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At least 595 records · Page 33Linked to original sources
PubMed1995

Regulation of porcine granulosa cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin and insulin-like growth factor I: synergism with follicle-stimulating hormone or protein kinase A agonist.

In species such as the pig and human, gonadal steroidogenesis is believed to be dependent upon the availability of low density lipoprotein (LDL) cholesterol. However, before ovulation, Graafian follicles are impermeant to lipoproteins in the LDL class. Thus, de novo cholesterol biosynthesis via the rate-determining enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is likely to provide a significant mechanism for generating sterol substrate for steroidogenesis by granulosa cells before follicular rupture. As serum-free monolayer culture of (swine) granulosa cells offers an in vitro model of hormonally responsive HMG-CoA reductase, we generated a (porcine) complementary DNA and homologous complementary RNA to investigate by sensitive and specific ribonuclease protection assay the hormonal regulation of HMG-CoA reductase gene expression in ovarian cells from immature Graafian follicles. Using reverse transcriptase-polymerase chain reaction, we cloned and sequenced a 238-base pair complementary DNA from porcine luteal tissue that encodes the catalytic region of HMG-CoA reductase. GenBank analysis of the DNA sequence homology between the pig and other species showed the greatest concordance with human (88%) and hamster (90%). Solution hybridization/ribonuclease protection analysis of total RNA isolated from serum-free monolayer cultures of porcine granulosa cells revealed that insulin (3 micrograms/ml) increased HMG-CoA messenger RNA (mRNA) concentrations corrected for constitutive 18S ribosomal RNA expression in a time-dependent fashion, with significant effects observed at 12 h and a 6-fold increase by 48 h. Recombinant human insulin-like growth factor I (IGF-I) peptide was able to mimic the action of insulin alone. Neither FSH (100 ng/ml) nor 8-bromo-cAMP (1 mM) had observable effects on HMG-CoA message accumulation at any time point studied. However, the combined action of either FSH and insulin or 8-bromo-cAMP and insulin resulted in synergistic increases in reductase mRNA by 31- and 17-fold, respectively. To assess the possible feedback effects of sterol on HMG-CoA gene expression, granulosa cells were treated with LDL. At physiological concentrations, LDL suppressed basal expression of HMG-CoA mRNA to levels below the control value. In addition, LDL inhibited insulin-stimulated HMG-CoA mRNA accumulation by 84% as well as the synergistic effects of insulin and FSH (by 94%) and of insulin and 8-bromo-cAMP (by 93%). We conclude that insulin alone or in combination with FSH or cAMP augments the accumulation of HMG-CoA reductase mRNA in ovarian (granulosa) cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1979

Interferon induction by viruses. II. Sindbis virus: interferon induction requires one-quarter of the genome--genes G and A.

We have measured the amounts of interferon formed by chick cells 'aged' in vitro in response to different amounts of infectious wild-type Sindbis virus. Our results suggest that one plaque-forming unit is enough to induce maximum interferon formation. With higher m.o.i. the yield of interferon is less. To inactivate the interferon-inducing activity of Sindbis virus, four times more u.v.-radiation was needed than to inactivate the infectivity of the virus. This suggests that only 25% of the virus genome need be intact in order to induce interferon. Temperature-sensitive Sindbis virus mutants from the three RNA+ complementation groups, C. D and E, gave rise to interferon in chick cells incubated at a non-permissive temperature, Similarly, mutants from two of the RNA- groups, B and F, gave rise to interferon, but not mutants from groups G and A. We conclude that no pre-formed inducer of interferon is present in Sindbis virus. It appears, however, that genes G and A represent a special one-quarter of the genome which must be functional in order to synthesize an interferon-inducing moiety. We suggest that this moiety is a double-stranded RNA molecule formed after synthesis of a segment of RNA complementary to the genome.

Animals↗
PubMed1995

Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity.

The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O-propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2'-fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides.

Base Sequence↗
PubMed1991

Inhibition of human immunodeficiency virus type 1 replication in human T cells stably expressing antisense RNA.

Human T-lymphoid cell lines with constitutive intracellular expression of antisense RNA complementary to a 407-bp sequence of the 5' leader-gag region of human immunodeficiency virus type 1 were established by using a nonretroviral expression vector. In cell lines with antisense RNA expression detectable by Northern (RNA) hybridization, human immunodeficiency virus type 1 replication was inhibited to 88% 10 days postinfection and this inhibition lasted 3 weeks postinfection.

Cell Line↗
PubMed1995

Regulation of oxytocin receptor messenger ribonucleic acid in the ventromedial hypothalamus by testosterone and its metabolites.

Oxytocin receptor (OR) binding in the ventromedial hypothalamus (VMH) is regulated by testosterone (T) and its metabolites, estrogen (E2) and dihydrotestosterone (DHT). Previous studies have reported that OR binding increases in the VMH in castrated male rats when they are replaced with T or E2 compared to that in vehicle-treated animals. DHT alone had no effect on OR binding, but when given in combination with E2 appeared to have a synergistic effect. This study was designed to determine whether these effects of steroid hormones on OR binding in the VMH are associated with changes in OR messenger RNA (mRNA) expression. Male rats were castrated or sham operated and given T propionate (TP), E2 benzoate (EB), DHT plus EB, or an oil vehicle. OR mRNA was assessed using a rat complementary RNA OR probe and in situ hybridization techniques. OR binding to tissue slices was quantified autoradiographically using an OR antagonist, [125I]d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)] ornithine vasotocin. These experiments showed that TP and EB increased both OR mRNA and OR binding in the VMH significantly above levels in vehicle-treated animals. However, animals given both EB and DHT exhibited significantly lower OR mRNA expression and OR binding in the VMH compared to those in animals treated with TP or EB alone. These data indicate that increases in VMH OR binding in response to gonadal steroids are accompanied by changes at the mRNA level that correspond well in magnitude and direction with those in the OR-binding sites.

Animals↗
PubMed2003

Synthesis and properties of RNA analogues having amides as interuridine linkages at selected positions.

Oligoribonucleotide analogues having amide internucleoside linkages (AM1: 3'-CH(2)CONH-5' and AM2: 3'-CH(2)NHCO-5') at selected positions have been synthesized and the thermal stability of duplexes formed by these analogues with complementary RNA fragments has been evaluated by UV melting experiments. Two series of oligomers with either 2'-OH or 2'-OMe vicinal to the amide linkages were studied. Monomeric synthons (3' and 5'-C amines and carboxylic acids) were synthesized as follows: For synthesis of the AM1 analogue, the known sequence of radical allylation followed by the cleavage of the double bond was adopted. For synthesis of the AM2 analogue, novel routes via addition of nitromethane followed by conversion of the nitro function to either amino or carboxyl groups were developed. Coupling of monomeric amines and carboxylic acids followed by protecting group manipulation and phosphonylation gave dimeric 3'-hydrogenphosphonate building blocks for oligonucleotide synthesis. Monomeric model compounds having 3'-amide and 2'-OH or 2'-OMe groups were also prepared and their conformational equilibrium was determined by (1)H NMR. The AM1 and AM2 models showed equal preferences for the North conformers (at 40 degrees C, 88-89% with 2'-OH, and 92-93% with 2'-OMe). At physiological salt concentration (0.1 M NaCl) the duplexes between AM1 modified oligonucleotides and RNA had stability similar to unmodified RNA-RNA duplexes (Delta t(m)= -0.2 to +0.7 degrees C per modification). However, the AM2 modification resulted in substantial stabilization of duplexes: Delta t(m)= +1 to +2.4 degrees C per modification compared to all RNA. A 2'-O-methyl vicinal to the AM2 linkage further increased the duplex stability. Our results suggest that RNA analogues having amide internucleoside bonds are very promising candidates for medicinal applications.

Amides↗
PubMed2002

Oligonucleotides comprised of alternating 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides and D-2'-deoxyribonucleosides (2'F-ANA/DNA 'altimers') induce efficient RNA cleavage mediated by RNase H.

Chimeric oligonucleotides comprised of alternating residues of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (2'F-ANA) and DNA were synthesized and evaluated for an important antisense property-the ability to elicit ribonuclease H (RNase H) degradation of complementary RNA. Experiments used both human RNase HII and Escherichia coli RNase HI. Mixed backbone oligomers comprising alternating three-nucleotide segments of 2'F-ANA and three-nucleotide segments of DNA were the most efficient at eliciting RNase H degradation of target RNA, and were significantly better than oligonucleotides entirely composed of DNA, suggesting that these mixed backbone oligonucleotides may be potent antisense agents.

Arabinonucleosides↗
PubMed2003

Isolation and characterisation of the rabies virus N degrees-P complex produced in insect cells.

When the nucleoprotein (N) of nonsegmented negative-strand RNA viruses is expressed in insect cells, it binds to cellular RNA and forms N-RNA complexes just like viral nucleocapsids. However, in virus-infected cells, N is prevented from binding to cellular RNA because a soluble complex is formed between N and the viral phosphoprotein (P), the N degrees -P complex. N is only released from this complex for binding to newly made viral or complementary RNA. We coexpressed rabies virus N and P proteins in insect cells and purified the N degrees -P complex. Characterisation by gel filtration, polyacrylamide gel electrophoresis, analytical ultracentrifugation, native mass spectroscopy, and electron microscopy showed that the complex consists of one N protein plus two P proteins, i.e., an N degrees -P(2) complex.

Animals↗
PubMedDate not supplied

NMR studies of fully modified locked nucleic acid (LNA) hybrids: solution structure of an LNA:RNA hybrid and characterization of an LNA:DNA hybrid.

LNA is a bicyclic nucleic acid analogue that contains one or more 2'-O,4'-C methylene linkage(s), which effectively locks the furanose ring in a C3'-endo conformation. We report here the NMR solution structure of a nonamer LNA:RNA hybrid and a structural characterization of a nonamer LNA:DNA hybrid, where the LNA strands are composed entirely of LNA nucleotides. This is the first structural characterization of fully modified LNA oligonucleotides. The high-resolution structure reveals that the LNA:RNA hybrid adopts an almost canonical A-type duplex morphology. The helix axis is almost straight and the duplex geometry is regular. This shows that fully modified LNA oligomers can hybridize with complementary RNA and form duplexes within the Watson-Crick framework. The LNA:DNA hybrid structurally resembles an RNA:DNA hybrid as shown by determination of deoxyribose sugar puckers and analysis of NOESY NMR spectra.

DNA↗
PubMed1996

Towards artificial ribonucleases: the sequence-specific cleavage of RNA in a duplex.

Lanthanide complexes covalently attached to oligonucleotides have been shown to cleave RNA in a sequence-specific manner. Efficient cleavage, however, is at present limited to single-stranded RNA regions, as RNA in a duplex is considerably more resistant to strand scission. To overcome this limitation, we have designed and synthesised artificial nucleases comprising lanthanide complexes covalently linked to oligodeoxyribonucleotides which cleave a partially complementary RNA at a bulged site, in the duplex region. Strand scission occurs at or near the bulge. Cleavage of the RNA target by the metal complex can be addressed via the major or the minor groove. In an example of a competitive situation, where the cleavage moiety has access to both a bulge and a single-strand region, transesterification at the bulge is favoured. Such artificial ribonucleases may find application as antisense agents and as tools in molecular biology. In addition, the results may have importance for the design of artificial ribonucleases which are able to act with catalytic turnover.

Autoradiography↗
PubMed1987

Control of gene expression in bacteriophage P22 by a small antisense RNA. II. Characterization of mutants defective in repression.

Phage P22 produces antirepressor protein early after infection from a transcript initiated at the Pant promoter. After the first few minutes of infection, transcription from Pant is repressed by a protein encoded by the arc gene. Antirepressor is not produced late in infection, even though the antirepressor gene, ant, is transcribed from the late operon promoter Plate. We describe the isolation of P22 mutants that synthesize antirepressor from the Plate transcript. The mutations inactivate a promoter Psar, which lies within the ant coding sequence and directs the synthesis of sar RNA, a small antisense regulatory RNA complementary to the ant ribosome binding site. Characterization of the Psar down-mutants shows that transcription from Psar interferes with synthesis of antirepressor from both the Plate and Pant transcripts. Since sar RNA represses synthesis of antirepressor in trans, we propose that sar RNA base-pairs with ant mRNA to inhibit antirepressor synthesis at a post-transcriptional level. The role and importance of sar RNA in P22 biology are discussed.

Gene Expression Regulation↗
PubMed1982

Newly formed mRNA lacking polyadenylic acid enters the cytoplasm and the polyribosomes but has a shorter half-life in the absence of polyadenylic acid.

Labeled adenovirus type 2 nuclear RNA molecules from cells treated with 3'-deoxyadenosine (3'dA) were earlier reported to lack polyadenylic acid [poly(A)], but to be correctly spliced in the nucleus (M. Zeevi et al., Cell 26:39-46, 1981). We have now found that the shortened mRNA molecules, lacking poly(A), can also be found in the cytoplasm of 3'dA-treated cells in association with the polyribosomes. In addition, the accumulation of labeled, nuclear adenovirus-specific RNA complementary to early regions 1a, 1b, and 2 of the adenovirus genome was approximately equal in 3'dA-treated and control cells. At the initial appearance of newly labeled adenovirus type 2 RNA (10 min) in the cytoplasm, there was one-half as much labeled RNA in 3'dA-treated cells as in the control. However, control cells accumulated additional mRNA in the cytoplasm very rapidly in the first 40 min of labeling, whereas the 3'dA-treated cells did not. Therefore, it appears that the correctly spliced, poly(A)- mRNA molecules that are labeled in the presence of 3'dA can be transported from the nucleus with nearly the same frequency and the same exit time as in control cells and can be translated in the cytoplasm but have a much shorter half-life than the poly(A)+ mRNA molecules from control infected cells. From these results it is suggested that the role of poly(A) may be entirely to increase the longevity of cytoplasmic mRNA.

Adenoviruses, Human↗
PubMed1985

Biochemical and electron microscopic studies of the transcription of vaccinia DNA by RNA polymerase from Escherichia coli: localization and characterization of transcriptional complexes.

We used the prokaryotic Escherichia coli RNA polymerase to determine if vaccinia DNA might provide recognition sites for the bacterial binding and initiation. Electron microscopic studies of the interaction of E. coli RNA polymerase with vaccinia DNA and molecular hybridization analysis of the transcription products formed after 3 or 5 min of in vitro incubation showed that: there were 30-40 sites on the template where the polymerase could bind and initiate cRNA synthesis; the entire coding capacity of the genome was utilized for cRNA synthesis; transcription was asymmetric; cRNA molecules were similar in size to the transcripts synthesized by the vaccinia virus RNA polymerase in vitro and in vivo; cRNA contains sequences in common with 'pre-early', 'early', and 'late' in vivo RNA; 'self-annealing' of cRNA in the presence or absence of RNA synthesized in vitro by the virion associated RNA polymerase showed that less than 1% dsRNA product could be detected suggesting that initially the same strand(s) was copied by the viral and bacterial enzymes; no differences in the frequency with which sequences represented in the Hind III fragments of vaccinia DNA were transcripted with time of in vitro incubation could be detected. These findings strongly suggest that the bacterial enzyme might recognize truly viral promotors. With extended in vitro incubations of the E. coli RNA polymerase with vaccinia DNA the control of transcription was found to diminish. This was correlated with an increase in the size of the transcripts and the synthesis of significant amounts of self-complementary RNA, indicating that symmetrical transcription was occurring. The dsRNA species recovered after self-annealing the cRNA from a 30 min in vitro reaction mixture were found to contain sequences which hybridized to some portion of all the Hind III restriction fragments of vaccinia DNA. The methods described here might be useful for the localization and characterization of promotor sequences in the genome of vaccinia virus, as well as for studies on sequence conservation between members of the Poxvirus genus.

Animals↗
PubMed1990

Combined use of immunocytochemistry and in situ hybridization to study beta thyroid-stimulating hormone gene expression in pituitaries of hypothyroid rats.

Immunocytochemistry and in situ hybridization were used to demonstrate beta thyroid-stimulating hormone (TSH) immunoreactivity and beta TSH messenger RNA (mRNA) in the same tissue section of rat pituitary. Sections (10 micronsP of 4% paraformaldehyde-fixed pituitaries from surgically thyroidectomized and intact male rats were first hybridized with a 32P-labelled complementary RNA beta TSH probe and were then immunostained for beta TSH. In both sets of animals beta TSH mRNA and beta TSH immunoreactivity were simultaneously localized to many thyrotrophs, although there was considerable heterogeneity in mRNA labelling intensity between individual cells. In hypothyroid rats more cells were positive with both probe and antiserum, and the intensity of mRNA labelling in most quantified by direct apposition of hybridized sections to autoradiography film, and the image grey levels were measured using an image analyser to convert them into equivalent amounts of radioactivity. In hypothyroid rats this amount was 22 times greater than in controls (control 4.77 +/- 0.84 attomoles mm-2; hypothyroid 107.06 +/- 14.2 attomoles mm-2; mean +/- SEM p less than 0.001). In situ hybridization has been used in combination with immunocytochemistry, to demonstrate beta TSH and mRNA and immunoreactivity at a cellular level in rats with different thyroid status.

Animals↗
PubMed1988

Interferon RNA of embryonic origin is expressed transiently during early pregnancy in the ewe.

Ovine trophoblast protein-1 (oTP-1), an interferon of embryonic origin, is produced during the peri-implantation period of early pregnancy. Secretion of oTP-1 is detectable between days 13 and 21, but not beyond. In this study, the levels of oTP-1 mRNA in embryos were analyzed to determine if they reflected the transient nature of oTP-1 production. Total cellular RNA (tcRNA) was isolated from embryos representing day 12 (n = 5), 14 (n = 7), 16 (n = 5), 18 (n = 6), 20 (n = 4), and 22 (n = 5) of pregnancy and spotted on nylon membranes. Complementary RNA was transcribed from a specific oTP-1 cDNA (550 base pairs) template and applied (16-1000 pg) to nylon membranes to develop a standard curve. The fixed RNA samples were then allowed to hybridize with the 32P-labeled oTP-1 cDNA. oTP-1 mRNA was not detectable on day 12, increased to high levels (3.6 +/- 1.6 ng/ug of embryo DNA) on day 14, decreased about 5-fold by day 16, 15-fold by day 18, 170-fold by day 20, and 200-fold by day 22 of pregnancy. At day 14 oTP-1 mRNA comprised 0.060 +/- 0.019% of the tcRNA and was more abundant than actin mRNA. Northern analyses of pooled tcRNA representing each day of pregnancy showed that the oTP-1 probe hybridized to a single class of mRNA (approximately 1.1 kilobases) and confirmed the results obtained with dot blots.

Actins↗
PubMed1997

The molecular anatomy of influenza virus RNA polymerase.

The genome of influenza virus is composed of eight RNA segments of negative polarity. The RNA-dependent RNA polymerase is associated with each viral RNA (vRNA) segment and in virus-infected cells, involved in both transcription, i.e. vRNA-directed synthesis of viral mRNA, and two step reactions of vRNA replication, i.e. vRNA-dependent synthesis of complementary RNA (cRNA) and cRNA-dependent synthesis of vRNA. The RNA polymerase is composed of three viral proteins, PB1, PB2 and PA. PB1 is the core subunit for not only the RNA synthesis but also the assembly of PB2 and PA into this multifunctional enzyme complex. PB1 alone is able to catalyze vRNA-dependent RNA synthesis, but PB2 is required for capped RNA-dependent transcription, both together forming the transcriptase. The third P protein, PA, and an as yet unidentified host factor(s) are involved in the conversion of RNA polymerase from transcriptase to replicase. The functional map is being made for both PB1 and PB2 proteins.

Animals↗
PubMed1993

cDNA cloning, characterization, and tissue-specific expression of human xanthine dehydrogenase/xanthine oxidase.

We isolated cDNAs encoding xanthine dehydrogenase (XD; xanthine:NAD+ oxidoreductase, EC 1.1.1.204) from a human liver cDNA library. The complete nucleotide sequence of human XD was determined; the deduced amino acid sequence encoded a protein of 1336 amino acid residues of M(r) 147,782. Human XD possessed many of the signature sequences typical of XDs from flies and rodents, including an unusual cysteine distribution, a potential 2Fe/2S binding site, and a putative molybdopterin cofactor binding domain. Analysis of potential NAD binding sites suggested a simple hypothesis for the conversion of human XD into the oxygen metabolite forming xanthine oxidase (XO; xanthine:oxygen oxidoreductase, EC 1.1.3.22). Using a human XD complementary RNA hybridization probe, we found a 5100-base RNA in human liver by RNA blot-hybridization analysis. This RNA exhibited tissue-specific distribution that may be pertinent to XD- and XO-mediated oxygen radical injury in ischemia/reperfusion and inflammation. A second 4500-base RNA was detected in some tissues and may arise through differential transcription termination.

Amino Acid Sequence↗
PubMed1976

Synthesis of RNA by mutants of vesicular stomatitis virus (Indiana serotype) and the ability of wild-type VSV New Jersey to complement the VSV Indiana ts G I-114 transcription defect.

The ability of certain vesicular stomatitis virus (VSV; Indiana serotype) temperature-sensitive (ts) mutants to synthesize intracellular viral complementary RNA (vcRNA) at permissive or nonpermissive temperatures for productive infections has been investigated. Mutants belonging to complementation groups II, III, and V synthesize RNA at nonpermissive temperature in amounts essentially equivalent to that obtained at permissive temperatures. Mutant ts G I-114 possesses a thermolabile transcriptase and does not synthesize vcRNA at 40 degrees C; however, mutants ts O I-5, O I-53, O I-78, and O I-80 possess thermostabile transcriptases that are capable of some vcRNA synthesis at 40 degrees C. All five group I mutants are defective in their secondary transcription ability at 40 degrees C. Wild-type VSV New Jersey virus is able to complement the transcription defect of ts G I-114 at 40 degrees C. This complementation is inhibited by puromycin, suggesting that a viral gene product of VSV New Jersey (e.g., its transcriptase or a transcriptase component) is involved. Mokola virus is not able to complement the ts G I-114 defect, although Mokola does synthesize vcRNA in infected cells (in the presence or absence of cycloheximide).

Cycloheximide↗