RESUMO
Recent genetic and biochemical studies have implicated cysteine-dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVD-aminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100- and 20-fold, respectively; but there was only a 1. 5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-biotin yllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one- and two-dimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Apoptose/efeitos dos fármacos , Núcleo Celular/enzimologia , Cisteína Endopeptidases/metabolismo , Citosol/enzimologia , Etoposídeo/toxicidade , Caspase 1 , Ativação Enzimática , Células HL-60 , HumanosRESUMO
Pemphigus vulgaris (PV) is mediated by autoantibodies to desmoglein 3, the pemphigus vulgaris antigen (PVA). PVA and an extracellular domain of PVA-Ig fusion protein (PV-Ig) can completely adsorb the blister-causing Abs from PV patient sera, suggesting that the extracellular segment of PVA might be sufficient to induce pathogenic Abs. To test this, we immunized rabbits with either PVA or its extracellular domain (EPVA) expressed in insect cells in our laboratory. When Igs were passively transferred from these rabbits into neonatal mice, anti-PVA, but not the anti-EPVA, induced blisters. To understand the basis for their differential pathogenic effects, we examined the properties of these sera. Both sera showed comparable ELISA titers and indirect immunofluorescence reactivity against monkey esophagus, a source of native PVA. Moreover, EPVA, like PVA adsorbed blister-causing Abs from sera of PV patients and rabbits immunized with PVA. In contrast, when IgG preparations were incubated with fura-2-AM (acetyloxymethyl ester)-loaded human keratinocytes in culture, only IgG from anti-PVA serum induced intracellular calcium mobilization. These data showed that PVA but not EPVA can elicit Abs that induced blisters in neonatal mice and mediate intracellular signaling through calcium mobilization.
Assuntos
Autoanticorpos/biossíntese , Doenças Autoimunes/imunologia , Vesícula/etiologia , Caderinas/imunologia , Epitopos/imunologia , Pênfigo/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Autoanticorpos/sangue , Autoanticorpos/imunologia , Doenças Autoimunes/sangue , Vesícula/imunologia , Caderinas/química , Caderinas/genética , Cálcio/metabolismo , Linhagem Celular , DNA Complementar/genética , Desmogleína 3 , Epitopos/química , Epitopos/genética , Humanos , Imunização Passiva , Técnicas de Imunoadsorção , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mariposas/citologia , Nucleopoliedrovírus/genética , Pênfigo/sangue , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Dobramento de Proteína , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes de Fusão/químicaRESUMO
Recently, we reported the development of a species-specific neurotensin analog that displays selective binding affinity at the rat and human neurotensin (NT) receptor, L-[3,2'-Nal11]NT(8-13) (where Nal is naphthylalanine) (NT19). We have developed another neurotensin analog, L-[3,1'-Nal11]NT(8-13), (NT34), that exhibits a 126-fold difference in binding affinities between the rat and human receptors. This compound differs from our previous reported species-specific ligand in the steric positioning of the naphthyl ring on the L-alanine side chain. For NT34, the observed Kd values at the rat and human neurotensin receptors were 0.046 and 5.8 nM, respectively. In stimulating phosphatidylinositol turnover, the observed EC50 values were 2.8 nM and 130 nM in rat and human, respectively. We constructed a series of chimeric rat/human neurotensin receptor genes and expressed them by transient transfection into human embryonic kidney (HEK-293) cells. Radioligand binding assays were then performed using neurotensin and NT34. Our results led us to propose a region of the neurotensin receptor that may be involved in determining species specificity, i. e. the transmembrane VI, the third extracellular loop, and transmembrane VII regions of the neurotensin receptor.
Assuntos
Neurotensina/metabolismo , Oligopeptídeos/metabolismo , Receptores de Neurotensina/química , Receptores de Neurotensina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Células CHO , Cricetinae , Primers do DNA , DNA Complementar , Humanos , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neurotensina/farmacologia , Oligopeptídeos/química , Fosfatidilinositóis/metabolismo , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
1. The metabolism of two compounds which undergo predominantly Phase I (propranolol) and Phase II (xamoterol) metabolism in vivo has been studied in isolated rat hepatocytes. 2. Propranolol was rapidly metabolized by rat hepatocytes to a number of metabolites which correlated well with those observed in vivo. The effect of saturable metabolism on the in vitro clearance of propranolol at high substrate concentrations was very similar to the changes observed in vivo. 3. Xamoterol was metabolized by rat hepatocytes to produce mainly xamoterol glucuronide, with the sulphate conjugate of xamoterol representing a minor component. The low rate of formation of xamoterol sulphate is probably due to the low affinity of xamoterol for the sulphotransferase enzyme, since supplementation with inorganic sulphate did not significantly alter the rate of sulphation; the sulphotransferase system of these hepatocytes was however shown to be active in the metabolism of phenol. 4. The correlations observed between the known routes of metabolism of propranolol and xamoterol in vivo and those observed in isolated hepatocytes support the utility of isolated hepatocytes as a predictive model of metabolic events in vivo.