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At least 631 records · Page 35Linked to original sources
PubMed1966

Studies in rapidly labelled ribonucleic acid in cell fractions and in tumour tissues.

1. Twenty minutes after injection of [(3)H]orotic acid into rats the rapidly labelled RNA from the liver is mainly associated with the nuclear fraction and little with the ribosomal cytoplasmic fraction. 2. The thermal denaturation of RNA from the fractions was not as reversible as that of the RNA extracted from whole liver. 3. Rapidly labelled RNA is synthesized by cells from a transplantable hepatoma when incubated in the presence of [(3)H]uridine and, after extraction and centrifugation, the label is present in three main fractions: one which sediments to the bottom of a gradient and is associated with DNA, a second which sediments to the heavy side of the 28s RNA, and a third which has a peak of activity between 28s RNA and 18s RNA and is associated with DNA. 4. After labelling and extraction of the RNA from Ehrlich ascites cells the distribution of radioactive components is similar to that of the material from the hepatoma cells. 5. The difference between the tumour cells and liver is due to some extent to the method of homogenizing the tissues and the nature of the components is discussed.

Animals↗
PubMed1993

Gene expression of insulin-like growth factor II in human intracranial meningioma.

BACKGROUND: Insulin-like growth factor II (IGF-II) is synthesized in the normal brain of adult humans predominantly in the choroid plexus and meninges and is secreted in the cerebrospinal fluid. The authors measured IGF-II transcripts and peptides in biopsy specimens from human intracranial tumors including astrocytomas, glioblastomas, and meningiomas. METHODS: The presence of IGF-II mRNA was analyzed in 12 human brain tumors by Northern analysis of total RNA extracted from tumor biopsies and by in situ hybridization of tissue sections. The amount of immunoreactive IGF-II was determined by radioimmunoassay of tumor extracts. RESULTS: Northern analysis of RNA from four meningiomas showed IGF-II mRNA of 6.0, 4.8, and 2.2 kb, and in situ hybridization revealed that meningioma tumor cells contained IGF-II mRNA. In contrast, biopsy specimens from four astrocytomas, one oligoastrocytoma, and four glioblastomas showed no IGF-II mRNA. Radioimmunoassay of IGF-II in tumor extracts showed that all tumors contained IGF-II (40-160 ng/g tissue). Two meningiomas contained the highest amounts of IGF-II (144 and 160 ng/g tissue). CONCLUSIONS: IGF-II mRNA is present in higher amounts in benign meningiomas than in malignant glioblastomas and astrocytomas, whereas the content of immunoreactive IGF-II is similar. On the basis of these findings, the authors believe that IGF-II may be involved in growth regulation of meningiomas.

Adult↗
PubMed1976

Effects of electrons and neutrons on the synthesis of RNA in resistant and sensitive strains of the slime-mould Dictyostelium discoideum and the modifying effect of caffeine.

RNA synthesis was investigated after irradiation in resistant and sensitive lines of the slime-mould Dictyostelium discoideum. When 3H adenine was used as a precursor to RNA, incorporation increased after irradiation in the resistant WT line but not in the sensitive line (gammas-13). The extent of RNA synthesis after irradiation was correlated with the shoulder width on the survival curve of the resistant line. When this was reduced by irradiating with neutrons, or treatment with caffeine RNA synthesis was also reduced. No preferential synthesis of one RNA species occurred; there was increased labelling in all RNA species after irradiation. Sucrose gradient analysis of ribosomal RNA extracted from irradiated cells and free of messenger RNA revealed no apparent difference in composition from that extracted from unirradiated cells. Increased RNA synthesis after irradiation may form part of the recovery processin the resistant cells.

Caffeine↗
PubMed2006

Detection of hepatitis A virus (HAV) in oysters (Crassostrea gigas).

Because shellfish (oysters, clams, and mussels) are filter-feeders, pathogens become concentrated within them, and human consumption of raw, or under-cooked shellfish can result in disease outbreaks. Identification of hepatitis A virus (HAV) in shellfish has been difficult for several reasons: the concentration of virions in shellfish tissues are very low, detection methods based on in vitro propagation are unreliable, recovery of virions from shellfish tissues is inefficient, and PCR inhibitors in shellfish tissues limit the success of RT-PCR. These facts underlie difficulties in determining cause and effect relationships between hepatitis A outbreaks and detection of HAV contamination in shellfish samples. We have developed a reliable and highly sensitive method for detection of HAV in oyster tissues at low levels (0.001 FFU/ml-fluorescent focus units per milliliter). Our method combines dissection of the gastrointestinal oyster tract, organic extraction before PEG precipitation, and RNA extraction with Trizol LS, followed by RT-PCR and hybridization using a digoxigenin-labeled HAV cDNA probe. Our results will benefit both public health officials concerned about hepatitis A infections caused by consumption of HAV-contaminated oysters and shellfish producers who require reliable methods for quality control of commercial oyster production.

Animals↗
PubMed1992

The gene of hepatocyte growth factor is expressed in fat-storing cells of rat liver and is downregulated during cell growth and by transforming growth factor-beta.

Hepatocyte growth factor (HGF) has been detected in non-parenchymal cells but not in hepatocytes. We performed Northern blot analysis of total RNA extracted from rat hepatocytes, Kupffer cells, endothelial cells and fat-storing (Ito-) cells. Total RNA was extracted from fat-storing cells at different times after isolation and from cells treated with different amounts of transforming growth factor beta. The RNA was hybridized with HGF, fibronectin-, and alpha-actin-specific cDNA probes, consecutively. We found an abundant amount of HGF mRNA in freshly isolated fat-storing cells, but not in other liver cells. The amount of the HGF transcripts decreases significantly in FSC during the time of culture, while fibronectin gene expression increases and alpha-actin gene expression as well. TGF-beta dramatically inhibits HGF gene expression, but causes an enhanced fibronectin mRNA level. Northern blot hybridisation of total RNA from CCl4-chronically damaged liver with HGF cDNA shows a significant increase of HGF mRNA during development of liver fibrosis. We suggest that in damaged liver either non-parenchymal cells, others than FSC, became able to express the HGF in vivo, or other mediators overcome the inhibitory effect of TGF-beta.

Actins↗
PubMed2005

DNA and RNA obtained from Bouin's fixed tissues.

BACKGROUND: The use in many countries of acid fixatives, such as Bouin's solution, has limited the use of archival tissue for molecular analysis. An acidic environment is one of the main causes of DNA degradation. Moreover, RNA extraction is difficult in these types of fixed tissues. AIMS: To amplify DNA and RNA from Bouin's fixed tissues. METHODS: DNA and RNA were extracted from 20 breast cancer samples that had been routinely fixed in Bouin's fixative. Amplification of several genes using primers that produced amplicons of different lengths was carried out using the polymerase chain reaction (PCR) for DNA (with and without restoration) and reverse transcription PCR for RNA. RESULTS: The acid environment of Bouin's fixative damaged both DNA and RNA. However, amplification was successful when the amplicon length was reduced to about 80 bp for RNA and 100-200 bp for DNA, especially if submitted to DNA reconstruction procedures. CONCLUSIONS: It is possible to recover and analyse DNA and RNA from Bouin's fixed and paraffin wax embedded tissues.

Acetic Acid↗
PubMed1969

Assembly of protein and nucleoprotein particles from extracted tobacco rattle virus protein and RNA.

Protein extracted from tobacco rattle virus by treatment with formic or acetic acid reassociated in vitro to form rings and tubular structures of indeterminiate lengths in 0.25 molar glycine at pH 7.5 to 9.5. When tobacco rattle virus RNA that had been extracted with phenol was incubated with the protein at 9 degrees C, particles with more regular lengths formed; these particles correspond in size and sedimentation properties to the short particles that normally accompany infection by tobacco rattle virus. The biological activity of the reconstitutted short particles was identical to that of native short particles.

Acetates↗
PubMed2006

Differential response of neuroblastoma cells to TRAIL is independent of PI3K/AKT.

BACKGROUND: In many human tumor cells, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through caspase activation, whereas activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway prevents apoptosis. We hypothesized that inhibition of PI3K/Akt would increase TRAIL-induced apoptosis in neuroblastoma cells. METHODS: SK-N-AS, SH-SY5Y, and IMR-32 neuroblastoma cells were cultured with either standard media or media with PI3K/Akt inhibitor for 24 hours. These cells were then exposed to 100 ng/mL of TRAIL for 90 minutes and harvested. Cells either underwent flow cytometric analysis of apoptosis, had protein extracted for Western blot, had RNA extracted for reverse transcription-polymerase chain reaction, or had cell lysates analyzed for caspase-3, -8, and -9. RESULTS: Baseline expression of TRAIL receptors and Akt varied among the cell lines. Inhibition of PI3K/Akt decreased caspase-3 activation in the AS and SY cells, but did not alter TRAIL-induced apoptosis in any of the cell lines. Activity of caspase-8 and -9 was also unaffected by PI3K/Akt attenuation. CONCLUSIONS: Inhibition of the PI3K/Akt pathway does not increase the sensitivity of neuroblastoma cell lines to TRAIL-induced apoptosis. Neuroblastoma is unique in that activation of the PI3K/Akt pathway is either not essential to its TRAIL resistance or counteracted because of the multiple repetitive pathways of TRAIL resistance.

Apoptosis↗
PubMed2002

Thyroglobulin mRNA quantification in the peripheral blood is not a reliable marker for the follow-up of patients with differentiated thyroid cancer.

BACKGROUND: The detection of serum thyroglobulin (Tg) by immunoassay is widely used to detect residual, recurring or metastatic thyroid carcinoma tIssue in patients with differentiated thyroid cancer (DTC) after total thyroidectomy and radioiodine therapy. However, this method requires thyroid hormone withdrawal to increase sensitivity and is limited by the interference of anti-Tg antibodies. To solve these problems, the detection of Tg mRNA from circulating thyroid cells by reverse transcription (RT)-PCR has been suggested as an alternative method. However, different previous reports show discrepant conclusions as to the clinical usefulness of Tg mRNA quantification. METHODS: We compared three methods of blood collection and RNA extraction, and quantified Tg mRNA (by real time RT-PCR) in the peripheral blood of a) probands without thyroid disease (n=42), patients with b) thyroid autonomy (n=15), c) Graves' disease (n=22), d) euthyroid goiter (n=6), and in DTC-patients after thyroidectomy and radioiodine therapy e) with (n=16) and f) without (n=37) metastasis. As the use of citrate blood in combination with a subsequent separation of mononuclear cells showed a significantly better RNA yield than the extraction of RNA from EDTA or citrate blood without the separation of mononuclear cells, this was the method used. Total RNA was reverse transcribed with random hexamer primers and Tg mRNA was amplified by real time RT-PCR using specific primers and hybridization probes. The Tg mRNA concentrations were normalized to beta-actin mRNA concentrations. RESULTS: Mean circulating Tg mRNA for each group detailed above, expressed as the ratio of Tg to beta-actin concentrations x 1000, were: a) 2.3 (range 0.03-70.89), b) 0.25 (range 0.02-0.55), c) 0.31 (range 0.05-1.36), d) 0.18 (range 0.08-0.35), e) 0.57 (range 0.03-3.03) and f) 0.17 (range 0.02-0.60). Furthermore, we found no correlation between serum Tg and Tg mRNA. CONCLUSIONS: In summary, our data do not show significant differences in Tg mRNA expression between the investigated groups. Therefore, the detection and quantification of Tg mRNA in peripheral blood is unlikely to be suitable for the follow-up of DTC.

Biomarkers, Tumor↗
PubMed1994

Characterization of a replicating and packaged defective RNA of avian coronavirus infectious bronchitis virus.

The Beaudette strain of IBV was passaged 16 times in chick kidney cells. Total cellular RNA was analyzed by Northern hybridization and was probed with 32P-labeled cDNA probes corresponding to the first 2 kb of the 5' end of the genome, but excluding the leader, and to the last 1.8 kb of the 3' end of the genome. A new, defective IBV RNA species (CD-91) was detected at passage 6. The defective RNA, present in total cell extract RNA and in oligo-(dT)30-selected RNA from passage 15, was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments. The oligonucleotides used were selected such that CD-91 RNA, but not the genomic RNA, would be amplified. Cloning and sequencing of the PCR products showed that CD-91 comprises 9.1 kb and has three regions of the genome. It contains 1133 nucleotides from the 5' end of the genome, 6322 from gene 1b corresponding to position 12,423 to 18,744 in the IBV genome, and 1626 from the 3' end of the genome. At position 749 one nucleotide, an adenine residue, was absent from CD-91 RNA. By Northern hybridization CD-91 RNA was detected in virions in higher amounts than the subgenomic mRNAs.

Base Sequence↗
PubMed1995

Generation of a defective RNA of avian coronavirus infectious bronchitis virus (IBV). Defective RNA of coronavirus IBV.

The Beaudette strain of IBV was passaged 16 times in chick kidney (CK) cells. Total cellular RNA was analyzed by Northern hybridization and was probed with 32P-labeled cDNA probes corresponding to the first 2 kb of the 5' end of the genome, but excluding the leader, and to the last 1.8 kb of the 3' end of the genome. A new, defective IBV RNA species (CD-91) was detected at passage six. The defective RNA, present in total cell extract RNA and in oligo-(dT)30-selected RNA from passage 15, was amplified by the reverse transcription-polymerase chain reaction (RT-PCR) to give four fragments. The oligonucleotides used were selected such that CD-91 RNA, but not the genomic RNA, would be amplified. Cloning and sequencing of the PCR products showed that CD-91 comprises 9.1 kb and has three regions of the genome. It contains 1133 nucleotides from the 5' end of the genome, 6322 from gene 1b corresponding to position 12423 to 18744 in the IBV genome and 1626 from the 3' end of the genome. At position 749 one nucleotide, an adenine residue, was absent from CD-91 RNA. By Northern hybridization CD-91 RNA was detected in virions in higher amounts than the subgenomic mRNAs.

Animals↗
PubMed2006

Hepatitis B virus RNA is measurable in serum and can be a new marker for monitoring lamivudine therapy.

BACKGROUND: Changes in the serum hepatitis B virus (HBV) RNA level during lamivudine therapy were compared to those in the serum HBV DNA and HBV core-related antigen (HBVcrAg) levels in 24 patients with chronic hepatitis B. METHODS: For measurement of HBV RNA, total nucleic acid was extracted from serum samples and treated with RNase-free DNase I. After cDNA synthesis from extracted RNA, HBV RNA was measured by real-time detection polymerase chain reaction. RESULTS: The peak fraction of HBV RNA in serum samples was consistent with peak fractions of HBV DNA and HBV core protein in a sucrose gradient analysis, indicating that HBV RNA was incorporated into virus particles. All levels of HBV DNA, HBV RNA, and HBVcrAg decreased gradually during lamivudine therapy (P < 0.001 for all). The amount of decrease from the start of lamivudine therapy was significantly higher for HBV DNA than for HBV RNA or HBVcrAg during 6 months of lamivudine therapy (P < 0.001 for all). However, a similar difference was not seen between HBV RNA and HBVcrAg levels during that period. The HBV RNA level was significantly correlated (P < 0.001 for all) with levels of HBV DNA and HBVcrAg both at the beginning and 2 months after the start of lamivudine therapy. CONCLUSIONS: HBV RNA is detectable in serum in a form indicating incorporation into virus particles, and its serum level might serve as a new viral marker with a significance different from that of HBV DNA in lamivudine therapy.

Adult↗
PubMed1997

Quantitative liver parameters of HCV infection: relation to HCV genotypes, viremia and response to interferon treatment.

BACKGROUND/AIMS: This study aimed to evaluate the relation between the number of hepatocytes positive for HCV antigens and the amount of HCV RNA in the liver and to evaluate the relationship between the above parameters and viremia levels, HCV genotype and response to interferon treatment. METHODS: This was a retrospective study on 31 consecutive patients with chronic HCV-related liver disease, selected on the basis of the availability of frozen liver tissue for both liver HCV antigens detection and liver HCV RNA quantitation. HCV antigens (immunohistochemistry), liver and plasma HCV RNA (competitive RT-PCR), and HCV genotype (commercial kit) were studied. RESULTS: A significant correlation (p=0.0005) was found between the amount of liver HCV RNA (log 10 copy/microg of extracted RNA) and the number of HCV-infected hepatocytes (scored from 0 to 3). These parameters were not significantly correlated with viremia levels. The highest liver HCV RNA levels and HCV antigen scores were found in patients infected with genotype 1b. Liver HCV RNA (median 541 x 10(3) vs 118 x 10(3) copy number/microg, p=0.031) and liver HCV antigens (mean score 2.3 vs 1.3, p=0.018) but not plasma HCV RNA (median 14956 x 10(3) vs 2885 [correction of 2.885] x 10(3) copy number/ml, ns) were significantly higher in patients not responding to interferon treatment compared to responders. CONCLUSIONS: The tissue parameters tested in this study were significantly correlated, shared the same clinical implications and predicted short-term response to interferon treatment better than viremia levels. We suggest that these tests should be included in the study protocol of patients under evaluation for interferon treatment, basing the choice on local facilities.

Adult↗
PubMed2006

Consensus guidelines for microarray gene expression analyses in leukemia from three European leukemia networks.

A plethora of studies have documented that gene expression profiling using DNA microarrays for various types of hematological malignancies provides novel information, which may have diagnostic and prognostic implications. However, to successfully use microarrays for this purpose, the quality and reproducibility of the whole procedure need to be guaranteed. Critical steps of the method are handling, processing and storage of the leukemic sample, purification of tumor cells (or lack thereof), RNA extraction methods, quality control of RNA, labeling techniques, hybridization, washing, scanning, spot filtering, normalization and initial interpretation, and finally the biostatistical analysis. These items have been extensively discussed and evaluated in different multi-center quality rounds within the three networks, that is, I-BFM-SG, the German Competence Network 'Acute and Chronic Leukemias' and the European LeukemiaNet. Based on the exchange of knowledge and experience between the three networks over the last few years, we have formulated guidelines for performing microarray experiments in leukemia. We confine ourselves to leukemias, but many of these requirements also apply to lymphomas or other clinical samples, including solid tumors.

Data Interpretation, Statistical↗
PubMed1995

The nucleotide sequence of RNA3 and RNA4 of olive latent virus 2.

Olive latent virus 2 (OLV2), a virus with particle shapes resembling those of alfalfa mosaic alfamovirus (AMV), has four major RNA species, two of which (RNA3 and RNA4) were completely sequenced. RNA3 was a bicistronic molecule containing two clear-cut ORFs, one of which (ORF1) coded for a 36.5 kDa polypeptide with conserved motifs of the '30K superfamily' movement proteins and the other (ORF2) encoded a 20 kDa polypeptide identified as the viral coat protein. RNA4, which was a little smaller than RNA3 (2078 nt versus 2438 nt), also differed from RNA3 in a few positions, but its in vitro translation did not produce any protein. By contrast, an additional RNA, 1042 nt in size with strong sequence homology with RNA3 and RNA4, was identified in RNA extracts from infected plants. This RNA, which may be a subgenomic form of RNA3 carrying the coat protein cistron, is apparently encapsidated to a very small extent, or not at all. Comparative computer-assisted analysis of virus-coded protein sequences disclosed homologies suggesting that OLV2 may belong to the family Bromoviridae, but as an entity separated from the currently known genera.

Amino Acid Sequence↗
PubMed1975

Transcription of DNAs of known sequence after injection into eggs and oocytes of Xenopus laevis.

1. When the synthetic polynucleotide, poly[d(A-T) - d(A-T)] is injected into the eggs and oocytes of Xenopus laevis, a stimulation of RNA synthesis results. Analysis of extracted RNA by high-voltage electrophoresis, shows that this stimulation of RNA synthesis is due to the production of poly[r(A-U)] transcripts. The rate of poly[r(A-U)] synthesis is calculated to be at least ten-fold greater in eggs than in oocytes. The amount of poly[r(A-U)] detectable in injected eggs has reached a maximum by 1.5 h after injection; in oocytes, however, poly[r(A-U)] continues to accumulate between the third and 16th hour after injection. The transcripts range in length from less than 80 nucleotides up to over 2000 nucleotides long. The co-injection of poly[d(A-T)] - d(A-T)] and alpha-amanitin into oocytes, has shown that the synthesis of poly[r(A-U)] is approximately 90% inhibited at a concentration of alpha-amanitin which has no effect on the capacity of the oocyte to synthesize ribosomal and 4-S RNA; thus the nucleoplasmic RNA polymerases IIa and/or IIb, are implicated as playing a major role in poly[r(A-U)] synthesis in oocytes. 2. When poly(dG) - poly(d-C), poly(dA), poly(dA) - poly(dT) and poly[d(I-C) - d(I-C)] are individually injected into eggs only poly[d(I-C) - d(I-C)] is transcribed as efficiently as poly[d(A-T) - d(A-T)]. 3. When calf thymus native or denatured DNA, polyoma, T2, T4 and phiX DNAs are individually injected into eggs only the injection of calf thymus native DNA causes a detectable stimulation of RNA synthesis. 4. The activities of crude preparations of egg and oocyte RNA polymerases are tested in vitro with different DNA templates. In contrast to the situation in vivo, it is found that poly[d(A-T1 - d(A-T)] is as efficiently transcribed in vitro by oocyte polymerase as by egg polymerase. Additionally poly[d(A-T) - d(A-T)] is transcribed ten-fold more efficiently in vitro than calf thymus native DNA. When poly(dA) - poly(dT), poly(dA), phiX, T2, and calf thymus denatured DNA are tested in vitro, only calf thymus denatured DNA is transcribed to a significant extent. The above results are discussed in relation to the known synthetic activities of Xenopus eggs and oocytes.

Animals↗
PubMed2006

Performance of the TruGene human immunodeficiency virus type 1 genotyping kit and OpenGene DNA sequencing system on clinical samples diluted to approximately 100 copies per milliliter.

The TruGene human immunodeficiency virus type 1 (HIV-1) genotyping kit/OpenGene DNA sequencing system (Bayer HealthCare, Tarrytown, NY) reliably produced clinically acceptable resistance profiles for reverse transcriptase and protease inhibitors on patient samples diluted to approximately 100 copies/ml following extraction with the QIAamp viral RNA minikit (QIAGEN Inc., Valencia, CA). One modification of the standard protocol was made to guarantee PCR amplification: a centrifugation step to concentrate virus was added before RNA extraction. For genotypic antiretroviral resistance testing, no significant differences in the identification and sensitivity of detection for codon mutations, base mutations, and multibase sites were found between the original and diluted samples.

Anti-HIV Agents↗
PubMed1998

Detection and sequence analysis of borna disease virus p24 RNA from peripheral blood mononuclear cells of patients with mood disorders or schizophrenia and of blood donors.

Borna disease virus (BDV) p24 RNA was detected in the peripheral blood mononuclear cells (PBMCs) of psychiatric patients and blood donors by nested reverse transcriptase PCR (RT-PCR). The prevalences of BDV p24 RNA in patients with mood disorders (4%) and schizophrenia (4%) were not significantly different from that in blood donors (2%). This finding was inconsistent with previous reports that showed either a high prevalence or absence of BDV p24 RNA in patients with psychiatric disorders. The differences in BDV p24 RNA prevalence in these studies may be due to differences in the criteria for positivity, the number of PBMCs used for RNA extraction, or the amount of RNA tested for nested RT-PCR or to laboratory contamination. Sequence analysis of BDV p24 RNA from the PBMCs of patients and blood donors showed a high nucleotide sequence conservation but definite nucleotide mutations compared with horse BDV p24 RNA sequences. In comparison with human BDV p24 RNA sequences previously reported from Japan and Germany, there were several positions with silent nucleotide mutations among these clones.

Adult↗