD and HD groups, peak LV pressures were decreased to a similar extent compared with the vehicle-treated group but stayed at higher levels than in the sham-operated group. In the LD group, both end-diastolic wall thickness (3.08 +/- 0.14 mm) and myocyte width (13.3 +/- 0.1 microm) decreased compared with those in the vehicle-treated group (3.67 +/- 0.19 mm and 15.3 +/- 0.1 microm, respectively; both P < 0.05). In the HD group, myocyte length was further decreased (HD: 82.6 +/- 2.6, LD: 94.1 +/- 2.9 microm; P < 0.05) in association with a reduction in LV midwall radius (HD: 3.36 +/- 0.12, LD: 3.60 +/- 0.14 mm; P < 0.05) and peak midwall fiber stress (HD: 69 +/- 8, LD: 83 +/- 10 x 10(3) dyn/cm2; P < 0.05). There was no significant difference in cardiac output among all groups. The AT1-receptor antagonist TCV-116 induced an inhibition of the development of pressure-overload hypertrophy. Morphologically, not only the width but also the length of myocytes was attenuated with TCV-116, leading to a reduction of midwall radius and hence wall stress, which in turn may contribute to a preservation of cardiac output.