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At least 901 records · Page 50Linked to original sources
PubMed2005

Development of a rapid assay for determining the relative abundance of bacteria.

A sandwich hybridization assay for high-throughput, rapid, simple, and inexpensive quantification of specific microbial populations was evaluated. The assay is based on the hybridization of a target rRNA with differentially labeled capture and detector probes. Betaproteobacterial ammonia-oxidizing bacteria (AOB) were selected as the target group for the study, since they represent a phylogenetically coherent group of organisms that perform a well-defined geochemical function in natural and engineered environments. Reagent concentrations, probe combinations, and washing, blocking, and hybridization conditions were optimized to improve signal and reduce background. The detection limits for the optimized RNA assay were equivalent to approximately 10(3) to 10(4) and 10(4) to 10(5) bacterial cells, respectively, for E. coli rRNA and RNA extracted from activated sludge, by using probes targeting the majority of bacteria. Furthermore, the RNA assay had good specificity, permitted discrimination of rRNA sequences that differed by a 2-bp mismatch in the probe target region, and could distinguish the sizes of AOB populations in nitrifying and nonnitrifying wastewater treatment plants.

Bacteria↗
PubMed1983

In vitro synthesis of subunits of bacterial luciferase in an Escherichia coli system.

RNA extracted from the luminescent bacterium Vibrio harveyi was translated in an Escherichia coli system. RNA from highly luminescent cells produced both alpha and beta subunits of luciferase in vitro, as confirmed by immunoprecipitation and partial proteolysis. RNA from cells before induction of luminescence did not direct luciferase synthesis.

Escherichia coli↗
PubMed2003

Improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the 5' noncoding regions of additional rhinovirus strains.

The isothermal nucleic acid sequence-based amplification (NASBA) system was applied for the detection of rhinoviruses using primers targeted at the 5' noncoding region (5' NCR) of the viral genome. The nucleotide sequence of the 5' NCRs of 34 rhinovirus isolates was determined to map the most conserved regions and design more appropriate primers and probes. The assay amplified RNA extracted from 30 rhinovirus reference strains and 88 rhinovirus isolates, it did not amplify RNA from 49 enterovirus isolates and other respiratory viruses. The assay allows one to discriminate between group A and B rhinoviruses. Sensitivities for the detection of group B and group A rhinoviruses was 20 and 200 50% tissue culture infective doses, respectively.

5' Untranslated Regions↗
PubMed1973

RNA synthesis in cells infected with herpes simplex virus. VII. Control of transcription and of transcript abundancies of unique and common sequences of herpes simplex virus 1 and 2.

Analysis of the kinetics of hybridization in liquid of labeled herpes simplex virus (HSV) 1 and 2 DNAs with excess unlabeled RNA extracted at 2 (early) and 8 (late) h postinfection revealed the following. (i) The RNA transcripts present in the HSV-1-infected cells at 2 and 8 h postinfection are complementary to 44 and 48% of HSV-1 DNA. The RNA transcripts present in the HSV-2-infected cells at 2 and 8 h postinfection are complementary to 21 and 50% of HSV-2 DNA. (ii) The transcripts present in 2-h HSV-1- or HSV-2-infected cells treated with cycloheximide are complementary to 44 and 45% of the respective DNAs. (iii) The RNA transcripts present in the HSV-1-infected cells at 2 h postinfection and in HSV-2-infected cells at 8 h postinfection form 2 classes, abundant and scarce, differing in molar concentrations. The RNA transcripts present in the HSV-2-infected cells at 2 h postinfection form only one abundance class. (iv) The transcripts present in the HSV-1-infected cells at 8 h postinfection are complementary to 24% of HSV-2 DNA and therefore 50% of the transcribed HSV-1 sequences are shared by the two viruses. Of the RNA sequences complementary to HSV-2 DNA, 13% arise from HSV-1 templates specifying abundant RNA and 11% arise from HSV-1 templates specifying scarce RNA. Thus, the DNA sequences shared in common by HSV-1 and HSV-2 DNAs constitute 71% of the HSV-1 templates specifying abundant RNA and 39% of sequences specifying scarce RNA.

Base Sequence↗
PubMed1994

Cloning and characterization of a novel RNA involved in cellular growth regulation.

During the course of antisense oligodeoxynucleotide (oligo) inhibition experiments investigating the role of insulin-like growth factor I (IGF-I) in the WI-38 cell cycle, we found that a sense-strand oligo (S oligo), used as a control, inhibited DNA synthesis 90 to 95%. S1 nuclease protection assays demonstrated that this S oligo formed intracellular duplexes with WI-38 RNA, and Northern (RNA) hybridization analyses demonstrated specific hybridization of this 32P-labeled S oligo to 1.8-, 2.3-, and 3.2-kb RNAs. We have cloned and sequenced a 2,251-bp cDNA, designated BB1, corresponding to the 2.3-kb RNA. Decoding of the BB1 cDNA sequence reveals several open reading frames arranged in a motif similar to that seen in proteins subject to translational control mechanisms. Homology searches of nucleic acid and protein data bases reveal no significant homology of BB1 with known sequences other than a 234-bp region in the BB1 5' untranslated region that shared 97% homology with a region in the 3' untranslated region of the human cdc42 mRNA. S1 nuclease protection analyses performed with IGF-I gene fragments and computer homology searches demonstrated that the BB1 RNA does not derive from transcription from the opposite strand of the IGF-I gene. Northern hybridization analyses of RNA extracted from serum-starved HeLa S3 cells demonstrated that steady-state BB1 RNA levels increased upon serum growth stimulation, with steady-state levels peaking 4 h after release from the block induced by serum starvation. Antisense oligo inhibition experiments using specific BB1 antisense oligos targeted to the putative open reading frames of the BB1 RNA reduce DNA synthesis of HeLa S3 cells to 15% of control levels, indicating that the BB1 RNA is essential for cell cycle traversal and, as such, encodes a growth-reguLating gene product.

Amino Acid Sequence↗
PubMed1996

Gene expression of hypothalamic somatostatin, growth hormone releasing factor, and their pituitary receptors in hypothyroidism.

Thyroid hormones are important to growth in mammals and have been shown to rapidly stimulate the rate of GH gene transcription. In this study, we investigated whether thyroid hormones modulate GH secretion through their effects on the gene expression of GRF, somatostatin (SS), GRF receptor, and receptor subtype 2 for SS (SSTR2). Male adult Sprague-Dawley rats were rendered hypothyroid with a single injection of propylthiouracil followed by methimazole in drinking water (0.02%) for 1 day to 12 weeks. Total RNA extracted from the anterior pituitary and hypothalamus was analyzed by Northern hybridization. GH messenger RNA (mRNA) level in the anterior pituitary was significantly reduced in the hypothyroid animals (P < 0.0001 vs. controls for all treatment duration > or = 1 week). An increase in hypothalamic GRF mRNA level, by 2- and 4-fold, respectively, was seen after 3 and 12 weeks of antithyroid treatment (both P < 0.001 vs. controls). Hypothalamic GRF content, studied in 12-week hypothyroid rats only, was decreased compared with controls (P < 0.05). A reduction in pituitary GRF receptor mRNA level was observed after 1 week of antithyroid treatment (P < 0.01 after 1 week, P < 0.001 after 3 weeks). Total hypothalamic SS content and SS mRNA level in hypothalamic fragments consisting predominantly of the paraventricular and periventricular nuclei became significantly decreased (P < 0.05 and P < 0.005 respectively) after 12-weeks of antithyroid treatment. The reduction in SS gene expression in the periventricular nuclei was confirmed by in situ hybridization. No significant change in the mRNA level of pituitary SSTR2 was observed up to 12 weeks of antithyroid treatment. In conclusion, we have demonstrated a reduction in the gene expression of GRF receptor and SS in the hypothyroid rat. Our results suggest that the changes in hypothalamic GRF and SS gene expression in hypothyroid rats may be compensatory in nature and are likely to be secondary to the reduction in GH synthesis and secretion in these animals. The reduction in basal and GRF-stimulated GH secretion in hypothyroidism can be explained by the observed reduction in GH and GRF receptor gene expression.

Animals↗
PubMed1988

Anterior determinants in embryos of Chironomus samoensis: characterization by rescue bioassay.

Embryos of Chironomus samoensis are programmed, by anterior u.v. irradiation, to form the abnormal body pattern 'double abdomen'. Most double abdomen embryos show a mirror-image duplication of abdominal segments in the absence of cephalic or thoracic segments. Such embryos can be 'rescued', i.e. restored to normal development, by microinjection of cytoplasm or RNA from unirradiated donor embryos. Most of the rescued embryos look completely normal and many of them hatch spontaneously. The rescuing activity decreases from the anterior to the posterior pole in the donor cytoplasm and must be delivered near the anterior pole of the recipient for maximum efficiency. Rescuing activity is present in total RNA extracted from whole, unirradiated embryos. Upon fractionation, the activity is associated with poly(A)+ RNA, with LiCl precipitate depleted of RNA smaller than 250 nucleotides (nt) and with a sucrose gradient fraction depleted of RNA larger than 500 nt. Corresponding fractions of RNA from Xenopus oocytes have no rescuing activity. The activity of Chironomus RNA is sensitive to u.v. irradiation with low fluence affecting less than 2% of the pyrimidine bases. Rescuing activity is present in cytoplasm until the blastoderm stage but disappears earlier from poly(A)+ RNA. Rescuing activity is also present, and localized, in cytoplasm of embryos from two related dipterans, Smittia sp. and Drosophila melanogaster, although the extent of rescue observed in Chironomus decreases with the phylogenetic distance between donor and recipient. The results of these and previous experiments indicate that dipteran embryos contain localized RNP particles acting as anterior determinants. In Chironomus, the activity of these particles seems to depend on the integrity of polyadenylated RNA of about 250 to 500 nt length.

Abdomen↗
PubMed1988

IL-2 production in human T lymphotropic virus I-infected leukemic T lymphocytes analyzed by in situ hybridization.

In situ hybridization studies were performed with 35S-labeled anti-sense RNA probes to study IL-2 mRNA expression in three human T lymphotropic virus I-infected T cell lines at the single cell level. In HuT 102, MT-2, and MT-4 cells, IL-2 mRNA-expressing cells were identified, occurring at frequencies of 2 x 10(-2), 8 x 10(-3), and 5 x 10(-3), respectively. In these cell lines, IL-2 mRNA was not detectable in RNA extracted from whole adult T cell leukemia cell populations because of dilution by other RNA species from the vast majority of cells that do not contain IL-2 mRNA. The data indicate the possibility of paracrine growth stimulation via IL-2 and its receptor even in those human T lymphotropic virus I-infected T cell populations that apparently lack IL-2 activity when analyzed by conventional assay procedures.

Cell Line↗
PubMed1977

[Presence in immunostimulated cells of an RNA molecule utilizable as template for reverse transcriptase of avian myeloblastosis virus (AMV)].

Cytoplasmic RNA extracted from antigen stimulated immunocompetant cells is transcribed in vitro into DNA by the RNA directed DNA polymerase from avian myeloblastosis virus, in the absence of any added primer. Cytoplasmic RNA from other organs of the same animal, from non-stimulated immunocompetent cells, or from cells in tissue culture is not transcribed in the absence of exogenous primer.

Animals↗
PubMed1996

Premature chain termination is a unifying mechanism for COL1A1 null alleles in osteogenesis imperfecta type I cell strains.

Nonsense and frameshift mutations, which predict premature termination of translation, often cause a dramatic reduction in the amount of transcript from the mutant allele (nonsense-mediated mRNA decay). In some genes, these mutations also influence RNA splicing and induce skipping of the exon that contains the nonsense codon. To begin to dissect how premature termination alters the metabolism of RNA from the COL1A1 gene, we studied nonsense and frameshift mutations distributed over exons 11-49 of the gene. These mutations were originally identified in 10 unrelated families with osteogenesis imperfecta (OI) type 1. We observed marked reduction in steady-state amounts of mRNA from the mutant allele in both total cellular and nuclear RNA extracts of cells from affected individuals, suggesting that nonsense-mediated decay of COL1A1 RNA is a nuclear phenomenon. Position of the mutation within the gene did not influence this observation. None of the mutations induced skipping of either the exon containing the mutation or, for the frameshifts, the downstream exons with the new termination sites. Our data suggest that nonsense and frameshift mutations throughout most of the COL1A1 gene result in a null allele, which is associated with the predictable mild clinical phenotype, OI type 1.

Alleles↗
PubMed1997

Immunotherapy of metastases with lymphocytes treated with exogenous RNA in mice bearing B16 melanoma.

Subline B16-F10, a variant cell line of B16 melanoma, is highly metastatic to the lung when injected intravenously into C57BL/6 mice. This experimental metastasis model was used to test the anti-tumor effect of exogenous RNA extracted from the lymphoid organs of immunized animals with B16-F10 cells. This RNA preparation is referred to as B16-RNA. Adoptive immunotherapy with lymphocytes treated with B16-RNA was effective in reducing significantly the number of pulmonary metastatic nodules. Lymphocytes incubated with medium alone or with RNA from non-immunized animals (N-RNA) were used as controls. The ability of B16-RNA in modulating antimetastatic activity of normal lymphocytes is abolished by hydrolysis with KOH. This finding indicates that the integrity of the polynucleotide chain is essential for the activity of B16-RNA. The anti-tumor effect of lymphocytes treated with B16-RNA was enhanced by incubation with a low dose of interleukin-2 (IL-2). A possible role of the double-stranded RNA dependent protein kinase in this phenomenon is discussed.

Animals↗
PubMed2004

Comparison of storage methods for reverse-transcriptase PCR amplification of rotavirus RNA from gorilla (Gorilla g. gorilla) fecal samples.

Detection of enteric viral nucleic acids in preserved gorilla fecal specimens was investigated using reverse transcription polymerase chain reaction (rt-PCR). A commercially available viral RNA extraction kit was used to isolate nucleic acids from captive gorilla fecal samples seeded with rotavirus and stored in ethanol, formalin, a commercial RNA preservation solution, guanidine thiocyanate buffer (GT), and samples dried in tubes containing silica gel. Nucleic acids were extracted at 1, 7, 70 and 180 days and used for rt-PCR amplification of specific rotavirus RNA sequences. Successful rt-PCR amplification of the target product varied according to storage conditions, and storage time. Only samples stored in GT gave 100% positive results at 180 days. It is recommended that fecal samples be collected in GT for viral RNA analysis.

Animals↗
PubMed2001

Inducible nitric oxide synthase and glomerular hemodynamics in rats with liver cirrhosis.

This study was designed to test the hypothesis that glomerular de novo expression of inducible nitric oxide synthase (iNOS) contributes to renal hemodynamic abnormalities in liver cirrhosis developed 3 wk after common bile duct ligature (CBDL). De novo expression of iNOS mRNA was detected by RT-PCR in RNA extracts from isolated CBDL rat glomeruli whereas no iNOS mRNA was found in control rat glomerular RNA. Immunohistochemical staining for iNOS was negative in control animals whereas, in CBDL rats, positive iNOS staining was detected in an apparently mesangial pattern in all glomeruli. Western blots of protein extracts from isolated glomeruli of CBDL rats, but not control animals, showed a prominent iNOS band of 130 kDa. Mean arterial pressure (MAP), renal plasma flow (RPF; p-aminohippurate clearance), and glomerular filtration rate (GFR; inulin clearance) were unaltered in CBDL rats, but the application of 4 mg/kg L-N(6)-(1-iminoethyl)lysine, a specific inhibitor of iNOS, reduced GFR and RPF significantly in CBDL rats, whereas control animals were not affected. Similar results were obtained with lipopolysaccharide (LPS)-pretreated animals, which were studied as a positive control for iNOS expression and as a model for recent iNOS induction. We conclude that de novo expression of iNOS occurs in glomeruli of rats with liver cirrhosis and that nitric oxide, generated by iNOS, contributes to the maintenance of glomerular filtration in the early state of this disease.

Animals↗
PubMed2000

Evaluation of automated nucleic acid extraction devices for application in HCV NAT.

BACKGROUND: To further improve the safety of the blood supply, various national blood transfusion organizations presently use or are in the process of implementing routine HCV NAT in minipools. According to the Committee for Proprietary Medicinal Products (CPMP) of the European Union, the HCV NAT detection limit of the assay should be 100 IU per mL (270 geq/mL) for testing initial plasma pools. Paul Ehrlich Institute (PEI) regulations stipulate that 5000 IU per mL (13,500 geq/mL) must be detected to calculate the amount contributed by individual donations composing the minipool. The sensitivity for HCV RNA extraction achieved by three commercially available laboratory kits was compared. STUDY DESIGN AND METHODS: Nucleic acids from 1-in-3 serial dilutions of an HCV RNA run control (Pelispy, CLB) were extracted with three kits (Cobas Amplicor, Roche Diagnostic Systems; BioRobot 9604, Qiagen; and NucliSens Extractor, Organon Teknika). HCV PCR of all extracts was performed using a second-generation Cobas Amplicor HCV test and the Cobas Amplicor analyzer. RESULTS: The manual Cobas Amplicor, the BioRobot 9604, and the NucliSens Extractor setups allow a 95-percent HCV RNA detection limit of 129, 82, and 12 geq per mL, respectively. The maximal pool size for the manual Cobas Amplicor, the BioRobot 9604, and the NucliSens Extractor kits that would still meet the PEI criteria for HCV NAT in minipools was calculated at 104, 164, and 1125 donations, respectively. CONCLUSION: All three HCV NAT kits evaluated meet the criteria set by CPMP and PEI. The highest sensitivity for HCV NAT screening can be achieved with the high-volume NucliSens Extractor method in combination with the Cobas Amplicor HCV v2.0 test on the Cobas Amplicor analyzer.

Blood Donors↗
PubMed1976

Message activity of influenza viral RNA.

The message activity of influenza virion RNA in the wheat germ cell-free protein-synthesizing system was investigated. RNA extracted from purified virions was found to direct the synthesis of a polypeptide that had the mobility of viral nucleocapsid protein on sodium dodecyl sulfate-polyacrylamide gels. Further characterization of the protein indicated it was not the nucleocapsid protein. No other polypeptides were detected. We conclude that influenza virion RNA is inactive as a template for the synthesis of virus-specific proteins.

Amino Acids↗
PubMed2005

Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors.

BACKGROUND: There has been much research into the use of RNA interference (RNAi) for the treatment of human diseases. Many viruses, including hepatitis B virus (HBV), are susceptible to inhibition by this mechanism. However, for RNAi to be effective therapeutically, a suitable delivery system is required. METHODS: Here we identify an RNAi sequence active against the HBV surface antigen (HBsAg), and demonstrate its expression from a polymerase III expression cassette. The expression cassette was inserted into two different vector systems, based on either prototype foamy virus (PFV) or adeno-associated virus (AAV), both of which are non-pathogenic and capable of integration into cellular DNA. The vectors containing the HBV-targeted RNAi molecule were introduced into 293T.HBs cells, a cell line stably expressing HBsAg. The vectors were also assessed in HepG2.2.15 cells, which secrete infectious HBV virions. RESULTS: Seven days post-transduction, a knockdown of HBsAg by approximately 90%, compared with controls, was detected in 293T.HBs cells transduced by shRNA encoding PFV and AAV vectors. This reduction has been observed up to 5 months post-transduction in single cell clones. Both vectors successfully inhibited HBsAg expression from HepG2.2.15 cells even in the presence of HBV replication. RT-PCR of RNA extracted from these cells showed a reduction in the level of HBV pre-genomic RNA, an essential replication intermediate and messenger RNA for HBV core and polymerase proteins, as well as the HBsAg messenger RNA. CONCLUSIONS: This work is the first to demonstrate that delivery of RNAi by viral vectors has therapeutic potential for chronic HBV infection and establishes the ground work for the use of such vectors in vivo.

Base Sequence↗
PubMed1981

Characterisation of viral RNA in cells infected with the murine coronavirus JHM.

The murine coronavirus JHM induces in Sac(-) cells seven major and two minor RNA species. These RNAs are polyadenylated and single stranded. Their sizes were estimated by electrophoresis in agarose gels containing methylmercury hydroxide. The mol. wts. for the major species are 6.67 x 10(6) for RNA of genome size, 3.42 x 10(6) for RNA 2, 2.76 x 10(6) for RNA 3, 1.35 x 10(6) for RNA 4, 1.19 x 10(6) for RNA 5, 0.93 x 10(6) for RNA 6 and 0.62 x 10(6) for RNA 7. The minor species have a size of 4.7 x 10(6) (RNA a) and 1.5 x 10(6) (RNA b). No essential difference in the number and proportion of each RNA species was found between total cytoplasmic RNA, polyadenylated cytoplasmic RNA and RNA extracted from pelleted polysomes, nor was any difference found during the infection cycle. The major RNA species are likely to be subgenomic mRNAs.

Animals↗
PubMed1991

RNA-mediated transfer of cellular immunity to a synthetic env antigen of the human immunodeficiency virus (HIV-1).

A sheep was immunized with a synthetic peptide corresponding to amino acid residues 586-606 of the precursor envelope protein GP-160 of the HIV-1 including a conserved epitope region of the GP-41 transmembrane protein in the mature viral particles, referred to as SM 284 HIV-1 [1]. It is demonstrated that immune RNA extracted from the lymphoid organs of the immunized animal (SM 284 HIV-1 I-RNA) was able to transfer immune cellular reactivity to SM 284 HIV-1 in vitro to human and rabbit lymphocytes and in vivo to Cebus apella monkeys. The transfer was detected by the leukocyte adherence inhibition test (LAI) as an indicator of cellular reactivity. One of the most relevant results was the demonstration that SM 284 HIV-1 I-RNA was able to induce cellular immunological memory in vivo in monkeys. These results may be relevant to delineate a new alternative for immunomodulation against HIV infection.

Amino Acid Sequence↗