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At least 487 records · Page 27Linked to original sources
PubMed2000

Inhibition of RNA transcription modulates magnesium-supplemented potassium cardioplegia protection.

BACKGROUND: Previously we reported that decreased postischemic functional recovery was associated with increased DNA fragmentation in the aged myocardium. Magnesium-supplemented potassium (K/Mg) cardioplegia ameliorated DNA fragmentation and enhanced post-ischemic functional recovery. We hypothesized that K/Mg cardioprotection might involve either an RNA- or a protein-dependent mechanism. METHODS: Aged rabbit hearts underwent Langendorff perfusion. Global ischemia hearts (GI) received 30 minutes of global ischemia and 60 minutes of reperfusion; K/Mg hearts received cardioplegia before global ischemia. To investigate the role of RNA and protein synthesis, K/Mg hearts were treated with alpha-amanitin or cycloheximide to inhibit RNA or protein synthesis. We also determined the quantity of DNA fragmentation and RNA/DNA ratio. RESULTS: Inhibition of RNA but not protein synthesis significantly decreased K/Mg cardioprotection and was associated with significantly decreased postischemic functional recovery (p < 0.05 versus K/Mg), increased DNA fragmentation, and decreased RNA/DNA ratio (p < 0.05 versus K/Mg). CONCLUSIONS: These results indicate that K/Mg cardioprotection in the aged myocardium was modulated by an RNA-dependent mechanism.

Animals↗
PubMed1983

Fourier phase analysis in non-cyclic dynamic studies.

Single harmonic Fourier amplitude and phase (FA/P) analyses of 129 radionuclide angiographic (RNA) studies were found to reflect accurately the sequence and quantity of perfusion determined in the independently interpreted RNA studies. In addition, the anatomic detailing in peripheral studies (hands, feet, etc.) was considered superior to that obtained in RNA studies.

Adult↗
PubMed1976

Comparison of the products of the reaction of 7-methylbenz(a)anthracene 5,6-oxide and RNA, with those formed in 7-methylbenz(a)anthracene-treated cells.

RNA was isolated by a phenol extraction method from mouse embryo cells treated in culture with either [G-3H]-7-methylbenz(a)anthracene or [G-3H]-7-methylbenz(a)anthracene 5,6-oxide (the K-region epoxide). The RNA was degraded to ribonucleosides, mixed with ultraviolet-absorbing quantities of the epoxide ribonucleoside products isolated from RNA that had reacted with 7-methylbenz(a)anthracene 5,6-oxide in aqueous ethanol solution, and chromatographed on a column of Sephadex LH-20 eluted with a methanol:water gradient. The 7-methyl-benz(a)anthracene 5,6-oxide ribonucleoside products formed in cells were identical to those formed in aqueous solution, although the relative amounts of the products varied. The majority of these epoxide-ribonucleoside products were not identical to the products formed in cells treated with the parent hydrocarbon. These results suggest that the major reactive form of 7-methylbenz(a)anthracene that binds to RNA in mouse embryo cells is not the K-region epoxide of this hydrocarbon.

Benz(a)Anthracenes↗
PubMed2002

Detection of epithelial tumour RNA in the plasma of colon cancer patients is associated with advanced stages and circulating tumour cells.

BACKGROUND: Although circulating tumour DNA has been detected in patients with different types of cancer, little is known of free RNA in cancer patients. AIMS: We investigated the presence of RNA from epithelial tumours in plasma from patients with colorectal carcinomas, and its correlation with tumour characteristics and circulating tumour cells. METHODS: beta-actin mRNA was analysed to assess the viability of plasma RNA in samples from 53 patients with colonic cancer and 25 controls. Subsequently, nested primers were used to detect the presence of cytokeratin 19 (CK19) and carcinoembryonic antigen (CEA) RNA in the same samples. Nine clinicopathological parameters were studied to correlate the molecular and clinical parameters. Additionally, we investigated for micrometastases in blood in 18 of these patients and in 10 of the controls samples. RESULTS: All samples had detectable quantities of beta-actin RNA. In the controls, one case (4%) was positive for CEA and five (20%) for CK19 RNA; of the 53 patients, 17 cases (32%) were positive for CEA and 39 (73.6%) for CK19 RNA. This was statistically significant (p=0.000001). Advanced stages (p=0.03) and soluble CEA status (p=0.03) were associated with the presence of CEA, CK19, or both RNAs in plasma. Lymph node metastases (p=0.06) and vascular invasion (p=0.07) were almost significant. On the basis of these results, we examined the possible presence of micrometastases in blood in several of these patients. The presence of plasma tumour RNA was found to be associated with circulating tumour cells in blood (p=0.04). CONCLUSIONS: Epithelial tumour RNA is detectable in plasma from colon cancer patients. This molecular event is associated with advanced stages and circulating tumour cells. Our results could offer new approaches in the diagnosis and monitoring of colon cancer.

Actins↗
PubMed1993

Increase in deoxyribonuclease activity in uraemic lymphocytes is caused by the cleavage of the largest polymerase I subunit.

Deoxyribonucleases and DNA-dependent RNA polymerase activities in T and B lymphocytes isolated from patients with chronic renal failure and control subjects were studied. The data clearly shows that the nuclease activity in T and B cells isolated from uraemic patients is remarkably enhanced when compared to the control cells. Concomitant with the enhancement in enzyme activity, the reduction in RNA polymerase I activity and quantity was observed. It was found that the increase in nuclease activity and quantity was limited to the group of relatively small nucleases with molecular weights ranging from 14 kDa to 18 kDa. It has been reported previously that these nucleases are among the cleavage products of the largest subunit of DNA-dependent RNA polymerase I. Thus we suggest that the depressed metabolic activity is a characteristic feature of the uraemic lymphocyte cells and the observed increased in DNase activity in those cells is a result of polymerase I degradation.

Adult↗
PubMed1992

A quantitative reverse transcriptase-polymerase chain reaction for HIV-1-specific RNA species.

The ability to evaluate the patterns and levels of human immunodeficiency virus type I (HIV-1)-specific RNA in latently and productively-infected cell lines, and primary human cells, is critical to the understanding of HIV-1 expression in cell cultures and possibly in vivo. We have developed a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), utilizing in vitro transcribed RNA standards, to evaluate the copy number per cell and per microgram of total cellular RNA of multiply-spliced, unspliced and total HIV-1-specific RNA species. The latently-infected monocytic and T-lymphocyte cell lines, U1 and ACH-2 respectively, are shown to express between 10(4) to 10(6) copies of total HIV-1-specific RNA per cell, based on the state of cellular stimulation. A dramatic increase of unspliced HIV-1-specific RNA in both the U1 cell line and the ACH-2 cell line is demonstrated by this quantitative RT-PCR, 24 h after stimulation with phorbol esters. These data suggest that a single integrated HIV-1 provirus can rapidly express large quantities of HIV-1-specific RNA. Quantitative RT-PCR, for HIV-1-specific transcripts, should prove extremely useful in evaluating retroviral load and pathogenesis in cell cultures and in vivo.

Base Sequence↗
PubMed1976

Studies on the synthesis of casein messenger RNA during pregnancy in the rabbit.

The quantity of casein mRNA in the rabbit mammary gland was assayed during the course of pregnancy, by translation of casein mRNA in a reticulocyte lysate and by hybridization to DNA complementary to purified casein mRNA. Both methods indicated that the major increases in the concentration of casein mRNA in both polysomal and total cellular RNA occurred between the 18th and 25th day of pregnancy. The change in casein mRNA concentration during this period coincided with a sharp rise in casein synthesis in mammary gland explants suggesting that the levels of casein mRNA determine the rate of casein synthesis in the mammary gland. The sensitivity of the hybridization assay made it possible to detect the presence of casein mRNA in virgin animals and during the very early stages of pregnancy. At day 5, casein mRNA was found associated with polysomes indication that there was probably some casein synthesis at this early stage of gestation. These results suggest that the hormones controlling lactogenesis in the rabbit may function by augmenting the rate of casein mRNA synthesis rather that initiating transcription of previously inactive genes.

Animals↗
PubMed1972

Ribonucleic acid synthesis of vesicular stomatitis virus. IV. Transcription by standard virus in the presence of defective interfering particles.

Exposure of vesicular stomatitis virus-infected Chinese hamster ovary cells to cycloheximide results in the complete transcription of virion ribonucleic acid (RNA) into only 28S and 13 to 15S viral-specific RNA species. These RNA are identical to viral messenger RNA by the following criteria: size, single-strandedness, complementarity to virion RNA, and formation of messenger ribonucleoproteins. This transcription represents the intracellular enzymatic activity of the virion-associated polymerase and is shown to be dependent on input multiplicity. Intracellular transcription differs from in vitro polymerase activity in having a temperature optimum of 34 to 37 C and in synthesizing 28S as well as 13 to 15S messenger RNA species. Addition of interfering quantities of defective T particles to these cycloheximide-treated cells, either an hour before or at the same time as standard B particles of vesicular stomatitis virus, does not alter the rate of transcription nor does it change the sucrose gradient pattern of the viral RNA species. From these results it is concluded that the RNA of defective T particles does not serve a transcriptive function and probably interferes through the replicative mechanism for virion RNA synthesis.

Adenosine↗
PubMed1988

Patterns of polyadenylation site selection in gene constructs containing multiple polyadenylation signals.

We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region. The signals used were from the simian virus 40 (SV40) late gene, the HSV tk gene, and an AATAAA-containing segment of the SV40 early region. This last fragment signals polyadenylation poorly in our constructs and not at all during SV40 infection. All plasmids contained the SV40 origin of replication. Plasmids were transfected into Cos-1 cells; after 48 h, cytoplasmic RNA was isolated and the quantity and 3'-end structure of tk mRNAs was analyzed by using S1 nuclease protection assays. In all constructs, all polyadenylation signals were used. Increasing the number of poly(A) signals 3' to the tk-coding region did not affect the total amount of polyadenylated RNA produced, even with the weakest signal. Increasing the distance between two signals caused an increase in the use of the 5' signal and a decrease in the use of the 3' signal. Changing the distance between the 5' cap and first signal did not affect signal use. Analyses of cytoplasmic mRNA stability, nuclear RNA distribution, and transcription in the polyadenylation signal region indicated that the distribution of tk RNAs ending at different poly(A) sites was the result of poly(A) signal choice, not other aspects of RNA metabolism. Four possible mechanisms of polyadenylation signal recognition are discussed.

Base Sequence↗
PubMed1977

Purified DNAs are transcribed after microinjection into Xenopus oocytes.

The possibility of using DNA-injected Xenopus laevis oocytes and eggs for studying the control of transcription in eukaryotes has been investigated. When purified DNA of simian virus 40 (SV40) is injected into Xenopus laevis oocytes, tritiated RNA precursors are incorporated into DNase-I-resistant, RNase-A- and alkali-sensitive material that hybridizes specifically to SV40 DNA. This viral transcription continues for at least 5 days and occurs only when the injected DNA is directed to the nucleus of the oocyte. The quantity of SV40-specific RNA produced is roughly proportional to the amount of DNA injected; above 1 ng per oocyte, most of the nonribosomal RNA made in successfully injected oocytes is virus-specific. Transcription also occurs, although at a lower efficiency, after injection of the DNA into unfertilized eggs. The DNAs of adenovirus 5, cloned Drosophila melanogaster histone genes, and even bacteriophage phiX174 replicative form, bacteriophage phi80plac, and the ColE1 plasmid are also transcribed after injection into oocytes or eggs.

Animals↗
PubMed1983

Methylations of adenosine residues (m6A) in pre-mRNA are important for formation of late simian virus 40 mRNAs.

Cycloleucine, a competitive inhibitor of methionine transferase was used to generate in vivo partially methylated mRNA in SV40-infected BSC-1 cells. Cycloleucine at 0.5 mg/ml causes more than a 30% decrease in internal m6As of late SV40 mRNA with only minor effect on the dimethyladenosine of the 5' caps m7GpppmAm. After treatment with 2 and 5 mg/ml of cycloleucine, internal m6As were reduced by 10- and 100-fold, respectively. The inhibition of BSC-1 mRNA methylations paralleled that observed for late SV40 mRNAs. In cells exposed to 2 mg/ml cycloleucine production of late SV40 mRNA was inhibited by 80% whereas the amount of SV40 nuclear RNA was only slightly reduced. Size fractionation of SV40 nuclear RNA from cycloleucine-treated cells revealed a loss of SV40 19 S RNA with a corresponding increase of fragmented RNA sedimenting between 11 to 5 S, so that the total amount of SV40 RNA in the nucleus was almost unchanged. Analysis of viral transcription complexes from cells treated with cycloleucine indicated that SV40 transcription was not affected by cycloleucine. SV40-transformed cells, in contrast to BSC-1 cells, were able to process and transport undermethylated RNA. When transformed cells were treated with 2 mg/ml cycloleucine no changes in quantities or size of cytoplasmic and nuclear RNA were detected. The data argues for a role of internal m6A moieties in modulating the processing-linked transport of mRNA from the nucleus to the cytoplasm of nontransformed cells. Transformed cells may escape these controls due to structural alterations in their perinuclear regions.

Adenosine↗
PubMed1974

Poliovirus infection and poly(A) sequences of cytoplasmic cellular RNA.

The influence of polio infection on Poly(A) sequences of cellular cytoplasmic RNA was investigated. In the presence of guanidine, cellular protein synthesis was still shut off after poliovirus infection, although there was no viral RNA synthesis. The Poly(A) of these cells was unchanged with respect to quantity, size, and linkage to cellular cytoplasmic RNA. This finding strongly suggests that the shut off of cellular protein synthesis is not caused by a change of the Poly(A) sequences of cellular mRNA.

Adenosine↗
PubMed1974

Measurement of the sequence complexity of cloned Moloney murine leukemia virus 60 to 70S RNA: evidence for a haploid genome.

The sequence complexity of the 60-70S RNA complex from Moloney murine leukemia virus (M-MuLV) was determined by measuring the annealing rate of radioactively labeled virus-specific DNA with M-MuLV 60-70S RNA in conditions of vast RNA excess. The M-MuLV RNA annealing rate, characterized by the quantity C(r)t((1/2)), was compared with the C(r)t((1/2)) values for annealing of poliovirus 35S RNA (2.6 x 10(6) molecular weight) with poliovirus-specific DNA and Sindbis virus 42S RNA (4.3 x 10(6) molecular weight) with Sindbis-specific DNA. M-MuLV-specific DNA was prepared in vitro by the endogenous DNA polymerase reaction of M-MuLV virions, and poliovirus and Sindbis virus DNAs were prepared by incubation of viral RNA and DNA polymerase purified from avian myeloblastosis virus and an oligo deoxynucleotide primer. The poliovirus and Sindbis virus DNAs were sedimented through alkaline sucrose gradients, and those portions of the DNA with sizes similar to the M-MuLV DNA were selected out for the annealing measurements. M-MuLV was cloned on NIH-3T3 cells because it appeared possible that the standard source of M-MuLV for these experiments was a mixture of viruses. The annealing measurements indicated a sequence complexity of approximately 9 x 10(6) daltons for the cloned M-MuLV 60-70S RNA when standardized to poliovirus and Sindbis virus RNAs. This value supports the hypothesis that each of the 35S RNA subunits of M-MuLV 60-70S RNA has a different base sequence.

Animals↗
PubMed1985

[Characteristics of non-nucleolar RNP structures in hepatocyte nuclei in the early stages of the stimulation of proliferation].

Quantitative changes of extranucleolar RNP-structures were studied on ultrathin cell sections of hepatocytes in the first hours after partial hepatectomy of guinea pigs. This purpose in mind, the sections were treated according to Bernhard. In 2.5 hours after the operation, the relation quantity of perichromatin and interchromatin fibrils increased, whereas the number of perichromatin and interchromatin granules decreased. Therefore the early response of hepatocytes to the proliferative stimulus represents not only intensification of the synthesis of a newly formed hnRNA but also enhancement of the processing and transport of this RNA. After 5 hours the relative quantity of both types of RNP-fibrils diminished to the control level, while the number of perichromatin granules was even smaller than that in the previous case. This points out that the transcription of the extranucleolar RNA becomes less active, and the processing and transport of the earlier synthetized RNA continues. Within the next time lapse (9 hours) the relative quantity of all the studied RNP-structures was low, the content of perichromatin granules being dramatically decreased as compared to that after 5 hours. A comparison of changes in RNP-structures with the degree of condensation of chromatin allows a conclusion that the decondensation of some part of condensed chromatin after 2.5 and 9 hours is functionally different. If initially it is required for the intensification of the template synthesis, thereafter it may be connected with the preparation for replication.

Animals↗
PubMed1975

Comparative effect of heparin on RNA synthesis of isolated rat-liver nucleoli and purified RNA polymerase A.

The polyanion heparin has been employed to study the interaction of rat liver DNA-dependent RNA polymerase A and its template under various conditions. Heparin very efficiently inhibits polymerase molecules, which are not bound to DNA or are associated with the template in a loose, i.e. non-specific fashion. Purified nucleoli, isloated from rat liver nuclei, contain RNA polymerase A in abundant quantities of which only a portion is bound in a transcriptional complex. Excess enzyme, which is contained in the nucleolus in a quasi free form, can be transferred to an exogenously added template and can be completely inhibited by the prior addition of heparin. The enzyme contained in a transcriptional complex, however, initiated in vivo and completing these RNA chains in vitro, is fully resistant to heparin. In contrast to these results it has been found that RNA polymerase A extracted from nuclei and purified by various chromatographic steps does not form heparin-resistant complexes, even after the enzyme has been bound to the DNA template. Moreover it has been found that purified RNA polymerase A transcribes truly native DNA extremely poorly, indicating that the enzyme is highly deficient in the act of initiation on duplex DNA. It is therefore questionable whether the interaction of the purified enzyme and isolated DNA represents binding to true initiation complexes as is observed in the intact nucleolus.

Animals↗
PubMed1995

Identification of vaccine-related polioviruses by hybridization with specific RNA probes.

We developed RNA probes for the identification of poliovirus isolates by blot hybridization. Two sets of vaccine strain-specific probes were prepared. They complemented variable genomic domains within (i) the 5'-untranslated region and (ii) the amino-terminal codons of VP1. An enterovirus group probe (EV/5UT) matching highly conserved 5'-untranslated region sequences was used to estimate the quantities of poliovirus (or enterovirus) RNA in the samples. Poliovirus sequences amplified from Sabin strain virion RNA templates by PCR were inserted into the pUC18 plasmid vector. The antisense PCR primer for each probe set contained sequences encoding a T7 promoter. Hybrids were detected by a sensitive nonisotopic method. RNA probes were labeled by incorporation of digoxigenin-uridylate into the transcripts. The binding of probe to immobilized poliovirus RNAs was visualized by hydrolysis of the chemiluminescent substrate 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2'-adamant ane) catalyzed by alkaline phosphatase conjugated to anti-digoxigenin (Fab) fragments. The specificities of the probes were evaluated with a panel of poliovirus isolates that had previously been characterized by sequence analysis. The RNAs of vaccine-related isolates hybridized with the appropriate probe sets. Wild polioviruses representing a broad spectrum of contemporary genotypes were recognized by the inabilities of their genomes to form stable hybrids with the Sabin strain-specific probes.

Base Sequence↗
PubMed1999

Passage of ribonucleic acid along the intestine of sheep.

Sheep (Flemish female x Texel male, 55 kg BW), fitted with a PVC cannula in the dorsal rumen and single T-shaped PVC cannulas in the proximal duodenum, distal duodenum, mid-jejunum and terminal ileum were fed hay or hay-concentrate diets at various levels of nitrogen and cell walls (NDF) (22 to 32 g N/d; 150 to 699 g NDF/d). Co-EDTA and Cr-NDF were used as markers to measure the flow rate of digesta. Ribonucleic acid (RNA) intestinal digesta and in rumen bacteria was determined with orcinol after extraction with sodium chloride, precipitation with tungstophosphoric acid and alkaline hydrolysis. The RNA:total N ratio in bacteria, harvested from the rumen, amounted to 0.70 (CV 4.4%). The apparent digestibility of RNA in different sections of the intestine was higher than of total N. About 6% of RNA entering the duodenum disappeared between the proximal and distal duodenum. At jejunum, the net disappearance of RNA amounted to 68% of the quantity which entered the proximal duodenum. A higher result of 71% was obtained at the ileum. Total net disappearance of RNA between the proximal duodenum and rectum averaged 75%. Sixteen percent of RNA leaving the ileum was apparently digested in the large intestine. The true digestibility of RNA between the proximal duodenum and the terminal ileum, as estimated by multiple regression analysis, amounted to 78%. Of the amount of RNA entering the ileum, 24% was of endogenous origin. At ileum, the RNA passage was positively related to the ileal flow of NDF (R2 = 0.67) and N (R2 = 0.94). The passage of RNA increased by 3 mg RNA per g ileal indigestible NDF. Ileal endogenous N consisting of approximately 2% of endogenous RNA-N. In conclusion, the digestion capacity in the first part of the small intestine is high. Rising flows of indigestible cell walls and nitrogen increase the loss of ileal RNA. Further, using RNA as a microbial marker to assess the amount of microbial protein entering the duodenum of ruminants, digesta samples should be collected immediately post pylorus at the proximal duodenum, in order to avoid underestimation of the microbial protein synthesis in the rumen.

Animals↗
PubMed1979

"sarc" sequence transcription in Moloney sarcoma virus-transformed nonproducer cell lines.

(3)H-labeled complementary DNA(sarc), complementary to the murine sarcoma virus (MSV)-specific portion of the Moloney MSV (M-MSV) genome, was prepared. M-MSV-specific RNA was then quantitated in the cytoplasm of several M-MSV-transformed, non-virus-producing, clonal NIH 3T3 cell lines. These lines, designated 71 N clones 5, 6, and 3, have been characterized previously by the degree to which they exhibit transformation properties and transcribe Moloney murine leukemia virus-related RNA (S. Salzberg and M. Green, J. Virol. 13:1001-1004, 1974; N. Tsuchida and M. Green, J. Virol. 14:587-591, 1974). By the criteria of cell morphology and agglutination by concanavalin A, cells of clone 5 are highly transformed, cells of clone 6 are almost normal in the sense that they resemble the parent NIH 3T3 cells, and cells of clone 3 are phenotypically intermediate. In the present study, the amounts of cytoplasmic MSV-specific RNA correlated well with the relative degrees of transformation of the cell lines, varying over 35-fold between the least transformed (clone 6) and most transformed (clone 5) lines. Superinfection of either clone 5 or clone 6 with Moloney murine leukemia virus resulted in a fivefold increase in the MSV-specific RNA in the cell cytoplasm. Evidence from (3)H-labeled complementary DNA:cell DNA hybridization studies indicated that the quantity of M-MSV-specific RNA in the nonproducer lines was not directly related to DNA provirus copy number in the cell DNA. Although clones 5 and 6 differ greatly in transformation characteristics and in MSV-specific RNA content, they each apparently contain about two copies of MSV-specific DNA sequence per haploid genome. Thus, factors such as site of provirus integration may be of primary importance in determining virus-specific transcription and cell transformation.

Cell Line↗