RESUMEN
Peroxisome proliferators (PPs) are a class of non-genotoxic chemicals that cause rodent liver enlargement and hepatocarcinogenesis. In primary rat hepatocyte cultures, PPs suppress spontaneous apoptosis and that induced by a number of pro-apoptotic stimuli such as transforming growth factor-beta(1). Tumour necrosis factor alpha (TNF-alpha) and the transcription factor NFkappaB have been implicated in the mode of action of PPs. TNF-alpha signalling to NFkappaB is thought to be responsible for many of the effects elicited by this cytokine. NFkappaB regulates gene expression in immunity, stress responses and the inhibition of apoptosis. Activation of NFkappaB requires the successive action of NFkappaB-inducing kinase and the phosphorylation of NFkappaB inhibitory proteins (IkappaB) by an IkappaB kinase (IKK) complex. The IKK2 subunit of IkappaB kinase is thought to be essential for NFkappaB activation and prevention of apoptosis. To determine whether IKK2 plays a role in the suppression of apoptosis by PPs, we expressed a dominant negative form of IKK2 (IKK2dn) in primary rat hepatocyte cultures. Infection with an adenovirus construct expressing IKK2dn caused apoptosis in control primary rat hepatocytes in the absence of exogenous TNF-alpha. Moreover, IKK2dn-induced apoptosis could not be rescued by addition of TNF-alpha or the peroxisome proliferator nafenopin. These results demonstrate a requirement for intracellular signalling pathways mediated by IKK2 in the suppression of apoptosis by the PP class of hepatocarcinogens.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/fisiología , Adenoviridae/genética , Animales , Apoptosis/fisiología , Regulación de la Expresión Génica , Quinasa I-kappa B , Proteínas Inhibidoras de la Apoptosis , Hígado/citología , Hígado/efectos de los fármacos , Hígado/enzimología , FN-kappa B/fisiología , Nafenopina/antagonistas & inhibidores , Nafenopina/toxicidad , Proliferadores de Peroxisomas/antagonistas & inhibidores , Proliferadores de Peroxisomas/toxicidad , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transducción Genética , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Full-length cytosolic phospholipase A2 (cPLA2) was cloned from U937 cells and polymorphonuclear leukocytes (PMNLs) while a naturally occurring variant of cPLA2, which lacks residues Val473-Ala749 but has a C-terminal extension of ILMNLSEYMLWMSKVKRFM (DcPLA2) was cloned from PMNLs and mononuclear leukocytes. We were unable to clone DcPLA2 from U937 cells. When cPLA2 and DcPLA2 were expressed in insect cells, both proteins were detected in cell lysates by SDS/PAGE as single bands of apparent molecular masses 100 kDa and 57 kDa, respectively. Full-length cPLA2 was phosphorylated stoichiometrically by p42 mitogen-activated protein (MAP) kinase in vitro at a similar rate to other physiological substrates of this protein kinase and the major site of phosphorylation was identified by amino acid sequencing as Ser505. [32P]Ser(P)505 in cPLA2 was only dephosphorylated at a slow rate by mammalian tissue homogenates. Protein phosphatases 2A, 2B and 2C all contributed significantly to the overall dephosphorylation of cPLA2. The phosphorylation of cPLA2 by p42 MAP kinase correlated with an approximately 1.5-fold increase in specific enzyme activity which was reversed by dephosphorylation.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Citosol/enzimología , Fosfolipasas A/genética , Fosfolipasas A/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Éteres Cíclicos/farmacología , Humanos , Insectos/citología , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/fisiología , Linfoma/enzimología , Linfoma/patología , Proteína Quinasa 1 Activada por Mitógenos , Datos de Secuencia Molecular , Neutrófilos/fisiología , Ácido Ocadaico , Fosfolipasas A/efectos de los fármacos , Fosfolipasas A2 , Fosfoproteínas Fosfatasas/metabolismo , Fosforilasas/efectos de los fármacos , Fosforilasas/metabolismo , Fosforilación , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Extractos de Tejidos , Células Tumorales CultivadasRESUMEN
Using the novel ligand [4,5-3H-Leu9]neurokinin A ([4,5-3H-Leu9] NKA) in a receptor binding assay, we characterized the pharmacology of a cloned neurokinin NK-2 receptor from human lung (hNK-2R), expressed in baculovirus-infected Sf-21 insect cells. Functional hNK-2R cDNA clones were isolated from human lung using a polymerase chain reaction-based methodology. hNK-2R was cloned into pAcYM1, a vector designed to couple expression to the polyhedrin promoter, and the recombinant baculovirus was isolated and used to infect Sf-21 insect cells. hNK-2R expression levels were monitored by Northern blots and 125-I-NKA binding assays. Isolates demonstrating the highest specific binding of 125-I-NKA were grown and membrane preparations from high-speed centrifugations were prepared from both hNK-2R-expressing and wild-type virus-infected cells. [3H]NKA bound in a protein-dependent, saturable (Bmax = 820 +/- 167 fmol/mg of protein), and highly specific (88 +/- 5%) manner to hNK-2R, but not to membranes from cells infected with wild-type virus (14 +/- 8%, 7 +/- 10 fmol/mg of protein). [3H]NKA binding was rapid (k1 = 0.085 nM-1 x min-1) and reversible (t1/2 = 4-5 min). Equilibrium binding experiments demonstrated binding to a mixture of receptors in high and low affinity states (Kd1 = 2.28 +/- 0.26 nM and Kd2 = 266 +/- 91 nM). Binding to hNK-2R was greatly enhanced (400%-600%) by Ca2+ and Mg2+ (EC50 values of 30 microM and 140 microM, respectively), whereas guanosine-5'-O-(3'-thio)triphosphate and guanosine-5'-(beta, gamma-imido)diphosphate were inhibitory. Competition experiments with agonists also demonstrated binding to high and low affinity states, with the following order of potency: NKA > [Nle10]NKA(4-10) > [beta-Ala8]NKA(4-10) >> substance P; Senktide and the NK-1 antagonist CP96,345 (10 microM) did not inhibit binding. Inhibition of binding by selective NK-2 antagonists was consistent with a single affinity state and demonstrated the following order of affinity: SR48,968 >> MEN10,376 > L659,877 > R396. These data suggest that infection of Sf-21 cells with baculovirus expression vector harboring the cDNA of hNK-2R resulted in expression of high affinity, G protein-coupled hNK-2R, with pharmacological selectivity compatible with the NK-2A receptor subtype.
Asunto(s)
Neuroquinina A/metabolismo , Receptores de Neurotransmisores/metabolismo , Animales , Baculoviridae/genética , Secuencia de Bases , Benzamidas/farmacología , Sitios de Unión , Northern Blotting , Línea Celular , Clonación Molecular , Proteínas de Unión al GTP/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , Mariposas Nocturnas , Neuroquinina A/análogos & derivados , Neuroquinina A/farmacología , Fragmentos de Péptidos/farmacología , Piperidinas/farmacología , Reacción en Cadena de la Polimerasa , Ensayo de Unión Radioligante , Receptores de Neuroquinina-2 , Receptores de Neurotransmisores/química , Receptores de Neurotransmisores/efectos de los fármacos , Receptores de Neurotransmisores/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Peroxisome proliferators are a diverse group of chemicals, including several hypolipidaemic drugs, that activate a nuclear hormone receptor termed the peroxisome proliferator activated receptor (PPAR). The peroxisomal enzyme acyl CoA oxidase (ACO) is the most widely used marker of peroxisome proliferator action. We have examined the 5' flanking region of the rat ACO gene for sequences that mediate the transcriptional effect of peroxisome proliferators and have identified an element located 570 bp upstream of the ACO gene that confers responsiveness to the hypolipidaemic peroxisome proliferator Wy-14,643. This peroxisome proliferator response element (PPRE) contains a direct repeat of the sequence motifs TGACCT and TGTCCT and binds PPAR. These data therefore indicate an important role of PPAR in mediating the action of peroxisome proliferators including the induction of ACO.
Asunto(s)
Anticolesterolemiantes/farmacología , Ácido Graso Desaturasas/genética , Microcuerpos/efectos de los fármacos , Oxidorreductasas/genética , Regiones Promotoras Genéticas , Pirimidinas/farmacología , Acil-CoA Oxidasa , Animales , Baculoviridae/genética , Secuencia de Bases , Sitios de Unión , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Humanos , Cinética , Metilación , Microcuerpos/fisiología , Microcuerpos/ultraestructura , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , TransfecciónRESUMEN
During a prospective study of infectious gastroenteritis in children under 2 years, 19 out of 390 patients (4.9%) were found to have Clostridium difficile cytotoxin in the faeces. In several there was no history of use of antibiotics. The symptoms of many infants with toxin settled spontaneously, but one child became acutely and severely ill and developed a toxic megacolon and five others required, and responded to, vancomycin. Cl difficile was cultured from the stools in 191 (49%) of the children. The highly significant increased prevalence of past use of antibiotics in 118 control patients was not associated with an increased incidence of either isolation of Cl difficile or presence of faecal cytotoxin. Cl difficile should not be overlooked as a cause of acute diarrhoea and vomiting in children under 2 years.