RESUMEN
The alpha 7 nicotinic receptor is reportedly a key element in the cholinergic anti-inflammatory pathway. Because a prototypical ligand for this receptor is nicotine, we studied the in vivo human response to bacterial endotoxin or lipopolysaccharide (LPS) in the context of nicotine or placebo pretreatment. Twelve adult male normal subjects were studied prospectively. Six received overnight transcutaneous nicotine administration by application of a standard patch (7 mg). Six hours later, all subjects were given an intravenous dose of endotoxin (2 ng/kg) and were evaluated for an additional 24 h for circulating levels of inflammatory biomarkers, vital signs and symptoms. The nicotine subjects had elevated blood levels of the nicotine metabolite, continine, prior to and throughout the 24-h post-endotoxin exposure phase. Subjects receiving nicotine exhibited a significantly lower temperature response as well as attenuated cardiovascular responses for 2.5-6 h after LPS exposure. In addition, increased circulating interkeukin (IL)-10 and cortisol levels were also noted in nicotine subjects. These data indicate an alteration in LPS-induced systemic inflammatory responses in normal subjects exposed to transcutaneous nicotine. In this model of abbreviated inflammation, nicotine exposure attenuates the febrile response to LPS and promotes a more prominent anti-inflammatory phenotype.
Asunto(s)
Endotoxinas/farmacología , Lipopolisacáridos/farmacología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Administración Cutánea , Adolescente , Adulto , Análisis de Varianza , Biomarcadores/sangre , Cotinina/sangre , Citocinas/sangre , Selectina E/sangre , Humanos , Hidrocortisona/sangre , Inflamación , Masculino , Estudios ProspectivosRESUMEN
Insight into the mechanisms of action of neurotrophic growth factors has been obtained through the identification and characterization of gene products that are regulated or modified at the transcriptional, translational, and/or posttranslational level in response to neurotrophin treatment. VGF (non-acronymic) was identified approximately 15 years ago as a nerve growth factor (NGF)-regulated transcript in rat PC12 pheochromocytoma cells. Subsequent studies have demonstrated that neurotrophins such as NGF and brain-derived neurotrophic factor induce vgf gene expression relatively rapidly in PC12 cells and cultured cortical neurons, respectively, in comparison to less robust regulation by epidermal growth factor (EGF) and insulin, growth factors which do not trigger the neuronal differentiation of PC12 cells. vgf gene expression is stimulated in vitro by NGF and the ras/map kinase signaling cascade through a CREB-dependent mechanism, while in vivo, VGF mRNA levels are regulated by neuronal activity, including long-term potentiation, seizure, and injury. Both the mRNA and encoded approximately 68-kDa protein (VGF) are selectively synthesized in neuroendocrine and neuronal cells. The predicted VGF sequence is rich in paired basic amino acid residues that are potential sites for proteolytic processing, and VGF undergoes regulated release from dense core secretory vesicles. Although VGF mRNA is synthesized widely, by neurons in the brain, spinal cord, and peripheral nervous system, its expression is particularly abundant in the hypothalamus. In addition, VGF peptides are found in hypophysial, adrenal medullary, gastrointestinal, and pancreatic endocrine cells, suggesting important neuroendocrine functions. Recent analysis of VGF knockout mice indeed demonstrates that VGF plays a critical role in the control of energy homeostasis. VGF knockout mice are thin, small, hypermetabolic, hyperactive, and relatively infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic pro-opiomelanocortin, neuropeptide Y, and agouti-related peptide expression. Coupled with the demonstration that VGF mRNA levels are induced in the normal mouse hypothalamic arcuate nuclei in response to fasting, important central and peripheral roles for VGF in the regulation of metabolism are suggested. Here we review previous studies of VGF in the broader context of its newly recognized role in the control of energy balance and propose several models and experimental approaches that may better define the mechanisms of action of VGF.
Asunto(s)
Metabolismo Energético/fisiología , Neuronas/metabolismo , Sistemas Neurosecretores/metabolismo , Proteínas/fisiología , Secuencia de Aminoácidos/genética , Animales , Regulación de la Expresión Génica/fisiología , Humanos , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso , Neuropéptidos , Proteínas/genética , Proteínas/metabolismo , Distribución TisularRESUMEN
Platinum-based chemotherapies cause the formation of DNA adducts and have profound effects on DNA. This study measured cis-diamminedichloroplatinum II (cisplatin) DNA adducts by 32P-radiolabeling DNA, enzymatically digesting radiolabeled DNA, separating the formed adducts on two-dimensional thin-layer chromatography, and quantitating the adducts with autoradiography and densitometry. HeLa DNA was incubated with cisplatin at varying concentrations (6.25-325 nM) and times (0 min to 72 hr). Cisplatin rapidly depurinated dGMP and dAMP (90%, 15-min incubation with 325 nM cisplatin). Partial depurination of dGMP (15%) and dAMP (25%) occurred with lower cisplatin concentrations at equal incubation times. A minimum of four new adducts, with relatively rapid migratory patterns, were detected at high cisplatin concentrations with short incubation times. These results indicate that the depurination of DNA correlates with DNA adduct formation and that the quantification of these adducts may be applicable to monitoring tumor and host cell response to cisplatin chemotherapy.
Asunto(s)
Adenosina Monofosfato/metabolismo , Antineoplásicos/farmacología , Cisplatino/farmacología , Aductos de ADN/análisis , ADN de Neoplasias/efectos de los fármacos , Guanosina Monofosfato/metabolismo , Cromatografía en Capa Delgada , Relación Dosis-Respuesta a Droga , Células HeLa/efectos de los fármacos , Humanos , Radioisótopos de FósforoRESUMEN
A >15-fold increase in vasoactive intestinal polypeptide (VIP) mRNA and VIP peptide levels occurred in primary chromaffin cells following exposure to the neurotrophic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)-27 with an EC50 of approximately 2 nM. PACAP induction of VIP expression was blocked by methoxyverapamil or by a combination of nimodipine and omega-conotoxin MVIIC, indicating a requirement for PACAP-initiated calcium entry through voltage-dependent calcium channels for regulation of VIP biosynthesis. Ascomycin, which inhibits calcineurin through formation of an ascomycin/FKBP12/calcineurin ternary complex, abolished the PACAP-evoked increase in VIP expression, whereas rapamycin, which also binds to FKBP12 but does not cause inhibition of calcineurin, did not. Cyclosporin A, which inhibits calcineurin through formation of a cyclosporin A/cyclophilin/calcineurin complex, also abolished PACAP-evoked VIP biosynthesis. These data indicate that PACAP regulates the expression of VIP via a signaling pathway that requires calcium influx and activation of calcineurin.
Asunto(s)
Médula Suprarrenal/metabolismo , Calcineurina/metabolismo , Calcio/metabolismo , Células Cromafines/metabolismo , Neuropéptidos/farmacología , Neurotransmisores/farmacología , Transcripción Genética/fisiología , Péptido Intestinal Vasoactivo/genética , Médula Suprarrenal/citología , Animales , Bovinos , Células Cultivadas , Células Cromafines/citología , Activación Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , ARN Mensajero/genética , Transcripción Genética/efectos de los fármacosRESUMEN
To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.
Asunto(s)
Metabolismo Energético/fisiología , Eliminación de Gen , Neuronas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Agresión/fisiología , Animales , Núcleo Arqueado del Hipotálamo/química , Núcleo Arqueado del Hipotálamo/citología , Núcleo Arqueado del Hipotálamo/metabolismo , Peso Corporal/fisiología , Catecolaminas/metabolismo , Ritmo Circadiano/fisiología , Ayuno/fisiología , Femenino , Fertilidad , Expresión Génica/fisiología , Gonadotropinas/metabolismo , Homeostasis/fisiología , Hibridación in Situ , Leptina , Masculino , Glándulas Mamarias Animales/química , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Crecimiento Nervioso , Neuronas/química , Neuropéptidos , Ovario/química , Ovario/metabolismo , Consumo de Oxígeno/fisiología , Fenotipo , Hipófisis/química , Hipófisis/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , ARN Mensajero/análisis , Tirotropina/genéticaRESUMEN
Vasoactive intestinal peptide (VIP) gene expression is highly restricted throughout the neuroaxis and regulated by extracellular factors that activate tyrosine- or serine/threonine-directed protein kinase pathways. Cytokine, cyclic AMP, and tissue-specific response elements on the VIP gene have been characterized. Those mediating responsiveness to protein kinase C have not. The endogenous VIP gene and a 5.2-kilobase pair (kb) VIP-luciferase reporter gene, are up-regulated by phorbol 12-myristate 13-acetate (PMA) in SK-N-SH neuroblastoma cells. PMA stimulation was abolished by deletion of sequences at -1.37 to -1.28 or -1.28 to -0.904 kb, but not by removal of the single phorbol ester response element (TRE; TGACTCA) located at -2.25 kb. Mutation of sites at -1.32 or -1.20 that mediate neurotrophin responsiveness of the VIP gene (Symes, A., Lewis, S., Corpus, L., Rajan, P., Hyman, S. E., and Fink, J. S. (1994) Mol. Endocrinol. 8, 1750-1763) each reduced PMA induction in SK-N-SH cells by >50%, and double mutation abolished it. The two mutations also reduced basal VIP reporter gene transcription in SH-EP neuroblastoma cells expressing VIP constitutively. Both cis-active elements bound pre-existing AP-1 proteins in SH-EP- or PMA-stimulated SK-N-SH cell nuclear extracts. The AP-1 complex at both sites contained a Fos-related protein with c-Jun in SH-EP cells and c-Fos with a Jun-related protein in SK-N-SH cells. Recruitment of combinatorially distinct AP-1 complexes to these elements may underlie cell type-specific regulation of the VIP gene.
Asunto(s)
Regulación de la Expresión Génica , Factor de Transcripción AP-1/genética , Transcripción Genética , Péptido Intestinal Vasoactivo/genética , Animales , Sitios de Unión/genética , Elementos Transponibles de ADN/genética , Mutación , Unión Proteica , Factor de Transcripción AP-1/metabolismo , Células Tumorales Cultivadas , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Pituitary adenylate cyclase activating polypeptide-27 (PACAP-27) caused a dose-dependent increase in met-enkephalin secretion and increased production of met-enkephalin peptide and proenkephalin A (PEnk) mRNA in bovine chromaffin cells, at concentrations as low as 300 pM. PACAP-38 was less potent than PACAP-27, but had similar effects. Vasoactive intestinal polypeptide (VIP) (1-100 nM) was without appreciable effect on either enkephalin secretion or biosynthesis, implicating PACAP type I receptors in PACAP-stimulated enkephalin secretion and synthesis. PACAP type I receptors can activate adenylate cyclase and stimulate phospholipase C through heterotrimeric G protein interactions, leading to increased intracellular cyclic AMP (cAMP), inositol triphosphate (IP3)-mediated calcium mobilization, and calcium- and diacylglycerol (DAG)-mediated protein kinase C (PKC) activation. Enkephalin secretion evoked by 10-100 nM PACAP-27 was not inhibited by 1 microM (-)-202-791, an L-type specific dihydropyridine calcium channel blocker, but was inhibited 65-80% by the arylalkylamine calcium channel blocker D600. Forty mM potassium-evoked secretion was inhibited > 90% by both D600 and (-)-202-791, 25 microM forskolin-induced secretion was blocked < 50% by D600 and was unaffected by (-)-202-791, and 100 nM phorbol myristate acetate (PMA)-induced secretion was unaffected by either D600 or (-)-202-791. Enkephalin biosynthesis was increased by 10 nM PACAP-27, as measured by increased met-enkephalin pentapeptide content and PEnk A mRNA levels. PACAP-, forskolin-, and PMA-stimulated enkephalin synthesis were not blocked by D600 or (-)-202-791. Elevated potassium-induced enkephalin biosynthesis upregulation was completely blocked by either D600 or (-)-202-791 at the same concentrations. PACAP acting through type I PACAP receptors couples calcium influx-dependent enkephalin secretion and calcium influx-independent enkephalin biosynthesis in chromaffin cells. Restriction of the effects of enhanced calcium influx to stimulation of secretion, but not of biosynthesis, is unique to PACAP. By contrast, potassium-induced enkephalin biosynthesis upregulation is completely calcium influx dependent, specifically via calcium influx through L-type calcium channels. We propose that subpopulations of voltage-dependent calcium channels are differentially linked to intracellular signal transduction pathways that control neuropeptide gene expression and secretion in chromaffin cells.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Células Cromafines/efectos de los fármacos , Encefalina Metionina/metabolismo , Neuropéptidos/farmacología , Glándulas Suprarrenales/citología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Canales de Calcio Tipo L , Bovinos , Células Cromafines/metabolismo , Colforsina/farmacología , AMP Cíclico/fisiología , Encefalina Metionina/biosíntesis , Encefalina Metionina/genética , Encefalinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Modelos Biológicos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Potasio/farmacología , Precursores de Proteínas/genética , Radioinmunoensayo , Acetato de Tetradecanoilforbol/farmacología , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Gliotoxin is produced by the fungus Aspergillus fumigatus. Aspergillus is widespread in the environment and this ubiquitous nature results in disease and co-carcinogenesis to be distributed world-wide. Gliotoxin contains an epipolythiodioxopiperazine (ETP) ring that is believed to be involved in redox reactions. The reactive oxygen species produced interact with DNA to form hydroxylated and other altered DNA products. To measure DNA adduct formation, we used 32P radiolabelling and, after enzymatic DNA digestion, separated adducts in two dimensions using thin-layer chromatography (2D-TLC), with ultimate autoradiography and densitometry. HeLa DNA was incubated with 0.1 mmol l-1 and 0.3 mmol l-1 of gliotoxin (and necessary redox agents) for 1 and 20 h. We found an increase in 6-hydro-5,6-dihydroxythymidine (thymine glycol) monophosphate [d(TG)MP] from 0.0% to 30.4%, an increase in 8-hydroxy-2'-deoxyguanidine monophosphate [8(OH)dGMP] from 0.0% to 4.2%, an increase in deoxynucleotide diphosphate (dNDP) from zero adducts to six DNA adducts, as well as an increase of other as yet unidentified adducts. Also, time exposure may have a greater effect than concentration based on a 20-h incubation with 0.3 mmol l-1 gliotoxin that completely obliterates the pyrimidines deoxythymidine 3'-monophosphate (dTMP) and deoxycytidine 3'-monophosphate (dCMP).
Asunto(s)
Aspergillus fumigatus/química , Aductos de ADN/análisis , Daño del ADN , Gliotoxina/farmacología , Cromatografía en Capa Delgada , ADN de Neoplasias/efectos de los fármacos , Células HeLa , Humanos , Exposición Profesional , Oxidación-Reducción , Radioisótopos de Fósforo , Especies Reactivas de OxígenoRESUMEN
Vasoactive intestinal peptide (VIP) is a neuromodulator expressed with great anatomical specificity throughout the nervous system. Cell-specific expression of the VIP gene is mediated by a tissue specifier element (TSE) located within a 2.7-kilobase (kb) region between -5.2 and -2.5 kb upstream from the transcription start site, and requires an intact promoter proximal VIP-CRE (cyclic AMP-responsive element) (Hahm, S. H., and Eiden, L. E. (1997) J. Neurochem. 67, 1872-1881). We now report that the TSE comprises a 425-base pair domain located between -4.7 and -4.2 kb containing two AT-rich octamer-like sequences. The 425-base pair TSE is sufficient to provide full cell-specific regulation of the VIP gene, when fused to the 5' proximal 1.55 kb of the VIP gene. Mutational analysis and gel shift assays of these octamer-like sequences indicate that the binding of proteins related to the ubiquitously expressed POU-homeodomain proteins Oct-1 and/or Oct-2 to these octamer-like sequences plays a central role for the function of the TSE. The TSE interacts with three additional discrete domains besides the cAMP response element, which are located within the proximal 1.55 kb of the VIP gene, to provide cell-specific expression. An upstream domain from -1.55 to -1.37 kb contains E-boxes and MEF2-like motifs, and deletion of this domain results in complete abrogation of cell-specific transcriptional activity. The region from -1.37 to -1. 28 kb contains a STAT motif, and further removal of this domain allows the upstream TSE to act as an enhancer in both SH-EP and HeLa cells. The sequence from -1.28 to -0.9 kb containing a non-canonical AP-1 binding sequence (Symes, A., Gearan, T., Eby, J., and Fink, J. S. (1997) J. Biol. Chem. 272, 9648-9654), is absolutely required for TSE-dependent cellspecific expression of the VIP gene. Thus, five discrete domains of the VIP gene provide a combination of enhancer and repressor activities, each completely contingent on VIP gene context, that together result in cell-specific transcription of the VIP gene.
Asunto(s)
Transcripción Genética , Péptido Intestinal Vasoactivo/genética , Secuencia de Bases , ADN , Genes Reporteros , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Células Tumorales CultivadasAsunto(s)
Calcio/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Neuropéptidos/biosíntesis , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Factor de Transcripción Activador 2 , Animales , Secuencia de Bases , Sitios de Unión , Cromogranina A , Cromograninas/biosíntesis , Cicloheximida/farmacología , Humanos , Péptido Intestinal Vasoactivo/biosíntesisRESUMEN
The cis-acting elements of the VIP gene important for basal and stimulated transcription have been studied by transfection of VIP-reporter gene constructs into distinct human neuroblastoma cell lines in which VIP transcription is constitutively high, or can be induced to high levels by protein kinase stimulation. The 5.2 kb flanking sequence of the VIP gene conferring correct basal and inducible VIP gene expression onto a reporter gene in these cell lines was systematically deleted to define its minimal components. A 425-bp fragment (-4656 to -4231) fused to the proximal 1.55 kb of the VIP promoter-enhancer was absolutely required for cell-specific basal and inducible transcription. Four additional components of the VIP gene were required for full cell-specific expression driven by the 425 bp TSE (region A). Sequences from -1.55 to -1.37 (region B), -1.37 to -1.28 (region C), -1.28 to -.094 (region D), and the CRE-containing proximal 94 bp (region E) were deleted in various combinations to demonstrate the specific contributions of each region to correct basal and inducible VIP gene expression. Deletion of region B, or mutational inactivation of the CRE in region E, resulted in constructs with low transcriptional activity in VIP-expressing cell lines. Deletion of regions B and C together resulted in a gain of transcriptional activity, but without cell specificity. All five domains of the VIP gene were also required for cell-specific induction of VIP gene expression with phorbol ester. Gelshift analysis of putative regulatory sequences in regions A-D suggests that both ubiquitous and neuron-specific trans-acting proteins participate in VIP gene regulation.
Asunto(s)
Regulación de la Expresión Génica , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Péptido Intestinal Vasoactivo/genética , Animales , Secuencia de Bases , Línea Celular , Elementos de Facilitación Genéticos , Humanos , Neuropéptidos/farmacología , Neuropéptidos/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Péptido Intestinal Vasoactivo/biosíntesisRESUMEN
The 5' flanking region of the human VAChT gene was sequenced to approx 5350 bases upstream of the initiating methionine codon of the VAChT open reading frame (orf). The 5' flanks of the human and rat cholinergic gene loci were compared to identify regions of local sequence conservation, and therefore of potential regulatory importance. Several discrete domains of high homology, including a cluster of far-upstream cis-active consensus motifs, a neuronally restrictive silencer element consensus sequence, and additional conserved sequences within the putative nerve growth factor response domain of the locus, were identified. The probable start of transcription of the VAChT gene was deduced from mapping of sequences of rat and human VAChT cDNAs onto the 5' flanking regions of the human and rat cholinergic gene loci. The actual utilization of a putative 5' VAChT exon in rat central nervous system (CNS) tissue was assessed by in situ hybridization histochemistry. RNA transcripts containing both VAChT and ChAT protein-coding sequences were abundant in spinal cord motoneurons, sympathetic preganglionic cells, basal forebrain, striatum, and cranial motor nuclei. R-exon-containing transcripts could be detected only at low levels in these cell groups, implying that most transcription of VAChT proceeds from a promoter downstream of the R-exon. To assess the structural requirements for expression of the VAChT gene without bias regarding the actual start of transcription, a 5' fragment of the human gene corresponding to approximately 3 kb of sequence extending upstream from within the presumed 5' untranslated region of VAChT itself was fused to a luciferase-encoding reporter and transfected into VAChT-expressing and nonexpressing human and rat cell lines. This portion of the VAChT gene provided strong promoter expression in both cholinergic and noncholinergic cell lines. Deletion of the putative neuronally restrictive silencer element (NRSE) resulted in enhanced transcription in all cell lines. Lack of differential expression of VAChT transcription in VAChT-expressing vs non-VAChT-expressing cell lines suggested that additional enhancer elements controlling cell-specific expression of the VAChT gene exist further upstream in the cholinergic locus 5' flank. Conservation of potential cis-active elements within a 1.4 kb sequence immediately upstream of the NRSE in both rat and human cholinergic gene loci suggests that this domain is required for cholinergic-specific regulation of VAChT and ChAT gene transcription.
Asunto(s)
Acetilcolina/genética , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas de Transporte de Membrana , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Proteínas de Transporte Vesicular , Animales , Secuencia de Bases , Northern Blotting , Proteínas Portadoras/química , Línea Celular , Genes Reporteros , Humanos , Datos de Secuencia Molecular , Nematodos , Reacción en Cadena de la Polimerasa , Ratas , Análisis de Secuencia de ADN , Transfección , Proteínas de Transporte Vesicular de AcetilcolinaRESUMEN
An upstream enhancer element [tissue specifier element (TSE)] located between 4.66 and 4.02 kb from the transcription start site is important for cell type-specific expression and phorbol ester induction of the vasoactive intestinal peptide (VIP) gene. An element located within 100 bases of the VIP promoter [the VIP cyclic AMP-responsive element (VIP-CRE)] confers cyclic AMP and phorbol ester responsiveness to heterologous promoters. The possibility that these two regions of the VIP gene function cooperatively to determine tissue-specific and second messenger-dependent expression of the VIP gene was addressed by assaying transcription from a VIP-luciferase reporter gene with progressive deletions from the 5' flanking sequence of the gene, with or without inactivation of the proximal VIP-CRE. Basal expression of the reporter gene in both SH-EP and SK-N-SH human neuroblastoma cells, which express endogenous VIP mRNA, was absolutely dependent on the presence of the upstream TSE. Full constitutive expression was also dependent on the intact VIP-CRE. Forskolin-mediated induction of the reporter gene in SH-EP and SK-N-SH cells was completely abolished by mutations in the VIP-CRE but not by deletion of the upstream sequence, indicating that the VIP-CRE alone determines cyclic AMP responsiveness. In contrast to reports that the VIP-CRE imparts 12-O-tetradecanoylphorbol 13-acetate (phorbol 12-myristate 13-acetate; PMA) responsiveness to heterologous promoters, PMA stimulation in SK-N-SH cells was independent of an intact VIP-CRE but dependent on a region between -2.5 kb and the VIP-CRE. Sequencing of the entire 5.2-kb VIP 5' flank revealed a consensus PMA-responsive element (TGACTCA) 2.25 kb upstream of the transcription start site that may represent the site imparting PMA responsiveness to the VIP gene.
Asunto(s)
AMP Cíclico/metabolismo , Regulación Neoplásica de la Expresión Génica , Regiones Promotoras Genéticas , Péptido Intestinal Vasoactivo/biosíntesis , Animales , Secuencia de Bases , Cartilla de ADN , Genes Reporteros , Células HeLa , Humanos , Luciferasas/biosíntesis , Datos de Secuencia Molecular , Neuroblastoma , Especificidad de Órganos , Células PC12 , Reacción en Cadena de la Polimerasa , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Mapeo Restrictivo , Transfección , Células Tumorales Cultivadas , Péptido Intestinal Vasoactivo/genéticaRESUMEN
A novel photoinduced electron-transfer reaction is reported in complexes between resting ferric state cytochrome c peroxidase (CcP) and several horse cytochrome c derivatives labeled at single lysine amino groups with [bis(bipyridine)](dicarboxybipyridine)ruthenium(II) (Ru-CC). Photoexcitation of Ru(II) in the 1:1 Ru-27-CC:CcP complex results in formation of a metal-to-ligand charge-transfer state, Ru(II*), which is a strong reducing agent and rapidly transfers an electron to the CC heme Fe(III) with rate constant k1 = 2.3 x 10(7) s-1. The resulting Ru(III) is a strong oxidizing agent with a redox potential of 1.3 V, and it oxidizes the indole ring of Trp-191 with rate constant k3 = 7 x 10(6) s-1. The cycle is completed by electron transfer from Fe(II) in CC to the Trp-191 radical in CcP with rate constant k4 = 6.1 x 10(4) s-1. The Ru group is located close to the interaction domain in the Ru-27-CC:CcP complex, allowing rapid electron transfer with both the heme in CC and Trp-191 in CcP. The electron-transfer reaction was not observed in CcP compound I, where Trp-191 is already oxidized to the radical, or in the W191F mutant, where the indole group is replaced with a phenyl group. The electron-transfer reaction was observed in CcP mutants modified at residues in the heme crevice, R48K, R48L, H52L, M230I, and M231I, but not in D235N which destabilizes the radical on Trp-191. Increasing the ionic strength results in an increase in the equilibrium dissociation constant K of the Ru-27-CC:CcP complex and an increase in the rate constant k5 for dissociation of the transient intermediate containing Fe(II) CC and the radical form of CcP. Both K and k5 were also increased significantly by the mutations D34N, E290N, and A193F involving residues located in the interaction domain of the crystalline complex between yeast CC and CcP [Pelletier & Kraut (1992) Science 258, 1748-1755]. This new method allows the study of the electron-transfer reaction between CC and the radical on Trp-191 in the complete absence of hydrogen peroxide, and it opens the possibility of measurements at low temperatures in frozen glasses or in crystals.
Asunto(s)
Grupo Citocromo c/química , Citocromo-c Peroxidasa/química , Rutenio/química , Animales , Caballos , Técnicas In Vitro , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Concentración Osmolar , Oxidación-Reducción , Fotoquímica , Fotólisis , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Triptófano/químicaRESUMEN
The kinetics of electron transfer from cytochrome c (CC) to yeast cytochrome c peroxidase (CcP) compound I were studied by flash photolysis and stopped-flow spectroscopy. Flash photolysis studies employed horse CC derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis-(bipyridine)ruthenium (Ru-CC). Initial electron transfer from Ru-CC reduced the indole radical on Trp-191 of CcP compound I [CMPI(IV,R.)], producing CMPII(IV,R). This reaction was biphasic for each of several Ru-CC derivatives, with rate constants which varied according to the position of the Ru label. For Ru-27-CC labeled at lysine 27, rate constants of 43,000 and 1600 s-1 were observed at pH 5.0 in 2 mM acetate. After reduction of the indole radical by Ru-CC, intramolecular electron transfer from Trp-191 to the oxyferryl heme in CMPII(IV,R) was observed, producing CMPII(III,R.). The rate constant and extent of this intramolecular electron transfer reaction were independent of both the protein concentration and the Ru-CC derivative employed. The rate constant decreased from 1100 s-1 at pH 5 to 550 s-1 at pH 6, while the extent of conversion of CMPII(IV,R) to CMPII(III,R.) decreased from 56% at pH 5 to 29% at pH 6. The reaction was not detected at pH 7.0 and above. The pH dependence of the rate and extent of this internal electron transfer reaction paralleled the pH dependence of the rate of bimolecular reduction of CMPII(IV,R) by native horse CC measured by stopped-flow spectroscopy at high ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Citocromo-c Peroxidasa/química , Animales , Cristalografía por Rayos X , Grupo Citocromo c/química , Compuestos Férricos/química , Proteínas Fúngicas/química , Caballos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Metionina/química , Oxidación-Reducción , Fotólisis , Triptófano/químicaRESUMEN
Site-directed mutants of cytochrome c peroxidase (CcP) were created to modify the interaction domain between CcP and yeast iso-1-cytochrome c (yCC) seen in the crystal structure of the CcP-yCC complex [Pelletier & Kraut (1992) Science 258, 1748-1755]. In the crystalline CcP-yCC complex, two acidic regions of CcP contact lysine residues on yCC. Mutants E32Q, D34N, E35Q, E290N, and E291Q were used to examine the effect of converting individual carboxylate side chains in the acidic regions to amides. The A193F mutant was used to test the effect of introducing a phenyl moiety at the point of closest contact between CcP and yCC in the crystal structure. Stopped-flow experiments carried out in 310 mM ionic strength buffer at pH 7 revealed that yCC initially reduced the indole radical on Trp-191 of the parent CcP compound I with a bimolecular rate constant ka = 2.5 x 10(8) M-1 s-1. A second molecule of yCC subsequently reduced the oxyferryl heme of compound II with a rate constant kb = 5 x 10(7) M-1 s-1. The bimolecular rate constants ka and kb were affected in parallel by each mutation examined. CcP mutants D34N and E290N that are closest to a complementary yCC lysine residue in the crystalline CcP-yCC complex gave the lowest values for ka and kb, which were 25-50% of the values of the CcP parent. Mutants E32Q and E291Q that are removed from the interaction domain gave the same ka and kb values as the CcP parent.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Grupo Citocromo c/química , Citocromo-c Peroxidasa/química , Animales , Cristalografía por Rayos X , Compuestos Férricos/química , Radicales Libres , Proteínas Fúngicas/química , Hemo/química , Caballos , Cinética , Oxidación-Reducción , Estructura Terciaria de Proteína , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
The reactions of recombinant cytochrome c peroxidase [CcP(MI)] and a number of CcP(MI) mutants with native and ruthenium-labeled horse ferrocytochrome c have been studied by stopped-flow spectroscopy and laser flash photolysis. At 100 mM ionic strength, pH 7.5, native horse ferrocytochrome c reduces the radical on the indole group of Trp-191 in cytochrome c peroxidase compound I (CMPI) with a second-order rate constant of 1.3 x 10(8) M-1 s-1. Ferrocytochrome c then reduces the oxyferryl heme Fe(IV) in CMPII with a rate constant of 2.0 x 10(6) M-1 s-1. The rate constant for the reduction of the radical is nearly independent of pH from 5 to 8, but the rate constant for reduction of the oxyferryl heme Fe(IV) increases 33-fold as the pH is decreased from 8 to 5. This increase in rate is correlated with the pH dependence of the electron transfer equilibrium between the radical and the oxyferryl heme Fe(IV) in the transient form of CMPII. The second-order rate constants for reduction of the radical and the oxyferryl heme in the mutants Y39F, Y42F, H181G, W223F, and Y229F are nearly the same as for wild-type CcP(MI). The intracomplex rate constants for reduction of the radical in these mutants by the ruthenium-labeled cytochrome c derivatives are also similar to that for CcP(MI). This rules out a direct role for these aromatic residues in electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Grupo Citocromo c/metabolismo , Citocromo-c Peroxidasa/metabolismo , Hemo/metabolismo , Animales , Radicales Libres , Caballos , Concentración de Iones de Hidrógeno , Cinética , Concentración Osmolar , Oxidación-Reducción , Fotólisis , Proteínas Recombinantes/metabolismo , Triptófano/metabolismoRESUMEN
Transforming growth factor-beta 1 (TGF-beta 1) rapidly activated phosphorylase in isolated rat hepatocytes (half-maximal rate of activation with approximately 0.1 ng/ml). Removal of Ca2+ from the external medium just before TGF-beta 1 addition markedly attenuated phosphorylase activation. TGF-beta 1 (1 ng/ml) produced a small increase in [Ca2+]i (approximately 10% increase after 30 s), which appears sufficient to account for phosphorylase activation. These observations indicate that activation of the TGF-beta 1 signal transduction system in hepatocytes is linked with a small increase in [Ca2+]i, and external Ca2+ may contribute in part to this increase.
Asunto(s)
Calcio/metabolismo , Hígado/enzimología , Fosforilasa a/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Calcio/farmacología , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Femenino , Hígado/citología , Fenilefrina/farmacología , Fosforilasa a/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
The reactions of yeast cytochrome c peroxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) [Ru(II)] were studied by flash photolysis. All of the derivatives formed complexes with cytochrome c peroxidase compound I (CMPI) at low ionic strength (2 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in electron transfer to the ferric heme group in cytochrome c, followed by electron transfer to the radical site in CMPI. This reaction was biphasic and the rate constants were independent of CMPI concentration, indicating that both phases represented intracomplex electron transfer from the cytochrome c heme to the radical site in CMPI. The rate constants of the fast phase were 5200, 19,000, 55,000, and 14,300 s-1 for the derivatives modified at lysines 13, 25, 27, and 72, respectively. The rate constants of the slow phase were 260, 520, 200, and 350 s-1 for the same derivatives. These results suggest that there are two binding orientations for cytochrome c on CMPI. The binding orientation responsible for the fast phase involves a geometry that supports rapid electron transfer, while that for the slow phase allows only slow electron transfer. Increasing the ionic strength up to 40 mM increased the rate constant of the slow phase and decreased that of the fast phase. A single intracomplex electron transfer phase with a rate constant of 2800 s-1 was observed for the lysine 72 derivative at this ionic strength. When a series of light flashes was used to titrate CMPI to CMPII, the reaction between the cytochrome c derivative and the Fe(IV) site in CMPII was observed. The rate constants for this reaction were 110, 250, 350, and 140 s-1 for the above derivatives measured in low ionic strength buffer.