RESUMEN

Alterations in cellular and extracellular matrix components play an important role during tumorigenesis; proteoglycans are included among these components. Ameloblastomas are odontogenic tumors distinguished as invasive and infiltrative neoplasms and are divided into different histological types, the most common of which are the unicystic ameloblastoma and the conventional ameloblastoma. The aim of this study was to identify the presence of two proteoglycans, perlecan and biglycan, in different types of ameloblastoma. Using immunohistochemistry, we determined the presence of both proteins in 28 unicystic ameloblastomas and 23 conventional ameloblastomas. We identified the cytoplasmic and nuclear presence of perlecan and the cytoplasmic presence of biglycan in both types of ameloblastoma. The mean values of immunoexpression were higher in the conventional type compared to the unicystic type. Neither the presence of biglycan in ameloblastomas nor the nuclear presence of perlecan in any odontogenic tumor has previously been reported. The differential immunoexpression of perlecan and biglycan in these types of ameloblastomas suggests their participation in the developmental process of these tumors.

Asunto(s)

Ameloblastoma , Biglicano/biosíntesis , Regulación Neoplásica de la Expresión Génica , Proteoglicanos de Heparán Sulfato/biosíntesis , Neoplasias Maxilomandibulares , Proteínas de Neoplasias/biosíntesis , Adulto , Ameloblastoma/clasificación , Ameloblastoma/metabolismo , Ameloblastoma/patología , Femenino , Humanos , Inmunohistoquímica , Neoplasias Maxilomandibulares/clasificación , Neoplasias Maxilomandibulares/metabolismo , Neoplasias Maxilomandibulares/patología , Masculino

RESUMEN

Poly(ADP-ribose) polymerase (PARP) inhibitors enhance the effect of DNA alkylating agents on BRCA1­ and BRCA2-deficient cell lines. The aim of this study was to analyze the effect of the PARP inhibitor nicotinamide (NAM) on breast cancer cells with different BRCA1 expression or function, such as BRCA1­deficient MDA-MB-436 cells, low expression BRCA1 MCF-7 cells, and the BRCA1 wild­type MDA-MB-231 cells, to demonstrate its effects as a chemo­ or radiosensitizing agent. PARP activity was analyzed in MDA-MB-436, MCF-7 and MDA-MB-231 breast cancer cells subjected or not to NAM. Inhibition of PARP by NAM in the presence of DNA damage was examined by Alexa Fluor 488 immunofluorescence. Crystal violet assays were used to test growth inhibition and the chemo­ and radiosensitization effects of NAM were investigated using clonogenic assays. Significant differences among data sets were determined using two-tailed ANOVA and Bonferroni tests. We demonstrated that NAM reduces PARP activity in vitro, and in cells subjected or not to DNA damage, it also reduces the viability of breast cancer cell lines and synergyzes the cytotoxicity of cisplatin in MDA-MB-436 and MCF-7 cells. Downregulation of PARP1 with siRNA led to modest growth inhibition, which was further increased by cisplatin. Nicotinamide also induced radiosensitization in MDA-MB-436 and MDA-MB-231 cells. In conclusion, NAM may be used as a chemo­ or radiosensitizing agent regardless of the BRCA1 status in breast cancer.

Asunto(s)

Neoplasias de la Mama/metabolismo , Cisplatino/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Niacinamida/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Apoptosis , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Daño del ADN , Sinergismo Farmacológico , Femenino , Humanos , Células MCF-7 , Radiación Ionizante

RESUMEN

The kinetic and metabolic behavior of an aerobic granular sludge to nitrify, denitrify and nitrify-denitrify was evaluated in batch cultures. In nitrification control, ammonium, 4-methylphenol and sulfide were consumed efficiently (∼100%) and recovered as NO3(-), CO2, S(0) and SO4(2-), respectively. In denitrification control, S(0) and nitrate were efficiently consumed and recovered as SO4(2-) and N2, respectively. Sequential nitrification-denitrification process was evaluated by applying oxic/anoxic conditions. Ammonium, 4-methylphenol and sulfide were oxidized to nitrate, CO2 and mainly S(0), respectively, under aerobic conditions. After that, anoxic conditions were established where S(0) reduced all nitrate to N2, with molecular nitrogen yield (YN2) of 1.03 ± 0.06 mg/mg NH4(+)-N consumed. This is the first study to show the capability of an aerobic granular sludge in simultaneous removal of ammonium, 4-methylphenol and sulfide by sequential nitrification-denitrification process in the same bioreactor.

Asunto(s)

Reactores Biológicos , Desnitrificación , Nitrificación , Compuestos de Nitrógeno/aislamiento & purificación , Fenoles/aislamiento & purificación , Compuestos de Azufre/aislamiento & purificación , Compuestos de Amonio/aislamiento & purificación , Técnicas de Cultivo Celular por Lotes , Biodegradación Ambiental , Cresoles/aislamiento & purificación , Sulfuros/aislamiento & purificación , Factores de Tiempo

RESUMEN

Ameloblastoma is the most frequent odontogenic tumor and is considered a benign, but locally invasive, neoplasm with variable clinico-pathological expression. Syndecan-1 is a cell surface proteoglycan that binds cells to the extracellular matrix and its expression is down-regulated in many cellular transformation models. The aims of this study were to examine the pattern of syndecan-1 expression, to evaluate the proliferating activity in a large series of solid/multicystic (SA) and unicystic ameloblastomas (UA), and to study its possible correlation to their biological behavior. Immunohistochemical studies were performed for syndecan-1 (clone MI15) and Ki-67 (clone MIB-1) in 120 ameloblastomas (75 SA and 45 UA). The salient finding was that expression of syndecan-1 was related to the histological subtype of tumors, as there was a lower expression in SA (40.2%) as compared to UA (49.7%) (p<0.05). These findings did not correlate with Ki-67 expression, as this was similar in both types of ameloblastomas. Our results suggest that the reduced expression of syndecan-1 supports the view that SA has a more aggressive biological behavior than the UA. The lack of correlation between reduction of the syndecan-1 and Ki-67 index may be due to the different histomorphologies of both types of ameloblastoma, and more studies are necessary to better understand the role of this protein in the biological behavior of these tumors.

Asunto(s)

Ameloblastoma/metabolismo , Antígeno Ki-67/metabolismo , Proteínas de Neoplasias/metabolismo , Sindecano-1/metabolismo , Adolescente , Adulto , Anciano , Niño , Preescolar , Regulación hacia Abajo , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mucosa Bucal/metabolismo , Germen Dentario/metabolismo , Adulto Joven

RESUMEN

The pituitary gonadotropins--luteinizing hormone and follicle-stimulating hormone--as well as the placental choriogonadotropin belong to the family of glycoprotein hormones. These structurally related hormones, which regulate several major reproductive functions of the body, are heterodimers consisting of a common alpha-subunit noncovalently bound to a beta-subunit. The N- and O-linked oligosaccharide chains of these gonadotropins play an important role in intracellular folding, assembly, secretion, metabolic clearance, and biological activity of the hormone. Gonadotropin glycosylation is a highly complex process; within the gonadotropes it is modulated by a variety of extrapituitary factors of hypothalamic and gonadal origin. In particular, estrogens and androgens appear to regulate terminal sialylation and/or sulfation of the oligosaccharide attachments and hence some functional properties of the gonadotropin molecule determined by these residues, i.e., metabolic clearance and in vivo biopotency. Through these extrapituitary inputs, the anterior pituitary may not only regulate the quantity but also the quality of the gonadotropin signal delivered to the gonads in a given physiologic or pathologic condition.

Asunto(s)

Hormonas Esteroides Gonadales/fisiología , Gonadotropinas Hipofisarias/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Secuencias de Aminoácidos , Andrógenos/farmacología , Andrógenos/fisiología , Animales , Secuencia de Carbohidratos , Castración , Gonadotropina Coriónica/química , Gonadotropina Coriónica/metabolismo , Enfermedades del Sistema Endocrino/metabolismo , Retículo Endoplásmico Rugoso/metabolismo , Estrógenos/farmacología , Estrógenos/fisiología , Femenino , Hormona Folículo Estimulante/química , Hormona Folículo Estimulante/metabolismo , Glicosilación , Hormonas Esteroides Gonadales/farmacología , Hormona Liberadora de Gonadotropina/fisiología , Gonadotropinas Hipofisarias/química , Humanos , Sistema Hipotálamo-Hipofisario/fisiología , Hormona Luteinizante/química , Hormona Luteinizante/metabolismo , Masculino , Mamíferos/fisiología , Tasa de Depuración Metabólica , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico/metabolismo , Oligosacáridos/metabolismo , Adenohipófisis/metabolismo , Placenta/metabolismo , Embarazo , Pliegue de Proteína , Ratas , Relación Estructura-Actividad , Tirotropina/fisiología , Hormona Liberadora de Tirotropina/fisiología

RESUMEN

Levonorgestrel (13beta-ethyl-17alpha-ethynyl-17beta-hydroxy-4-gonen-3-one), a potent contraceptive progestin stimulates growth and proliferation of cultured breast cancer cells through a receptor-mediated mechanism, even though levonorgestrel does not bind to the oestrogen receptor (ER). To assess whether the oestrogen-like effects induced by this synthetic progestin are exerted via its metabolic conversion products, we studied the binding affinity of three A-ring levonorgestrel derivatives to the ER and their capability to transactivate an oestrogen-dependent yeast system co-transfected with the human ER gene and oestrogen responsive elements fused to a beta-galactosidase reporter vector. The results demonstrated that the 3beta,5alpha reduced levonorgestrel derivative and to a lesser extent its 3alpha isomer interact with the oestrogen receptor, with a significantly lower relative binding affinity (2.4% and 0.4%, respectively) than that of oestradiol (100%), while levonorgestrel does not. Both levonorgestrel metabolites were able to activate, in a dose-dependent manner, the beta-galactosidase reporter gene in the yeast expression system, an effect that was precluded by a steroidal antioestrogen. The oestrogenic potency of levonorgestrel metabolites was significantly lower (750-fold) than that of oestradiol. Furthermore, high doses of 3beta,5alpha levonorgestrel (2.5 mg/day/6 days) induced an increase of oestrogen-dependent progestin receptor in the anterior pituitary of castrated rats. The overall data offer a plausible explanation for the weak oestrogenic effects induced by high, non-pharmacological doses of levonorgestrel.

Asunto(s)

Anticonceptivos Femeninos/farmacología , Estrógenos/farmacología , Levonorgestrel/farmacología , Animales , Unión Competitiva , Anticonceptivos Femeninos/metabolismo , Relación Dosis-Respuesta a Droga , Estradiol/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Estrógenos/metabolismo , Femenino , Fulvestrant , Humanos , Levonorgestrel/análogos & derivados , Levonorgestrel/metabolismo , Adenohipófisis/efectos de los fármacos , Adenohipófisis/metabolismo , Ratas , Ratas Wistar , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/efectos de los fármacos , Receptores de Progesterona/metabolismo , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , beta-Galactosidasa/efectos de los fármacos , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

Gestodene (13beta-ethyl-17alpha-ethynyl-17beta-hydroxy-4,5-gonadien-3-one), the most potent progestin ever synthesized, stimulates breast cancer cell growth through an oestrogen receptor-mediated mechanism, and its use in hormonal contraception has been associated with side effects attributable to oestrogenic actions. These observations have remained controversial, since gestodene does not bind to the oestrogen receptor or exert oestrogen-like activities. Recently, we have demonstrated that non-phenolic gestodene derivatives interact with oestrogen receptors and induce oestrogenic effects in cell expression systems. To assess whether gestodene is biotransformed to metabolites with intrinsic oestrogenic potency, [3H]- and [14C]-labelled gestodene were incubated in vitro with rat anterior pituitary, hypothalamus and ventral prostate homogenates under different experimental conditions. The most remarkable finding was the isolation and identification of 3beta,5alpha-tetrahydrogestodene and 3alpha,5alpha-tetrahydrogestodene as metabolic conversion products of gestodene, presumably with 5alpha-dihydrogestodene as intermediate. The overall results seem to indicate that the weak oestrogenic effects attributable to gestodene could be mediated by its tetrahydro metabolites.

Asunto(s)

Hipotálamo/metabolismo , Norpregnenos/química , Norpregnenos/metabolismo , Adenohipófisis/metabolismo , Próstata/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Animales , Biotransformación , Anticonceptivos Sintéticos Orales/química , Anticonceptivos Sintéticos Orales/metabolismo , Anticonceptivos Sintéticos Orales/farmacocinética , Femenino , Concentración de Iones de Hidrógeno , Hipotálamo/enzimología , Masculino , NADP/metabolismo , Norpregnenos/farmacocinética , Adenohipófisis/enzimología , Congéneres de la Progesterona/química , Congéneres de la Progesterona/metabolismo , Congéneres de la Progesterona/farmacocinética , Próstata/enzimología , Ratas , Ratas Wistar , Testosterona/metabolismo

RESUMEN

The aim of present study is the analysis of monoamines concentrations changes in the anterior, medium and posterior hypothalamus, as well as changes in serum gonadotropins levels, ovarian steroids and follicular growth during the prepubertal development of the female rat. Noradrenergic activity in the anterior, medium and posterior hypothalamus reached highest level at day 13 after birth, followed by a subsequent decrease from day 15 to 19 and an increase on days 22 and 27 postnatal. At day 1, neural activity in the medium hypothalamus was higher than the activity in the anterior and posterior hypothalamus. Serotoninergic activity in three portions of the hypothalamus was higher throughout the prepubertal development. Follicle-stimulating hormone and luteinizing hormone serum levels increased between days 11 and 17 and decreased from day 19 to 36. The concentration of 17beta-estradiol was consistently low throughout the prepubertal development and increased at day 39 after birth. These results indicate that during the prepubertal development of the rat, the three regions of the hypothalamus show significant changes in the monoaminergic neural activity. There is an inverse relationship between the noradrenergic activity on the anterior and medium hypothalamus and serotoninergic activity in the posterior hypothalamus with ovarian steroids during sexual maturation. These changes may be linked to the development of the neuroendocrine processes that modulate gonadotropin secretion and ovarian function.

Asunto(s)

Monoaminas Biogénicas/metabolismo , Hipotálamo Anterior/metabolismo , Hipotálamo Medio/metabolismo , Hipotálamo Posterior/metabolismo , Maduración Sexual/fisiología , Animales , Estradiol/sangre , Femenino , Hormona Folículo Estimulante/sangre , Gonadotropinas/metabolismo , Hormona Luteinizante/sangre , Tamaño de los Órganos/fisiología , Folículo Ovárico/metabolismo , Ovario/metabolismo , Ratas , Útero/metabolismo

RESUMEN

Gestodene (17 alpha-ethynyl-13 beta-ethyl-17 beta-hydroxy-4, 15-gonadien-3-one) is the most potent synthetic progestin currently available and it is widely used as a fertility regulating agent in a number of contraceptive formulations because of its high effectiveness, safety and acceptability. The observation that contraceptive synthetic progestins exert hormone-like effects other than their progestational activities, prompted us to investigate whether gestodene (GSD) administration may induce oestrogenic effects, even though the GSD molecule does not interact with intracellular oestrogen receptors (ER). To assess whether GSD may exert oestrogenic effects through some of its neutral metabolites, a series of experimental studies were undertaken using GSD and three of its A-ring reduced metabolites. Receptor binding studies by displacement analysis confirmed that indeed GSD does not bind to the ER, whereas its 3 beta,5 alpha-tetrahydro reduced derivative (3 beta GSD) interacts with a relative high affinity with the ER. The 3 alpha,5 alpha GSD isomer (3 alpha GSD) also binds to the ER, though to a lesser extent. The ability of the A-ring reduced GSD derivatives to induce oestrogenic actions was evaluated by the use of two different molecular bioassays: (a) transactivation of a yeast system co-transfected with the human ER alpha (hER alpha) gene and oestrogen responsive elements fused to the beta-galactosidase reporter vector and (b) transactivation of the hER alpha-mediated transcription of the chloramphenicol acetyl transferase (CAT) reporter gene in a HeLa cells expression system. The oestrogenic potency of 3 beta GSD was also assessed by its capability to induce oestrogen-dependent progestin receptors (PR) in the anterior pituitary of castrated female rats. The results demonstrated that 3 beta GSD and 3 alpha GSD were able to activate, in a dose-dependent manner, the hER alpha-mediated transcription of both the beta-galactosidase and the CAT reporter genes in the yeast and HeLa cells expression systems respectively. In both assays the 3 beta derivative of GSD exhibited a significantly greater oestrogenic effect than its 3 alpha isomer, while unchanged GSD and 5 alpha GSD were completely ineffective. Neither 3 beta GSD nor 3 alpha GSD exhibited oestrogen synergistic actions. Interestingly, the pure steroidal anti-oestrogen ICI-182,780 diminished the transactivation induced by 3 beta GSD and 3 alpha GSD in the yeast expression system. Furthermore, administration of 3 beta GSD resulted in a significant increase of oestrogen-dependent PR in the anterior pituitaries of castrated rats in comparison with vehicle-treated animals. The characteristics of the 3 beta GSD-induced PR were identical to those induced by oestradio benzoate. The overall results demonstrate that 3 beta GSD and its 3 alpha isomeric alcohol specifically bind to the ER and possess a weak intrinsic oestrogenic activity, whereas unmodified GSD does not. The data contribute to a better understanding of the GSD mechanism of action and allow the hypothesis to be advanced that the slight oestrogenlike effects attributable to GSD are mediated by its non-phenolic, tetrahydro reduced metabolites.

Asunto(s)

Anticonceptivos Sintéticos Orales/farmacología , Norpregnenos/farmacología , Congéneres de la Progesterona/farmacología , Animales , Unión Competitiva , Anticonceptivos Sintéticos Orales/metabolismo , Femenino , Células HeLa , Humanos , Norpregnenos/metabolismo , Oxidación-Reducción , Adenohipófisis/efectos de los fármacos , Adenohipófisis/metabolismo , Congéneres de la Progesterona/metabolismo , Ratas , Ratas Wistar , Receptores de Estradiol/metabolismo , Receptores de Progesterona/biosíntesis , Saccharomyces cerevisiae/genética , Activación Transcripcional/efectos de los fármacos

RESUMEN

Follicle-stimulating hormone (FSH) is synthesized by the anterior pituitary gland in multiple molecular forms. Increased acidic/sialylated FSH charge isoforms are associated with conditions characterized by a low oestrogen output. In the present study, we analysed the dynamics of the changes in mRNA levels of the enzyme Galbeta1,3[4]GlcNAc alpha2,3-sialyltransferase (2,3-STase) (one of the enzymes that incorporate sialic acid residues into the FSH molecule) in intact and ovariectomized rats. The anterior pituitaries of 4-day regularly cyclic adult female Wistar rats were obtained at 1000 h on the days of pro-oestrus (P), oestrus (O), dioestrus 1 (D1) and dioestrus 2 (D2), at 0200 h, 1400 h, 1800 h and 2200 h on D1, at 1800 h on day of O and at 1000 h after 7, 14, 21, 28 and 45 days of oophorectomy performed on the morning of P. Total RNA was isolated from each gland and the 2,3-STase levels were measured by Northern blot hybridization analysis employing a 346-base pair cDNA probe encoding for a non-conserved amino acid sequence of the catalytic domain of the enzyme. Maximal levels of the enzyme mRNA were detected at 1000 h on D1; thereafter, they progressively decreased by 60% during the ensuing 24 h, reaching the lowest concentration values (26% of the maximally observed level on D1) at 1000 h on day of P and remaining unchanged during the morning of O. Administration of the potent oestradiol receptor antagonist ICI 182,780 at 1000 h on D1 completely reverted the time-dependent decrease in 2,3-STase mRNA levels observed during the afternoon of D1, whereas oestradiol benzoate administered at 1000 h on day of O significantly reduced the enzyme mRNA levels (to 21% of the levels detected in vehicle-treated controls). In ovariectomized rats, the alpha2,3-STase mRNA progressively increased from day 21 to day 45 post castration. Administration of oestradiol benzoate on day 28 after oophorectomy significantly reduced the 2,3-STase mRNA levels (to 36% of the levels detected in vehicle-injected controls); ICI 182,780 partially counteracted this oestradiol-mediated effect. The dynamics of these changes in 2,3-STase mRNA levels partially correlated with changes in the relative abundance of the FSH charge isoforms separated by preparative chromatofocusing of anterior pituitary extracts, particularly in glands obtained during the morning of P and O. These data demonstrate for the first time that pituitary 2,3-STase is a hormonally-regulated enzyme and that the changes in transcription and/or stability of its mRNA may be involved, in part, in the post-translational processing of the FSH molecule during certain physiological conditions.

Asunto(s)

Estrógenos/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Adenohipófisis/enzimología , ARN Mensajero/genética , Sialiltransferasas/genética , Animales , Secuencia de Bases , ADN Complementario , Estradiol/sangre , Femenino , Hormona Folículo Estimulante/sangre , Ratas , Ratas Wistar , beta-Galactosida alfa-2,3-Sialiltransferasa

RESUMEN

In the present studies we analysed the main physicochemical and biological properties of the several isoforms of human pituitary follicle-stimulating hormone (hFSH). Extracts of total anterior pituitary glycoproteins from adult donors were submitted to chromatofocusing and several forms of immunoactive hFSH with isoelectric points (pI) ranging from 7.6 to 3.8 were identified. An additional isoform was detected after passing through the chromatofocusing column a 1.0 M NaCl solution (salt peak). Each hFSH isoform or pool of neighbouring isoforms (pI value 7.6-7.1, pool I, 1.5 +/- 0.13% of total immunoactivity recovered; pI value 5.9-5.3, pool II, 8.9 +/- 1.6% of total; pI value 5.0-4.7, pool III, 14.4 +/- 1.4% of total; pI value 4.5-4.1, pool IV, 54.8 +/- 4.9% of total; pI value 3.9-3.8, pool V, 3.67 +/- 0.9% of total; salt peak, pool VI, 16.8 +/- 4.8% of total) eluted as single hFSH peaks after Sephadex G-100 exclusion chromatography (apparent Mr 60,000). Even though hFSH present within each pool was recognized by a receptor preparation, the receptor-binding activity expressed as the radioreceptor assay:radioimmunoassay (RRA/RIA) activity ratio varied with the pI value of the particular hFSH isoform tested; starting from a pI value of 5.9, the receptor-binding activity of hFSH decreased from 4.25 +/- 0.28 to 1.17 +/- 0.14, as the pI value of the corresponding isoform declined. A similar trend was observed when the potency of each isoform was assessed by an in vitro bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Hormona Folículo Estimulante/análogos & derivados , Animales , Bioensayo , Cromatografía en Gel , Hormona Folículo Estimulante/aislamiento & purificación , Hormona Folículo Estimulante/farmacocinética , Semivida , Humanos , Punto Isoeléctrico , Masculino , Radioinmunoensayo , Ensayo de Unión Radioligante , Ratas , Ratas Endogámicas , Extractos de Tejidos

RESUMEN

In the present study, we investigated the biological characteristics of different molecular forms of chorionic gonadotrophin (HCG) secreted by the human cytotrophoblast during its morphological and functional differentiation in culture. Highly purified cytotrophoblasts were prepared from term placentae and cultured for 24 to 96 h in the absence or presence of 8-bromo-3',5'-cAMP. Media were collected at 24 h intervals and the secreted isoforms of HCG were then separated by polyacrylamide gel isoelectric focusing (pH range 8.0-3.0) and quantified by radioimmunoassay. The secretion of HCG was significantly increased by 8-bromo-cAMP (from 23.5 +/- 6.3 ng/ml at 24 h to 1619 +/- 835.8 ng/ml at 96 h; controls, 9.3 +/- 0.1 ng/ml at 24 h and 26.6 +/- 3.5 ng/ml at 96 h, mean +/- SD). Analysis of media concentrates from cAMP-stimulated cultures by isoelectric focusing revealed the presence of several distinct peaks of HCG within the pH range of 7.3-4.8; major peaks consistently exhibited isoelectric points (pI) of 7.3-7.0 (peak 1), 5.6-5.4 (peak 2) and 5.1-4.8 (peak 3). The relative HCG content of the most acidic peak (as % of total on gel) progressively increased with time of exposure to the cAMP analogue (from 19.8 +/- 1.6% at 24 h to 34.4 +/- 4.3% at 96 h, mean +/- SEM, P less than 0.01). HCG recovered from peak 1 exhibited the highest receptor-binding capacity and in-vitro biological potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Gonadotropina Coriónica/química , Gonadotropina Coriónica/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismo , Bioensayo , Células Cultivadas , Gonadotropina Coriónica/análisis , Gonadotropina Coriónica Humana de Subunidad beta , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Humanos , Focalización Isoeléctrica , Neuraminidasa , Fragmentos de Péptidos/análisis , Radioinmunoensayo , Ensayo de Unión Radioligante

RESUMEN

Anterior pituitary glands were removed from neonatally androgenized (100 micrograms testosterone propionate) female rats and normal controls at 5, 10, 18, 21, 30, 60 and 90 days of age, and the multiple forms of FSH present within them were separated by chromatofocusing (pH range 7.5-4.0). Additional pituitary glands from intact adult males (90 days old) were also studied for comparative purposes. All animal groups exhibited multiple forms of immunoactive FSH within a pH range of 7.5-4.0, as well as an additional FSH form obtained after the addition of 1.0 mol NaCl/l to the chromatofocusing column (salt peak). In animals 5-30 days old (controls and androgenized) the majority of FSH applied to the chromatofocusing columns was recovered within the salt peak (45-85% of total FSH immunoactivity recovered). However, as the animals aged, more FSH immunoactivity focused within less acidic regions (isoelectric point (pI) 5.9-5.0); pituitaries from animals 60 days old contained the greatest proportion of FSH focused within this pH range (controls, 39.2 +/- 0.6%; androgenized, 23.1 +/- 0.9% of total immunoactivity recovered; P less than 0.03 vs animals 30 days old for both experimental groups). This shift towards less acidic FSH was attenuated in androgenized animals compared with the controls (P less than 0.01). In control adult rats, the chromatofocusing distribution pattern of pituitary FSH varied according to the day of the oestrous cycle. Pituitary extracts from control rats decapitated during the morning of pro-oestrus, oestrus and day 1 of dioestrus exhibited the highest proportion of immunoactive FSH (23.2-28.8% of total) focused within a pH range of 5.9-5.0, whilst only 10.4-11.6% of FSH from androgenized rats and those on day 1 of dioestrus was recovered within this pH range (P less than 0.05). In control animals decapitated during the morning of pro-oestrus and oestrus, 10-26% of FSH focused within the most alkaline region (pI 7.5-6.0); the chromatofocusing pattern of pituitary FSH from the neonatally androgenized animals was characteristic, in that no more than one peak (1.5 +/- 0.5% of total) was detected in this alkaline region. In the adult male rats, the majority of pituitary FSH eluted from the chromatofocusing columns within a pH of 4.9-4.0 (52.4 +/- 1.2% of total FSH immunoactivity) and the salt peak (pH less than 4.0) (33.1 +/- 2.4 of total). All FSH isoforms obtained after chromatofocusing represented alpha and beta dimers as disclosed by size exclusion chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Hormona Folículo Estimulante/análisis , Adenohipófisis/análisis , Testosterona/farmacología , Animales , Cromatografía por Intercambio Iónico , Femenino , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Masculino , Folículo Ovárico/efectos de los fármacos , Adenohipófisis/efectos de los fármacos , Embarazo , Radioinmunoensayo , Ratas , Ratas Endogámicas

RESUMEN

Anterior pituitary (AP) glands were removed from adult female rats at different times throughout the estrous cycle, and the isohormones of follicle-stimulating hormone (FSH) present within them were separated by isoelectric focusing in polyacrylamide gels (PAGE-IEF; pH range 3.0-8.0). Gel eluents were analyzed for FSH content by radioimmunoassay (RIA) and radioreceptor assay (RRA). All AP samples exhibited several peaks of FSH immunoactivity within a pH range of 6.2-4.0; the major peak constantly exhibited an isoelectric point (pI) of 4.9-4.5. To quantify differences in the IEF pattern of AP-FSH between the pituitaries collected during the different days of the cycle, each PAGE-IEF profile was divided into 7 regions (pI 7.0-6.3 = Area 1, 6.2-5.5 = Area 2, 5.4-5.0 = Area 3, 4.9-4.5 = Area 4, 4.4-4.0 = Area 5, 3.9-3.5 = Area 6, and less than 3.5 = Area 7), and the amount of FSH present within each was determined. In all APs collected at 0900 h of diestrus 1 (D1) and 2 (D2), proestrus (P), and estrus (E); at 1300 h of D1, D2 and E; at 2200 h of P; and at 0200 h of E, the majority of FSH immunoactivity (37-55% of total FSH on gel) focused within Area 4, whereas Areas 2 and 3 contained minor amounts of FSH activity (11-26% and 14-24%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Estro/fisiología , Hormona Folículo Estimulante/análisis , Adenohipófisis/análisis , Animales , Cromatografía , Electroforesis en Gel de Poliacrilamida , Femenino , Focalización Isoeléctrica , Adenohipófisis/fisiología , Radioinmunoensayo , Ensayo de Unión Radioligante , Ratas