RESUMO
Flow cytometry was used to demonstrate the presence of beta-lactoglobulin (betaLG) receptors on living murine hybridoma MARK-3 cells using a fluorescein isothiocyanate-betaLG conjugate (FITC-betaLG: molar ratio of 5:1). A site occupation curve was produced using a shift in the mean channel fluorescence at various concentrations of FITC-betaLG. The binding of labelled ligand was concentration dependent and was inhibited by unlabelled betaLG. The on-rate constant was 3.2x10(2) M(-1) min(-1) and the off-rate constant was 0.002 min(-1). Scatchard plot analysis gave a dissociation constant (K(d)) of 44+/-21x10(-7) and 39+/-24x10(-5) M (n=3). Flow cytometry indicated that at least 15% of the FITC-betaLG were internalized for 5 min and that internalization was temperature- and time-dependent. The internalization was confirmed by 3-D fluorescence microscopy (CELLScan system).
Assuntos
Citometria de Fluxo , Hibridomas/química , Lactoglobulinas/metabolismo , Receptores de Superfície Celular/análise , Animais , Bovinos , Linhagem Celular , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Camundongos , Receptores de Superfície Celular/metabolismoRESUMO
Circulatory shock and its treatment have been compared to a whole-body ischemia and reperfusion with activation of oxygen-derived free radicals. A pilot study had suggested a selenium redistribution in this context. To verify this hypothesis, an experimental study was designed. Temporary occlusion of the superior mesenteric artery was performed in 18 male adult Wistar rats using clamping for 0, 10, and 20 min. Hemodynamic and biochemical data were assessed before clamping and 20 min after release of the mesenteric blood flow. After release, mean arterial pressure decreased, plasma lactate increased, and erythrocyte glutathione peroxidase decreased. Plasma and erythrocyte selenium did not change; however, a slight decrease in plasma selenium was observed when related to hematocrit (to take into account the fluid balance). Erythrocyte-reduced glutathione did not change. In contrast, liver and kidney selenium increased, whereas reduced glutathione decreased in kidney, but not in liver after 20 min of clamping as compared to the sham-operated group. These results suggest that, after temporary intestinal ischemia, the changes in selenium and reduced glutathione observed in blood and tissues, like liver or kidney, could be related to a redistribution pattern in selenium metabolism during shock injury.
Assuntos
Antioxidantes/metabolismo , Hemodinâmica , Intestinos/irrigação sanguínea , Isquemia/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Selênio/metabolismo , Animais , Pressão Sanguínea , Eritrócitos/metabolismo , Radicais Livres/sangue , Radicais Livres/metabolismo , Glutationa/sangue , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Isquemia/sangue , Isquemia/metabolismo , Rim/metabolismo , Lactatos/sangue , Fígado/metabolismo , Masculino , Artéria Mesentérica Superior , Especificidade de Órgãos , Projetos Piloto , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/metabolismo , Selênio/sangueRESUMO
Chinese hamster ovary (CHO-KI) cells were cotransfected with a plasmid pcDNAI containing the human preproinsulin-like growth factor II cDNA linked downstream to the human cytomegalovirus promoter and with a plasmid containing the neomycin resistance gene (pMAM-neo). CHO neo+ were selected by growth in medium supplemented with G418 geneticin. After amplification, the neomycin-resistant clones were screened for IGF-II production. IGF-II produced was identified by dot blot and quantified by ELISA. The clones C24, C40 and C94 secreted IGF-II at about 350-400 ng per 10(6) cells per day. DNA analysis of C24 and C40 CHO cells by PCR demonstrated the presence of the IGF-II construct in the transfected cells, presumably integrated into the chromosomal DNA. IGF-II produced by CHO cells and purified by RP-HPLC was a mitogen for MCF-7 stimulating mitosis 2-fold.
Assuntos
Fator de Crescimento Insulin-Like II/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Sequência de Bases , Células CHO/metabolismo , Cricetinae , DNA Complementar/análise , DNA Complementar/genética , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes/isolamento & purificaçãoAssuntos
Cromatografia Gasosa/métodos , Lactulose/urina , Manitol/urina , Humanos , Técnicas In VitroRESUMO
To assess the clinical value of urinary trypsin inhibitory activity (UTIA) in elderly people, a prospective study was carried out over 4 months in our internal medicine department. Two hundred and forty-three patients of more than 60 years of age were included. A positive correlation was observed between UTIA and serum creatinine (p < 10(-3)). In the population with serum creatinine of less than 133 mumol/l (200 patients), UTIA was independent of age, sex and serum creatinine. UTIA was compared with seven serum inflammatory proteins titrated on patient admission. The principal interest of UTIA determination appeared in bacterial infections. UTIA was significantly increased in this group (p < 10(-4)). However, a positive correlation was proved only with C-reactive protein (CRP) (p = 9 x 10(-4)). Nevertheless, CRP appeared to be the best marker of bacterial infectious diseases after receiver operating characteristic curves analysis.