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At least 1,189 records · Page 66Linked to original sources
PubMed2000

Expression of transforming growth factor-alpha mRNA in livers of patients with chronic viral hepatitis and hepatocellular carcinoma.

BACKGROUND: Transforming growth factor-alpha (TGFalpha) is an important autocrine growth factor of hepatocytes. The authors evaluated the roles of TGFalpha in chronic viral hepatitis (CVH) and hepatocellular carcinoma (HCC). METHODS: The authors measured the amounts of TGFalpha mRNA in liver tissues from 18 patients with HCC, 31 patients with CVH, and 7 normal controls. " Hot-start" reverse transcription-polymerase chain reaction (RT-PCR) using oligo-dT and specific primers detected TGFalpha mRNA in total cellular RNA extracted from liver tissues. The levels of TGFalpha mRNA were determined by the end point titers of serial, two-fold dilutions of cDNA. The amounts of hepatitis B virus RNA (HBV-RNA) in livers of patients with chronic hepatitis B also were measured by Northern blot hybridization. RESULTS: TGFalpha mRNA levels were extremely higher in patients with HCC compared with patients with CVH and normal controls, and the levels in patients with CVH also were elevated compared with normal controls. The levels of TGFalpha mRNA were overexpressed in the underlying livers of patients with HCC compared with patients with CVH, although they were lower than those found in HCC tissues. The levels of TGFalpha mRNA were higher in samples from patients with chronic hepatitis B than in samples from patients with chronic hepatitis C. The levels of TGFalpha mRNA were not correlated with serum alanine aminotransferase or HBV-RNA levels in liver tissues in patients with chronic hepatitis B. However, the expression of TGFalpha mRNA tended to be higher in the livers of patients with raised serum alpha-fetoprotein levels. CONCLUSIONS: The overexpression of TGFalpha mRNA in the liver seems to be associated with the regeneration of hepatocytes rather than hepatic necrosis or viral replication. Also, it may be related closely to the development or progression of HCC, especially in the livers of patients with chronic hepatitis B.

Adult↗
PubMed1984

The genome of simian hemorrhagic fever virus.

Techniques are described for preparing intact Simian Hemorrhagic Fever (SHF) virus RNA. SHF RNA extracted by proteinase K digestion in the presence of sodium dodecyl sulphate (SDS) has a sedimentation coefficient of 49 S compared with a reference figure of 47 S for Sindbis RNA. Purified SHF RNA in cesium sulphate gradient has a buoyant density of 1.63 g/ml similar to that of Sindbis RNA. This result leads to the conclusion that SHF RNA is single stranded. This is supported by results on RNAse sensitivity and analyses on sucrose gradients under different ionic strength conditions. Electrophoresis analyses on both hydroxymethylmercuric acid or formaldehyde containing gels, gave a value of about 5.5 X 10(6) daltons for the molecular weight of SHF RNA. No evidence of subunit structure was found. From these results, we conclude that the SHF virus genome is a single continuous chain of about 15,000 ribonucleotides.

Animals↗
PubMed1984

Detection of genomic-length soybean mosaic virus RNA on polyribosomes of infected soybean leaves.

Soybean mosaic virus (SMV)-related RNAs were examined in both polyribosomal and nonpolyribosomal fractions of systemically infected soybean leaves. Viral RNAs were detected by Northern blot hybridization analysis using two cloned SMV-cDNAs representing different regions of the viral genome as hybridization probes. Genomic length SMV-RNA (Mr of 3.3 X 10(6] was found in specific association with EDTA-sensitive polyribosomes of infected leaves, indicating that it functions as a messenger RNA in these cells. A smaller SMV-related RNA (Mr of 1.6 X 10(6] was sometimes detected in the polyribosomal fraction; however, reconstruction experiments indicate that this RNA is a breakdown product of the genomic-length RNA, generated during cell fractionation or RNA extraction. Two other SMV-related RNAs with Mr of 2.0 and 0.78 X 10(6) were sometimes detected in infected cells and were not generated from genomic SMV-RNA or intact virus particles in reconstruction experiments. However, these RNAs were exclusively associated with the EDTA-resistant, nonpolyribosomal fraction of infected cells. These data suggest that genomic-length SMV-RNA is the only viral RNA which is translated in these infected plants.

Cloning, Molecular↗
PubMed2004

Expression levels for many genes in human peripheral blood cells are highly sensitive to ex vivo incubation.

Monitoring of gene and protein expression in peripheral blood cells has significant potential for improving the diagnosis and therapy of many human diseases. As genomic-scale microarray and proteomic technologies are applied to peripheral blood, it is important to consider the variables that may affect interpretation of data. Here we report experiments performed to identify genes that are particularly sensitive to ex vivo handling prior to RNA extraction for gene expression microarrays or quantitative real-time RT-PCR assays. We examined Affymetrix gene expression in samples from eight normal individuals where blood was processed for RNA either immediately after blood draw or the next day following overnight incubation. These studies identified hundreds of genes that are sensitive to ex vivo handling of blood, and suggest that this is an important variable to consider when designing and interpreting human PBMC experiments.

Blood Specimen Collection↗
PubMed1974

Multiple gene sites for 5S and 18 plus 28S RNA on chromosomes of Glyptotendipes barbipes (Staeger).

Ribosomal RNAs (28 + 18S and 5S) and 4S RNA extracted from the chironomid Glyptotendipes barbipes were iodinated in vitro with (125)I and hybridized to the salivary gland chromosomes of G. barbipes and Drosophila melanogaster. Iodinated 18 + 28 S RNA labeled three puffed sites with associated nucleoli on chromosomes IR, IIL, and IIIL of G. barbipes and the nucleolar organizer of Drosophila. Labeled 5S RNA hybridized to three sites on chromosome IIIR, two sites on chromosome IIR and one site in a Balbiani ring on chromosome IV of Glyptotendipes. Most of the label produced by this RNA was localized seven bands away from the centromere on the right arm of chromosome III, and we consider this to be the main site complementary to 5S RNA in the chironomid. This same RNA preparation specifically labeled the 56 EF region of chromosome IIR of Drosophila which has been shown previously to be the only site labeled when hybridized with homologous 5S RNA. Hybridization of G. barbipes chromosomes with iodinated 4S RNA produced no clearly localized labeled sites over the exposure periods studied.

Animals↗
PubMed2001

Effects of dietary polyunsaturated fatty acids on in vivo splenic cytokine mRNA expression in layer chicks immunized with Salmonella typhimurium lipopolysaccharide.

Effects of dietary polyunsaturated fatty acids (PUFA) on immune responses in poultry have been reported. However, effects on the underlying mechanisms, such as the role of cytokines, have not been documented because the necessary tools were lacking. Recently, primer sets for chicken interleukin (IL)-1beta, IL-2, interferon-gamma (IFN-gamma), myelomonocytic growth factor (MGF), and transforming growth factor (TGF)-beta2 have become available. Therefore, in the present study we first examined the in vivo effects of an inflammatory challenge with Salmonella typhimurium lipopolysaccharide (LPS) on cytokine profiles in growing laying-type chicks. Second, we examined whether dietary fat sources affected the observed cytokine profiles. Two hundred forty chicks were assigned in a 2 x 4 factorial design of treatments, with injection with LPS or saline and dietary fat source as factors. Factors were i.v. injection with S. typhimurium LPS or saline (control) and four dietary fat sources: corn oil, linseed oil, menhaden oil, and tallow. Two hours after injection, birds were killed, and their spleens were removed for RNA extraction. Reverse transcription polymerase chain reactions with primer sets for chicken IL-1beta, IL-2, IFN-gamma, MGF, TGF-beta2, and beta-actin were performed with RNA samples pooled by pen. The expression of cytokine mRNA was expressed relative to the level of beta-actin mRNA. Interleukin-1 (P < 0.001), MGF (P < 0.0001), IL-2 (P < 0.001), and IFN-gamma (P < 0.001) mRNA expressions were enhanced by challenge with LPS. Immunization treatment had no effect on TGF-beta2 or beta-actin expression. Dietary treatment did not affect mRNA expression of IL-1, MGF, IFN-gamma, TGF-beta2, or beta-actin. Interleukin-2 expression in LPS-injected birds that were fed the fish-oil-enriched diet was enhanced (P = 0.05). The present study indicates that in vivo effects of immune challenge on cytokine mRNA expression can be measured in poultry. The observation that mRNA level of IL-2, but not the mRNA levels of IFN-gamma or MGF, is enhanced by dietary fish oil at 2 h suggests that dietary PUFA at this moment initially affected naïve T lymphocytes.

Actins↗
PubMed1993

Myf-4 does not mediate AChR receptor subunit mRNA expression in thymic tissues.

We investigated whether a MyoD gene family member plays a role in the expression of AChR or AChR-like proteins in human thymus. We amplified from thymic tissue, mRNA of subunits of the fetal- and adult-type AChR, and Myf-4, a gene product that appears to regulate AChR expression. RNA extracted from paraffin-embedded thymic tissue of five myasthenics and five nonmyasthenics was subjected to reverse transcription followed by polymerase chain reaction using primers specific for these mRNAs. Thymic RNA from eight of ten patients contained transcripts of the alpha- and epsilon-subunits (specific for the adult-type AChR). Presence of these transcripts did not correlate with thymic pathology or clinical presentation. No gamma-subunit (specific for the fetal-type AChR) or Myf-4 transcripts were found. Our results indicate that mRNA for the adult-type AChR is expressed in thymic tissue and is expressed by mechanisms not involving Myf-4. Thymic AChR subunits may be the primary epitope that initiates the immune response in MG against the adult-type AChR.

Adult↗
PubMed2005

Evaluation of the COBAS Hepatitis C Virus (HCV) TaqMan analyte-specific reagent assay and comparison to the COBAS Amplicor HCV Monitor V2.0 and Versant HCV bDNA 3.0 assays.

Performance characteristics of the COBAS hepatitis C virus (HCV) TaqMan analyte-specific reagent (TM-ASR) assay using the QIAGEN BioRobot 9604 for RNA extraction were evaluated and compared to the COBAS Amplicor HCV Monitor V2.0 (Amplicor) and Versant HCV bDNA 3.0 (Versant) assays using clinical samples. Calibration of TM-ASR using Armored RNA allowed determination of the distribution of HCV RNA in clinical samples, using 22,399 clinical samples. Limit of detection, linearity, and inter- and intraassay assay precision were determined for the TM-ASR assay using multiple clinical specimen panels across multiple determinations. Genotype specificity for the TM-ASR assay was determined using samples with different HCV RNA genotypes evaluated and compared against predetermined results. Contamination control of the TM-ASR assay was evaluated using pools of HCV RNA-positive and -negative samples tested in a checkerboard pattern over 12 runs of 96 samples. Correlation of the TM-ASR, Amplicor, and Versant assays was determined using 100 paired clinical samples and Deming regression analysis. The TM-ASR performed well with respect to linearity, precision, and contamination control. The correlation between TM-ASR and the Amplicor and Versant assays was poor, with large differences between assay results for individual samples. Calibration of the TM-ASR assay with Armored RNA allowed for a wide dynamic range and description of the distribution of HCV RNA in clinical samples.

DNA, Viral↗
PubMed1977

DNA synthesized in the hepatitis B Dane particle DNA polymerase reaction.

Radioactive DNA was prepared in extensive (4 h) Dane particle DNA polymerase reactions. In different experiments the amount of new DNA, determined by the amount of nucleotide incorporation into an acid-insoluble form, was between 29 and 45% of the total circular DNA isolated from Dane particle preparations after the reaction. DNA reassociation kinetics were used to determine the complexity of the newly synthesized DNA. In different experiments COt1/2 values, corresponding to between 625 and 1,250 nucleotide pairs, were obtained for the radioactive Dane particle DNA. These results suggest that a unique region (or regions), corresponsing to approximately one-fourth to one-half of the circular Dane particle DNA template, was copied one time during the reaction. DNA and RNA extracted from hepatitis B virus-infected liver but not from uninfected liver accelerated the rate of reassociation of radioactive DNA from Dane particles. These Dane particle DNA base sequences were found in alkali-stable, rapidly sedimenting DNA from infected liver as well as in DNA sedimenting at a rate similar to the DNA extracted from Dane particles. These findings are consistent with Dane particle DNA being hepatitis B virus DNA that is integrated into high-molecular-weight cellular DNA and transcribed into RNA in infected liver.

Base Sequence↗
PubMed2000

Detection of sialomucin complex (MUC4) in human ocular surface epithelium and tear fluid.

PURPOSE: To evaluate human ocular surface epithelium and tear fluid for the presence of sialomucin complex (MUC4), a high-molecular-weight heterodimeric glycoprotein composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis assays were used to identify sialomucin complex RNA in ocular surface epithelia. Immunoprecipitation and immunoblot analysis were used to identify immunoreactive species in human tears and in the corneal and conjunctival epithelia using antibodies specific for carbohydrate and peptide epitopes on the sialomucin complex subunits. Immunofluorescence staining was used to detect sialomucin complex in frozen sections and impression cytology specimens of human cornea and conjunctival epithelia. RESULTS: ASGP-1- and ASGP-2-specific sequences were amplified from RNA extracted from both conjunctival and corneal epithelial biopsies by RT-PCR. Sialomucin complex transcripts were also detected in these tissues by Northern blot analysis, with a greater level of RNA detected in the peripheral than the central corneal epithelium. Sialomucin complex was immunoprecipitated from tear fluid samples and both corneal and conjunctival epithelia and detected by immunoblot analysis with specific anti-ASGP-1 and anti-ASGP-2 antibodies. The ASGP-1 peptide antibody HA-1 stained the full thickness of the corneal and conjunctival epithelia. In contrast, antibody 15H10, which reacts against a carbohydrate epitope on ASGP-1, stained only the superficial epithelial layers of these tissues. No staining was observed in the conjunctival goblet cells. CONCLUSIONS: Sialomucin complex was originally identified in rat mammary adenocarcinoma cells and has recently been shown to be produced by the ocular surface epithelia of rats. Furthermore, it has been identified as the rat homologue of human MUC4 mucin. The present studies show that it is expressed in the stratified epithelium covering the surface of the human eye and is present in human tear fluid. Expression of a carbohydrate-dependent epitope on the mucin subunit (ASGP-1) of sialomucin complex occurs in a differentiation-dependent fashion. Sialomucin complex joins MUC1 as another membrane mucin produced by the human ocular surface epithelia but is also found in the tear fluid, presumably in a soluble form, as found on the rat ocular surface.

Antibody Specificity↗
PubMed2003

Gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells.

OBJECTIVE: To study the gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells. METHODS: Total RNA extracted from human bone marrow derived mesenchymal stem cells and tendon cells underwent reverse transcription, and the products were labeled with alpha-(32)P dCTP. The cDNA probes of total RNA were hybridized to cDNA microarray with 1176 genes, and then the signals were analyzed by Atlas Image analysis software Version 1.01a. RESULTS: Fifteen genes associated with cell proliferation and signal transduction were up-regulated, and one gene that takes part in cell-to-cell adhesion was down-regulated in tendon cells. CONCLUSION: The 15 up-regulated and one down-regulated genes may be beneficial to the orientational differentiation of mesenchymal stem cells into tendon cells.

Bone Marrow Cells↗
PubMed1991

[Quantification of mRNA coding for enterocyte fatty acid binding proteins (FABP) in rats: effect of high lipid diet and starvation].

We have tested in adult rat the specificity and the sensitivity of two cDNA probes derived from recombinant plasmids pJG19 and pJG418. The Northern blot analysis of total RNA extracted from small bowel reveals 0.9 and 0.7 kb mRNA corresponding respectively to the I and L-FABPc mRNA. A linear relation occurs between the amounts of input total RNA and the quantities of specific mRNA found by hybridization with cDNA probes (r = 0.991 and r = 0.998 for I and L-FABPc respectively). We have also evaluated the effect of drastic nutritional status: a high fat diet containing 45% of lipids and a starvation of three days. A two fold increase in I and L-FABPc mRNA occurs in starved rats. By contrast, only the I-FABPc mRNA are slightly enhanced after the hyperlipidic diet (50%). These preliminary results, which must be confirmed by further experiments, suggest a differential nutritional regulation of I and L-FABPc gene expression in rat intestine.

Animals↗
PubMed2006

Impact of RNA degradation on gene expression profiles: assessment of different methods to reliably determine RNA quality.

DNA microarray technology enables investigators to measure the expression of several 1000 mRNA species simultaneously in a biological specimen. However, the reliability of the microarray technology to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. Thus, it is of critical importance to standardize sample-handling protocols and to perform a quality assessment of RNA preparations. In this report, 59 human tissue samples were used to evaluate the relationships between RNA quality and gene expression. From Affymetrix GeneChip array data analysis of these samples, we compared the performance of the 28S/18S ratio, two computer methods (RIN and degradometer) and our in-house RNA quality scale (RQS) in assessing RNA quality. The optimal RNA reliability threshold was determined for each method using statistical discrimination measures. We showed that RQS, RIN and degradometer have a similar capacity to detect reliable RNA samples whereas the 28S/18S ratio leads to a misleading categorization. Furthermore, we developed a new approach, based on clustering analyses of full chip expression, to control RNA quality after hybridization experiments. The combination of these methods, allowing monitoring of RNA quality prior to and after the hybridization experiments, ensured reliable and reproducible microarray data.

Cluster Analysis↗
PubMed1975

Bacteriophage SPO1 DNA- and RNA-directed protein synthesis in vitro: comparison with in vivo control.

A cell free protein synthesizing system, derived from E. coli, is shown to be a quantitiative assay system for messenger RNA extracted from B. subtilis infected with bacteriophage SPO1. DNA-directed protein synthesis in this system is shown to be limited mostly to those proteins whose messages are contained in early RNA. A phage induced enzyme, dCMP deaminase, is shown to be dependent on appearance of a class mRNA made in vivo in response to new initiations of transcription dependent on prior synthesis of phage induced protein. Control of the enzyme synthesized in the cell free system is contrasted with in vivo control, and an estimate of "read-through" by RNA polymerase in vitro is presented.

Bacillus subtilis↗
PubMed1981

The RNA of the human syncytium-forming (foamy) virus.

Human syncytium-forming (foamy) virus was labeled with 3H-uridine and banded isopycnically in sucrose gradients (buoyant density = 1.16 to 1.18 g/cm3). Viral RNA extracted from the banded virus was analyzed either by rate zonal separation in sucrose gradients or by polyacrylamide-agarose gel electrophoresis. The results indicated that purified HSFV contains a 60S RNA component plus several smaller molecular weight RNA components. On dissociation with heat, smaller RNA structures were released from the 60S component. These results indicate that the genome of HSFV, like the other members of the Retroviridae family, is composed of an aggregate of several RNA species.

Centrifugation, Isopycnic↗
PubMed1994

Rapid detection of African horsesickness virus by the reverse transcriptase polymerase chain reaction (RT-PCR) using the amplimer for segment 3 (VP3 gene).

The complete sequence of the major core protein (VP3) gene of African horsesickness virus serotype 4 (AHSV-4; vaccine strain) was determined by analysis of a complete cDNA clone representing segment 3. The RNA was 2,789 bp long and a comparison of its sequence with that of bluetongue virus serotype 10 (BTV-10) revealed 58% nucleotide similarity. Based on these data, the reverse transcriptase-polymerase chain reaction (RT-PCR) technique was applied to the specific detection of AHSV using a pair of primers designed for AHSV-4 VP3 gene. Approximately 230 bp of PCR products were amplified by RT-PCR from the total RNA extracts (mRNA and dsRNA) of Vero cells infected with eight serotypes of AHSV. No product was observed analogous to other orbiviruses. The supernatant of the infected cell culture fluid without any RNA purification was also suitable as a template for RT-PCR after being denatured at 94 degrees C for 5 min. The sensitivity of this method was between 10(0) and 10(1) TCID50 when viral RNA from the supernatant of infected cell culture was subjected to RT-PCR. The whole procedure for detecting the virus RNA by RT-PCR could be carried out within 5 h. The RT-PCR with AHSV VP3 gene as a target was found to be a simple, highly specific and sensitive assay for AHSV.

African Horse Sickness Virus↗
PubMed2003

Analysis of in vitro immunization: generation of cytotoxic T-lymphocytes against allogeneic melanoma cells with tumor lysate-loaded or tumor RNA-transfected antigen-presenting cells.

In this study we compared several protocols for in vitro induction of cytotoxic T lymphocytes (CTL) from naïve HLA-A*0201(+) peripheral blood mononuclear cells (PBMC) against allogeneic melanoma cells. As immunization material we compared: (1) the lysate of apoptotic or live melanoma tumor cells [MSM-M14 (M14) and MSM-M3 (M3)]; and (2) total RNA extracted from the same melanoma cell lines, either unconjugated or conjugated with a charged carrier (DMRIE-C). Overall killing activity was very similar in CTL induced by tumor lysate or RNA. CTL induced by both methods preferentially killed an HLA class I-matched M14 melanoma cell line rather than HLA class I-unmatched M3 cells. Cytotoxicity could be partially blocked by anti-HLA class I antibodies. There were no significant differences in cytotoxicity and in other clonal characteristics in CTL lines induced by a lysate of apoptotic bodies as compared to lines induced by lysate of viable cells. However, CTL induced by DMRIE-C-bound total RNA demonstrated superior cytotoxicity when compared with CTL induced by unconjugated total RNA. Polyclonal CTL induced by tumor lysate contained a substantial percentage of tyrosinase(368-376 370N) tetramer-positive cells and demonstrated specific killing activity against tyrosinase(368-376 370N) peptide-labeled T2 cells, comparable to cytotoxicity of the CTL developed against this peptide alone. In contrast, there were no detectable tyrosinase(368-376 370N)-tetramer positive cells and no specific anti-tyrosinase peptide(368-376 370N) response in polyclonal CTL induced by immunization with tumor RNA. These data demonstrate that both total tumor RNA and tumor lysate are effective for inducing of cytotoxic anti-melanoma CTL, but tyrosinase(368-376 370N) specific cells were detected only in lysate-induced CTL cultures. This suggests that nature of the antigens present in tumor lysate might be different from those in tumor RNA.

Antigen-Presenting Cells↗
PubMedDate not supplied

Changes in the pattern of RNA synthesis in mouse limb cartilage during early development.

Patterns of RNA synthesis using tritiated uridine were studied in organ cultures of mouse embryonic limb cartilage and epiphyseal cartilage from mice 5 days of age. The cold phenol-sodium dodecyl sulfate extracted RNA was characterized by methylated albumin kieselguhr chromatography and polyacrylamide gel electrophoresis. The general pattern of synthesis indicates the persistence of primarily low molecular weight tRNA and a diminishing synthesis of rRNA in the older cartilage. Data show that the increased radioactivity in the low molecular weight fraction is not due to the accumulation of rRNA breakdown products. These findings are discussed in relation to other age-related studies on RNA biosynthesis.

Animals↗