RESUMEN

The groggy rat (strain name; GRY) exhibits ataxia, an unstable gait, and paroxysmal severe extension of the entire body. Adults show a reduction in size of the cerebellum and presynaptic and axon terminal abnormalities of Purkinje cells. These neurological abnormalities are inherited in an autosomal recessive manner, and the causative mutation has been named groggy (gry). In this study, we mapped gry on rat chromosome 19 and found a nonconservative missense (M251K) mutation in the alpha(1A) subunit of the P/Q-type voltage-gated Ca(2+) channel gene (Cacna1a) within the gry-critical region. This mutation was located at a highly conserved site close to the ion-selective pore and led to the shortening of the inactivation phase of the Ca(2+) channel current without a change of peak current density or current-voltage relationship in whole cell patch recordings of the recombinant Ca(2+) channel expressed in HEK cells. It has been well established that mice with a mutation at Cacna1a such as tottering and leaner show absence seizures. The Cacna1a-mutant GRY rat also exhibited absence-like seizures from 6 to 8 weeks of age, which were characterized by bilateral and synchronous 7-8 Hz spike-and-wave discharges concomitant with sudden immobility and staring, on cortical and hippocampal EEGs. The pharmacological profile of the seizures was similar to that of human absence epilepsy: the seizures were inhibited by ethosuximide and valproic acid but not phenytoin. Thus, the GRY rat with P/Q-type Ca(2+) channel disorders is a useful model for studying absence epilepsy and Cacna1a-related diseases.

Asunto(s)

Ataxia/genética , Encéfalo/metabolismo , Canales de Calcio Tipo P/genética , Canales de Calcio/genética , Epilepsia Tipo Ausencia/genética , Mutación Missense/genética , Animales , Ataxia/metabolismo , Ataxia/fisiopatología , Encéfalo/fisiopatología , Canales de Calcio/química , Canales de Calcio Tipo P/química , Línea Celular , Membrana Celular/química , Membrana Celular/genética , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Electroencefalografía , Epilepsia Tipo Ausencia/metabolismo , Epilepsia Tipo Ausencia/fisiopatología , Femenino , Hipocampo/metabolismo , Hipocampo/fisiopatología , Humanos , Masculino , Potenciales de la Membrana/genética , Técnicas de Placa-Clamp , Ratas , Ratas Mutantes

RESUMEN

Dictyostelium is a favored model for studying problems in cell and developmental biology. To comprehend the genetic potential and networks that direct growth and multicellular development, we are performing a large-scale analysis of Dictyostelium cDNAs. Here, we newly determine 7720 nucleotide sequences of cDNAs from the multicellular, slug stage (S) and 10 439 from the unicellular, vegetative stage (V). The combined 26 954 redundant ESTs were computer assembled using the PHRAP program to yield 5381 independent sequences. These 5381 predicted genes represent about half of the estimated coding potential of the organism. One-third of them were classified into 12 functional categories. Although the overall classification patterns of the V and S libraries were very similar, stage-specific genes exist in every category. The majority of V-specific genes function in some aspect of protein translation, while such genes are in a minority in the S-specific and common populations. Instead, genes for signal transduction and multicellular organization are enriched in the population of S-specific genes. Genes encoding the enzymes of basic metabolism are mainly found in the common gene population. These results therefore suggest major differences between growing and developing Dictyostelium cells in the nature of the genes transcribed.

Asunto(s)

ADN Complementario/análisis , Dictyostelium/crecimiento & desarrollo , Dictyostelium/genética , Genes Protozoarios , Animales , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Genes de Cambio , Datos de Secuencia Molecular

RESUMEN

We have newly established a sensitive, two-site enzyme immunoassay system for neurotrophin-4 (NT-4) and investigated its tissue distribution in the rat nervous system. The minimal limit of detection of the assay is 0.3 pg/0.2 mL of assay mixture. Concentrations of NT-4 were found to be extremely low in all brain regions, irrespective of the animal age, the highest level being found in the brain stem of 40-day-old rats, at 0.12 ng/g wet weight. NT-4 levels in young adult rats were significantly lower in the thalamus and higher in the olfactory bulb, neocortex, hypothalamus and brain stem than respective levels in 1-week-old rats. NT-4 immunoreactivity was strong in large neurons of the red nucleus and pontine reticular nucleus as well as the locus coeruleus, and moderate in cells in the mesencephalic trigeminal nucleus and interstitial nucleus of the medial longitudinal fasciculus. In the rat embryo, stong staining of NT-4 was detected in cells of regions corresponding to the midbrain/pons from E11.5 through E15.5. The intensity was decreased after E13.5 when the cytoplasm of cells in the medulla oblongata, fibers of the cerebellar primordium, and both cells and fibers of the dorsal root ganglion were also stained. Concentrations of NT-4 were detected in regions including the hindbrain and the dorsal root ganglion. Immunoblotting of NT-4-immunoreactive proteins extracted from these two regions revealed a band corresponding to mature NT-4 with a molecular mass of approximately 14 kDa. Kainic acid and another glutamte agonist, (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid did not affect NT-4 levels in the hippocampus. The present results show NT-4 to be localized in very limited brain cells and fibers from the embyonic period through to the young adult, suggesting specific roles in brain functions.

Asunto(s)

Tronco Encefálico/metabolismo , Ganglios Espinales/metabolismo , Factores de Crecimiento Nervioso/biosíntesis , Neuronas/metabolismo , Factores de Edad , Animales , Especificidad de Anticuerpos , Tronco Encefálico/citología , Tronco Encefálico/embriología , Factor Neurotrófico Derivado del Encéfalo/análisis , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Corteza Entorrinal/efectos de los fármacos , Corteza Entorrinal/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Ganglios Espinales/citología , Ganglios Espinales/embriología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Factores de Crecimiento Nervioso/análisis , Neuronas/citología , Especificidad de Órganos , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Testículo/química , Testículo/metabolismo

RESUMEN

The Dictyostelium stalk cell inducer differentiation-inducing factor (DIF) directs tyrosine phosphorylation and nuclear accumulation of the STAT (signal transducer and activator of transcription) protein Dd-STATc. We show that hyperosmotic stress, heat shock and oxidative stress also activate Dd-STATc. Hyperosmotic stress is known to elevate intracellular cGMP and cAMP levels, and the membrane-permeant analogue 8-bromo-cGMP rapidly activates Dd-STATc, whereas 8-bromo-cAMP is a much less effective inducer. Surprisingly, however, Dd-STATc remains stress activatable in null mutants for components of the known cGMP-mediated and cAMP-mediated stress-response pathways and in a double mutant affecting both pathways. Also, Dd-STATc null cells are not abnormally sensitive to hyperosmotic stress. Microarray analysis identified two genes, gapA and rtoA, that are induced by hyperosmotic stress. Osmotic stress induction of gapA and rtoA is entirely dependent on Dd-STATc. Neither gene is inducible by DIF but both are rapidly inducible with 8-bromo-cGMP. Again, 8-bromo-cAMP is a much less potent inducer than 8-bromo-cGMP. These data show that Dd-STATc functions as a transcriptional activator in a stress-response pathway and the pharmacological evidence, at least, is consistent with cGMP acting as a second messenger.

Asunto(s)

GMP Cíclico/análogos & derivados , Dictyostelium/metabolismo , Proteínas Protozoarias/fisiología , Transducción de Señal , Transactivadores/fisiología , Transporte Activo de Núcleo Celular , Animales , Northern Blotting , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Proteínas Luminiscentes/metabolismo , Modelos Biológicos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Ósmosis , Estrés Oxidativo , Fosforilación , Transporte de Proteínas , Proteínas Protozoarias/metabolismo , Factores de Transcripción STAT , Factores de Tiempo , Transactivadores/metabolismo , Transcripción Genética , Tirosina/metabolismo

RESUMEN

The dentate gyrus of the hippocampus, generating new cells throughout life, is essential for normal recognition memory performance. Reduction of brain-derived neurotrophic factor (BDNF) in this structure impairs its functions. To elucidate the association between BDNF levels and hippocampal neurogenesis, we first conducted a search for compounds that stimulate endogenous BDNF production in hippocampal granule neurons. Among ion channel modulators tested, riluzole, a neuroprotective agent with anticonvulsant properties that is approved for treatment of amyotrophic lateral sclerosis, was highly effective as a single dose by an intraperitoneal injection, causing a rise in BDNF localized in dentate granule neurons, the hilus, and the stratum radiatum of the CA3 region. Repeated, but not single, injections resulted in prolonged elevation of hippocampal BDNF and were associated with increased numbers of newly generated cells in the granule cell layer. This appeared due to promoted proliferation rather than survival of precursor cells, many of which differentiated into neurons. Intraventricular administration of BDNF-specific antibodies blocked such riluzole effects, suggesting that BDNF increase is necessary for the promotion of precursor proliferation. Our results suggest the basis for a new strategy for treatment of memory dysfunction.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Hipocampo/metabolismo , Riluzol/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Animales , Anticuerpos/administración & dosificación , Anticuerpos/farmacología , Química Encefálica , Factor Neurotrófico Derivado del Encéfalo/análisis , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Inyecciones , Inyecciones Intraventriculares , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Riluzol/administración & dosificación , Riluzol/antagonistas & inhibidores , Riluzol/inmunología , Bloqueadores de los Canales de Sodio/administración & dosificación , Bloqueadores de los Canales de Sodio/antagonistas & inhibidores , Células Madre/citología , Células Madre/efectos de los fármacos

RESUMEN

A distinct feature of development in the simple eukaryote Dictyostelium discoideum is an aggregative transition from a unicellular to a multicellular phase. Using genome-wide transcriptional analysis we show that this transition is accompanied by a dramatic change in the expression of more than 25% of the genes in the genome. We also show that the transcription patterns of these genes are not sensitive to the strain or the nutritional history, indicating that Dictyostelium development is a robust physiological process that is accompanied by stereotypical transcriptional events. Analysis of the two differentiated cell types, spores and stalk cells, and their precursors revealed a large number of differentially expressed genes as well as unexpected patterns of gene expression, which shed new light on the timing and possible mechanisms of cell-type divergence. Our findings provide new perspectives on the complexity of the developmental program and the fraction of the genome that is regulated during development.

Asunto(s)

Dictyostelium/crecimiento & desarrollo , Dictyostelium/genética , Animales , Dictyostelium/citología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genoma de Protozoos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reproducibilidad de los Resultados , Transcripción Genética

RESUMEN

We have previously isolated several cell-type-enriched mRNAs of Dictyostelium discoideum. Since the temporal pattern of appearance and cell-type-enrichment of these RNAs were examined only by determining their accumulation, it was unclear whether their accumulation is regulated at the transcription level or the post-transcriptional level. To distinguish between these two possibilities, we examined the temporal and cell-type-enriched transcription of several of these genes by nuclear run-on assay. The results suggest that some genes are controlled in both temporal accumulation and cell-type-enrichment at the transcriptional level, but post-transcriptional regulation is also important for regulating cell-type enrichment in the case of some other genes.

RESUMEN

A considerable number of postnatally-viable microphthalmic offspring with optic nerves completely absent were obtained by X-irradiation at a dose of 100 R in pregnant rats on gestational day 10.5. Thirteen of 15 mi-crophthalmic eyes examined displayed histological features characteristic of aplasia of the optic nerve: complete absence of optic papilla, nerve fiber layer and retinal blood vessels, and great reduction in the number of ganglion cells. The remaining 2 eyes showed the histological features of hypoplasia of the optic nerve. The present experimental system may afford suitable materials for postnatal patho-genetic studies and also for various physiological and behavioral studies of aplasia of the optic nerve.

RESUMEN

The differentiation processes of Dictyostelium discoideum cells under the conditions which favored either stalk or spore cell formation were examined by the use of prestalk- and prespore-specific antibodies. In stalk cell-forming conditions, cells reactive with prestalk-specific monoclonal antibody (C1) increased rapidly early in development and later differentiated into stalk cells. No or only a few cells became reactive with prespore-specific monoclonal (B6) and polyclonal (antispore) antibodies. Despite the fact that most cells terminally became spores under spore cell-forming conditions, cells were first stained with the C1 antibody before becoming reactive with the B6 antibody. Unlike the case of normal development where cells coincidentally become reactive with the B6 and antispore antibodies, the appearance of the cells reactive with the latter was either delayed or suppressed. In conclusion, under either spore or stalk cell-forming conditions, the appearance of the prestalk antigen preceded that of the prespore one, which is consistent with normal development.

RESUMEN

In pre-primitive streak-stage rat egg cylinders, both the embryonic and extraembryonic ectodermal cells projected cytoplasmic protrusions through gaps in the basal lamina and formed intimate cell-to-cell contact with the primitive endodermal cells. The 70 Å microfilaments were considered to participate in the production of these cytoplasmic protrusions. However, direct cell contact mediated by adherent junctions was occasionally found between the embryonic or extraembryonic ectodermal cells and the primitive endodermal cells. It has been proposed that these cell-to-cell contacts may play a role either in the supporting effect of primitive endodermal cells in the maintenance of cellular organization of the ectodermal cells, or in the facilitation of transport of nutritive materials from the primitive endodermal cells to both types of ectodermal cells.

RESUMEN

The processes of differentiation of the presumptive cells (prespore and prestalk cens) into mature spores, stalk and basal-disc cells in Dictyotelium discoideum was investigated. The number of stalk and disc cells in pre-labeled culminating cell masses was estimated by determining the radioactivity of the undissociable fraction separated by filtration from the dissociable fraction containing presumptive cells and spores. Changes in the proportion of amoeboid cells stainable with fluorescein-conjugated antispore serum and encapsulated spores were also followed in the dissociable fraction. Formation of stalk and disc cells began at 17 hr of development and was completed at 26 hr, while formation of morphologically identifiable spores began at 18 hr and was completed at 20 hr, long before completion of stalk formation. At the onset of culmination, unstained cells abruptly increased with an accompanying decrease of stained cells, when unstained rear-guard cells appeared in the hind region. Although some of the rear-guard cells soon differentiated into basal-disc cells, the rest remained amoeboid in the upper part of the spore mass (sorus) after complete formation of a fruiting body. Despite the presence of the amoeboid cells in mature sori, the proportion of the sorus to the stalk and disc of a fruiting body was approximately equal to that of stained (prespore) to unstained (prestalk) cells in a migrating slug.

RESUMEN

Ultrastructural changes of the nucleolus in mitotic embryonic ectodermal cells of 7 1/2-day and 7 2/3-day rat embryos were examined. It was found that the nucleolus was broken down into small fragments during late prophase and metaphase, and that some of these fragments persisted in the cytoplasm of telophase cell (persistent nucleoli). No interphase embryonic ectodermal cells contained persistent nucleoli. Persistent nucleoli were also found in telophase cells of extraembryonic ectoderm, extraembryonic visceral endoderm and parietal endoderm of the embryos, but they disappeared in interphase cells. Persistent nucleoli in telophase cells tended to decrease in size with embryonic age, and they had almost completely disappeared in neuroectodermal cells of the telencephalon in 14 1/2-day embryos. They were concluded to be remnants of disappearing nucleoli in embryonic cells that were cycling too rapidly to permit their nucleoli to disappear completely.

RESUMEN

Embryonic ectodermal cells of rat embryos were examined by light and electron microscopy during the early stage of neurulation. Before the onset of neurulation (day 9-6 hr embryos), the cells underwent certain characteristic ultrastructural changes; that is, apical cytoplasmic protrusions and free spherules appeared, numerous vacuoles were formed in the cytoplasm, mitochondria showed ballooning, and the endoplasmic reticulum became dilated. The amniotic cells derived from the embryonic ectoderm exhibited the same ultrastructural changes, but those from the extraembryonic mesoderm did not. Embryonic mesodermal cells and neuroectodermal cells also did not show these changes. In the middle stage of neurulation (day 9-12 hr embryos), the embryonic ectodermal cells and the amniotic cells derived from the embryonic ectoderm assumed a flat squamous shape. None of the ultrastructural changes observed in day 9-6 hr embryos were noted in these cells. The functional significance of the production of apical cytoplasmic protrusions and free spherules in the embryonic ectodermal cells and amniotic cells is discussed in relation to similar phenomena reported to occur in other cell types.

RESUMEN

Changes in agglutinability of Dictyostelium discoideum cells with Concanavalin A (Con A) during the course of development were investigated. The agglutinability of the cells was assayed under conditions where no spontaneous cell agglutination occurred. It was found that there was a progressive decrease in Con A-induced agglutinability during development: a decrease to half from exponentially growing cells to preaggregation cells, and to sixth in disaggregated slug cells. Pronase-BAL treatment of preaggregation cells did not enhance their agglutinability with Con A. The amounts of sites available for binding Con A were determined with preaggregation and slug cells. Cells were incubated at 4°C and in the presence of NaN3 to avoid possible endocytosis of Con A. No significant differences in numbers of Con A-binding sites per unit area of cell surface was detected among preaggregation cells, those treated with pronase and BAL and cells disaggregated from slugs by similar treatment. It was thus concluded that the decrease in Con A-induced agglutinability during development is not attributable to changes in the numbers of Con A-binding sites.