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At least 1,495 records · Page 83Linked to original sources
PubMed1983

Amplification and hormone-regulated expression of a mouse mammary tumor virus-Eco gpt fusion plasmid in mouse 3T6 cells.

Mouse 3T6 cells were transformed with a chimeric DNA plasmid, pSVMgpt, in which the mouse mammary tumor virus (MMTV) promoter was fused to the Escherichia coli gene encoding xanthine-guanine phosphoribosyl transferase (Eco gpt). The transformants exhibited glucocorticoid-inducible expression of Eco gpt. With limiting xanthine concentrations, conditions were established in which cell growth became hormone dependent. Cells selected for their ability to grow in limiting concentrations of both xanthine and glucocorticoids contained amplified levels of Eco gpt DNA, and expression of Eco gpt remained glucocorticoid inducible in these amplified cells. Thus, amplification of the MMTV promoter region in itself did not abolish hormonal responsiveness of a gene. In addition to increased levels of Eco gpt DNA, some of the selected cells also exhibited increased levels (two- to threefold) of glucocorticoid receptors. Lastly, we found that excessive expression of Eco gpt is toxic to 3T6 cells; by maintaining low hormone levels and, therefore, low levels of expression, we were able to select cells with amplified Eco gpt. Thus, the MMTV promoter may be of general utility in expressing genes whose products may be lethal if they are produced in excessive quantities.

Animals↗
PubMed1990

Comparison of cytokine effects on mouse pancreatic alpha-cell and beta-cell lines. Viability, secretory function, and MHC antigen expression.

Cytokine effects on permanent cell lines of transformed mouse pancreatic alpha- and beta-cells were compared. The beta-tumor cell 1 (beta TC1) line (from an adenoma created in transgenic mice expressing the SV40 large T-antigen oncogene under control of the rat insulin II promoter) produced insulin predominantly, although small quantities of intracellular glucagon (100:1 insulin to glucagon) were detectable by radioimmunoassay. The alpha TC1 line (from an adenoma created in transgenic mice expressing the SV40 large T-antigen oncogene under control of the rat preproglucagon promoter) produced not only glucagon but also considerable quantities of insulin (4:1 glucagon to insulin) and preproinsulin mRNA. We therefore cloned alpha TC1 cells and obtained 12 glucagon-producing clonal cell lines that did not produce levels of insulin detectable by radioimmunoassay. Analysis by Northern blotting of total RNA from two lines, alpha TC1 clones 6 and 9, confirmed the absence of preproinsulin mRNA. No somatostatin or pancreatic polypeptide was detected by immunohistochemical staining in alpha TC1 clones 6 or 9 or beta TC1 cells. Rat recombinant gamma-interferon (IFN-gamma; 5-250 U/ml) or mouse recombinant interleukin 1 (IL-1; 1-25 U/ml) individually inhibited DNA synthesis in beta TC1 cells after 3 days of treatment. The two cytokines in combination acted synergistically to further depress DNA synthesis and increase cytotoxicity. In contrast, alpha TC1 clone 9 cells were not sensitive to inhibition of DNA synthesis by each cytokine individually, although glucagon synthesis was inhibited. The combination of these cytokines caused marked inhibition of DNA and glucagon syntheses in alpha TC1 clone 9 cells. alpha TC1 clone 9 cells were somewhat more resistant to the cytotoxic action of the combined cytokines than were beta TC1 cells. Incubation with 50 U/ml IFN-gamma induced class II MHC molecules (I-Ab, I-Ad, and I-Ed) and enhanced the constitutive expression of class I molecules (H-2Kb and H-2Kd) on the cell surfaces of beta TC1, uncloned alpha TC1, and alpha TC1 clones 6 and 9. Thus, these cell lines are heterozygous for MHC alleles derived from both parental strains used in the construction of the transgenic mice [C57BL/6J (H-2b) and DBA/2J (H-2d)]. Class II gene transcription induced by IFN-gamma was confirmed in beta TC1 and alpha TC1 clone 9 cells by Northern blot analysis with A alpha-, A beta-, E alpha, and E beta-DNA probes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2004

Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA microarray.

AIM: To characterize the gene expression profiles associated with activation of mouse hepatic stellate cell (HSC) and provide novel insights into the pathogenesis of hepatic fibrosis. METHODS: Mice HSCs were isolated from BALB/c mice by in situ perfusion of collagenase and pronase and single-step density Nycodenz gradient. Total RNA and mRNA of quiescent HSC and culture-activated HSC were extracted, quantified and reversely transcribed into cDNA. cDNAs from activated HSC were labeled with Cy5 and cDNAs from the quiescent HSC were labeled with Cy3, which were mixed with equal quantity, then hybridized with cDNA chips containing 4000 genes. Chips were washed, scanned and analyzed. Increased expression of 4 genes and decreased expression of one gene in activated HSC were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: A total of 835 differentially expressed genes were identified by cDNA chip between activated and quiescent HSC, and 465 genes were highly expressed in activated HSC. The differentially expressed genes included those involved in protein synthesis, cell-cycle regulation, apoptosis, and DNA damage response. CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis and proliferation were up-regulated in activated HSC. cDNA microarray is an effective technique in screening for differentially expressed genes between two different situations of the HSC. Further analysis of the obtained genes will help understand the molecular mechanism of activation of HSC and hepatic fibrosis.

Animals↗
PubMed1976

Changes in the activities of nucleoside triphosphatases and nucleoside kinases in Vero cells infected with Simian virus 40.

Changes in the activities of three enzymes involved in nucleotide metabolism were studied following infection of Vero cells with SV40. The results showed that SV40 infection enhanced the activities of uridine phosphokinase and UMP phosphokinase involved in nucleotide synthesis and suppressed the activity of nucleoside triphosphatases involved in nulceotide degradation. These SV40-induced enzyme activity changes probably served to somehow increase the quantity of nucleotides for virus proliferation.

Animals↗
PubMed1994

Intrathyroidal cytokine gene expression profiles in autoimmune thyroiditis.

Cytokines are thought to mediate the initiation and perpetuation of autoimmune thyroiditis. However, this concept is mainly based on in vitro findings and to date only interleukin (IL)-6 and interferon-gamma (IFN-gamma) have been detected in Graves' disease in vivo. The cytokine pattern produced by T-helper (Th) cells has important regulatory effects on the nature of the immune response. We therefore determined these cytokine mRNAs in Graves' disease and Hashimoto's thyroiditis. RNA was extracted by cesium chloride gradient centrifugation from the thyroid tissue of 12 patients undergoing thyroid resection for Graves' disease and from two patients being treated for Hashimoto's thyroiditis. Two patients with parathyroid adenomas and one patient with a goiter were used as controls. RNA was also extracted from normal human thyroid epithelial cells in primary culture. The cDNAs were prepared by reverse transcription and amplified for IL-2, -4, -5, -6 and -10 and IFN-gamma by polymerase chain reaction. All the cytokine mRNAs were detected in the Hashimoto's thyroid glands in large quantities. Six of the 12 Graves' disease thyroid glands showed, when compared with controls, an increased accumulation of transcripts for: IFN-gamma, IL-2, -4 and -10 or IL-2, -4 and IFN-gamma or IL-2 and IFN-gamma or IFN-gamma alone, each in one case or IL-2 alone in two cases. These cytokine profiles were not representative of a Th1 or Th2 phenotype. Increased amounts of cytokine mRNA in thyroid glands from Graves' disease patients were mostly associated with high microsomal antibody titres and/or prominent intrathyroidal lymphocytic infiltration.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2004

Evaluation of a TaqMan PCR assay to detect rabies virus RNA: influence of sequence variation and application to quantification of viral loads.

Published assays that use TaqMan PCR are consistently sensitive, rapid, and readily transferable. Here we describe a TaqMan PCR-based method for the detection of rabies virus (RV) RNA in tissue samples. We show that the method has an acceptable linear range, is both sensitive and specific, and, importantly, correlates with the concentration of infectious virus. In addition, the levels of RV-specific amplification are adjustable according to the levels of an endogenous control (beta-actin mRNA), allowing the calculation of comparable quantities. We tested the capacity of this assay to cope with target sequence variations. The number of sequence mismatches between gene-specific oligonucleotides and the target sequence significantly affects amplification (P < 0.001), and point mutations at the center of the probe can result in false-negative results through the prevention of probe binding and subsequent fluorescence. This study demonstrates that the genetic heterogeneity of RVs may prove a serious obstacle in the development of a diagnostic assay based on TaqMan PCR; however, the quantification of RV levels may prove to be a valuable application of this assay.

Animals↗
PubMed1994

Semi-quantitative analysis of DNA topoisomerase-I mRNA level using reverse transcription-polymerase chain reaction in cancer cell lines: its relation to cytotoxicity against camptothecin derivative.

Expression of DNA topoisomerase (Topo)-I-mRNA in various cancer cell lines was detected using the reverse transcription-polymerase chain reaction (RT-PCR) method. The cytoplasmic polyadenylated RNA isolated from cancer cell lines was reverse-transcribed and the complementary DNA was amplified by PCR primed with Topo-I specific primers. Fidelity of the amplified sequence was confirmed by restriction endonuclease digestion and Southern blot hybridization. The level of Topo-I mRNA was correlated positively with the cytotoxicity of a Topo-I inhibitor, a camptothecin derivative. This RT-PCR method may be applicable to the assessment of sensitivity of cells to Topo-I targeted drugs, especially when only small quantities of cell samples are available.

Adenocarcinoma↗
PubMed2001

Quantitative mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry.

RNA viruses exist as quasispecies, heterogeneous and dynamic mixtures of mutants having one or more consensus sequences. An adequate description of the genomic structure of such viral populations must include the consensus sequence(s) plus a quantitative assessment of sequence heterogeneities. For example, in quality control of live attenuated viral vaccines, the presence of even small quantities of mutants or revertants may indicate incomplete or unstable attenuation that may influence vaccine safety. Previously, we demonstrated the monitoring of oral poliovirus vaccine with the use of mutant analysis by PCR and restriction enzyme cleavage (MAPREC). In this report, we investigate genetic variation in live attenuated mumps virus vaccine by using both MAPREC and a platform (DNA MassArray) based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Mumps vaccines prepared from the Jeryl Lynn strain typically contain at least two distinct viral substrains, JL1 and JL2, which have been characterized by full length sequencing. We report the development of assays for characterizing sequence variants in these substrains and demonstrate their use in quantitative analysis of substrains and sequence variations in mixed virus cultures and mumps vaccines. The results obtained from both the MAPREC and MALDI-TOF methods showed excellent correlation. This suggests the potential utility of MALDI-TOF for routine quality control of live viral vaccines and for assessment of genetic stability and quantitative monitoring of genetic changes in other RNA viruses of clinical interest.

Animals↗
PubMed1975

Influence of diet and age on ribonucleic acid, protein and free amino acid levels of rat skeletal muscle.

The relationship of dietary protein to RNA, protein and free amino acids in the growth of rat gastrocnemii (GN) muscles over 4, 8, and 12 week periods, was investigated in male weanling rats. The N content of casein diets, C-1, C-2, and C-3 was 0.76, 1.41 and 2.88% while that of the supplemented wheat (SW) diet was 2.5%, respectively. The decrease in concentration of RNA with age was significantly influenced by diet. Accumulation of RNA over a period of time was also influenced by diet, for the C-1 diet permitted only limited accumulation and the C-2 and SW diets restricted accumulation to within 80% of that of the C-3 diet. While protein concentration was affected more by age than by diet, accumulation was influenced by diet, greatest quantities being found in C-3 and SW rats throughout the experimental period. Changes in free amino acid concentration of GN muscles also occurred with age and reflected quantitatively the C-2 and C-3 dietary amino acid patterns. The high levels of certain amino acids in C-1 rats, however, were typical of those found during periods of restrictive protein intake. All criteria of measurement indicated growth and development were greatly restricted by the C-1 diet, less restricted by the C-2 diet while the C-3 diet permitted normal growth.

Aging↗
PubMed2001

Diel variation of the RNA/DNA ratios in Crassostrea angulata (Lamarck) and Ruditapes decussatus (Linnaeus 1758) (Mollusca: Bivalvia).

The aim of this study was to investigate the effect of time of day on RNA/DNA ratios among fed and starved Crassostrea angulata and Ruditapes decussatus juveniles. Sampling to investigate the day and night condition of juveniles was carried out for 48 h. A highly sensitive method for nucleic acid quantification was applied to bivalves. The results suggest that there is some variation in nucleic acid quantities with the time of the day. For the two species analysed, the RNA/DNA ratio was particularly high during the night and was higher in the fed animals. The results seem to indicate that there is some endogenous rhythm in the production of RNA. If there are diel changes in RNA/DNA ratios, it follows that average RNA/DNA ratios can be unrepresentative if there is any day or night bias in sampling.

Journal Article↗
PubMed1987

Expression of cytoskeletal protein 4.1 during avian erythroid cellular maturation.

We have isolated a cDNA clone encoding part of protein 4.1, an integral component of the erythrocyte cytoskeleton. The recombinant was isolated by immunological screening of a chicken erythroid lambda gt11 cDNA library using a monoclonal antibody directed against protein 4.1. DNA blot analysis shows that the gene is present as a single copy per haploid chicken genome, while RNA blot analysis reveals the presence of a single mRNA of 7 kilobases in reticulocytes. Message of the same size (in reduced amounts) is also present in an erythroleukemic cell line transformed by avian erythroblastosis virus and is also present in vastly reduced quantities in nonerythroid hemopoietic cells. Immunoblotting and immunofluorescence experiments show that a subset of the chicken 4.1 variant proteins is preferentially expressed during in vitro differentiation of chicken erythroleukemic cells. These data indicate that the gene is both actively transcribed and translated during early erythroid cellular maturation.

Animals↗
PubMed1989

Use and applications of nucleic acid probes in the clinical laboratory.

The use and availability of nucleic acid probes are significantly greater than they were three to five years ago. This increased utilization has resulted because of numerous changes in molecular genetic procedures. First, the sources of probes have shifted from cloned genomic and cDNA fragments to single-strand oligonucleotides and RNA or "riboprobes." These newer probes offer defined composition and capability to create separate sense and anti-sense strands. Second, an increased variety of labeling techniques provide more ways to incorporate isotopic and nonisotopic species into these nucleic acid fragments than before. Third, the advent of nucleic acid amplification forecasts the ability to detect sub-attomol quantities of target nucleic acids, which could not be detected by classical hybridization methods. Fourth, more-sensitive signaling systems (e.g., chemiluminescence) now available will also contribute to the lowering of detection limits. Fifth, alternative sample-processing methods will shorten the time required to perform these assays. Finally, coupling these newer molecular probe procedures with automation will lead to improved precision and reliability and promote their introduction into the clinical laboratory as routine procedures. Additional factors such as clinical correlation, cost analysis, etc. will stimulate clinical laboratory personnel to participate further in development and use of these procedures.

Clinical Laboratory Techniques↗
PubMed1998

[The effect of ribozyme RzDS on duck hepatitis B virus].

To study the effect of ribozyme in vivo, the hammerhead ribozyme RzDS was designed and synthesized, which aims at the pregenome RNA of DHBV(LJ-76) at the 736 site of the genome. The RzDS cleaved efficiently its substrate in vitro. We constructed pJ-RzDS, in which RzDS was inserted into pJ120 plasmid under the control of PII promoter. After homogenous recombination with the vaccinia virus, Tian Tan strain, we got the recombinant(V-RzDS) which carried RzDS. V-RzDS was injected into nestling Beijing ducks preinfected with DHBV. On the tenth day after intravenous injection, DHBV DNA and DHBsAg in serum were detected. The quantity of DHBV DNA and DHBsAg were evidently lower than that of the controls, when V-RzDS injection was simultaneously given with DHBV. It suggests that RzDS can block replication and expression of DHBV in vivo.

Animals↗
PubMed1986

Control of cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance.

Cachectin (tumor necrosis factor) is a macrophage hormone strongly implicated in the pathogenesis of endotoxin-induced shock. The availability of a DNA probe complementary to the cachectin messenger RNA (mRNA), as well as a specific antibody capable of recognizing the cachectin gene product, has made it possible to analyze the regulation of cachectin gene expression under a variety of conditions. Thioglycollate-elicited peritoneal macrophages obtained from mice contain a pool of cachectin mRNA that is not expressed as protein. When the cells are stimulated with endotoxin, large quantity of additional cachectin mRNA is produced, and immunoreactive cachectin is secreted. Macrophages from mice of the C3H/HeJ strain do not produce cachectin in response to endotoxin. A dual defect appears to prevent cachectin expression. First, a diminished quantity of cachectin mRNA is expressed in response to low concentrations of endotoxin. Second, a post-transcriptional defect prevents the production of cachectin protein. Macrophages from endotoxin-sensitive mice do not produce cachectin if they are first treated with dexamethasone, apparently for similar reasons. These findings give new insight into the nature of the C3H/HeJ mutation and suggest an important mechanism by which glucocorticoids may act to suppress inflammation.

Animals↗
PubMed1998

Large quantity production with extreme convenience of human SDF-1alpha and SDF-1beta by a Sendai virus vector.

We describe a robust expression of human stromal cell-derived factor-1alpha (SDF-1alpha) and SDF-1beta, the members of CXC-chemokine family, with a novel vector system based upon Sendai virus, a non-segmented negative strand RNA virus. Recombinant SDF-1alpha and SDF-1beta were detected as a major protein species in culture supernatants, reached as high as 10 microg/ ml. This remarkable enrichment of the products allowed us to use even the crude supernatants as the source for biological and antiviral assays without further concentration nor purification and will thus greatly facilitate to screen their genetically engineered derivatives.

Anti-HIV Agents↗
PubMed1993

On the association of DNA primase activity with the nuclear matrix in HeLa S3 cells.

We have reinvestigated the association of DNA primase activity with the nuclear matrix prepared from exponentially growing HeLa S3 cells. We have found that 25-30 per cent of the nuclear primase activity resists extraction with 2 M NaCl and digestion with Dnase I. Unlike previous investigations, done with the same cell line, the results showed that nuclear matrix-bound DNA primase activity represented less than 10 per cent of the total cell activity. Association of high levels of primase activity with the nuclear matrix was strictly dependent on a 37 degrees C incubation of isolated nuclei prior to subfractionation. Evidence was obtained that the method used for preparing nuclei can have a dramatic effect on the amount of primase activity which is recovered both in the postnuclear supernatant and in isolated nuclei, thus seriously affecting the interpretation of the results about the quantity of DNA primase activity bound to the nuclear matrix.

Cell Compartmentation↗