RESUMEN
The activity regenerating luciferin from the luminescent product oxyluciferin was found in the protein fraction of a lantern extract from Photinus pyralis. The protein, luciferin-regenerating enzyme (LRE), was purified to homogeneity by ammonium sulfate precipitation followed by successive column chromatography on Ultrogel AcA34, S-Sepharose FF, Q-Sepharose FF, TSKgel super Q 5pw and TSKgel G3000 SW(XL). This enzyme was a single polypeptide with a molecular mass of 38 kDa. LRE converted oxyluciferin to 2-cyano-6-hydroxybenzothiazole and thioglycolic acid. In the presence of d-cysteine, 2-cyano-6-hydroxybenzothiazole was turned over into luciferin. The same activities were detected in the extracts from two Japanese fireflies, Luciola cruciata and Luciola lateralis. We have cloned a cDNA encoding LRE from poly(A)+ RNA of the lantern of P. pyralis using reverse transcription-polymerase chain reaction, 5'-RACE (rapid amplification of cDNA ends) and 3'-RACE. The primary structure of LRE from P. pyralis deduced from the nucleotide sequence was shown to consist of 308 amino acids with a molecular weight of 33,619. The cDNA was successfully expressed under the control of the tac promoter in Escherichia coli.
Asunto(s)
Escarabajos/enzimología , Luciferina de Luciérnaga/química , Luciferina de Luciérnaga/genética , Indoles , Luciferasas/biosíntesis , Luciferasas/química , Pirazinas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Clonación Molecular , Cisteína/química , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Luciferina de Luciérnaga/aislamiento & purificación , Modelos Químicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Temperatura , Tioglicolatos/química , Factores de TiempoRESUMEN
Sodium ascorbate induced cytotoxicity against human glioblastoma T98G cells in RPMI1640 medium supplemented with fetal bovine serum or human serum samples was studied. Several human serum samples significantly reduced the cytotoxic activity of sodium ascorbate, regardless of sex, age or the disease of the serum donor with or without heat-inactivation of the serum. ESR spectroscopy revealed that this serum effect was not simply due to the alteration of the ascorbyl radical intensity, produced from sodium ascorbate. The present study suggests that the apoptosis-inducing activity of sodium ascorbate might be significantly affected by human serum.
Asunto(s)
Esclerosis Amiotrófica Lateral/sangre , Ácido Ascórbico/toxicidad , Neoplasias Encefálicas/sangre , Supervivencia Celular , Medios de Cultivo , Neoplasias Gastrointestinales/sangre , Adulto , Anciano , Anciano de 80 o más Años , Animales , Bovinos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ácido Deshidroascórbico/análogos & derivados , Ácido Deshidroascórbico/análisis , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Glioblastoma , Humanos , Masculino , Persona de Mediana Edad , Valores de Referencia , Células Tumorales CultivadasRESUMEN
A cDNA and genomic gene encoding calmodulin were isolated from Aspergillus oryzae using a part of the calmodulin gene from A. nidulans as a hybridization probe. The gene was in a 3.4-kb SphI fragment and Southern-blot analysis of genomic DNA suggested the existence of a single copy of the calmodulin gene in A. oryzae. The nucleotide sequence analysis showed that the gene consists of five introns and six exons. Although the nucleotide sequence homology with that of A. nidulans was not so high (68%), the deduced amino acid sequence was 100% and 84% identical with calmodulin of A. nidulans and chicken, respectively. The cDNA encoding A. oryzae calmodulin was expressed under the control of the GAL1 promoter in the calmodulin null mutant (cmd1) of yeast, Saccharomyces cerevisiae, and could function as a calmodulin gene.
Asunto(s)
Aspergillus oryzae/enzimología , Calmodulina/genética , Secuencia de Aminoácidos , Animales , Aspergillus nidulans/genética , Aspergillus oryzae/genética , Secuencia de Bases , Pollos , Clonación Molecular , ADN Complementario/aislamiento & purificación , Datos de Secuencia Molecular , Mutación , Saccharomyces cerevisiae , Homología de Secuencia de AminoácidoRESUMEN
We studied the effect of hemodilutional autotransfusion on coagulative-fibrinolytic dynamics and metabolic response. Hemodilutional solutions used were Dextran-40 (group A) and Salin-HES (group B). The activated partial thromboplastin time (APTT) immediately after hemodilution was prolonged in both groups. Prothrombin time (PT) and hepaplastin decreased significantly, and a remarkable variation was observed particularly in group A. The results suggest that effect on fibrinolytic dynamics is not exerted in either group because antithrombin-III (AT-III) and fibrinogen decreased significantly, while fibrinogen degradation products (FDP) are within normal ranges, and plasminogen as well as alpha 2-plasmin inhibitor (alpha 2-PI) decreased significantly. On the other hand, all other parameters such as lactic acid level, pyruvic acid level in blood, lactic acid pyruvic acid ratio, and blood glucose level were elevated during surgery, but no difference was observed regarding these parameters between the two groups.