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Regulation of neurotrophin and trkA, trkB and trkC tyrosine kinase receptor messenger RNA expression in kindling.

Levels of messenger RNA for nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and the tyrosine kinase receptors trkA, trkB and trkC have been studied using in situ hybridization in the rat brain 2 h and four weeks after kindling-induced seizures. Epileptiform activity evoked by hippocampal stimulation and exceeding 70 s lead to a concomitant and transient increase of brain- derived neurotrophic factor, nerve growth factor, trkB and trkC messenger RNA expression in dentate granule cells after both focal and generalized seizures. Brain-derived neurotrophic factor messenger RNA levels were also increased bilaterally in the CA1-CA3 regions, amygdala and the piriform, entorhinal, perirhinal, retrosplenial and temporal cortices after generalized seizures. The magnitude of the increases was similar throughout the development of kindling and in the fully kindled brain. No changes of trkA messenger RNA were observed. In amygdalar kindling, elevated brain-derived neurotrophic factor messenger RNA levels developed more rapidly in the amygdala-piriform cortex than after stimulation in the hippocampus but changes in the hippocampal formation were only seen in few animals. Intraventricular 6-hydroxydopamine or a bilateral fimbria-fornix lesion did not alter basal expression or seizure-evoked changes in messenger RNA levels for neurotrophins or trk receptors but increased the number of animals exhibiting elevated levels after the first stimulation, probably due to a prolongation of seizure activity. Both in sham-operated and fimbria-fornix-lesioned rats seizure activity caused a marked reduction of neurotrophin-3 messenger RNA levels in dentate granule cells. The results indicate that activation of the brain-derived neurotrophic factor gene, at least in dentate granule cells, is an "all-or-none" type of response and dependent on the duration but not the severity of seizures or the stage of kindling epileptogenesis. Changes in brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and trkB and trkC were observed concomitantly in the dentate gyrus, which suggests that seizure activity sets in motion a cascade of genomic events possibly mediated via a common mechanism. Since altered messenger RNA levels outside hippocampus were detected only for brain-derived neurotrophic factor, neurotrophin and trk gene expression in these regions seems to be regulated differently.

Amygdala↗

Immune challenge-stimulated hypophysiotropic corticotropin-releasing hormone messenger RNA expression is associated with an induction of neurotensin messenger RNAs without alteration of vasopressin messenger RNAs.

The corticotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus are the final common pathway of the neuroendocrine adaptative response to a variety of stressors. To meet varied homeostatic needs, corticotropin-releasing hormone neurons exhibit a marked phenotypical plasticity, enabling them to rapidly modify their neuroendocrine output. In particular, they synthesize the neuropeptides vasopressin and neurotensin. Under many experimental circumstances, it is observed that corticotropin-releasing hormone and vasopressin are regulated in parallel, whereas the expression of neurotensin seems dissociated, in these neurons, evoking different transcriptional control over the co-existing neuropeptides depending on the adaptative response required. Using radioactive and dual-label in situ hybridization techniques, we have studied the respective expression of paraventricular corticotropin-releasing hormone, vasopressin and neurotensin messenger RNAs in the context of an immune challenge. A single intraperitoneal injection of the endotoxin lipopolysaccharide was administered to adult male rats that were killed 8 h later. Compared to control animals, lipopolysaccharide-injected rats showed elevated plasma corticosterone (614+/-65 vs 185+/-40 ng/ml in control) and increased expression of paraventricular corticotropin-releasing hormone messenger RNA (+200%); expression of neurotensin messenger RNA was induced in about one-third of corticotropin-releasing hormone neurons, whereas vasopressin messenger RNA expression remained unchanged. Therefore, in this experimental context and at the time-point examined, co-existing corticotropin-releasing hormone and vasopressin appeared differentially expressed, and an additional stimulus (inflammation) is demonstrated to result in neurotensin expression in neuroendocrine corticotropin-releasing hormone neurons. Neurotensin may be released in the pituitary portal blood to trigger pituitary response associated with mobilization of the immune system.

Animals↗

Purification of the cleavage and polyadenylation factor involved in the 3'-processing of messenger RNA precursors.

Polyadenylation of messenger RNA precursors requires the nucleotide sequence AAUAAA and two factors: poly(A) polymerase and a specificity factor termed cleavage and polyadenylation factor (CPF). We have purified CPF from calf thymus and from HeLa cells to near homogeneity. Four polypeptides with molecular masses of 160, 100, 73, and 30 kDa cofractionate with CPF activity. Glycerol gradient centrifugation and gel filtration indicate that these four proteins form one large complex with a sedimentation constant of 12 S, a Stokes radius near 100 A, and a native molecular mass near 500 kDa. Purified CPF binds specifically to an RNA that contains the AAUAAA sequence. Mutation of the AAUAAA sequence inhibits CPF binding as well as polyadenylation. Purified CPF contains only trace amounts of RNA and does not react with antibodies against common epitopes of small nuclear ribonucleoprotein particles. Thus, contrary to previous indications, CPF does not appear to be a small nuclear ribonucleoprotein particle.

Animals↗

Organ and species specificity of the messenger RNA transport factor.

The transport of messenger RNA from isolated rat liver nuclei is dependent on a 35,000 dalton protein localized in the cytoplasm. The tissue and species specificity of this protein are reported. The factor from female rat liver or from rat brain, kidney, or hepatoma supports the transport of messenger RNA from male rat liver nuclei. Messenger RNA is also transported in response to the factor from beef and chicken liver. The cytosols from rat erythrocytes, amoeba and yeast were inactive in the rat liver system. The results indicate that the messenger RNA transport factor is not specific for the nucleotide sequence of the coding portion of the messenger RNA and that it is not organ or species specific within the vertebrates.

Amoeba↗

PROTEIN SYNTHESIS DURING DEVELOPMENT: CONTROL THROUGH MESSENGER RNA.

Utilization of long-lived messenger RNA appears to be the exception rather than the rule in cells which are differentiating and synthesizing large amounts of specialized product at the same time. The fact that polyribosomes synthesize protein after RNA synthesis is turned off by actinomycin D is used to demonstrate messenger RNA of long half-life. The data suggest that most tissues examined have short-lived messenger RNA's, but the ocular lens can synthesize protein after an incubation of 24 hours in 40 microg of actinomycin D per milliliter. A common basis for the presence of long-lived messenger RNA in the cells of the lens, the feather, and in reticulocytes is discussed.

Animals↗

Estradiol-induced synthesis of vitellogenin. III. The isolation and characterization of vitellogenin messenger RNA from avian liver.

The messenger RNA of the hormone-induced protein vitellogenin was isolated from the liver of estrogen-treated roosters. Starting from total polysomal RNA, the vitellogenin messenger was purified 67-fold by oligo (dT)-cellulose chromatography and sizing on a sucrose gradient. The messenger was translated in vitro into a 170 000 dalton polypeptide chain, having the immunochemical characteristics of vitellogenin. From electrophoretic and immunochemical analysis of the in vitro product of translation at least 63% of the messenger activity of the RNA preparation could be attributed to vitellogenin mRNA. Gel electrophoresis of the most purified fraction revealed residual contamination with the larger ribosomal RNA species. The molecular weight of the messenger RNA molecule, obtained by contour length measurements in the electron microscope, lies between 2.5 - 10(6) and 2.8 - 10(6).

Animals↗

Yeast mutant, rna 1, affects the entry into polysomes of ribosomal RNA as well as messenger RNA.

The entry of newly labeled ribosomal subunits and mRNA into polysomes was examined in the yeast mutant rna1. The entry of both types of RNA into polysomes is inhibited rapidly at the restrictive temperature. Analysis of the labeling of the ATP pool and the kinetics of synthesis and processing of mRNA at the restrictive temperature leads to the conclusion that the primary defect in the mutant affects transport of both ribosomes and messenger across the nuclear membrane.

Adenosine Triphosphate↗

Myosin heavy chain messenger RNA from myogenic cell cultures.

The appearance of messenger RNA for myosin heavy chains in chick-embryo myogenic cell cultures was investigated. Total polyribosomes were isolated from cultures at various times of development and were purified in sucrose step gradients. These polysomes were either extracted with phenol or were treated with puromycin. The ribonucleoprotein particles and ribosomal subunits released by puromycin were fractionated on sucrose gradients. RNA from polysomes or from puromycin-dissociated subunits was fractionated on oligo(dT)-cellulose columns, and the bound and unbound RNA was assayed for activity of myosin heavy chain messenger RNA in a rabbit reticulocyte cell-free system. RNA stimulating myosin heavy-chain synthesis was found predominantly in the unbound fractions of the oligo(dT)-cellulose columns. After puromycin treatment of polysomes, the myosin heavy chain messenger RNA, which sediments at 18-26 S, was associated with a ribonucleoprotein particle sedimenting between 30 and 40 S. Myosin heavy chain messenger RNA was obtained from cultures containing well-developed myotubes and from cultures undergoing myogenic cell fusion. This messenger RNA was not detectable in early, unfused cultures, or in later cultures in which myogenic cell fusion had been prevented by treatment with ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. These experiments demonstrate that messenger RNA for myosin heavy chains becomes associated with ribosomes only after myogenic cell fusion has begun.

Acetates↗

Partial purification of a progesterone-inducible messenger RNA (avidin) from hen oviduct.

The messenger RNA (mRNA) for avidin, which represents less than 0.05% of the total cellular proteins, was partially purified from hen oviduct, and the presence of avidin mRNA was shown to depend upon prior stimulation by progesterone. A total nucleic acid extract was subjected to oligo (dT)-cellulose chromatography, followed by Sepharose 4B chromatography, preparative agarose gel electrophoresis, and sucrose gradient centrifugation. The relative purity of each preparation was assessed by translation in a wheat-germ system; avidin messenger RNA activity was measured by specific immunoprecipitation of synthesized proteins. Avidin mRNA was separated from the bulk of the total messenger RNA activity of the oviduct and from all ribosomal RNAs to produce greater than a 1000-fold enrichment of avidin mRNA activity compared with total cellular RNA. Based on the translation assay, the most highly purified fraction contained about 2.5% avidin messenger RNA. Avidin mRNA activity was absent in partially purified mRNA obtained from estrogen-stimulated chick oviducts, but was detected in oviducts following progesterone administration.

Animals↗

GTP-binding proteins Gi and Go transplanted onto Xenopus oocyte by rat brain messenger RNA.

After injection with messenger RNA (mRNA) isolated from rat brain, Xenopus laevis oocytes acquired electrophysiological responsiveness to externally perfused acetylcholine (ACh) or serotonin (5-HT), and elevated responsiveness to internally applied guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). Compared with the membranes of native oocytes, those of mRNA-injected oocytes contained increased amounts of 39 and 41 kDa proteins, which could be [32P]ADP-ribosylated by pertussis toxin (PTX). The amplitude of the GTP gamma S response and the amounts of the 39 and 41 kDa proteins increased in a parallel manner for at least 3 days following mRNA injection. Current responses to internally applied GTP gamma S showed properties common to those of responses to ACh or 5-HT perfusion: both responses had reversal potentials close to the Cl- potential, were mimicked by intracellular injection of IP3, desensitized by a large dose of IP3, and inhibited by a simultaneous injection of neomycin or EGTA. Incubation of mRNA-injected cells with PTX inhibited both the 5-HT response and the [32P]ADP-ribosylation of the 39 and 41 kDa proteins in a parallel, dose-dependent manner. After pretreatment of oocytes with PTX followed by mRNA injection, the levels of the 39 and kDa proteins and the 5-HT response appeared to be similar to those of non-treated cells injected with mRNA, whereas no detectable amounts of these proteins were induced when PTX-pretreated cells were analyzed under the same conditions without mRNA injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗