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At least 865 records · Page 48Linked to original sources
PubMed1993

Cloning and functional expression of a brain G-protein-coupled ATP receptor.

A cDNA encoding a novel member of the G-protein-coupled receptor (GCR) superfamily, an ATP receptor, has been isolated from an embryonic chick whole brain cDNA library by hybridization screening. The encoded protein has a sequence of 362 amino acids (41 kDa) and shares no more than 27% amino acid identity with any known GCR. When expressed as a complementary RNA (cRNA) in Xenopus oocytes a slowly-developing inward current was observed in response to application of ATP. The pharmacology of this expressed protein defines it as a P2Y purinoceptor.

Adenosine Triphosphate↗
PubMed1996

SV40 T1-mRNA trans-splicing and translation requires that the in vitro synthesized cRNA is capped before microinjection.

The purpose of this investigation was to study the effect on cap structure for trans-splicing in mammalian cells. The early SV40 Bst/Bam pre-mRNA (cRNA) was synthesized in vitro in both capped (cap-Bst/Bam-cRNA) and non-capped (Bst/Bam-cRNA) versions and microinjected into the nuclei of TC7 cells. Trans-splicing was monitored by immunofluorescence staining (T1-antigen) and by RT-PCR analysis. Cap-Bst/Bam-cRNA was trans-spliced with high efficiency, but not the Bst/Bam-cRNA molecules. Northern blot analysis revealed that both the capped and uncapped cRNA molecules had similar stability in the microinjected cells. The coinjected m7G(5')ppp(5')G cap analog did not inhibit the trans-splicing reaction in vivo and did not prevent nuclear export of the mRNA.

Animals↗
PubMed1996

In vitro synthesized SV40 cRNA is trans-spliced after microinjection into the nuclei of mammalian cells.

We present for the first time experimental evidence that in vitro synthesized RNA (cRNA) is trans-spliced after microinjection into the nuclei of mammalian tissue culture cells. The template used for cRNA synthesis was the early SV40 BstXl/BamHI DNA fragment. This DNA fragment encodes exclusively for the second T-antigen exon and contains the intact small t-antigen intron. To generate the corresponding mRNA (T1-mRNA) by trans-splicing, the cells utilize a 5' cryptic splice site located within the second T-antigen exon of one cRNA molecule which is spliced to the small t-antigen 3' splice site of another cRNA molecule. Formation of the T1-mRNA by trans-splicing was confirmed by RT-PCR analysis and DNA sequencing. Efficient trans-splicing required that competitive small t-antigen cis-splicing be inhibited by deletion of the small t-antigen 5' splice site. The T1-mRNA was not generated when the cryptic 5' splice site was mutated.

Animals↗
PubMed1994

Excess iron into hepatocytes is required for activation of collagen type I gene during experimental siderosis.

BACKGROUND/AIMS: Liver fibrosis and cirrhosis represent common pathological findings in humans with iron overload. This study was undertaken to assess whether in vivo targeting of iron to liver parenchymal or nonparenchymal cells would differently affect collagen gene activity. METHODS: Rats were treated with an iron diet or intramuscular injections of iron dextran, and in situ hybridization analyses on liver samples were performed. RESULTS: These iron treatments determined parenchymal or reticuloendothelial cell iron overload, respectively. The typical distribution of iron into different liver cells was documented by histochemistry and confirmed by in situ hybridization analysis with a ferritin L complementary RNA probe. In iron-fed rats, in situ hybridization analysis identified a signal for collagen type I messenger RNA into nonparenchymal cells in zones I and II. In rats with nonparenchymal cell iron overload, no activation of collagen gene expression was detected into or near iron-laden nonparenchymal cells. These findings were also confirmed by quantitative Northern blot analysis. CONCLUSIONS: The results of this study indicate that, regardless of the total hepatic iron burden, selective localization of iron into liver cells (i.e., parenchymal cells) is required for the activation of collagen gene during long-term iron overload in rodents.

Animals↗
PubMed1985

Template-dependent RNA polymerase from black beetle virus-infected Drosophila melanogaster cells.

Infection of cultured cells of Drosophila melanogaster with black beetle virus (BBV) induces an RNA polymerase that is bound to cellular particulate material in a complex with a template RNA. We have solubilized the polymerase by treatment of the relevant particulates with detergents such as dodecyl-beta-D-maltoside. The polymerase activity was made dependent upon exogenous RNA by destruction of the endogenous template RNA with micrococcal nuclease. Addition of BBV RNA1 or RNA2 induced synthesis of full-length negative-strand RNA isolated as a double-stranded complex with the added RNA. Newly synthesized plus strands were also detected in the RNA2 complexes. Certain other viral RNAs also induced synthesis of their negative strands.

Animals↗
PubMed1977

A very large repeating unit of mouse DNA containing the 18S, 28S and 5.8S rRNA genes.

The organization of the 18S, 28S and 5.8S rRNA genes in the mouse has been elucidated by mapping with restriction endonucleases Eco RI, Hind III and Bam HI. Ribosomal DNA fragments were detected in electrophoretically fractionated digests of total nuclear DNA by in situ hybridization with radioiodinated rRNAs or with complementary RNA synthesized directly on rRNA templates. A map of the rDNA which includes 13 restriction sites was constructed from the sizes of rDNA fragments and their labeling by different probes The map indicates that the rRNA genes lie within remarkably large units of reiterated DNA, at least 44,000 base pairs long. At least two, and possibly four, classes of repeating unit can be distinguished, the heterogeneity probably residing in the very large nontranscribed spacer region. The 5.8S rRNA gene lies in the transcribed region between the 18S and 28S genes.

Animals↗
PubMed1993

Expression of endothelin-1 in lungs of patients with cryptogenic fibrosing alveolitis.

The vasoconstrictor and mitogenic peptide endothelin-1 (ET-1) is believed to play a part in fibrosis and collagen production. We examined expression of ET-1 in lung tissue from 52 patients with interstitial lung fibrosis, of whom 45 had cryptogenic fibrosing alveolitis (CFA), 10 had CFA and concomitant pulmonary hypertension, and 7 had non-specific focal fibrosis. 17 normal unused donor lungs were studied as controls. Immunohistochemistry and in-situ hybridisation were done with polyclonal antisera to ET-1 and its precursor big ET-1, and complementary RNA probes for preproET-1. Normal lung tissue and that from patients with focal fibrosis expressed very little ET-1. By contrast, there was striking expression of ET-1 in lung tissue from patients with CFA. Immunostains for ET-1 and big ET-1 and expression of ET-1 mRNA were most prominent in airway epithelium and type II pneumocytes, particularly those lining areas of young granulation tissue. ET-1-like immunoreactivity and mRNA were also present in pulmonary vascular endothelial cells, particularly in specimens from patients with pulmonary hypertension. In all patients, there was a significant correlation between ET-1-like immunoreactivity and histological parameters of disease activity (r = 0.78, 95% CI 0.65-0.87, p < 0.001). These findings suggest a possible role for cell-specific expression of ET-1 in the pathogenesis of CFA and associated pulmonary hypertension.

Biomarkers↗
PubMed1991

Co-localization of proenkephalin mRNA using cRNA probes and a cell-type-specific immunocytochemical marker for intact astrocytes in vitro.

To determine whether cultured astrocytes express opioid gene mRNA, a method was developed for co-localizing a cell-type specific immunocytochemical marker for astrocytes, glial fibrillary acidic protein (GFAP), and proenkephalin mRNA in situ hybridization signal using high affinity cRNA probes. GFAP immunoreactivity and proenkephalin mRNA hybridization reaction were examined in intact glial cell preparations from neonatal mice that were cultured for 4-6 days prior to fixation. The double labelling method described herein permits the unambiguous identification of mRNA expression in specific populations of intact cultured cells using cell type-specific markers.

Animals↗
PubMed1995

PGP9.5, a ubiquitin C-terminal hydrolase; pattern of mRNA and protein expression during neural development in the mouse.

PGP9.5 is a neurone and neuro-endocrine specific ubiquitin carboxyl terminal hydrolase estimated to form 1-2% of total brain protein. We have examined the temporal and spatial distribution of PGP9.5 mRNA and protein in the developing mouse embryo. These studies show that PGP9.5 is present at high levels in all differentiated neurones throughout the central and peripheral nervous systems at all stages of development. The mRNA is detected in the neural tube 1 day prior to the protein and before neuronal differentiation is underway. Both mRNA and protein are present during the initial appearance of motor and sensory neurones, prior to their peak production. PGP9.5 immunoreactivity was detected using microwave pretreatment of sections in order to unmask epitopes. In general unmasking led to an overall enhancement of immunoreactivity although in some sites, for example the dorsal root and cranial nerve ganglia and the bundle of His, there was evidence for anatomical variation in the distribution of masked versus unmasked protein. The very early expression of PGP9.5 suggests that there is a role for ubiquitin hydrolases in the differentiation of neurone precursors as well as in the differentiated neurone.

Animals↗
PubMed1988

Interspersed repetitive DNA from Plasmodium falciparum.

An interspersed repetitive DNA element from the Camp strain of the human malaria parasite Plasmodium falciparum was cloned and sequenced. The element is repeated at least 11 times in the genome, is a minimum of 2.0 kilobases long and maps to eight or more different parasite chromosomes. The cloned DNA includes a 1.0 kilobase open reading frame. RNA complementary to the repetitive element is found within blood stage forms of the parasite, but only in the most mature forms. Searches of DNA and protein sequence databases for homology to the element were unsuccessful. Sizes of restriction enzyme fragments which hybridize to the element show considerable variation between strains, indicating that the repeat family represented by this element could be transposable.

Amino Acid Sequence↗
PubMed1996

Calbindin localization in African giant rat kidney (Cricetomys gambianus).

Cricetomys gambianus are rodents living in savanna and follow area. They can live with restricted drinking water eating fresh food. Therefore their kidney may have some adaptive mechanisms for ion/water homeostasis compared to usual laboratory rats. In this study we have looked for calbindin, an intracellular calcium binding protein previously found in distal convoluted tubules from all mammalian species that have been studied and able to increase, in vitro, Ca2+ reabsorption. We have shown by using in situ hybridization, immunoblotting and immunohistochemistry that calbindin was expressed in three different portions of the distal nephron of the African giant rat. Calbindin was found in distal convoluted tubules, in cortical collecting tubules and in outer medullary collecting ducts. By contrast, in laboratory rat, calbindin was only found in distal convoluted tubules and undetectable in medullary collecting ducts. Thick ascending limb of Henle's loop were calbindin negative as shown by double immunolabelling using anti-uromucoid (Tamm-Horsfall protein). As previously shown in laboratory rat and rabbit, transcellular Ca2+ movement seems to be facilitated by calbindin in renal tubules segments predominantly actively transporting Ca2+, it may be suggested that in African giant rat, outer medullary collecting ducts may also actively transport Ca2+. As calretinin, another intracellular calcium binding protein highly homologous to calbindin but whose function is still conjectural has been suspected to be expressed in kidney, we have looked and not found any calretinin in both adult rat species.

Animals↗
PubMed1985

Dysdifferentiative nature of aging: age-dependent expression of mouse mammary tumor virus and casein genes in brain and liver tissues of the C57BL/6J mouse strain.

Many aspects of the aging process could be the result of cells slowly drifting away from their proper state of differentiation. This possibility has been studied by searching for an age-dependent increase in the expression of specific genes in tissues where expression of these genes would not normally be expected. In these studies, cDNA probes of specific genes are used in a DNA X NA hybridization assay to detect possible complementary RNA sequences in tissues of different-aged animals. Using this technique in past experiments, a qualitative increase in the RNA sequence complexity of mouse leukemia virus (MuLV) and a quantitative increase in the amount of alpha- and beta-globin RNA were found with increasing age in the brain and liver of the C57BL/6J mouse strain. We report here a similar age-dependent qualitative increase in the RNA sequence complexity for mouse mammary tumor virus (MMTV) but no quantitative or qualitative age-dependent change in casein RNA sequences for the same tissues and mouse strain.

Aging↗
PubMed1995

Detection by in situ hybridization of hepatitis C virus positive and negative RNA strands using digoxigenin-labeled cRNA probes in human liver cells.

In situ hybridization was performed using cRNA probes on human liver biopsies to localize both positive and negative RNA strands of hepatitis C virus. From the 5' non-coding region of the viral genome, 210 bp, were amplified by reverse transcriptase-polymerase chain reaction and cloned in a plasmid. Probes were produced by in vitro transcription, and labeled using digoxigenin-11-UTP. Positive HCV-RNA strands were detected in all 20 of the patients analyzed, whereas negative strands were detected in only nine patients, as confirmed using computerized image analysis. Both probes labeled the cytoplasm of hepatocytes with a perinuclear intensification. Few of the mononuclear cells infiltrating the portal connective space contained positive HCV-RNA strands only. Stacks of dilated endoplasmic reticulum cisternae were observed by electron microscopy and their relationship with the infection was discussed. This study confirmed that non-radioactive in situ hybridization represents a useful tool to analyze the localization and replication of hepatitis C virus in liver tissue.

Biopsy↗
PubMed1988

Expression of the gene for preproatriopeptin in the central nervous system of the rat.

Atriopeptin (AP) is a peptide hormone synthesized and secreted by the atria of the heart that participates in the regulation of fluid and electrolyte balance. AP-like materials have been detected immunologically in neurons in the central nervous system of the rat. In this study, we have used a solution hybridization-nuclease protection assay to determine whether the brain of the rat contains RNA coding preproatriopeptin, the atrial biosynthetic precursor of AP, and to study the regional distribution of preproatriopeptin mRNA in the brain. We have found that the brain contains mRNA identical to the atrial messenger RNA for preproatriopeptin. AP mRNA is differentially distributed in the brain; the highest concentration was found in the hypothalamus, followed by the cortex and septum, hippocampus, midbrain, spinal cord, olfactory bulb, striatum, and pons and medulla. Very low levels were found in the cerebellum, while no detectable AP mRNA was observed in retina, anterior pituitary, or rat liver. The presence of AP mRNA in the brain demonstrates that neurons have the capacity to synthesize preproatriopeptin identical to that produced in the heart. Bioactive peptides produced from this precursor may be endogenous central neuromodulators as well as a circulating hormone.

Animals↗
PubMed1988

Prodynorphin gene expression in spinal cord is enhanced after traumatic injury in the rat.

Levels of mRNAs coding for prodynorphin (Pro-Dyn) and proenkephalin (Pro-Enk) as well as the levels of immunoreactive (ir)-dynorphin (Dyn) and (ir)-Met-enkephalin (Met-Enk) were measured in the spinal cord of rats, 65 h following transection or injury of the spinal cord at the T6 spinal segment. Levels of both Pro-Dyn mRNA and of ir-Dyn were significantly increased between 60 and 150%, above control levels in the whole spinal cord, whereas those of Pro-Enk mRNA and of ir-Met-Enk remained unchanged. The increase in spinal levels of Pro-Dyn mRNA were highest in the areas close to the side of transection and indicate an involvement of the Pro-Dyn opioid system in the response to spinal injury and transection.

Animals↗
PubMed1991

The genes encoding nerve growth factor and its receptor are expressed in the developing female rat hypothalamus.

Nerve growth factor (NGF), its messenger RNA (mRNA) and its receptor protein and mRNA are found in several brain regions. Little attention has been given to the possibility that the hypothalamus, which controls the endocrine system, may produce NGF and/or express NGF receptors. This would indicate the existence in this brain area of neuronal populations which are either target for NGF-responsive cells or sensitive to NGF, respectively. Blot hybridization of polyadenylated (poly(A)+) RNA to a mouse NGF cRNA probe revealed the presence of NGF mRNA in both the suprachiasmatic region (henceforth, called preoptic area [POA]) and medial basal hypothalamus (MBH) of developing female rats. The mRNA was already detectable on fetal day 18 and reached maximal levels around postnatal day 3 (MBH) and 12 (POA), declining thereafter. This pattern was temporally different than that of areas known to produce NGF, i.e., the neocortex (Cc) and hippocampus (Hc). In agreement with previous reports, NGF mRNA levels in these areas were negligible before birth, became maximal between the second and third week of postnatal life and decreased moderately thereafter. The concentration of NGF protein, measured by a two-site enzyme immunoassay, was 3 times higher in the POA than in the MBH at an infantile age (day 12), increasing 2-fold in the POA of juvenile animals (day 28) but not in the MBH. This developmental pattern was similar to that seen in the Hc, though the NGF concentrations were significantly lower in the POA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Cellular distribution in the rat telencephalon of mRNAs encoding for the alpha 3 and alpha 4 subunits of the nicotinic acetylcholine receptor.

Pharmacological and electrophysiological studies provide evidence for the involvement of different nicotinic acetylcholine receptor isoforms in rat neocortical and hippocampal signal transduction. Yet, rather little is known on the cellular localization of these isoforms. With the availability of isoform specific nucleic acid probes and sensitive non-isotopic detection systems, nicotinic receptors can be studied on the mRNA level in individual neurons. In this way, we have paradigmatically studied the distribution of the alpha 3 and alpha 4 isoform mRNAs of the nicotinic receptor in the rat telencephalon. In the cerebral cortex, alpha 3 transcripts were mainly located in pyramidal neurons of layers V and VI and in some non-pyramidal cells in layer IV, while alpha 4 mRNA was detected in different types of neurons located in almost all layers. In the hippocampus, local distribution of both transcripts was comparable. Only very few labeled neurons were observed in the dentate gyrus. In the CA region, the specific mRNAs were detected in pyramidal perikarya and individual neurons in the strata oriens and lacunosum-moleculare. Our data show that the applied method is sufficiently sensitive and isoform-selective in order to study the differential expression of nicotinic receptors on the cellular level in the mammalian brain.

Animals↗