RESUMEN
Cocaine- and amphetamine-regulated transcript (CART) is a neurotransmitter and hormone, involved in the regulation of e.g. food intake, body weight, reward and addiction, and stress response. CART has also been found to affect insulin secretion and beta cell morphology, both in vivo and in vitro. Furthermore, CART affects regulation of the cardiovascular system and helps to modulate vascular tone. The present study evaluated the local effect of CART on the pancreatic and islet circulation and function. CART (25 µg/h) or saline, combinations of CART and endothelin-A receptor antagonist (BQ123; 100 µg/kg), and glucose (2 g/kg) were intravenously infused in Sprague Dawley rats followed by blood flow measurements using a microsphere technique. Separately, CART-infused animals underwent an intravenous glucose tolerance test (ivGTT). The direct effect of CART on insulin release was investigated using isolated islets from Sprague Dawley rats. CART reduced islet blood flow, without reduction in total pancreatic blood flow. The normal glucose-induced islet blood flow increase was diminished by CART, albeit still present. Simultaneously, CART had no effect on systemic-, intestinal- or renal blood flow. The endothelin-A receptor antagonist BQ123 together with CART had no pancreatic vascular effects. We found that CART has pronounced vascular constrictive actions restricted to the pancreatic islet circulation but had no effect on insulin release neither in vivo nor in vitro. The mechanisms behind the vascular effects are still unknown, but may reflect a direct action on pancreatic blood vessels.
Asunto(s)
Aminoácidos/genética , Secreción de Insulina/genética , Islotes Pancreáticos/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Ácidos Nicotínicos/genética , Receptor de Endotelina A/genética , Anestésicos/farmacología , Animales , Glucemia/efectos de los fármacos , Glucemia/genética , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Cromo , Antagonistas de los Receptores de la Endotelina A/farmacología , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/genética , Insulina/metabolismo , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/irrigación sanguínea , Islotes Pancreáticos/metabolismo , Masculino , Páncreas/irrigación sanguínea , Páncreas/efectos de los fármacos , Péptidos Cíclicos/farmacología , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/efectos de los fármacosRESUMEN
The genetic mechanisms that determine the size of the adult pancreas are poorly understood. Imprinted genes, which are expressed in a parent-of-origin-specific manner, are known to have important roles in development, growth and metabolism. However, our knowledge regarding their roles in the control of pancreatic growth and function remains limited. Here we show that many imprinted genes are highly expressed in pancreatic mesenchyme-derived cells and explore the role of the paternally-expressed insulin-like growth factor 2 (Igf2) gene in mesenchymal and epithelial pancreatic lineages using a newly developed conditional Igf2 mouse model. Mesenchyme-specific Igf2 deletion results in acinar and beta-cell hypoplasia, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy, suggesting that the mesenchyme is a developmental reservoir of IGF2 used for paracrine signalling. The unique actions of mesenchymal IGF2 are demonstrated by the absence of any discernible growth or functional phenotypes upon Igf2 deletion in the developing pancreatic epithelium. Additionally, increased IGF2 levels specifically in the mesenchyme, through conditional Igf2 loss-of-imprinting or Igf2r deletion, leads to pancreatic acinar overgrowth. Furthermore, ex-vivo exposure of primary acinar cells to exogenous IGF2 activates AKT, a key signalling node, and increases their number and amylase production. Based on these findings, we propose that mesenchymal Igf2, and perhaps other imprinted genes, are key developmental regulators of adult pancreas size and function.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/genética , Mesodermo/crecimiento & desarrollo , Páncreas/crecimiento & desarrollo , Comunicación Paracrina/genética , Células Acinares/metabolismo , Células Acinares/patología , Aminoácidos/genética , Animales , Linaje de la Célula/genética , Cromo , Metilación de ADN/genética , Femenino , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica/genética , Impresión Genómica/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Ratones , Ácidos Nicotínicos/genética , Páncreas/citología , Páncreas/metabolismo , Embarazo , ARN Largo no Codificante/genéticaRESUMEN
Poly(ADP-ribosyl)ation is a posttranslational modification of proteins, which is mainly catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) by using NAD(+) as substrate and is directly triggered by DNA strand breaks. Under mild genotoxic stress poly(ADP-ribose) (PAR) formation plays an important role in DNA repair whereas severe genotoxic stress and the ensuing overactivation of PARP-1 induce cellular NAD(+) depletion, energy failure and ultimately cell death. We are interested in studying the consequences of moderately enhanced enzymatic activity under conditions of DNA damage. Here we chose supplementation of cells with the NAD(+) precursor nicotinic acid (NA) as a strategy. In order to reliably assess PAR accumulation in living cells we first developed a novel, sensitive flow-cytometric method for the rapid analysis of poly(ADP-ribose) accumulation (RAPARA). Our data showed that ex vivo supplementation of human peripheral blood mononuclear cells (PBMC) with low concentrations of NA significantly raised cellular NAD(+) levels by 2.1-fold. Upon X-irradiation or exposure to hydrogen peroxide or N-methyl-N'-nitro-N-nitrosoguanidine, PAR accumulation was significantly increased and sustained in NA-supplemented cells. Furthermore, NA-supplemented PBMC displayed significantly higher cell viability due to a lower rate of necrotic cell death. In summary, ex vivo supplementation of human PBMC with NA increases cellular NAD(+) levels, boosts the cellular poly(ADP-ribosyl)ation response to genotoxic treatment, and protects from DNA-damage-induced cell death.
Asunto(s)
Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Adenosina Difosfato Ribosa/genética , Adenosina Difosfato Ribosa/metabolismo , Adulto , Células Sanguíneas/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células/metabolismo , Daño del ADN , Reparación del ADN/efectos de los fármacos , Femenino , Humanos , Masculino , Metilnitronitrosoguanidina/farmacología , Persona de Mediana Edad , NAD/genética , NAD/metabolismo , Neutrófilos/metabolismo , Niacina , Ácidos Nicotínicos/genética , Poli Adenosina Difosfato Ribosa/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
A vitamin B(6) degradative pathway has recently been identified and characterized in Mesorhizobium loti MAFF303099. One of the enzymes on this pathway, 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO), is a flavin-dependent enzyme and catalyzes the oxidative ring-opening of 2-methyl-3-hydroxypyridine-5-carboxylic acid to form E-2-(acetamino-methylene)succinate. The gene for this enzyme has been cloned, and the corresponding protein has been overexpressed in Escherichia coli and purified. The crystal structure of MHPCO has been solved to 2.1 A using SAD phasing with and without the substrate MHPC bound. These crystal structures provide insight into the reaction mechanism and suggest roles for active site residues in the catalysis of a novel oxidative ring-opening reaction.
Asunto(s)
Alphaproteobacteria/enzimología , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Ácidos Nicotínicos/química , Ácidos Nicotínicos/metabolismo , Sitios de Unión/genética , Catálisis , Clonación Molecular , Cristalografía por Rayos X , Dimerización , Escherichia coli/genética , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Histidina/química , Enlace de Hidrógeno , Ligandos , Oxigenasas de Función Mixta/genética , Modelos Moleculares , Ácidos Nicotínicos/genética , Ácidos Nicotínicos/aislamiento & purificación , Oxidación-Reducción , Unión Proteica/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato/genética , UltracentrifugaciónRESUMEN
The M4 muscarinic acetylcholine (ACh) receptor (mAChR) is a potential therapeutic target but characterized by a lack of subtype-selective ligands. We recently generated "designer receptors exclusively activated by a designer drug" (DREADDs), which contained mutations of two conserved orthosteric-site residues (Y113C/A203G in the M4 mAChR) that caused a loss of ACh activity but a gain in responsiveness to clozapine-N-oxide (CNO). The current study characterized the interactions of the wild type and the M4 DREADD with a range of agonists, antagonists, and the recently discovered M4 mAChR allosteric potentiator, 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298). LY2033298 displayed positive binding cooperativity with ACh, neutral cooperativity with the antagonist, [3H]quinuclidinyl benzilate, and agonism for activation of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 at the wild-type M4 mAChR. LY2033298's cooperativity with clozapine or CNO was weakly positive with respect to binding but profoundly negative with respect to LY2033298 signaling. Although the DREADD mutations increased the binding and function of clozapine-like compounds, all other agonists lost the ability to activate the mutant; for the orthosteric agonists ACh and pilocarpine, this was due partly to a reduced affinity, whereas the affinity of LY2033298 or the atypical agonist 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride was unaltered. The interaction between LY2033298 and clozapine-like compounds reverted to neutral cooperativity on the DREADD, whereas LY2033298 caused a striking functional rescue of ACh potency and efficacy at the DREADD. These results provide conclusive evidence for the retention of a functional allosteric site on the M4 DREADD and highlight a role for residues Tyr113 and Ala203 in the transmission of cooperativity.
Asunto(s)
Ácidos Nicotínicos/metabolismo , Ácidos Nicotínicos/farmacología , Receptor Muscarínico M4/fisiología , Tiofenos/metabolismo , Tiofenos/farmacología , Acetilcolina/química , Acetilcolina/metabolismo , Acetilcolina/farmacología , Regulación Alostérica/fisiología , Sitio Alostérico/fisiología , Animales , Células CHO , Clozapina/análogos & derivados , Clozapina/química , Clozapina/metabolismo , Clozapina/farmacología , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Biológicos , Estructura Molecular , Mutación , Ácidos Nicotínicos/química , Ácidos Nicotínicos/genética , Fosforilación/efectos de los fármacos , Quinuclidinil Bencilato/metabolismo , Quinuclidinil Bencilato/farmacología , Ensayo de Unión Radioligante , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inhibidores , Transducción de Señal , Tiofenos/químicaRESUMEN
The orphan receptor ChemR23 is a G-protein coupled receptor (GPCR) with homology to neuropeptide and chemoattractant receptors. Tazarotene, a synthetic retinoid activating retinoic acid receptor (RAR), up-regulates tazarotene-induced gene-2 (TIG2). The function and molecular target of this protein are now described. By means of reverse pharmacology screening using a peptide library generated from human hemofiltrate, we have isolated and identified TIG2 as the natural ligand of ChemR23 and report the specific molecular form of the bioactive, circulating TIG2, representing the amino-acid residues 21 to 154 of the 163 amino acid-containing prepropeptide. Based on the expression pattern of ChemR23 and TIG2, the physiological role in bone development, immune and inflammatory responses and the maintenance of skin is now being investigated.
Asunto(s)
Ácidos Nicotínicos/genética , Receptores de Quimiocina/metabolismo , Receptores de Ácido Retinoico/sangre , Receptores de Ácido Retinoico/genética , Secuencia de Aminoácidos , Animales , Células CHO , Calcio/análisis , Calcio/metabolismo , Cricetinae , Fluorometría/métodos , Hemofiltración , Humanos , Ligandos , Datos de Secuencia Molecular , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/química , Receptores de Quimiocina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , TransfecciónAsunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , Ácidos Nicotínicos/genética , Hiperplasia Prostática/genética , Neoplasias de la Próstata/genética , Proteínas/genética , Animales , Perfilación de la Expresión Génica , Genes Supresores de Tumor , Humanos , Masculino , Proteínas de la Membrana , Proteínas de Neoplasias/metabolismo , Ácidos Nicotínicos/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , ARN Mensajero/metabolismo , Retinoides/genética , Retinoides/metabolismoRESUMEN
BACKGROUND: Prostate cancer is the most common noncutaneous male cancer and one of the least understood malignant diseases. Identifying key genetic factors involved in the metastasis of prostate cancer cells is critical. In this study, we used selective subtractive differential gene display to identify a gene whose decreased expression may contribute to the growth and expansion of prostate cancer. METHODS: We used 192 primer pair combinations and polymerase chain reaction technology to identify genes expressed in the benign prostate cell line PNT-2 but not in the malignant prostate cancer cell lines LNCaP, Du-145, PC-3, or PC-3M. The tazarotene-induced gene 1 (TIG1) was chosen for further study. TIG1 expression in normal tissues and cell lines was analyzed by northern blot and in normal and tumor prostate tissue sections by in situ hybridization. The in vitro invasiveness (migration through extracellular matrix) and in vivo tumorigenicity (growth in nude mice) were assessed for the highly malignant PC-3M cell line transfected with TIG1 or control cDNA. All statistical tests were two-sided. RESULTS: TIG1 mRNA was expressed in a variety of normal tissues other than prostate tissue. TIG1 mRNA was detected in all 10 normal human prostate tissues and all 51 benign prostatic hyperplastic tissues analyzed but in only four of 51 malignant prostate tissues analyzed. Compared with vector-transfected cells, transfection of PC-3M cells with TIG1 decreased in vitro invasiveness from 14.7% to 3.7%, (mean difference = 11%; 95% confidence interval [CI] = 9.2% to 12.8%, P<.001) and decreased in vivo tumorigenicity from an average tumor weight of 1.31 g to 0.55 g, (mean difference = 0.76 g; 95% CI = 0.43 to 1.09 g, P<.001). CONCLUSION: TIG1 may be a tumor suppressor gene whose diminished expression is involved in the malignant progression of prostate cancer.
Asunto(s)
Ácidos Nicotínicos/genética , Hiperplasia Prostática/genética , Neoplasias de la Próstata/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación in Situ , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Ácidos Nicotínicos/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , ARN Mensajero/metabolismo , Retinoides/genética , Retinoides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales CultivadasRESUMEN
Retinoids affect keratinocyte differentiation and modulate the expression of many epidermal proteins, among them cellular retinoic acid-binding protein II and the family of cytokeratins. The upregulation of the former protein is a well-known phenomenon, whereas the retinoid-induced regulation of epidermal keratin expression is more complex and only partially understood. We studied the effect of topical retinoids on the expression in healthy skin of cellular retinoic acid-binding protein II, tazarotene-induced genes 1 and 2, several epidermal keratins (K1, K2e, and K10), and two mucous keratins (K4 and K13) known to appear in epidermis under certain abnormal conditions. Reverse transcription-polymerase chain reaction experiments showed that the K4 expression was the one most overtly induced by 2 wk of open treatment with 0.05% of retinoic acid and tazarotene. Using real-time quantitative polymerase chain reaction (TaqMan) and normalization of the mRNA values to beta-actin, the increase in K4 was found to be 100-1000-fold. In comparison, the expression of K13 and cellular retinoic acid-binding protein II was increased 10-50-fold, the K1 and K10 mRNA levels remained unchanged, and the K2e level decreased by a factor of 100-1000. In parallel biopsies, immunohistochemistry showed no change in K1, K2e, or K10 staining, but a strong de novo appearance of K4 in the granular layer after retinoid treatment. In a separate study, occlusive application of 0.025% retinoic acid in four healthy subjects produced a maximal K4 mRNA signal after 48 h and strong K4 staining after 80 h. Finally, a dose-response study showed that the de novo appearance of K4 can be utilized as a sensitive test for retinoid bioactivity in epidermis in vivo.
Asunto(s)
Queratinas/fisiología , Tretinoina/farmacología , Administración Tópica , Adulto , Biomarcadores/análisis , Relación Dosis-Respuesta a Droga , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Queratinas/genética , Masculino , Persona de Mediana Edad , Ácidos Nicotínicos/genética , ARN Mensajero/análisis , Receptores de Ácido Retinoico/genética , Retinoides/administración & dosificación , Retinoides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/química , Piel/metabolismo , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Retinoids exert their biologic effects through two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which belong to the superfamily of steroid/thyroid hormone nuclear receptors. By using a subtraction hybridization approach, we have identified a cDNA sequence TIG2 (Tazarotene-induced gene 2), whose expression is up-regulated by the treatment of skin raft cultures by an RAR beta/gamma-selective anti-psoriatic synthetic retinoid tazarotene [AGN 190168/ethyl 6-[2-(4,4-dimethylthiochroman-6-yl)-ethynyl] nicotinate]. The retinoid-mediated up-regulation in the expression of TIG2 was confirmed by Northern blot analysis. Upon sequencing, TIG2 was found to be a cDNA whose complete sequence was not in the GenBank and EMBL data bases. The TIG2 cDNA is 830 bp long and encodes a putative protein product of 164 amino acids. TIG2 is neither expressed nor induced by tazarotene in primary keratinocyte and fibroblast cultures. Thus, TIG2 is expressed and induced by tazarotene only when keratinocytes and fibroblasts form a tissue-like 3-dimensional structure. We further demonstrate that RAR-specific retinoids increase TIG2 mRNA levels. In contrast, neither RXR-specific retinoids nor 1,25-dihydroxyvitamin D3 increased TIG2 levels. Finally, we demonstrate that TIG2 is expressed at high levels in nonlesional psoriatic skin but at lower levels in the psoriatic lesion and that its expression is up-regulated in psoriatic lesions after topical application of tazarotene.
Asunto(s)
Ácidos Nicotínicos/genética , Fenómenos Fisiológicos de la Piel , Administración Tópica , Secuencia de Aminoácidos , Secuencia de Bases , Calcitriol/farmacología , Células Cultivadas , Mapeo Cromosómico , Cromosomas Humanos Par 17/genética , ADN Complementario/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Ácidos Nicotínicos/administración & dosificación , Psoriasis/genética , ARN Mensajero/metabolismo , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/fisiología , Receptores X Retinoide , Homología de Secuencia de Ácido Nucleico , Piel/citología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Regulación hacia ArribaRESUMEN
The nicotinic acid phosphoribosyltransferase locus pncB was located on the Salmonella typhimurium linkage map counterclockwise relative to pyrC. P22 and P1 transductional analyses revealed linkage of pncB with aroA and pyrD, indicating a pncB map position of approximately 20 map units. The results of these cotransduction experiments also indicated that the genetic map distance between gal and pyrD is greater than the published 2.2 map units.