RESUMEN
Antibodies, due to their high specificities and retention, represent potential beta cell imaging agents, however their slow clearance from the blood may preclude their use. Antibody fragments (Fabs) have much higher clearance and if they can be made with similar binding characteristics, would be more efficacious agents. An existing beta cell specific antibody (K14D10) and its Fab were evaluated with a previously developed screening assay. The Fab and the intact immunoglobulin (IgG) had similar affinities (6 - 20 nM), binding sites (300 000 - 700 000 sites/cell), and binding kinetics (t (1/2) = 8 - 18 minutes) for beta cells. However, the cellular specificity was far below the estimated requisite values needed to overcome the very low beta cell mass in the pancreas. The Fab cleared the blood twice as fast as the IgG, but did not preferentially accumulate into pancreas. Thus, generation of Fabs from IgGs with high beta cell binding and blood clearance appears feasible, but in order for molecules to be useful for tracking beta cell mass, antibodies of greater cellular specificity will have to be used.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Diabetes Mellitus Tipo 1/inmunología , Inmunoconjugados/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Islotes Pancreáticos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacocinética , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos , Secuencia de Bases , Sitios de Unión de Anticuerpos , Inmunoconjugados/farmacocinética , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/farmacología , Radioisótopos de Yodo , Datos de Secuencia Molecular , Unión Proteica , Ratas , Ratas Endogámicas F344 , Distribución TisularRESUMEN
The orphan nuclear receptor Nurr1 is essential for development of midbrain dopamine (DA) cells. In Nurr1-deficient mice, DA precursor cells fail to migrate normally, are unable to innervate target areas, and only transiently express DA cell marker genes. In the search for Nurr1-regulated genes that might explain this developmental phenotype, we found that expression of the receptor tyrosine kinase Ret is deregulated in these cells of Nurr1-deficient embryos. In addition, our analyses establish Nurr1 as an early marker for the dorsal motor nucleus (DMN) of the vagus nerve. Interestingly, Ret expression is absent also in these cells in Nurr1-targeted mice. Neuronal innervation of vagus nerve target areas appeared normal apart from a subtle disorganization of the DMN-derived nerve fibers. In conclusion, regulation of Ret by Nurr1 in midbrain DA neurons and in the DMN has implications for both embryonal development and adult physiology in which signaling by neurotrophic factors plays important roles.
Asunto(s)
Proteínas de Unión al ADN , Dopamina/metabolismo , Proteínas de Drosophila , Bulbo Raquídeo/metabolismo , Proteínas de Transporte de Membrana , Mesencéfalo/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Citoplasmáticos y Nucleares/deficiencia , Factores de Transcripción/deficiencia , Nervio Vago/metabolismo , Proteínas de Transporte Vesicular , Acetilcolinesterasa/metabolismo , Aldehído Oxidorreductasas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Femenino , Feto , Regulación de la Expresión Génica/fisiología , Inmunohistoquímica , Hibridación in Situ , Masculino , Bulbo Raquídeo/citología , Bulbo Raquídeo/embriología , Mesencéfalo/citología , Mesencéfalo/embriología , Ratones , Ratones Noqueados , Neuronas/citología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Proteínas Proto-Oncogénicas c-ret , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Retinal-Deshidrogenasa , Factores de Transcripción/genética , Nervio Vago/citología , Nervio Vago/embriología , Proteínas de Transporte Vesicular de Acetilcolina , Vísceras/embriología , Vísceras/inervaciónRESUMEN
Dopamine cells are generated in the ventral midbrain during embryonic development. The progressive degeneration of these cells in patients with Parkinson's disease, and the potential therapeutic benefit by transplantation of in vitro generated dopamine cells, has triggered intense interest in understanding the process whereby these cells develop. Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons. However, the mechanism by which Nurr1 promotes dopamine cell differentiation has remained unknown. In this study we have used a dopamine-synthesizing cell line (MN9D) with immature characteristics to analyze the function of Nurr1 in dopamine cell development. The results demonstrate that Nurr1 can induce cell cycle arrest and a highly differentiated cell morphology in these cells. These two functions were both mediated through a DNA binding-dependent mechanism that did not require Nurr1 interaction with the heterodimerization partner retinoid X receptor. However, retinoids can promote the differentiation of MN9D cells independently of Nurr1. Importantly, the closely related orphan receptors NGFI-B and Nor1 were also able to induce cell cycle arrest and differentiation. Thus, the growth inhibitory activities of the NGFI-B/Nurr1/Nor1 orphan receptors, along with their widespread expression patterns both during development and in the adult, suggest a more general role in control of cell proliferation in the developing embryo and in adult tissues.
Asunto(s)
Encéfalo/citología , Dopamina/química , Proteínas del Tejido Nervioso , Retinoides/farmacología , Factores de Transcripción/química , Animales , Bromodesoxiuridina/metabolismo , Ciclo Celular , Diferenciación Celular , División Celular , Línea Celular , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Dimerización , Fase G1 , Genes Reporteros , Humanos , Inmunohistoquímica , Hibridación in Situ , Proteínas Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Fenotipo , Plásmidos/metabolismo , Unión Proteica , Receptores de Ácido Retinoico/metabolismo , Receptores de Esteroides , Receptores de Hormona Tiroidea , Receptores X Retinoide , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
The orphan nuclear receptor NURR1 was previously demonstrated to be required for the generation of mesencephalic dopamine (DA) cells. However, even in the absence of NURR1, which is normally expressed as cells become postmitotic, neuronal differentiation is induced and expression of several genes detected in developing dopamine cells appears normal during early stages of development. These include the homeobox transcription factors engrailed and Ptx-3 as well as aldehyde dehydrogenase 2, here defined as the earliest marker identified in developing DA cells, expressed already in mitotic DA progenitors. We have used the expression of these dopaminergic markers, retrograde axonal tracing, and apoptosis analyses to study the fate of the DA progenitor cells in the absence of NURR1. We conclude that NURR1 plays a critical role in the maturation, migration, striatal target area innervation, and survival of differentiating mesencephalic DA cells.
Asunto(s)
Aldehído Deshidrogenasa/genética , Proteínas de Unión al ADN , Mesencéfalo/citología , Neuronas/enzimología , Células Madre/enzimología , Factores de Transcripción/genética , Aldehído Deshidrogenasa Mitocondrial , Animales , Animales Recién Nacidos , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Dopamina/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Proteínas de Homeodominio/genética , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Mutantes , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , ARN Mensajero/análisis , Células Madre/citología , Transcripción Genética/fisiologíaRESUMEN
In response to injury of the central nervous system, astrocytes become reactive and express high levels of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP), vimentin, and nestin. We have shown that astrocytes in mice deficient for both GFAP and vimentin (GFAP-/-vim-/-) cannot form IFs even when nestin is expressed and are thus devoid of IFs in their reactive state. Here, we have studied the reaction to injury in the central nervous system in GFAP-/-, vimentin-/-, or GFAP-/-vim-/- mice. Glial scar formation appeared normal after spinal cord or brain lesions in GFAP-/- or vimentin-/- mice, but was impaired in GFAP-/-vim-/- mice that developed less dense scars frequently accompanied by bleeding. These results show that GFAP and vimentin are required for proper glial scar formation in the injured central nervous system and that some degree of functional overlap exists between these IF proteins.
Asunto(s)
Astrocitos/fisiología , Lesiones Encefálicas/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Proteínas del Tejido Nervioso , Traumatismos de la Médula Espinal/metabolismo , Vimentina/genética , Animales , Astrocitos/química , Astrocitos/ultraestructura , Lesiones Encefálicas/genética , División Celular/fisiología , Cicatriz/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Epéndimo/citología , Epéndimo/metabolismo , Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Nestina , Traumatismos de la Médula Espinal/genética , Vasodilatación/fisiología , Vimentina/metabolismo , Heridas Punzantes/genética , Heridas Punzantes/metabolismoRESUMEN
Nurr1, an orphan receptor of the nuclear receptor superfamily, is widely expressed in the central nervous system (CNS) including brain regions where dopaminergic neurons are abundant. Recent analyses of Nurr1 null mutant mice have shown that Nurr1 is essential for the development and survival of midbrain dopaminergic neurons. However, other dopaminergic neuronal populations do not seem to be affected by ablation of the Nurr1 gene. The purpose of the present study was to investigate the degree of co-existence of Nurr1 mRNA and tyrosine hydroxylase (TH) immunoreactivity in the brain of adult mice to better characterize the selective effects of Nurr1 on catecholaminergic neurons. Our results indicate that the majority of TH-immunoreactive neurons in the substantia nigra (SN; 96%), ventral tegmental area (VTA; 95%), retrorubral field (91%), olfactory bulb (85%), linear nucleus raphe (91%) and central grey (61%) express Nurr1. In contrast, dopaminergic cells of the paraventricular and periventricular hypothalamic nucleus showed only a few Nurr1/TH double labeled neurons, while TH-immunoreactive neurons in the arcuate nucleus and zona incerta did not express Nurr1 mRNA. Nurr1 expression was also excluded from (nor)adrenergic neurons of the brainstem. In conclusion, Nurr1 transcripts were not found in all CNS catecholaminergic neurons. Nurr1 expression was confined to periglomerular and midbrain dopaminergic neurons. These results suggest that within the adult mouse brain, Nurr1 may participate in dopaminergic functions of the olfactory bulb and midbrain.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Unión al ADN , Dopamina/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores NuclearesRESUMEN
The family of Tyr/Thr protein phosphatases, called dual-specificity phosphatases, have been implicated in the feedback regulation of the MAP kinase cascade by dephosphorylating the MAP kinases. Using low stringent cDNA screening we have isolated a chicken homologue of the CL100 phosphatase also called MAP kinase phosphatase 1 (MKP-1). The chicken MKP-1 has 84% and 85.5% identity to the rat and human amino acid sequence, respectively. Using RNase protection assay and in situ hybridization we have found that MKP-1 mRNA is expressed at low levels in most tissues during development. In embryonic dorsal root and sympathetic ganglia MKP-1 mRNA expression increases with age. The expression in large cells in dorsal root ganglia suggests that it is neurons which express MKP-1 mRNA. We also show that MKP-1 mRNA is induced in dissociated embryonic sympathetic neurons after nerve growth factor stimulation. In addition, our results show that MKP-1 mRNA is induced after NGF stimulation of fibroblasts expressing the NGF receptor TrkA, suggesting that MKP-1 is upregulated after activation of the TrkA receptor. These data show that the MKP-1 gene is regulated in a tissue and temporal specific fashion with strong expression in the developing peripheral ganglia, and suggest that the activation of MKP-1 mRNA expression by NGF is a ubiquitously induced response to TrkA activation, independent of the cellular origin or type on which the TrkA receptor is active.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Inmediatas-Precoces/genética , Factores de Crecimiento Nervioso/farmacología , Fosfoproteínas Fosfatasas , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/biosíntesis , Regulación hacia Arriba/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Encéfalo/embriología , Embrión de Pollo , Pollos , Clonación Molecular , ADN Complementario/aislamiento & purificación , Fosfatasa 1 de Especificidad Dual , Ganglios Simpáticos/metabolismo , Humanos , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/efectos de los fármacos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Ratas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
To study the magnitude of agreement between a short self-administered food frequency questionnaire and a 7-day weighed dietary record regarding the consumption of fibre and fat, we collected information from 92 randomly selected middle-aged Swedish men. The participants first recorded all foods and drinks consumed over seven consecutive days by means of a digital scale. One month after the 7-day weighed record had been completed, a self-administered food frequency questionnaire on habitual consumption of foods containing fibre or fat during the preceding month was sent to the subjects. The 20 largest contributors of fibre and fat in the diet accounted for 71% and 52% of the total intake, respectively, based on the 7-day weighed record. The estimated mean consumption of fibre was 19.0 grams per day based on the 7-day weighed record and 18.3 grams per day based on the food frequency questionnaire. Corresponding estimated mean consumption of fat was 88.6 grams per day versus 46.9 grams per day. Men with high physical activity and low BMI, respectively, had higher intake of fibre and fat. These differences were seen for both dietary measurement methods but were more marked using the 7-day weighed record. The agreement between methods for each individual was also assessed. Based on five categories of fibre consumption, 61% of the respondents in the highest quintile according to the 7-day weighed record were classified in one of the two highest quintiles according to the food frequency questionnaire. The corresponding figure for fat intake was 56%. We conclude that the short self-administered food frequency questionnaire used in the present study can assess the absolute intake of fibre, but not of fat, with good precision. Also, the ranking of individuals in broad categories of consumption of fibre and fat was not largely misclassified based on this short questionnaire.
Asunto(s)
Registros de Dieta , Grasas de la Dieta/administración & dosificación , Fibras de la Dieta/administración & dosificación , Conducta Alimentaria , Adulto , Índice de Masa Corporal , Ingestión de Energía , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , SueciaRESUMEN
With the end of the Cold War and establishment of relations with Russia, the opportunity for nurses to meet their Russian colleagues is now available. The authors, with an international group of nurses, visited Russia to learn about their healthcare system and nursing practice. The authors found Russian healthcare system severely lacking in the technology and resources available in other industraialized nations. Within this system, the nurse's role is significantly different from nursing in the American healthcare system. Nurses practice within a system of severe shortages and provide nursing care dependent upon physicians.
Asunto(s)
Atención a la Salud/organización & administración , Intercambio Educacional Internacional , Atención de Enfermería/organización & administración , Humanos , Federación de RusiaRESUMEN
An analysis was conducted on the association between weight and overactivity in a sample of 148 mentally retarded adults. The results indicate an inverse relationship between weight and degree of overactivity.