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At least 1,135 records · Page 63Linked to original sources
PubMed1999

Detailed localization of aquaporin-4 messenger RNA in the CNS: preferential expression in periventricular organs.

We have performed a detailed in situ hybridization study of the distribution of aquaporin-4 messenger RNA in the CNS. Contrary to expectation, we demonstrate that aquaporin-4 is ubiquitously expressed in the CNS. Strong hybridization labeling was detected in multiple olfactory areas, cortical cells, medial habenular nucleus, bed nucleus of the stria terminalis, tenia tecta, pial surface, pontine nucleus, hippocampal formation and multiple thalamic and hypothalamic areas. A low but significant hybridization signal was found, among others, in the choroid plexus of the lateral ventricles, ependymal cells, dorsal raphe and cerebellum. Overall, a preferential distribution of aquaporin-4 messenger RNA-expressing cells was evident in numerous periventricular organs. From the distribution study, the presence of aquaporin-4 messenger RNA-expressing cells in neuronal layers was evident in neuronal layers including the CA1 -CA3 hippocampal pyramidal cells, granular dentate cells and cortical cells. Further evidence of neuronal expression comes from the semicircular arrangement of aquaporin-4 messenger RNA-expressing cells in the bed nucleus of the stria terminalis and medial habenular nucleus exhibiting Nissl-stained morphological features typical of neurons. Combined glial fibrillary acidic protein immunohistochemistry and aquaporin-4 messenger RNA in situ hybridization demonstrated that aquaporin-4 messenger RNA is expressed by glial fibrillary acidic protein-lacking cells. We conclude that aquaporin-4 messenger RNA is present in a collection of structures typically involved in the regulation of water and sodium intake and that aquaporin-4 water channels could be the osmosensor mechanism responsible for detecting changes in cell volume by these cells.

Animals↗
PubMed2000

Electroconvulsive stimuli enhance both neuropeptide Y receptor Y1 and Y2 messenger RNA expression and levels of binding in the rat hippocampus.

Repeated electroconvulsive stimulations and other seizure modalities produce an increase in neuropeptide Y synthesis and local release in the rat hippocampus, and perhaps as a consequence, a change in the concentration of neuropeptide Y binding sites in the same region. The aim of the present study was to determine possible changes in the expression of neuropeptide Y receptor subtypes affected by repeated stimulations in the hippocampus. Rats were exposed to 14 daily stimulations, and the brains were removed 24h after the last stimulation. For in vitro receptor autoradiography and in situ hybridisation histochemistry, the brains were frozen, sectioned, and levels of neuropeptide Y binding sites and messenger RNA expressions were determined quantitatively on sections from the same animals. In order to determine the contribution of different neuropeptide Y receptor subtypes, serial sections were incubated with either 125I-labelled peptide YY alone or the same radio-labelled peptide mixed with an excess of a number of displacing compounds with affinity for either neuropeptide Y receptor subtype Y1, Y2, or both. Binding studies revealed that the majority of peptide YY binding sites was represented by Y2, and that electroconvulsive stimulations reduced the binding capacity or the concentration of this receptor. A prominent reduction of Y1-preferring binding sites was determined in the dentate gyrus, and to a lesser extent in the CA1 and CA3 regions. Similarly, the treatment produced a significant reduction of Y2-preferring binding sites in the CA1 and CA3 region, but not in the granular cell layer of the dentate gyrus. Using semi-quantitative in situ hybridization, Y1 receptor messenger RNA level in the granular cell layer of the dentate increased by the stimulations. In the same region, Y2 receptor messenger RNA was expressed in low to undetectable amounts, but after the repeated stimulations, this transcript was found in moderate to high levels. These data suggest that the neuropeptide Yergic system in the dentate gyrus and the pyramidal cell layer are affected by the treatment, and that this includes both Y1 and Y2 receptor subtypes. Because levels of messenger RNA and binding are distinctly regulated, the turnover of both Y1 and Y2 molecules is strongly increased under electroconvulsive stimulations, suggesting that the intrahippocampal neuropeptide Yergic neurotransmission is also increased under the stimulations.

Animals↗
PubMed1989

Human papillomavirus messenger RNA expression in adenocarcinoma in situ of the uterine cervix.

Over recent years, the association between human papillomavirus (HPV) and squamous cell carcinoma of the cervix and its precursors has been well established, largely as a result of advances in the techniques of molecular biology. A similar association between HPV and invasive adenocarcinoma of the cervix has also been recently demonstrated; however, little work has been published on the relationship between the precursor lesion, adenocarcinoma in situ (ACIS), and HPV. We have therefore undertaken an in situ hybridization study of 22 cases of known cervical ACIS using probes for HPV messenger RNA expression. Seventeen of these had residual ACIS in the blocks studied, 15 of which (88.6%) expressed HPV messenger RNA. Five cases were positive for HPV 16 and 10 cases for HPV 18. Early invasive adenocarcinoma was present with ACIS in three cases and one case had early invasion plus cervical intraepithelial neoplasia (CIN III). These invasive lesions showed a similar type and degree of HPV expression as the in situ component. One section had only residual CIN III, which was positive for HPV type 18. Four cases had only minor glandular atypias (less than ACIS) in the studied sections, and all four of these were negative for HPV expression. None of the normal endocervical epithelia in any of the sections were positive by this technique. In summary, the finding of HPV messenger RNA expression in nearly 90% of cervical ACIS supports a possible role for these viruses in the pathogenesis of glandular neoplasms of the uterine cervix.

Adenocarcinoma↗
PubMed1983

A rapid method for analysis of the products of in vitro translation of messenger RNA.

A rapid and sensitive method for assaying the production of a specific protein during translation of messenger RNA is described. The method involves running the samples mixed with carrier protein in antibody-containing agarose and determining the radioactivity in the rocket-shaped immunoprecipitates which form. The radioactivity detected is shown to be qualitatively and quantitatively dependent on the amount of immunoreactive protein produced in translation and can thus be used as a measure of specific protein production. The ability of the method to rapidly analyze many samples in parallel is of particular use in screening large numbers of recombinant clones for the ability to bind a specific messenger RNA and affect its translation in vitro.

Albumins↗
PubMed1992

Distribution of cells expressing vasoactive intestinal peptide/peptide histidine isoleucine-amide precursor messenger RNA in the rat brain.

The distribution of cells expressing vasoactive intestinal peptide/peptide histidine isoleucine-amide precursor messenger RNA was investigated in the rat brain and pituitary by in situ hybridization using a synthetic 35S-labeled oligonucleotide probe. Detection of labeled neurons by light-microscopic radioautography revealed a selective repartition of the messenger RNA-expressing cells. Several major vasoactive intestinal peptide/peptide histidine isoleucine-amide messenger RNA-containing cell groups were demonstrated including layers II-VI of the cerebral cortex, the suprachiasmatic nucleus and various thalamic structures such as the ventrolateral, posterior, lateral reticular, paracentralis and gelatinosus nuclei. Positive cells, to a lesser extent, were also found in the limbic system, medial preoptic area, superior and inferior colliculi as well as in the central gray matter. They were totally absent in the pituitary and the pineal gland of normal rats. The results of the present study provide a detailed mapping of neurons expressing vasoactive intestinal peptide/peptide histidine isoleucine-amide messenger RNA in the adult rat brain. The predominance of vasoactive intestinal peptide/peptide histidine isoleucine-amide messenger RNA-containing neurons in the cerebral cortex, suprachiasmatic nucleus and thalamus suggest that vasoactive intestinal peptide is mainly involved in the control of cortical informations, circadian rhythms and sensory perception in agreement with several physiological data.

Animals↗
PubMed2006

Expression of heme oxygenase-1 protein and messenger RNA in permanent cerebral ischemia in rats.

BACKGROUND: Carbon monoxide has been regarded as a gaseous molecular messenger like nitric oxide. PURPOSE: To clarify the role of heme oxygenase-1 in the permanent cerebral ischemia at the protein and mRNA level. METHODS: The expression of heme oxygenase-1 protein and messenger RNA was investigated at different time points following MCAO using immunohistochemistry, Western blotting, RT-PCR, and Northern blotting. RESULTS: Increased HO 1immunoreactivity was detected in hippocampal and cortical neurons after 1 hour of ischemia, and was also observed in astroglial cells. After 12 hours of ischemia, HO-1 was found in both neurons and glia in cerebral cortex and thalamus, and in striatal glia cells. Western blotting analysis show the expression of HO-1 protein in cortical neurons reached the peak after 12 hours of occlusion and decreased gradually, but was still detected at day 7 post-occlusion. The expression of messenger RNA was examined in the brains of rats subjected to permanent cerebral ischemia by semi-quantitative RT-PCR and Northern blotting. HO-1 mRNA transcription could be detected 1 hour after occlusion. After 1 to 6 hours of occlusion, the expression of HO-1 rose rapidly, reaching a peak at 12 hours post-occlusion, decreased gradually, and lasted until day 7 of occlusion. Although HO activity of cerebral tissue can be detected in both sham-operated group and operated groups, the HO activity in operated groups is much stronger than that in sham-operated group. CONCLUSIONS: The induction of HO-1 protein may protect cerebral tissues from ischemic damage.

Analysis of Variance↗
PubMed1977

Differential translation of rat liver albumin messenger RNA in a wheat germ cell-free system.

Rat liver polysomal RNA directs the synthesis of albumin in a wheat germ cell-free system. The in vitro product has the same mobility in polyacrylamide-sodium dodecyl sulfate gels as purified rat liver albumin and can be immune precipitated with rabbit anti-rat liver albumin serum. The incorporation of [35S] methionine into total protein in an in vitro system increased linearly with increasing messenger RNA concentration until a saturation plateau was reached. The synthesis of albumin compared to total protein increased continually with increasing RNA concentration. Addition of the polynucleotide poly(A) to the cell-free system inhibited incorporation, presumably through interference with the initiation process, and increased the relative albumin synthesis. Translation of albumin messenger RNA was also resistant to inhibition by high levels of potassium acetate where total incorporation was cut in half. The results suggest that the messenger RNA for albumin, in addition to the mRNAs for the beta chain of globin and for the immunoglobulin polypeptides, is particularly efficient in initiating protein synthesis.

Albumins↗
PubMed1984

The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors.

We have investigated the role of small nuclear ribonucleoprotein particles (snRNPs) in the in vitro splicing of messenger RNA precursors by a variety of procedures. Removal of the U-type snRNPs from the nuclear extracts of HeLa cells with protein A-Sepharose-coupled human autoimmune antibodies leads to complete loss of splicing activity. The inhibition of splicing can be prevented by saturating the coupled antibodies with purified nucleoplasmic U snRNPs prior to incubation with nuclear extract. We further demonstrate that an intact 5' terminus of U1 snRNA is required for the functioning of U1 snRNP in the splicing reaction. Antibodies directed against the trimethylated cap structure of the U snRNAs inhibit splicing. Upon removal of the first eight nucleotides of the U1 snRNA in the particles by site-directed hydrolysis with ribonuclease H in the presence of a synthetic complementary oligodeoxynucleotide splicing is completely abolished. These results are in strong support of current models suggesting that a base-pairing interaction between the 5' terminus of the U1 snRNA and the 5' splice site of a mRNA precursor is a prerequisite for proper splicing.

Antigen-Antibody Complex↗
PubMed2001

Regional distribution of glycine receptor messenger RNA in the central nervous system of zebrafish.

We report the cloning of the zebrafish beta subunit of the glycine receptor and compare the anatomical distribution of three glycine receptor subunit constituents in adult zebrafish brain (alphaZ1, alphaZ2 and betaZ) to the expression pattern of homologous receptor subunits (alpha1, alpha2 and beta) in the mammalian adult CNS. Non-radioactive hybridization was used to map the distribution of the alphaZ1, alphaZ2 and betaZ glycine receptor subunit messenger RNAs in the adult zebrafish brain. The anterior-posterior expression gradient found in adult zebrafish brain was similar to that reported in mammalian CNS. However, the glycine receptor transcripts, notably the alphaZ1 subunit, were more widely distributed in the anterior regions of the zebrafish than in the adult mammalian brain. The isoform-specific distribution pattern was less regionalized in zebrafish than in the rat mammalian CNS. Nevertheless, there was some regionalization of alphaZ1, alphaZ2 and betaZ transcripts in the diencephalic and mesencephalic nuclei where different sensory and motor centers express either alphaZ1/betaZ or alphaZ2 subunits. In contrast to the widespread distribution of the beta subunit in adult mammalian brain, alphaZ2 messenger RNA presented the widest expression territory of all three glycine receptor subunits tested. alphaZ2 messenger RNA was expressed in the absence of alphaZ1 and betaZ messenger RNA in the outer nuclear layer of the retina, the inferior olive and the raphe of the medulla oblongata, as well as in the nucleus of Cajal of the medulla spinalis. In contrast, an identified central neuron of the reticular formation, the Mauthner cell, expresses all three glycine receptor subunits (alphaZ1, alphaZ2 and betaZ). This report extends the already described glycine receptor expression in the vertebrate CNS and confirms the importance of glycine-mediated inhibition in spinal cord and brainstem.

Amino Acid Sequence↗
PubMed1993

A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus.

A quantitative three-dimensional analysis of nuclear components involved in precursor messenger RNA metabolism was performed with a combination of fluorescence hybridization, immunofluorescence, and digital imaging microscopy. Polyadenylate [poly(A)] RNA-rich transcript domains were discrete, internal nuclear regions that formed a ventrally positioned horizontal array in monolayer cells. A dimmer, sometimes strand-like, poly(A) RNA signal was dispersed throughout the nucleoplasm. Spliceosome assembly factor SC-35 localized within the center of individual domains. These data support a nuclear model in which there is a specific topological arrangement of noncontiguous centers involved in precursor messenger RNA metabolism, from which RNA transport toward the nuclear envelope radiates.

Cell Nucleus↗
PubMed1994

Cannabinoid receptor binding and messenger RNA expression in human brain: an in vitro receptor autoradiography and in situ hybridization histochemistry study of normal aged and Alzheimer's brains.

The distribution and density of cannabinoid receptor binding and messenger RNA expression in aged human brain were examined in several forebrain and basal ganglia structures. In vitro binding of [3H]CP-55,940, a synthetic cannabinoid, was examined by autoradiography in fresh frozen brain sections from normal aged humans (n = 3), patients who died with Alzheimer's disease (n = 5) and patients who died with other forms of cortical pathology (n = 5). In the structures examined--hippocampal formation, neocortex, basal ganglia and parts of the brainstem--receptor binding showed a characteristic pattern of high densities in the dentate gyrus molecular layer, globus pallidus and substantia nigra pars reticulata, moderate densities in the hippocampus, neocortex, amygdala and striatum, and low densities in the white matter and brainstem. In situ hybridization histochemistry of human cannabinoid receptor, a ribonucleotide probe for the human cannabinoid receptor messenger RNA, showed a pattern of extremely dense transcript levels in subpopulations of cells in the hippocampus and cortex, moderate levels in hippocampal pyramidal neurons and neurons of the striatum, amygdala and hypothalamus, and no signal over dentate gyrus granule cells and most of the cells of the thalamus and upper brainstem, including the substantia nigra. In Alzheimer's brains, compared to normal brains, [3H]CP-55,940 binding was reduced by 37-45% in all of the subfields of the hippocampal formation and by 49% in the caudate. Lesser reductions (20-24%) occurred in the substantia nigra and globus pallidus, internal segment. Other neocortical and basal ganglia structures were not different from control levels. Levels of messenger RNA expression did not differ between Alzheimer's and control brains, but there were regionally discrete statistically significant losses of the intensely expressing cells in the hippocampus. The reductions in binding did not correlate with or localize to areas showing histopathology, estimated either on the basis of overall tissue quality or silver staining of neuritic plaques and neurofibrillary tangles. Reduced [3H]55,940 binding was associated with increasing age and with other forms of cortical pathology, suggesting that receptor losses are related to the generalized aging and/or disease process and are not selectively associated with the pathology characteristic of Alzheimer's disease, nor with overall decrements in levels of cannabinoid receptor gene expression.

Aged↗
PubMed1979

Isolation of rat alpha1-fetoprotein messenger RNA from Morris hepatoma 7777.

A double-antibody procedure has been developed for the isolation of alpha1-fetoprotein (AFP)-synthesizing polysomes from Morris hepatoma 7777. The polyadenylic acid-containing RNA, subsequently purified by differential sedimentation on sucrose gradient and oligodeoxythymidylic acid-cellulose chromatography, migrates as a single 21S component in polyacrylamide gel electrophoresis; in a cell-free translation system, it yields a peptide product immunoprecipitable by anti-rat AFP antiserum, but not by anti-rat albumin, and which migrates slightly faster than serum AFP on sodium dodecyl sulfate-urea-polyacrylamide gels. This messenger RNA fraction was used for the synthesis of a radioactive complementary DNA. In hybridization assays, the complementary DNA reassociated with its purified template at a Cr0t1/2 [product of RNA concentration (mol of nucleotides per liter) X half-time (sec)] of 1.5 X 10(-2). By constitute 3,2, and less than 0.01% of total polyadenylic acid-containing polysomal RNA of Morris hepatoma 7777, 10-day-old-rat liver, and adult rat liver, respectively. The high specificity of the polysome immunoprecipitation system, the electrophoretic homogeneity of the isolated messenger RNA fraction, its selective translation into AFP, and the specificity of the hybridization probe indicate that the procedure described yields a highly purified rat AJP messenger RNA.

Animals↗
PubMed1985

Quantification of thyroglobulin messenger RNA by in situ hybridization in differentiated thyroid cancers. Difference between well-differentiated and moderately differentiated histologic types.

Thyroglobulin messenger RNA (mRNA) was located and quantified in tissue sections of differentiated human thyroid cancers by in situ hybridization using cloned complementary DNA probes. The cells of the well-differentiated follicular and papillary forms contained similar levels of thyroglobulin mRNA, corresponding to about 2000 copies per cell. In contrast, cells of moderately differentiated thyroid cancers contained about two to three times less thyroglobulin mRNA. It was also found that thyroglobulin mRNA was present almost exclusively in polyribosomes under the form of heavy polyribosomes actively synthesizing thyroglobulin. It is suggested that in situ hybridization method allows localization of specific mRNA in differentiated thyroid cancers and correlation with the level of differentiation of the cells.

Autoradiography↗
PubMed1974

Cell-free translation of messenger RNA of simian virus 40: synthesis of the major capsid protein.

Extracts of wheat germ are capable of synthesizing the major capsid protein of simian virus 40. Poly(A)-containing RNA from BS-C-1 cells infected with simian virus 40 directed the synthesis of a novel polypeptide that migrates in polyacrylamide gels together with the major capsid polypeptide of simian virus 40, VP-1. The patterns of the major tryptic peptides of purified VP-1 and the novel polypeptide synthesized in vitro were identical after two-dimensional paper electrophoresis. The novel polypeptide was not synthesized in response to poly(A)-rich RNA from uninfected cells or from virus-infected cells treated with cytosine arabinoside. Messenger RNA from infected cells purified by selective hybridization to DNA of simian virus 40 directs the synthesis of a major polypeptide of electrophoretic mobility similar to that of VP-1 of simian virus 40. This approach should prove useful in identifying additional products specified by DNA tumor viruses.

Adenine Nucleotides↗
PubMed2006

Prolyl-tRNA(Pro) in the A-site of SecM-arrested ribosomes inhibits the recruitment of transfer-messenger RNA.

Translational pausing can lead to cleavage of the A-site codon and facilitate recruitment of the transfer-messenger RNA (tmRNA) (SsrA) quality control system to distressed ribosomes. We asked whether aminoacyl-tRNA binding site (A-site) mRNA cleavage occurs during regulatory translational pausing using the Escherichia coli SecM-mediated ribosome arrest as a model. We find that SecM ribosome arrest does not elicit efficient A-site cleavage, but instead allows degradation of downstream mRNA to the 3'-edge of the arrested ribosome. Characterization of SecM-arrested ribosomes shows the nascent peptide is covalently linked via glycine 165 to tRNA(3Gly) in the peptidyl-tRNA binding site, and prolyl-tRNA(2Pro) is bound to the A-site. Although A-site-cleaved mRNAs were not detected, tmRNA-mediated ssrA tagging after SecM glycine 165 was observed. This tmRNA activity results from sequestration of prolyl-tRNA(2Pro) on overexpressed SecM-arrested ribosomes, which produces a second population of stalled ribosomes with unoccupied A-sites. Indeed, compensatory overexpression of tRNA(2Pro) readily inhibits ssrA tagging after glycine 165, but has no effect on the duration of SecM ribosome arrest. We conclude that, under physiological conditions, the architecture of SecM-arrested ribosomes allows regulated translational pausing without interference from A-site cleavage or tmRNA activities. Moreover, it seems likely that A-site mRNA cleavage is generally avoided or inhibited during regulated ribosome pauses.

Binding Sites↗
PubMed2001

Human cytomegalovirus immediate-early messenger RNA in blood of pregnant women with primary infection and of congenitally infected newborns.

Human cytomegalovirus (HCMV) immediate-early messenger RNA (IEmRNA) in sequential blood samples from 32 pregnant women with primary infection and from 14 congenitally infected newborns was qualitatively investigated by nucleic acid sequence-based amplification. IEmRNA was detected in 100%, 75%, 36.3%, 22.2%, and 0% of samples collected 1, 2, 3, 4-6, and >6 months after onset of primary HCMV infection, respectively, showing 83.7% sensitivity and 92.2% specificity, compared with results of quantitative DNAemia (detection of viral DNA in blood). In infected newborns, IEmRNA was positive in 100% of samples collected 1-7 days (median, 1.5 days) and in 46.4% of samples collected 27-260 days (median, 88 days) after birth, showing 75.7% sensitivity and 100% specificity, compared with DNAemia results. IEmRNA was not detected in HCMV-immune individuals with remote or recurrent HCMV infection or in uninfected newborns. IEmRNA determination appears to be a valuable tool for early diagnosis of both primary and congenital HCMV infection.

Adolescent↗
PubMed1979

Post-transcriptional control of messenger RNA diversity in frog embryos.

The control of mRNA diversity during frog development has been investigated. Nuclear and messenger RNA from the early neurula and larval stages of Rana pipiens were hybridized in vast excess to labeled single-copy DNA and resistance to S1 nuclease was measured. Mixtures of RNA populations were also hybridized with single-copy DNA as a measurement of sequence overlap. Neurula and larval nuclear RNA hybridize to 11.3% and 12.1% of the single copy DNA. A mixture of both nuclear RNA populations hybridizes to 10.8% of the DNA, indicating a great amount of sequence overlap between the two populations. The mRNA complexity almost doubles during this developmental period from 4.7% of the single-copy DNA complexity at the early neurula to 8.7% at the larval stage. Mixtures of nuclear and messenger RNA were used to hybridize single-copy DNA and the results indicate that mRNA sequences present on neurula polysomes, but not on larval polysomes, are found in larval nuclei. Furthermore, mRNA sequences found on larval polysomes, but not on neurula polysomes, are found in the neurula nuclei. The data indicate that post-transcriptional events appear to play a role in the qualitative control of mRNA diversity during development.

Animals↗