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PubMed2001

Quantitation of hepatitis C virus RNA in saliva and serum of patients coinfected with HCV and human immunodeficiency virus.

The presence and the quantity of hepatitis C virus (HCV) RNA were investigated in saliva and serum of patients infected with both HCV and human immunodeficiency virus (HIV). Paired serum and saliva samples were collected from 59 HIV-HCV coinfected patients. HCV RNA was detected by nested-PCR, using primers derived from the 5' non-coding region of HCV, and positive results were quantified using the b-DNA method. HCV RNA was detected in the saliva of 22/59 (37.3%) patients, with a mean level of 1.15 x 10(6) genome equivalents/ml; there was no correlation of salivary positivity with immune status (CD4 cell count), age or HIV risk group, but there was with gender (19/38 [50%] positive results in male, compared to 3/21 [14.3%] in female, P = 0.007). HCV RNA was detected in the serum of 45/59 (76.3%) patients at a higher level (mean of 2.52 x 10(7) genome equivalents/ml) compared to saliva. Positivity was not correlated with age, gender or CD4 + cell count. There was a correlation between qualitative saliva and serum results (P = 0.003), but not between quantifications (P = 0.57). This first study reporting significant amounts of HCV RNA in saliva could have important implications for HCV epidemiology.

Adult↗

PubMed1977

Cell-free modulation of proinsulin synthesis.

In vivo, glucose preferentially stimulates proinsulin biosynthesis; at least part of this process is independent of new RNA synthesis and is accompanied by increases in the overall rate of polypeptide chain initiation. The cell-free translation of proinsulin messenger RNA is very sensitive to changes in the protein-synthesizing system. Proinsulin synthesis is preferentially inhibited by the addition of increasing quantities of polyadenylate-containing RNA from the fetal bovine pancreas or by the addition of the drug, aurintricarboxylic acid, which blocks polypeptide chain initiation. These results suggest that proinsulin messenger RNA completes less efficiently for rate controlling initiation factors. We propose that glucose stimulates proinsulin biosynthesis by allowing the less competitive proinsulin messenger RNA to be translated more efficiently.

Aurintricarboxylic Acid↗

PubMed1976

Actions of aldosterone on polyadenylated ribonucleic acid and Na+ transport in the toad bladder.

Polyadenylated ribonucleic acid [poly(A)(+)-RNA] has been isolated from the cytoplasm of the epithelium of the urinary bladder of the toad (Bufo marinus) by oligo-(deoxythymidylate)cellulose chromatography. Aldosterone increased the incorporation of [3H]uridine, and of [3H]uridine and [3H]adenosine (given in combination) into 7S, 12S, and 18S poly(A)(+)-RNA during the first 30 min of the action of the hormone, as defined by either a pulse or pulse-chase sequence. The quantity of cytoplasmic poly(A)(+)-RNA that hybridized to [3H]poly(uridine) was also increased by aldosterone. These results are consistent with the inference of induction of messenger RNA synthesis. This effect was most marked during the first 30 min of the action of the hormone.

Aldosterone↗

PubMed1973

Messenger RNA for hepatic tryptophan oxygenase: its partial purification, its translation in a heterologous cell-free system, and its control by glucocorticoid hormones.

Messenger RNA from rat liver was partially purified by chromatography on cellulose on the basis of its poly(A) content. Microgram amounts of this RNA stimulate protein synthesis manyfold in a heterologous cell-free system, derived from Krebs ascites cells supplemented with reticulocyte initiation factors. The messenger RNA directs the initiation, synthesis, and release of a product that was identified as complete subunits of hepatic tryptophan oxygenase (EC 1.13.1.12) by immunoprecipitation with monovalent antibodies prepared against homogeneous tryptophan oxygenase and subsequent sodium dodecyl sulfate-polyacrylamide electrophoresis of the solubilized immunoprecipitate. This may represent the first complete translation in a heterologous system of a mammalian messenger RNA coding for an enzyme protein. Analysis of the messenger RNA content of the liver after glucocorticoid administration demonstrates that the hormonally enhanced rate of synthesis of tryptophan oxygenase is accompanied by an increased quantity of its corresponding messenger RNA.

Animals↗

PubMed1977

Studies of human histone messenger RNA. I. Methods for the isolation and partial characterization of RNA fractions containing human histone message from HeLa S3 polyribosomes.

Large quantities of nonpolyadenylated [poly(A(-))] 4 to 18 S RNA were isolated from the polyribosomes of S phase HeLa S3 cells and were fractionated into multiple discrete RNA components by continuous elution preparative electrophoresis. Previous studies have shown that treatment os S phase HeLa cells with cytosine arabinoside inhibits DNA replication and causes translatable histone messenger RNA (mRNA) species to disappear from cytoplasmic polyribosomes (Borun, T. W., Scharff, M.D., and Robbins, E. (1967) Proc. Natl. Acad. Sci. U.S.A. 58, 1977-1983; Gallwitz, D., and Mueller, G. C. (1969) J. Biol. Chem. 244, 5948-5952; Borun, T. W., Gabrielli, F., Ajiro, K., Zweidler, A., and Baglioni, C. (1975) Cell 4, 59-67; Gallwitz, D. (1975) Nature 257, 247-248). In the present study it was found that cytosine arabinoside treatment does not appreciably affect major 7.5 to 8 S RNA species but does cause the disappearance of 8.6 to 13 S RNA components from preparative electrophoresis elution profiles of S phase polyribosomal 4 to 18 S RNA. Base ratio analysis of the 8.6 to 13 S putative histone mRNA species indicates that they are GC-rich but not like the HeLa 18 or 28 S rRNA in base composition.

Chromatography, Affinity↗

PubMed2004

Estimation of ribosomal RNA transcription rate in situ.

Traditionally, the rate of transcription is measured by metabolic labeling (e.g., the run-on assay), which can be carried out only in isolated or cultured cells. It has been difficult if not impossible to assess the rate of transcription of a gene in a specific cell type in situ. We show here that the quantity of 47S precursor ribosomal RNA (pre-rRNA), which correlates positively with the rate of rRNA transcription as measured by the run-on assay, can serve as an indicator for the rate of its transcription. We adopted this method as an in situ hybridization procedure to demonstrate its validity in vivo. The notion of using the quantity of the primary transcript as an indicator of the rate of transcription has the potential application in monitoring the rate of messenger RNA transcription in single cells within a tissue of complex cellular composition.

Animals↗

PubMed2000

Extraction of total RNA from a high pigment content plant. Marigold (Tagetes erecta).

The Mexican marigold (Tagetes erecta) produces inflorescences of intense yellow color that contain high levels of xanthophylls, particularly lutein, which makes it a suitable model for the study of carotenoid biosynthesis and regulation throughout the development of the inflorescences. However, these studies require the recovery of total RNA from floral buds and inflorescences at different developmental stages, each of which presents specific extraction problems. Four protocols were tested, but only through the modification of one of them was it possible to obtain total RNA of sufficient quality and quantity to perform RT-PCR and Northern blots and to construct a cDNA library. This article presents the modified protocol for the recovery of total RNA from carotenoid-rich plant tissues.

Asteraceae↗

PubMed2002

Association of plasma viral RNA load with prognosis in cats naturally infected with feline immunodeficiency virus.

We measured the quantity of plasma feline immunodeficiency virus (FIV) RNA using a real-time sequence detecting system. Plasma viral RNA load was shown to correlate with the clinical stage, survival time, and disease progression in naturally FIV-infected cats. The present study indicates that the plasma viral RNA load can be used as a clinical marker representing the impairment of the immune system and predicting the clinical outcome in FIV-infected cats.

Animals↗

PubMedDate not supplied

Serum HIV-1 RNA load to predict CD4+ T-cell depletion in asymptomatic patients.

The temporal association between the increase in viral replication and the depletion in CD4+ T cells in HIV-1 infection is not yet clear. To investigate this phenomenon HIV-1 RNA was quantified in several frozen sera from 20 asymptomatic HIV-1 infected patients in the 2 years preceding CD4+ T cell depletion of 50% or more, and compared with 20 HIV-1 infected paired patients who were stable in the same period. In each group, no statistically significant variation in the mean HIV-1 RNA titre was found between the last checkup and the one 24 months earlier. The mean HIV-1 RNA titre was 10(3.86) copies/ml in the non-progressor group and 10(5.12) copies/ml in the progressor group. These data support the view that the quantity of circulating HIV-1 RNA is an early predictor of disease progression that is relatively constant during the asymptomatic period of HIV-1 infection.

Adult↗

PubMed1982

Increased amounts of double-stranded RNA in the cytoplasm of rat liver following treatment with carcinogens.

Cytoplasmic RNA was isolated from control rats or at 48 hr following carcinogen administration. A significant quantity (0.6%) of double-stranded RNA was present in control preparations, as assessed by digestion with RNase III. Following carcinogen treatment of rats, this percentage increased significantly. The results suggest that the altered transport of repetitive sequences which is induced by carcinogen treatment involves alterations in the metabolism of double-stranded RNA sequences.

2-Acetylaminofluorene↗

PubMed1984

Mitochondrial ribosomal RNA genes of yeast: their mutations and a common nuclear suppressor.

Due to the absence of repetition of the rRNA genes in S. cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system. We describe here a novel thermosensitive mutation, ts1297, localized by rho- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene. Defective mutations of the small rRNA have not been reported so far. In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced. The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal. However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca2+ in place of Mg2+. The 50S particles seem to be completely degraded under normal conditions of extraction with Mg2+. The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU101. The SU101 mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene. These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits.

Cell Nucleus↗

PubMed1992

Correlation between the serum level of hepatitis C virus RNA and disease activities in acute and chronic hepatitis C.

The influence of viremia on hepatic injury in patients infected with hepatitis C virus was examined by analysis of the relationship between alanine aminotransferase activity and the amount of hepatitis C virus RNA in sequential serum samples from I untreated patient with acute hepatitis C and 3 untreated patients with chronic hepatitis C. Semiquantitative analysis by the competitive-reverse-transcription/polymerase-chain-reaction method indicated that the quantity of hepatitis C virus RNA in the serum affected the disease activities of acute and chronic hepatitis C through their natural clinical courses in all these patients. The nucleotide sequence encoding the putative envelope region of the viral genome in the patient with acute hepatitis C was examined. Blood samples taken serially at 2 times of exacerbation of the hepatitis revealed 2 nucleotide mutations, resulting in changes of predicted amino acid residues. This finding suggests that nucleotide mutations in the envelope region of the viral genome may be responsible for the recurrent hepatic injury attributed to recurrence of viremia in patients with hepatitis C. From these aspects, the serial divergence of the virus genome in infected individuals, especially in the region encoding the viral envelope protein, may possibly play an important role in developing chronic infection of hepatitis C virus.

Amino Acid Sequence↗

PubMed1961

The kinetics of the synthesis of ribosomal RNA in E. coli.

The kinetics of the synthesis of ribosomal RNA in E. coli has been studied using C(14)-uracil as tracer. Two fractions of RNA having sedimentation constants between 4 and 8S have kinetic behavior consistent with roles of precursors. The first consists of a very small proportion of the RNA found in the 100,000 g supernatant after ribosomes have been removed. It has been separated from the soluble RNA present in much larger quantities by chromatography on DEAE-cellulose columns. The size and magnitude of flow through this fraction are consistent with it being precursor to a large part of the ribosomal RNA.A fraction of ribosomal RNA of similar size is also found in the ribosomes. This fraction is 5 to 10 per cent of the total ribosomal RNA and a much higher proportion of the RNA of the 20S and 30S ribosomes present in the cell extract. The rate of incorporation of label into this fraction and into the main fractions of ribosomal RNA of 18S and 28S suggests that the small molecules are the precursors of the large molecules. Measurements of the rate of labeling of the 20, 30, and 50S ribosomes made at corresponding times indicate that ribosome synthesis occurs by concurrent conversion of small to large molecules of RNA and small to large ribosomes.

Escherichia coli↗

PubMed1995

Dilated cardiomyopathy associated with hepatitis C virus infection.

BACKGROUND: Myocarditis is thought to be commonly caused by various viruses, and accumulating evidence links viral myocarditis with the eventual development of dilated cardiomyopathy. In many cases, however, the evidence is only circumstantial, and direct conclusive proof is not available. Polymerase chain reaction (PCR) has been used to detect enterovirus RNA in myocardial tissue, but the wide discrepancy in results emphasizes the need for further study. METHODS AND RESULTS: We investigated hepatitis C virus infection in patients with dilated cardiomyopathy. The presence, type, and quantity of hepatitis C virus RNA were evaluated in the sera, and the presence of positive and negative strands of hepatitis C virus RNA in the heart was investigated with the PCR technique. Anti-hepatitis C virus antibody was present in the sera of 6 of 36 patients (16.7%) with dilated cardiomyopathy and in 1 of 40 patients (2.5%) with ischemic heart disease, showing a statistically significant (P < .05) difference. At an earlier time, acute myocarditis was suspected in 3 patients who had developed acute onset of heart failure, and the diagnosis was confirmed by endomyocardial biopsy in 1 patient. Hepatitis C virus RNA was present in the sera of 4 of the 6 patients, and all 4 had hepatitis C virus type II. The copy number of hepatitis C virus RNA in the serum was 8 x 10(2) to 2 x 10(3) genomes per 1 mL serum. Positive strands of hepatitis C virus were found in the hearts of 3 patients, and negative strands of hepatitis C virus were detected in the heart of 1 patient. CONCLUSIONS: The results suggest that hepatitis C virus infection is frequently found in patients with dilated cardiomyopathy and that hepatitis C virus is an important causal agent in the pathogenesis of the disease. Antiviral therapy against hepatitis C virus may be indicated in these patients.

Adult↗

PubMed2000

Development of a semi-quantitative assay to detect full-length CYP2C19 RNA.

Cytochrome P450 2C19 (CYP2C19) is the enzyme responsible for the metabolism of a number of drugs, including anticonvulsants and antidepressants. We have developed a semi-quantitative competitive RT-PCR assay to estimate the degree of expression of the full-length CYP2C19 message. This assay used a known quantity of internally deleted CYP2C19 RNA to quantitate the RT-PCR products of the CYP2C19 transcript in the RNA extracted from tissues. We determined that this method is sensitive and reproducible in assaying for CYP2C19 RNA in human livers. The lowest detectable amount of competitor RNA was 0.166 fg or 270 copies of CYP2C19 competitor RNA. Using human liver samples containing 3-23 x 10(5) copies of CYP2C19 RNA, we found the assay to be reproducible with a coefficient of intra- and interday variation of 11% and 20%, respectively. Using this assay, we measured full-length CYP2C19 RNA in 10 human livers. We found the CYP2C19 transcripts range from 0.1-23 x 10(5) copies/microgram liver total RNA. The analysis of CYP2C19 transcripts for liver of *1 and *2 genotypes, based on restriction enzyme digest analysis of RT-PCR products, suggests that only normal (*1), not the variant (*2) copy of full-length CYP2C19 RNA, was detectable in these livers. We report for the first time the quantification of full-length CYP2C19 RNA in livers.

Alleles↗

PubMed1983

Preliminary characterization of coxsackievirus B3 temperature-sensitive mutants.

Prototype temperature-sensitive (ts) mutants of a coxsackievirus B3 parent virus capable of replication to similar levels at 34 or 39.5 degrees C were examined for the nature of the temperature-sensitive event restricting replication in HeLa cells at 39.5 degrees C. The ts mutant prototypes represented three different non-overlapping complementation groups. The ts1 mutant (complementation group III) synthesized less than 1% of the infectious genomic RNA synthesized by the coxsackievirus B3 parent virus at 39.5 degrees C and was designated an RNA- mutant. Agarose gel analysis of glyoxal-treated RNA from cells inoculated with ts1 virus revealed that cell RNA synthesis continued in the presence of synthesis of the small amount of viral RNA. This mutant was comparatively ineffective in inducing cell cytopathology and in directing synthesis of viral polypeptides, likely due to the paucity of nascent genomes for translation. The ts5 mutant (complementation group II) directed synthesis of appreciable quantities of both viral genomes (RNA+) and capsid polypeptides; however, assembly of these products into virions occurred at a low frequency, and virions assembled at 39.5 degrees C were highly unstable at that temperature. Shift-down experiments with ts5-inoculated cells showed that capsid precursor materials synthesized at 39.5 degrees C can, after shift to 34 degrees C, be incorporated into ts5 virions. We suggest that the temperature-sensitive defect in this prototype is in the synthesis of one of the capsid polypeptides that cannot renature into the correct configuration required for stability in the capsid at 39.5 degrees C. The ts11 mutant (complementation group I) also synthesized appreciable amounts of viral genomes (RNA+) and viral polypeptides at 39.5 degrees C. Assembly of ts11 virions at 39.5 degrees C occurred at a low frequency, and the stability of these virions at 39.5 degrees C was similar to that of the parent coxsackievirus B3 virions. The temperature-sensitive defect in the ts11 prototype is apparently in assembly. The differences in biochemical properties of the three prototype ts mutants at temperatures above 34 degrees C may ultimately offer insight into the differences in pathogenicity observed in neonatal mice for the three prototype ts mutants.

Cytopathogenic Effect, Viral↗

PubMed1990

Estimation of cellular DNA content in cell lysates suitable for RNA isolation.

Methods presently available for the isolation of RNA are incompatible with conditions necessary for the measurement of either DNA or cell number, resulting in infrequent quantitation of messenger RNA in relation to the quantity of cells studied. In the present studies, a microfluorometric method has been modified from previous techniques to permit the quantitation of DNA in cell lysates prepared using an acid guanidinium thiocyanate-phenol (AGTP) solution from which RNA can subsequently be isolated. The lysate is incubated in alkaline EDTA, then neutralized with KH2PO4, followed by the addition of the fluorochrome bisbenzimidazole (Hoechst 33258), and measurement of fluorescence. DNA content is comparable in measurements by the present technique and by the diphenylamine method on parallel samples. DNA content per cell for human cells measured with this technique is comparable to that previously reported using other methods. The use of AGTP solution results in stability of measurable DNA in cell lysates for periods of at least 10 weeks (permitting batching of samples and retrospective measurement) and stability of fluorescence for at least 20 h after the addition of bisbenzimidazole making the timing of fluorescence measurement less critical. The technique described should permit quantitation of messenger RNA in relation to DNA (and hence indirectly to cell number) on a routine basis.

Benzimidazoles↗

PubMed1976

Neurospora crassa cytoplasmic ribosomes; ribosomal ribonucleic acid synthesis in the wild type.

The biosynthesis of ribosomal ribonucleic acid (rRNA) In wild-type Neurospora crassa growing at 25 degrees C was investigated by continuous-labeling and pulsechase experiments using [5-3H]uridine. The results of these experiments suggest the following precursor-product relationships: the first RNA molecule to be synthesized in significant quantities is the 2.4 X 10(6)-dalton (2.4-Mdal) ribosomal precursor RNA. This RNA is cleaved to produce two species of RNA with weights of 0.7 and 1.4-Mdal. The former is the mature 17S rRNA of the 37S ribosomal subunit. The 1.4-Mdal RNA is subsequently cleaved to produce the mature 1.27-Mdal (25S) and 61,000-dalton (5.8S) rRNA's of the 60S ribosomal subunit. In the maturation process, approximately 15 to 20% of the 2.4-Mdal ribosomal precursor rRNA molecule is lost. As in other eukaryotes that have been examined, 5S rRNA is not derived from this precursor molecule.

Kinetics↗