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Regional variations in contractile responses to prostaglandins and prostanoid receptor messenger ribonucleic acid in pregnant baboon uterus.

OBJECTIVES: The aims of this study were to compare (1) the contractile responses of the lower uterine segment and fundus to prostaglandins, (2) expression of genes encoding prostanoid receptors in myo-metrium from different regions of the uterus, and (3) the distribution of expression of genes encoding prostanoid receptors (P receptors) in key intrauterine tissues. STUDY DESIGN: Cesarean hysterectomy was performed in 8 pregnant baboons, not in labor, in the last third of pregnancy. Contractile responses of fresh tissue were quantified in a superfusion system. Polyadenylated ribonucleic acid was extracted from frozen tissue and gene expression was quantified by Northern blot analysis with complementary deoxyribonucleic acid probes. RESULTS: Prostaglandin E2 contracted strips of myometrium from the fundus but had no significant effect on strips from lower uterine segment. Prostaglandin F2 alpha contracted myometrium from both regions equally. Compared with fundus tissue, lower uterine segment tissue had greater expression of EP2 receptor messenger ribonucleic acid, less expression of EP3 receptor messenger ribonucleic acid, but similar levels of EP4 receptor and FP receptor messenger ribonucleic acid. EP2 receptor, EP3 receptor, and EP4 receptor messenger ribonucleic acids were also detected in cervix, decidua, and chorion. EP2 receptor messenger ribonucleic acid was most abundant in the cervix, EP3 receptor messenger ribonucleic acid was most abundant in the myometrium, and EP4 receptor messenger ribonucleic acid was most abundant in the decidua. CONCLUSIONS: The reduced contractile response of lower uterine segment tissue to prostaglandin E2 is paralleled by greater inhibitory EP2 receptor expression and less contractile EP3 receptor expression, a pattern similar to that seen in the cervix. Drugs with selective activity at prostanoid receptor types and subtypes are likely to allow safer and more effective control of the uterus and cervix than native prostaglandins.

Animals↗

Evidence for messenger ribonucleic acid of an ammonium-inducible glutamate dehydrogenase and synthesis, covalent modification, and degradation of enzyme subunits in uninduced Chlorella sorokiniana cells.

The cells of Chlorella sorokiniana cultured in nitrate medium contain no detectable catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH). However, several lines of experimental evidence indicated that the NADP-GDH messenger ribonucleic acid was present at high levels and was being translated in uninduced cells. First, binding studies with 125I-labeled anti-NADP-GDH immunoglobulin G and total polysomes isolated from uninduced and induced cells showed that NADP-GDH subunits were being synthesized on polysomes from both types of cells. Second, when polyadenylic acid-containing ribonucleic acid was extracted from polysomes from uninduced and induced cells and placed into a messenger ribonucleic acid-dependent in vitro translation system, NADP-GDH subunits were synthesized from the ribonucleic acid from both sources. Third, when ammonia was added to uninduced cells, NADP-GDH antigen accumulated without an apparent induction lag. Fourth, by use of a specific immunoprecipitation procedure coupled to pulse-chase studies with [35S]sulfate, it was shown that the NADP-GDH subunits are rapidly synthesized, covalently modified, and then degraded in uninduced cells.

Ammonium Sulfate↗

Expression and regulation of the messenger ribonucleic acid encoding the prostaglandin F(2alpha) receptor in the rat myometrium during pregnancy and labor.

OBJECTIVE: We examined expression of messenger ribonucleic acid encoding the prostaglandin F(2alpha) receptor in the rat myometrium throughout late gestation and its regulation by progesterone and mechanical stretch. STUDY DESIGN: Normal pregnant rats were killed on gestational day 15, 22, or 23 (during labor) or 1 day post partum. The effects of progesterone on prostaglandin F(2alpha) receptor messenger ribonucleic acid levels were investigated by daily injections of progesterone (4 mg) from day 20 of normal gestation or from day 17 in rats bilaterally ovariectomized on day 17. To investigate the effects of myometrial stretch, unilaterally pregnant rats underwent either sham surgery or placement of a polyvinyl tube 3 mm in diameter in the nongravid uterine horn on day 15 or 18 and were killed 5 days later. Prostaglandin F(2alpha) receptor messenger ribonucleic acid levels in the myometrium were determined by a semiquantitative reverse transcription-polymerase chain reaction method. RESULTS: Myometrial prostaglandin F(2alpha) receptor messenger ribonucleic acid levels significantly increased during both term and ovariectomy-induced preterm labor. This increase was blocked by progesterone. In rats with unilateral pregnancies prostaglandin F(2alpha) receptor messenger ribonucleic acid levels in the nongravid horns were similar to those in the contralateral gravid horns on day 20 and during labor regardless of whether they were stretched by a 3-mm tube. CONCLUSION: Increased myometrial expression of prostaglandin F(2alpha) receptor messenger ribonucleic acid during term and preterm labor is temporally associated with progesterone withdrawal but is not dependent on mechanical stretch of the myometrium.

Animals↗

The binding of transfer ribonucleic acids to 5 S and 5.8 S eukaryotic ribosomal ribonucleic acid-protein complexes.

Rat liver 5 S and 5.8 S rRNAs were oxidized with periodate and the 3' termini were coupled to Sepharose 4B through an adipic acid dihydrazide spacer. Ribosomal proteins were passed through the nucleic acid affinity columns to form ribonucleoprotein complexes containing the nucleic acid and the proteins that bind to it (5 S . L6, L7, L19; and 5.8 S . L6, L19, S9, S13). Pure isoaccepting species of yeast initiator-tRNA (tRNAfMet) and elongator-tRNAs (tRNAmMet, and tRNAPhe) were chromatographed on the ribosomal ribonucleoprotein affinity columns. The three rRNAs were bound to the 5 S and 5.8 S ribosomal ribonucleoprotein complexes. The elongation and initiation ternary complexes, EF-1 alpha . GTP . Phe-tRNAPhe, and eIF-2 . GTP . Met-tRNAfMet, also were bound to both ribosomal ribonucleoprotein affinity columns, whereas the binary complex EF-1 alpha . GTP and puromycin were not.

Animals↗

Presence of ribonucleic acid in human spermatozoa: differences in content between normal and abnormal spermatozoa.

OBJECTIVE: Our purpose was to determine whether there are any differences in total ribonucleic acid content between normal and abnormal human spermatozoa. STUDY DESIGN: Spermatozoa were obtained from men undergoing routine semen analysis at a university-based reproductive genetics laboratory. Specimens were classified as normal or abnormal according to World Health Organization criteria. Total ribonucleic acid was removed by acid-phenol extraction, and ribonucleic acid expression levels were determined by spectrophotometric analysis. RESULTS: Abnormal spermatozoa were found to have significantly more ribonucleic acid (0.14 +/- 0.02 mg/10(6) spermatozoa) than normal spermatozoa (0.05 +/- 0.01 mg/10(6) spermatozoa; P <.001). CONCLUSION: Ribonucleic acid content is significantly altered in abnormal spermatozoa, and this alteration may be the result of some defect in the posttranscriptional pathway.

Humans↗

Ribonucleic acid biosynthesis in adult and infant rat brain in vitro.

The rate of biosynthesis of ribonucleic acid (as judged by the rate of incorporation of uridine into ribonucleic acid) in infant and adullt rat brain cortex slices, incubated (aerobically in tile presenice of various substrates, is directly proportionial to the adenosine triphosphate concentration. This suggests that the adenosine triphosphate concentration is one of the factors involved in the control of ribonucleic acid biosynthesis in infant and adult rat brain. Acetoacetate or beta-hydroxybutyrate is about 70 percent as effective as glucose, with both infant and adult brain, for the promotion of ribonucleic acid biosynthesis. but they are considerably mnore effective than succinate in infant brain than in adult brain.

Acetoacetates↗

Genetic mapping of a linked cluster of ribosomal ribonucleic acid genes in Bacillus subtilis.

A ribosomal ribonucleic acid gene set consisting of genes for 16S, 23S, 5S, and 4S ribonucleic acid species has been genetically mapped to a position between the markers recG13 and abrB74 on the Bacillus subtilis chromosome and designated rrnA. A ribosomal mutation, ksgA, was found to be linked to rrnA. This places rrnA in a region of the chromosome where ribosome-related genes occur but that is not directly adjacent to the major cluster of ribosome-related markers.

Bacillus subtilis↗

Isolation and characterization of a double-stranded ribonucleic acid from Penicillium chrysogenum.

Double-stranded ribonucleic acid has been obtained from cells of the fungus Penicillium chrysogenum. This ribonucleic acid appears to be associated with mycophage and is an efficient inducer of interferon Its. extraction and partial purification are discussed, and evidence for its double-stranded and ribosidal nature is reviewed. The implications of viral nucleic acid in the life processes of fungi are considered.

Animals↗

Distinctive ribonucleic acid patterns of human rotavirus subgroups 1 and 2.

The ribonucleic acid migration patterns of 7 subgroup 1 and 16 subgroup 2 human rotaviruses recovered from four geographic areas were compared. The subgroup 1 ribonucleic acid patterns had strikingly slower-moving segments 10 and 11, suggesting a correlation between the ribonucleic acid pattern and the subgroup specificity.

Electrophoresis, Polyacrylamide Gel↗

Separation of isoacceptor cysteine transfer ribonucleic acids of bakers' yeasts.

Isoacceptor species of certain amino acid-specific transfer ribonucleic acids (tRNAs) were fractionated by gel permeation chromatography using Sephadex G-100. The separation is attributed to the 20% ethanol-1% NaCl solvent and to the characteristics of Sephadex. Isoacceptor tRNAs specific for cysteine, arginine, phenylalanine, and histidine were recovered from commercial tRNA of yeast by this method. Highly purified cysteine-specific tRNA, obtained by a method which would not be expected to separate isoacceptor molecules when fractionated by this procedure, was shown to contain two cysteine isoacceptor tRNAs.

Amino Acyl-tRNA Synthetases↗

Diffusion-enhanced energy transfer shows accessibility of ribonucleic acid polymerase inhibitor binding sites.

Rifamycin and Cibacron Blue F3GA are powerful inhibitors of Escherichia coli deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase. In addition, both inhibitors strongly absorb visible light, making them suitable for use as acceptors in energy-transfer experiments. Transfer of energy to these acceptors from freely diffusing energy donors with long excited-state lifetimes (approximately 10(-3) s) depends strongly on whether donor and acceptor can make direct intermolecular contact. We observe that the rate constant for energy transfer from a small terbium chelate to enzyme-bound rifamycin is 1 X 10(7) M-1 s-1, which is about half as large as the rate constant observed for free rifamycin in solution. This relatively small change upon binding indicates that enzyme-bound rifamycin is highly accessible to small molecules in the solvent. In the case of Cibacron Blue, under conditions where approximately 90% of this inhibitor is bound to RNA polymerase, the small amount of unbound inhibitor accounts for practically all of the observed energy transfer. This implies that enzyme-bound Cibacron Blue is relatively inaccessible to energy donors in the solution. The dependence of energy transfer on the accessibility of the acceptor is illustrated by using simple geometric models. synthesis of a stable, electrically neutral terbium chelate which can be efficiently excited with UV radiation is also described.

Anthracenes↗

Differential inhibition of various deoxyribonucleic and ribonucleic acid polymerases by suramin.

The inhibitory effects of hexasodium sym-bis(m-aminobenzoyl-m-amino-p-methylbenzoyl-1-naphthylamino-4,6, 8-trisulfonate)carbamide (trivial name: suramin) on the activities of various deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) polymerases from mammalian cells, bacteria and retrovirus were examined and compared with each other. Among the various DNA and RNA polymerases tested, the activities of DNA primase, DNA polymerase alpha, reverse transcriptase and Escherichia coli RNA polymerase were strongly inhibited by suramin, while the activities of other enzymes including DNA polymerases beta and gamma, terminal deoxynucleotidyl-transferase and DNA polymerase I were relatively resistant to inhibition by this drug. The inhibition by suramin of DNA polymerase alpha from KB cells and Rauscher murine leukemia virus (RLV) reverse transcriptase was due to competition with the respective template primer (activated DNA for alpha polymerase and (rA)n.(dT)12-18 for reverse transcriptase) for the template.primer-binding site of the enzyme, while the inhibition of DNA primase and E.coli RNA polymerase was due to competition with the ribonucleoside triphosphate substrate. The inhibition constants (Ki) of suramin were determined to be 2.6 microM, 0.35 microM, 0.54 microM and 0.70 microM for DNA primase, DNA polymerase alpha, RLV reverse transcriptase and E. coli RNA polymerase respectively. The observed inhibitions of these polynucleotide-synthesizing enzymes by suramin seem to explain, at least in part, an as yet unknown mechanism of trypanocidal action of this drug.

DNA Primase↗

Genetic and physical studies of lambda transducing bacteriophage carrying the beta subunit gene of the Escherichia coli ribonucleic acid polymerase.

The prophage lambdac1857 was inserted into the bfe gene located near rif (the structural gene for the beta subunit of deoxyribonucleic acid [DNA]-dependent ribonucleic acid polymerase) on the Escherichia coli chromosome. Induced lysates (low-frequency transducing lysates) of such a lysogen contained defective lambda phage particles (lambdadrif+) that can specifically transduce the wild-type rif+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harboring both the wild-type and the mutant rif genes were isolated. Rec+ derivatives of these heterogenotes produce high-frequency transducing lysates that contain lambdadrif+ and normal active phages at a ratio of 1 to 2. The results of marker rescue experiments and of density determination with several transducing phages indicate that most of the late genes are deleted and replaced by a segment of the chromosomal DNA carrying the bfe-rif region. The length of the chromosomal segment seems to vary between approximately 0.5 and 0.6% of the total bacterial DNA among the three independently isolated lambdadrif+ phages. Electron microscopy of heteroduplex DNA consisting of one strand from lambdadrif+-6 and the other from lambdaimm-21 phages directly confirmed that most of the phage DNA of the "left arm" was replaced by the bacterial DNA. The heteroduplex study also demonstrated that the integration of prophage lambda into the bfe region occurred at the normal cross-over point within the phage attachment site.

Centrifugation, Density Gradient↗

Preparations and properties of ribonucleic acid polymerase from Acinetobacter calcoaceticus.

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) from Acinetobacter calcoaceticus was purified to apparent homogeneity and its properties were compared with those of the Escherichia coli B enzyme. The molecular weights of the two native active enzymes as well as their alpha and beta subunits appeared to be similar. No subunit corresponding to that of sigma from E. coli was found, and furthermore no separation between the beta subunits could be detected by gel electrophoresis. A number of different DNAs were transcribed by the enzyme from A. calcoaceticus. Maximal RNA synthesis occurred at pH 8.7, 10 mM Mg2+, or 0.3 mM Mn2+ and at a total ionic strength of 0.1. Higher ionic strengths led to increasing inhibition of transcription and at mu = 0.4 complete inhibition was observed. The mechanism of inhibition of salt was not related to the initiation event as observed with T4 core RNA polymerase (R.Kleppe, 1975). In an attempt to understand the mechanism of inhibition by salt, the effect of ionic strength on the sedimentation properties of the enzyme was investigated. At low ionic strength, enzyme species with sedimentation coefficients, s20,w, of 5.8S, 12.4S, and 19.3S were present. In buffers with higher ionic strengths the relative amounts of the 12.4S species decreased. It is suggested, therefore, that the inhibition of activity at higher salt concentrations is caused by a decrease in concentration of the active enzyme species.

Acinetobacter↗

Inhibition of bacterial conjugation by ribonucleic acid and deoxyribonucleic acid male-specific bacteriophages.

Both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) male-specific phages, with an F-specific host range, inhibited the bacterial mating process of Escherichia coli. DNA phages prevented the formation of mating pairs but had no effect on mating pairs once they were formed. A step in RNA phage infection, prior to RNA penetration, prevented the formation of mating pairs and, in addition, prevented a fraction of existing mating pairs from completing the mating process. These findings are compatible with the hypothesis that donor cells have a single surface structure involved in both conjugation and male-phage adsorption and that this element is the F pilus.

Binding Sites↗

Isolation of phi X174 specific messenger ribonucleic acids in vivo and identification of their 5' terminal nucleotides.

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) hybridization in formamide at low temperature was applied to hybridization of PhiX174 replicative form DNA and in vivo PhiX174 specific messenger RNA (mRNA) with some modification. We found that PhiX174 mRNA up to molecular weight 1.2 x 10(6) could be hybridized to and eluted from DNA without apparent breakage of phosphodiester bonds and the 5' terminal guanosine triphosphate and adenosine triphosphate of the RNA. By alkali hydrolysis of the purified in vivo PhiX174 mRNA and subsequent thin-layer chromatography of the digest, we isolated the 5' terminal nucleotides and identified them as 2'- or 3'-monophosphate guanosine 5'-triphosphate (pppGp) and 2'- or 3'-monophosphate adenosine 5'-triphosphate (pppAp). By comparing the in vitro and in vivo synthesized PhiX174 mRNA, a difference in the pppAp-pppGp ratio was observed. In the in vitro RNA, this ratio was 1.5, whereas in the in vivo RNA it was 5.5.

Adenosine Monophosphate↗