RESUMEN
PURPOSE: The aim of our paper was to validate a testicular biopsy procedure that simplifies handling, processing, and cryopreservation, while at the same time optimizes sperm motility before freezing and after thawing. METHODS: Two prospective studies were conducted to verify, optimize, and understand the virtues of pre-freeze testicular tissue IVC at different temperatures (21, 30, or 37 °C). Testicular tissue was obtained from clinical specimens designated for whole tissue cryopreservation (i.e., intact mass of tubules) and/or for fresh use in IVF-ICSI cycles. Whole testicular biopsy pieces (1-3 mm(3)) were diluted in glycerol containing freeze solutions, slow cooled to 4 °C and then rapidly frozen in LN2 vapor. Fresh and post-thaw testicular biopsy tissue were evaluated for changes in the quantity (%) and pattern of motility (I-IV: twitching to rapid progression, respectively) over a 1 week duration. The clinical effectiveness of IVC-cryopreserved whole testicular biopsy tissue was also validated analyzing fresh embryo transfers. RESULTS: More reliable recovery of motile testicular sperm was achieved using whole tissue freeze preservation combined with IVC (24-96 h) post-acquisition at an incubation temperature of 30 °C compared to ambient temperature (21 °C) or 37 °C. Up to 85 % of the pre-freeze motility was conserved post-thaw (+3 h) for easy ICSI selection. Sperm longevity was optimized to fresh tissue levels by implementing testicular biopsy sucrose dilution post-thaw. Favorable clinical outcomes were proven using frozen-thawed testicular biopsy sperm for ICSI. CONCLUSIONS: By employing minimal tissue manipulation, integrating pre-freeze IVC processing at 30 °C and the freezing of whole testicular biopsy tissue, we have reduced the labor and improved the efficacy of processing testicular tissue for freeze-preservation and subsequent ICSI use.
Asunto(s)
Criopreservación/métodos , Fertilización In Vitro/métodos , Motilidad Espermática/fisiología , Testículo/fisiología , Congelación , Humanos , Técnicas In Vitro/métodos , Masculino , Oligospermia/fisiopatología , Inyecciones de Esperma Intracitoplasmáticas/métodos , Espermatozoides/patología , Espermatozoides/fisiología , Sacarosa/farmacología , Testículo/patologíaRESUMEN
The aim of the present study was to test a new hypothesis that brain cytochrome P450 reductase (CPR) and CPR-dependent enzymes play important roles in behavioral performance. A mouse model with brain neuron-specific deletion of the Cpr gene (brain-Cpr-null) was recently generated. Brain-Cpr-null mice and wild-type (WT) littermates were compared in a variety of behavioral assays. Notable differences were found in the exploratory behavior assay: for both males and females, activity in the center of the chamber was significantly higher for brain-Cpr-null than for WT mice on days 2 and 3 of the assay, although no significant difference was found between the two groups in anxiety-like behavior in the elevated zero maze. Furthermore, in the fear-conditioning assay, brain-Cpr-null mice exhibited significantly less activity suppression than did WT controls. This deficit in activity suppression was not accompanied by any difference between WT and brain-Cpr-null mice in nociceptive responses to foot shocks. Abnormal activity suppression was also observed in both male and female brain-Cpr-null mice during the contextual memory test. However, in the Morris water maze assay, the brain-Cpr-null and WT mice were indistinguishable, indicating normal spatial memory in the mutant mice. These data collectively indicate a novel role of the Cpr gene in fear conditioning and memory.
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Conducta Animal/fisiología , Encéfalo/enzimología , Miedo/fisiología , Memoria/fisiología , NADPH-Ferrihemoproteína Reductasa/metabolismo , Animales , Condicionamiento Clásico , Femenino , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Noqueados , NADPH-Ferrihemoproteína Reductasa/genéticaRESUMEN
Huntington's disease is a neurodegenerative disorder, caused by an elongation of CAG repeats in the huntingtin gene. Mice with an insertion of an expanded polyglutamine repeat in the mouse huntingtin gene (knock-in mice) most closely model the disease because the mutation is expressed in the proper genomic and protein context. However, few knock-in mouse lines have been extensively characterized and available data suggest marked differences in the extent and time course of their behavioral and pathological phenotype. We have previously described behavioral anomalies in the open field as early as 1 month of age, followed by the appearance at 2 months of progressive huntingtin neuropathology, in a mouse carrying a portion of human exon 1 with approximately 140 CAG repeats inserted into the mouse huntingtin gene. Here we extend these observations by showing that early behavioral anomalies exist in a wide range of motor (climbing, vertical pole, rotarod, and running wheel performance) and non-motor functions (fear conditioning and anxiety) starting at 1-4 months of age, and are followed by progressive gliosis and decrease in dopamine and cyclic AMP-regulated phosphoprotein with molecular weight 32 kDa (DARPP32) (12 months) and a loss of striatal neurons at 2 years. At this age, mice also present striking spontaneous behavioral deficits in their home cage. The data show that this line of knock-in mice reproduces canonical characteristics of Huntington's disease, preceded by deficits which may correspond to the protracted pre-manifest phase of the disease in humans. Accordingly, they provide a useful model to elucidate early mechanisms of pathophysiology and the progression to overt neurodegeneration.
Asunto(s)
Conducta Animal/fisiología , Enfermedad de Huntington/patología , Enfermedad de Huntington/psicología , Actividad Motora/fisiología , Neostriado/patología , Animales , Animales Recién Nacidos , Ansiedad/genética , Ansiedad/psicología , Condicionamiento Psicológico/fisiología , Emociones/fisiología , Miedo , Femenino , Técnicas de Sustitución del Gen , Suspensión Trasera/fisiología , Suspensión Trasera/psicología , Enfermedad de Huntington/genética , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Luz , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades Neurodegenerativas/patología , Equilibrio Postural/fisiología , Desempeño Psicomotor/fisiologíaRESUMEN
OBJECTIVE: To determine if finasteride can reduce symptoms in men with a clinical diagnosis of chronic nonbacterial prostatitis (National Institutes of Health, NIH, category IIIA chronic pelvic pain syndrome, CPPS) compared with placebo. PATIENTS AND METHODS: Men (76) with category IIIA CPPS enrolled in four North American prostatitis research centres were randomized after a 2-week placebo run-in to finasteride or placebo for 6 months. The primary efficacy variable was a subjective overall assessment (SOA); the secondary efficacy variables included the NIH chronic prostatitis symptom index (NIH-CPSI) and safety data. Patients were assessed at screening, baseline (after the 2-week placebo run-in), 3 and 6 months. RESULTS: Sixty-four patients had at least one assessment on medication (31 placebo, 33 finasteride); 75% of the finasteride and 54% of the placebo group had at least a mild improvement (defined as > 25% improvement in SOA), and 44% and 27%, respectively, a moderate or marked improvement (>50% improvement in SOA). The trend was similar in the NIH-CPSI scores. Five patients in the finasteride and seven in the placebo group reported medication-related adverse events. CONCLUSION: This randomized placebo-controlled pilot study suggests that finasteride was of benefit for some men with category IIIA CPPS, but the results do not justify recommending finasteride as monotherapy, except for men who also have benign prostatic hyperplasia. A larger, properly powered study, possibly evaluating combination with other therapies or specifically in men with prostatitis and benign prostatic hyperplasia, is required to confirm any clinical benefit.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Finasterida/uso terapéutico , Dolor Pélvico/prevención & control , Prostatitis/tratamiento farmacológico , Adulto , Anciano , Enfermedad Crónica , Inhibidores Enzimáticos/efectos adversos , Finasterida/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Resultado del TratamientoRESUMEN
To determine whether neurons lacking huntingtin can participate in development and survive in postnatal brain, we used two approaches in an effort to create mice consisting of wild-type cells and cells without huntingtin. In one approach, chimeras were created by aggregating the 4-8 cell embryos from matings of Hdh (+/-) mice with wild-type 4-8 cell embryos. No chimeric offspring that possessed homozygous Hdh (-/-) cells were obtained thereby, although statistical considerations suggest that such chimeras should have been created. By contrast, Hdh (-/-) ES cells injected into blastocysts yielded offspring that were born and in adulthood were found to have Hdh (-/-) neurons throughout brain. The Hdh (-/-) cells were, however, 5-10 times more common in hypothalamus, midbrain, and hindbrain than in telencephalon and thalamus. Chimeric animals tended to be smaller than wild-type littermates, and chimeric mice rich in Hdh (-/-) cells tended to show motor abnormalities. Nonetheless, no brain malformations or pathologies were evident. The apparent failure of aggregation chimeras possessing Hdh (-/-) cells to survive to birth is likely attributable to the previously demonstrated critical role of huntingtin in extraembryonic membranes. That Hdh (-/-) cells in chimeric mice created by blastocyst injection are under-represented in adult telencephalon and thalamus implies a role for huntingtin in the development of these regions, whereas the neurological dysfunction in brains enriched in Hdh (-/-) cells suggests a role for huntingtin in adult brain. Nonetheless, the lengthy survival of Hdh (-/-) cells in adult chimeric mice indicates that individual neurons in many brain regions do not require huntingtin to participate in normal brain development and to survive.
Asunto(s)
Encéfalo/patología , Quimera/genética , Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/deficiencia , Neuronas/metabolismo , Proteínas Nucleares/deficiencia , Animales , Conducta Animal , Encéfalo/embriología , Encéfalo/metabolismo , Diferenciación Celular , Movimiento Celular , Supervivencia Celular , Genes Reporteros , Genotipo , Proteína Huntingtina , Hipotálamo/metabolismo , Hipotálamo/patología , Mesencéfalo/metabolismo , Mesencéfalo/patología , Ratones , Actividad Motora/genética , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/genética , Neuronas/patología , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Especificidad de Órganos/genética , Rombencéfalo/metabolismo , Rombencéfalo/patología , Células Madre , Tasa de Supervivencia , Telencéfalo/metabolismo , Telencéfalo/patología , Tálamo/metabolismo , Tálamo/patologíaRESUMEN
Recent studies have shed new light on how the physical association between sister cells is severed at the end of cytokinesis while the membrane is resealed. Comparisons with yeast suggest that daughter cell shape may feed back to regulate cytokinesis through the Bub2 checkpoint system.
Asunto(s)
Proteínas de Ciclo Celular , División Celular , Proteínas de Saccharomyces cerevisiae , Animales , Tamaño de la Célula , Proteínas Fúngicas/fisiología , Humanos , Microtúbulos/ultraestructuraRESUMEN
Grief and bereavement are frequent concerns of primary care physicians. This article outlines grief in terminally ill patients and discusses interventions. Also included in this article is a review of the process of anticipatory grief and mourning and a discussion on normal and complicated grief. A portion of this article also covers grief in children and discusses interventions for grieving children.
Asunto(s)
Actitud Frente a la Muerte , Medicina Familiar y Comunitaria/métodos , Pesar , Atención Primaria de Salud/métodos , Cuidado Terminal/métodos , Cuidado Terminal/psicología , Adaptación Psicológica , Adulto , Actitud Frente a la Salud , Niño , Mecanismos de Defensa , Familia/psicología , Humanos , Relaciones Médico-Paciente , Relaciones Profesional-Familia , Psicología Infantil , Apoyo SocialRESUMEN
Conditional gene repair mutations in the mouse can assist in cell lineage analyses and provide a valuable complement to conditional gene inactivation strategies. We present a method for the generation of conditional gene repair mutations that employs a loxP-flanked (floxed) selectable marker and transcriptional/translational stop cassette (neostop) located within the first intron of a target gene. In the absence of Cre recombinase, expression of the targeted allele is suppressed generating a null allele, while in the presence of Cre, excision of neostop restores expression to wild-type levels. To test this strategy, we have generated a conditional gene repair allele of the mouse Huntington's disease gene homolog (HDH:). Insertion of neostop within the HDH: intron 1 generated a null allele and mice homozygous for this allele resembled nullizygous HDH: mutants and died after embryonic day 8.5. In the presence of a cre transgene expressed ubiquitously early in development, excision of neostop restored HDH: expression and rescued the early embryonic lethality. A simple modification of this strategy that permits the generation of conventional gene knockout, conditional gene knockout and conditional gene repair alleles using one targeting construct is discussed.
Asunto(s)
Silenciador del Gen , Mutagénesis/genética , Proteínas Virales , Alelos , Animales , Northern Blotting , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Ingeniería Genética/métodos , Genotipo , Proteína Huntingtina , Integrasas/genética , Integrasas/metabolismo , Masculino , Ratones , Ratones Noqueados , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN/genética , ARN/metabolismo , Recombinación Genética , TransfecciónRESUMEN
After DNA replication, cells condense their chromosomes in order to segregate them during mitosis. The condensation process as well as subsequent segregation requires phosphorylation of histone H3 at serine 10. Histone H3 phosphorylation initiates during G2 in pericentric foci prior to H3 phosphorylation in the chromosome arms. Centromere protein A (CENP-A), a histone H3-like protein found uniquely at centromeres, contains a sequence motif similar to that around H3 Ser10, suggesting that CENP-A phosphorylation might be linked to pericentric initiation of histone H3 phosphorylation. To test this hypothesis, we generated peptide antibodies against the putative phosphorylation site of CENP-A. ELISA, western blot and immunocytochemical analyses show that CENP-A is phosphorylated at the shared motif. Simultaneous co-detection demonstrates that phosphorylation of CENP-A and histone H3 are separate events in G2/M. CENP-A phosphorylation occurs after both pericentric initiation and genome-wide stages of histone H3 phosphorylation. Quantitative immunocytochemistry reveals that CENP-A phosphorylation begins in prophase and reaches maximal levels in prometaphase. CENP-A phosphoepitope reactivity is lost during anaphase and becomes undetectable in telophase cells. Duplication of prekinetochores, detected as the doubling of CENP-A foci, occurs prior to complete histone H3 phosphorylation in G2. Mitotic phosphorylation of histone H3-family proteins shows tight spatial and temporal control, occurring in three phases: (1) pericentric H3 phosphorylation, (2) chromosome arm H3 phosphorylation and (3) CENP-A phosphorylation at kinetochores. These observations reveal new cytological landmarks characteristic of G2 progression.
Asunto(s)
Autoantígenos , Proteínas Cromosómicas no Histona/metabolismo , Fase G2 , Histonas/metabolismo , Mitosis , Secuencia de Aminoácidos , Línea Celular , Proteína A Centromérica , Proteínas Cromosómicas no Histona/química , Histonas/química , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Fosforilación , Homología de Secuencia de AminoácidoRESUMEN
Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.
Asunto(s)
Autoantígenos , Proteínas Cromosómicas no Histona/metabolismo , Citocinas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Aurora Quinasa B , Aurora Quinasas , Western Blotting , Caenorhabditis elegans/metabolismo , Línea Celular , Centrómero/fisiología , Proteína A Centromérica , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/genética , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Histonas/metabolismo , Humanos , Mitosis , Mutagénesis Sitio-Dirigida , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , TransfecciónRESUMEN
Inactivation of the mouse homologue of the Huntington disease gene (Hdh) results in early embryonic lethality. To investigate the normal function of Hdh in the adult and to evaluate current models for Huntington disease (HD), we have used the Cre/loxP site-specific recombination strategy to inactivate Hdh expression in the forebrain and testis, resulting in a progressive degenerative neuronal phenotype and sterility. On the basis of these results, we propose that huntingtin is required for neuronal function and survival in the brain and that a loss-of-function mechanism may contribute to HD pathogenesis.
Asunto(s)
Encéfalo/metabolismo , Marcación de Gen , Infertilidad Masculina/etiología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Enfermedades Neurodegenerativas/etiología , Proteínas Nucleares/antagonistas & inhibidores , Testículo/metabolismo , Proteínas Virales , Alelos , Animales , Encéfalo/patología , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Huntingtina , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Integrasas/genética , Integrasas/metabolismo , Longevidad/genética , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/análisis , Datos de Secuencia Molecular , Trastornos del Movimiento/etiología , Trastornos del Movimiento/genética , Trastornos del Movimiento/metabolismo , Proteínas del Tejido Nervioso/fisiología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Proteínas Nucleares/fisiología , Fenotipo , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Secuencias Reguladoras de Ácidos Nucleicos , Espermatogénesis/genética , Testículo/patologíaRESUMEN
Huntington's disease is a devastating progressive neurodegenerative illness characterized by massive neuronal loss in the striatum. It is caused by the presence of an expanded CAG repeat in the gene encoding huntingtin, a protein of unknown function. We have examined the expression of neurotransmitters and other antigens present in striatal neurons with immunohistochemistry, and the level of expression of mRNAs encoding enkephalin, substance P, and glutamic acid decarboxylases with quantitative in situ hybridization histochemistry, in the striatum of two mouse models of Huntington's disease: transgenic animals expressing exon 1 of the human huntingtin gene with 144 CAG repeats and "knock-in" mice containing a chimeric mouse/human exon 1 with 71 or 94 CAG repeats inserted by homologous targeting. Although the transgenic (but not the knock-in) mice were previously shown to display prominent huntingtin- and ubiquitin-containing nuclear inclusions in striatal neurons, in situ nick translation followed by emulsion autoradiography did not reveal any DNA damage in striatum or cortex in these mice. Immunolabeling for calbindin D 28K, enkephalin, substance P, glutamic acid decarboxylases (M(r) 65,000 or 67,000, GAD65 and GAD67), somatostatin, choline acetyltransferase, parvalbumin, and glial fibrillary acidic protein were remarkably similar in transgenic, knock-in, and wild-type mice. Both transgenic and knock-in mice, however, showed a marked decrease in the level of expression of enkephalin mRNA in striatal neurons without significant decreases in mRNAs encoding substance P, GAD65, or GAD67. The data indicate that decreased expression of enkephalin mRNA may be an early sign of neuronal dysfunction due to the Huntington's disease mutation.
Asunto(s)
Cuerpo Estriado/metabolismo , Encefalinas/genética , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , ARN Mensajero/metabolismo , Animales , Biomarcadores , Calbindinas , Corteza Cerebral/metabolismo , Cuerpo Estriado/citología , Daño del ADN/genética , Modelos Animales de Enfermedad , Encefalinas/metabolismo , Femenino , Glutamato Descarboxilasa/biosíntesis , Glutamato Descarboxilasa/genética , Homocigoto , Proteína Huntingtina , Isoenzimas/biosíntesis , Isoenzimas/genética , Masculino , Ratones , Ratones Noqueados , Ratones Mutantes Neurológicos , Actividad Motora/genética , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/enzimología , Proteínas Nucleares/genética , Proteína G de Unión al Calcio S100/biosíntesis , Sustancia P/biosíntesis , Sustancia P/genética , Repeticiones de Trinucleótidos/genéticaAsunto(s)
Encéfalo/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Regulación de la Expresión Génica , Integrasas/genética , Recombinación Genética , Transgenes , Proteínas Virales , Animales , Encéfalo/enzimología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Ratones , Ratones TransgénicosRESUMEN
Huntingtin-associated protein 1 (HAP1) interacts with the product of the Huntington's disease gene. To investigate the function of Hap1 in development and in the adult mouse, we have examined the expression of Hap1 by northern analysis and in situ hybridization histochemistry. Hap1 expression is first detected in the embryonic day 8.5 (E8.5) neuroepithelium. Expression persists throughout development, predominantly in the brain and spinal cord, and to a lesser extent in enteric neurons and abdominal sympathetic ganglia. In the adult, Hap1 expression is detected not only in the brain but also in the ovary, testis, and the intermediate lobe of the pituitary.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Animales , Northern Blotting , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario y Fetal , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Especificidad de ÓrganosRESUMEN
To explore the potential of a simple and rapid approach for ubiquitous conditional gene disruption, we have generated Cre-producer mouse transgenic lines (Hs-cre1, 6 and 7) expressing a recombinase transgene (cre) from a heat shock gene promoter and tested their performance in Cre-mediated excision of target DNA in crosses with Cre-responder strains carrying loxP-modified alleles of the genes encoding the Huntington's disease gene homolog (Hdh), the epidermal growth factor receptor (Egfr), and the type 1 insulin-like growth factor receptor (Igf1r). Analyses of progeny possessing various transgene/reporter combinations showed that cre expression can occur without heat shock in early embryos, but this constitutive transcription is stochastic and transgene dependent. Thus, Hs-cre1 behaves predominantly as a "deleter" strain, since the majority of progeny (approximately 70-85%) exhibit complete recombination, regardless of reporter locus. Lines Hs-cre6 and Hs-cre7, however, function successfully as "mosaicking" strains because, in addition to two extreme classes of progeny with 0% or 100% recombination, they generate an intermediate class of mosaics exhibiting various degrees of partial DNA excision. Notably, the frequency of offspring in each class varies between reporters, but mosaic embryos are consistently obtained in adequate numbers (approximately 30-60%). The Hs-cre6 transgene is also inducible and can be used to introduce mosaicism into adult tissues at preselected developmental times by heat shock treatment of mice with 0% recombination in tail DNA. By bypassing the lethality resulting from some gene knockouts, mosaic embryos and mice make particular mutational analyses possible and are also very useful for the identification of cell lineage-specific gene functions.
Asunto(s)
Proteínas de Choque Térmico/genética , Integrasas/genética , Regiones Promotoras Genéticas/genética , Transgenes/genética , Proteínas Virales , Animales , Embrión de Mamíferos/metabolismo , Femenino , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Calor , Masculino , Ratones , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Embarazo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Distribución TisularRESUMEN
Huntington's disease (HD) is a neurodegenerative disease associated with polyglutamine expansion in huntingtin, a widely expressed protein. The function of huntingtin is unknown although huntingtin plays a fundamental role in development since gene targeted HD (-) (/-)mouse embryos die shortly after gastrulation. Expression of huntingtin is detected in spleen and thymus but its role in hematopoiesis has not been examined. To determine the function of huntingtin and to provide insight into potential pathologic mechanisms in HD, we analyzed the role of huntingtin in hematopoietic development. Expression of huntingtin was analyzed in a variety of hematopoietic cell types, and in vitro hematopoiesis was assessed using an HD ( +/-)and several HD( -) (/-)embryonic stem (ES) cell lines. Although wild-type, HD ( +/-)and HD( -) (/-)ES cell lines formed primary embryoid bodies (EBs) with similar efficiency, the numbers of hematopoietic progenitors detected at various stages of the in vitro differentiation were reduced in HD ( +/-)and HD( -/-)() ()ES cell lines examined. Expression analyses of hematopoietic markers within the EBs revealed that primitive and definitive hematopoiesis occurs in the absence of huntingtin. However, further analysis using a suspension culture in the presence of hematopoietic cytokines demonstrated a highly significant gene dosage-dependent decrease in proliferation and/or survival of HD ( +/-)and HD( -) (/-)cells. Enrichment for the CD34(+)cells within the EB confirmed that the impairment is intrinsic to the hematopoietic cells. These obser- vations suggest that huntingtin expression is required for the generation and expansion of hematopoietic cells and provides an alternative system in which to assess the function of huntingtin.
Asunto(s)
Hematopoyesis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Células Madre/metabolismo , Animales , Southern Blotting , Western Blotting , Línea Celular , Células Cultivadas , Quimera , Embrión de Mamíferos , Humanos , Proteína Huntingtina , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Hyperprolactinemia from a pituitary adenoma is a rare cause of erectile dysfunction. Men with erectile dysfunction who are found to have a low testosterone level should have a measurement of their prolactin level. Treatment consists of lowering the prolactin level by medication or surgery, or both. Bromocriptine, a dopamine agonist, is efficacious in lowering elevated prolactin levels and can simultaneously shrink these pituitary tumors. With large tumors, transphenoidal surgery may be used to debulk/remove the tumor. Post-treatment prolactin levels can be used to monitor the efficacy of treatment.
RESUMEN
OBJECTIVES: The National Institutes of Health (NIH) category III chronic prostatitis syndromes (nonbacterial chronic prostatitis and prostatodynia) are common disorders with few effective therapies. Bioflavonoids have recently been shown in an open-label study to improve the symptoms of these disorders in a significant proportion of men. The aim of this study was to confirm these findings in a prospective randomized, double-blind, placebo-controlled trial. METHODS: Thirty men with category IIIa and IIIb chronic pelvic pain syndrome were randomized in a double-blind fashion to receive either placebo or the bioflavonoid quercetin 500 mg twice daily for 1 month. The NIH chronic prostatitis symptom score was used to grade symptoms and the quality-of-life impact at the start and conclusion of the study. In a follow-up unblind, open-label study, 17 additional men received 1 month of a supplement containing quercetin, as well as bromelain and papain (Prosta-O), which enhance bioflavonoid absorption. RESULTS: Two patients in the placebo group refused to complete the study because of worsening symptoms, leaving 13 placebo and 15 bioflavonoid patients for evaluation in the blind study. Both the quercetin and placebo groups were similar in age, symptom duration, and initial symptom score. Patients taking placebo had a mean improvement in NIH symptom score from 20.2 to 18.8 (not significant), while those taking the bioflavonoid had a mean improvement from 21.0 to 13.1 (P = 0.003). Twenty percent of patients taking placebo and 67% of patients taking the bioflavonoid had an improvement of symptoms of at least 25%. In the 17 patients who received Prosta-Q in the open-label study, 82% had at least a 25% improvement in symptom score. CONCLUSIONS: Therapy with the bioflavonoid quercetin is well tolerated and provides significant symptomatic improvement in most men with chronic pelvic pain syndrome.
Asunto(s)
Prostatitis/tratamiento farmacológico , Quercetina/uso terapéutico , Adulto , Anciano , Enfermedad Crónica , Método Doble Ciego , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
We used two mouse models of Huntington's disease (HD) to examine changes in glutamate receptor sensitivity and striatal electrophysiology. One model, a transgenic, consisted of mice expressing exon 1 of the human HD gene and carrying 141-157 CAG repeat sequences (R6/2 line). The second model, a CAG repeat "knockin," consisted of mice with different lengths of CAG repeats (CAG71 and CAG94 repeats). The effects of glutamate receptor activation were examined by visualizing neurons in brain slices with infrared videomicroscopy and differential interference contrast optics to determine changes in somatic area (cell swelling). Striatal and cortical neurons in both models (R6/2 and CAG94) displayed more rapid and increased swelling to N-methyl-D-aspartate (NMDA) than those in controls. This effect was specific as there were no consistent group differences after exposure to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or kainate (KA). Intracellular recordings revealed that resting membrane potentials (RMPs) in the R6/2 transgenics were significantly more depolarized than those in their respective controls. RMPs in CAG94 mice also were more depolarized than those in CAG71 mice or their controls in a subset of striatal neurons. Confirming previous results, R6/2 mice expressed behavioral abnormalities and nuclear inclusions. However, CAG71 and CAG94 knockins did not, suggesting that increased sensitivity to NMDA may occur early in the disease process. These findings imply that NMDA antagonists or compounds that alter sensitivity of NMDA receptors may be useful in the treatment of HD.