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At least 487 records · Page 27Linked to original sources
PubMed2005

Differential expression of the duplicated cellular retinoic acid-binding protein 2 genes (crabp2a and crabp2b) during zebrafish embryonic development.

The cellular retinoic acid-binding protein 2 (CRABP2) is believed to be involved in regulating access of retinoic acid to nuclear retinoic acid receptors. We have determined the cDNA sequence and the genomic organization of the duplicated crabp2 gene (crabp2b) in zebrafish. The crabp2b cDNA was 522bp in length and encodes a polypeptide consisting of 146 amino acids. Radiation hybrid mapping assigned the crabp2b gene to zebrafish linkage group 19. The comparison of the mapped human CRABP2 gene, zebrafish crabp2a and zebrafish crabp2b genes revealed that human chromosome 1 has a syntenic relationship to zebrafish linkage groups 16 and 19. Reverse transcription-polymerase chain reaction (RT-PCR) detected crabp2b mRNA in total RNA extracted from whole adult zebrafish, but not in any of the adult zebrafish tissues examined. The crabp2a mRNA was detected in total RNA extracted from whole adult zebrafish, adult zebrafish muscle, testes, and skin and to a lesser extent in heart, ovary and brain. No crabp2a mRNA-specific product was detected in kidney, liver or intestine of the adult zebrafish. Whole mount in situ hybridization detected crabp2b and crabp2a mRNA in a number of structures known to require retinoic acid signaling during embryonic development. The crabp2b mRNA was detected in the central nervous system, branchial arches, pectoral fins, retina (dorsal to the lens), epidermis and otic vesicle of the developing zebrafish. The crabp2a transcripts were detected by whole mount in situ hybridization in the central nervous system, epidermis, proliferative zone of the retina, intestinal bulb, oesophagus, pectoral fins and branchial arches during zebrafish embryonic development.

Amino Acid Sequence↗
PubMed2001

Rapid methods to extract DNA and RNA from Cryptococcus neoformans.

Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (10(9) cells), RNA appears undegraded, making it suitable for molecular manipulations.

Centrifugation↗
PubMed1977

Specificity of antitumor immune reactions mediated by xenogeneic immune RNA.

Evidence that xenogeneic immune RNA (I-RNA) mediated specific cytotoxic immune responses against human tumor-associated antigens was obtained from in vitro studies in two autologous melanoma systems. In these systems, malignant melanoma target cells, matching normal fibroblast target cells, lymphocyte effector cells, and melanoma and normal skin tissue used to immunize RNA donor animals were derived from the same autochthonous hosts. When incubated with autologous lymphocytes, I-RNA extracted from the lymphoid organs of donor animals immunized with melanoma tissue mediated immune reactions against autologous melanoma target cells in vitro. I-RNA from animals immunized with normal skin tissue from autochthonous hosts did not increase the cytotoxicity of autologous lymphocytes for autologous melanoma cells. Using autologous fibroblasts as target cells, we detected no increase in cytotoxicity when autologous lymphocytes were incubated with RNA from animals immunized either with melanoma tissue or normal skin tissue from the autochthonous host. By contrast, when allogeneic lymphocytes were used as effector cells, RNA extracted from animals immunized either with melanoma tissue or normal skin mediated cytotoxic immune reactions against melanoma target cells and normal fibroblast target cells derived from the same patient.

Antigens, Neoplasm↗
PubMed1991

The location of the 5' end of the potato leafroll luteovirus subgenomic coat protein mRNA.

Northern blot analysis of nucleic acid from potato plant tissues and tobacco protoplasts infected with a Scottish isolate of potato leafroll luteovirus (PLRV) detected the 6 kb genomic RNA and one subgenomic RNA species of about 2.7 kb; RNA extracted from virus particles contained only the genomic species. Blotting with small defined probes suggested that the location of the 5' end of the subgenomic RNA was between 2380 and 2510 nucleotides from the 3' end of the PLRV genome (between 3370 and 3500 nucleotides from the 5' end of PLRV Dutch isolate RNA). When RNA extracted from PLRV-infected or mock-inoculated protoplasts was used as the template for primer extension using primers complementary to the sequence at, or upstream of, the initiation codon of the coat protein gene, a single major infection-specific product was detected. A primer complementary to the sequence between 162 and 179 nucleotides upstream of the coat protein AUG yielded a product of 56 nucleotides. Thus, the subgenomic RNA has a leader sequence of 212 nucleotides, is 2505 nucleotides in length and starts at a position equivalent to 3376 nucleotides from the 5' end of the PLRV-Dutch genome, 11 nucleotides upstream of the termination codon of the putative polymerase gene. The nucleotide sequence immediately downstream of this position closely resembles that of the 5' end of the PLRV genomic RNA.

Base Sequence↗
PubMedDate not supplied

A simple single-tube procedure of PCR assay for the detection of hepatitis C virus RNA.

We developed a simple single tube procedure using the PCR (polymerase chain reaction) for the detection of hepatitis C virus RNA. The entire reaction from RNA extraction to PCR occurred in one tube; this was made possible by the use of more than 1,000 micrograms/ml of proteinase K for RNA extraction, instead of the acid-guanidinium thiocyanate-phenol-chloroform method. All necessary reagents were added to the tube, and PCR products were not removed from the tube until the end of PCR, although opening of the tube during the procedure could not be avoided. Therefore, cross-contamination which might theoretically take place during transfer of products between tubes never occurred. This method detected about 1 chimpanzee infectious dose of HCV in H strain plasma.

Base Sequence↗
PubMed1997

Hepatitis C virus infection of salivary gland epithelial cells. Lack of evidence.

BACKGROUND: Hepatitis C virus genome (HCV-RNA) has been detected in whole salivary gland tissue of chronically infected patients. However, contamination of the tissue by plasma or blood cells was not excluded by the previous reports. AIMS: To assess whether HCV infects the salivary gland epithelial cells in patients with chronic HCV liver disease. METHODS: Twenty unselected patients with chronic active hepatitis (11 cases) or active cirrhosis (nine cases) were examined. Serum and saliva samples were obtained from all patients, 12 of whom (seven, chronic active hepatitis; five, active cirrhosis) underwent salivary gland biopsy. PCR for HCV-RNA was performed on RNA extracted from serum, saliva and salivary gland epithelial cells collected by isokinetic gradient separation after trypsin digestion of whole salivary gland tissue. Saliva samples were also examined for the presence of secretory IgA anti-HCV by gel chromatography and ELISA testing. RESULTS: HCV-RNA was detected in all sera with titers ranging from 5.42 x 10(5) genome equivalents/ml to 123.2 x 10(5) genome equivalents/ml. Thirteen patients were infected with genotype 1b, four patients had genotype 1a, two patients had genotype 2a and one patient was unclassifiable. Low titer HCV-RNA (<2 x 10(5) genome equivalents/ml) was detected in 3/20 saliva samples (15%) from highly viremic patients infected with 1b genotype. RNA extracted from salivary gland epithelial cells consistently tested negative for HCV-RNA. In addition, all saliva specimens tested negative for secretory-IgA (S-IgA) anti-HCV, even after a 10-fold concentration of the samples. CONCLUSIONS: There was no evidence that HCV infects the salivary gland epithelial cells in our viremic patients with HCV chronic liver disease. Low level HCV-RNA in saliva is most probably due to virus spillover from blood.

Adult↗
PubMed1995

In situ localization and quantification of mRNA for 92-kD type IV collagenase and its inhibitor in aneurysmal, occlusive, and normal aorta.

Ninety-two-kilodalton type IV collagenase (MMP-9) is present in aortic aneurysms and may be important to the pathogenesis of this disease. Alteration in expression of MMP-9 or its inhibitor, the tissue inhibitor of metalloproteinase type 1 (TIMP-1), could increase degradation of extracellular matrix and lead to aneurysm formation. The purpose of this study was (1) to measure tissue levels of MMP-9 and TIMP-1 mRNA in aneurysmal (AAA), atherosclerotic occlusive (AOD), and normal (NL) human infrarenal aorta; (2) to test for their expression by cultured AAA and NL vascular smooth muscle cells (VSMCs); and (3) to locate in situ the cells responsible for mRNA production within AAA, AOD, and NL aortic wall. Total RNA extracted from AAA (n = 8), AOD (n = 8), and NL (n = 7) tissue was subjected to Northern analysis. Signals for MMP-9 and TIMP-1 were normalized to alpha-tubulin. Mean values +/- SEM were compared by ANOVA. NL and AAA VSMCs were cultured, passaged, and grown to confluence before RNA extraction and Northern analysis. In situ hybridization with digoxigenin-labeled RNA probes localized cells responsible for MMP-9 and TIMP-1 mRNA expression within sections of AAA (n = 5), AOD (n = 2), and NL (n = 2) aorta. MMP-9 mRNA levels were significantly greater in AAA (0.855 +/- 0.180) than NL (0.046 +/- 0.23) (P < .02), but differences between AOD (0.406 +/- 0.196) and AAA or AOD and NL were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneurysm↗
PubMed2002

Differential amplifying RT-PCR: a novel RT-PCR method to differentiate mRNA from its DNA lacking intron.

A major problem with reverse transcription-polymerase chain reaction (RT-PCR) is that the products amplified from RNA and DNA are not distinguishable. The use of a primer set targeted for an intron-containing sequence or RNA extract without contaminating DNA has been established to overcome this problem. However, an intron sequence does not always exist in the target region and complete removal of DNA during RNA extraction is not practical. For these reasons, we developed differential amplifying RT-PCR (DART-PCR) based on differences in reaction temperatures between RT and PCR, as well as in architectures between RNA and DNA. DART-PCR was designed to differentiate products amplified from RNA and DNA regardless of the presence of introns. DART-PCR can be readily applied for diagnosis of active infection of human cytomegalovirus by detection of glycoprotein B mRNA, which gene lacks introns. Other advantages of DART-PCR were as follows: there is no need for separation of RNA from DNA, simultaneous/differential detection in a single tube and possibility of determination of relative amounts of RNA and DNA.

Cell Line↗
PubMed1979

In vitro transfer of cellular immunity in experimental allergic orchitis by means of immune RNA.

Ribonucleic acid (RNA) extracts were obtained from lymph nodes of guinea-pigs that had previously been immunized with a purified testicular antigen emulsified in Freund's complete adjuvant. The RNA extracts were incubated with normal guinea-pig peritoneal exudate cells (NGP-PEC). After treatment, the NGP-PEC cells showed specific inhibition of migration when tested with the specific antigen. No inhibition of migration was observed when cells were tested with an unrelated antigen or when the cells were incubated with RNA obtained from animals immunized with adjuvant alone. Failure of inhibition of migration was also observed when the 'immune' RNA was degraded with RNAse. The appearance of this I-RNA in the immunized guinea-pigs correlates with the appearance of delayed hypersensitivity in vivo.

Animals↗
PubMed1994

[Comparison of histology, immunohistochemistry, RT-PCR, in situ hybridization, and in situ RT-PCR for demonstration of hepatitis C virus in paraffin-embedded liver biopsies].

To compare immunohistochemical and molecular methods for the detection of hepatitis C virus (HCV) infection in archival liver biopsies we analyzed formalin-fixed and paraffin-embedded liver specimens of 10 patients with serologically confirmed HCV infection. Methods employed included indirect FITC-immunofluorescence, reverse-transcriptase polymerase chain reaction (RT-PCR) using extracted RNA and Southern blotting with chemiluminescence-based detection, non-radioactive in situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes, direct in situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and indirect in situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. (Gastroenterology 1993; 104-595) together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies are representative; (2) the commercially available mAB TORDJI-22 appears to cross-react with non-HCV epitopes; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV-RNA by RT-PCR of extracted total RNA is more sensitive than indirect in situ RT-PCR or ISH; and (5) direct in situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. Further studies are required to define both optimal methods for sample processing and improvements of protocols, in order to increase detection sensitivity and specificity of HCV infection by immunohistochemical and molecular methods.

Biopsy↗
PubMed1981

Transcription and processing of adenoviral RNA by extracts from HeLa cells.

Concentrated extracts from HeLa cells prepared by the procedure of Sugden and Keller [Sugden, B. & Keller, W. (1973) J. Biol. Chem. 248, 3777-3788] can synthesize RNA molecules of discrete lengths in the presence of DNA templates carrying complete viral transcription units. Accurate initiation of transcription was shown to occur at the "major late" promoter and at the promoter for the "early region III" transcription unit of the adenovirus 2 genome. A cloned fragment of adenovirus 2 DNA containing the early region III transcription unit directs the synthesis of discrete RNA species, many of which correspond in length to spliced early region III mRNAs isolated from adenovirus 2-infected HeLa cells. As shown by hybridization/nuclease S2 analysis, RNA splicing and the formation of specific 3' ends are also taking place in vitro.

Adenoviruses, Human↗
PubMed1993

Isolation of RNA from cells in culture using Catrimox-14 cationic surfactant.

Traditional RNA isolation methods use chaotropic agents and anionic detergents to lyse cells and solubilize nucleic acids. In contrast, the cationic surfactant, Catrimox-14, lyses cells and simultaneously precipitates RNA, thereby protecting it from RNases. We describe and compare four methods for extracting RNA from cultured cells that differ in the technique used to extract the RNA from the precipitate. The first uses a high-salt solution (guanidinium isothiocyanate). In the second, the RNA is extracted with a polar solvent (formamide). The third involves conversion of the RNA to the sodium salt by treatment of the precipitate in situ with sodium acetate in ethanol. The fourth uses 2 M lithium chloride to convert the RNA in the pellet to the lithium salt in situ. We applied these methods to human leukemia cells growing in culture and each method resulted in excellent yields of RNA (typically 23 micrograms/million K562 cells, 13 micrograms/million HL-60 cells) over a wide range of cell concentrations (1 x 10(5) - 3 x 10(7)/ml) and of good to excellent quality as judged by agarose electrophoresis and UV absorbance data (OD260/280 1.90-2.05). The advantages and limitations of each method are discussed.

Acetates↗
PubMed1978

Immunofluorescent detection of nuclear double-stranded RNA in situ in Vero and mosquito cells.

Double-stranded RNA (dsRNA) was detected in situ by indirect immunofluorescence with antibodies to dsRNA. It was seen in nuclei of Vero and Aedes albopictus cells, but not in BHK cells, KB cells, chick embryo fibroblasts, or HeLa cells. Reactive dsRNA was present in the nucleoplasm, but not in nucleoli or cytoplasm. Extracted RNA from the whole cell contained from 0.08 percent (BHK) to 0.46 percent (HeLa) dsRNA, as estimated by serological methods. This dsRNA, found in molecules having the size distribution of heterogeneous nuclear RNA, did not renature rapidly after denaturation. The quantity of dsRNA in total extracted RNA did not correlate with the presence or absence of nuclear staining in situ.

Cell Line↗
PubMed2002

Fully automated detection of hepatitis C virus RNA in serum and whole-blood samples.

In this study, we established a fully automated molecular assay for qualitative detection of hepatitis C virus (HCV) in serum and whole-blood samples and compared it with conventional molecular assays, including manual HCV RNA extraction protocols. Whole-blood samples were collected from patients with and without chronic HCV infection in EDTA tubes and nucleic acid stabilization tubes (NASTs). Prior to HCV RNA extraction, the HCV Internal Control (IC), derived from the COBAS AMPLICOR HCV test, version 2.0 (Roche Molecular Diagnostics), was added. The new assay was based on an automated extraction protocol on the MagNA Pure LC instrument (Roche Applied Science), followed by automated reverse transcription, amplification, hybridization, and detection on the Cobas Amplicor analyzer (Roche Molecular Diagnostics). The detection limit of the new assay was found to be similar to those of conventional molecular assays. In clinical samples, 100% agreement between the new assay and conventional methods was observed. The introduced amount of IC was detected in 45 of 45 serum samples, 41 of 45 EDTA tube whole-blood samples, and 43 of 45 NAST whole-blood samples. Retesting led to more frequent IC detection. The fully automated molecular assay was found to be suitable for detection of HCV RNA in different kinds of sample materials. It may be recommended for use in the high-throughput routine molecular diagnostic laboratory.

Hepacivirus↗