Total RNA Synthesis Research Articles

Regulation of gene expression during the vegetative incompatibility reaction in Podospora anserina. Characterization of three induced genes.

Vegetative incompatibility in fungi limits the formation of viable heterokaryons. It results from the coexpression of incompatible genes in the heterokaryotic cells and leads to a cell death reaction. In Podospora anserina, a modification of gene expression takes place during this reaction, including a strong decrease of total RNA synthesis and the appearance of a new set of proteins. Using in vitro translation of mRNA and separation of protein products by two-dimensional gel electrophoresis, we have shown that the mRNA content of cells is qualitatively modified during the progress of the incompatibility reaction. Thus, gene expression during vegetative incompatibility is regulated, at least in part, by variation of the mRNA content of specific genes. A subtractive cDNA library enriched in sequences preferentially expressed during incompatibility was constructed. This library was used to identify genomic loci corresponding to genes whose mRNA is induced during incompatibility. Three such genes were characterized and named idi genes for genes induced during incompatibility. Their expression profiles suggest that they may be involved in different steps of the incompatibility reaction. The putative IDI proteins encoded by these genes are small proteins with signal peptides. IDI-2 protein is a cysteine-rich protein. IDI-2 and IDI-3 proteins display some similarity in a tryptophan-rich region.

A New Stable Isotope Method Enables the Simultaneous Measurement of Nucleic Acid and Protein Synthesis In Vivo in Mice

We developed a method based on the incorporation of 13C2-units derived from [U-13C]glycine that allows the simultaneous quantification of tissue protein and RNA synthesis in vivo. Two groups of 26 mice were fed diets containing a high (HF diet) or a low quantity of fiber (LF diet). After 6 d, [U13C]glycine was added to the diet and groups of four mice were killed after 2, 4, 6, 8, 12 and 24 h. Hepatic and intestinal mucosal free and RNA-bound purine nucleosides were extracted and enzymically degraded to allantoin. Allantoin was degraded to glyoxylate, which was then reductively aminated to glycine, which contains the two 13C-atoms incorporated via de novo synthesis. Ingestion of the HF diet was associated with significantly (P < 0.05) higher rates of total RNA synthesis in both the liver ( HF diet, 29%/d; LF diet, 21%/d) and mucosa (HF diet, 27%/d; LF diet, 17 %/d). The mean rates of RNA synthesis in each tissue were significantly (P < 0.01) lower than the respective rates of protein synthesis (liver, 67%/d; mucosa, 74%/d). The isotopic enrichment of the free purine nucleotide pool increased rapidly and exponentially, but the steady-state value was substantially (P < 0.001) lower than that of the RNA-bound purines. The results suggest that the free nucleotide pool consists of two kinetically distinct compartments, one of which is small and has a rapid rate of turnover. This, we propose, acts as the RNA precursor pool. The other is large, has a low rate of turnover and, we believe, is the pool of adenosine triphosphate involved in cellular energetics.

Effect of DNA-interacting drugs on phage T7 RNA polymerase.

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

Granulocyte-Macrophage Colony-Stimulating Factor Enhances EBV-Induced Synthesis of Chemotactic Factors in Human Neutrophils

We have recently demonstrated that EBV binds to human neutrophils and stimulates a wide range of activities, including homeotypic aggregation, total RNA synthesis, and expression of the chemokines IL-8 and macrophage inflammatory protein-1alpha (MIP-1alpha). Neutrophil function is also known to be modulated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have therefore investigated the modulation of EBV-induced activation of human neutrophils by GM-CSF. Treatment of neutrophils with GM-CSF before EBV activation enhanced the production of both MIP-1alpha and IL-8. The IL-8 produced under these conditions was biologically active as determined in the calcium mobilization assay. GM-CSF was also found to increase the ability of EBV to prime neutrophils for increased leukotriene B4 (LTB4) synthesis. Prior treatment of GM-CSF with neutralizing Abs inhibited these effects. GM-CSF also increased the specific binding of FITC-EBV to the neutrophil surface, as evaluated by fluorocytometry. Local production of GM-CSF in tissues invaded by EBV could therefore serve to potentiate a host defense mechanism directed toward the destruction of the infectious virus via increased production of chemotactic factors. Since both IL-8 and MIP-1alpha are reported to be chemoattractants in vitro for T cells and T and B cells, respectively, the ability of EBV to induce their production by neutrophils may enhance its ability to infect B and T lymphocytes via increased recruitment to sites of infection.

Paracetamol increases sensitivity to ultraviolet (UV) irradiation, delays repair of the UNG-gene and recovery of RNA synthesis in HaCaT cells

We have studied the effect of low levels of paracetamol (0.3 and 1.0 mM) on gene-specific DNA repair, recovery of total RNA synthesis and cytotoxicity after exposure of human keratinocyte cells (HaCaT) to ultraviolet (UV) irradiation. Repair of cyclobutane pyrimidine dimers (CPDs) was measured in the transcriptionally active uracil-DNA glycosylase ( UNG) and c-MYC loci. Repair of both strands in the UNG gene was consistently lower in the presence of paracetamol, but this reduction reached significance only at 8 h after irradiation and no dose-response was observed. For the c-MYC gene, we found no significant effect of paracetamol on the repair of CPDs, possibly because UV-irradiation is known to induce transcription of the c-MYC gene and enhanced transcription coupled repair might counteract a negative effect of paracetamol on global genome repair. A dose-dependent delay in the recovery of total RNA synthesis after UV exposure was observed in the presence of paracetamol, which also caused a 20% increase in UV-induced cytotoxicity after 24 h. Paracetamol had no significant effect on either RNA synthesis or cell survival in the absence of UV after 24 h, but reduced cell survival by ∼10% (at 0.3 mM) and 50% (at 1.0 mM) after 96 h exposure. Our results demonstrate that paracetamol may inhibit gene-specific repair of CPDs by affecting global genome repair and that different genes may be differentially affected.

Mitotic repression of RNA polymerase II transcription is accompanied by release of transcription elongation complexes.

Nuclear RNA synthesis is repressed during the mitotic phase of each cell cycle. Although total RNA synthesis remains low throughout mitosis, the degree of RNA polymerase II transcription repression on specific genes has not been examined. In addition, it is not known whether mitotic repression of RNA polymerase II transcription is due to polymerase pausing or ejection of transcription elongation complexes from mitotic chromosomes. In this study, we show that RNA polymerase II transcription is repressed in mammalian cells on a number of specific gene regions during mitosis. We also show that the majority of RNA polymerase II transcription elongation complexes are physically excluded from mitotic chromosomes between late prophase and late telophase. Despite generalized transcription repression and stripping of RNA polymerase II complexes from DNA, arrested RNA polymerase II ternary complexes appear to remain on some gene regions during mitosis. The cyclic repression of transcription and ejection of RNA polymerase II transcription elongation complexes may help regulate the transcriptional events that control cell cycle progression and differentiation.

Changes in RNA and Protein Metabolism Associated with Alterations in the Germination Efficiency of Sunflower Seeds

Precise knowledge of seed quality after harvest and during storage is of particular importance for seed producers. We analysed different sunflower seed lots (Helianthus annuusL.) characterized by extremes of germination ability. We used RNA analysis to study possible changes in gene expression in seeds unable to germinate. Total RNA content was very small in dry seeds showing a low germination ability. Capacity for total RNA synthesis at the onset of imbibition was also reduced in these seeds. In addition, correlations were found between these parameters and germination ability at 19 °C. We demonstrated a high correlation between the amount of total RNA in the dry seed, the capacity of RNA synthesis at the onset of imbibition and the seed moisture content at the time of the harvest. The ability of dry seed mRNAs to be translatedin vitrowas also reduced and seven polypeptides, from stored mRNAs, were characteristic of the cotyledons from high germinability seeds. Germination can thus be affected at several levels including membrane, enzymatic and nucleic acid deteriorations.

Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli. Purification and characterization of the recombinant protein.

Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII, and LbIII) that are encoded by at least two genes. We have cloned and sequenced the gene that encodes for LbII (lbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR. Primers were designed using the sequence of the soybean lbc gene. The lbII gene is 679 bp in length and codes for a predicted protein of 145 amino acids. Using sequences of the cowpea lbII gene for the synthesis of primers and total nodule RNA as the template, we cloned a cDNA for LbII into a constitutive expression vector (pEMBL19+) and then expressed it in Escherichia coli. Recombinant LbII (rLbII) and native LbII (nLbII) from cowpea nodules were purified to homogeneity using standard techniques. Properties of rLbII were compared with nLbII by partially sequencing the proteins and by sodium dodecyl sulfate- and isoelectric focusing polyacrylamide gel electrophoresis, western-blot analysis using anti-soybean Lba antibodies, tryptic and chymotryptic mapping, and spectrophotometric techniques. The data showed that the structural and spectral characteristics of rLbII and nLbII were similar. The rLbII was reversibly oxygenated/deoxygenated, showing that it is a functional hemoglobin.

Defects of Insulin and IGF-1 Action at Receptor and Postreceptor Level in a Patient with Type A Syndrome of Insulin Resistance

The action of insulin and IGF-1 in comparison to non-diabetic controls was studied in cultured fibroblasts of a patient with an inherited syndrome of insulin resistance (Type A syndrome). Insulin binding was reduced due to decreased receptor affinity, but sequence analyses revealed no alterations of splicing or primary insulin receptor (IR) structure. Most likely due to the IR affinity defect analyses of signal transduction pathways showed an impairment of insulin action on glucose uptake, total RNA synthesis and phosphorylation as well as activity of MAP-kinase. In addition inducibility of c-fos mRNA level was strongly impaired by insulin and IGF-1, but comparable to controls by PDGF indicating a postreceptor defect. In conclusion, we provide evidence that genetic syndromes of insulin resistance can be associated with both, receptor and postreceptor defects.

ω-Conotoxin-sensitive Ca 2+ voltage-gated channels modulate protein secretion in cultured rat Sertoli cells

Recent results have demonstrated that substantial calcium influx in rat Sertoli cells is mediated by cation channels of both L- and N-type. In this report we have investigated the possible role of such channels in the protein secretion of immature rat Sertoli cell monolayers. The blocking of N-type voltage-gated channels by ω-conotoxin ( ω-CTX) GVIA results in a 50–60% inhibition of the protein secretion in the culture medium while total protein and RNA synthesis are not affected. The same extent of protein secretion inhibition is obtained in FSH-stimulated Sertoli cells. L-type voltage-gated channels apparently are not involved in such a modulation. These data, showing that a major fraction of secreted proteins from cultured rat Sertoli cells is Ca 2+ dependent, represent the first evidence of a physiological role of voltage-operated Ca 2+ channels in mammalian testis.

Directional random oligonucleotide primed (DROP) global amplification of cDNA: its application to subtractive cDNA cloning.

We describe a method of global PCR amplification of cDNA such that the strand sense is maintained. The products of this process are random primed fragments ranging in size from 100 to 500 bp which facilitates uniform PCR amplification of total cDNA. Directional incorporation of a T7 RNA polymerase initiator/promoter sequence allows efficient synthesis of total sense RNA from this material and the use of a biotinylated primer permits the separation of single-stranded cDNA. Isolation of these products from different cell types provides a renewable source of target single-stranded cDNA and driver RNA from limited cell numbers and we demonstrate their use for subtractive hybridisation cloning of differentially expressed cDNAs.

Functional antagonism between IL-2 and PGA 1 or PGJ 2 in the control of proliferation of human cord blood-derived mononuclear cells

Cyclopentenone prostaglandins PGA 1 and PGJ 2 induce growth arrest at the G 1/S interphase of the cell cycle in tumour cell lines. Notably, PGE, the precursor molecule of PGA, downregulates the interleukin (IL)-2-dependent proliferation of lymphocytes. Therefore the IL-2/IL-2 receptor system and relative signal transduction is a possible target of the antiproliferative effect of PGA/PGJ. In the present study the PGA 1/PGJ 2-dependent growth inhibition of IL-2-stimulated primary human cord blood mononuclear cells (CBMCs) was found to be mediated by interference with the IL-2 proliferative signal. Both prostaglandins (PGs) inhibited the synthesis of total RNA and protein in IL-2 stimulated cells. PGA 1 and even more PGJ 2 downregulated the expression of IL-2 receptor α (CD25 phenotype). IL-2 partly reversed this effect. Moreover, suppression of IL-2-stimulated cells was not the result of PG-mediated activation of apoptosis. On the contrary, PGs reduced both apoptosis and the high expression of c-Jun detectable in CBMCs spontaneously. Cyclin A/Cdk2 complexes regulate G 1/S transition during the cell cycle. In IL-2-stimulated cells, the levels of Cdk2 were found to be lower in PG-treated cells than those detected in controls. In conclusion, cyclopentenone PGs inhibit CBMCs spontaneous or IL-2-dependent proliferation in part by interfering with the I L-2 pathway.

Effect of cycloheximide and mRNA synthesis inhibition on death of trophically deprived ciliary ganglion neurons in culture.

1. The relationship between cycloheximide (CHX) and RNA synthesis inhibitors on trophic-deprived neuronal survival was studied with the use of primary cultures of stage (St) 34 chick ciliary ganglion (CG) neurons, to analyze the biological process of neuronal death caused by trophic factor withdrawal. Tissue culture conditions were refined by characterizing the additional medium components required to obtain 100% survival, for at least 1 wk, in the presence of an eye extract [choroid, ciliary body, iris, and pigment epithelium (CIPE)] as a trophic support for the neurons. Highly enriched neuronal cultures almost devoid of nonneuronal cells were used. 2. The time at which trophically deprived neurons cannot be rescued by the addition of trophic support, "commitment point," was established to be between 11 and 17 h after trophic deprivation. 3. CHX, an inhibitor of protein translation, reduced 3H-leucine incorporation by 90-95%, at a concentration of 10-100 micrograms/ml. The effect of the RNA transcription blockers actinomycin D (Act-D), alpha-amanitin, and 5.6 dichlorobenzimidazole riboside (DRB) on 3H-uridine incorporation into macromolecules was evaluated. Total RNA synthesis was inhibited by 10-25% by alpha-amanitin, whereas Act-D and DRB inhibited 80-97.5% of the 3H-uridine incorporation. 4. The effect of short- and long-term incubation with CHX on neuronal survival was analyzed. Continuous application of CHX promoted survival for 2-3 days, but thereafter neurons died regardless of whether CIPE was present or absent. Application of CHX for 6 h from the onset of the culture was enough to delay the commitment point up to 24 h after plating, and the addition of CIPE at this time maintained survival and promoted differentiation of CHX treated neurons. 5. The RNA transcription blockers Act-D, alpha-amanitin, and DRB were applied to both trophically deprived and trophically supported neurons, and the survival of each was evaluated. Neither drug was effective in supporting the survival of trophically deprived neurons in culture, and in most cases neurons even when cultured with CIPE died within 1-2 days in the presence of either drug. 6. Experiments using both CHX and mRNA synthesis blockers were performed to determine the effect of blocking mRNA transcription in trophically deprived neurons rescued by CHX. The addition of mRNA synthesis inhibitors precluded the effect of CHX on neuronal survival. 7. The effect of CHX (20 micrograms/ml) on RNA and protein synthesis was studied by measuring the incorporation of radiolabeled metabolic precursors (3H-leucine or 3H-uridine) into macromolecules. A 95% reduction in the protein synthesis was observed after 1 h of application of the drug, and by 24 h, 3H-leucine incorporation was reduced to 15-20% of the control values. Wash out of CHX after 6 h of incubation produced a recovery of protein synthesis up to 50% of control values 18 h later. CHX did not affect the synthesis of RNA for up to 12 h; however, it impaired the ability of the cell to take up metabolic precursors. 8. In conclusion, the present results support the hypothesis that the CHX effect on neuronal survival is due to its ability to induce the expression of survival or protective genes rather than to block the expression of killer proteins. This view is supported by 1) the 24-h delay of the commitment point following the short-term application of CHX, 2) the impaired ability of CHX to rescue trophic-deprived neurons by the addition of mRNA synthesis blockers, and 3) the fact that neuronal survival caused by trophic factors like CIPE, is blocked by blocking RNA transcription.

Cobalt exerts opposite effects on erythropoietin gene expression in rat hepatocytes in vivo and in vitro.

This study investigates the effects of hypoxia and of cobalt on erythropoietin (EPO) gene expression in hepatocytes in vivo and in vitro in neonatal, juvenile, and adult rats. With the use of the ribonuclease protection assay to quantify RNA, both hypoxia (0.1% CO or 9% O2) and cobalt (60 mg/kg) elicit production of increased amounts of EPO mRNA in neonatal and juvenile rat liver in vivo. In vitro hepatocyte EPO gene expression could be reproducibly stimulated by hypoxia (3% O2) but not by cobaltous chloride (50-150 microM) within 2-20 h. Conversely, cobalt substantially attenuated the rise of EPO mRNA levels in response to hypoxia. This inhibitory effect of cobalt was mimicked by zinc but not by other metals. CO attenuated the rise of EPO mRNA levels in vitro in response to hypoxia; this inhibitory effect coincided with an inhibition of total RNA synthesis as determined by [3H]uridine incorporation. The lack of specific inhibitory effects of CO and of specific stimulatory effects of cobalt on hepatocyte EPO gene expression in vitro suggests that a specific heme oxygen sensor may be less important than in hepatoma cells.

Epstein-Barr virus modulates de novo protein synthesis in human neutrophils

Neutrophils and macrophages represent the first line of defense against microbial invaders. However, the role of phagocytes in host response to viral infection is poorly understood. We have previously shown that Epstein-Barr virus (EBV) interacts with human monocytes and modulates cytokine production in this cell type, but its effects on neutrophils are still unknown. In the present study, we investigated the presence of EBV receptor (CR2 or CD21) on neutrophils by cytofluorometry using five different anti-CD21 monoclonal antibodies (MoAbs), as well as fluoroscein isothiocyanate-EBV (FITC-EBV). Whereas no significant amount of neutrophils reacted with anti-CD21 MoAbs, studies with FITC-EBV indicated that viral particles bind to 30% of cells (in some individuals, EBV binds to more than 50% of neutrophils). This interaction is specific as it was completely inhibited by nonconjugated virus or with labeled virus preincubated with neutralizing MoAbs. After EBV treatment, cellular aggregation was observed in neutrophil cultures, an indication that neutrophils were activated. Although EBV did not induce respiratory burst activity in neutrophils, pretreatment with infectious particles enhanced (priming effect) the fMLP-induced O2- release in neutrophils. Instead of restricting our analysis to specific cytokine genes, we investigated the effects of EBV on neutrophil transcriptional events in general. The effect of this virus on de novo synthesis of total cellular RNA was first investigated by measuring the incorporation of [5-3H] uridine into total RNA. The results showed that RNA synthesis in neutrophils was significantly increased (2.3- to 21.3-fold) by EBV compared with the unstimulated controls. Live and UV-inactivated virus markedly induced RNA synthesis, whereas heat-inactivated virus lost this ability. Induction of RNA transcription was EBV specific, as an EBV-neutralizing antiserum abolished this effect. Induction of protein synthesis was also studied by measuring the incorporation of [35S] methionine and [35S] cysteine into secreted and intracellular proteins in neutrophils incubated with EBV. The synthesis of both secreted and cytoplasmic proteins was induced by EBV. One- and two-dimensional gel electrophoresis analysis showed that EBV modulates protein synthesis, because activation of the synthesis of certain proteins was accompanied by the inhibition of others. Interleukin-1 beta (IL-1 beta) and IL-1 receptor antagonist (IL-1Ra) synthesis was found to be induced by EBV. Therefore, modulation of host-response proteins such as IL-1Ra could be one of the many mechanisms by which this virus avoids rejection.(ABSTRACT TRUNCATED AT 400 WORDS)

Epstein-Barr Virus Modulates De Novo Protein Synthesis in Human Neutrophils

Abstract Neutrophils and macrophages represent the first line of defense against microbial invaders. However, the role of phagocytes in host response to viral infection is poorly understood. We have previously shown that Epstein-Barr virus (EBV) interacts with human monocytes and modulates cytokine production in this cell type, but its effects on neutrophils are still unknown. In the present study, we investigated the presence of EBV receptor (CR2 or CD21) on neutrophils by cytofluorometry using five different anti-CD21 monoclonal antibodies (MoAbs), as well as fluoroscein isothiocyanate-EBV (FITC-EBV). Whereas no significant amount of neutrophils reacted with anti-CD21 MoAbs, studies with FITC-EBV indicated that viral particles bind to 30% of cells (in some individuals, EBV binds to more than 50% of neutrophils). This interaction is specific as it was completely inhibited by nonconjugated virus or with labeled virus preincubated with neutralizing MoAbs. After EBV treatment, cellular aggregation was observed in neutrophil cultures, an indication that neutrophils were activated. Although EBV did not induce respiratory burst activity in neutrophils, pretreatment with infectious particles enhanced (priming effect) the fMLP-induced O2-release in neutrophils. Instead of restricting our analysis to specific cytokine genes, we investigated the effects of EBV on neutrophil transcriptional events in general. The effect of this virus on de novo synthesis of total cellular RNA was first investigated by measuring the incorporation of [5–3H] uridine into total RNA. The results showed that RNA synthesis in neutrophils was significantly increased (2.3- to 21.3-fold) by EBV compared with the unstimulated controls. Live and UV-inactivated virus markedly induced RNA synthesis, whereas heat-inactivated virus lost this ability. Induction of RNA transcription was EBV specific, as an EBV-neutralizing antiserum abolished this effect. Induction of protein synthesis was also studied by measuring the incorporation of [35S] methionine and [35S] cysteine into secreted and intracellular proteins in neutrophils incubated with EBV. The synthesis of both secreted and cytoplasmic proteins was induced by EBV. One- and two-dimensional gel electrophoresis analysis showed that EBV modulates protein synthesis, because activation of the synthesis of certain proteins was accompanied by the inhibition of others. Interleukin-1 beta (IL-1 beta) and IL-1 receptor antagonist (IL-1Ra) synthesis was found to be induced by EBV. Therefore, modulation of host-response proteins such as IL-1Ra could be one of the many mechanisms by which this virus avoids rejection.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutathione depletion in human T lymphocytes: analysis of activation-associated gene expression and the stress response.

Glutathione depletion achieved by continuous exposure of mitogen-activated human T lymphocytes to L-buthionine-(S,R)-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, leads to a marked inhibition of the proliferative response. Concanavalin A-activated T cells treated with buthionine sulfoximine failed to exhibit the increase in glutathione content normally observed in activated T cells and were depleted of cellular glutathione over 4 days of culture. On Day 3 of culture, DNA synthesis was inhibited by greater than 75%. In addition, total RNA synthesis was dramatically reduced in the glutathione-depleted cells being inhibited by 26, 61, and 82% on Days 2, 3, and 4, respectively. Despite this global reduction in RNA synthesis, no specific effects on mRNA expression of a number of critical T cell genes required for activation and/or proliferation were detected. In contrast to a recent report of GSH depletion leading to down-regulation of ras mRNA expression in a number of transformed cell lines, glutathione depletion did not influence N-ras mRNA expression in T lymphocytes. No influence of glutathione depletion on the induction of histone mRNA expression was observed. However, consistent with previous studies on regulation of histone mRNA expression, histone transcript levels were reduced when DNA synthesis was markedly inhibited. A cellular stress response, characterized by an increase in mRNA levels of the two stress response genes, HSP70 and gadd 153, was evident in glutathione-depleted unstimulated cells. Additionally, in these cells at 48 hr, we observed a 3.5-fold increase in the steady-state level of mRNA encoding the catalytic subunit of gamma-glutamylcysteine synthetase, the enzyme inhibited by buthionine sulfoximine.

N-ras mRNA expression is unaffected in glutathione-depleted cells of hematopoietic origin.

Glutathione (GSH) depletion in mitogen-stimulated T lymphocytes has been shown to markedly inhibit their proliferative response. This block in proliferation is associated with a significant reduction in total RNA and DNA synthesis; however, the specific mechanism involved in this inhibition of proliferation is unknown. Miller et al. have reported that lowering intracellular GSH levels by greater than 30%, in murine and human tumor cell lines of non-hematopoietic origin, leads to down-regulation of HA-, Ki- and N-ras oncogene expression [Miller. A.C., Gafner, J., Clark, E.P. and Samid, D. (1993) Mol. Cell Biol., 13, 4416-4422]. The reduction in ras transcript levels correlated with the extent of GSH depletion and was independent of the specific mode of oncogene activation. Since the activity of p21(ras) is thought to be involved in pathways of T cell activation, we set out to determine whether down-regulation of ras expression in T cells could be the mechanism by which T cell proliferation was inhibited in GSH-depleted T lymphocytes. Despite reducing the GSH level of concanavalin A-activated human peripheral blood mononuclear cells by 66%, no effect on ras mRNA expression was observed. Similarly, no reduction of ras transcript levels were detected in a human T cell line (Jurkat) or in a human monocytic cell line (THP-1) depleted of glutathione. Our results demonstrate that the mechanism by which GSH depletion inhibits T cell proliferation does not appear to involve a decrease in ras mRNA expression. In addition, our results suggest that differences in the regulation of ras mRNA expression may exist between lymphoid/monocytic cells of non-hematopoietic origin.

Transcriptional regulation of Na/K-ATPase by corticosteroids, glycyrrhetinic acid and second messenger pathways in rat kidney epithelial cells.

Corticosteroid regulation of Na/K-ATPase is of key importance in the modulation of Na+ transport across renal tubular epithelia. In amphibian renal cells, aldosterone induction of Na/K-ATPase alpha 1 and beta 1 subunit gene transcription is mediated by an indirect mechanism dependent on the synthesis of a labile protein. In mammalian target cells, while both mineralo- and glucocorticoids increase the levels of Na/K-ATPase alpha 1 and beta 1 subunit mRNA and enzyme activity, they are diminished by glycyrrhetinic acid (GE), the active ingredient of licorice. To investigate the mechanisms underlying the regulation of mammalian renal Na/K-ATPase, levels of alpha 1 and beta 1 mRNA were measured in rat kidney epithelial (NRK-52E) cells treated with a range of concentrations of aldosterone, corticosterone and GE in the presence of a specific inhibitor of mRNA synthesis, dichlororibofuranosylbenzimidazole (DRB), an inhibitor of total RNA synthesis, actinomycin D (ActD), and the protein synthesis inhibitor cycloheximide (CHX). In addition, GE was co-incubated with the sodium channel antagonist benzamiloride (BZ). The increase in both alpha 1 and beta 1 mRNA levels following aldosterone and corticosterone was completely abolished by treatment with ActD and DRB, while CHX did not affect this response. Similarly, the GE-induced decrease in alpha 1 and beta 1 mRNA was also completely abolished by ActD and DRB, but not by CHX or by BZ. The half-lives of alpha 1 and beta 1 mRNA in these cells (means +/- S.E.M., n = 4), estimated from the rate of mRNA decay in the presence of DRB, were 6.8 +/- 0.3 and 4.8 +/- 0.2 h respectively. This was unaffected by GE. The inhibitory action of GE on alpha 1 and beta 1 mRNA levels was accompanied by a dose-dependent decrease in levels of intracellular cAMP (means +/- S.E.M., n = 4) from 395 +/- 28 fmol cAMP/microgram total cell protein to between 275 +/- 19 fmol/micrograms total cell protein (0.1 microM GE) and 78 +/- 11 fmol/micrograms total cell protein (10 microM GE). This was abolished following down-regulation of protein kinase C by prolonged treatment with the phorbol ester tetradecanoylphorbol-13-acetate (TPA), and by pertussis toxin (PT), but not by cholera toxin (CT). Indeed, subunit mRNA levels were increased by 8-bromo-cAMP (2.2-fold) and stimulators of adenylate cyclase activity, i.e. forskolin (2.1-fold), PT (2.1-fold) and CT (1.9-fold), but not by TPA.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholinergic regulation of amylase gene expression in the rat parotid gland. Inhibition by two distinct post-transcriptional mechanisms.

Stimulation of the beta-adrenergic or cholinergic muscarinic receptors are the principal mechanisms by which parotid salivary secretion is regulated in vivo. In this study we have examined the effects of cholinergic stimulation on amylase gene expression in dispersed rat parotid cells. [3H]Leucine incorporation into amylase and total protein was inhibited by carbamylcholine. Within 5 min of its addition, 10 microM carbamylcholine induced a 50-60% reduction in the rate of amylase synthesis which was sustained for more than 2 h. Blockade of the muscarinic receptor with atropine 8 min after addition of 10 microM carbamylcholine reversed the carbamylcholine-induced inhibition of amylase synthesis. When cells were exposed to carbamylcholine for 2 h before addition of atropine, there was only a slight reversal of inhibition. Carbamylcholine had no significant effect on the rate of total RNA synthesis but caused a progressive loss of amylase mRNA. After 2 h, amylase mRNA in cells treated with 10 microM carbamylcholine was 46% of control levels. Actinomycin D (5 micrograms/ml) lowered amylase mRNA by 8%; cycloheximide and phorbol 12-myristate 13-acetate had no effect. Isoprenaline (isoproterenol; at a concentration of 10 microM), which is an inducer of amylase gene transcription, elevated the amylase mRNA content by 30% after 2h. The calcium ionophore A23187 mimicked the effect of carbamylcholine by inhibiting [3H]leucine incorporation into amylase and lowering amylase mRNA content. The results suggest that acute stimulation of the muscarinic cholinergic receptor inhibits amylase biosynthesis in parotid cells not only by rapid attenuation of translation but also by causing a gradual loss of amylase mRNA, apparently by a Ca(2+)-dependent destabilization of the mRNA.