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R N Kurl

Publications and source records attributed to R N Kurl.

At least 19 recordsLinked to original sources
PubMed1997

RNA-RNA interaction and gene splicing.

The precise excision of intervening sequences during RNA splicing is an interesting example of the high degree of specificity involved in biosynthesis processes. Self-splicing RNA precursors achieve this specificity primarily through intramolecular interactions whereas all other types of RNA splicing requires interaction between cellular factors and specific recognition signals in the RNA precursor. About twelve years ago, the in vitro splicing system was developed and a general scheme of the pre-mRNA was proposed (Hernandez and Keller, 1983; Krainer et al., 1984; Lin et al., 1985; Padgett et al., 1984; Ruskin et al., 1984). A fundamental question in the splicing field is how the 5' and 3' splice sites are recognized and paired during the splicing reaction. Recent work in the splicing field has established that a network of RNA interactions may form the structural foundation of the spliceosomes. Possible solutions to many unsolved puzzles are getting attention. RNA-RNA interactions now appear to underlie many aspects of substrate recognition, reaction partner juxtaposition and catalysis. In this article we have presented the latest mechanisms involved in the pre-mRNA splicing and their implication in applied research including cancer.

RNA↗
PubMed1996

Promoter of the canine tracheobronchial mucin gene.

The mucin gene is up-regulated in diseases such as cystic fibrosis (CF) and asthma. To understand the mechanisms involved in transcriptional regulation of mucin gene expression we have characterized the region of the mucin gene up-stream of the transcriptional start site and analysed the cis-acting elements required for mucin promoter activity. We isolated clones from a dog genomic library containing the promoter region for the tracheobronchial mucin gene (TBM). The authenticity of the promoter was tested by nucleotide sequencing, primer extension analysis, electrophoretic mobility shift assay (EMSA) and reporter gene expression analysis. The canine TBM promoter is different from housekeeping gene promoters (as it is not rich in GC content and contains TATA- and CAAT-like sequences) and different from that of regulatory genes (because it contains many TATA- and CAAT-like sequences and multiple transcriptional initiation sites). Reporter gene analysis using canine TBM promoter-chloramphenicol acetyltransferase (CAT) fusion plasmids established the regions responsible for promoter activity and verified the positions of the major mucin transcriptional initiation sites. Reporter gene analysis also established that a region of the canine TBM promoter and first exon containing all of the transcriptional initiation sites is more active in mucin expressing cells (e.g. CT1 cells-immortalized canine tracheal epithelial cells, human CFT1 cells-immortalized tracheal epithelial cells from a CF subject, or HBE1 cells-immortalized tracheal epithelial cells from non-CF subject) than in mucin non-expressing cells (COS7, 3T3), suggesting cell specificity. The promoter region contained cAMP response element (CRE) sequences, and the TBM gene transcription was enhanced when cAMP analogs were added to transfected cells. EMSA indicated the presence of at least two DNA binding proteins in CT1 cells. This is the first report describing the characterization of a TBM gene promoter. The information obtained in the present studies will be valuable in understanding mucin gene regulation in normal and pathological conditions.

Animals↗
PubMed1996

2,3,7,8-tetrachlorodibenzo-p-dioxin modulates expression of the prostaglandin G/H synthase-2 gene in rat thymocytes.

As an approach to understanding the molecular mechanism(s) of thymic gene expression mediated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we investigated the effect of TCDD on expression of prostaglandin G/H synthase-2 (PGHS-2) in rat thymocytes by reverse transcription-polymerase chain reaction. Incubation of thymocytes with increasing doses of TCDD resulted in inhibition of PGHS-2 gene expression in a concentration-dependent manner, with an IC50 of 10 nM. In contrast, TCDD had no appreciable effect on expression of glyceraldehyde phosphate dehydrogenase. Because the xenobiotic-responsive element is conserved in the PGHS-2 promoter from several animal species, it seems likely that inhibition of PGHS-2 expression by TCDD may occur at the level of transcription. To test this hypothesis in cultured thymocytes, we characterized the Ah receptor in the thymoma cell line WEHI 7.1. Reverse transcription-polymerase chain reaction experiments indicated that TCDD inhibited PGHS-2 expression in this cell line. Sucrose density gradient centrifugation experiments indicated that WEHI 7.1 cytosol exhibited 9 to 10S ligand-binding activity characteristic of the Ah receptor. The viability of WEHI 7.1 cells incubated with TCDD was comparable to that of control cells, whereas dexamethasone induced toxicity in a concentration-dependent manner. Transient transfection experiments using PGHS-2 promoter fragments ligated into a chloramphenicol acetyltransferase reporter plasmid suggested that TCDD inhibits PGHS-2 transcription, and deletion of the xenobiotic-responsive element failed to exhibit this repression. These results demonstrate that TCDD is a potent inhibitor of PGHS-2 gene expression, and they represent the first mechanistic evidence for TCDD-dependent inhibition of transcription in thymocytes.

Animals↗
PubMed1995

Primary structure of the canine U1 snRNA.

The nucleotide (nt) sequence of the canine U1 snRNA was determined. It exhibited significant homology (90-98%) with known U1 sequences. The RNA can be folded according to the secondary structure previously proposed for the U1 snRNA. It contained the conserved sequence UUACCUG in loop A (nt 6-12), required for the recognition of the 5' splice site, and the sequence UGCACU in loop B (nt 68-73), required for recognition of the U1-70K protein. The U1 snRNA was localized in the nucleus and its transcription was sensitive to alpha-amanitin, suggesting that it is transcribed by RNA polymerase II. Southern analysis revealed that the canine genome possesses 5-10 copies of U1 snRNA-encoding genes.

Animals↗
PubMed1995

Differential effect of heat shock on RNA metabolism in human Burkitt's lymphoma B-cell lines.

Thermal stress induces expression of a family of heat shock proteins which may regulate the synthesis of various cellular genes. We investigated the effect of heat shock on polyadenylation in Epstein-Barr Virus (EBV) negative and EBV transformed human Burkitt's lymphoma (BL) B-cell lines. Incubation of the BL B-cell line P3HR-1, carrying the defective EBV genome [EBV nuclear antigen-2 gene deletion] at 46 degrees C for 15 min increased nuclear poly(A) polymerase (PAP) activity. Thereafter, enzymatic activity declined and at 60 min it was reduced to about 50% of that observed in cells incubated at 37 degrees C. In contrast, no significant increase in PAP activity was observed at 15 min or thereafter in an EBV- BL cell line, ST-486, in response to elevated temperature. Furthermore, no heat shock mediated change in nuclear poly(A)-specific endonuclease activity was observed in either P3HR-1 or ST-486 cells suggesting a specific effect on PAP activity. However, thermal stress dependent increase in c-myc expression was detected only in P3HR-I cells. These results suggest an association between EBV transformation and enhanced expression of c-myc and PAP activity. To further determine the role of EBV, and EBV- BL cell line, BL-30, and BL-30 cells infected in vitro with a wild type strain of EBV, BL-30/B95-8, were investigated. BL-30/B-95-8, unlike the parental BL-30 cells, exhibited c-myc and PAP gene upregulation at 15 min but were downregulated at 60 min following exposure of cells to elevated temperatures. These results suggest that infection of human B-cells with EBV is associated with their ability to respond to thermal stress by increased PAP activity which may stabilize mRNA through enhanced polyadenylation.

Burkitt Lymphoma↗
PubMed1994

2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated gene expression in the immature rat thymus.

To examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thymic gene expression in vitro, freshly isolated rat thymocytes were incubated with 10 nM TCDD, and reverse transcriptase polymerase chain reaction experiments were performed using primers specific for prostaglandin G/H synthase (PGHS) and glyceraldehyde 3-phosphate dehydrogenase. TCDD selectively repressed PGHS gene expression, with maximal inhibition occurring within 60 min. Gel retardation assays demonstrated that dioxin transiently induced binding of the ubiquitous transcription factor NF kappa B to its cognate response element at early time points. However, TCDD had little ability to induce transformation of the Ah receptor to the xenobiotic responsive element in thymic cytosol. These results indicate that TCDD exerts changes in thymocyte gene expression prior to inducing toxicity.

Animals↗
PubMed1994

An inhibitory factor in rat thymus which interferes with binding of cytosol Ah receptor to xenobiotic responsive element.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to induce aryl hydrocarbon hydroxylase in several tissues in the rat. This effect of TCDD is mediated by the Ah receptor. However, TCDD does not significantly enhance aryl hydrocarbon hydroxylase activity in the rat thymus. Analysis of 3H-TCDD bound Ah receptor on sucrose gradients revealed the presence of radioactivity sedimenting at 8-9S and 5-6S in the cytosol and nuclei of thymus respectively. Incubation of rat thymic cytosol with TCDD increased protein kinase C dependent phosphorylation, which reached saturation levels at high concentrations of TCDD, but the TCDD bound receptor did not appreciably retard 32P-labeled XRE-3 as determined by electrophoretic mobility shift assay. Incubation of rat hepatic cytosol with TCDD also increased protein kinase C dependent phosphorylation in a concentration dependent manner as well as retarded the mobility of 32P-labeled XRE far more than that observed with thymic cytosol. The ability of the Ah receptor to bind to XRE was temperature dependent. Incubation of thymic cytosol either with okadaic acid or with sodium fluoride did not increase the binding of the Ah receptor to XRE. On mixing hepatic cytosol with thymic cytosol prior to the electrophoretic mobility shift assay, the binding of the hepatic Ah receptor to XRE was reduced in a concentration-dependent manner. A similar effect was observed when renal cytosol was used instead of hepatic cytosol. Thymic cytosol also reduced the binding of the hepatic glucocorticoid receptor to its cognate responsive element. Thymic cytosol did not alter the topology/integrity of XRE. These data suggest that the thymus contains a factor(s) which interferes with the binding of AhR to its cognate responsive element.

Animals↗
PubMed1993

Inhibition of Ah (dioxin) receptor transformation by 9-hydroxy ellipticine. Involvement of protein kinase C?

9-Hydroxy ellipticine (9-OHE), a metabolite of the anti-neoplastic agent ellipticine, is known to bind the aryl hydrocarbon (Ah) receptor in rat lung cytosol and to inhibit aryl hydrocarbon hydroxylase activity (AHH) in rat hepatic microsomes. In this study, the effects of 9-OHE on the transformation of the rat hepatic cytosolic Ah receptor to a form that binds the xenobiotic responsive enhancer element-3 (XRE-3) of the cytochrome P4501A1 gene was investigated. Sucrose density gradient analysis of [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding in rat hepatic cytosol indicated that 9-OHE inhibited binding of the radiolabeled ligand to the Ah receptor with an IC50 of 90 microM. Gel retardation assays revealed that at low concentrations of 9-OHE the Ah receptor bound to XRE-3, as was the case with the TCDD-liganded receptor. However, in the presence of high concentrations of 9-OHE, the Ah receptor failed to transform to a form that could bind to XRE-3. In vitro studies indicated that incubation of rat hepatic cytosol with TCDD resulted in concentration-dependent increases in levels of protein kinase C (PKC) mediated phosphorylation as compared to vehicle-treated extracts. Furthermore, 9-OHE concentrations that exhibited agonist activity with respect to Ah receptor transformation did not alter PKC phosphorylation in hepatic cytosol, whereas higher concentrations exhibited significant concentration-dependent decrease in PKC-mediated phosphorylation. These results demonstrate that the antagonistic effect of 9-OHE observed at high concentrations is due to inhibition of Ah receptor-XRE complex formation, a phenomenon that correlates with alterations in PKC activity.

Animals↗
PubMed1993

Serum cross-reactive thymosin alpha 1 levels in rats during induction of mammary carcinoma with 7,12-dimethylbenz[a]anthracene: short- and long-term effects.

The levels of serum cross-reactive thymosin alpha 1 (CRT alpha 1) were measured at various time intervals during the course of development of mammary tumors in female Sprague-Dawley rats intubated with 7,12-dimethylbenz[a]-anthracene (DMBA; 10 mg/rat). Matched control rats were also tested simultaneously. An increase in CRT alpha 1 in DMBA-treated animals was observed within 2 h of DMBA treatment. Thereafter the levels of CRT alpha 1 in the serum of the DMBA-treated rats remained elevated for another 3 weeks prior to declining to control levels. Levels remained stationary until an increase in serum CRT alpha 1 was observed at 9-11 weeks post-DMBA treatment. This correlated with the time when mammary tumors were either palpable or observed. Levels of CRT alpha 1 fell at 13 weeks but remained slightly elevated until sacrifice due to tumor burden at 18 weeks.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1993

Early effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on rat thymocytes in vitro.

TCDD is known to induce thymic atrophy in several mammalian species through activation of programmed cell death, or apoptosis. To investigate the time course of events which precede TCDD-induced thymic apoptosis in vitro, experiments were performed with thymocytes isolated from immature rats. Peak accumulation of both total and specifically bound [3H]TCDD was observed at 60 min post incubation. Incubation of cells with 10 nM TCDD resulted in significant increases in RNA polymerase activity and incorporation of [3H]uridine at 30 min, indicating increased RNA synthesis in response to TCDD. TCDD-induced stimulation of [3H]uridine incorporation was not significantly altered in the presence of cycloheximide, while this effect was abrogated in the presence of actinomycin D. Incubation of thymocytes with 10 nM TCDD also stimulated the activity of poly(A)polymerase, the enzyme catalyzing mRNA polyadenylation, at time points beyond 30 min. No significant increases in [35S] incorporation were observed in cells treated with 10 nM TCDD, although analysis of detergent and high salt extracted nuclear proteins by SDS-PAGE and coomassie blue staining revealed the increased abundance of at least two proteins with molecular masses of 52,000 and 42,000 Da, respectively. These studies reveal that thymocyte nuclei rapidly accumulated TCDD in vitro, leading to increased RNA synthesis, poly(A)polymerase activity and protein synthesis. These events correlate closely with the process of programmed cell death.

Animals↗
PubMed1993

Stimulation of protein kinase C by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat thymocytes.

Exposure of immature thymocytes to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10 nM) transiently increased detergent extractable protein kinase C (PKC) activity. The increase was apparent at 7.5 min after exposure of cells to TCDD. At later time points, PKC activity returned to control values. Addition of TCDD to cytosol significantly increased phospholipid-stimulated PKC activity. Thus, stimulation of PKC activity may be one of the early events affected by TCDD.

Animals↗
PubMed1993

Human lung enolase: cloning and sequencing of cDNA and its inducibility with dexamethasone.

Corticosteroids used orally and intravenously lead to lung diseases and vascular disorder. To investigate whether enolase levels are also changed by treatment with synthetic steroid dexamethasone (as alteration in the enolase levels have been correlated with lung cancer) we performed the following studies. A cDNA library was prepared from poly(A) mRNA extracted from human lung fibroblast cells. cDNA clone HLE1 containing 1.7 kb insert coding for enolase was isolated. Its identity was confirmed by (a) translation of the hybrid selected mRNA and (b) nucleotide sequence analysis of the insert and comparison with known enolase sequences from other species. The lung enolase is coded by a polypeptide of 458 amino acid residues (mr = 49.5 kD). Nucleotide sequencing and derived amino acid sequence data suggest that the cloned enolase is non-neuronal isoform of enolase (NNE). In lung fibroblast cells, dexamethasone caused remarkable increase in the abundance of the enolase mRNA, which was concentration and time dependent. The induction by dexamethasone required de novo RNA synthesis but not de novo protein synthesis.

Amino Acid Sequence↗
PubMed1991

Serum poly(A)polymerase levels in rats during induction of mammary carcinoma with 7,12-dimethylbenz[a]anthracene.

Levels of activity of poly(A)polymerase (polynucleotide adenylyl transferase), the enzyme responsible for the addition of poly(A) tracts to the 3'-termini of eukaryotic mRNAs were assayed in the serum of rats during induction of mammary carcinoma by intragastric administration of 7,12-dimethylbenz[a]anthracene (DMBA, 10 mg/rat). An appreciable rise in the levels of poly(A)polymerase coincided with the growth of palpable DMBA-induced mammary carcinoma, which then dropped sharply 2 weeks later when the tumor incidence reached its peak of 65%.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

Effect of feeding a choline-deficient diet on the hepatic nuclear response to tryptophan in the rat.

In earlier studies the acute administration of tryptophan (TRP) to rats was reported to induce enhanced in vivo [14C]orotate-labeled hepatic nuclear RNA release in vitro. This change was considered to possibly be related to the induction of more and larger gamma-glutamyl transpeptidase-positive foci in the livers of rats treated with diethylnitrosamine and fed long-term elevated TRP in a choline-supplemented (CS) but not in a choline-deficient (CD) diet (comparisons with respective controls). In this study we investigated whether feeding a CD compared to a CS diet for 1 week would affect selected hepatic nuclear responses to TRP. Rats fed the CS but not the CD diet and tube-fed TRP 10 min before being killed revealed enhanced labeled hepatic nuclear RNA release in vitro. In all experiments, comparisons were made with the control groups (rats fed the CS or stock diet). When rats were fed elevated TRP (2%) in the diets (CS or CD) for 1 week, labeled hepatic nuclear RNA release was increased with the CS + TRP but not with the CD + TRP diet group. [3H]TRP binding to hepatic nuclei in vitro revealed no change in the CS + TRP group, decreased in the CD group, and markedly increased in the CD + TRP group in comparison with the control (CS) group. Hepatic nuclear nucleoside triphosphatase activity was increased only in the CS + TRP group while hepatic nuclear poly(A) polymerase activity was increased in the CS + TRP and in the CD +/- TRP groups. Serum cholesterol and triglycerides were decreased in the CD group and increased to control levels in the CD + TRP group.

Animals↗
PubMed1988

Nuclear envelope glycoprotein with poly(A) polymerase activity of rat liver: isolation, characterization, and immunohistochemical localization.

A protein with poly(A) polymerase activity has been identified and isolated from hepatic nuclear envelopes of rats to near homogeneity. The ability of the enzyme to bind to concanavalin A-agarose and to be eluted from the column with methyl alpha-D-mannopyranoside (0.2 M) as well as the inhibitory effects of alpha-mannosidase suggested that it was a glycoprotein. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP, and an oligonucleotide primer. The enzyme activity with Mn2+ was about 20-fold higher than that with Mg2+. Several known inhibitors adversely affected poly(A) polymerase activity. The enzyme has a molecular weight of 64,000 when analyzed by polyacrylamide gel electrophoresis under denaturing conditions and has a sedimentation coefficient of 4.5 S. Immunohistochemical studies using polyclonal antibodies raised against the purified enzyme revealed that the antigen was localized in the nuclear membranes.

Adenosine Triphosphate↗
PubMed1988

Poly(A) polymerase activity in murine serum. Elevation in animals with proliferative changes.

Sera of normal rats contain polynucleotide adenylyltransferase [poly(A) polymerase] activity. The enzymatic activity has been optimized with regard to primer concentration, ion requirements, kinetics, and protein. Results based on inclusion of inhibitors in the assay system show that the enzyme is poly(A) polymerase. High levels of the enzymatic activity were prevalent in sera of (a) BUF/SimfBR rats bearing sc transplanted hepatomas; (b) Sprague-Dawley rats with hepatoma cells grown in ascites; (c) partially hepatectomized Sprague-Dawley rats; and (d) MRL/lpr mice, which are in a massive lymphoproliferative autoimmune state.

Animals↗
PubMed1988

Identification and immunohistochemical localization of a tryptophan binding protein in nuclear envelopes of rat liver.

A tryptophan binding protein which was identified by binding studies has been purified to apparent homogeneity from rat liver nuclear envelopes. Two affinity matrices, namely, concanavalin A-agarose and tryptophan-agarose, were utilized for purification of the binding protein. Findings with lectin affinity chromatography suggested that the binding entity was a glycoprotein since it could be eluted off the column with methyl alpha-D-mannopyranoside (0.2 M). Eluates from both columns, when electrophoresed separately (under denaturing conditions) on polyacrylamide gels, revealed the presence of a protein with an apparent molecular weight of approximately 33,000-34,000 which is the same as that observed when covalently bound (i.e., crosslinked) [3H]-tryptophan is analyzed on polyacrylamide gels under denaturing conditions and then autoradiographed. Polyclonal antibodies raised against the binding protein recognized polypeptides with molecular weights of 64,000 and 33,000-34,000 when analyzed by the Western blot technique, suggesting that the protein was probably a dimer. Immunohistochemical studies revealed that the antigen is localized in the nuclear membranes, thereby corroborating our biochemical premise that the binding protein was present in the nuclear envelopes.

Animals↗