RESUMEN
OBJECTIVE: To report the rationale, recruitment, design, dietary intervention and baseline characteristics of participants in the Medi-RIVAGE study (Mediterranean Diet, Cardiovascular Risks and Gene Polymorphisms). DESIGN: A randomised, parallel trial comparing a new nutritional programme with a conventional programme. SETTING: Centre for Detection and Prevention of Arteriosclerosis, Timone University Hospital, Marseille, France, and collaborating teams. SUBJECTS: Two hundred and twelve male and female volunteers with at least one cardiovascular risk factor. INTERVENTION: A Mediterranean-type diet characterised mainly by the quality of fatty acids, amount of fish, vegetable foodstuffs and fibre was proposed and compared with a usually prescribed, low-fat/cholesterol diet. Body mass index, fasting lipids and lipoproteins, apolipoproteins, glucose, insulin and homocysteine were the main outcome measures. Gene polymorphisms of interest were determined. RESULTS: Characteristics of men in the two arms were comparable with regard to sociodemographic variables, and clinical and biological cardiovascular risk factors. There were few differences between the groups of women (cholesterol-related parameters, P<0.05). There was no difference between arms in allelic distribution of the gene polymorphisms studied. Saturated fat and protein intakes were high while carbohydrate and fibre intakes were low, but with no difference between arms. Overall, the nutritional markers were comparable in both arms with few exceptions. Correlations between nutritional intakes and plasma nutrient levels ranged from 0.19 (beta-carotene) to 0.47 (folate). CONCLUSIONS: The comparability of the two arms is notable and warrants a low risk of biases. Current diet departs from the traditional Mediterranean one. The assessment of nutritional intake is validated by correlations obtained between dietary intake and relevant biomarkers. This will be important to estimate participant compliance and to analyse intervention data.
Asunto(s)
Arteriosclerosis/prevención & control , Dieta con Restricción de Grasas , Dieta Mediterránea , Adulto , American Heart Association , Arteriosclerosis/etiología , Biomarcadores/sangre , Índice de Masa Corporal , Colesterol/efectos adversos , Colesterol/sangre , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/efectos adversos , Femenino , Francia , Humanos , Metabolismo de los Lípidos , Lípidos/genética , Masculino , Persona de Mediana Edad , Evaluación Nutricional , Polimorfismo Genético , Prevención Primaria/métodos , Factores de RiesgoRESUMEN
In hepatocytes, the amount of the Spot 14 (S14) protein is closely related to the full expression of enzymes involved in the glycolytic and lipogenic pathways. In the present study we address the role played by this protein in the control of transcription of the L-type pyruvate kinase (L-PK) gene in primary hepatocytes. We show that human S14, which by itself does not bind to the L-PK promoter, physically interacts with the human chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) and induces the switch of this factor from a repressor to an activator. However, the enhancing activity of S14 and COUP-TF1 depends on the presence of a proximal GC-rich box (the L0 element) that specifically binds nuclear proteins from the livers of rats fed a glucose-rich diet. Moreover, the L0 element, which strongly binds dephosphorylated specificity protein 1 (Sp1), loses all affinity when this factor is phosphorylated by cAMP-dependent protein kinase. Mutations that affect binding of Sp1 and nuclear proteins to the L0 box also decrease basal transcription and impair glucose responsiveness of the promoter. These results therefore shed light on the mechanism by which the S14 protein, whose concentration rapidly rises after glucose intake, contributes to the full activity of the L-PK promoter.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas/química , Proteínas/metabolismo , Piruvato Quinasa/genética , Factor de Transcripción Sp1/química , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Sitios de Unión , Factor de Transcripción COUP I , Núcleo Celular/metabolismo , Células Cultivadas , Pollos , AMP Cíclico/metabolismo , Glucosa/metabolismo , Glutatión Transferasa/metabolismo , Hepatocitos/metabolismo , Humanos , Masculino , Mutación , Proteínas Nucleares , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Regiones Promotoras Genéticas , Unión Proteica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
The dopamine D3 receptor gene is of potential interest in the physiopathology of affective disorder because of its expression pattern in brain structures controlling various aspects of behaviour, cognition and emotions. Moreover, it encodes for a receptor protein that is a target for psychotropic drugs, which turn out to be efficient in the treatment of this disorder. Two polymorphisms have been described at this locus (the Bal I and the Msp I Restriction Fragment Length Polymorphisms) that are useful in genetic studies. We therefore researched these polymorphisms in 60 patients suffering from bipolar affective disorder who were compared with 60 healthy volunteers. No statistical difference was observed between the whole patient sample versus the controls. However, one subgroup [homozygous for the (2-2) Bal I polymorphism] exhibits a characteristic clinical pattern consisting of: manic monopolar form of bipolar disorder, low age of onset and initiation by an acute delusional episode. A gender distribution difference for the Bal I polymorphism (chi 2 = 6.61, degrees of freedom = 1, P = 0.01) was then noted, the bipolar females being preferentially heterozygous, and the males homozygous. These results could involve the dopamine D3 receptor locus as a minor effect gene in the manic depression condition.
Asunto(s)
Trastorno Bipolar/genética , Proteínas del Tejido Nervioso/genética , Polimorfismo de Longitud del Fragmento de Restricción , Receptores de Dopamina D2/genética , Adulto , Edad de Inicio , Alelos , Trastorno Bipolar/clasificación , Trastorno Bipolar/epidemiología , Desoxirribonucleasa HpaII , Desoxirribonucleasas de Localización Especificada Tipo II , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Proteínas del Tejido Nervioso/fisiología , Receptores de Dopamina D2/fisiología , Receptores de Dopamina D3 , Población Blanca/genéticaRESUMEN
Procedures to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels have already been described but are not universally accepted, and many investigators are skeptical about quantification. To be able to accurately monitor gene therapy, we developed a quantitative multistandard RT-PCR method. This was based on the observation that the CFTR and ribosomal phosphoprotein PO (PR-PO) genes have retained important sequence homologies between rat and human species, allowing the use of rat RNA as an internal standard. A mixture of rat and human RNAs is simultaneously reverse-transcribed in one reaction tube and amplification of CFTR leads to rat and human amplificates with identical sizes which will be discriminated by restriction analysis. PR-PO is analyzed similarly and serves as a control of template loading. RT-PCR of different amounts of RNAs gave similar CFTR/PR-PO ratios, with a coefficient variation below 10%. This technique was applied to a cell line of cystic fibrosis tracheal gland serous cells (CF-KM4) incubated with a recombinant adenovirus containing the CFTR cDNA. Kinetics and dose dependency of transgene expression could be accurately quantified. This method is precise, reproducible, and very simple and could be applied to monitor gene therapy in minute amounts of tissue such as biopsies from cystic fibrosis patients.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Adenoviridae/genética , Animales , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , ARN Mensajero/análisis , Ratas , Transfección/métodosRESUMEN
Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.
Asunto(s)
Diabetes Mellitus Experimental/enzimología , Isoenzimas/metabolismo , Hígado/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Secuencia de Bases , Glucemia/análisis , Peso Corporal , Cartilla de ADN , Diabetes Mellitus Experimental/sangre , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/metabolismo , Isoenzimas/antagonistas & inhibidores , Hígado/metabolismo , Masculino , Lípidos de la Membrana/metabolismo , Ouabaína/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , EstreptozocinaRESUMEN
We describe here new software able to retrieve from protein or nucleic acid databases, the sequence corresponding to a protein or peptide whose only amino acid composition and molecular weight are known. This algorithm is particularly devoted to the retrieval of partial sequences, a task that other available software performs poorly. Its accuracy for the attribution of a protein fragment to a sequence could represent an easy and economical first tool upstream the use of more sophisticated and expensive methods in proteomic research. SPAC (Sequence Protein Alignment with Composition) is a shareware available software on web site http://www.univ-tln.fr/ approximately grillas/SPAC/. This web site also presents help and a detailed description of the algorithm and interface.
Asunto(s)
Aminoácidos/análisis , Péptidos/química , Programas Informáticos , Algoritmos , Animales , Bases de Datos Factuales , Humanos , Peso Molecular , Fragmentos de Péptidos/química , Proteínas/química , Reproducibilidad de los Resultados , Alineación de Secuencia/métodos , Alineación de Secuencia/estadística & datos numéricosRESUMEN
The ability of two 15-ketosubstituted sterols, 5alpha-cholest-8(14)-en-3beta-ol-15-one and 3beta-(2-hydroxyethoxy)-5alpha-cholest-8(14)-en-15-one, to alter the mRNA levels of 3-hydroxy-3-methylglutaryl-CoA reductase, low density lipoprotein receptor, and oxysterol binding protein was studied and compared with the effects of 25-hydroxycholesterol in Hep G2 cells. All three oxysterols decreased the level of HMG CoA reductase mRNA at concentrations of 10-30 microM, although 25-hydroxycholesterol was effective at concentrations of 1-3 microM. 25-Hydroxycholesterol lowered the level of LDL receptor mRNA more efficiently after 8 hours than after 24 hours of incubation, whereas 15-ketosterols did not decrease the mRNA level of the LDL receptor. The transcriptions of HMG CoA reductase and LDL receptor genes are therefore independently regulated by 15-ketosterols in Hep G2 cells. In addition, the level of the oxysterol binding protein mRNA is not affected by oxysterols in Hep G2 cells.
Asunto(s)
Regulación de la Expresión Génica , Hidroximetilglutaril-CoA Reductasas/genética , Hígado/metabolismo , Receptores de LDL/genética , Esteroles/metabolismo , Línea Celular , Colesterol/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , ARN Mensajero/análisis , Receptores de Esteroides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
In the murine Ob 17 preadipocyte cell line, the thyroid hormone T3 is an adipogenic factor necessary at an early stage for differentiation into adipocyte. We demonstrate here that this T3 dependence may involve a transient expression (at both the messenger RNA and the protein levels) of c-ErbA beta-type receptors (T3R), although a large body of T3R remained the product of the c-erbAalpha gene, as previously described. c-ErbAbeta1 (and not beta2) expression emerged significantly at growth arrest, peaked 2 days later, and almost disappeared in maturing adipocytes. This expression is related to the presence of T3, as total deprivation of culture medium from T3 prevented it, and the addition of 1.5 nM T3 to preconfluent cultures was able to restore it. When cells were cultured in the presence of T3 and thus were able to differentiate, the c-erbAbeta peak was accompanied by sequential rapid increases in CAAT/enhancer-binding protein-delta(C/EBPdelta), peroxisome proliferator-activated-gamma receptor (PPARgamma), and C/EBPalpha gene expressions. On the contrary, under thyroid hormone-deprived culture conditions that result in nondifferentiation of the preadipocytes, c-erbAbeta1, PPARgamma, and the large C/EBPalpha expressions were blunted, and a moderate early increase in c-erbAalpha1 transcripts was sustained for a longer period. Addition of T3 to T3-deprived preconfluent cells restored PPARgamma and C/EBPalpha expressions. Taken together, the results highlight the important role of T3 in the adipogenesis of Ob 17 cells through the involvement of both beta1 and alpha1 T3R subtypes.
Asunto(s)
ARN Mensajero/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Células Madre/citología , Células Madre/metabolismo , Adipocitos/citología , Animales , Diferenciación Celular/fisiología , Línea Celular , Regulación del Desarrollo de la Expresión Génica/fisiología , Ratones , Receptores Citoplasmáticos y Nucleares/genética , Factores de Tiempo , Transcripción Genética/fisiología , Triyodotironina/genética , Triyodotironina/fisiologíaRESUMEN
Liver fatty acid-binding protein (L-FABP) is a small cytoplasmic molecule highly expressed in the liver. Since L-FABP exhibits affinities for several biliary components, its presence in bile was explored by Western blotting and competitive ELISA in various mammalian species. A L-FABP-like immunoreactivity was consistently found in both hepatic and gallbladder bile. A close molecular identity between this 14 kDa biliary protein and the purified L-FABP was assessed by immunological analyses and high performance capillary electrophoresis. Pharmacological induction of hepatic L-FABP biosynthesis led to a similar increase in biliary L-FABP levels showing a close relationships between the cytosolic and biliary contents of this protein. Finally, a correlation between the presence of L-FABP in bile and both bile flow and bile acid release was found. These data suggest an output of L-FABP in bile in normal conditions which might be coupled with the physiological release of biliary components.
Asunto(s)
Bilis/metabolismo , Proteínas Portadoras/metabolismo , Ácidos Grasos/metabolismo , Hígado/metabolismo , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Proteínas Supresoras de Tumor , Animales , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Citosol/metabolismo , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Vesícula Biliar/metabolismo , Humanos , Inmunoquímica , Masculino , Ratones , Peso Molecular , Proteína P2 de Mielina/química , Proteína P2 de Mielina/aislamiento & purificación , Ratas , Ratas WistarRESUMEN
The cellular distribution of sodium channel beta2 subunit mRNA was examined in the central nervous system from adult Wistar rats using a non-radioactive in situ hybridization method with digoxigenin-labeled cRNA probes. The expression of the subunit was strong in cerebral and cerebellar cortex, in medulla oblongata and in the spinal cord whereas heterogeneous in hippocampus. The distribution was evaluated in hippocampus and cerebral cortex from 1 to 72 h after kainate injection and compared to control rats using densitometric analysis. In these areas, a transient increase was seen 1 h after the drug administration, followed, in the hippocampus, by a significant decrease. These variations differ from those we previously reported for alpha subunits and might play a role in cellular excitability changes occurring in the course of seizures.
Asunto(s)
Canales de Calcio Tipo L , Canales de Calcio/biosíntesis , Sistema Nervioso Central/metabolismo , Agonistas de Aminoácidos Excitadores/toxicidad , Activación del Canal Iónico , Ácido Kaínico/toxicidad , ARN Mensajero/biosíntesis , Convulsiones/metabolismo , Animales , Encéfalo/anatomía & histología , Encéfalo/citología , Encéfalo/metabolismo , Canales de Calcio/genética , Sistema Nervioso Central/anatomía & histología , Sistema Nervioso Central/citología , Hibridación in Situ , Cinética , Masculino , ARN Complementario/genética , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
Several lines of evidence underscore a possible role of voltage-gated Na+ channels (NaCH) in epilepsy. We compared the regional distribution of mRNAs coding for Na+ channel alpha subunit I, II and III in brains from control and kainate-treated rats using non-radioactive in situ hybridization with subtype-specific digoxigenin-labelled cRNA probes. Labelling intensity was evaluated by a densitometric analysis of digitized images. Heterogeneous distribution of the three Na+ channel mRNAs was demonstrated in brain from adult control rats, which confirmed previous studies. Subtype II mRNAs were shown to be abundant in cerebellum and hippocampus. Subtype I mRNAs were also detected in these areas. Subtype III mRNAs were absent in cerebellar cortex, but significantly expressed in neurons of the medulla oblongata and hippocampus. The three subtypes were differentially distributed in neocortical layers. Subtype II mRNAs were present in all of the layers, but mRNAs for subtypes I and III were concentrated in pyramidal cells of neocortex layers IV-V. During kainate-induced seizures, we observed an increase in Na+ channel II and III mRNA levels in hippocampus. In dentate gyrus, subtype III mRNAs increased 3 h after KA administration to a maximum at 6 h. At this latter time, a lower increase in NaCh III mRNAs was also recorded in areas CA1 and CA3. NaCh III overexpression in dentate gyrus persisted for at least 24 h. In the same area, NaCh II mRNAs were also increased with a peak 3 h after KA injection and a return to control levels by 24 h. No changes in NaCh I mRNAs were seen. The KA-induced up-regulation in NaCh mRNAs probably resulted in an increase in hippocampal neuronal excitability.
Asunto(s)
Encéfalo/metabolismo , Ácido Kaínico , ARN Mensajero/metabolismo , Convulsiones/inducido químicamente , Convulsiones/metabolismo , Canales de Sodio/genética , Animales , Corteza Cerebelosa/metabolismo , Cerebelo/metabolismo , Giro Dentado/metabolismo , Digoxigenina , Hipocampo/metabolismo , Hibridación in Situ , Bulbo Raquídeo/metabolismo , Sondas ARN , Ratas , Ratas WistarRESUMEN
Hepatitis G virus (HGV) and hepatitis GB virus (GBV-C) have been reported as possible causes of non-A-E transfusional hepatitis. To assess the prevalence of hepatitis G virus infection in haemophiliacs we retrospectively investigated the presence of viral RNA in 92 patients with and without HCV infection. HGV/GBV-C RNA was reverse transcribed and amplified with primers from the 5' non-coding region of the genome. RNA was detected in 16/92 patients (17.4%). Restriction enzyme analysis revealed that the 16 patients belonged to the HGV-like genotype. Serology with E2-specific antibodies demonstrated that HGV viraemia underestimates previous infection by HGV. 33 patients were positive for HGV; all but two have cleared HGV RNA. 47/92 patients had a marker of prior infection by HGV. No difference between HGV RNA positive and negative patients was observed concerning age, diagnosis, HIV and HCV status. Previous HBV infection correlated with the frequency of HGV infection. There was no difference in alanine aminotransferase levels between HGV positive and negative patients. All 18 patients exposed to only virally inactivated plasma-derived concentrates were negative for both HGV RNA and anti E2 antibodies. Prior exposure to untreated concentrates correlated with HGV viraemia (P=0.03), HGV seropositivity (P=0.0002), and markers of HGV infection (P<0.0001). In haemophiliacs with a past exposure to non-inactivated concentrates, persistence of HCV RNA (53/74 patients) was more frequent than HGV RNA persistence (16/74 patients) although HGV viraemia is more frequent than HCV viraemia in blood donors. This may be related to a greater ability of individuals to clear HGV infection and suggests that hepatitis G virus infection in multi-transfused patients has a better outcome than infection with other blood-borne viruses.
Asunto(s)
Flaviviridae/genética , Hemofilia A/epidemiología , Hepatitis Viral Humana/epidemiología , ARN Viral/aislamiento & purificación , Adolescente , Adulto , Anciano , Niño , Preescolar , Hemofilia A/virología , Hepatitis Viral Humana/genética , Humanos , Persona de Mediana Edad , Prevalencia , Estudios RetrospectivosRESUMEN
BACKGROUND/AIMS: Electroneutral absorption of NaCl by the gallbladder mucosa is likely to depend at least in part on a Na+/H+ exchanger. In intestine and colon, absorption due to Na+/H+ exchanger is explained by the presence of specific isoforms of the exchanger, the NHE-3 isoform and possibly the NHE-2 isoform. The aim of the present work was to determine whether the mRNAs coding for NHE-2 and NHE-3 are expressed in epithelial cells of human gallbladder. METHODS: Total RNAs from human gallbladder were subjected to reverse transcription-polymerase chain reaction using specific primers. No message was observed with NHE-2 specific primers, showing that NHE-2 isoform plays no role in gallbladder absorption. With NHE-3 specific primers, a 239 bp cDNA fragment was obtained and showed a high homology with the NHE-3 isoform, confirming the presence of NHE-3 in the gallbladder wall. This fragment was cloned in a pLitmus vector in order to produce cRNA probes by in vitro transcription. Cellular localization of the NHE-3 mRNA was studied on cryostat sections using the cRNA probes labeled with Digoxigenin-11-UTP, controls included assays with sense probe, antibodies without probe and RNaseA treated tissue. A specific staining of the NHE-3 mRNAs was found to be strictly localized to the gallbladder epithelial cells. RESULTS/CONCLUSIONS: Expression of NHE-3 in the gallbladder was found only in the absorptive epithelial cells. The NHE-3 isoform of the Na+/H+ exchanger is likely to be involved in water and electrolyte absorption from bile.
Asunto(s)
Vesícula Biliar/metabolismo , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Epitelio/metabolismo , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Membrana Mucosa/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Homología de Secuencia de Ácido Nucleico , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , Transcripción GenéticaRESUMEN
Conventional risk factors have very low predictive power in identifying haemodialysis patients at high risk of vascular accidents. A role for apolipoprotein E isotypes was looked for in a small, but rigorously defined, cohort of longterm haemodialysis patients. In individuals with high vascular risk, as identified by higher common carotid intima/media thickness, we found an excess of apolipoprotein E4 alleles. This preliminary result requires confirmation in large patient cohorts.
Asunto(s)
Alelos , Apolipoproteínas E/genética , Diálisis Renal/efectos adversos , Enfermedades Vasculares/etiología , Enfermedades Vasculares/genética , Adulto , Anciano , Apolipoproteína E4 , Apolipoproteínas E/fisiología , Arteriosclerosis/etiología , Arteriosclerosis/genética , Arteriosclerosis/patología , Arteria Carótida Común/diagnóstico por imagen , Arteria Carótida Común/patología , Estudios de Cohortes , Femenino , Humanos , Lípidos/sangre , Masculino , Persona de Mediana Edad , Factores de Riesgo , Ultrasonografía , Enfermedades Vasculares/patologíaRESUMEN
In a previous report, we showed that physiological concentrations of calcitriol (1 alpha,25-(OH)2 vitamin D3 or VD), markedly stimulated the terminal adipose differentiation of Ob 17 preadipocytes cultured under standard conditions with fetal calf serum (FCS), and increased the differentiating effect of triiodothyronine (T3) reported as a necessary adipogenic factor in these cells. Here, we demonstrate, for the first time, that VD is an intrinsic strong adipogenic factor for the Ob 17 preadipocytes cultured in thyroid hormone-deprived medium (adipogenic concentrations: 0.025-0.25 nM in the presence of stripped FCS, 1-10 pM under serum-free conditions). VD action was potentiated by the coaddition of either T3, or arachidonic acid, two agents which also bear proper adipogenic properties. The efficient concentration ranges of other vitamin D3 metabolites suggest a mediation through the VD nuclear receptor (VDR). An expression of the VDR gene is here demonstrated in the Ob 17 cells, and evidence is given that VDR mRNA level increased during the differentiation process and that this increase is moderately amplified under long term treatment with adipogenic concentrations of VD. Our results strongly suggest that adipose differentiation is under the control of different closely related nuclear receptors acting at an early preadipocyte step and probably in an interchangeable manner depending on the availability of their respective ligands. The existence of an interplay between these receptors in exerting their adipogenic action is suggested.
Asunto(s)
Tejido Adiposo/citología , Tejido Adiposo/efectos de los fármacos , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Triyodotironina/farmacología , Tejido Adiposo/metabolismo , Animales , Secuencia de Bases , Calcitriol/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Cartilla de ADN/genética , Regulación de la Expresión Génica , Ratones , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Triyodotironina/metabolismoRESUMEN
Subtypes I, II and III of sodium channel alpha-subunit mRNAs were analyzed in adult rat brain areas after kainate-induced seizures. Tissue samples were microdissected from occipital neocortex, CA1 and CA3 hippocampus areas and dentate gyrus. Three reverse transcriptase-polymerase chain reaction (RT-PCR) protocols were undertaken to amplify these mRNAs. Amplification products were then distinguished after digestion by restriction enzymes, electrophoresis separation and densitometric analysis of gel profiles. PCR 1 evidenced the relative percentage of mRNAs I, II and III as well as neonatal II and III subtype isoforms, which resulted from an alternative splicing. PCR 2 and 3 were performed to focus on the neonatal vs. adult ratio in II and III subtypes, respectively. Seizures were shown to induce an increase in both neonatal subtypes, which suggested an alteration at the splicing level. These changes exhibited a peculiar brain regional distribution, the maximal effect being observed in dentate gyrus and hippocampus CA1 area. In situ hybridization experiments, using a digoxigenin-labeled oligonucleotide probe-specific for neonatal II and III mRNAs, confirmed this increase in neonatal mRNA subtypes. These changes were transient, reaching a maximum 6 h after drug injection, then disappearing between 12 and 48 h. They were prevented by a pre-treatment of animals by MK-801, a non-competitive antagonist of NMDA receptors. This work, thus, suggested that KA-induced seizures can be accompanied by transient alteration in the splicing pattern of sodium channel alpha-subunit mRNAs which resulted in an increase in expression of their neonatal isoforms within localized areas of adult rat brain.
Asunto(s)
Hipocampo/efectos de los fármacos , ARN Mensajero/genética , Convulsiones/metabolismo , Canales de Sodio/genética , Animales , Animales Recién Nacidos , Maleato de Dizocilpina/uso terapéutico , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Código Genético , Hipocampo/metabolismo , Hibridación in Situ , Ácido Kaínico , Masculino , Fármacos Neuroprotectores/uso terapéutico , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Transcripción GenéticaRESUMEN
The intricate regulation of Spot 14 expression in rat lipogenic tissues has provided a useful tool in studying nutritional and hormonal factors involved in transcription. To gain insight into its function and its possible involvement in human lipid disorders, we cloned human and mouse Spot 14 genes that shared with the rat gene a strong homology concerning the deduced amino acid sequence (81 and 94%, respectively) as well as the promoter region. The mouse promoter was characterized by transfection studies, while quantitative RT-PCR and in situ hybridization experiments showed that Spot 14 is expressed in human liver and, at a high level, in multiple symmetric lipomatosis nodules.
Asunto(s)
Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN , Humanos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de TranscripciónRESUMEN
In previous reports, we showed that T3 is required for terminal differentiation of the murine Ob 17 preadipocytes, and that it partially down-modulates the abundance of its own nuclear receptor sites (T3R). We also reported that a profound depletion of the T3R was produced by all-trans-retinoic acid at concentrations that inhibit adipose differentiation. Here, we report that calcitriol (VD), which activates a nuclear receptor (VDR) closely related to the T3R and retinoid receptors, also markedly affects nuclear T3 binding and T3-induced differentiation of Ob 17 cells. Within a nearly physiological concentration range (0.1-2.5 nM), calcitriol profoundly down-modulated T3R abundance without altering the affinity for T3. The T3R depletion was a fast event, sustained under VD and reversed within 48 h of VD withdrawal. The order of efficient concentration ranges of VD and analogs suggests an involvement of the VDR. The T3R-depleting effect of VD was observed at every stage of adipose differentiation and was additive to the depleting effect of T3. Within the 0.1-2.5 nM VD concentration range, the c-erbA alpha and -alpha 1 messenger RNA levels (only c-erbA alpha gene products were detected in these cells) were poorly decreased; VD also did not alter a protein band specifically detected with specific anti-c-erbA alpha 1 antibodies in Western blots of nuclear extracts. VD accelerated the T3R disappearance rate; the results suggest that this would probably involve sequestration, rather than degradation, events. Interestingly, calcitriol added to the culture medium of Ob 17 preadipocytes markedly influenced the adipose differentiation, exerting a clear-cut stimulation at levels of 0.25 nM or less and profound inhibition at concentrations above 0.25 nM. Both effects were observed provided that VD was added within an early critical period of the differentiation process, as we previously reported for T3. The stimulations caused by low concentrations of VD and 1.5 nM T3 were additive. Increasing the VD concentration produced a progressive attenuation, then a suppression, of the stimulating effect of T3. Comparative analyses of VD-related changes in adipose differentiation and T3R abundance suggest that a correlation may exist between optimal differentiation and a partial depletion of the T3R, whereas a profound depletion of the T3R occurred at inhibitory concentrations of VD. The present results sustain the concept that T3R play a role in the differentiation of Ob 17 preadipocytes. Moreover, the results suggest that there may be a T3 receptor site concentration optimal for efficient differentiation. A regulation of this concentration involves ligands of other closely related receptors and, thus, probably the interplays that exist between these receptors.
Asunto(s)
Adipocitos/citología , Tejido Adiposo/citología , Calcitriol/farmacología , Regulación hacia Abajo , Receptores de Hormona Tiroidea/metabolismo , Triyodotironina/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Ratones , Biosíntesis de Proteínas , Receptores de Hormona Tiroidea/efectos de los fármacos , Receptores de Hormona Tiroidea/genética , Células Madre/citología , Triyodotironina/farmacologíaRESUMEN
We present here a computational method based on the analysis of amino acid composition for performing comparisons between proteins. This user-friendly and reliable test is aimed at rapidly identifying, from data-base subsets, sequences--if necessary, partial sequences--which share similar amino acid compositions to the input composition (deduced from experimental results). Apparent molecular weight (as determined by SDS-PAGE) and artefactual modifications due to the experimental determination of the amino acid composition are taken into account to perform the comparison. This program thus constitutes a useful tool in searching for the probable identification of either non-sequenced proteins or peptides from hydrolysed proteins.