RESUMEN
An efficient and accurate method for controlled in vivo transgene modulation by site-directed recombination is described. Seven transgenic mouse founder lines were produced carrying the murine lens-specific alpha A-crystallin promoter and the simian virus 40 large tumor-antigen gene sequence, separated by a 1.3-kilobase-pair Stop sequence that contains elements preventing expression of the large tumor-antigen gene and Cre recombinase recognition sites. Progeny from two of these lines were mated with transgenic mice expressing the Cre recombinase under control of either the murine alpha A-crystallin promoter or the human cytomegalovirus promoter. All double-transgenic offspring developed lens tumors. Subsequent analysis confirmed that tumor formation resulted from large tumor-antigen activation via site-specific, Cre-mediated deletion of Stop sequences.
Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Integrasas , Oncogenes , Proteínas Virales , Animales , Secuencia de Bases , Catarata/genética , ADN Nucleotidiltransferasas/genética , Neoplasias del Ojo/genética , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Recombinación Genética , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
The isolation and sequencing of the core peptide (beta-amyloid) found in the plaques of patients with Alzheimer's disease has allowed the identification of a cDNA for the precursor protein. Using a human cDNA clone for this beta-amyloid material, we have identified an homologous mRNA (3.8 kb) in brain tissue obtained from 8 additional species. We have also determined its distribution in 7 brain regions and 12 organs obtained from rodents. A prominent, second mRNA species (2.2 kb) has been identified in rat non-neuronal tissues. The beta-amyloid gene is amply expressed in the brain of all vertebrates tested and in most rodent organs, indicating that it encodes a highly conserved and ubiquitous protein.
Asunto(s)
Enfermedad de Alzheimer/patología , Amiloide/análisis , Química Encefálica , Precursores de Proteínas/análisis , ARN Mensajero/análisis , Amiloide/genética , Péptidos beta-Amiloides , Animales , Bovinos , Pollos , Cricetinae , Perros , Cobayas , Humanos , Inmunoensayo , Ratones , Conejos , RatasRESUMEN
We have investigated the effect of dopamine receptor blockade on striatal proenkephalin mRNA and protachykinin mRNA by Northern gel analysis and by in situ hybridization histochemistry. Chronic haloperidol treatment resulted in a 3.5 fold increase in striatal proenkephalin mRNA and a 30% decrease in protachykinin mRNA (no apparent change in alpha-tubulin mRNA was observed). The changes in mRNA levels for protachykinin and proenkephalin were uniform throughout the caudate-putamen of the rat as determined by in situ hybridization histochemistry. The results imply that altering receptor-mediated neurotransmitter functions can lead to profound, specific, and long-lasting alterations in neuronal gene expression.
Asunto(s)
Cuerpo Estriado/metabolismo , Encefalinas/genética , Haloperidol/farmacología , Neuropéptidos/genética , Precursores de Proteínas/genética , ARN Mensajero/metabolismo , Receptores Dopaminérgicos/fisiología , Taquicininas , Animales , Secuencia de Bases , Cuerpo Estriado/efectos de los fármacos , ARN Mensajero/genética , Ratas , Receptores Dopaminérgicos/efectos de los fármacosRESUMEN
Immunocytochemical studies have shown that adrenalectomy produces changes in the content and distribution of [arginine-8]vasopressin (AVP) immunoreactivity in the paraventricular nucleus of the hypothalamus. The purpose of this study was to determine whether manipulation of adrenal hormones affects the levels of AVP mRNA. In situ hybridization assays with highly specific synthetic oligodeoxyribonucleotide probes and immunocytochemistry were used to detect the distribution of AVP mRNA and AVP-immunoreactive perikarya. AVP mRNA is codistributed with AVP immunoreactivity in the posterior magnocellular subdivision of the paraventricular nucleus and its accessory nuclei, the supraoptic nucleus and the suprachiasmatic nucleus. In adrenalectomized rats, the density and distribution of the hybridization signal were increased in the paraventricular nucleus; a 2-fold increase in the area comprising the signal was observed. At the cellular level, silver grains were detected in corticotropin-releasing-factor-immunoreactive neurons throughout the medial parvocellular subdivision of the paraventricular nucleus. No changes were seen in the distribution of AVP mRNA in the supraoptic or suprachiasmatic nuclei. Treatment with dexamethasone prevented the increase in AVP mRNA produced by adrenalectomy. In contrast, adrenalectomy did not alter the hybridization signal obtained with a probe for alpha-tubulin mRNA. These results suggest, at the cellular level, that adrenalectomy induces a glucocorticoid-sensitive stimulation of AVP mRNA synthesis in the central nervous system. Thus, considerable plasticity in gene expression is retained in the hypothalamus of the adult rat.
Asunto(s)
Arginina Vasopresina/biosíntesis , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , ARN Mensajero/biosíntesis , Adrenalectomía , Animales , Arginina Vasopresina/genética , Marcadores Genéticos , Masculino , Neurosecreción/efectos de los fármacos , Oligodesoxirribonucleótidos/síntesis química , Núcleo Hipotalámico Paraventricular/metabolismo , Ratas , Ratas EndogámicasRESUMEN
In summary, this study supports previous research and suggests that the high-turnover profile can be described by the following characteristics (the reader is reminded that these characteristics describe the sample in this study and cannot be readily applied to the population of all nurse's aides): Individuals who appeared likely to terminate employment in one year were less than 28 years of age, not married, and trained or educated beyond the job's requirements. Previous employment histories of individuals who left their jobs within one year indicated that they were more likely to have had less than 18 months in previous positions, and worked in two or more nursing homes within a three-year period. Nursing home management may find it helpful to refer to high-turnover characteristics when screening and selecting applicants for the position of nurse's aide. Through careful selection of employees, nursing homes can expect a reduced rate of turnover, which may, in turn, improve the quality of resident care.
Asunto(s)
Asistentes de Enfermería/provisión & distribución , Casas de Salud , Administración de Personal , Selección de Personal , Reorganización del Personal , Factores de Edad , Humanos , Estadística como Asunto , Recursos HumanosRESUMEN
Procedures are presented for routine evaluation of antibody specificity, titre, and quantitation of antigen levels in tissue extracts without the use of radiolabeled probes. A colorimetric, enzyme-linked immunosorbent assay (ELISA) is described for general use with neuropeptides, using neurotensin as a primary example. These assays use rabbit anti-neurotensin immune serum which is colorimetrically identified after combination with an alkaline phosphatase-conjugated, affinity purified, goat anti-rabbit IgG and reaction with the chromogenic substrate, p-nitrophenyl phosphate. Because the principle of these methods can be adapted for use with various proteins and neuropeptides, they should find widespread applicability in neurobiology.
Asunto(s)
Química Encefálica , Ensayo de Inmunoadsorción Enzimática , Técnicas para Inmunoenzimas , Neurotensina/análisis , Fosfatasa Alcalina , Animales , Cromatografía Líquida de Alta Presión , Sueros Inmunes , Neurotensina/inmunología , Nitrofenoles/farmacología , Compuestos Organofosforados/farmacología , Conejos , Ratas , Factores de TiempoRESUMEN
The discrete anatomical distribution of arginine vasopressin and corticotropin releasing factor (CRF) immunoreactivity in the paraventricular nucleus (PVN) of the rat hypothalamus is altered after adrenalectomy. Not only is the immunostaining of both peptides enhanced, but vasopressin immunoreactivity, normally confined to the magnocellular subdivision, becomes clear in a large percentage of CRF neurones in the parvocellular subdivision. These changes in immunoreactivity may reflect changes in post-translational events, peptide metabolism or genomic activity that lead indirectly or directly to the enhanced expression of vasopressin. Here we report that levels of transcripts homologous to vasopressin messenger RNA increase in the PVN after adrenalectomy, in parallel with increases in vasopressin immunoreactivity. In fact, after adrenalectomy, vasopressin mRNA can be detected in CRF-immunoreactive neurones. These results indicate that a considerable degree of plasticity is retained by the adult neuronal genome of the rat and that this plasticity may be modulated by the endocrine environment.
Asunto(s)
Adrenalectomía , Hormona Liberadora de Corticotropina/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Vasopresinas/metabolismo , Animales , Hormona Liberadora de Corticotropina/genética , Regulación de la Expresión Génica , Masculino , Plasticidad Neuronal , Hibridación de Ácido Nucleico , Ratas , Ratas Endogámicas , Vasopresinas/genéticaRESUMEN
The postnatal ontogeny of neurotensin (NT) in the medial preoptic area (MPO) of the rat was quantitatively investigated using immunocytochemical and neurochemical techniques. On postpartum day 0, NT-like immunoreactive neurons could not be identified within the MPO by peroxidase-antiperoxidase histochemistry and, in fact, did not appear until 9 days postpartum. After this age, the number of NT cells in the MPO increased ontogenetically, as did concentrations of NT in MPO extracts measured by radioimmunoassay and enzyme-linked immunosorbent assay. In organotypic MPO explants derived from neonatal rats, NT-like immunoreactivity was detected only after the explants were maintained 14 days in vitro, after which, the number of NT-like immunoreactive somata increased as the time in vitro was extended. These findings indicate that a substantial amount of differentiation occurs postnatally in the MPO both in vivo and in vitro and, more specifically, that the ontogenetic expression of NT may be an intrinsic property of the MPO.
Asunto(s)
Neurotensina/análisis , Área Preóptica/análisis , Factores de Edad , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnicas para Inmunoenzimas , Masculino , Neurotensina/metabolismo , Área Preóptica/crecimiento & desarrollo , Área Preóptica/metabolismo , Radioinmunoensayo , Ratas , Ratas EndogámicasRESUMEN
The distribution of mRNA with high sequence homology to somatostatin mRNA within the periventricular hypothalamus of rat was assessed using in situ hybridization techniques with synthetic oligodeoxyribonucleotide probes, complementary to the 3' coding region of rat somatostatin mRNA. The probes (22- and 24-mers) were 5'-end labeled using T4 polynucleotide kinase and gamma-32P-ATP. They were used either individually or after ligation with T4 DNA ligase to form a 46-mer. Serial tissue sections (less than 10 microns) were taken from the level of the preoptic/anterior hypothalamus through the paraventricular hypothalamus. In situ hybridizations were conducted at room temperature in hybridization buffer. Neurons immunoreactive with antiserum raised against somatostatin were identified in alternate sections using standard immunocytochemical procedures. The anatomical location of the hybridization signal was determined by autoradiography. Our results show that the peri- and paraventricular hypothalamus is rich in transcripts putatively coding for somatostatin and that these transcripts are co-distributed with neurons immunoreactive with antisomatostatin immunoglobulin.