RESUMEN
Denaturing high performance liquid chromatography (DHPLC) is a novel high-capacity technique for gene mutation scanning. We have assessed the sensitivity and specificity of this method for analysis of the full coding sequence of the hamartin (TSC1) gene in 20 tuberous sclerosis patients, whose TSC1 genes previously had been studied by single strand conformation polymorphism analysis and protein truncation assay. All eight sequence variants previously identified were adequately detected by DHPLC. Additionally, this approach picked up three polymorphisms, one of which (IVS13-55 C>G) was hitherto unreported, therefore serving as proof of principle for this technique. Thus, DHPLC appears to be a highly sensitive method with advantages in terms of flexibility, fragments size analysis, cost and time and labor sparing, compared to classical approaches of mutation scanning.
Asunto(s)
Genes Supresores de Tumor/genética , Proteínas/genética , Esclerosis Tuberosa/genética , Cromatografía Líquida de Alta Presión/métodos , ADN/sangre , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Desnaturalización de Ácido Nucleico/genética , Polimorfismo Genético/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de TumorRESUMEN
The regulatory effects of angiotensin II (AngII) on its receptor subtypes, AT1 and AT2, were studied using cultured bovine adrenal cells (BAC), which express both receptor subtypes, and PC12W and R3T3 cells, which express only AT2 receptors. In BAC, AngII caused a decrease in AT1- and AT2-binding sites and their corresponding messenger RNAs (mRNAs), but with different kinetics. AT1-binding sites decreased by more than 50% within the first 3 h, whereas AT1 mRNA started to decline after a lag period of 3 h. Both AT2-binding sites and mRNA remained stable within the first 6 h of AngII treatment. Then, AT2 mRNA decreased rapidly with an apparent half-life of 2-3 h, whereas AT2-binding sites declined with an apparent half-life of about 16 h. Measurement of transcription rate and mRNA half-life by the [3H]uridine-thiouridine method revealed that AngII reduced by 90% the rate of AT1 transcription, but had no effect on AT1 mRNA half-life, whereas it slightly reduced AT2 transcription, but markedly reduced AT2 mRNA stability. All of the effects of AngII on both AT1 and AT2 receptors were blocked by losartan, indicating that they were mediated exclusively through the AT1 receptor. In PC12W cells, AngII was unable to modify AT2-binding sites or mRNA. Moreover, in BAC, [125I]AngII was internalized through the AT1 receptor, whereas occupancy of AT2 receptors in either BAC or PC12W did not produce internalization of the hormone. These results indicate that AngII, through the AT1 receptor, down-regulates both AT1 and AT2, but by different mechanisms; AT1 receptor is regulated through internalization-degradation of the occupied receptor and inhibition of transcription, whereas AT2 receptor is regulated mainly by decreasing the stability of its mRNA. Moreover, the phorbol ester phorbol 12-myristate 13-acetate mimicked most of the effects of AngII in BAC and decreased both AT2-binding sites and mRNA on PC12W cells, indicating that the hormonal regulation of both AT1 and AT2 receptors is mediated through protein kinase C activation.
Asunto(s)
Angiotensina II/farmacología , Regulación hacia Abajo , Receptores de Angiotensina/metabolismo , Zona Fascicular/metabolismo , Angiotensina II/metabolismo , Animales , Sitios de Unión , Bovinos , Células Cultivadas , Activación Enzimática , Semivida , Cinética , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Receptores de Angiotensina/genética , Transcripción GenéticaRESUMEN
The nuclear protooncogenes have been implicated in the coordinate regulation of gene expression during cell proliferation and differentiation. Previous work has shown that LH and human h CG as well as several growth factors including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), transforming growth factor-beta, and insulin-like growth factor-I play a role in Leydig cell differentiated functions. To evaluate the possibility that protooncogenes mediate long term effects of these factors, their action on the levels of c-fos, c-jun, jun-B, and c-myc messenger (m) RNAs was studied. hCG (10(-9) M) produced a time-dependent increase in c-fos (9-fold), jun-B (18-fold) and c-myc (5-fold) mRNA levels but did not affect c-jun. The concentration of hCG required for half-maximal stimulation (ED50 = 7 +/- 4 x 10(-12) M) was similar to that required to induce half-maximal testosterone production. At optimal concentrations, the effects of EGF and bFGF on c-fos and jun-B mRNAs were lower than those induced by hCG, but their effects on c-myc mRNA were higher. In addition, they stimulated c-jun. Moreover, EGF and bFGF potentiated the effects of hCG on c-fos and jun-B, whereas hCG potentiated the action of growth factors on c-jun. Transforming growth factor-beta increased only jun-B mRNA, whereas insulin-like growth factor I increased c-fos, jun-B, and c-myc but less effectively than hCG. Lastly, the phorbol ester phorbol 12-myristate 13-acetate increased the level of the four protooncogene mRNAs, and its effects on c-fos and c-myc were significantly higher than those produced by hCG. These data indicate that the regulation of protooncogene mRNAs in normal Leydig cells is multifactorial. They also show differential responsiveness of the members of the Jun family to several factors. Our results are consistent with the hypothesis that the Fos and Jun families of regulatory proteins could play a role in mediating long term responses to the complex array of hormones and growth factors to which Leydig cells are exposed in vivo.
Asunto(s)
Gonadotropina Coriónica/farmacología , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genes myc , Sustancias de Crecimiento/farmacología , Células Intersticiales del Testículo/fisiología , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/efectos de los fármacos , ARN Mensajero/genética , Factores de Transcripción/genética , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Cinética , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-fos , Proteínas Proto-Oncogénicas c-jun , ARN Mensajero/efectos de los fármacos , ARN Mensajero/aislamiento & purificación , Porcinos , Testosterona/biosíntesis , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
In the present work we have investigated the effects of several growth factors on the expression of Angiotensin II (A-II) receptors subtype AT1 and their pertussis toxin-insensitive coupling to G-proteins in bovine adrenal fasciculata-reticularis cells (BAC). Insulin, Insulin-like growth factor and basic Fibroblast growth factor increased AT1 receptors (mRNA and binding sites) as well as the alpha subunit of Gq (mRNA and protein) and G11 (protein). These changes were associated with an enhanced A-II-induced inositol phosphate accumulation and cortisol production. In contrast, Transforming growth factor beta 1, which reduced slightly AT1 binding sites, but not the level of alpha q or alpha 11 proteins, did not change the A-II-induced inositol phosphate accumulation. However, this factor, as previously reported, markedly reduced cortisol production.
Asunto(s)
Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/metabolismo , Sustancias de Crecimiento/farmacología , Receptores de Angiotensina/metabolismo , Glándulas Suprarrenales/citología , Animales , Bovinos , Células Cultivadas , Proteínas de Unión al GTP/metabolismo , Hidrocortisona/biosíntesis , Fosfatos de Inositol/metabolismo , Insulina/farmacología , ARN Mensajero/metabolismo , Receptores de Angiotensina/efectos de los fármacosRESUMEN
The regulatory effects of angiotensin-II (AngII) and several growth factors, including insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), and transforming growth factor beta1 (TGFbeta1) on the AngII subtype 2 (AT2) receptor were studied using R3T3 cells, a mouse fibroblast cell line that expresses only AT2 receptors. AngII increased (in a time- and dose-dependent manner) AT2 binding sites but had no effects on AT2 messenger RNA (mRNA) levels. At maximal concentration (10(-7) M) AngII caused a 4-fold increase of AT2 receptor number. In contrast, IGF-1 increased (3- to 4-fold), whereas bFGF and TGFbeta1 decreased (by about 90% and 80%, respectively) AT2 receptor and mRNA levels. Moreover, AngII potentiated the effect of IGF-1 on receptor number, but not on AT2 mRNA levels, and significantly reduced the inhibitory action of bFGF and TGFbeta1 on AT2 binding sites but not on AT2 mRNA levels. None of these factors modified AT2 mRNA half-life. The potential effects of these factors on transcription of the AT2 gene were measured by means of nuclear run-on assays. IGF-1 increased the rate of transcription by about 2.5-fold, whereas bFGF and TGFbeta1 reduced it by 90 and 80%, respectively. In contrast, AngII did not modify either the basal or IGF-1-stimulated transcription rate. Finally, AngII alone or together with IGF-1, but not IGF-1 alone, increased the attachment of AT2 mRNA to polysomal fractions. The present findings demonstrate that the main mechanism by which AngII regulates the AT2 receptor is by increasing the rate of AT2 mRNA translation, whereas the stimulatory (IGF-1) or inhibitory (bFGF and TGFbeta1) effects of these growth factors on AT2 expression are exerted at the transcriptional level.
Asunto(s)
Angiotensina II/farmacología , Regulación de la Expresión Génica , Sustancias de Crecimiento/farmacología , Receptores de Angiotensina/genética , Autorradiografía , Sinergismo Farmacológico , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Hibridación in Situ , Factor I del Crecimiento Similar a la Insulina/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ribosomas/química , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Previous work has shown that corticotropin (ACTH) and angiotensin-II (A-II), in addition to their acute steroidogenic effects, exert long-term influences on adrenal cell differentiated function, stimulatory or inhibitory, respectively. Certain nuclear proto-oncogenes have been implicated in the regulation of gene expression in many cell systems. We have investigated the effects of ACTH and A-II on the levels of c-fos, c-jun, and jun-B messenger RNAs (mRNAs), in bovine and ovine (OAC) adrenal fasciculata cells. In both cell types ACTH produced time- (maximum at 1 h) and dose-dependent (ED50 congruent to 10(-12) M) increase in c-fos (2- to 4-fold) and jun-B (10- to 20-fold) mRNA levels but did not affect c-jun. The concentrations required to induce half-maximal mRNA accumulation and cortisol production were similar. A-II also produced a dose-dependent increase in c-fos and jun-B mRNAs but also in c-jun in both cell types, despite the fact that OAC are resistant to the steroidogenic action of the hormone. The stimulatory effects of A-II on c-fos mRNA were higher than those produced by ACTH, whereas the effects on jun-B were similar but ACTH abolished (OAC) or decreased (bovine adrenal fasciculata cells) the stimulatory effects of A-II on c-jun mRNA. The effects of ACTH and A-II on cortisol production and proto-oncogene mRNAs were in part mimicked by 8 Bromo-cAMP and the phorbol ester phorbol-12-myristate-13 acetate plus calcium ionophore A23187, respectively. In the presence of cycloheximide, which blocks the steroidogenic effects of both hormones, proto-oncogene mRNAs were superinduced by both hormones. This result, together with the fact that dexamethasone failed to affect the mRNA levels suggests that the stimulatory effects of ACTH and A-II on proto-oncogene expression were not related to an autocrine/intracrine action of cortisol. Taken together, these findings show that the proto-oncogene mRNAs in normal adrenal cells are regulated by ACTH and A-II, acting through different intracellular pathways. They also demonstrate differential responsiveness of the Jun family to both hormones. Thus, the opposite long-term action of ACTH and A-II on adrenal cell differentiated function could be mediated by its different initial effects on proto-oncogene expression, in particular in the members of the Jun family.
Asunto(s)
Corteza Suprarrenal/citología , Hormona Adrenocorticotrópica/farmacología , Angiotensina II/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/genética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Corteza Suprarrenal/química , Corteza Suprarrenal/efectos de los fármacos , Animales , Northern Blotting , Calcimicina/farmacología , Bovinos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Activación Enzimática/fisiología , Glucocorticoides/farmacología , Proteína Quinasa C/metabolismo , Proteína Quinasa C/fisiología , Proteínas Quinasas/metabolismo , Proteínas Quinasas/fisiología , ARN Mensajero/análisis , Ovinos , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
Interactions between signal transducing systems may be important in the integrated control of cellular processes in basal and hormonally regulated cells. The cultured bovine adrenal fasciculata cell provides a model to study the interactions between the cAMP and calcium-sensitive phospholipid dependent protein kinase C. In this study, angiotensin II (A-II) and phorbol ester (PMA) potentiated the stimulatory actions of ACTH in a dose-dependent manner on cAMP production. At maximal concentrations, A-II and PMA also potentiated the effects of cholera toxin and forskolin on cAMP production. Both staurosporine, a protein kinase C inhibitor, and desensitization of protein kinase C by a 24-h pretreatment with PMA blunted the effect of PMA, but only partially inhibited (34%) the effect of A-II. Neither nifedipine, a specific calcium channel antagonist, nor pretreatment of cells with pertussis toxin modified the amplifying effects of A-II or PMA. In contrast, trifluoperazine, a calmodulin inhibitor, reduced the potentiating effect of A-II by about 35%, but association with staurosporine blunted its effects. Moreover, the steroidogenic effects of ACTH plus A-II were more than additive, but this synergism was blunted in the presence of both inhibitors. In conclusion, PMA and A-II potentiated agonist-induced cAMP production by bovine adrenal fasciculata cells. The data suggest that the effects of PMA were mediated exclusively by protein kinase C, whereas those of A-II were mediated by both protein kinase C and calmodulin.
Asunto(s)
Glándulas Suprarrenales/citología , Glándulas Suprarrenales/metabolismo , Angiotensina II/farmacología , Calmodulina/fisiología , AMP Cíclico/metabolismo , Proteína Quinasa C/fisiología , Glándulas Suprarrenales/fisiología , Hormona Adrenocorticotrópica/farmacología , Alcaloides/farmacología , Animales , Canales de Calcio/fisiología , Bovinos , Células Cultivadas , Toxina del Cólera/farmacología , Colforsina/farmacología , AMP Cíclico/fisiología , Relación Dosis-Respuesta a Droga , Proteínas de Unión al GTP/fisiología , Hidrocortisona/metabolismo , Nifedipino/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Estaurosporina , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Trifluoperazina/farmacologíaRESUMEN
The diterpene, forskolin, stimulated cAMP accumulation about 15-fold over basal levels in purified mouse Leydig cells; however, it remained far less potent than hCG. Simultaneous addition of forskolin and hCG resulted in a striking synergistic stimulation of cAMP production. In contrast, forskolin-enhanced testosterone accumulation was never synergistic with that produced by maximal concentrations of hCG. hCG (3 X 10(-9) M) lowered about 6-fold the ED50 for forskolin-elicited cAMP accumulation and increased the maximal response to forskolin about 16-fold. Conversely, forskolin 10(-6) M) reduced the ED50 for hCG 2-fold but had a much smaller effect (2-3-fold) on maximal response. Moreover, pretreatment with hCG induced only a homologous desensitization of adenylate cyclase, whereas the enzyme became partially resistant to both hCG and forskolin in cells pretreated with forskolin. The homologous hCG-induced desensitization and the partial heterologous one induced by forskolin suggest that more than the catalytic unit of the cyclase is required for the diterpene activation.
Asunto(s)
Adenilil Ciclasas/metabolismo , Gonadotropina Coriónica/farmacología , Diterpenos/farmacología , Células Intersticiales del Testículo/enzimología , Animales , Células Cultivadas , Colforsina , AMP Cíclico/biosíntesis , Sinergismo Farmacológico , Tolerancia a Medicamentos , Masculino , Ratones , Ratones Endogámicos BALB C , Testosterona/biosíntesisRESUMEN
Previous studies have suggested that the expression of angiotensin type 2 receptor was inversely related to cell proliferation. We examined the effects of insulin-like growth factor (IGF-1), basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGFbeta1) and fetal calf serum (FCS) on cell proliferation and AT2 binding sites and mRNA level in PC12W (rat pheochromocytoma cell line) and R3T3 (mouse fibroblast cell line) which express abundant AT2 receptors. In both cell lines, serum deprivation markedly increased both AT2 receptor number and mRNA. However, in the absence of serum cell proliferation continued in PC12W and R3T3 at late passages (R3T3 LP) but not at early passages (R3T3 EP). In PC12W, none of the three growth factors studied stimulated cell proliferation, but TGFbeta1 and more particularly bFGF markedly reduced AT2 expression. In R3T3 LP, IGF-1 and bFGF, but not TGFbeta1, slightly stimulated cell proliferation, but the three factors, specially bFGF, reduced AT2 expression. In contrast, in R3T3 EP, the three growth factors significantly increased cell proliferation, but whereas TGFbeta1 and bFGF markedly reduced AT2 binding sites and mRNA, IGF-1 caused the opposite effects. These results indicate that regulation of AT2 expression is not correlated with cell proliferation and appears to be more complex than initially suspected. In addition, they show that the same factor can have an opposite effect on AT2 expression in the same cell line depending upon the cell passage.
Asunto(s)
Angiotensina II , Sustancias de Crecimiento/farmacología , Receptores de Angiotensina/análisis , Animales , Sitios de Unión , Sangre , División Celular , Línea Celular , Fibroblastos , Ratones , Células PC12 , ARN Mensajero/análisis , Ratas , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/genéticaRESUMEN
From his first years a child showed signs of a primary and rapidly developing muscular dystrophy. The diagnosis was established by an increased serum CK level and by electromyography and muscle biopsies. Afterwards this child developed a severe renal deficiency which needed binephrectomy and the graft of a normal kidney. During the few months just after the graft, the disability increased and the patient could not stand upright by himself. Later on, he gradually became able to walk on his own and without bracing. He could climb stairs and stand up from the floor. The CK activity returned to normal. At present, 4 years after the graft (the patient is 16 years), the improvement of his functional abilities is constant, although the CK activity has increased again. In this article we give evidence that this patient suffers from a primary muscular dystrophy. We discuss the type of dystrophy concerned. We believe that it is the graft of a normal kidney which was responsible for the improvement observed, and not the physiotherapy or the drugs administered after the graft.
Asunto(s)
Fallo Renal Crónico/cirugía , Trasplante de Riñón , Distrofias Musculares/fisiopatología , Adolescente , Niño , Preescolar , Humanos , Fallo Renal Crónico/complicaciones , Masculino , Músculos/patología , Distrofias Musculares/complicaciones , Distrofias Musculares/patología , Trasplante HomólogoRESUMEN
Diphenoloxidases, enzymes which accelerate the auto-oxidation of epinephrine and Dopa, have been described by one of us in blood platelets. Earlier we identified these enzymes in different animal species and particularly in human red blood cells. With the object of localising these enzymes and of understanding their function in vivo, we separated the ghosts of red blood cells according to the method described by Fairbanks G., Steck, T.L. and Wallach, D.F.H. (1971) (Biochemistry 1, 2606) and using the protease inhibitors diisopropylfuorophosphate (DFP) (10(-3) M) and 6-aminocaproic acid 10(-2) M in sodium phosphate buffer, 5 X 10(-3) M (pH 8). These ghosts, totally free of haemoglobin, were first of all pulverised in liquid nitrogen then treated ultrasonically. The supernant shows the presence of a band of diphenoloxidase activity on starch gel electrophoresis and two bands on isoelectrofocusing in polyacrylamide gel pH 5 to pH 8 after incubation with 0.02 M Dopa, 0.076 M Tris, 0.005 M citric acid, 0.004 M magnesium, pH 8.7 for 2 h at 37 degrees C. These enzymes differ from (Na, K)-ATPase in that they are neither inhibited by DFP (10(-1) M) nor by EDTA (10(-2) M) but are inhibited by lead acetate 10(-2) M. Like (Na, K)-ATPase diphenoloxidases are present at membranes level. The role in vivo of these diphenoloxidases in ATPase activity of red blood cells is discussed.
Asunto(s)
Catecol Oxidasa/sangre , Membrana Eritrocítica/enzimología , Eritrocitos/enzimología , Adenosina Trifosfatasas/sangre , Ácido Aminocaproico/farmacología , Catecol Oxidasa/aislamiento & purificación , Humanos , Isoflurofato/farmacología , CinéticaRESUMEN
An oxidative activity has been demonstrated in different tissues of various animals species (man, rabbit, hen, rat, mouse) measuring it by means of adrenaline, by starch gel electrophoresis and acrylamide gel electrofocusing, followed by incubation of these gel with DOPA. Kidney, liver and erythrocytes of rabbit, rat, mouse and hen, human erythrocytes and platelets display a high oxidative activity. This activity, which is not completely inhibited by KCN, is found in one band on starch gel electrophoresis and in two distinct bands on electrofocusing in the case of platelets and erythrocytes of man, rabbit and rat. The starch gel electrophoretic migration and the electrofocusing pattern are tissue and species dependent.
Asunto(s)
Catecol Oxidasa/análisis , Animales , Plaquetas/enzimología , Encéfalo/enzimología , Pollos , Cianuros/farmacología , Electroforesis en Gel de Poliacrilamida , Electroforesis en Gel de Almidón , Eritrocitos/enzimología , Humanos , Intestinos/enzimología , Focalización Isoeléctrica , Riñón/enzimología , Hígado/enzimología , Pulmón/enzimología , Ratones , Músculos/enzimología , Miocardio/enzimología , Conejos , Ratas , Bazo/enzimologíaRESUMEN
PIP: Abortion in sows may be complete, or much more often partial, since the average litter size is about 10. This review describes the clinical and serological findings, mode of transmission and recommended treatment for the most common parasitic, fungal, mycotoxin, deficient, and toxic causes of abortion in sows. The most likely possibilities in France are brucellosis, leptospirosis, Aujeszky virus, mycotoxin, or dietary deficiencies. The bacterialtion in French sows are Brucella species, Leptospira species, E. coli, streptococci, staphylococci, Pseudomonas, Hemophilus, Corynebacterium pyogenes, salmonellae, Listeria monocytogenes, Mycobacteriumr fetus, Chlamydia and Mycoplasma bovi genitalium. Toxoplasma gondii are parasites known to cause abortion in sows. Mycotoxins from Fusarium species may contaminate feed. Noninfectious causes include poisoning from nitrates, nitrites, estrogens, or insecticides and deficiencies of Vitamins-A, -B, in abortion.^ieng
Asunto(s)
Abortivos , Animales de Laboratorio , Aborto Inducido , Servicios de Planificación Familiar , InvestigaciónAsunto(s)
Catecol Oxidasa/sangre , Eritrocitos/enzimología , Monofenol Monooxigenasa/sangre , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Electroforesis en Gel de Almidón , Epinefrina/metabolismo , Hemoglobinas , Humanos , Focalización Isoeléctrica , Monofenol Monooxigenasa/aislamiento & purificaciónRESUMEN
The effects of fibroblast growth factor (FGF) on testicular aromatase activity has been studied using primary cultures of porcine Leydig cells. After culture for 3 days in the absence or presence of FGF, the ability of the cells to produce estrogen was examined in a 4h-test period in which either (a) hCG (10(-9) M) or (b) androstenedione (3 x 10(-6) M) was added to the medium. FGF produced a 3- to 20-fold increase in estrogen formation from endogenous or exogenous substrate during the test period, in spite of a marked decrease (approximately equal to 60%) in [125I]-hCG binding and no significant change in testosterone concentration. Stimulation of estrogen secretion by FGF was dose-(ED50 approximately equal to 2 ng/ml) and time-dependent, the first and maximal effects were observed after 12h and 48h, respectively. Preliminary tests with several other factors (insulin, EGF, TGF-beta, FSH and hCG) showed that hCG alone directly stimulated aromatase activity. From these findings a role is suggested for FGF as a paracrine/autocrine agent in the control of estrogen secretion by Leydig cells.
Asunto(s)
Aromatasa/metabolismo , Estrógenos/biosíntesis , Factores de Crecimiento de Fibroblastos/farmacología , Células Intersticiales del Testículo/enzimología , Animales , Células Cultivadas , Gonadotropina Coriónica/farmacología , Relación Dosis-Respuesta a Droga , Insulina/farmacología , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Porcinos , Testosterona/biosíntesisRESUMEN
The present work was done to investigate the cell localization of testicular aromatase activity and its regulation in immature pig testis using an in vitro model. Leydig cells and Sertoli cells were isolated from immature pig testes and cultured alone or together in the absence or presence of human chorionic gonadotropin (hCG) or porcine follicle-stimulating hormone (pFSH) for 2 days. At the end of incubation, the amounts of testosterone (T), estrone sulfate (E1S) and estradiol (E2) were measured. Then the cells were incubated for 4 h in the presence of saturating concentrations of delta 4-androstenedione (3 microM) and the amounts of E1S and E2 were measured again (aromatase activity). The ability of Sertoli cells to produce estrogens was very low and neither hCG nor pFSH had any significant effect. hCG stimulated, in a dose-dependent manner, the secretion of T and E1S by Leydig cells cultured alone as well as the aromatase activity of these cells. The main estrogen produced by Leydig cells was E1S. pFSH also stimulated the above parameters of Leydig cell function; this may have been due to the contamination of this hormone with luteinizing hormone (LH). Coculture of Leydig cells with Sertoli cells without gonadotropins had very small effects on T and E1S production and on aromatase activity. However, treatment of coculture with increasing concentrations of hCG had a dramatic effect on Leydig cell functions. For each hCG concentration, the amounts of T and E1S secreted, as well as the aromatase activity of the coculture, were 2- to 3-fold higher than those of Leydig cells cultured alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aromatasa/metabolismo , Gonadotropinas/fisiología , Células Intersticiales del Testículo/enzimología , Células de Sertoli/fisiología , Androstenodiona/metabolismo , Animales , Células Cultivadas , Gonadotropina Coriónica/farmacología , Estradiol/metabolismo , Estrona/análogos & derivados , Estrona/metabolismo , Hormona Folículo Estimulante/farmacología , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/metabolismo , Masculino , Células de Sertoli/citología , Porcinos , Testosterona/metabolismoRESUMEN
The Epstein-Barr virus-soluble (S) antigen extracted from RAJI cells was characterized by sucrose gradient centrifugation, gel filtration, and ion-exchange chromatography. The sedimentation coefficient was estimated to be 8.5S corresponding to a molecular weight of 180,000. The S antigen binds to DEAE-A25 ion exchanger from which it can be eluted with 0.3 M NaCl in Tris buffer (pH 7.2). All fractions which contained complement-fixing S antigen also inhibited the anticomplement immunofluorescence reaction as used to detect the Epstein-Barr virus-associated nuclear antigen. These results are consistent with the hypothesis that the S and Epstein-Barr virus-associated nuclear antigens are either a single antigen or that both activities are present on the same molecule.
Asunto(s)
Antígenos Virales , Herpesvirus Humano 4/inmunología , Antígenos Virales/análisis , Línea Celular , Núcleo Celular/inmunología , SolubilidadRESUMEN
Using human sera having antibodies against Epstein-Barr virus (EBV) early antigens (EA) but lacking antibodies directed against the EB nuclear antigen (EBNA), the detection of EBV/EA was possible both by the anticomplement immunofluorescent test (ACIF) and by the classical complement fixation test (CF). The latter is particularly suitable for biochemical studies on EA.
Asunto(s)
Antígenos Virales/aislamiento & purificación , Herpesvirus Humano 4/inmunología , Anticuerpos Antivirales , Formación de Anticuerpos , Línea Celular , Pruebas de Fijación del Complemento , Técnica del Anticuerpo Fluorescente , MétodosRESUMEN
The acute and the long-term (24 h) effects of protein kinase C activators, phorbol 12 myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol, and the calcium ionophore A23187 on cultured pig Leydig cell functions were investigated. None of these drugs modified basal cAMP production, but they induced a small (3-4-fold) increase in testosterone secretion. The stimulatory effects of human choriogonadotropin (hCG; 1 nM) on both cAMP and testosterone productions were inhibited by short-term incubation with these drugs. In addition, they suppressed the stimulation of testosterone output by forskolin and 8-bromo-adenosine 3',5'-monophosphate, whereas the forskolin-dependent cAMP production was unaffected. The inhibitory effects of PMA on hCG stimulation of both cAMP and testosterone were due mainly to a decrease of the Vmax without modification of the ED50. Moreover, PMA did not modify the binding of 125I-hCG. Pretreatment of Leydig cells with the three drugs for 24 h induced more pronounced modifications, such as a reduction in the number of hCG binding sites and a decreased responsiveness to hCG and forskolin, the testosterone production being drastically reduced. The effects of PMA were dose- and time-dependent; however, the concentration of PMA required to induce half-maximal effects on hCG receptors (10 nM) was about one order of magnitude higher than those required to reduce cAMP and testosterone productions. Further, the inhibitory effects on cAMP and testosterone secretions appeared within the first 3 h, whereas the hCG receptor number remained constant for at least 8 h. It appears therefore, that the main alteration responsible for the steroidogenic refractoriness of PMA-treated Leydig cells is located beyond cAMP formation. Moreover, since conversion of exogenous pregnenolone to testosterone by control and PMA-treated cells was similar, the alteration was probably located before pregnenolone formation. Kinetic studies with 125I-hCG showed that the rate of internalization of the hormone-receptor complexes was similar in control cells and in PMA-treated cells, suggesting that the decline in receptor number observed in the latter group after an 8-h delay is not due to an increased rate of internalization nor to sequestration of the internalized receptors inside the cells. Since cycloheximide blocked the effects of PMA on hCG down-regulation, it is likely that the phorbol esters and 1-oleoyl-2-acetyl-sn-glycerol induce the synthesis of some proteins which blocked the recycling of internalized receptors. A similar hypothesis has been put forward recently to explain the hCG-induced down regulation.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Calcimicina/farmacología , Células Intersticiales del Testículo/efectos de los fármacos , Proteína Quinasa C/fisiología , Animales , Células Cultivadas , AMP Cíclico/biosíntesis , Diglicéridos/farmacología , Activación Enzimática/efectos de los fármacos , Masculino , Receptores de HL/efectos de los fármacos , Porcinos , Testosterona/biosíntesis , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Prenatal diagnosis of phenylketonuria (PKU) is now available owing to restriction fragment length polymorphism (RFLP) of the phenylalanine hydroxylase gene. Biopsies of chorionic villi were carried ou at 11 weeks gestation in 2 families who had previously a child with the classical form of PKU and who were considered informative after DNA studies. Fetal DNA was extracted and studied with restriction enzymes selected according to the study of each family. Hybridization studies suggested that one fetus was affected by the disease and that the second was normal. Termination of pregnancy was carried out in the first case; however, study of the fetus could not be performed. In the second family, the pregnancy resulted in the delivery of a normal child, as shown by normal phenylalanine level at birth.