RESUMEN
PURPOSE: To gain new insight into the roles of cruciferous vegetable-derived bioactive phytochemicals in bone cells, we investigated the effects of indole-3-carbinol (I3C) on cell proliferation and differentiation in estradiol (E2)-exposed calvarial osteoblasts that were obtained from neonatal rats. METHODS: Osteoblast activity was assessed by analyzing cellular DNA, cell-associated osteocalcin (OC) levels and alkaline phosphatase (AP) activity. We also examined [(3)H]-estrone (E1) metabolism and estrogen-agonistic and estrogen-antagonistic activities of 2-hydroxy (OH) E1 and 2-OHE2 and their capacity to displace [(3)H]-E2 at ER binding sites using competition studies. RESULTS: I3C did not affect on cellular DNA, OC levels or AP activity. However, I3C completely inhibited E2-induced increases in cell proliferation and differentiation in neonatal rat osteoblasts. Metabolic studies demonstrated that I3C promoted the conversion of [(3)H]-E1 to 2-OHE1 and 2-OHE2 and those higher rates of conversion (twofold-threefold) were archived when a higher dose of I3C was applied. Proliferation and differentiation studies showed that 2-OHE2 but not 2-OHE1 inhibited E2-induced increases in cell proliferation and differentiation via an ER-mediated mechanism. Likewise, Esr1 was expressed at high level than Esr2. 2-OHE1 showed no activity or affinity for ER. CONCLUSIONS: This study is the first to show that a bioactive compound derived from cruciferous vegetables, I3C, abolishes the E2-mediated stimulation of cell activities including, proliferation and differentiation, in rat osteoblasts and increases the 2-hydroxylation of E1, resulting in the formation of inactive and anti-estrogenic metabolites. These results suggest that in neonatal rat osteoblasts, the anti-estrogenic effect of I3C is mediated by 2-OHE2 through ER-α.
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Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Antagonistas del Receptor de Estrógeno/farmacología , Indoles/farmacología , Osteoblastos/efectos de los fármacos , Animales , Animales Recién Nacidos , Anticarcinógenos/farmacología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Estradiol/farmacología , Femenino , Osteoblastos/citología , Osteoblastos/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Estrógenos/química , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The human ether à go-go 1 potassium channel (hEAG1) is required for cell cycle progression and proliferation of cancer cells. Inhibitors of hEAG1 activity and expression represent potential therapeutic drugs in cancer. Previously, we have shown that hEAG1 expression is downregulated by calcitriol in a variety of cancer cells. Herein, we provided evidence on the regulatory mechanism involved in such repressive effect in cells derived from human cervical cancer. Our results indicate that repression by calcitriol occurs at the transcriptional level and involves a functional negative vitamin D response element (nVDRE) E-box type in the hEAG1 promoter. The described mechanism in this work implies that a protein complex formed by the vitamin D receptor-interacting repressor, the vitamin D receptor, the retinoid X receptor, and the Williams syndrome transcription factor interact with the nVDRE in the hEAG1 promoter in the absence of ligand. Interestingly, all of these transcription factors except the vitamin D receptor-interacting repressor are displaced from hEAG1 promoter in the presence of calcitriol. Our results provide novel mechanistic insights into calcitriol mode of action in repressing hEAG1 gene expression.
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Calcitriol/farmacología , Canales de Potasio Éter-A-Go-Go/genética , Receptores de Calcitriol/genética , Neoplasias del Cuello Uterino/genética , Elemento de Respuesta a la Vitamina D/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Regulación hacia Abajo , Ensayo de Cambio de Movilidad Electroforética , Femenino , Humanos , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Maternal obesity (MO) impairs maternal and offspring health. Mechanisms and interventions to prevent adverse maternal and offspring outcomes need to be determined. Human studies are confounded by socio-economic status providing the rationale for controlled animal data on effects of maternal exercise (MEx) intervention on maternal (F0) and offspring (F1) outcomes in MO. HYPOTHESIS: MO produces metabolic and endocrine dysfunction, increases maternal and offspring glucocorticoid exposure, oxidative stress and adverse offspring outcomes by postnatal day (PND) 36. MEx in part prevents these outcomes. METHODS: F0 female rats ate either control or obesogenic diet from weaning through lactation. Half of each group wheel ran (from day 90 of life through pregnancy beginning day 120) providing four groups (n=8/group)--(i) controls, (ii) obese, (iii) exercised controls and (iv) exercised obese. After weaning, PND 21, F1 offspring ate a control diet. Metabolic parameters of F0 prepregnancy and end of lactation and F1 offspring at PND 36 were analyzed. RESULTS: Exercise did not change maternal weight. Before breeding, MO elevated F0 glucose, insulin, triglycerides, cholesterol, leptin, fat and oxidative stress. Exercise completely prevented the triglyceride rise and partially increases glucose, insulin, cholesterol and oxidative stress. MO decreased fertility, recovered by exercise. At the end of lactation, exercise returned all metabolic variables except leptin to control levels. Exercise partially prevented MO elevated corticosterone. F1 offspring weights were similar at birth. At PND 36, MO increased F1 male but not female offspring leptin, triglycerides and fat mass. In controls, exercise reduced male and female offspring glucose, prevented the offspring leptin increase and partially the triglyceride rise. CONCLUSIONS: MEx before and during pregnancy has beneficial effects on the maternal and offspring metabolism and endocrine function occurring with no weight change in mothers and offspring indicating the importance of body composition rather than weight in evaluations of metabolic status.
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Lactancia/metabolismo , Leptina/sangre , Obesidad/metabolismo , Preñez , Efectos Tardíos de la Exposición Prenatal/metabolismo , Adiposidad , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Glucemia/metabolismo , Dieta Alta en Grasa , Femenino , Resistencia a la Insulina/fisiología , Masculino , Fenómenos Fisiologicos Nutricionales Maternos , Condicionamiento Físico Animal , Embarazo , Ratas , Ratas Wistar , DesteteRESUMEN
Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm-ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein-protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.
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Proteínas del Huevo/genética , Glicoproteínas de Membrana/genética , Receptores de Superficie Celular/genética , Capacitación Espermática/genética , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Reacción Acrosómica/efectos de los fármacos , Adulto , Anticuerpos/farmacología , Sitios de Unión , Unión Competitiva , Proteínas del Huevo/metabolismo , Proteínas del Huevo/farmacología , Epidídimo/citología , Epidídimo/efectos de los fármacos , Epidídimo/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacología , Unión Proteica , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal , Capacitación Espermática/efectos de los fármacos , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Zona Pelúcida/efectos de los fármacos , Glicoproteínas de la Zona PelúcidaRESUMEN
The objective of this investigation was to analyse the carriage rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faecal samples of healthy humans in Tunisia and to characterise the recovered isolates. One hundred and fifty samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 µg/ml) for ESBL-positive E. coli recovery. The characterisation of ESBL genes and their genetic environments, detection of associated resistance genes, multilocus sequence typing (MLST) and phylogroup typing were performed by polymerase chain reaction (PCR) and sequencing. The presence and characterisation of integrons and virulence factors were studied by PCR and sequencing. ESBL-positive E. coli isolates were detected in 11 of 150 faecal samples (7.3%) and one isolate/sample was further characterised. These isolates contained the blaCTX-M-1 (ten isolates) and blaTEM-52c genes (one isolate). The ISEcp1 (truncated by IS10 in four strains) and orf477 sequences were found upstream and downstream, respectively, of all bla (CTX-M-1) genes. Seven different sequence types (STs) and three phylogroups were identified among CTX-M-1-producing isolates [ST/phylogroup (number of isolates)]: ST58/B1 (3), ST57/D (2), ST165/A (1), ST155/B1 (1), ST10/A (1), ST398/A (1) and ST48/B1 (1). The TEM-52-producing isolate was typed as ST219 and phylogroup B2. Six ESBL isolates contained class 1 integrons with the gene cassettes dfrA17-aadA5 (five isolates) and dfrA1-aadA1 (one). Healthy humans in the studied country could be a reservoir of CTX-M-1-producing E. coli.
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Portador Sano/epidemiología , Portador Sano/microbiología , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/enzimología , Escherichia coli/aislamiento & purificación , beta-Lactamasas/genética , Adolescente , Adulto , Anciano , Animales , Niño , Preescolar , Escherichia coli/genética , Femenino , Voluntarios Sanos , Humanos , Lactante , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Prevalencia , Análisis de Secuencia de ADN , Túnez/epidemiología , Adulto JovenRESUMEN
BACKGROUND: The present study sought to evaluate the possibility of using the splenic artery for arterialization of a living donor liver graft. PATIENTS AND METHODS: In the period between August 2004 and April 2006, we performed 31 adult-to-adult living donor liver transplantations. In 27 patients (group A), the right or left hepatic artery was used to arterialize the graft, whereas in the other four cases (group B), we used the recipient splenic artery. RESULTS: The Model for End-stage Liver Disease (MELD) score of the patients averaged 17 (17.2 and 15.2 for groups A and B, respectively) ranging between 7 and 28. We did not observe pancreatitis, splenic infarction, or other complications related to ligation of the splenic artery. Two cases (6.4%) of arterial complication were observed, both in group A patients. CONCLUSION: The use of the splenic artery is a safe, practical alternative for arterial reconstruction in living donor liver transplantation procedures, when the hepatic artery is not adequate or in cases of portal hypertension with splenomegaly.
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Trasplante de Hígado/métodos , Donadores Vivos , Arteria Esplénica/cirugía , Adulto , Femenino , Humanos , Circulación Hepática , Fallo Hepático/cirugía , Masculino , Procedimientos de Cirugía Plástica , Estudios RetrospectivosRESUMEN
The diversity of structures carrying the aac(6')-aph(2") gene was studied in 46 high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium clinical strains recovered in a Tunisian hospital during the period 2000-2003. The inclusion of the aac(6')-aph(2") gene within the Tn4001 composite element or in its truncated forms (lacking the IS256 at the right, the left or at both sides of the aac(6')-aph(2") gene) was investigated by PCR and sequencing. The aac(6')-aph(2") gene was included in the composite Tn4001 element in 19 of 34 high-level gentamicin-resistant E. faecalis strains (56%) and in 1 of 12 E. faecium strains (12%). A truncated form of Tn4001 lacking IS256 at the left-hand (in 10 E. faecalis and 8 E. faecium), at the right-hand (3 E. faecalis and 2 E. faecium) or at both sides of the aac(6')-aph(2") gene (in 2 E. faecalis and 1 E. faecium) was also detected in 26 of our enterococci. The transference by conjugation of the aac(6')-aph(2") gene, associated with other resistance genes, was demonstrated in seven of the high-level gentamicin-resistant E. faecalis strains.
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Acetiltransferasas/genética , ADN Bacteriano/genética , Enterococcus faecalis/genética , Enterococcus faecium/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Conjugación Genética , Elementos Transponibles de ADN/genética , Farmacorresistencia Bacteriana/genética , Gentamicinas/farmacología , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Infecciones por Bacterias Grampositivas/microbiología , Humanos , TúnezRESUMEN
Previous work in [3H]inositol-labelled GH3 pituitary tumor cells stimulated with thyrotropin-releasing hormone (TRH) reported the existence of at least ten distinct [3H]inositol-containing substances which were identified as different inositol mono-, bis- and tris-phosphate isomers [1]. Here a complete kinetic study of the dephosphorylation pathways of the second messenger Ins(1,4,5)P3 is reported in GH3 cell homogenates, identifying a new intermediate, Ins(4,5)P2, in the metabolism of the second messenger. in vitro results obtained with exogenous substrates are compared with in vivo results obtained measuring levels of the endogenous [3H]inositol-labelled isomers that participate in the dephosphorylation pathways of Ins(1,4,5)P3 in resting and TRH-stimulated GH3 cells. The effect of Li+ on the activity of the different phosphatases involved in these pathways is studied as well.
Asunto(s)
Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Animales , Línea Celular , Inositol Polifosfato 5-Fosfatasas , Cinética , Litio/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Neoplasias Hipofisarias/metabolismo , Ratas , Hormona Liberadora de Tirotropina/farmacología , Tritio , Células Tumorales Cultivadas/metabolismoRESUMEN
Red wine amino acids and volatile compounds were analyzed before and after malolactic fermentation carried out by four different starter cultures of the species Oenococcus oeni and Lactobacillus plantarum. The purpose of this study was to determine whether differences can be attributed to the lactic acid bacteria strain used in this important step of the wine-making process. The malolactic cultures selected for this study were indigenous wine lactic acid bacteria strains. The data were evaluated using different multivariate analysis techniques. Results showed different malolactic behaviors for O. oeni and L. plantarum and significant metabolic differences between both species. A degree of diversity was found within each lactic acid bacteria group, since wines presented specific characteristics depending on the lactic acid bacteria strain used. In all cases, malolactic fermentation seemed to modify the amino acid and volatile composition of the wine.
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Aminoácidos/análisis , Fermentación , Lactobacillus plantarum/metabolismo , Leuconostoc/metabolismo , Malato Deshidrogenasa/metabolismo , Vino/análisis , Alcoholes/análisis , Ésteres/análisis , Ácidos Grasos Volátiles/análisis , VolatilizaciónRESUMEN
It has previously been demonstrated that 19-nor contraceptive progestins undergo in vivo and in vitro enzyme-mediated A-ring double bond hydrogenation. Bioconversion of 19-nor progestins to their corresponding tetrahydro derivatives results in the loss of progestational activity and acquisition of estrogenic activities and binding to the ER. Herein, we report subtype-selective differences in ligand binding and transcriptional potency of nonphenolic synthetic 19-nor derivatives between ER alpha and ER beta. In this study, we have examined both ER- and PR-mediated transcriptional activity of a number of A-ring chemically reduced derivatives of norethisterone and Gestodene. Double bond hydrogenation decreased the transcriptional potency of norethisterone and Gestodene through both PR isoforms with a 100- to 1,000-fold difference, respectively. In terms of the effects of norethisterone and Gestodene and their corresponding 5 alpha-dihydro (5 alpha-norethisterone and 5 alpha-Gestodene), or 3 alpha,5 alpha-tetrahydro or 3 beta,5 alpha-tetrahydro derivatives (3 alpha,5 alpha-norethisterone/3 alpha,5 alpha-Gestodene and 3 beta,5 alpha-norethisterone/3beta,5 alpha-Gestodene, respectively) on estrogen-mediated transcriptional regulation, the 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene showed the highest induction when HeLa cells were transiently transfected with an expression vector for ER alpha. This activity could be inhibited with tamoxifen. These compounds did not activate gene transcription via ER beta, and none of them showed antagonistic activities through either ER subtype. The 3 beta,5 alpha-tetrahydro derivatives of both norethisterone and Gestodene were active in other cells in addition to HeLa cells and activated reporter expression through the oxytocin promoter. In summary, two ER alpha selective agonists have been identified. These compounds, with ER alpha vs. ER beta selective agonist activity, may be useful in evaluating the distinct role of these receptors as well as in providing useful insights into ER action.
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Progestinas/metabolismo , Receptores de Estrógenos/agonistas , Animales , Células CHO , Cricetinae , Receptor alfa de Estrógeno , Células HeLa , Humanos , Estructura Molecular , Noretindrona/metabolismo , Noretindrona/farmacología , Norpregnenos/metabolismo , Norpregnenos/farmacología , Oxidación-Reducción , Receptores de Estrógenos/clasificación , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/fisiología , Activación Transcripcional/fisiologíaRESUMEN
Steroid hormone-binding globulin in human serum displays different isoelectric focusing (IEF) patterns among individuals, suggesting genetic variation in the gene for this extracellular steroid carrier protein. Analysis of allele frequencies and family studies suggested the existence of two codominant alleles of the gene. Subsequent determination of the molecular basis of a variant of the gene was carried out using DNA from homozygous individuals from a single Belgian family. It was of interest to characterize other variant individuals to determine whether all variants identified by IEF phenotyping were caused by the same mutation or whether other mutations occurred in the gene in different populations. Previous studies identified Mexican subjects who were heterozygous for the variant IEF phenotype. Denaturing gradient gel electrophoresis was used to localize the mutation in these subjects and to purify the variant allele for DNA sequence analysis. The results show that the mutation in this population is identical to that identified in the Belgian family, and no other mutations were detected in the gene. These data represent the first analysis of steroid hormone-binding globulin gene variation in heterozygous subjects and further support the conclusion of biallelism of the gene worldwide.
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Genes , Heterocigoto , Globulina de Unión a Hormona Sexual/genética , Secuencia de Bases , Electroforesis/métodos , Exones , Variación Genética , Humanos , Sondas Moleculares/genética , Datos de Secuencia Molecular , Mutación PuntualRESUMEN
We have demonstrated the selective secretion of high mol wt PRL series (big big PRL) in women with hyperprolactinemia and normal ovarian function. This observation suggests that big big PRL is immunologically similar, but biologically less active, than monomeric or little PRL. In this study we determined the molecular size heterogeneity of immunoreactive PRL in the serum from two ovulatory hyperprolactinemic women (subjects A and B) who had large amounts of serum big big PRL during a menstrual cycle and/or gestation. Serum samples obtained throughout the menstrual cycle (days 6, 10, 14, 17, 23, and 28, taking as day 1 the first day of bleeding) and pregnancy (weeks 7, 9, 11, 15, 20, 25, 30, 34, and 38) were fractionated by gel filtration chromatography. PRL was identified in column eluates by specific RIA. Two additional pregnant women, one with a bromocriptine-treated PRL-secreting adenoma (subject C), and a normal woman (subject D) were studied. Big big PRL was the predominant species throughout the different phases of the menstrual cycle in subject B, comprising 70-80% of the total immunoreactive PRL. Most of the remainder was big PRL, and little PRL was present in only small amounts (6-12%) during the luteal phase. During their pregnancies, the serum PRL in subjects A and B initially was mostly big big PRL, but later in gestation the PRL composition shifted from the high mol wt variants to little PRL. The infant's cord (subject A) and peripheral (subject B) serum at birth contained appreciable quantities of big big and big PRL, respectively. These results indicate that structural changes in PRL occur during pregnancy and the menstrual cycle which are probably influenced by the hormonal environment. In addition, the occurrence of larger mol wt PRL species in the serum of the infant of a hyperprolactinemic mother suggests that the presence of high proportions of big big PRL in the serum is genetically determined.
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Hiperprolactinemia/sangre , Ciclo Menstrual , Ovario/fisiología , Embarazo/sangre , Prolactina/sangre , Adulto , Cromatografía en Gel , Estradiol/sangre , Femenino , Sangre Fetal/análisis , Humanos , Hiperprolactinemia/genética , Recién Nacido , Peso Molecular , Progesterona/sangre , Prolactina/genéticaRESUMEN
Although accumulating data show that placenta is able to synthesize 1,25-dihydroxyvitamin D3, the presence of cytochrome P(450) enzyme capable of converting 25-hydroxyvitamin D3 (250HD(3)) to the biologically active form of vitamin D in this tissue, has not been yet clearly established. In this study, we have investigated the presence of 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-(OH)ase) gene expression products in cultured human syncytiotrophoblast. Total RNA was isolated from cultured placental cells and subjected to Northern blots or RT-PCR by using 1alpha-(OH)ase-specific primers. The amplified complementary DNA fragments were analyzed by gel electrophoresis and nucleotide sequencing. Total RNA from kidney HEK 293 cells was subjected to reverse transcriptase reaction, and a 298-bp complementary DNA 1alpha-(OH)ase probe was generated by PCR. Primary cultures of human syncytiotrophoblasts exhibited 1alpha-(OH)ase activity, and a transcript for this gene could be demonstrated in these cells. Northern blot analysis revealed the presence of a 2.5-kb product, similar in size to that previously reported in kidney. RT-PCR analysis demonstrated the presence of a single transcript with nucleotide sequence identical to that previously reported for human 1alpha-(OH)ase complementary DNA clones. In addition, data are presented which suggest that differentiation of cytotrophoblast to the syncytial state was not necessary for this gene to be expressed, which may indicate a role of this enzyme all through pregnancy. The overall results of this study provide evidence for the presence of 1alpha-(OH)ase in the human placenta, suggesting that conversion of 25OHD(3) to 1,25-dihydroxyvitamin D3 in the trophoblast is most probably attributed to an enzymatic 1alpha-hydroxylation reaction.
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25-Hidroxivitamina D3 1-alfa-Hidroxilasa/biosíntesis , Células Gigantes/enzimología , Trofoblastos/enzimología , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Adulto , Secuencia de Bases , Células Cultivadas , ADN Complementario/biosíntesis , ADN Complementario/genética , Femenino , Regulación Enzimológica de la Expresión Génica/genética , Células Gigantes/citología , Humanos , Riñón/metabolismo , Datos de Secuencia Molecular , Placenta/metabolismo , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Insulin-like growth factor I (IGF-I) stimulates renal and placental 1,25-dihydroxyvitamin D [1,25-(OH)2D] and is considered an important regulator of fetal growth. As 1,25-(OH)2D and birth weight are low in preeclampsia, this study was undertaken to determine whether circulating levels of IGF-I were associated with serum 1,25-(OH)2D concentrations in preeclamptic (PE group) and normotensive (NT group) pregnancies. Maternal and umbilical cord serum levels of IGF-I and 1,25-(OH)2D were significantly (P < 0.01) lower in the PE group than in the NT group. The concentrations of these two hormones correlated significantly in the umbilical cord (P < 0.05) and in the maternal (P < 0.001) compartments of the PE and NT groups, respectively. The amount of IGFBP-3 was 64% lower whereas that of IGFBP-1 was 2.9-fold higher in umbilical cord serum of the PE group compared with the NT group. In addition, maternal and umbilical cord serum IGF-I correlated significantly (P < 0.05) with weight and length at birth only in the PE group. In conclusion, the results of this study indicate that circulating IGF-I and 1,25-(OH)2D levels in both maternal and umbilical cord compartments are low in preeclampsia. Furthermore, this study suggests a differential regulatory effect of IGF-I on 1,25-(OH)2D synthesis and fetal growth depending on the presence or absence of preeclampsia.
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Calcitriol/sangre , Sangre Fetal/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Preeclampsia/sangre , Adulto , Peso al Nacer , Presión Sanguínea , Femenino , Edad Gestacional , Humanos , Recién Nacido , Recién Nacido Pequeño para la Edad Gestacional/sangre , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Preeclampsia/fisiopatología , Embarazo , Proteinuria , Valores de ReferenciaRESUMEN
A 20-yr-old phenotypical male with a 46, XY chromosome complement, a hernia uteri inguinale, and bilateral anorchia was studied. Eunochoidal body proportions, infantile type of male external genitalia with empty scrotum, underdeveloped sexual characteristics, and delayed bone age suggested the existence of inadequate testicular function. Extremely low levels of circulating testosterone and a lack of response to hCG stimulation was found. Persistently elevated blood levels of LH and FSH with an adequate pituitary response to an iv bolus of synthetic LRH was demonstrated, thus indicating inadequate endocrine gonadal function as well as functional integrity of the hypothalamic-pituitary unit. At the time of an inguinal hernioplasty, a small but well developed uterus was removed. No gonads were found within the true pelvis, inguinal canals, or along the anatomical pathways of testicular descent. A cord-like structure found in the left inguinal canal contained only fibrous tissue without gonadal elements. It is proposed that the occurrence of two altered events during embryogenesis, failure of Müllerian duct regression and late testicular regression, may explain the underlying defect in this unusual abnormality of sexual differentiation.
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Trastornos del Desarrollo Sexual/patología , Disgenesia Gonadal/patología , Conductos Paramesonéfricos/patología , Testículo/anomalías , Adulto , Gonadotropina Coriónica , Trastornos del Desarrollo Sexual/sangre , Trastornos del Desarrollo Sexual/complicaciones , Hormona Folículo Estimulante/sangre , Hormona Liberadora de Gonadotropina , Hernia Inguinal/complicaciones , Humanos , Hormona Luteinizante/sangre , Masculino , Síndrome , Testosterona/sangreRESUMEN
Four postpubertal 46 XY male patients with an inherited form of bilateral gynecomastia were studied to delineate the nature of the disease. Normal serum FSH and moderately elevated serum LH with concomitantly increased circulating levels of testosterone (T) and estradiol (E2) were found persistently in all cases in blood samples drawn at frequent intervals. LRH pituitary stimulation resulted in an exaggerated LH response and a normal FSH response. Chronic administration of T-cyclopentylate failed to decrease serum LH levels. The peripheral conversion rate of androstenedione to estrone was within normal limits. All patients had low ejaculate volumes with relatively normal spermatozoa counts. Testicular biopsies revealed normal Leydig cells and complete spermatogenesis. Urological examination disclosed that the prostate gland was extremely small. The breast tissue demonstrated the presence of tubular structures as well as the specific binding of [3H]T and [3H]dihydrotestosterone (DHT), which was inhibited by nonlabeled T, DHT, E2, and progesterone, but not by cortisol. The pedigree suggested a recessive X-linked inherited trait. A patient with a nonfamilial form of gynecomastia served as a control in all studies. These data were interpreted as demonstrating that this inherited type of gynecomastia represents the mildest expression of the androgen resistance syndromes and, therefore, belongs to the type 1 form of familial incomplete male pseudohermaphroditism.
Asunto(s)
Trastornos del Desarrollo Sexual/genética , Estradiol/sangre , Hormona Folículo Estimulante/sangre , Ginecomastia/genética , Hormona Luteinizante/sangre , Testosterona/sangre , Adolescente , Trastornos del Desarrollo Sexual/fisiopatología , Femenino , Hormona Liberadora de Gonadotropina , Ginecomastia/fisiopatología , Humanos , Cinética , Masculino , Linaje , Síndrome , Testículo/patologíaRESUMEN
We have evaluated the production of PRL by human peripheral mononuclear cells (PBMNC) from normal subjects and patients with systemic lupus erythematosus (SLE). Conditioned medium prepared from basal and Con-A-stimulated PBMNC was assessed for the presence of PRL-like by its ability to stimulate growth of PRL-responsive Nb2 rat lymphoma cells. In the presence or absence of Con-A, SLE PBMNC secrete significantly higher (P < 0.001) amounts of bioactive PRL-like species than normal cells. Growth of Nb2 cells by conditioned medium was inhibited with specific antiserum to human PRL. Western blotting using a polyclonal antibody to human PRL revealed a single 60-kDa PRL-like species in both normal and SLE PBMNC extracts, the immunoreactivity of which was preferentially found in SLE subjects. With the use of reverse transcription-PCR an expected 633-bp band was observed, and its similarity to pituitary PRL was further confirmed by Southern blot analysis with human PRL complementary DNA as a probe. We conclude that a high molecular mass PRL-like species is synthesized and secreted by PBMNC, and patients with SLE have an increased secretion of lymphocyte-derived PRL-like material.
Asunto(s)
Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/sangre , Prolactina/metabolismo , Adolescente , Adulto , Animales , Bioensayo , Southern Blotting , Western Blotting , Células Cultivadas , Concanavalina A/farmacología , Medios de Cultivo Condicionados , Femenino , Expresión Génica , Humanos , Linfoma/patología , Peso Molecular , Reacción en Cadena de la Polimerasa , Prolactina/genética , Prolactina/farmacología , ARN Mensajero/análisis , RatasRESUMEN
We describe the reconstitution and purification of a membrane-associated phosphoinositide-specific phospholipase C (PIC) from turkey erythrocyte ghosts. This PIC is responsive to a G-protein coupled to P2y purinergic receptors which are expressed in turkey erythrocytes. Reconstitution is achieved by adding partially purified PIC to [3H]inositol-prelabelled turkey erythrocyte membranes depleted of their endogenous PIC (acceptor membranes). PIC activity is associated with a 52 kDa polypeptide on SDS-polyacrylamide gel electrophoresis. Addition of a 307-fold purified enzyme to the acceptor membranes has no effect on basal PIC activity, but markedly increases the response to GTP gamma S and P2y-purinergic receptor activation.
Asunto(s)
Membrana Eritrocítica/enzimología , Proteínas de Unión al GTP/metabolismo , Receptores Purinérgicos/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Calcio/farmacología , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Citosol/enzimología , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas/efectos de los fármacos , Membrana Eritrocítica/ultraestructura , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Peso Molecular , Fosfatidilinositoles/metabolismo , Pavos , Fosfolipasas de Tipo C/aislamiento & purificaciónRESUMEN
Anterior pituitary glands were removed from male rats at 5, 10, 15, 18, 21, 28, 30, 40, 50 and 90 days of age, and the multiple forms of FSH present within them were separated by polyacrylamide gel-isoelectric focusing (PAGE-IEF; pH range 3.0-8.0). Gel eluents were analysed for FSH content by radioimmunoassay (RIA) and a specific radioreceptor assay (RRA). All pituitaries studied exhibited one or more peaks of immunoactive FSH within a pH range of 7.0-3.0; the major peak exhibited an isoelectric point (pI) of 4.9-4.0. Between 25 and 56% of anterior pituitary FSH obtained from rats 5-30 days old focused within a pH range of 4.9-4.5, whilst in older animals (greater than or equal to 40 days) this pH range contained 17-27% of the total FSH recovered. In contrast, in animals 40-90 days old, the greatest proportion of immunoactive FSH (42-62% of the total immunoactivity recovered) focused within a pH range of 4.4-4.0; further, only these groups of animals exhibited a significant proportion of anterior pituitary FSH with a pI less than or equal to 3.9. Between 14 and 21% of total FSH from 5- to 30-day-old rats focused within a pH range of 5.4-5.0, whereas in older animals this pH range contained 6-9% of the total FSH recovered. These shifts in FSH pI occurred at the time of appearance of spermiogenesis, at 45 days of age. Although the ratio of the concentration of FSH measured by RRA to that measured by RIA declined as the pI of the anterior pituitary FSH decreased throughout a pH range of 7.0-4.0, the most acidic FSH molecules (pI less than 4.0) showed an abrupt increase in that ratio. These results demonstrate that the transition from sexual immaturity to adulthood is accompanied by qualitative changes of intracellular pituitary FSH. They contrast with previous findings in female rats in which a shift to less acidic anterior pituitary FSH forms was detected at the time of vaginal opening, thus indicating the existence of a sexual dichotomy in terms of the action of gonadal steroids on the type of FSH molecule synthesized by the anterior pituitary gland.
Asunto(s)
Hormona Folículo Estimulante/metabolismo , Adenohipófisis/metabolismo , Maduración Sexual , Animales , Electroforesis en Gel de Poliacrilamida , Hormona Folículo Estimulante/sangre , Focalización Isoeléctrica , Isomerismo , Masculino , Ensayo de Unión Radioligante , Ratas , Ratas Endogámicas , Testosterona/sangreRESUMEN
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.