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At least 829 records · Page 46Linked to original sources
PubMed1987

Use of oligodeoxynucleotide probes for quantitative in situ hybridization to actin mRNA.

We have employed an analytical approach for the development of an in situ hybridization methodology using synthetic oligodeoxynucleotide probes for actin messenger RNA detection in cultures of chicken fibroblasts and myoblasts. The methodology developed shows that oligonucleotides can complement the use of nick-translated probes in specific situations. Since they can be made to specific nucleic acid regions independent of restriction enzyme sites, they may be the most convenient approach for analysis of gene families among which sequences are highly conserved. However, it was found that oligonucleotides synthesized to different regions of a messenger RNA behave in situ with differing efficiencies, indicating that not all target sequences are equivalent. Therefore it was necessary to screen several oligonucleotide probes to a target molecule to find the optimal one. The convenience of using synthetic DNA probes makes it worthwhile to explore some of these characteristic properties so as to increase the sensitivity of this approach beyond its application to targets in high abundance.

Actins↗
PubMed1991

Altered pathogenicity in the liver induced by a mouse hepatitis virus type 3 thermosensitive mutant.

Intraperitoneal inoculation into sensitive BALB/c mice of D85, a thermosensitive (ts) mutant, provokes acute hepatitis followed by recovery of the mice. The ts mutant was able to replicate in the liver. However, the maximal viral titre was obtained 2 days later than was the case with the wild-type (wt) MHV 3 infection; the viral antigens remained localized within small foci and no invasion of the entire liver was observed. The hepatocytes infected with D85 showed strong steatosis similar to that induced by wt virus, but the other lesions induced by MHV 3 (closing of endothelial cell fenestrae and hepatocytolysis) were not seen. An important feature noticed with the D85 mutant concerned the establishment, in the surviving animals, of persistent infection: this phenomenon was demonstrated by the decrease of viral titre in the liver, viral RNA detection, and the fact that viral antigens gradually decreased until the 3rd month post-infection.

Animals↗
PubMed1992

[Second generation hepatitis-C virus test and polymerase chain reaction in anti-C 100 negative patients with chronic non-A, non-B hepatitis].

The aim of our study was to evaluate whether a negative HCV test of the first generation (HCV-ELISA 1) using the antigen C100-3 excludes chronic HCV infection, or whether patients exist who are negative for antibodies to C100-3 in spite of chronic hepatitis C. 27 patients with histologically proven chronic non-A, non-B hepatitis, all of whom were HCV-ELISA 1 negative, were tested by the HCV test systems of the second generation (Ortho-HCV-ELISA 2 and Chiron-HCV-RIBA 2) based on the distinct HCV antigens 5-1-1, C100-3, C33c and C22-3. To determine the presence of viremia, serum samples were also tested for HCV-RNA with "nested" PCR. 10 of 27 patients proved to be persistently negative when tested with the second generation assays. One patient showed low grade reactivity by HCV-ELISA 2, but non-reactivity by HCV-RIBA 2. In none of these 11 patients was HCV-RNA detected. 16 (60%) of 27 patients negative with HCV-ELISA 1 were positive with HCV-ELISA 2. HCV-RIBA 2 detected antibodies to the structural core antigen C22-3 in all of these 16 patients and antibodies to the non-structural antigen C33c in 14 of them, while antibodies to 5-1-1 or C100-3 were not found in any of these cases. 10 (63%) of the 16 HCV-ELISA 1 negative, but HCV-ELISA 2 and HCV-RIBA 2 positive patients were positive for HCV-RNA by "nested" PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2004

Profile-based detection of microRNA precursors in animal genomes.

MOTIVATION: MicroRNAs (miRNA) are essential 21-22 nt regulatory RNAs produced from larger hairpin-like precursors. Local sequence alignment tools such as BLAST are able to identify new members of known miRNA families, but not all of them. We set out to estimate how many new miRNAs could be recovered using a profile-based strategy such as that implemented in the ERPIN program. RESULTS: We constructed alignments for 18 miRNA families and performed ERPIN searches on animal genomes. Results were compared to those of a WU-BLAST search at the same E-value cutoff. The two combined approaches produced 265 new miRNA candidates that were not found in miRNA databases. About 17% of hits were ERPIN specific. They showed better structural characteristics than BLAST-specific hits and included interesting candidates such as members of the miR-17 cluster in Tetraodon. Profile-based RNA detection will be an important complement of similarity search programs in the completion of miRNA collections.

Algorithms↗
PubMed1999

Comparison of human immunodeficiency virus 1 DNA polymerase chain reaction and qualitative and quantitative RNA polymerase chain reaction in human immunodeficiency virus 1-exposed infants.

BACKGROUND: HIV-1 RNA PCR is a widely available and sensitive assay but has not been studied for use in early diagnosis of HIV-1 infection in infants. METHODS: Research HIV-1 DNA PCR and HIV-1 RNA PCR were performed on peripheral blood mononuclear cells and plasma, respectively, from 284 blood samples from 204 infants. A commercially available HIV-1 quantitative RNA PCR was also performed on plasma from the 132 samples from HIV-1-infected infants and 22 of the samples from HIV-1-uninfected infants. RESULTS: Sensitivities of all assays varied with infant age. HIV-1 DNA PCR had a sensitivity of 27% in the < or = 3-week age group (n = 11) whereas qualitative and quantitative RNA PCR had sensitivities of 64 and 55%, respectively (P not significant). Each assay had a sensitivity of 96.2% at 4 to 6 weeks (n = 26) and 100% at > or = 7 weeks of age (n = 95). Specificity of HIV-1 DNA PCR for all age groups was 100%, whereas specificities of qualitative and quantitative RNA PCR assay were 96.1 and 95.5%, respectively. CONCLUSIONS: HIV-1 RNA PCR may offer a slight advantage in sensitivity over DNA PCR in the diagnosis of HIV infection in young infants. Positive RNA results can be found in a small number of infants who are not HIV-1-infected. HIV-1 RNA detection should not be routinely used alone for the diagnosis of HIV infection in young infants.

Chi-Square Distribution↗
PubMedDate not supplied

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

Animals↗
PubMed2001

RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples.

The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseudomonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RNAlater, and held in cold storage for 3 months was compared. Fluorometric determination of the RNA extracted from Synechococcus and Pseudomonas cells indicated that the cell storage treatments tested were equally effective at maintaining their total RNA content. There was not any detectable decrease in the quantity of RNA isolated from the preserved samples during storage. Intact mRNA transcripts of the RuBisCO (rbcL) and nir genes were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved bacterial cells throughout 3 months of storage. In contrast, RT-PCR failed to amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/or unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly. In addition, RNAlater is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples.

Bacteria↗
PubMed2000

Diagnosis of HIV infection in children.

Many advances have been made in the area of HIV diagnostics. Commercially available virologic assays are sensitive and specific for the early detection of HIV in perinatal infection. The timing of the transmission of HIV from mother to child (in utero, at the time of birth, or postnatally by breast-feeding) is a critical consideration in the appropriate diagnosis of infants. Several algorithms can be used to define early infection and the potential timing of acquisition of infection that combine different assays and timing of specimens. The use of virologic assays, including HIV DNA PCR and HIV RNA detection methods and culture, can define and rule out infection in infants less than 18 months of age. Serologic diagnostic methods, including HIV ELISA, immunofluorescence, and western blot assays, can be used to diagnose infants more than 18 months of age, when transplacental antibody has disappeared in uninfected HIV-exposed infants. The challenge of the early and accurate diagnosis of perinatally HIV-exposed infants is the use of new assays to detect different HIV subtype infections that are prevalent in developing countries. Rapid, simple, and inexpensive serologic and virologic assays are being developed for worldwide use.

Child↗
PubMed1996

Epidemiology of hepatitis C virus infection in chronic haemodialysis.

There is a high prevalence of hepatitis virus infection in haemodialysis patients (17.7% of patients on the EDTA registry). That prevalence varies according to countries with a North-South gradient but also according to institutions or to the mode of treatment: haemodialysis in a unit or at home, peritoneal dialysis. Post-transfusional contamination was the number one risk factor before 1991. The incidence of infection in haemodialysis units is also variable, which reveals the risk of nosocomial transmission between patients. Strict observance of universal precautions against nosocomial infections reduces the contamination risk. The relevancy of separating patients is controversial. Other risk factors exist in haemodialysis patients, especially in those with heavier medical records, multiple surgery, endoscopy. Diagnostic anti-HCV antibody screening has improved in terms of specificity and sensitivity. HCV RNA detection by PCR confirms that most HCV antibody-positive patients are also viraemic and potentially contaminant; it permits detecting recent contamination. Virus genotyping is epidemiologically relevant because it offers the possibility to trace infections, and may also have therapeutical interest. Prevalence is greater in personnel than in the general population, which underlines the necessity to observe universal precautions.

Antibodies, Viral↗
PubMed1992

5' end-dependent translation initiation of hepatitis C viral RNA and the presence of putative positive and negative translational control elements within the 5' untranslated region.

Hepatitis C virus (HCV) is a distant relative of pestiviruses and flaviviruses, but it has a 5' untranslated region (UTR) with some features structurally similar to that of picornaviruses. In order to test the role of the 5' UTR in controlling the expression of the HCV polyprotein, we fused full-length or deleted versions of the 5' UTR of HCV-1 RNA to chloramphenicol acetyl transferase (CAT) mRNA to monitor CAT activity in vivo. We found: (1) the full-length 5' UTR of HCV-1 RNA is translationally inactive while 5' deletions which mimic a 5' subgenomic RNA detected in vivo are active, (2) an efficient cis-acting element which represses translation is found at the 5' terminus, (3) a putative element which enhances translation is found near the 3' terminus of the 5' UTR, (4) additional cis-acting elements including small open reading frames (ORFs) upstream from the putative enhancer element downregulate translation. We did not find evidence supporting the existence of an internal ribosome entry site in the 5' UTR of HCV-1 RNA. These data suggest that HCV may employ a distinctive translation control strategy such as the generation of subgenomic viral mRNA in infected cells. Translational control of HCV might be responsible for some of the characteristic pathobiology seen in viral infection.

Base Sequence↗
PubMed1970

Immunochemical measurement of double-stranded RNA of uninfected and arbovirus-infected mammalian cells.

A quantitative complement fixation assay which specifically measures double-stranded RNA has been used to study this RNA extracted from uninfected and arbovirus-infected cells. The double-straned RNA of the uninfected BHK-21 cells sedimented in the 12S region in sucrose gradients. The double-stranded RNA of Sinbis virus-infected cells, as measured immunochemically, included a predominant peak at 12S, a smaller 18S peak, and polydisperse material extending into the 26-30S region. All classes of this RNA detected immunochemically showed a sharp thermal denaturation curve, and increased in amount progressively during infection, with the 12S peak predominant at all times.

Animals↗
PubMed2001

Increased AST and GGT activity as marker of RT-PCR inhibition in RNA extracts from peripheral blood.

The presence of HCV RNA in PMBC, with simultaneous absence of the virus in the plasma (7.8%), suggests that blood is better material for HCV RNA detection than plasma or serum in the diagnostic procedures of patients with chronic hepatitis C as well as in monitoring the antiviral therapy. We studied 111 patients with chronic hepatitis C (anti-HCV+) and elevated level of at least one biochemical marker: AST, ALAT, GGT, AP and bilirubin. Inhibition of amplification was 2% in plasma and 34% in whole blood samples. We applied modification of extraction to reduce the inhibitory effect on PCR, by introducing additional purification of the RNA extract in the Chomczyński method. After our modification of extraction was applied, inhibition was reduced to 1%. In attempt to identify such inhibitory markers that would label the samples, in which additional RNA extract purification should be applied, we analysed the activity of AST and ALAT enzymes, the key markers for parenchymal liver damage; GGT and AP, the markers for cholestatic hepatitis as well as bilirubin. We observed that the increased GGT and AST activities were correlated with the inhibition of RT-PCR. This correlation was statistically significant; for AST (Mann-Whitney test p = 0.09654 and Kolmogorow-Smirnow test p = 0.01543) and for GGT (Mann-Whitney test p = 0.02419 and Kolmogorow-Smirnow test p = 0.01921).

Alanine Transaminase↗
PubMed2005

Vertical transmission of HIV in Ghanaian women diagnosed in cord blood and post-natal samples.

HIV RNA detection in the newborn is the main diagnostic tool for vertical transmission. Most infections are thought to occur peri- or post-natally, hence preventive antiviral therapy administered days before and during delivery. This study used cord blood for molecular diagnosis, examined viral load and HIV-1 subtypes as determinants of transmission, and compared molecular variability of maternal, cord blood, and post-natal quasispecies. Ninety-seven seropositive mother-cord blood paired plasmas from Ghana were tested for HIV RNA. Viral load was quantified and a subgroup of 45 random women samples was typed and subtyped. HIV-1 from infected pairs was cloned, sequenced, and analyzed phylogenetically. The prevalence of HIV infection in pregnant women was 3.3%. 13/97 cord blood samples (13.5%) contained HIV RNA. No correlation between either viral load at labor (range 10(3)-10(7)) or HIV-1 subtype and in utero transmission was found. In both transmitting and non-transmitting mothers, 56% of HIV-1 strains were CRF02_AG. In three pairs, maternal and cord blood quasispecies were closely related, suggesting late pregnancy or perinatal transmission, while in four pairs, genetic distances suggested transmission earlier during gestation. Maternal viral load and genotype did not correlate with HIV-1 pre-natal transmission. HIV infection during gestation appears relatively frequent.

Female↗
PubMed2003

MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms.

MicroRNAs (miRNAs) are endogenously encoded small noncoding RNAs, derived by processing of short RNA hairpins, that can inhibit the translation of mRNAs bearing partially complementary target sequences. In contrast, small interfering RNAs (siRNAs), which are derived by processing of long double-stranded RNAs and are often of exogenous origin, degrade mRNAs bearing fully complementary sequences. Here, we demonstrate that an endogenously encoded human miRNA is able to cleave an mRNA bearing fully complementary target sites, whereas an exogenously supplied siRNA can inhibit the expression of an mRNA bearing partially complementary sequences without inducing detectable RNA cleavage. These data suggest that miRNAs and siRNAs can use similar mechanisms to repress mRNA expression and that the choice of mechanism may be largely or entirely determined by the degree of complementary of the RNA target.

Base Sequence↗
PubMed1979

Synthesis of a 600-nucleotide-long plus-strand DNA by virions of Moloney murine leukemia virus.

A discrete, 600-nucleotide-long plus-strand DNA has been identified among the products of reverse transcription by virions of Moloney murine leukemia virus. Its polarity was shown by hybridization to minus-strand DNA. It appears to be copied from the right end of minus-strand DNA because (i) its restriction endonuclease cleavage pattern corresponds to the redundant 600-base segment found at either end of the ultimate double-stranded reverse transcription products, (ii) its synthesis is actinomycin D sensitive, and (iii) its synthesis begins during the first hour of a reverse transcription reaction when only the right-hand end of minus-strand DNA is available as template. We therefore call this DNA plus-strong-stop DNA by analogy with the minus-strong-stop DNA copied from the left end of the viral RNA. Both strong-stop DNAs are made early during in vitro reactions and decline in concentration later, consistent with postulated roles as initiators of long minus- and plus-strand DNA. Unlike minus-strong-stop DNA, plus-strong-stop DNA remains as a double-stranded nucleic acid after its synthesis, as shown by S1 nuclease resistance. A primer to initiate plus-strong-stop DNA synthesis has not been identified; the product found thus far has no detectable RNA attached to it.

Chromosome Mapping↗
PubMed2000

Assessment of genotype and molecular evolution of hepatitis C virus in formalin-fixed paraffin-embedded liver tissue from patients with chronic hepatitis C virus infection.

Drawbacks of hepatitis C virus (HCV) RNA detection in paraffin-embedded liver tissue have satisfactorily been solved by RT-PCR amplification of the 5'non-coding region (5'NCR). However, detection of this highly conserved region does not provide information on epidemiological or pathogenetic aspects of HCV infection. This study explores whether other functionally important genetic regions of HCV, such as the hypervariable region 1 (HVR-1) and the interferon sensitivity-determining region (ISDR), can be retrieved from paraffin-embedded liver specimens by RT-PCR, and whether the amplified material is suitable for further molecular analyses. RT-PCR amplification of 5'NCR, HVR-1, and ISDR was assessed in RNA extracted from 50 formalin-fixed, paraffin-embedded liver specimens, including 23 needle liver biopsies (11 from patients with non-A, non-B chronic hepatitis diagnosed between 1971 and 1985, 8 from subjects with normal liver histology and 4 from sequential biopsies from a patient with HCV recurrence after liver transplantation), and 27 liver explants from patients undergoing transplantation between 1988 and 1996 (16 with HCV-related cirrhosis and 11 with other disorders). The 5'NCR was successfully amplified in 8 of 11 (73%) non-A, non-B chronic hepatitis biopsies and in all of the specimens from patients with serological documentation of HCV infection. There were no false-positive results. HCV genotype was identified by RFLP analysis of the 5'NCR in the 13 cases analyzed. HVR-1 and ISDR were amplified in 24 of 28 (86%) samples, which were positive for the 5'NCR. Efficient amplification was inversely related to the time of storage. The evolutionary changes of HVR-1 and ISDR were successfully analyzed by direct sequencing of amplificates from the explanted liver and from the sequential liver biopsies in a patient with HCV infection recurrence after transplantation. These observations indicate that paraffin-embedded liver tissue, even when stored for more than 20 years, is appropriate for advanced studies on the molecular biology of HCV.

Amino Acid Sequence↗
PubMed2002

Genomic structure of newly identified paralogue of RNA helicase II/Gu: detection of pseudogenes and multiple alternatively spliced mRNAs.

RNA helicase II/Gu (RH-II/Gu or DDX21) is a DEAD-box enzyme that localizes to the nucleoli and may be involved in ribosomal RNA synthesis or processing. It has two paralogues, RH-II/Gualpha and RH-II/Gubeta, both genes of which are on chromosome 10. Their similar genomic structures suggest the two genes arose by gene duplication. Both genes are expressed at higher levels in some normal human tissues compared to matching tumor tissues. Pseudogenes for RH-II/Gubeta exist on chromosomes 2, 3 and 4. No pseudogene was identified for RH-II/Gualpha. Both exon inclusion and exon skipping were found to post-transcriptionally regulate RH-II/Gubeta gene expression. No alternative splicing was identified for RH-II/Gualpha. Overall, the results suggest that the two paralogues of RH-II/Gu arose by gene duplication but the resulting genes are differentially regulated.

3T3 Cells↗
PubMed1997

An atypical verrucous carcinoma of the tongue arising in a patient with oral lichen planus associated with hepatitis C virus infection.

Recently, a clinical relationship between oral lichen planus (OLP) and hepatitis C virus (HCV) infection has been suggested, but the role of this virus on the course of OLP is unknown. We report an unusual lingual localisation of verrucous carcinoma arising in a patient with histologically confirmed OLP and HCV infection diagnosed with second generation enzyme-linked immunosorbent assay and recombinant immunoblot assay. Serum HCV-RNA detected using nested reverse transcriptase polymerase chain reaction (RT-PCR) confirm HCV replication. No classical risk factors associated with verrucous carcinoma were present.

Aged↗