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PubMed1998

Identification of glutathione as a driving force and leukotriene C4 as a substrate for oatp1, the hepatic sinusoidal organic solute transporter.

oatp1 is an hepatic sinusoidal organic anion transporter that mediates uptake of various structurally unrelated organic compounds from blood. The driving force for uptake on oatp1 has not been identified, although a role for bicarbonate has recently been proposed. The present study examined whether oatp1-mediated uptake is energized by efflux (countertransport) of intracellular reduced glutathione (GSH), and whether hydrophobic glutathione S-conjugates such as leukotriene C4 (LTC4) and S-dinitrophenyl glutathione (DNP-SG) form a novel class of substrates for oatp1. Xenopus laevis oocytes injected with the complementary RNA for oapt1 demonstrated higher uptake of 10 nM [3H]LTC4 and 50 microM [3H]DNP-SG, and higher efflux of [3H]GSH (2.5 mM endogenous intracellular GSH concentration). The oatp1-stimulated LTC4 and DNP-SG uptake was independent of the Na+ gradient, cis-inhibited by known substrates of this transport protein and by 1 mM GSH, and was saturable, with apparent Km values of 0.27 +/- 0.06 and 408 +/- 95 microM, respectively. Uptake of [3H]taurocholate, an endogenous substrate of oatp1, was competitively inhibited by DNP-SG. Of significance, oatp1-mediated taurocholate and LTC4 uptake was cis-inhibited and trans-stimulated by GSH, and [3H]GSH efflux was enhanced in the presence of extracellular taurocholate or sulfobromophthalein, indicating that GSH efflux down its large electrochemical gradient provides the driving force for uptake via oatp1. The stoichiometry of GSH/taurocholate exchange was 1:1. These findings identify a new class of substrates for oatp1 and provide evidence for GSH-dependent oatp1-mediated substrate transport.

Animals↗

PubMed1996

Forskolin stimulation of water and cation permeability in aquaporin 1 water channels.

Aquaporin 1, a six-transmembrane domain protein, is a water channel present in many fluid-secreting and -absorbing cells. In Xenopus oocytes injected with aquaporin 1 complementary RNA, the application of forskolin or cyclic 8-bromo- adenosine 3',5'-monophosphate increased membrane permeability to water and triggered a cationic conductance. The cationic conductance was also induced by direct injection of protein kinase A (PKA) catalytic subunit, reduced by the kinase inhibitor H7, and blocked by HgCl2, an inhibitor of aquaporin 1. The cationic permeability of the aquaporin 1 channel is activated by a cyclic adenosine monophosphate-dependent mechanism that may involve direct or indirect phosphorylation by PKA.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Heterologous expression of a P2x-purinoceptor in rat chromaffin cells detects vesicular ATP release.

A cloned P2x-purinoceptor was transiently expressed in single isolated rat adrenal chromaffin cells and evaluated for the detection of released ATP. After cytoplasmic injection of the P2x complementary RNA (cRNA; 4-24 h), application of ATP produced an inwardly rectifying current over the voltage range -130 to -10 mV as measured by the whole cell patch-clamp technique. The dose-response curve for ATP was sigmoidal with a 50% effective concentration of 18. 2 microM. Suramin, a P2x-antagonist, attenuated the ATP-induced current. Depolarizing voltage pulses to 0 mV or application of histamine, stimuli that trigger exocytosis, resulted in the appearance of suramin-sensitive spontaneous transient inward currents (at -60 mV) that resembled excitatory postsynaptic currents although they were slower in time course. Concurrent detection of catecholamine release with a carbon fiber electrode often showed coincidence of the amperometric current with the synaptic currentlike events suggesting that ATP and catecholamines were released from the same vessicle. These data demonstrate that expression of a P2x-purinoceptor in chromaffin cells produces a functional autoreceptor capable of detecting vesicular release of ATP. In combination with carbon fiber amperometry, simultaneous vesicular release of two neurotransmitters from a single chromaffin cell could be monitored. The P2x-purinoceptor, however, produced a regenerative effect on release apparently resulting from the high Ca2+ permeability of the receptor. Thus modification of the P2x-purinoceptor would be required before the system could be applied to examining processes involved in stimulus-release coupling.

Adenosine Triphosphate↗

PubMed1977

Human studies following animal models of tumorigenesis by oncornaviruses.

Similarities have been observed for some time between oncornavirus-induced malignancies in laboratory animals and leukemias and solid tumors in man. Particles similar to type C oncornaviruses have been detected by electron microscopy both in cells or plasma from leukemia patients and in solid-tumor human malignancies such as Hodgkin's lymphoma, lymphosarcomas, and sarcomas. Likewise, particles resembling type B oncornaviruses in shape and appearance have been found in human breast cancer. In neither case has the infectious nature of the particles been confirmed. However, DNA synthesized in vitro by the enzyme of murine mammary tumor virus was found to hybridize with polysomal RNA obtained from human mammary adenocarcinomas. The presence of RNA complementary to RNA from the Rauscher strain of murine leukemia virus has been observed in other human malignancies unrelated to breast cancer. It has also been found that cells of patients with myelogenous leukemia possess an oncornaviral-type reverse transcriptase that is distinguishable from other cell DNA polymerases and serologically related to the reverse transcriptase of primate oncornaviruses.

Animals↗

PubMedDate not supplied

[Transcription of influenza virus genome. Location and dependence on the nuclear primer function].

The synthesis of the complete and incomplete transcripts (the templates in genome replication and mRNA, respectively) of influenza A WSN virus in chicken fibroblasts was analyzed by gel electrophoresis analysis of the duplexes formed between virion RNA and complementary RNA. Three steps in the transcription could be defined: 1) primary transcription when similar amounts of mRNA of all the genes are accumulated; 2) early secondary transcription when mRNA of NS gene is synthesized in larger amounts than that of other genes and 3) late secondary transcription when the amplification of transcription from all the genes is performed. The synthesis of complete transcripts starts during or after primary transcription. When actinomycin D was added to infected cells, the synthesis of incomplete transcripts was inhibited to a larger degree then that of complete transcripts. Most of incomplete transcripts was observed within the cell nucleic while the complete transcripts were found in the nuclei and cytoplasm, suggesting that the synthesis of incomplete transcripts is located in the nuclei. alpha-Amanitin blocked the synthesis of incomplete transcripts without interfering with that of complete transcripts. These data suggest that the synthesis of complete transcripts does not require the synthesis of cell mRNA as primers in transcription.

Amanitins↗

PubMedDate not supplied

[Hybridase cleavage of RNA. V. Complementary precision of cleavage].

The efficiency of the cleavage of RNA involved in perfect as well as imperfect hybrid duplexes composed of three components: (1) homogeneous RNA's or polyribonucleotides; (2) corresponding complementary synthetic oligodeoxyribonucleotides; (3) E. coli RNase H was investigated. The predominant RNA hydrolysis was shown to take place within the perfect hybrid duplexes formed by the target RNA and the complementary oligodeoxyribonucleotide probes. RNase H was found to cleave effectively a number of imperfect hybrid duplexes containing a central base pair mismatch.

Autoradiography↗

PubMed1979

In vivo transcription and protein synthesis capabilities of bunyaviruses: wild-type snowshoe hare virus and its temperature-sensitive group I, group II, and group I/II mutants.

The in vivo primary and secondary transcription capabilities of wild-type snowshoe hare (SSH) virus and certain of its temperature-sensitive (ts) mutants have been analyzed. The results obtained agree with in vitro studies (Bouloy et al., C.R. Acad. Sci. Paris 280:213-215, 1975; M. Bouloy and C. Hannoun, Virology 69:258-264, 1976; M. Ranki and R. Pettersson, J. Virol. 16:1420-1425, 1975) which have shown that bunyaviruses are negative-stranded RNA viruses with a virion RNA-directed RNA polymerase. The in vivo transcription studies have demonstrated that in the presence of protein synthesis inhibitors (puromycin or cycloheximide) SSH virus can synthesize viral complementary RNA (primary transcription) throughout the infection cycle. The increased levels of viral complementary RNA obtained in the absence of protein synthesis inhibitors (secondary transcription) were not markedly reduced if cells were pretreated with actinomycin D (5 mug/ml), alpha-amanitin (25 mug/ml), or rifampin (100 mug/ml), although progeny virus yields were reduced by up to 80% in the actinomycin D- and rifampin-treated cells. The in vivo transcription capabilities of SSH group I ts mutants at temperatures which were nonpermissive (40 degrees C) for virus replication gave values comparable to those obtained at permissive temperatures (33 degrees C). The SSH group I mutants appear, therefore, to be RNA-positive mutant types. When compared with their transcription capabilities at 33 degrees C, the in vivo transcription abilities of four SSH group II ts mutants (and one double group I/II ts mutant) were found to be more impaired at 40 degrees C than those of the SSH group I ts mutants or wild-type SSH virus at 40 degrees C, although the viral complementary RNA synthetic capabilities of these group II (and group I/II) mutants at 40 degrees C were significantly higher than their primary transcription capabilities (as measured at 33 degrees C in the presence of puromycin or cycloheximide). It was concluded, therefore, that these SSH group II (and double group I/II) ts mutants have an intermediate RNA phenotype. Hybridization studies using (32)P-labeled individual L, M, and S viral RNA species of SSH virus have demonstrated the presence of viral complementary RNA to all three species in extracts of cells infected with SSH ts II-30 and incubated at 33 degrees C (primary and secondary transcription) or 40 degrees C, a nonpermissive temperature for its replication. The results of pulse-labeled in vivo protein analyses indicated that greater quantities of intracellular N protein (coded for by S RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978]) than G1 and G2 polypeptides (coded for by M RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-776, 1979]) were present in extracts of cells infected with wild-type SSH virus. In extracts of SSH group I, II, or I/II ts mutant-infected cells incubated at 33 degrees C, N and G1, and for the group II mutant-infected cells, G2, viral polypeptides were detected, whereas in extracts obtained from group I or II mutant virus-infected cells incubated at 40 degrees C, low levels of N and G1 polypeptides were evident.

Animals↗

PubMed1986

In situ hybridization versus northern analysis: working towards the correlation of two quantitative techniques for opioid and vasopressin mRNAs in the rat hypothalamus and pituitary.

Several physical and technical aspects of in situ hybridization are examined to help ascertain to what extent the total specific mRNA population of interest in a tissue section is actually detected using complementary RNA probes. Two experimental paradigms, Rot curve hybridization analysis and varying the tissue section thickness, indicate that complementary RNA (cRNA) probe hybridization to a specific mRNA in a tissue section can be made saturable, but remains so only for those mRNAs within a limited distance from the surface of the section. Direct comparison with Northern analysis, however, shows that in situ hybridization still gives comparable relative quantitative results for mRNA levels changing due to physiological stimulation.

Kinetics↗

PubMed1991

Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to exons.

The objective of the experiments described in this paper was to determine the feasibility of inhibition of pre-mRNA splicing by antisense RNA in vitro. Three different types of antisense RNA were utilized: antisense RNA complementary to the spliced RNA molecule; antisense RNA complementary to the unprocessed mRNA precursor molecule; and antisense RNA complementary to the 5' and 3' splice junctions. Whereas antisense RNA complementary to mRNA had little effect on splicing, antisense RNAs complementary to mRNA precursor or to splice junctions strongly inhibited splicing of pre-mRNA molecule. The results obtained indicate that the inhibitory effect is most likely due to hybrid formation between pre-mRNA and antisense RNA molecules and that antisense RNA complementary to the exon portion but not to the intron portion of splice junction exhibit an inhibitory effect. This inhibition can be overcome by bringing together 5' and 3' splice junctions via hybrid formation with antisense RNA complementary to the spliced RNA molecule.

Animals↗

PubMed1984

Cloning and sequencing of the preprotoxin-coding region of the yeast M1 double-stranded RNA.

Complementary DNA (cDNA) copies of the M1-1, toxin-coding region of the yeast M1 double-stranded RNA (dsRNA) have been cloned and sequenced. These sequences, in combination with the known terminal sequence of M1-1 dsRNA, identify a translation reading frame for a 316 amino acid protein of 34.7 kd, similar in size to the preprotoxin produced from M1 dsRNA by in vitro translation. Potential glycosylation sites in the preprotoxin peptide are identified. Based on its methionine content the extracellular yeast toxin appears to be contained within the C-terminal region of the precursor.

Amino Acid Sequence↗

PubMed1984

Gene expression during Drosophila melanogaster oogenesis: analysis by in situ hybridization to tissue sections.

The pattern of gene expression for specific genes during oogenesis has been examined by in situ hybridization to RNA in sectioned ovarian preparations. The accumulation (1) of RNA complementary to heat-shock genes, both before and after heat shock; (2) of RNA complementary to three lambda phage recombinants which contain sequences expressed during oogenesis; and (3) of RNA complementary to the rudimentary gene have been analyzed. Gene expression in the ovaries is found to be dependent on both the cell type and the developmental stage of the ovarian chamber. In the nurse cells, the onset of vitellogenesis (stages 8-11) appears to be accompanied by a substantial activation of many different genes and there is a pronounced increase in the level of accumulation of complementary RNAs. Moreover, much of the RNA synthesized in nurse cells during vitellogenesis is ultimately transported into the growing oocyte where it accumulates. It is likely that this accumulation of RNA provides a source of gene products, either as message or proteins, for embryogenesis.

Animals↗

PubMed1974

Effect of interferon upon the primary and secondary transcription of vesicular stomatitis and influenza viruses.

The synthesis of viral complementary RNA by vesicular stomatitis virus in permissive cells can be distinguished operationally into two phases, primary transcription, which can be observed in cycloheximide treated cells, and secondary transcription, which represents the further synthesis of viral complementary RNA obtained in untreated cells. Pretreatment of mouse L cells or chicken embryo fibroblasts with the homologous interferon and, for chicken embryo fibroblast cells, poly(rI):poly(rC), inhibits the production of infectious virus and reduces the synthesis of viral complementary RNA to levels comparable to that obtained during primary transcription. Treatment of mouse L cells with interferon plus cycloheximide also gives levels of vesicular stomatitis viral complementary RNA synthesis comparable to that observed with the cycloheximide treatment alone. Similar results have been obtained for interferontreated cells subsequently infected with influenza virus (strain WSN). The results are interpreted as indicating that inhibition of virus development by interferon does not act at the level of primary transcription but rather at an intermediate step between primary and secondary transcription, such as viral protein synthesis.

Animals↗

PubMed1997

Peptidylarginine deiminase of the hair follicle: characterization, localization, and function in keratinizing tissues.

The enzyme peptidylarginine deiminase (PAD; EC 3.5.3.15) is responsible for the formation of protein-bound citrulline, a major amino acid in the inner root sheath (IRS) and in the medulla of the hair follicle. From mainly biochemical evidence, it is known that the substrate for the enzyme is trichohyalin and that trichohyalin granules gradually disappear to form a matrix with intermediate-like filaments in the IRS cells. In the medulla, the granules aggregate into large masses without filaments. The proteins in both the IRS and medulla are finally cross-linked by transglutaminase. A corollary of the apparent central role of PAD acting on the trichohyalin protein in these processes is that it should be present in the IRS and medulla cells, coincident with trichohyalin. Hair-follicle PAD has not previously been isolated. In the current study, the enzyme was isolated from wool follicles of adult sheep and peptide sequences were used to design DNA primers for the synthesis of PCR products from follicle mRNA. Subsequently, a PAD-specific complementary RNA probe and a trichohyalin complementary RNA probe were prepared for localization studies by in situ hybridization in wool follicles and the epithelia of the rumen, embryonic hoof, and tongue papillae. The experiments have revealed a striking co-expression of PAD and trichohyalin in all of these tissues. The amino acid sequence of the wool-follicle PAD molecule has been deduced from sequencing of the cloned PCR products.

Amino Acid Sequence↗

PubMed1998

Partial cloning and differential expression of ryanodine receptor/calcium-release channel genes in human tissues including the hippocampus and cerebellum.

Cellular Ca2+ signalling is an important factor in the control of neuronal metabolism and electrical activity. Although the roles of Ca2+-release channels are well established for skeletal and cardiac muscle, less is known about their expression and roles in the central nervous system, especially in the human brain. We have isolated partial complementary DNAs derived from the human ryanodine receptor Ca2+-release channel genes (ryr1, ryr2 and ryr3), and examined their expression in the human hippocampus and cerebellum. For comparison, we have included in our analysis an inositol trisphosphate Ca2+-release channel type I complementary RNA probe. All four messenger RNAs show widespread distribution in the human hippocampus, where ryr2 is the most abundant isoform, and all four are expressed in the human cerebellum. However, striking differences were seen between ryr and inositol trisphosphate Ca2+-release channel type I complementary RNA expression in the cerebellum, with inositol trisphosphate Ca2+-release channel type I messenger RNA being largely restricted to, and very highly expressed, in Purkinje cells, whereas ryr1, ryr2 and ryr3 were all expressed predominantly in the granular layer. The widespread expression of ryr isoforms in the human hippocampus and cerebellum suggests that ryanodine receptor proteins may have a central role in Ca2+ signalling and Ca2+ homeostasis in the human central nervous system. These may include roles in fundamental processes like synaptic plasticity. Furthermore, these Ca2+-release channels may be involved in pathogenic processes such as excitotoxicity, where excessive rises in intracellular Ca2+ concentration mediate neuronal cell death.

Aged↗

PubMed1979

Genome complexities of the three mRNA species of snowshoe hare bunyavirus and in vitro translation of S mRNA to viral N polypeptide.

The genome complexities of the principal intracellular viral complementary RNA species of the snowshoe hare bunyavirus have been analyzed by duplex analyses involving hybridization of complementary RNA to individual 32P-labeled viral RNA species (large, L; medium, M; and small, S), recovery of nuclease-resistant duplexes, and determination of the oligonucleotide fingerprints of the protected 32P-labeled viral sequences. The result for the M RNA (which codes for the glycoproteins G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-770, 1979) indicates that there is a single polycistronic M mRNA. Similar results were obtained for the L and S RNA species. In vitro translation studies with the S complementary RNA species of snowshoe hare virus as well as melted purified S duplexes substantiate earlier genetic and molecular studies (J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978; J. Gentsch, D. H. L. Bishop, and J. F. Obijeski, J. Gen. Virol. 34-257-268, 1977), which indicate that S mRNA codes for the virion nucleocapsid protein N.

Animals↗

PubMed1994

Molecular subtyping of Borrelia burgdorferi in erythema migrans and acrodermatitis chronica atrophicans.

Recently, three subtypes of Borrelia burgdorferi have been identified: Borrelia burgdorferi sensu stricto, Borrelia garinii, and the VS 461 group of Borrelia burgdorferi. These subtypes differ by nucleotide sequence variations within several Borrelia burgdorferi specific genes and most likely by their pathogenetic potential. To assess whether different subtypes of Borrelia burgdorferi might be associated with different cutaneous manifestations and clinical courses of Lyme disease, lesional skin biopsies from 35 patients with erythema migrans and 18 patients with acrodermatitis chronica atrophicans were analyzed. A Borrelia burgdorferi specific gene segment encoding a 26-kD protein with subtype specific nucleotide sequence variations was amplified by a nested polymerase chain reaction technique. For molecular subtyping, the products were transcribed into complementary RNA. Upon polyacrylamide gel electrophoresis, complementary RNA molecules separate into several metastable conformational forms resulting in patterns of bands highly specific for the nucleotide sequence of the transcribed molecules. In biopsy specimens of erythema migrans, the VS 461 subtype was detected in 28 of 35 and the Borrelia garinii subtype in six of 35 cases. In one of 35 cases of erythema migrans Borrelia burgdorferi sensu stricto as well as Borrelia garinii was detected. In contrast, in all 18 biopsies of acrodermatitis chronica atrophicans, only the VS 461 subtype was identified. This subtype is rarely found in the USA, where acrodermatitis chronica atrophicans is almost unknown. These data indicate that acrodermatitis chronica atrophicans might be closely associated with the VS 461 group of Borrelia burgdorferi.

Acrodermatitis↗

PubMed1975

Hybridization of pigeon globin messenger RNA with complementary DNA synthesized in vitro by reverse transcription: influence of the homopolymeric regions.

The kinetics of hybridization of pigeon globin messenger RNA with complementary cDNA synthesized by means of AMV reverse transcriptase is complex. Addition of poly A or poly U in excess to the reaction mixture normalized the kinetics. It is concluded that association of the complementary homopolymeric regions of mRNA and cDNA accelerates the complex formation between heteropolymeric sequences in a fraction of the molecules.

Adenine Nucleotides↗

PubMed1995

Toward the therapeutic editing of mutated RNA sequences.

If RNA editing could be rationally directed to mutated RNA sequences, genetic diseases caused by certain base substitutions could be treated. Here we use a synthetic complementary RNA oligonucleotide to direct the correction of a premature stop codon mutation in dystrophin RNA. The complementary RNA oligonucleotide was hybridized to a premature stop codon and the hybrid was treated with nuclear extracts containing the cellular enzyme double-stranded RNA adenosine deaminase. When the treated RNAs were translated in vitro, a dramatic increase in expression of a downstream luciferase coding region was observed. The cDNA sequence data are consistent with deamination of the adenosine in the UAG stop codon to inosine by double-stranded RNA adenosine deaminase. Injection of oligonucleotide-mRNA hybrids into Xenopus embryos also resulted in an increase in luciferase expression. These experiments demonstrate the principle of therapeutic RNA editing.

Adenosine Deaminase↗