RESUMEN
Six murine monoclonal antibodies against morphine were produced using N-(4-aminobutyl)normorphine as a hapten. Most of the antibodies obtained distinguished the substituents at the 3 and 6 positions of morphine. This property of the antibodies led to a reduction in cross-reactivity with codeine, morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) to negligible levels. However, one of the antibodies distinguished the substituent only at the 3 position of morphine, which cross-reacted with M-6-G, naloxone and naltrexone. In the competitive inhibition enzyme-linked immunosorbent assay, morphine was detected at concentrations as low as circa 100 pg/ml.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Codeína/inmunología , Morfina/inmunología , Animales , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Partial complementary DNA (cDNA) for thymidine phosphorylase (dThdPase) was cloned by means of a polymerase chain reaction. There was complete sequence identity between the amino acid sequence deduced from the nucleotide sequence of a clone (288 nucleotides) and the residues of platelet-derived endothelial cell growth factor (PD-ECGF). The amino acid sequence of all four peptide fragments from purified human dThdPase could be aligned with that of PD-ECGF. Our data indicate that residues 125-244 of PD-ECGF are identical to the sequence of human dThdPase. The molecular weights of human dThdPase and recombinant PD-ECGF (rPD-ECGF) that lacks 10 amino acids at the amino terminal were 55 and 52 kDa, respectively. Anti-PD-ECGF antibody recognized dThdPase, and anti-dThdPase antibody recognized rPD-ECGF. rPD-ECGF had dThdPase activity and its specific activity was similar to that of purified human dThdPase. dThdPase activity and molecules were detected in COS cells transfected with human PD-ECGF cDNA, but not in nontransfected cells. The sizes of PD-ECGF and dThdPase in the transfected COS cells were identical. These data suggest that human dThdPase is identical to PD-ECGF.
Asunto(s)
Timidina Fosforilasa/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Complementario/genética , Humanos , Immunoblotting , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Timidina Fosforilasa/metabolismo , TransfecciónRESUMEN
A new 1,4-dihydropyridine derivative, CS-905, currently under development as a Ca antagonist, showed a gradual onset and long duration in its antihypertensive effect upon single oral administration. The partition coefficient for CS-905 was measured along with 11 known dihydropyridine Ca antagonists by the HPLC method to clarify its mode of action. The log PHPLC for CS-905 was, 5.18, almost the same as that of manidipine and this value is the highest among the measured drugs. Its pharmacokinetic profile in SHR was also discussed.
Asunto(s)
Ácido Azetidinocarboxílico/análogos & derivados , Bloqueadores de los Canales de Calcio , Dihidropiridinas , Animales , Ácido Azetidinocarboxílico/farmacocinética , Ácido Azetidinocarboxílico/farmacología , Presión Sanguínea/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacocinética , Bloqueadores de los Canales de Calcio/farmacología , Cromatografía Líquida de Alta Presión , Dihidropiridinas/farmacocinética , Dihidropiridinas/farmacología , Masculino , Octanoles , Ratas , Ratas Endogámicas SHR , Solubilidad , AguaRESUMEN
Three stable hybridoma cell lines producing monoclonal antibodies specific for human liver manganese superoxide dismutase were established, and one monoclonal antibody, PG 11, was chosen for immunochemical studies. Immunoblotting demonstrated that the monoclonal antibody binds exclusively to the manganese superoxide dismutase. Immunohistochemical studies indicated that the enzyme is localized in the matrix of human liver mitochondria. To localize antibody-binding epitope, synthetic peptides of the NH2-terminal (residues 1-16) and COOH-terminal (residues 182-189, 190-196, and 182-196) parts of the enzyme were synthesized, and then their effects on the binding were studied using an enzyme-linked immunosorbent assay method. All of the above COOH-terminal peptides inhibited the binding whereas the NH2-terminal ones did not, indicating that PG 11 recognizes several peptides of COOH termini of manganese superoxide dismutase. This is the first report of monoclonal antibodies against human manganese superoxide dismutase with a distinct epitope and of the immunocytochemical demonstration of manganese superoxide dismutase.
Asunto(s)
Anticuerpos Monoclonales , Hígado/enzimología , Superóxido Dismutasa/metabolismo , Animales , Complejo Antígeno-Anticuerpo/análisis , Línea Celular , Técnica del Anticuerpo Fluorescente , Humanos , Hibridomas/inmunología , Immunoblotting/métodos , Inmunohistoquímica , Cinética , Hígado/ultraestructura , Ratones , Microscopía Electrónica , Plasmacitoma , Superóxido Dismutasa/inmunologíaRESUMEN
Effects of Tumor Necrosis Factor (TNF), Interleukin-1 (IL-1), Interleukin-6 (IL-6) and Interferon-gamma (IFN-gamma) on the expression of Mn-superoxide dismutase (Mn-SOD) protein were investigated in human hepatoma cells, Hu-H1, which revealed resistance to the cytotoxicity of TNF and IL-1. Both TNF and IL-1 enhanced the Mn-SOD production to the level of 30- to 40-fold. IL-6 also increased the enzyme protein to 2- to 3-fold of the basal level without any cell proliferative effect. A specific antibody against IL-6 almost completely inhibited the induction of Mn-SOD. IL-6, as well as TNF and IL-1, appears to play some role in the Mn-SOD protein expression in human hepatoma cells.
Asunto(s)
Interleucina-1/farmacología , Interleucina-6/farmacología , Superóxido Dismutasa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Anticuerpos , Carcinoma Hepatocelular , División Celular/efectos de los fármacos , Línea Celular , Replicación del ADN/efectos de los fármacos , Inducción Enzimática , Humanos , Interferón gamma/farmacología , Cinética , Neoplasias Hepáticas , Proteínas Recombinantes/farmacología , Timidina/metabolismoRESUMEN
Marked increase in protein levels of Mn-superoxide dismutase (Mn-SOD) was found in TNF-resistant cell lines after treatment with Tumor Necrosis Factor (TNF). No such increase was observed in Cu,Zn-superoxide dismutase (Cu, Zn-SOD) protein in either TNF-resistant or sensitive cells. These results support the data that the Mn-SOD is one of the rescue proteins required for resistance to TNF cytotoxicity in these cell lines (Wong et al., Cell 58, 923-931, 1990). Mn-SOD was also responsive to TNF stimulation in KURAMOCHI, a human ovarian adenocarcinoma cell line. This may explain our previous result that Mn-SOD protein is highly expressed in epithelial ovarian cancer (Ishikawa et al. Cancer Res. 50, 2538-2542, 1990).