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At least 595 records · Page 33Linked to original sources
PubMed2000

UCN-01 (7-hydroxystaurosporine) enhances 5-fluorouracil cytotoxicity through down-regulation of thymidylate synthetase messenger RNA.

UCN-01 (7-hydroxystaurosporine) is a newly developed cell cycle inhibitor known to have several modes of action, including inhibition of cyclin-dependent kinase, induction of p21 and suppression of pRb phosphorylation. In order to test a combination therapy of UCN-01 and 5-fluorouracil (5-FU), growth inhibition of CRL 1420 (MIA PaCa-2; undifferentiated pancreatic carcinoma) by four different treatments was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The treatments used were UCN-01 alone, 5-FU alone, 5-FU followed by UCN-01 (5-FU/UCN-01) and UCN-01 followed by 5-FU (UCN-01/5-FU). We also assessed changes in thymidylate synthetase (TS) mRNA levels, TS activity, and 5-FU incorporation by RNA (F-RNA) for each treatment. Although treatment with UCN-01 alone, 5-FU alone, and 5-FU/UCN-01 inhibited CRL 1420 growth in a concentration-dependent manner, treatment with UCN-01/5-FU inhibited the growth of CRL 1420 synergistically at less than 1 microg/ml drug concentration. The down-regulation of TS mRNA by UCN-01 resulted in stable total TS and decreased free TS, and UCN-01/ 5-FU resulted in enhanced thymidylate synthetase inhibition rate (TSIR) compared to UCN-01 alone and 5-FU/UCN-01. This increased TSIR due to UCN-01 pretreatment was accompanied by elevated F-RNA concentrations in the UCN-01/5-FU treatment. The suppression of TS mRNA and TS activity by UCN-01 may lead to higher sensitivity of tumor cells to 5-FU and may explain the synergistic antitumor effect of UCN-01/5-FU. In conclusion, low concentrations of UCN-01 (from 0.01 to 1 microg/ml) may be clinically useful, affording low cytotoxicity of UCN-01, while enhancing the antitumor effect of 5-FU.

Alkaloids↗
PubMed1976

Separate pyrimidine-nucleotide pools for messenger-RNA and ribosomal-RNA synthesis in HeLa S3 cells.

Kinetic analyses of mRNA and 28-S RNA labeling [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleosidase attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA. The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 muM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.

Cell Division↗
PubMed1980

Studies on the intracellular distribution of Sindbis messenger RNA in infected chick embryo fibroblasts. 2. Non-parallel distribution of 26-S RNA and ribosomes within microsomal subfractions.

The submicrosomal distribution of membrane-associated viral 26-S RNA in chick embryo fibroblasts infected with Sindbis virus was studied. Infected chick embryo fibroblasts were homogenized in the presence of low amounts of EDTA and fractionated by differential centrifugation. Analysis of postmitochondrial supernatants by isopycnic flotation on continuous sucrose gradients showed that membrane-associated 26-S RNA and ribosomes were not distributed in parallel, with an enrichment in 26-S RNA in the light microsomal subfractions. This distribution could not be explained by adsorption artifacts. Analysis of the distribution of microsomal constituents on sucrose gradients after treatment with digitonin ruled out the possibility that the 26-S RNA might be associated with Golgi or plasma membranes. The attachment of viral RNAs to endoplasmic reticulum membranes is discussed in relation to its possible role in viral morphogenesis.

Animals↗
PubMed1980

Messenger RNA coding for phenylalanine ammonia-lyase. Characterization and partial purification from cell suspension cultures of Petroselinum hortense.

The mRNA coding for phenylalanine ammonia-lyase was partially purified from irradiated cell suspension cultures of parsley (Petroselinum hortense). The product of cell-free translation of the mRNA in a reticulocyte lysate was isolated by immunoprecipitation and compared with the native enzyme subunit. Evidence for the identity, or at least a great similarity, of both was provided by tryptic-peptide and gel-electrophoretic analyses. Under partially denaturing conditions, phenylalanine ammonia-lyase mRNA sedimented as a 20--21-S molecule in a sucrose gradient and had an apparent molecular weight of about 1.05 x 10(6) on a polyacrylamide gel. Approximately two-thirds of the polynucleotide sequence of the mRNA were estimated to be required as coding sequence for the enzyme. We suggest that phenylalanine ammonia-lyase mRNA is unlikely to code for more than of three coordinately induced enzymes.

Ammonia-Lyases↗
PubMed1982

Messenger RNA for human tissue plasminogen activator.

A human melanoma cell line (Bowes), which secretes extrinsic (tissue-type) plasminogen activator, was used as a source for the preparation of mRNA for extrinsic plasminogen activator. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)-rich RNA fraction was isolated by affinity chromatography on oligo(dT)-cellulose. This preparation was translated by oocytes into (a) protein(s) that had biological activity in the assay for extrinsic plasminogen activator. On sucrose gradient centrifugation the translatable fraction of the RNA migrated with a sedimentation coefficient of approximately 19 S, a value compatible with the molecular weight of extrinsic plasminogen activator (70 000). The translation product was characterized as being similar to or identical with authentic extrinsic plasminogen activator by the following criteria: (a) serological cross-reactivity with purified extrinsic plasminogen activator in neutralization and immunoprecipitation reactions; (b) plasminogen dependency of fibrinolytic activity and (c) apparent molecular weight of 70 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis.

Animals↗
PubMed1984

Formation of a 22S mRNA X rRNA X protein complex during translation of globin messenger RNA.

Poly(A)-rich RNA was isolated from rabbit reticulocyte polyribosomes by affinity chromatography on oligo(dT)-cellulose and fractionated in sucrose gradients under non-denaturing conditions. Most of the translatable RNA sedimented in sucrose gradients both as free 9S mRNA and as a 22S complex containing 18S ribosomal RNA and a protein of Mr 66 000. The complex was characterized by identification of the translation products. Experiments with both labelled globin mRNA and Mr-66 000 protein indicate that the complex is not an artefact, but rather that it is formed during the initiation of protein synthesis. The Mr-66 000 protein appears to be a component of the 48S pre-initiation complex and recycles before 80S complex formation.

Animals↗
PubMed1986

Characterization of messenger RNA from the cerebral cortex of control and Alzheimer-afflicted brain.

A detailed comparative study of RNA transcripts isolated from the neocortex of control and Alzheimer postmortem brains was made to determine whether morphological changes in the chromatin of Alzheimer neurons and glia, which we reported earlier, are accompanied by changes in the products of transcription. A number of parameters were determined including the yields of total and mRNA per gram of tissue, the relative proportions of polyadenylated [poly(A)+] mRNA in the total RNA, the size distribution of the transcripts and the length of their poly(A) tails, and the nature of their in vitro translation products. The levels of endogenous RNase activity were also measured. The effect of the agonal process on the transcript complement was examined by Northern blotting of a cloned human heat-shock cDNA to total human brain RNA. Our results reveal that the yields of total RNA, unadenylated mRNA, and poly(A) tail lengths from Alzheimer neocortex samples do not differ significantly from those of control and non-Alzheimer dementia neocortex. On the other hand we find a significant reduction in the levels and proportion of poly(A)+ mRNA in the Alzheimer samples as compared to control brain samples. Quantitative rather than qualitative differences were observed in the in vitro translation products when programmed with control and Alzheimer mRNA. No differences were found in the levels of RNase activity between control and Alzheimer samples. Heat-shock mRNA transcripts were detected in brain samples from patients in whom fever was associated with death. The direct correlation of reduced poly(A)+ mRNA and chromatin condensation in Alzheimer neocortex suggests a cause-and-effect relationship. Whether all transcribed genes are affected or only a specific subset has yet to be determined.

Aged↗
PubMed1992

On the stability of messenger RNA and ribosomal RNA in the brains of control human subjects and patients with Alzheimer's disease.

The levels of the mRNAs encoding the G protein subunits GS alpha, G beta 1, and G beta 2 were measured by northern blotting in the frontal cortex and hippocampus of control subjects and of patients with a clinical and histopathological diagnosis of dementia of the Alzheimer type (DAT). There was no significant difference, in either brain region, between the control and DAT groups for any of the G protein mRNAs measured. The degree of intersubject variability was very high, e.g., GS alpha mRNA in the frontal cortex (mean optical density +/- SD) was 405 +/- 342 in the control group versus 305 +/- 207 in the DAT group. The extent of generalised RNA degradation was assessed by detecting the breakdown products of 28S rRNA. RNA degradation was present in tissue samples from every human subject studied. The extent of 28S rRNA degradation in each subject was found to be related to the levels of G protein mRNA detected. The degree of RNA degradation in human subjects was found to be very variable and unaffected by the presence of DAT. RNA degradation correlated poorly with postmortem interval and this was confirmed by a controlled study of postmortem degradation in rat tissue. The possibility that the relative hypoxia and ischaemia in patients immediately before death could influence RNA degradation is discussed. The variable extent of RNA degradation means that great care must be taken to ensure the validity of RNA analyses undertaken in human postmortem brain, particularly when techniques are employed (such as in situ hybridisation) that themselves give no indication of RNA integrity.

Aged↗