RESUMEN
BACKGROUND: Students evaluations of their learning experiences can provide a useful source of information about clerkship effectiveness in undergraduate medical education. However, low response rates in clerkship evaluation surveys remain an important limitation. This study examined the impact of increasing response rates using a compulsory approach on validity evidence. METHODS: Data included 192 responses obtained voluntarily from 49 third-year students in 2014-2015, and 171 responses obtained compulsorily from 49 students in the first six months of the consecutive year at one medical school in Lebanon. Evidence supporting internal structure and response process validity was compared between the two administration modalities. The authors also tested for potential bias introduced by the use of the compulsory approach by examining students' responses to a sham item that was added to the last survey administration. RESULTS: Response rates increased from 56% in the voluntary group to 100% in the compulsory group (P < 0.001). Students in both groups provided comparable clerkship rating except for one clerkship that received higher rating in the voluntary group (P = 0.02). Respondents in the voluntary group had higher academic performance compared to the compulsory group but this difference diminished when whole class grades were compared. Reliability of ratings was adequately high and comparable between the two consecutive years. Testing for non-response bias in the voluntary group showed that females were more frequent responders in two clerkships. Testing for authority-induced bias revealed that students might complete the evaluation randomly without attention to content. CONCLUSIONS: While increasing response rates is often a policy requirement aimed to improve the credibility of ratings, using authority to enforce responses may not increase reliability and can raise concerns over the meaningfulness of the evaluation. Administrators are urged to consider not only response rates, but also representativeness and quality of responses in administering evaluation surveys.
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Prácticas Clínicas , Educación de Pregrado en Medicina , Evaluación de Programas y Proyectos de Salud/métodos , Estudiantes de Medicina , Encuestas y Cuestionarios , Adulto , Análisis de Varianza , Sesgo , Análisis Factorial , Femenino , Humanos , Líbano , Masculino , Reproducibilidad de los Resultados , Facultades de MedicinaRESUMEN
Benefits associated with lowered serum DHT levels after 5α-reductase inhibitor (5AR-I) therapy in men have contributed to a misconception that circulating DHT levels are an important stimulus for androgenic action in target tissues (e.g., prostate). Yet evidence from clinical studies indicates that intracellular concentrations of androgens (particularly in androgen-sensitive tissues) are essentially independent of circulating levels. To assess the clinical significance of modest elevations in serum DHT and the DHT/testosterone (T) ratio observed in response to common T replacement therapy, a comprehensive review of the published literature was performed to identify relevant data. Although the primary focus of this review is about DHT in men, we also provide a brief overview of DHT in women. The available published data are limited by the lack of large, well-controlled studies of long duration that are sufficiently powered to expose subtle safety signals. Nonetheless, the preponderance of available clinical data indicates that modest elevations in circulating levels of DHT in response to androgen therapy should not be of concern in clinical practice. Elevated DHT has not been associated with increased risk of prostate disease (e.g., cancer or benign hyperplasia) nor does it appear to have any systemic effects on cardiovascular disease safety parameters (including increased risk of polycythemia) beyond those commonly observed with available T preparations. Well-controlled, long-term studies of transdermal DHT preparations have failed to identify safety signals unique to markedly elevated circulating DHT concentrations or signals materially different from T.
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Dihidrotestosterona , Inhibidores de 5-alfa-Reductasa/farmacología , Animales , Dihidrotestosterona/sangre , Dihidrotestosterona/química , Dihidrotestosterona/metabolismo , Dihidrotestosterona/farmacología , Femenino , Humanos , Masculino , Próstata/efectos de los fármacos , Próstata/metabolismo , Neoplasias de la Próstata/sangre , Caracteres Sexuales , Testosterona/farmacología , Testosterona/fisiologíaRESUMEN
OBJECTIVE: To determine whether liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of total (TT) and free (FT) testosterone is more specific than extraction chromatography-radioimmunoassay (RIA) for distinguishing women with polycystic ovary syndrome (PCOS) from controls and whether differing cutoff values should be used depending on the setting. DESIGN: Prospective case-control cohort study. SETTING: Tertiary care center and reference laboratory. INTERVENTION(S): Blood sampling. PATIENT(S): One hundred patients with PCOS and 100 controls. MAIN OUTCOME MEASURE(S): Circulating TT measured by RIA and LC-MS/MS and FT measured by equilibrium dialysis using RIA or LC-MS/MS-generated TT values. RESULT(S): T measurement by RIA and LC-MS/MS similarly differentiated patients with PCOS from controls; detection of PCOS was higher for FT for both methods. TT values demonstrated greater overlap between patients with PCOS and controls than did FT for both RIA (80% vs. 42% overlap) and LC-MS/MS (52% vs. 67% overlap). A lower cutoff value by LC-MS/MS was better suited for the study of patients seen in the clinical (referred) setting (35 and 4.0 ng/dL for TT and FT, respectively) than in the screening of a general population (50 and 5.0 ng/dL for TT and FT, respectively). CONCLUSION(S): Extraction chromatography-RIA and LC-MS/MS measurements of T have similar performance for differentiating patients with PCOS from healthy controls; LC-MS/MS may be preferable given its relative ease of automation. Compared with FT, measurement of TT has relatively limited value for distinguishing PCOS from normal. Finally, different cutoff values should be considered depending on the clinical/investigative setting, with higher values being used in the study of biased (e.g., clinical or referred) populations.
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Cromatografía Liquida/métodos , Síndrome del Ovario Poliquístico/sangre , Síndrome del Ovario Poliquístico/diagnóstico , Testosterona/sangre , Adolescente , Adulto , Biomarcadores/sangre , Cromatografía Liquida/normas , Femenino , Humanos , Persona de Mediana Edad , Guías de Práctica Clínica como Asunto , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto JovenRESUMEN
BACKGROUND AND OBJECTIVES: Conventional lipid profiles usually cannot predict cardiovascular outcomes in chronic disease states. We hypothesized that novel lipoprotein subfraction concentrations and LDL particle size measurements better predict mortality in maintenance hemodialysis (MHD) patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Mortality-predictability of LDL particle diameter and lipoprotein subfraction concentrations, measured by novel ion mobility, was examined in a cohort of 235 hemodialysis patients who were followed for up to 6 years using Cox models with adjustment for important covariables. RESULTS: Patients were 54 ± 14 years old (mean ± SD) and included 45% women with total, LDL and HDL cholesterol levels of 143 ± 42, 76 ± 29, and 37 ± 12 mg/dl, respectively. Over 6 years, 71 patients (31%) died. Conventional lipid profile was not associated with mortality. The death hazard ratio (HR, 95% confidence interval) of the highest versus lowest quartiles of very small and large LDL particle concentrations were 2.43 (1.03 to 5.72) and 0.38 (0.15 to 0.96), respectively. Across increasing quartiles of LDL particle diameter, death HRs were 1.00, 0.93 (0.46 to 1.87), 0.43 (0.21 to 0.89), and 0.45 (0.31 to 1.00), respectively. CONCLUSIONS: Whereas conventional lipid profile cannot predict mortality in MHD patients, larger novel LDL particle diameter or higher large LDL particle concentrations appear predictive of greater survival, whereas higher very small LDL particle concentration is associated with higher death risk. Examining lipoprotein subfraction modulation in chronic diseases is indicated.
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HDL-Colesterol/sangre , LDL-Colesterol/sangre , Diálisis Renal/mortalidad , Adulto , Anciano , Índice de Masa Corporal , Femenino , Humanos , Masculino , Persona de Mediana Edad , Tamaño de la Partícula , Modelos de Riesgos ProporcionalesRESUMEN
Our understanding of the molecular mechanisms responsible for follicular thyroid cell oncogenesis has been advanced significantly in recent years. Specific genetic alterations and the molecular pathways they affect have been associated with particular histologic subtypes of well-differentiated thyroid cancer and are now being evaluated for their utility as clinical tools with diagnostic, prognostic and even therapeutic relevance. This paper focuses on the most common and clinically relevant genetic alterations shown to be consistently associated with well-differentiated thyroid carcinoma. We review the impact of recent molecular and technological advances on thyroid cancer standard of care and the practice of clinical medicine.
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Adenocarcinoma Folicular , Neoplasias de la Tiroides , Adenocarcinoma Folicular/diagnóstico , Adenocarcinoma Folicular/patología , Adenocarcinoma Folicular/terapia , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Humanos , Técnicas de Diagnóstico Molecular , Mutación , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box/genética , Pronóstico , Nivel de Atención , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/terapia , Proteínas ras/genéticaRESUMEN
BACKGROUND: For management and treatment of secondary hypertension, plasma renin activity (PRA) assay is considered an essential diagnostic tool. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to PRA offering improvements in laboratory workflow and throughput. During development, we observed a substantial number of clinical samples that have strong degradation activity toward angiotensin (Ang) I during generation. A preliminary characterization of this degradation activity was performed, and we provide here a method by which this degradation can be monitored via the addition of an isotope-labeled degradation standard. METHODS: Automated online sample extraction coupled with HPLC was used to isolate Ang I and internal standard from plasma. The effluent from the analytical column was directed to a triple quadrupole MS operated in selected reaction monitoring mode, monitoring the a(5) and b(5) product ions from the [M+3H](+3) precursors. Routine analysis could be achieved with as little as 150 µL plasma. RESULTS: We identified both C-terminal and N-terminal degradation products of Ang I using isotope-labeled peptides as controls and substrates. In 2%-5% of patient samples, the degradation essentially eliminated any Ang I produced during generation. CONCLUSIONS: Our method requires reduced sample handling when compared with an RIA and eliminates the need for extended generation times for samples with low renin activity. Degradation of Ang I during generation appears to be a confounding variable in the interpretation of results from some clinical samples. Samples with profound degradation activity can be identified using a degradation standard that is added at the start of generation.
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Angiotensina I/sangre , Péptido Hidrolasas/sangre , Renina/sangre , Secuencia de Aminoácidos , Angiotensina I/normas , Cromatografía Líquida de Alta Presión , Humanos , Oligopéptidos/sangre , Radioinmunoensayo , Estándares de Referencia , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Vitamin D has been added to calcium-fortified orange juice. It is unknown whether vitamin D is as bioavailable from orange juice as it is from supplements. OBJECTIVES: The objective was to compare the bioavailability of vitamin D(2) and vitamin D(3) from orange juice with that from vitamin D(2) and vitamin D(3) supplements. A secondary aim was to determine which form of vitamin D is more bioavailable in orange juice. DESIGN: A randomized, placebo-controlled, double-blind study was conducted in healthy adults aged 18-84 y (15-20/group) who received 1000 IU vitamin D(3), 1000 IU vitamin D(2), or placebo in orange juice or capsule for 11 wk at the end of winter. RESULTS: A total of 64% of subjects began the study deficient in vitamin D (ie, 25-hydroxyvitamin D [25(OH)D]) concentrations <20 ng/mL). Analysis of the area under the curve showed no significant difference in serum 25(OH)D between subjects who consumed vitamin D-fortified orange juice and those who consumed vitamin D supplements (P = 0.084). No significant difference in serum 25(OH)D(3) was observed between subjects who consumed vitamin D(3)-fortified orange juice and vitamin D(3) capsules (P > 0.1). Similarly, no significant difference in serum 25(OH)D(2) was observed between subjects who consumed vitamin D(2)-fortified orange juice and vitamin D(2) capsules (P > 0.1). No significant overall difference in parathyroid hormone concentrations was observed between the groups (P = 0.82). CONCLUSION: Vitamin D(2) and vitamin D(3) are equally bioavailable in orange juice and capsules.
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Bebidas , Colecalciferol/administración & dosificación , Citrus sinensis , Ergocalciferoles/administración & dosificación , Alimentos Fortificados , Frutas , Deficiencia de Vitamina D/dietoterapia , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Disponibilidad Biológica , Calcio/sangre , Colecalciferol/sangre , Colecalciferol/farmacocinética , Suplementos Dietéticos , Método Doble Ciego , Ergocalciferoles/sangre , Ergocalciferoles/farmacocinética , Femenino , Humanos , Persona de Mediana Edad , Hormona Paratiroidea/sangre , Deficiencia de Vitamina D/sangre , Deficiencia de Vitamina D/metabolismo , Adulto JovenRESUMEN
Accurate measurement of testosterone concentration is of critical importance when diagnosing and treating male hypogonadism, congenital adrenal hyperplasia, premature or delayed puberty, and androgen excess in polycystic ovary syndrome or other virilizing conditions. However, some assays have inherent limitations and biases that affect measurement of low-testosterone values. Therefore, we developed a highly specific online mass spectrometry method. Sera were extracted online using high-turbulence flow liquid chromatography coupled to analytical HPLC and atmospheric pressure chemical ionization tandem mass spectrometry (HTLC-APCI-MS/MS). Analyte ions were monitored by multiple reaction monitoring (MRM). Total analysis time was 1.15 min per sample when using the multiplexing system. Testosterone concentrations were measured directly from 150 microL of serum or plasma without derivatization or liquid-liquid extraction. The lower limit of quantification was 0.3 ng/dL, and the assay was linear up to 2000 ng/dL. The method compared very well with an established RIA: y=1.02x+1.5, r(2)=0.994. Comparison with a platform immunoassay confirmed the previously reported ICMA positive bias at low concentrations. Male and female adult and pediatric reference ranges were developed for this very sensitive and accurate high-throughput LC-MS/MS method. This method is suitable for measuring the expected low-testosterone concentrations seen in women, children, and hypogonadal males and for monitoring testosterone suppressive therapy in prostate cancer patients.
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Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodos , Testosterona/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Radioinmunoensayo , Valores de Referencia , Globulina de Unión a Hormona Sexual/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: Whereas epidemiological studies show that levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) predict incident cardiovascular disease (CVD), there is limited evidence relating lipoprotein subfractions and composite measures of subfractions to risk for CVD in prospective cohort studies. METHODS AND RESULTS: We tested whether combinations of lipoprotein subfractions independently predict CVD in a prospective cohort of 4594 initially healthy men and women (the Malmö Diet and Cancer Study, mean follow-up 12.2 years, 377 incident cardiovascular events). Plasma lipoproteins and lipoprotein subfractions were measured at baseline with a novel high-resolution ion mobility technique. Principal component analysis (PCA) of subfraction concentrations identified 3 major independent (ie, zero correlation) components of CVD risk, one representing LDL-associated risk, a second representing HDL-associated protection, and the third representing a pattern of decreased large HDL, increased small/medium LDL, and increased triglycerides. The last corresponds to the previously described "atherogenic lipoprotein phenotype." Several genes that may underlie this phenotype-CETP, LIPC, GALNT2, MLXIPL, APOA1/A5, LPL-are suggested by SNPs associated with the combination of small/medium LDL and large HDL. CONCLUSIONS: PCA on lipoprotein subfractions yielded three independent components of CVD risk. Genetic analyses suggest these components represent independent mechanistic pathways for development of CVD.
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Enfermedades Cardiovasculares/sangre , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Lipoproteínas VLDL/sangre , Adulto , Anciano , Biomarcadores/sangre , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Estudios de Cohortes , Femenino , Humanos , Transporte Iónico , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Valor Predictivo de las Pruebas , Probabilidad , Pronóstico , Estudios Prospectivos , Medición de RiesgoRESUMEN
BACKGROUND & AIMS: Biomarkers are being developed as alternatives to liver biopsy for predicting liver fibrosis in patients with chronic hepatitis C. Hepascore uses noninvasive serum markers and has been validated in Australian and European populations for predicting different degrees of fibrosis. This study validated this test in a U.S. population. METHODS: Patients with chronic hepatitis C virus infection were assigned to training (n = 203) or validation (n = 188) sets. Liver fibrosis was staged according to the METAVIR scoring system. The Hepascore algorithm uses data on age, sex, as well as total bilirubin, gamma-glutamyl transferase, alpha2-macroglobulin, and hyaluronic acid levels. RESULTS: The ability of Hepascore to predict significant fibrosis (F2-4) as determined by the area under the receiver operating curve was similar in training (0.83) and validation sets (0.81) and was comparable to results seen in previous studies. A cutoff score of > or =0.55 was best for predicting significant fibrosis, with a sensitivity and specificity of 82% and 65% and positive and negative predictive values of 70% and 78%. When compared with 2 simple indices, FIB-4 (age, platelets, AST, and ALT) and APRI (AST/platelet ratio index), Hepascore performed better at excluding advanced fibrosis by using a low cutoff score but worse at predicting fibrosis by using a high cutoff score. An algorithm with Hepascore followed by FIB-4 or APRI spared 103 of 391 individuals a liver biopsy and missed advanced fibrosis in only 1 patient. CONCLUSIONS: Hepascore accurately predicted likelihood of developing fibrosis and could alleviate the need for liver biopsy in a subset of patients.
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Hepatitis C Crónica/complicaciones , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/patología , Índice de Severidad de la Enfermedad , Adulto , Factores de Edad , Bilirrubina/sangre , Biomarcadores , Femenino , Humanos , Ácido Hialurónico/sangre , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Suero/química , Factores Sexuales , Estados Unidos , alfa-Macroglobulinas/análisis , gamma-Glutamiltransferasa/sangreRESUMEN
BACKGROUND: Though mass spectrometry (MS) assays are increasingly used for routine clinical measurements of serum total testosterone (TT), information about the variability of results is limited. This study assessed the variability of TT measurement results from routine MS assays. METHODS: Twenty serum samples (12 females, 8 males) were analyzed on 2 days by seven high performance liquid chromatography (HPLC), and one gas chromatography (GC)-tandem mass spectrometry (HPLC-MS/MS, GC-MS/MS) assays. Two samples (male and female) were provided in five replicates to assess the within-run variability. Results were compared against those obtained at National Institute of Standards and Technology (NIST). The within- and between-laboratory variability was assessed for each sample. Comparisons to the NIST results were performed using bias plot and Deming regression analysis. RESULTS: The overall coefficient of variation of the results obtained with MS assays was <15%CV at >1.53 nmol/L and <34%CV at 0.3 nmol/L. The between-assay variability was the major contributor to the overall variability. The assay precision was the highest (<3%CV) with assays using liquid-liquid extraction for sample preparation or GC-MS/MS. The mean percent difference to the reference assay was 11%. The slopes of Deming regression analysis of the MS assays were between 0.903 and 1.138 (correlation coefficient: >0.996). TT concentrations for one assay were above the measurement range. CONCLUSIONS: The variability of TT measurement results among MS assays is substantially smaller than that reported for immunoassays. The type of sample preparation may affect assay precision. Standardizing assays can further reduce the variability of measurement results.
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Espectrometría de Masas/métodos , Testosterona/sangre , Adulto , Anciano , Cromatografía Líquida de Alta Presión , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Laboratorios/normas , Masculino , Persona de Mediana Edad , Valores de Referencia , Análisis de Regresión , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
BACKGROUND: Current methods for measuring the concentrations of lipoprotein particles and their distributions in particle subpopulations are not standardized. We describe here and validate a new gas-phase differential electrophoretic macromolecular mobility-based method (ion mobility, or IM) for direct quantification of lipoprotein particles, from small, dense HDL to large, buoyant, very-low-density lipoprotein (VLDL). METHODS: After an ultracentrifugation step to remove albumin, we determined the size and concentrations of lipoprotein particles in serum samples using IM. Scan time is 2 min and covers a particle range of 17.2-540.0 A. After scanning, data are pooled by totaling the particle number across a predetermined size range that corresponds to particular lipoprotein subclasses. IM results were correlated with those of standard methods for cholesterol and apolipoprotein analysis. RESULTS: Intra- and interassay coefficients of variation for LDL particle size were <1.0%. The intra- and interassay variation for LDL and HDL particle subfraction measurements was <20%. IM-measured non-HDL correlated well with apolipoprotein B (r = 0.92). CONCLUSIONS: The IM method provides accurate, reproducible, direct determination of size and concentration for a broad range of lipoprotein particles. Use of this methodology in studies of patients with cardiovascular disease and other pathologic states will permit testing of its clinical utility for risk assessment and management of these conditions.
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Lipoproteínas/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Femenino , Humanos , Lipoproteínas HDL/sangre , Lipoproteínas IDL/sangre , Lipoproteínas LDL/sangre , Lipoproteínas VLDL/sangre , Masculino , Tamaño de la Partícula , Estándares de Referencia , Reproducibilidad de los Resultados , Manejo de Especímenes , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
CONTEXT: Two reports suggested that vitamin D2 is less effective than vitamin D3 in maintaining vitamin D status. OBJECTIVE: Our objective was to determine whether vitamin D2 was less effective than vitamin D3 in maintaining serum 25-hydroxyvitamin D levels or increased the catabolism of 25-hydroxyvitamin D3. SUBJECTS AND DESIGN: This was a randomized, placebo-controlled, double-blinded study of healthy adults ages 18-84 yr who received placebo, 1000 IU vitamin D3, 1000 IU vitamin D2, or 500 IU vitamin D2 plus 500 IU vitamin D3 daily for 11 wk at the end of the winter. RESULTS: Sixty percent of the healthy adults were vitamin D deficient at the start of the study. The circulating levels of 25-hydroxyvitamin D (mean+/-sd) increased to the same extent in the groups that received 1000 IU daily as vitamin D2 (baseline 16.9+/-10.5 ng/ml; 11 wk 26.8+/-9.6 ng/ml), vitamin D3 (baseline 19.6+/-11.1 ng/ml; 11 wk 28.9+/-11.0 ng/ml), or a combination of 500 IU vitamin D2 and 500 IU vitamin D3 (baseline 20.2+/-10.4 ng/ml; 11 wk 28.4+/-7.7 ng/ml). The 25-hydroxyvitamin D3 levels did not change in the group that received 1000 IU vitamin D2 daily. The 1000 IU dose of vitamin D2 or vitamin D3 did not raise 25-hydroxyvitamin D levels in vitamin D-deficient subjects above 30 ng/ml. CONCLUSION: A 1000 IU dose of vitamin D2 daily was as effective as 1000 IU vitamin D3 in maintaining serum 25-hydroxyvitamin D levels and did not negatively influence serum 25-hydroxyvitamin D3 levels. Therefore, vitamin D2 is equally as effective as vitamin D3 in maintaining 25-hydroxyvitamin D status.
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Colecalciferol/administración & dosificación , Ergocalciferoles/administración & dosificación , Vitamina D/análogos & derivados , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Vitamina D/sangreRESUMEN
Glycogen synthase kinase-3beta (GSK-3beta) is a serine/threonine kinase that plays a role in glycogen synthesis by inhibiting glycogen synthase (GS) through phosphorylation. We hypothesized that GSK-3beta by virtue of its role in glycogen synthesis through the inhibition of GS will play a role in the preparation of the endometrium for blastocyst implantation. Immunohistochemical (IHC) analysis and Western blot analysis (WBA) detected GSK-3beta in the endometrium, myometrium, Fallopian tube and ovary. WBA showed more than 5-fold higher endometrial expression of the phosphorylated GSK-3beta (pGSK-3beta) isoform (inactive) in the secretory phase as compared with the proliferative phase (P < 0.001), whereas no differences in total GSK-3beta expression were detected. IHC analysis confirmed the WBA and showed marked expression of pGSK-3beta predominantly in glandular epithelial cells in early and mid secretory endometrium with scant expression during the proliferative phase. In in vitro experiments using human endometrial-derived epithelial cell line (HES), progesterone did not alter total GSK mRNA or protein expression. However, progesterone induced a dose-dependent increase in the expression of pGSK-3beta, which could be blocked by RU486. Cyclic expression of GSK-3beta's active and inactive forms in the endometrium suggests that sex hormones regulate the expression of this enzyme. In vitro experiments demonstrate that progesterone through receptor-mediated mechanisms induces phosphorylation of endometrial GSK-3beta.
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Endometrio/enzimología , Glucógeno Sintasa Quinasa 3/metabolismo , Ciclo Menstrual/metabolismo , Progesterona/metabolismo , Adulto , Línea Celular , Relación Dosis-Respuesta a Droga , Endometrio/citología , Endometrio/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Femenino , Glucógeno/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Antagonistas de Hormonas/farmacología , Humanos , Inmunohistoquímica , Mifepristona/farmacología , Fosforilación , Progesterona/antagonistas & inhibidoresRESUMEN
Premature ovarian failure (POF) is characterized by elevated gonadotropins and amenorrhea in women aged <40 years. In a Lebanese family with five sisters who received the diagnosis of POF, we established linkage to the long arm of the X chromosome (between Xq21.1 and Xq21.3.3), using whole-genome SNP typing and homozygosity-by-descent mapping. By sequencing one candidate gene within that region, POF1B, we identified a point mutation localized in exon 10. This substitution of a nucleotide (G-->A), at position 1123, results in an arginine-->glutamine mutation of the protein sequence at position 329 (mutation R329Q). All the affected family members were homozygous for the mutation, whereas the unaffected members were heterozygous. Because POF1B shares high homology with the tail portion of the human myosin, we assessed the ability of both wild-type and mutant POF1B proteins to bind nonmuscle actin filaments in vitro. We found that the capacity of the mutant protein to bind nonmuscle actin filaments was diminished fourfold compared with the wild type, suggesting a function of POF1B in germ-cell division. Our study suggests that a homozygous point mutation in POF1B influences the pathogenesis of POF by altering POF1B binding to nonmuscle actin filaments.
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Actinas/metabolismo , Insuficiencia Ovárica Primaria/genética , Proteínas/metabolismo , Secuencia de Bases , Cromosomas Humanos X , Cartilla de ADN , Exones , Femenino , Ligamiento Genético , Heterocigoto , Homocigoto , Humanos , Masculino , Proteínas de Microfilamentos , Linaje , Mutación Puntual , Unión ProteicaRESUMEN
Age-related decline in male sex hormones is a direct consequence of testicular aging. These changes in the hormonal complement cause physiological disturbances affecting the quality of life for millions of aging men. To assess the influence on testicular aging of pituitary adenylate cyclase-activating peptide (PACAP), a polypeptide that regulates testicular steroidogenesis in vitro, we compared the testicular structure and function between C57BL/6 wild-type and PACAP-/- male mice, at 4 and 15 months of age. We show that, in 4-month-old PACAP-/- mice, steroidogenesis (evaluated by levels of testosterone, steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase, and P450c17) was impaired. However, the testicular structure of these animals was not affected. At 15 months of age, wild-type testis displayed typical signs of aging (patchy seminiferous tubules, germ cell depletion, and vacuolization), whereas testicular structure was remarkably well conserved in PACAP-/- animals. The depletion of germ cells found in wild-type animals was associated with a higher content of peroxynitrites, a marker of reactive oxygen species, and a higher number of apoptotic cells compared with PACAP-/- mice. Our results show that testicular aging is delayed in PACAP-/- animals. Because the expression levels of steroidogenic factors are low and constant over time in knockout animals, a proposed mechanism for the protection against testicular degeneration is that production of reactive oxygen species, a byproduct of steroidogenesis that induces apoptosis, is down-regulated in PACAP-/- animals.
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Envejecimiento/metabolismo , Envejecimiento/patología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/deficiencia , Testículo/metabolismo , Testículo/patología , Envejecimiento/genética , Animales , Apoptosis , Secuencia de Bases , ADN Complementario/genética , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Especies Reactivas de Oxígeno/metabolismo , Esteroide 17-alfa-Hidroxilasa/metabolismo , Esteroides/biosíntesisRESUMEN
Klinefelter syndrome (XXY males) is the most common sex chromosome aneuploidy. XXY mice were generated by using a four-generation breeding scheme that involves the use of a structurally rearranged Y chromosome, Y*, yielding approximately 50% of the live-born male offspring in the fourth generation with a XXY karyotype. Adult XXY mice have small testes, decreased plasma T levels, and elevated plasma FSH levels. The testes of adult XXY mice contained small seminiferous tubules with intraepithelial vacuolization and absence of germ cells, whereas Leydig cells appeared to be more abundant than their XY littermates. Androgen receptor immunoexpression was localized in Leydig cells and peritubular myoid cells in both XY and XXY mice. Androgen receptor immunoexpression was abundant in the Sertoli cells of XY mice but nearly absent in those of XXY mice. The testicular phenotype was marked by a 23.1% decrease in testis weights in XXY pups beginning at d 7 after birth. Gonocyte numbers were similar in XY and XXY mice at d 1 of age, followed by a 62.6% decrease in the number of gonocytes in the XXY mice on d 3 and further progressive loss in spermatogonia by d 5 and 7. On d 10, only a few spermatogonia remained in the XXY mice. To determine whether the phenotype of XXY mice extended into the neurobehavioral domain, studies were conducted demonstrating impairment of learning and memory function in XXY mice. We conclude that adult XXY mice have testicular failure and learning deficits, similar to its human counterpart, Klinefelter syndrome.
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Modelos Animales de Enfermedad , Síndrome de Klinefelter/genética , Síndrome de Klinefelter/fisiopatología , Ratones Mutantes , Testículo/patología , Factores de Edad , Animales , Conducta Animal , Peso Corporal , Condicionamiento Clásico , Femenino , Síndrome de Klinefelter/patología , Masculino , Ratones , Tamaño de los Órganos , Fenotipo , Receptores Androgénicos/metabolismo , Testículo/fisiopatología , Testosterona/sangreRESUMEN
PURPOSE: The purpose of this study is to summarize new data on etiology and clinical features of Klinefelter syndrome in order to derive research priorities. METHODS: This study was conducted using critical reviews of selective topics, emphasizing less well-recognized clinical findings. RESULTS AND CONCLUSIONS: The phenotype of the prototypic 47,XXY case is well recognized: seminiferous tubule dysgenesis and androgen deficiency. Less well appreciated is the varied expressivity of 47,XXY Klinefelter syndrome, in particular neurological/cognitive perturbations like language and behavioral problems. Effective therapies are available. Reproductive technologies allow 47,XXY men to sire offspring through intracytoplasmic sperm injection (ICSI); however, genetic counseling is complex and success is low. Behavioral and expressive language difficulties are amenable to treatment by androgen therapy and psychological help. Early treatment may be imperative for optimal outcome.
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Investigación Biomédica , Síndrome de Klinefelter/genética , Asesoramiento Genético , Humanos , Pruebas de Inteligencia , Síndrome de Klinefelter/psicología , Desarrollo del Lenguaje , MasculinoRESUMEN
Glycogen synthase kinase-3 beta (GSK-3 beta) regulates cell metabolism, cell cycle, and cell fate through the phosphorylation of a diverse array of substrates. Herein, we provide evidence that supports a role for GSK-3 in mammalian meiosis and spermatogenesis. Immunostaining of testis sections showed that while GSK-3 alpha was ubiquitous in the seminiferous tubules, GSK-3 beta was expressed in premeiotic type B spermatogonia, in both meiotic preleptotene and leptotene spermatocytes, as well as in Sertoli cells in both the mouse and rat. Thus, GSK-3 beta is expressed in germ cells entering meiosis. In addition, intense immunoreactivity was detected in rat step 6 though 11 spermatids. In situ hybridization (ISH) in rat testis confirmed the immunostaining pattern in leptotene and spermatids and showed a GSK-3 beta messenger RNA (mRNA) signal in some pachytene spermatocytes. The restricted pattern of expression suggests cell-specific regulation of Gsk-3 beta mRNA. To determine whether GSK-3 is required for meiosis entry, rat stage VIIa seminiferous tubule segments were cultured with selective small-molecule GSK-3 inhibitors. These compounds markedly and dose-dependently suppressed meiotic synthesis (S)-phase DNA. Since a yeast GSK-3 homolog, Rim11p (regulator of inducer of meiosis), is pivotal to meiosis entry, we tested whether GSK-3 beta complements Rim11p function in meiosis. Rim11p phosphorylates transcription factors Ume6p (unscheduled meiotic gene expression) and Ime1p (inducer of meiosis) to induce meiosis entry. Overexpression of murine GSK-3 beta in a rim11 mutant yeast failed to rescue the sporulation defect. Our finding that GSK-3 beta interacted only with Ume6p but not with IME1 in a yeast 2-hybrid assay suggests that noncomplementation reflects partial divergence in substrate specificity. This work provides the basis for future studies of GSK-3 beta signaling in mammalian meiosis and spermatogenesis.