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PubMed2004

Detection of RNA in purified cytomegalovirus virions.

Five viral RNA transcripts have recently been detected in purified virions of human cytomegalovirus (HCMV) strain AD169, a well-characterized member of the family Herpesviridae [Science 288 (2000) 2373]. While the function of these transcripts and/or the proteins they encode remains to be elucidated, it is not known whether these transcripts are unique to strain AD169 or are present in other HCMV strains. The objective of this study was to determine if these RNAs are present in other HCMV laboratory strains (Towne and Davis), and a low passage clinical isolate (CL203). These strains of CMV were purified by sequential ultracentrifugation through 20% D-sorbitol and glycerol-potassium tartarate gradients and the morphology and infectivity of the virions confirmed by electron microscopy and inoculation into cell culture. When RNA extracted from the purified virions was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) the UL 21.5 and TRL/IRL 2-5 transcripts were detected in virions of HCMV strains AD169, Davis, Towne and CL203. The presence of the UL 21.5 and TRL/IRL 2-5 RNA transcripts in all strains tested demonstrates that the packaged transcripts occurs in all strains of HCMV suggesting that they may have a relevant role in the biology of this virus.

Cytomegalovirus↗
PubMed1992

Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay.

We compared four primer sets from conserved regions of the hepatitis C virus (HCV) genome for their ability to detect HCV RNA in a "nested" cDNA polymerase chain reaction assay on sera from 114 anti-HCV antibody-positive individuals from around the world. The different primer sets had equivalent sensitivity, detecting less than 1 chimpanzee ID50 (dose that infects 50%) when tested against reference strain H of HCV. We tested equal amounts of RNA extracted from the serum of each individual with the four primer sets. The set derived from two highly conserved domains within the 5' noncoding (NC) region of the HCV genome, which also share significant similarity with Pestivirus 5' NC sequences, was the most effective at detecting HCV RNA. All samples positive for HCV RNA with any other primer set were also positive with the primer set from the 5' NC region, and the latter was at least 3 times more likely to detect HCV infection than a primer set from within the nonstructural protein 3-like gene region (P less than 0.001). We had no false positive results in greater than 500 negative controls interspersed among the test samples. The 5' NC region primer set detected HCV-specific RNA, verified by high-stringency Southern blot hybridization and DNA sequencing, in 100% of 15 acute and 33 chronic non-A, non-B hepatitis patients from the United States, Europe, and Asia and 10 hepatocellular carcinoma patients from Africa and Asia that tested negative for the hepatitis B virus-encoded surface antigen. In conclusion, use of an appropriate primer set is crucial for detecting HCV RNA in the serum of infected individuals.

Base Sequence↗
PubMed1989

Rat brain regional preproenkephalin A messenger RNA levels are altered in genetic hypertension.

Considerable neuroanatomical and pharmacological evidence suggests that preproenkephalin A-derived peptides, particularly methionine-enkephalin, are involved in regulation of the cardiovascular system in both physiological and pathological states. In this study, we used a rat preproenkephalin A complementary DNA to determine whether proenkephalin A-derived peptides participate in the pathogenesis of hypertension as reflected by brain regional messenger RNA levels. Complementary DNA clones of the rat preproenkephalin A mRNA and rat small myelin basic protein mRNA were hybridized to total RNA extracted from hypothalamus, pons-medulla, thoracic cord, midbrain, and cerebellum of 3 1/2-week-old and 12-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. In 3 1/2-week and 12-week animals there were no differences in the levels of myelin basic protein messenger RNA between the two groups in any brain region. At 3 1/2 weeks, preproenkephalin A mRNA levels did not differ between normotensive and hypertensive strains. In contrast, at 12 weeks preproenkephalin A mRNA levels were increased in hypothalamus, midbrain, thoracic cord, and cerebellum of SHR relative to WKY. Preproenkephalin A mRNA was significantly reduced in the pons-medulla of SHR relative to WKY. Our findings provide evidence that alterations in brain regional preproenkephalin A mRNA levels are associated with the development of spontaneous hypertension in the rat.

Aging↗
PubMed1997

Chronic variant of myocarditis associated with hepatitis C virus infection.

BACKGROUND: Although molecular biological studies suggest a pathogenic link between enterovirus infection and dilated cardiomyopathy (DCM), the frequency of detection of enteroviral RNA is not consistently high in myocardial tissue from patients with DCM. A recent study has suggested that hepatitis C virus (HCV) may also be involved in the development of DCM. METHODS AND RESULTS: We performed genomic analysis for HCV in three patients with chronic active myocarditis. In all three patients, serum aminotransferase activities remained within normal ranges until the terminal stage of heart failure. At necropsy, all three livers showed evidence of tissue damage caused by chronic congestion, and one liver had evidence of chronic hepatitis. Routinely processed, paraffin-embedded tissue blocks of myocardium and liver were analyzed. Renal specimens were also analyzed to exclude the possibility of myocardial contamination with HCV material from the circulating blood. RNA extracted from the heart, liver, and kidney was subjected to strand-specific reverse transcription and amplified by semi-nested polymerase chain reaction. The target nucleotide sequence was a 178-bp fragment of the highly conserved 5'-noncoding region. Both positive- (genomic) and negative-strand RNA (replicative intermediates) were present in myocardial and liver tissue samples from all three patients. However, negative-strand RNA was undetectable in renal tissue from one patient. CONCLUSIONS: These findings suggest that HCV replicated in myocardial tissue of these patients with myocarditis. Thus, HCV infection may contribute to the development of this unusual form of myocarditis.

Chronic Disease↗
PubMed1984

Induction of interleukin 1 messenger RNA and translation in oocytes.

Macrophages and P388D1 murine cultured cells produce interleukin 1 (lymphocyte-activating factor) when stimulated with lipopolysaccharide or other agents. Poly A+ RNA extracted from either type of stimulated cells and injected into Xenopus oocytes causes synthesis of material with the biologic and biochemical properties of interleukin 1. It potentiates lectin-mediated thymocyte proliferation, and it has the molecular dimensions of interleukin 1, as determined by gel exclusion chromatography. RNA from either cell type causes the synthesis of interleukin 1 with an isoelectric point of 4.8 to 5.0. RNA prepared from unstimulated macrophages or P388D1 cells does not cause interleukin 1 production by oocytes. We conclude that the amount of interleukin 1 mRNA increases greatly after stimulation of either cell type, and oocytes carry out any modifications of the polypeptide necessary for activity. The kinetics of interleukin 1 mRNA accumulation and of interleukin 1 production by macrophages and P388D1 cells are compared.

Animals↗
PubMed1985

Transcription of repeat DNA in vitro by RNA polymerase III in cytosol extracts is inhibited when the extract is of the same species as the DNA.

Cytosol extracts containing RNA Polymerase III were obtained from cultured human (HeLa) and bovine (MDBK) cells. Human extract efficiently transcribed genomic bovine DNA and cloned bovine alu-type repeat DNA while genomic human DNA and cloned human alu repeat DNA were inefficient templates. Conversely, genomic bovine DNA and cloned bovine alu-type repeat DNA were less effective templates for bovine extract than were genomic human DNA and alu repeat DNA. These results suggest that species specific factor(s) present in mammalian cells are responsible for the observed in vivo inhibition of transcription of most alu-type repeat DNA.

Animals↗
PubMed1995

Messenger RNA expression for a TSH receptor variant in the thymus of a two-year-old child.

We recently described the presence of a thyroid-stimulating hormone receptor (TSH-R) variant in orbital tissues. Although the presence of this TSH-R variant could provide the antigenic link between the thyroid and the orbit in Graves' disease and thyroid-associated ophthalmopathy (TAO), the etiopathophysiological significance of this finding remains to be elucidated. Graves' disease and TAO are autoimmune diseases which are likely to be caused by a breakdown of tolerance. We therefore investigated the presence of this variant TSH-R transcript in human thymus. Using primers specific for this variant in reverse polymerase chain reaction (PCR) experiments, Southern blotting, and sequencing of the PCR products we demonstrate the presence of this transcript in RNA extracted from normal human thymus of a 2-year-old child. We were also able to amplify this variant TSH-R transcript from a normal human thyroid cDNA library, but not from an epithelial thymus library. The presence of the variant TSH-R transcript in RNA prepared from normal human thymus suggests that induction of immunological tolerance against this variant TSH-R transcript in the thymus is possible. A lack of tolerance induction for this variant TSH-R transcript could provide an explanation for a possible antigenic link between Graves' disease and TAO.

Blotting, Southern↗
PubMed1998

Polysome-like structures in the chromatoid body of rat spermatids.

A procedure for isolating the chromatoid body from the testis of 40-day-old rats was developed. Electron-microscopical analysis indicated that about 70% of the isolated organelles were chromatoid bodies, while the remaining structures corresponded to dense bodies and probably to satellites. Negative staining of the isolated organelles revealed the presence of polysome-like structures in about 20% of the chromatoid bodies suggesting that the polysomes were not due to contamination with cytoplasmic polysomes. Moreover, the presence of RNA in the stroma of the chromatoid body was confirmed by RNAse-gold staining. Preliminary electrophoretic analysis of the RNA extracted from the organelles revealed the presence of a complex population of RNAs including 5.8 and 5 S ribosomal RNAs but no tRNA.

Animals↗
PubMed1990

Detection of cytomegalovirus nucleic acid sequences in pancreas in type 2 diabetes.

Nucleic acid sequences specific for human cytomegalovirus (CMV) were found in samples of pancreatic tissue from patients with non-insulin-dependent (type 2) diabetes mellitus. RNA extracted from paraffin-embedded or fresh-frozen specimens from 14 of 32 (44%) diabetic patients but from none of 49 non-diabetic controls reacted with 10 kb (pJN201) or 6.6 kb (pCM3) probes of human CMV immediate-early or late gene products, respectively. The RNA from the 32 diabetic patients did not react with nucleic acid probes for mumps, rubella, or coxsackie B viruses. In-situ nucleic acid hybridisation on tissues from 5 randomly selected human-CMV-positive patients showed that the human CMV signal was localised primarily in the islets of Langerhans and not in exocrine cells. Despite the clear viral nucleic acid signal in tissues of human-CMV-positive patients, there were no morphological injuries to the islets, no inflammatory cells in the islets, and no perivascular inflammatory cell cuffing. These findings suggest a possible association of human CMV with type 2 diabetes in human beings.

Aged↗
PubMed1991

Preproenkephalin mRNA and enkephalin in normal and denervated adrenals in the Syrian hamster: comparison with central nervous system tissues.

The distribution and characteristics of preproenkephalin (PPenk) mRNA and enkephalin-containing (EC) peptides are compared in CNS and adrenal tissues from Syrian hamsters and Sprague-Dawley rats. Total cellular RNA extracts from both rat and hamster tissues produce a single hybridization band of PPenk mRNA of approximately 1500 bases when analyzed by Northern blot hybridization. Quantitation by solution hybridization reveals that in the hamster the highest levels of PPenk mRNA are found in adrenal (16.3 +/- 1.4 pg equivalents/micrograms RNA (mean +/- S.E.M.)) and striatum (13.3 +/- 0.7), followed by hypothalamus (0.8 +/- 0.2), and hippocampus (0.4 +/- 0.2). In the rat the highest levels of PPenk mRNA are in the striatum (35 +/- 2 pg/micrograms RNA) followed by the hypothalamus (3.0 +/- 0.5), hippocampus (0.3 +/- 0.1) and adrenal (0.18 +/- 0.04). Thus, the rank order of abundance of PPenk mRNA is similar in these CNS tissues for rat and hamster. The hamster adrenal levels are more than 90-fold greater than those of the rat. The abundance of EC peptides in both hamster and rat tissues mirror the rank order found with PPenk mRNA. Hamster adrenal contains the highest level of EC peptides (441 +/- 37 pmol/mg protein (mean +/- S.E.M.)) which is more than 400-fold greater than that of the rat adrenal and 8- to 12-fold greater than that found in rat and hamster striatum or hypothalamus. Both size exclusion chromatography and Western blot analysis indicate that EC peptides in hamster adrenal are predominantly large proenkephalin-like peptides with approximately 6 copies of Met- and 1 copy of Leu-enkephalin and that included in their number is a prominent EC peptide with a molecular weight of 34 kDa. Unilateral denervation of the hamster adrenal results in a time-dependent ipsilateral decrease in EC peptide and PPenk mRNA levels. Thus, by day 8 postsurgery, PPenk mRNA levels have declined by an average of 80% while EC peptides are reduced by 68% when compared to the innervated contralateral adrenal. These results demonstrate the great abundance of PPenk mRNA and EC peptides in the hamster adrenal. They also demonstrate the apparent need for transsynaptic impulse activity to maintain the high steady-state levels of PPenk and EC peptides. These characteristics of the hamster adrenal system provide opportunities for physiological and pharmacological investigations of the regulation of proenkephalin gene expression.

Adrenal Glands↗
PubMed1995

A new factor from Escherichia coli affects translocation of mRNA.

Reconstitution of protein synthesis from purified translation factors on ribosomes from Escherichia coli has revealed the requirement for a protein, W, that affects chain elongation and is essential to reconstitute the process (Ganoza, M. C., Cunningham, C., and Green, R. M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1648-1652). We report that W has no effect on initiation complex formation by 30 or 70 S ribosomes or on the association of ribosomal subunits, peptide bond synthesis, or binding Ala-tRNA, which is the second amino acid of the coat protein of the MS2 RNA virion. W has a pronounced effect on tripeptide synthesis, and is obligatory for the synthesis of the coat protein or of the hexapeptide encoded by f2am3 RNA. Extracts from a temperature-sensitive mutant of the translocase, EF-G, were purified free of the W protein and were used to score for translocation defects. W is required for binding Ser-tRNA, the third N-terminal amino acid of the MS2 or f2 RNA coat protein to ribosomes bearing fMet-Ala-tRNA, as well as for the ejection of deacyl-tRNA from ribosomes, which occurred concomitant with the binding of the Ser-tRNA. We propose that W functions by ejecting tRNAs from ribosomes in a step that precedes the movement of mRNA during translocation.

Bacterial Proteins↗
PubMed1976

Characterization of polysomes from Xenopus liver synthesizing vitellogenin and translation of vitellogenin and albumin messenger RNA's in vitro.

1. Conditions are described for the isolation of polysomes from the liver of Xenopus laevis. The method involves homogenization of liver in 0.2 M Tris-HCl pH 8.5, treatment with 2% Triton X-100 and subsequent sucrose density gradient fractionation of polysomes from a 10000 X g supernatant. 2. Vitellogenin synthesis was induced in male Xenopus liver by oestradiol treatment. Polysomes were isolated and vitellogenin-synthesizing polysomes characterized by their association with monospecific 125 I-labelled rabbit anti-vitellogenin antibody and by reaction with rabbit anti-vitellogenin immunoglobulins followed by indirect immunoprecipitation with goat anti-rabbit antibody. 3. Changes in liver polysome content following oestrogen treatment of male Xenopus are correlated with the appearance of vitellogenin synthesis using an organ culture assay. 4. RNA extracted from livers of oestradiol-treated male Xenopus and from purified polysomes is shown to code for the synthesis of vitellogenin-specific immunoprecipitable polypeptides in a rabbit reticulocyte cell-free protein-synthesizing system, a major component having a molecular weight of 210000. Xanopus liver RNA is also shown to code for the synthesis of an albumin-specific immunoprecipitable polypeptide of 74000 molecular-weight which coelectrophoresed with Xenopus albumin.

Albumins↗
PubMed2004

Study of the ecology of fresh sausages and characterization of populations of lactic acid bacteria by molecular methods.

In this study, a polyphasic approach was used to study the ecology of fresh sausages and to characterize populations of lactic acid bacteria (LAB). The microbial profile of fresh sausages was monitored from the production day to the 10th day of storage at 4 degrees C. Samples were collected on days 0, 3, 6, and 10, and culture-dependent and -independent methods of detection and identification were applied. Traditional plating and isolation of LAB strains, which were subsequently identified by molecular methods, and the application of PCR-denaturing gradient gel electrophoresis (DGGE) to DNA and RNA extracted directly from the fresh sausage samples allowed the study in detail of the changes in the bacterial and yeast populations during storage. Brochothrix thermosphacta and Lactobacillus sakei were the main populations present. In particular, B. thermosphacta was present throughout the process, as determined by both DNA and RNA analysis. Other bacterial species, mainly Staphylococcus xylosus, Leuconostoc mesenteroides, and L. curvatus, were detected by DGGE. Moreover, an uncultured bacterium and an uncultured Staphylococcus sp. were present, too. LAB strains isolated at day 0 were identified as Lactococcus lactis subsp. lactis, L. casei, and Enterococcus casseliflavus, and on day 3 a strain of Leuconostoc mesenteroides was identified. The remaining strains isolated belonged to L. sakei. Concerning the yeast ecology, only Debaryomyces hansenii was established in the fresh sausages. Capronia mansonii was initially present, but it was not detected after the first 3 days. At last, L. sakei isolates were characterized by randomly amplified polymorphic DNA PCR and repetitive DNA element PCR. The results obtained underlined how different populations took over at different steps of the process. This is believed to be the result of the selection of the particular population, possibly due to the low storage temperature employed.

Animals↗
PubMed1990

Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens.

The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.

Base Sequence↗
PubMed1996

Hepatitis C virus genotypes in the United States: epidemiology, pathogenicity, and response to interferon therapy. Collaborative Study Group.

OBJECTIVE: To study 1) the geographic distribution and clinical significance of hepatitis C virus (HCV) genotypes in the United States and 2) the influence of HCV genotypes on response to interferon therapy. DESIGN: Hepatitis C virus genotype was determined in 179 stored serum samples obtained from patients who were positive for antibody to HCV and for HCV RNA by using polymerase chain reaction. SETTING: Tertiary referral centers in four geographic regions of the United States. PATIENTS: Patients who visited medical centers in the Midwest (50 patients), Northeast (42 patients), Southeast (35 patients), and West (52 patients). MEASUREMENTS: Chaotropic lysis and isopropanol precipitation were used to extract RNA from serum. Polymerase chain reaction was done on the NS5 region and was followed by automated direct sequencing and genotyping of desalted amplification products. RESULTS: 104 patients (58%) had subtype 1a; 38 (21%) had subtype 1b; 4 (2%) had subtype 2a; 23 (13%) had subtype 2b; 8 (5%) had subtype 3a; and 2 (1%) had subtype 4a. Examination of the known risk factors for acquiring HCV showed no association between genotype and mode of acquisition (blood transfusion, injection drug use, employment at a health care facility) or histologic findings at presentation (mild active hepatitis, moderately active hepatitis, or cirrhosis). Sixty-eight percent of patients with genotype 1a, 80% of patients with genotype 1b, and 37% of patients with genotype 2a or 2b had severe hepatitis. Thirteen of 46 (28%) patients with genotype 1a and 4 of 15 (26%) patients with genotype 1b had a complete biochemical response after 6 months of interferon therapy. In contrast, 10 of 14 (71%) patients with genotype 2a or 2b had a complete response to interferon therapy. Five of 39 (13%) patients with genotype 1a, 1 of 14 (7%) patients with genotype 1b, and 2 of 11 (18%) patients with genotype 2a or 2b had a sustained biochemical response. CONCLUSIONS: In the United States, HCV genotypes 1a and 1b are the predominant genotypes in patients with chronic hepatitis C. Genotype is not correlated with mode of virus acquisition or with histologic findings at presentation. Patients with HCV genotype 1a or 1b had more severe liver disease and lower rates of response to interferon therapy than did patients with HCV genotype 2a or 2b. These findings may have implications for predicting outcome and selecting patients for interferon treatment.

Adult↗
PubMed1981

Keyhole-limpet haemacyanin and immune ribonucleic acid immunotherapy of murine transitional cell carcinoma.

Transitional cell carcinoma is known to be an immunogenic tumour. This immunogenicity has been the basis of a search for effective immunotherapeutic agents and for the evaluation in this study of two additional agents, keyhole-limpet haemacyanin (KLH) and immune ribonucleic acid (RNA) extract. The results in this animal model showed KLH to be a potent non-specific stimulant of the immune response which caused both a reduction in tumour growth and a prolongation of animal survival (p = 0.01). No anti-tumour effects were observed with either local or systemic RNA.

Animals↗
PubMed2003

Complete nucleotide sequence and genome organization of a Chinese isolate of tobacco bushy top virus.

The complete nucleotide sequence of a Chinese isolate of tobacco bushy top virus (TBTV), designated TBTV-Ch, was determined from cDNA generated from double-stranded RNA extracted from diseased tobacco. The genome is 4152 nucleotides (nt) in size, contains four putative open reading frames (ORFs) and untranslated regions of 10 nt and 645 nt at the 5' and 3' ends, respectively. In genome organization and in the amino acid sequence of its potential products, the RNA of TBTV-Ch is similar to other umbraviruses sequenced to date. The results suggested that TBTV should be regarded as a definitive species of the genus Umbravirus.

Base Sequence↗
PubMed1995

Quantitative distribution of the delta opioid receptor mRNA in the mouse and rat CNS.

We have used a sensitive solution hybridization assay that employs a riboprobe obtained from the mouse delta opioid receptor (DOR) coding sequence to quantitate the relative abundance of DOR mRNA in the central nervous system (CNS) of the adult mouse and rat. In brain Poly A+ RNA extracts this riboprobe hybridized to a single 10 kb transcript from mouse and two transcripts, one of 12 and the other of 4.5 kb in size from rat. In mouse CNS the highest levels of DOR mRNA were found in the caudate-putamen at 3.3 +/- 0.1 (SEM) pg/micrograms RNA. DOR mRNA levels in the range from 2.6 to 2.1 pg/micrograms RNA were measured in frontal cortex, nucleus accumbens, whole brain and olfactory tubercle. Spinal cord, periaqueductal gray and hippocampus had DOR mRNA levels in the range from 1.8 to 1.5 pg/micrograms RNA, while medial thalamus and cerebellum had the lowest levels (0.5 pg/micrograms RNA). These results correlate with the reported relative distribution of DOR mRNA in mouse using an in situ hybridization technique. In rat CNS, the highest levels of DOR mRNA were measured in caudate-putamen at 2.3 +/- 0.1 pg equivalents/micrograms RNA. Whole brain, cerebral cortex, olfactory bulb and brain stem had levels in the range from 1.5 to 0.9 pg equivalents/micrograms RNA while the lowest DOR mRNA levels were measured at 0.5 pg equivalents/micrograms RNA or less in thalamus, hippocampus, substantia nigra and cerebellum. This study demonstrates the ability of solution hybridization assays to quantitate homologous (mouse) as well as similar but heterologous (rat) DOR mRNA levels.

Animals↗