RESUMEN
The antioxidant and hepatoprotective actions of Terminalia catappa L. collected from Okinawa Island were evaluated in vitro and in vivo using leaves extract and isolated antioxidants. A water extract of the leaves of T. catappa showed a strong radical scavenging action for 1,1-diphenyl-2-picrylhydrazyl and superoxide (O(2)(.-)) anion. Chebulagic acid and corilagin were isolated as the active components from T. catappa. Both antioxidants showed a strong scavenging action for O(2)(.-) and peroxyl radicals and also inhibited reactive oxygen species production from leukocytes stimulated by phorbol-12-myristate acetate. Galactosamine (GalN, 600 mg/kg, s.c.,) and lipopolysaccharide (LPS, 0.5 microg/kg, i.p.)-induced hepatotoxicity of rats as seen by an elevation of serum alanine aminotransferase, aspartate aminotransferase and glutathione S-transferase (GST) activities was significantly reduced when the herb extract or corilagin was given intraperitoneally to rats prior to GalN/LPS treatment. Increase of free radical formation and lipid peroxidation in mitochondria caused by GalN/LPS treatment were also decreased by pretreatment with the herb/corilagin. In addition, apoptotic events such as DNA fragmentation and the increase in caspase-3 activity in the liver observed with GalN/LPS treatment were prevented by the pretreatment with the herb/corilagin. These results show that the extract of T. catappa and its antioxidant, corilagin are protective against GalN/LPS-induced liver injury through suppression of oxidative stress and apoptosis.
Asunto(s)
Antioxidantes/farmacología , Fitoterapia , Extractos Vegetales/farmacología , Sustancias Protectoras/farmacología , Terminalia , Animales , Antioxidantes/administración & dosificación , Antioxidantes/uso terapéutico , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Radicales Libres/química , Galactosamina , Glucósidos/administración & dosificación , Glucósidos/farmacología , Glucósidos/uso terapéutico , Taninos Hidrolizables , Lipopolisacáridos , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Hojas de la Planta , Sustancias Protectoras/administración & dosificación , Sustancias Protectoras/uso terapéutico , Ratas , Ratas Sprague-Dawley , Taninos/administración & dosificación , Taninos/farmacología , Taninos/uso terapéuticoRESUMEN
The modifying effects of dietary administration of an herb, Terminalia catappa (TC), were investigated on rat colon carcinogenesis induced by a carcinogen azoxymethane (AOM). The number of aberrant crypt foci (ACF) and beta-catenin accumulated crypts (BCACs) in the colon, and proliferating cell nuclear antigen (PCNA) labelling index in the colonic epithelium were examined in a total of 36 male F344 rats. All animals were randomly divided into five experimental groups (4-10 rats in each group). At 6 weeks of age, rats in groups 1, 2 and 3 were given s.c. injections of AOM once a week for 2 weeks at a concentration of 20 mg/kg body weight. One week before the first injection of AOM, rats in groups 2 and 3 were fed a diet containing 0.02 and 0.1% TC, respectively, throughout the experiment. Rats in group 4 were fed a diet containing 0.1% TC. Rats in group 5 were served as untreated controls. All animals were sacrificed at the experimental week 5 after the start of the experiment. Oral administration of TC at both doses significantly decreased the numbers of both ACF/colon/rat (P<0.05 for 0.02% TC, P<0.005 for 0.1% TC) and BCAC/cm/rat (P<0.05 for both 0.02 and 0.1% TC), when compared with the control group (group 1). Colonic PCNA labelling index in groups 2 and 3 was also significantly lower than that in group 1 (P<0.001 for 0.02% TC, P<0.005 for 0.1% TC). These results suggest that TC has a potent short-term chemopreventive effect on biomarkers of colon carcinogenesis and this effect may be associated with the inhibition of the development of ACF and BCACs.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias del Colon/prevención & control , Extractos Vegetales/farmacología , Terminalia/química , Administración Oral , Animales , Azoximetano/administración & dosificación , Azoximetano/toxicidad , Carcinógenos/administración & dosificación , Carcinógenos/toxicidad , Transformación Celular Neoplásica , Quimioprevención , Enfermedades del Colon/inducido químicamente , Enfermedades del Colon/prevención & control , Enfermedades del Colon/veterinaria , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/veterinaria , Masculino , Fitoterapia/veterinaria , Extractos Vegetales/administración & dosificación , Antígeno Nuclear de Célula en Proliferación/sangre , Distribución Aleatoria , Ratas , Ratas Endogámicas F344RESUMEN
Aim of the study was to evaluate in vivo antioxidant action of medicinal herb Rhodococcum vitis-idaea (Rh.v) on galactosamine (GalN)-induced rat liver toxicity. The results showed that the hepatotoxicity and oxidative stress induced by GalN (700 mg/kg, s.c.) after 24 h evidenced by an increase in serum alanine aminotransferase and glutathione (GSH) S-transferase activities, and lipid peroxidation in liver homogenate were significantly inhibited, when 10 times diluted Rh.v. extract (5 ml/kg, i.p.) was given to rats 12 and 1 h before GalN treatment demonstrating that the extract of Rh.v is a potent antioxidant and protective against GalN-induced hepatotoxicity. The main antioxidant compound of the herb water extract used in the experiment was determined as arbutin, which possess 8% of dry weight of the herb. The electron spin resonance (ESR) spectrometer analysis revealed that the arbutin isolated from Rh.v exhibited strong superoxide and hydroxyl radical scavenging ability.
Asunto(s)
Antioxidantes/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Ericaceae , Fitoterapia , Extractos Vegetales/farmacología , Sustancias Protectoras/farmacología , Animales , Antioxidantes/administración & dosificación , Antioxidantes/uso terapéutico , Relación Dosis-Respuesta a Droga , Galactosamina , Masculino , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Hojas de la Planta , Sustancias Protectoras/administración & dosificación , Sustancias Protectoras/uso terapéutico , Ratas , Ratas WistarRESUMEN
A 64-year-old male with treated Parkinson's disease underwent mechanical valve replacement for aortic valve regurgitation. The antiparkinsonian drugs for internal use were interrupted on the morning of the operative day. After the operation, the patient developed fervescence, muscle rigidity, hidropoiesis and a rise in creatine kinase. The patient was diagnosed as neuroleptic malignant syndrome and given medication dantrolene sodium and antiparkinsonian drugs on the 5th postoperative day. The symptom of neuroleptic malignant syndrome disappeared on 12 postoperative days. As the stress of open heart surgery with extracorporeal circulation trigger off neuroleptic malignant syndrome, the patient with Parkinson's disease need early beginning of antiparkinsonian drugs on account of prevention of neuroleptic malignant syndrome after operation.
Asunto(s)
Insuficiencia de la Válvula Aórtica/cirugía , Válvula Aórtica/cirugía , Implantación de Prótesis de Válvulas Cardíacas , Síndrome Neuroléptico Maligno/etiología , Enfermedad de Parkinson/tratamiento farmacológico , Complicaciones Posoperatorias , Humanos , Masculino , Persona de Mediana Edad , Periodo PosoperatorioRESUMEN
Free radical scavenging action of Limonium wrightii O. kunthe was examined in vitro and in vivo by using electron spin resonance spectrometer and chemiluminescence analyzer. A water extract of L. wrightii showed a strong scavenging action for the 1,1-diphenyl-2-picrylhydrazyl, or superoxide anion and moderate for hydroxyl radical. The extract also depressed production of reactive oxygen species from polymorphonuclear leukocytes stimulated by phorbor-12-mysistate acetate and inhibited lipid peroxidation in rat liver microsomes. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Gallic acid was identified as the active component of L. wrightii with a strong free radical scavenging action. Our results demonstrate the free radical scavenging action of L. wrightii and that gallic acid contributes to these actions.
Asunto(s)
Antioxidantes/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Depuradores de Radicales Libres/farmacología , Peroxidación de Lípido/efectos de los fármacos , Fitoterapia , Extractos Vegetales/farmacología , Plumbaginaceae , Alanina Transaminasa/sangre , Animales , Antioxidantes/administración & dosificación , Antioxidantes/uso terapéutico , Aspartato Aminotransferasas/sangre , Tetracloruro de Carbono , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Depuradores de Radicales Libres/administración & dosificación , Depuradores de Radicales Libres/uso terapéutico , Inyecciones Intraperitoneales , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Ratones , Microsomas Hepáticos/efectos de los fármacos , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Ratas , Ratas Sprague-DawleyRESUMEN
Blood cysts of the heart are extremely rare in adults and usually involve valves or the left ventricle. Although two cases of blood cysts in the right atrium in adults have been reported, a cyst combined with a disorder of the valves has never been reported. We report a 52-year-old woman with a blood cyst that generated from the right atrial septum. Furthermore, the patient had regurgitation of both the mitral and tricuspid valves and then underwent surgical excision of the blood cyst, mitral valve plasty and tricuspid valve annuloplasty. We believe that it is possible to diagnose blood cysts with echocardiography, CT and magnetic resonance imaging. Echocardiography showed the cyst as a circle without a complete inner free-echo. CT and magnetic resonance imaging showed a mass with a non-enhanced inner structure. Furthermore, the latter showed a cyst that was enhanced by T1- but not T2-weighted images, indicating that the content of the cyst was a persistent substance such as blood. Concerning the generation of blood cysts, we hypothesize that heteroplastic growth arising from primitive pericardial mesothelium causes disorders of valves and blood cysts.
Asunto(s)
Cardiomiopatías/cirugía , Quistes/cirugía , Insuficiencia de la Válvula Mitral/cirugía , Insuficiencia de la Válvula Tricúspide/cirugía , Sangre , Cardiomiopatías/complicaciones , Cardiomiopatías/diagnóstico , Quistes/complicaciones , Quistes/diagnóstico , Ecocardiografía , Femenino , Humanos , Imagen por Resonancia Magnética , Persona de Mediana Edad , Insuficiencia de la Válvula Mitral/complicaciones , Insuficiencia de la Válvula Mitral/diagnóstico , Tomografía Computarizada por Rayos X , Insuficiencia de la Válvula Tricúspide/complicaciones , Insuficiencia de la Válvula Tricúspide/diagnósticoRESUMEN
Serine 89 of the inorganic pyrophosphatase (PPase) subunit from thermophilic bacterium PS-3 (PS-3) was replaced with glycine, alanine, threonine, glutamic acid, or aspartic acid by the PCR-mutagenesis method with Mut-1 in order to determine the contribution of this serine residue to the thermostability and structural integrity of the enzyme molecule. S89G, S89A, and S89T showed reduced catalytic activity, whereas S89D and S89E showed increased enzyme activity. S89G, S89A, and S89T as well as the wild-type PPase were stable in the presence of 5 mM MgCl(2) at 70 degrees C for 1 h, but were inactivated rapidly with time at 80 degrees C. On the contrary, S89D and S89E were stable at 80 degrees C, showing more than 95% of the original activity after 1 h incubation. The wild-type PPase, S89D and S89E were each a hexamer before and after incubation at 80 degrees C for 1 h, while S89G and S89A comprised a mixture of a hexamer and a trimer both before and after incubation at 80 degrees C for 1 h. On the other hand, S89T was a mixture of a hexamer, a trimer and a monomer, and it was partially precipitated during heat treatment at 80 degrees C. The CD spectra of the recombinant enzymes in the far-ultraviolet region were the same as that of the wild-type PPase, whereas those of S89G, S89A, and S89T as well as the wild-type PPase were markedly different after heat treatment, although those of S89D and S89E did not change. The present study suggested that local small change(s) in the network of interactions among amino acid residues on replacement at position 89 led to the PS-3 PPase molecule being unable to form a hexamer from trimers or to dissociate into monomers in some cases without a significant change in the backbone conformation. It was also suggested that the partial disordering of the conformation of PS-3 PPase caused by heat depended on the degree of hydrophilicity in the vicinity of position 89.
Asunto(s)
Sustitución de Aminoácidos , Bacterias/enzimología , Estabilidad de Enzimas , Pirofosfatasas/química , Pirofosfatasas/metabolismo , Serina/metabolismo , Bacterias/genética , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Calor , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Conformación Proteica , Pirofosfatasas/genética , Serina/genética , Relación Estructura-ActividadRESUMEN
A novel antibiotic, CJ-15,801 (I), was isolated from the fermentation broth of a fungus, Seimatosporium sp. CL28611. The structure was determined to be a pantothenic acid analog having an alpha,beta-unsaturated carboxylic acid moiety by spectroscopic analyses. The compound inhibits the growth of multi-drug resistant (MDR) Staphylococcus aureus strains with MIC ranging from 6.25 to 50 microg/ml.
Asunto(s)
Antibacterianos/aislamiento & purificación , Ácido Pantoténico/aislamiento & purificación , Antibacterianos/química , Antibacterianos/farmacología , Fermentación , Pruebas de Sensibilidad Microbiana , Ácido Pantoténico/análogos & derivados , Ácido Pantoténico/química , Ácido Pantoténico/farmacologíaRESUMEN
We report a case of mucosa-associated lymphoid tissue (MALT) lymphoma of the rectum that regressed spontaneously. A 76-year-old man visited our hospital because of positive faecal occult blood testing. Colonoscopic examination revealed a slightly yellowish protruded lesion with a grooved depression in the lower rectum and two flat elevations in the upper rectum. Microscopic and immunohistological studies led to a diagnosis of MALT lymphoma. As the patient exhibited severe renal dysfunction and angina pectoris, the lesions were left untreated. Three months later, the protruded lesion became flat and the other lesions became unclear. He was followed up closely with endoscopy, but no relapse of these lesions was detected 19 months after the diagnosis.
Asunto(s)
Linfoma de Células B de la Zona Marginal/patología , Regresión Neoplásica Espontánea , Neoplasias del Recto/patología , Anciano , Biopsia , Colonoscopía , Diagnóstico Diferencial , Humanos , Linfoma de Células B de la Zona Marginal/diagnóstico por imagen , Masculino , Cintigrafía , Neoplasias del Recto/diagnóstico por imagen , Tomografía Computarizada por Rayos XRESUMEN
A 78-year-old male who had a bronchial asthma underwent coronary artery bypass grafting (CABG) using the left internal thoracic artery and the radial artery. The patient could not be weaned from the cardiopulmonary bypass because the radial artery which anastomosed to the obtuse marginal artery (OM) had a spasm after CABG. An additional bypass using a long saphenous vein to OM was carried out immediately. It brought a weaning from cardiopulmonary bypass. If the cardiac function after CABG is insufficient in patients with bronchial asthma, CABG must be re-done immediately, considering that they cause the arterial spasm more than patients without bronchial asthma.
Asunto(s)
Asma/complicaciones , Puente de Arteria Coronaria , Enfermedad Coronaria/cirugía , Arteria Radial/trasplante , Espasmo/etiología , Anciano , Puente Cardiopulmonar , Humanos , Masculino , VasoconstricciónRESUMEN
Three groups of Rap1-specific guanine nucleotide exchange factors including C3G, CalDAG-GEFI, and Epac/cAMP-GEFI/II have been identified to date. In the present study, we report a new Rap1 guanine nucleotide exchange factor which we have named GFR (guanine nucleotide exchange factor for Rap1). GFR shows close sequence similarity to EPAC/cAMP-GEFI/II although GFR lacks a cAMP binding domain and contains a nuclear localization signal. We demonstrated that GFR can activate Rap1 but not H-Ras in 293T cells and that the cdc25 domain of GFR is required for the activation of Rap1. Northern blot analysis suggested that GFR mRNA is strongly expressed in the brain. In transfected HeLa cells, GFR has been found to be localized in the nuclei.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Nucleótidos de Guanina/metabolismo , Proteínas/química , Secuencia de Aminoácidos , Encéfalo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/química , Factores de Intercambio de Guanina Nucleótido , Nucleótidos de Guanina/química , Células HeLa , Humanos , Riñón/metabolismo , Datos de Secuencia Molecular , Proteínas/metabolismo , ARN Mensajero/análisis , Homología de Secuencia de Aminoácido , Distribución Tisular , Transfección , Proteínas de Unión al GTP rap , Factores de Intercambio de Guanina Nucleótido ras , ras-GRF1RESUMEN
C3G is a guanine nucleotide exchange factor for Rap1 and is activated by the expression of Crk adaptor proteins. We found that expression of CrkI in COS cells induced significant tyrosine phosphorylation of C3G. To understand the mechanism by which C3G is phosphorylated and activated by Crk, we constructed a series of deletion mutants. Deletion of the amino terminus of C3G to amino acid 61 did not remarkably affect either tyrosine phosphorylation or Crk-dependent activation of C3G. When C3G was truncated to amino acid 390, C3G was still phosphorylated on tyrosine but was not effectively activated by CrkI. Deletion of the amino terminus of C3G to amino acid 579 significantly reduced the Crk-dependent tyrosine phosphorylation of C3G and increased GTP-bound Rap1 irrespective of the presence of CrkI. We substituted all seven tyrosine residues in this region, amino acids 391-579, for phenylalanine for identification of the phosphorylation site. Among the substitution mutants, the C3G-Y504F mutant, in which tyrosine 504 was substituted by phenylalanine, was remarkably less activated and phosphorylated than the wild type. All the other substitution mutants were activated and tyrosyl-phosphorylated by the expression of CrkI. Thus, CrkI activates C3G by the phosphorylation of tyrosine 504, which represses the cis-acting negative regulatory domain outside the catalytic region.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas , Tirosina/metabolismo , Animales , Células COS , Dominio Catalítico , Factores de Intercambio de Guanina Nucleótido , Fosforilación , Fosfotirosina/metabolismo , Proteínas Quinasas/metabolismo , Prenilación de Proteína , Proteínas Proto-Oncogénicas c-crk , Relación Estructura-Actividad , Proteínas de Unión al GTP rap , Factores de Intercambio de Guanina Nucleótido ras , Dominios Homologos srcRESUMEN
A 48-year-old male with constrictive pericarditis was reoperated through median sternotomy 13 years after pericardiectomy through left thoracotomy. Extensive pericardiectomy is the most important point for constrictive pericarditis. Therefore, we have recently chosen the median sternotomy approach which has advantages over the left thoracotomy approach. Namely, the cardiopulmonary bypass is performed in the former approach immediately if the posterior pericardiectomy is difficult or the coronary arterial injury happens during decortication.
Asunto(s)
Pericardiectomía , Pericarditis Constrictiva/cirugía , Procedimientos Quirúrgicos Torácicos/métodos , Toracotomía , Humanos , Masculino , Persona de Mediana Edad , Reoperación , Esternón/cirugía , Factores de TiempoRESUMEN
The effects of calcium and magnesium ions in cardioplegic solutions on cardioprotection and intracellular calcium ion handling during ischemia and reoxygenation were investigated in cultured neonatal rat myocardial cells. Myocytes were subjected to simulated ischemia for 60 min at 37 degrees C in hyperkalemic cardioplegic solutions containing various concentrations of calcium and magnesium ions, followed by 30 min of reoxygenation. For each Ca2+ concentration (0.1, 0.6, 1.2, or 2.4 mM), the Mg2+ concentration was either 0, 1.2, 8, or 16 mM. The increase in intracellular Ca2+ concentration during ischemia and reoxygenation was suppressed by the addition of magnesium ion, independent of cardioplegic Ca2+ concentration. The recovery of spontaneous contraction rate and enzyme leakage (creatine phosphokinase and lactate dehydrogenase) during both ischemia and reoxygenation correlated with the degree of inhibition of intracellular Ca2+ accumulation. However, in the 0.1 mM Ca2+ groups in which the Mg2+ concentration was greater than 8 mM, the intracellular Ca2+ concentration increased during reoxygenation in a dose-dependent fashion of Mg2+, and was associated with increased enzyme leakage. The findings suggest that in immature cardiac myocytes, the concentrations of Ca2+ and Mg2+ present in cardioplegic solutions control the intracellular Ca2+ concentration during ischemia and reoxygenation, which, in turn, influences the cardioprotective effect of the cardioplegic solution.
Asunto(s)
Calcio/metabolismo , Soluciones Cardiopléjicas/farmacología , Magnesio/farmacología , Isquemia Miocárdica/metabolismo , Miocardio/metabolismo , Animales , Animales Recién Nacidos , Calcio/farmacología , Creatina Quinasa/metabolismo , Relación Dosis-Respuesta a Droga , Corazón/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Isquemia Miocárdica/tratamiento farmacológico , Reperfusión Miocárdica , Miocardio/citología , Oxígeno/fisiología , Ratas , Ratas Wistar , Sarcolema/efectos de los fármacos , Sarcolema/metabolismoRESUMEN
Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1 in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Receptores ErbB/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Dominios Homologos src , Animales , Células COS , Catálisis , Factor de Crecimiento Epidérmico/metabolismo , Proteína Adaptadora GRB2 , Factores de Intercambio de Guanina Nucleótido , Proteínas Proto-Oncogénicas c-crk , Proteínas de Unión al GTP rap , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
The thermophilic bacterium PS-3 ppa gene encoding inorganic pyrophosphatase (PPiase) has been cloned and sequenced. The deduced amino acid sequence was exactly the same as that determined on Edman degradation (Ichiba, T., Takenaka, O., Samejima, T. and Hachimori, A., J. Biochem. 108: 572-578, 1990) except that amino acid-70 is threonine instead of proline and two more amino acids, Asn-Lys, are present at the carboxyl terminus. The structural gene containing BamH I and Hind III restriction cleaving sites at the 5' and 3' ends, respectively, was amplified by the PCR method. Escherichia coli JM109 was transformed with a synthetic DNA, and we found that thermophilic ppa was expressed in E. coli, as judged on enzyme assaying, SDS-PAGE and immune assaying.
Asunto(s)
Bacterias/enzimología , Clonación Molecular , Pirofosfatasas/genética , Secuencia de Aminoácidos , Bacterias/genética , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Regulación Enzimológica de la Expresión Génica , Pirofosfatasa Inorgánica , Datos de Secuencia MolecularRESUMEN
The degradation of ornithine decarboxylase (ODC) catalyzed by the 26 S proteasome is accelerated by antizyme, an ODC inhibitory protein induced by polyamines. Previously, we have found another possible regulatory protein of ODC degradation, antizyme inhibitor. Antizyme inhibitor binds to the antizyme with a higher affinity than that of ODC, releasing ODC from ODC-antizyme complex. We report here the cDNA sequence of rat heart antizyme inhibitor. The deduced sequence of the protein is highly similar to, but distinct from, sequences of ODCs from various species. Antizyme inhibitor contains amino acid residues required for formation of active sites of ODC, but it completely lacks ODC activity. Antizyme inhibitor has no homology with peptide sequence in the mammalian ODC carboxyl terminus, which is needed for rapid turnover of ODC. It inhibits antizyme-dependent ODC degradation, but, unlike ODC, its degradation is not accelerated by antizyme.
Asunto(s)
Inhibidores de la Ornitina Descarboxilasa , Ornitina Descarboxilasa/química , Complejo de la Endopetidasa Proteasomal , Biosíntesis de Proteínas , Proteínas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Inhibidores Enzimáticos , Expresión Génica , Mamíferos , Datos de Secuencia Molecular , Miocardio/metabolismo , Sistemas de Lectura Abierta , Péptido Hidrolasas/metabolismo , Proteínas/metabolismo , ARN Mensajero/biosíntesis , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The effects of calcium (Ca) on a hyperkalemic cardioplegic solution for continuous cardioplegia were examined in an isolated perfused working rat heart model. The coronary arteries were perfused with a modified Krebs-Henseleit bicarbonate buffer (K-H) solution, containing various concentrations of Ca (0.1, 0.6, 1.2, and 2.5 mmol/l) and a high concentration of potassium (20 mmol/l), for 180 min, after which cardiac arrest was induced at 37 degrees C for 180 min. Cardiac function and creatine kinase (CK) were measured. In the control group, K-H solution was infused in place of the cardioplegic solution, and cardiac arrest was not induced. No significant differences were observed between the groups infused with the K-H solution containing Ca concentrations of 0.6, 1.2, and 2.5 mmol/l in the percent recovery of aortic flow (82.1 +/- 2.9%, 80.6 +/- 2.0%, and 71.5 +/- 3.7% (mean +/- SEM) respectively) or in the recovery of other indices of cardiac function, or in CK leakage. There were also no significant differences in the recovery of cardiac function and CK leakage between these groups and the control group. In the Ca 0.1 mmol/l group, however, the characteristic Ca paradox was observed. These findings suggest that if the Ca concentration in a cardioplegic solution is higher than 0.6 mmol/l during continuous cardioplegia, excellent cardioprotective effects will be achieved.
Asunto(s)
Calcio/farmacología , Soluciones Cardiopléjicas/química , Paro Cardíaco Inducido/métodos , Daño por Reperfusión Miocárdica/prevención & control , Animales , Soluciones Cardiopléjicas/farmacología , Creatina Quinasa/metabolismo , Glucosa/química , Masculino , Miocardio/metabolismo , Miocardio/ultraestructura , Perfusión , Ratas , Ratas Wistar , Trometamina/químicaRESUMEN
An ornithine decarboxylase antizyme cDNA was obtained from Xenopus laevis liver and its sequence was determined. The cDNA consists of two major open reading frames as found in mammalian antizymes, which require +1 ribosomal frameshifting for its translation. Sequences important for frameshifting, namely the frameshift site and downstream stimulatory pseudoknot determined in the rat mRNA, are conserved.