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At least 649 records · Page 36Linked to original sources
PubMed2002

Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress.

Reactive oxygen species are required for cell proliferation but can also induce apoptosis. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. Here we show that the PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans, and we conclude that this model also extends to mammalian systems.

Animals↗
PubMed1998

One-tube multiplex RT-PCR of BCR-ABL transcripts in analysis of patients with chronic myeloid leukaemia and acute lymphoblastic leukaemia.

We describe a one-tube multiplex reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of bcr-abl fusion mRNA in analysis of patients with chronic myeloid leukaemia and acute lymphoblastic leukaemia. The assay provides a quick and reliable method for the detection and analysis of chromosome translocations resulting in formation of the fusion proteins p210 (b3a2/b2a2) and p190 (e1a2). The method is based on the use of magnetic beads and sequence-specific reverse transcription primers. By combining direct mRNA isolation, reverse transcription and first-stage PCR we have reduced the number of manipulations, maintained sensitivity, and minimized the risk of contamination. A nested primer strategy is used to secure sensitivity. We also introduce a competitive one-tube RT-PCR to be able to monitor the relative quantity of transcripts using in vitro transcribed RNA as competitor.

Cloning, Molecular↗
PubMed1979

Heterogeneity of virus particles in measles virus.

A heterogeneous population of virions is generated by measles virus-infected cells. These particles are partially separable by sucrose density centrifugation into three peaks. Each population is stable and contains infectious particles. The particles of all three populations contain at least six polypeptide species that differ between particle populations only in quantity. All three populations contain a 50S RNA species, and the heaviest density peak also contains an additional species of 43S RNA. The difference between these results and previous studies with measles virions will be discussed.

Animals↗
PubMed1975

The utilization of nucleotides by animal cells.

We have extended our studies on the toxicity of several arabinosyl nucleotides to L cells. In contrast to a short-term toxic effect of 9-beta-D-arabinofuranosyladenine (araA), both 9-beta-D-arabinofuranosyladenine 5'-phosphate (araAMP) and 9-beta-D-arabinofuranosyladenine 3',5'-cyclic phosphate produced sustained killing of the fibroblasts. 9-beta-D-Arabinofuranosyladenine 2',5'-cyclic phosphate and 9-beta-D-arabinofuranosyladenine-N1-oxide-5'-phosphate were not toxic, whereas 9-beta-D-arabinofuranosylhypoxanthine 5'-phosphate and adenosine 3',5'-cyclic phosphate had relatively slight effects on cell growth. 2',3'-Dideoxyadenosine 5'-phosphate was lethal to L cells, whereas 2',3'-dideoxyadenosine inhibited growth only slightly. The survival time of mice bearing Ehrlich ascites carcinoma was prolonged beyond controls equally well by treatment with either araAMP or araA. To explore the mechanism of nucleotide toxicity, we studied the metabolism of exogenous [3H, 32P]-araAMP. The double-labeled compound entered the cells very slowly and accumulated in both acid-soluble and acid-insoluble materials. 32P and 3H were associated with the adenine nucleotide fractions in ratios similar to that of the exogenous compound. After dephosphorylation of the acid-soluble fraction, greater than 85% of the 3H was associated with araA. Following degradation of the adenine-containing triphosphates with apyrase, all of the 32P and 97% of the 3H were associated with araAMP. Only small quantities of each label were associated with RNA and DNA. Following enzymatic degradation of DNA to 5'-monophosphates, most of both 3H and 32P were associated with the dAMP fraction in a ratio that was similar to that of the exogenous araAMP. After dephosphorylation of the 5'-monophosphates, greater than 90% of the 3H was contained in arabinosyl nucleosides. DNA was also enzymatically degreded to 3'-monophosphates. 3H was found primarily in the 3'-adenine nucleotide fraction with a slight amount in the nucleoside, whereas 32P was distributed among all the 3'-mononucleotides. After dephosphorylation of the 3'-monophosphates, 93% of the 3H was associated with araA. These results indicate that a small amount of araAMP penetrated the cell as the intact nucleotide, was phosphyorlated to the triphosphate and was subsequently incorporated within DNA.

Adenosine↗
PubMed1979

Wheat embryo ribonucleates. XIV. Mass isolation of mRNA from wheat germ and comparison of its translational capacity with that of mRNA from imbibing wheat embryos.

Commercially milled wheat germ is shown to be a convenient source material for facile recovery of mass (milligram) quantities of highly purified poly(A)-rich RNA. This poly(A)-rich RNA is efficiently translated in a nuclease-treated extract of rabbit reticulocytes. By sucrose density gradient fractionation of bulk poly(A)-rich RNA from wheat germ, it has been possible to show that there is a direct relationship between the molecular weights of the polypeptide products of cell-free synthesis and the molecular weights of the wheat mRNA molecules which program their synthesis. As assessed by SDS -- polyacrylamide gel electrophoresis, the same array of polypeptides is synthesized when nuclease-treated reticulocyte extract is programmed by poly(A)-rich RNA from either commercially supplied or laboratory-prepared wheat embryos. Significantly, there are gross quantitative if not qualitative differences between the translational capacities of poly(A)-rich RNA from dry and imbibing wheat embryos, and the possible importance of these differences for interpreting a changing pattern of polypeptide synthesis in imbibing wheat embryos is the subject of a brief discussion.

Animals↗
PubMed2002

Comparison of different labeling methods for two-channel high-density microarray experiments.

In this report we evaluate three methods for labeling nucleic acids to be hybridized to a cDNA microarray: direct labeling, indirect amino-allyl labeling, and the dendrimer labeling method (Genisphere). The dendrimer method requires the smallest quantity of sample, 2.5 microg of total RNA compared with 20 microg with the direct or indirect methods. Therefore, we wanted to know whether the performance of the dendrimer method is comparable to the other methods, or whether significant information is lost. Performance can be considered in terms of sensitivity, dynamic range, and reproducibility of the quantitative signals for gene intensity. We compared the three labeling methods by generating three sets of eight self-to-self hybridizations using the same total RNA sample in all cases ("replicate study"). In our analysis, we controlled for the effects of print-tip and background subtraction biases. We also performed a smaller study, namely, a dilution series study with five dilution points per labeling method, to evaluate one aspect of predictive ability. From the replicate study, the dendrimer method appeared to perform as well, and often better, with respect to reproducibility and ability to detect expression. However, in the dilution series study, this method was outperformed by the other two in terms of predictive ability and did not perform very well. These findings are helping to guide our decisions on what labeling method to use for subsequent studies, based on the purpose of a specific study and its limitations in terms of available material.

Fluorescent Dyes↗
PubMed2005

Accurate discrimination of Barrett's esophagus and esophageal adenocarcinoma using a quantitative three-tiered algorithm and multimarker real-time reverse transcription-PCR.

Esophageal adenocarcinoma (EA) is increasing faster than any other cancer in the U.S. In this report, we first show that EA can be distinguished from normal esophagus (NE) and esophageal squamous cell carcinoma by plotting expression values for EpCam, TFF1, and SBEM in three-dimensional Euclidean space. For monitoring progression of Barrett's esophagus (BE) to EA, we developed a highly sensitive assay for limited quantities of tissue whereby 50 ng of RNA are first converted to cDNA using 16 gene-specific primers. Using a set of training tissues, we developed a novel quantitative three-tiered algorithm that allows for accurate (overall accuracy = 61/63, 97%) discrimination of BE versus EA tissues using only three genes. The gene used in the first tier of the algorithm is TSPAN: samples not diagnosed as BE or EA by TSPAN in the first tier are then subjected to a second-tier analysis using ECGF1, followed by a third-tier analysis using SPARC. Addition of TFF1 and SBEM to the first tier (i.e., a five-gene marker panel) increases the overall accuracy of the assay to 98% (62/63) and results in mean molecular diagnostic scores (+/- SD) that are significantly different between EA and BE samples (3.19 +/- 1.07 versus -2.74 +/- 1.73, respectively). Our results suggest that relatively few genes can be used to monitor progression of BE to EA.

Adenocarcinoma↗
PubMed1989

Role of molecular biology in hypertension research. State of the Art lecture.

In this article we will examine the potential impact of molecular biology on hypertension research. We will review the available molecular techniques, which include gene cloning, transient and stable expressions, as well as the use of transgenic animals. To facilitate our discussion, we will focus primarily on research of the renin gene. Renin provides a useful model that illustrates the power of biotechnology in providing detailed structural and biochemical information on a complex protein that exists in low quantities in vivo. Studies of its messenger RNA and gene expression have resulted in an improved understanding of the biology of the renin system and in generating new hypotheses. These approaches can be generalized to studies of other vasoactive hormones, contractile protein, and other gene products related to cardiovascular regulation. To elucidate the role of a specific gene in genetic hypertension, we will discuss the use of genetic markers in cosegregation or linkage analysis. Finally, we will examine the potential of transgenic animals in the study of regulation of gene expression in the whole animal and the contribution of selective genes to hypertension. We believe that molecular biology complements the biochemical and physiological approaches and provides new opportunities for furthering our concept of hypertension mechanisms.

Animals↗
PubMed1985

Gene expression in Xenopus embryogenesis.

This article considers some aspects of the storage of macromolecules in the oocyte of Xenopus laevis and the activation of previously unexpressed genes during early embryogenesis. The large quantity and complex nature of poly(A)+ RNA accumulated in the egg provides the cleavage embryo with a supply of mRNA sufficient to sustain protein synthesis for several hours of development. Onset of gene activity at the midblastula transition (MBT) leads to the synthesis and accumulation of molecules of various RNA classes, including tRNAs, rRNAs, mRNAs and mitochondrial RNAs. At gastrulation the poly(A)+ RNA population is still qualitatively similar to that of the egg but some sequences not present in egg RNA have accumulated by this time. Through the use of a subtractive cDNA cloning procedure we have prepared a library of sequences that represent genes activated for the first time between MBT and gastrula. A study of several of these cDNA clones suggests that genes in this class are restricted in their activity to embryonic and tadpole stages.

Animals↗
PubMed1977

Biological and biochemical characterization of a latent subacute sclerosing panencephalitis (SSPE) virus infection in tissue culture.

The present investigation describes the biological and biochemical properties of a persistent SSPE virus infection. Persistently infected cells were derived by cocultivation of infected brain cells and uninfected Vero cells, and cultures were maintained by normal subculturing methods. No infectious virus was ever released from these cultures, and all attempts to induce infectious virus release were unsuccessful. Biological assays showed that infected cells contained nucleocapsid and salt-dependent hemagglutinin antigens, whereas the normal hemagglutinin appeared not to be present. Electron microscopic examination demonstrated the presence of both intranuclear and cytoplasmic nucleocapsids together with the release of virus particles (defective?) from the cell membrane. Biochemical analysis demonstrated that approximately 90% of the intracellular genomic RNA was defective or subgenomic although a small quantity of infectious genomes was present. It is proposed that the large quantities of defective genomes in the infected cells are the major factor in the maintenance of this persistent infection.

Animals↗
PubMed1995

Experimental brain injury induces expression of interleukin-1 beta mRNA in the rat brain.

Cytokines have been shown to be induced following a variety of central nervous system (CNS) insults, and may play a role in the pathophysiological sequelae of CNS injury. In the present study, we characterized the regional expression of interleukin-1 beta (IL-1 beta) mRNA in specific brain regions following experimental lateral fluid-percussion traumatic brain injury (TBI) in rats. Adult Sprague-Dawley rats (n = 42) were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and subjected to lateral fluid-percussion brain injury of moderate severity (2.4 atm.) centered over the left parietal cortex, or 'sham' treatment (anesthesia and surgery without injury). Animals were sacrificed at 1, 6 and 24 h post injury, brains were removed, and tissue samples of left (injured) parietal cortex (LC), corresponding area in the contralateral right cortex (RC), cortex adjacent to injured parietal cortex (LA), corresponding adjacent area in the right cortex (RA), left hippocampus (LH) and right hippocampus (RH) were prepared. Total RNA was isolated and Northern blot hybridization was performed and the quantity of brain tissue IL-1 beta mRNA is presented as percent relative radioactivity of IL-1 beta positive macrophage RNA which was loaded on same gel.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Evaluation of methods for duration of preservation of RNA quality in rat liver used for transcriptome analysis.

In The Toxicogenomics Project, about 150 chemicals are administered to rats, and gene expression in the liver analyzed by Affymetrix GeneChip and stored in the database. As the quality of RNA greatly influences the accuracy of gene expression data, conditions of the storage of the sample are very important. Recently, an RNA stabilization solution, RNAlater, has become commercially available. In this study, the new storage method was compared with the traditional storage method (stored in freezer or liquid nitrogen) under various conditions by looking at the degradation of RNA assessed by its total yield, OD260/280 ratio, 28S/18S ratio, and quantity of beta-actin. It was confirmed that RNAlater preserved the liver tissue sample by maintaining the quality of RNA for one year (in liquid N(2) or -80 degrees C), for 3 days (4 degrees C), or for 2 hr (room temperature) without degradation of RNA. Quality of RNA samples dissolved in buffer RLT and stored at -20 degrees C tended to decrease, but samples stored at -80 degrees C were almost equivalent to those stored in liquid nitrogen. In conclusion, we recommend the following procedure for preservation of liver tissue for extraction of RNA: 1) tissues removed should be put into chilled RNAlater as soon as possible; 2) samples in RNAlater must be stored overnight or longer at 4 degrees C and can be left for as long as 2 weeks without freezing; 3) samples in RNAlater can be stored for at least one year under less than -20 degrees C and 4) samples dissolved in buffer RLT can be preserved at least for one year under -80 degrees C.

Actins↗
PubMed2004

Detection of cell-free nucleic acids in bronchial lavage fluid supernatants from patients with lung cancer.

The aim of this study was to determine whether nucleic acids are detectable in cell-free bronchial lavage supernatants, and whether it is possible to find alterations in this DNA and RNA of genes known to be present in lung tumour cells. DNA was isolated from cell-free lavage supernatants from 30 and RNA from 25 lung cancer patients. The DNA was examined for microsatellite alterations (MA) and the RNA analysed for the expression of seven tumour-associated genes. Intact DNA and mRNA could be isolated from all cell-free bronchial lavage supernatants. MA were found in lavage supernatants of 12/30 patients and in lavage cells of 6/30 patients. Altogether alterations were found in 14/30 patients. Analyses of tumour-associated gene expression showed positive results, with at least one marker in the lavage supernatants of all 25 patients. Thus, we could demonstrate, for the first time, that it is possible to isolate intact DNA and RNA from cell-free bronchial lavage supernatants. Their quantity and quality is sufficient for further amplification by polymerase chain reaction (PCR)/reverse transcriptase (RT)-PCR. Altogether, tumour-associated changes were detected in DNA samples from 47% of the patients and in RNA samples from all of the patients analysed.

Aged↗
PubMed1983

The white pock mutants of rabbit poxvirus: V. In vitro translation of early host range mutant mRNA.

The abortive infections of pig kidney (PK) cells by both RP mu hr23 and RP mu hr31, two early (DNA minus) white pock (mu) host range (hr) mutants of rabbit poxvirus (RPV), are characterized by the in vivo inhibition of both host and viral protein synthesis by 10 hr postinfection although viral RNA synthesis continues. Further analysis reveals that large quantities of functional viral mRNA can be isolated from PK cells abortively infected with RP mu hr23 and translated in vitro throughout the 10-hr period of infection, even though these mRNAs are almost totally inactive in vivo. The in vitro translation of accumulated mRNA isolated from PK cells abortively infected by RP mu hr31 shows a quite different pattern where the maximum amount of RNA translatable in vitro is found at 6 hr postinfection with lesser amounts at 10 hr postinfection. Although early or prereplicative viral proteins are detected with each mutant both in vivo and after in vitro translation of isolated RNA, few, if any, late proteins are observed under any conditions.

Animals↗
PubMed1980

[Asymmetric histogram of nucleolar nucleic acid concentration as a potential index of ribosomal DNA amplification in rabbit cerebellar Purkinje cells].

Purkinje's cells from the rat cerebellum were exposed to UV cytophotometry in order to evaluate the nucleic acid content in the nucleoli and perinucleolar chromatin. The study revealed an asymmetrical histogram of RNA distribution in the Purkinje's cell nucleoli. The cells with an increased RNA content in the nucleolus displayed the elevated quantities of nucleic acids in the perinuclear chromatin. A relationship of the phenomenon under study and the hypothesis of ribosomal DNA amplification in Purkinje's cells is discussed.

Animals↗
PubMed1966

Dissociation of cellular functions in Bacillus cereus by 5-fluorouracil.

Reich, Melvin (The George Washington University School of Medicine, Washington, D.C.), and H. George Mandel. Dissociation of cellular functions in Bacillus cereus by 5-fluorouracil. J. Bacteriol. 91:517-523. 1966.-5-Fluorouracil (FU) produced a marked inhibition of growth and deoxyribonucleic acid (DNA) synthesis in Bacillus cereus 569H. Protein and ribonucleic acid (RNA) synthesis were not specifically inhibited, and proceeded at the rate of turbidometric increase of the cells. Cell-wall synthesis, respiration, and penicillinase production continued in the presence of FU at essentially the control rate. The addition of equimolar concentrations of uracil and FU prevented growth inhibition but did not restore DNA synthesis. The addition of thymidine with FU did not relieve growth inhibition but did restore the DNA content to normal. Thymidine supplementation also increased the quantity of FU, but not uracil, incorporated into RNA and the acid-soluble fraction. The data indicate that inhibition of growth can be dissociated from inhibition of DNA synthesis and that more DNA is present in normal cells than is needed for growth and reproduction.

Adenine↗
PubMed2002

A novel method for quantitation of human von Willebrand factor messenger RNA.

We have developed a novel assay for quantitation of human von Willebrand factor (vWF) messenger RNA (mRNA) using the ABI 7700 quantitative PCR system (Applied Biosystems, Warrington, Cheshire, UK). To allow accurate measurement of the vWF mRNA concentration in samples, we have used 18S ribosomal RNA as an internal control. The specificity of the assay was demonstrated by polyacrylamide gel electrophoresis and ABI Prism BigDye terminator cycle sequencing of the PCR product. We have also assessed the assay by using RNA from both a hepatocyte cell line which does not express vWF mRNA, and an endothelial cell line which does express vWF mRNA. By mixing varying amounts of these RNAs together, we were able to adjust the vWF quantity in known increments while keeping the 18S ribosomal RNA internal control constant. The quantitation of vWF mRNA correlated with the proportion of endothelial cell RNA (r(2)=0.912, p<0.001).

Humans↗
PubMed1995

RNA editing in higher plant mitochondria: analysis of biochemistry and specificity.

RNA editing alters genomically encoded cytidines to uridines posttranscriptionally in higher plant mitochondria. Most of these editing events occur in translated regions and consequently alter the amino acid sequence. In Oenothera berteriana more than 500 editing sites have been detected and the total number of editing sites exceeds 1000 sites in this mitochondrial genome. To identify the components involved in this process we investigated the factors determining the specificity of RNA editing and the apparent conversion of cytidine to uridine residues. The possible biochemical reactions responsible for RNA editing in plant mitochondria are de- or transamination, base substitution and nucleotide replacement. In order to discriminate between these different biochemical mechanisms we followed the fate of the sugar-phosphate backbone by analysing radiolabeled nucleotides after incorporation into high molecular mass RNA. Plant mitochondria were supplied with [alpha-32P]CTP to radiolabel CMP residues in newly synthesized transcripts. Radiolabeled mtRNA was extracted and digested with nuclease P1 to hydrolyse the RNA to monophosphates. The resulting monophosphates were analysed on one- and two-dimensional TLC systems to separate pC from pU. Radiolabeled pU was detected in increasing quantities during the course of incubation. These results suggest that RNA editing in plant mitochondria involves either a deamination or a transglycosylation reaction. The editing product was identified as uridine and not as a hypermodified nucleotide which is recognized as uridine. Similar results have been obtained by incubating in vitro transcribed mRNAs with mitochondrial lysates indicating that RNA editing and transcription is not directly linked in plant mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Evolution↗