RESUMEN
The current treatment for glioblastoma includes temozolomide (TMZ) chemotherapy, yet the mechanism of action of TMZ is not thoroughly understood. Here, we investigated the TMZ-induced changes in the proteome of the glioma-derived cell line (U251) by 2D DIGE. We found 95 protein spots to be significantly altered in their expression after TMZ treatment. MS identified four upregulated spots: aspartyl tRNA synthetase glutathione synthetase, interleukin-1 receptor-associated kinase-4 (IRAK4), and breast carcinoma amplified sequence-1 and one downregulated spot: optineurin. TMZ-induced regulation of these five genes was validated by RT-qPCR and Western blot analysis. RNAi-mediated knockdown of IRAK4, an important mediator of Toll-like receptors signaling and chemoresistance, rendered the glioma cells resistant to TMZ. High levels of IRAK4 induced upon TMZ treatment resulted in IRAK1 downregulation and inhibition of NFkB pathway. Endogenous IRAK4 protein, but not transcript levels in glioma cell lines, correlated with TMZ sensitivity. Thus, we have identified several TMZ-modulated proteins and discovered an important novel role for IRAK4 in determining TMZ sensitivity of glioma cells through its ability to inhibit Toll-like receptor signaling and NFkB pathway.
Asunto(s)
Dacarbazina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , Glioma/tratamiento farmacológico , Glioma/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Proteoma/efectos de los fármacos , Antineoplásicos Alquilantes/farmacología , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Análisis por Conglomerados , Dacarbazina/farmacología , Dimetilsulfóxido , Electroforesis en Gel Bidimensional , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/análisis , Quinasas Asociadas a Receptores de Interleucina-1/genética , FN-kappa B/metabolismo , Proteoma/análisis , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Temozolomida , Receptores Toll-Like/metabolismoRESUMEN
The "J wave" (also referred to as "the Osborn wave,""the J deflection," or "the camel's hump") is a distinctive deflection occurring at the QRS-ST junction. In 1953, Dr. John Osborn described the "J wave" as an "injury current" resulting in ventricular fibrillation during experimental hypothermia. Although "J Wave" is supposed to be pathognomonic of hypothermia, it is seen in a host of other conditions such as hypercalcemia, brain injury, subarachnoid hemorrhage, cardiopulmonary arrest from over sedation, the Brugada syndrome, vasospastic angina, and idiopathic ventricular fibrillation. However, there is paucity of literature data as regards to ischemic etiology of "J Wave." In this article, we present a case where "J waves" were probably induced by ischemia. We also discuss the mechanism of ischemia-induced "J wave" accentuation and its prognostic implications.
Asunto(s)
Electrocardiografía , Isquemia Miocárdica/fisiopatología , Enfermedad Aguda , Adulto , Humanos , Masculino , Isquemia Miocárdica/patologíaRESUMEN
Isopenicillin N synthase (IPNS) from Streptomyces clavuligerus catalyses the oxidative cyclization of the acyclic tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine into isopenicillin N. All four of the cysteine residues found in this enzyme were mutated individually into serine residues, either by the polymerase chain reaction or by single-strand site-directed mutagenesis. Functional analysis of these single mutants showed that the C104S mutant lost more than 96% of its activity, while the remaining C37S, C142S, and C251S mutants each lost 30-50% of their activity. Treatment with the thiol-group-specific reagent N-ethylmaleimide confirmed the importance of the cysteine 104 residue. Activity analysis of an IPNS triple mutant (C37S, C142S, and C251S), prepared by recombining fragments of the IPNS-encoding pcbC gene from each of the three single mutants, showed that it had lost more than 90% of its activity. Conformational analysis by circular dichroism spectroscopy indicated that the IPNS triple mutant was structurally different from the wild type, suggesting that the loss of activity may be due to conformational changes rather than active site modifications.
Asunto(s)
Oxidorreductasas/genética , Streptomyces/genética , Secuencia de Bases , Cisteína/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oxidorreductasas/metabolismo , Streptomyces/enzimología , Relación Estructura-ActividadRESUMEN
Recombinant isopenicillin N synthase from Streptomyces clavuligerus was produced in the form of inactive inclusion bodies in Escherichia coli. These inclusion bodies were solubilized by treatment with 5 M urea under reducing conditions. Optimization of refolding conditions to recover active isopenicillin N synthase indicated that a dialysis procedure carried out at a protein concentration of about 1.0 mg ml(-1) gave maximal recovery of active isopenicillin N synthase. Solubilized isopenicillin N synthase of more than 95% purity was obtained by passing this material through a DEAE-Trisacryl ion exchange column. Expression studies conducted at different temperatures indicated that isopenicillin N synthase was produced predominantly in a soluble, active form when expression was conducted at 20 degrees C, and accounted for about 20% of the total soluble protein. This high-level production facilitated the purification of soluble isopenicillin N synthase to near homogeneity in four steps. Characterization of the purified soluble and solubilized isopenicillin N synthase revealed that they are very similar.
Asunto(s)
Escherichia coli/metabolismo , Transferasas Intramoleculares , Isomerasas/aislamiento & purificación , Proteínas de Unión a las Penicilinas , Streptomyces/enzimología , Escherichia coli/genética , Cuerpos de Inclusión/metabolismo , Isomerasas/biosíntesis , Isomerasas/química , Isomerasas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Streptomyces/genética , TemperaturaRESUMEN
The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein.
Asunto(s)
Oxidorreductasas/genética , Streptomyces/enzimología , Streptomyces/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/genética , Expresión Génica , Genes Bacterianos , Datos de Secuencia Molecular , PlásmidosAsunto(s)
Cardiopatías Congénitas/cirugía , Medicina Militar , Adulto , Coartación Aórtica/cirugía , Estenosis de la Válvula Aórtica/cirugía , Conducto Arterioso Permeable/cirugía , Defectos del Tabique Interatrial/cirugía , Defectos del Tabique Interventricular/cirugía , Humanos , India , Estenosis de la Válvula Pulmonar/cirugíaRESUMEN
Out of 709 consecutive patients with isolated secundum atrial septal defect, the pulmonary artery systolic pressure was greater than 50 mm Hg in 118 patients (17%). Pulmonary hypertension was present in 13% of patients under 10 years and in 14% aged 11 to 20 years. The Eisenmenger reaction was present in 9% of the 709 patients. The frequency of the Eisenmenger reaction was high in young patients and was not significantly different in patients in the first and second decades as compared to older patients. None of our patients with pulmonary hypertension resided at high altitude. The high frequency of pulmonary hypertension in our young patients cannot be satisfactorily explained. Autopsy studies suggest that in some, pulmonary hypertension is due to the persistence of the fetal pulmonary vascular pattern.
