Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3′-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3′-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

Salicylates and sulfasalazine, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 of NFκB

The antiinflammatory action of aspirin generally has been attributed to direct inhibition of cyclooxygenases (COX-1 and COX-2), but additional mechanisms are likely at work. These include aspirin’s inhibition of NFκB translocation to the nucleus as well as the capacity of salicylates to uncouple oxidative phosphorylation (i.e., deplete ATP). At clinically relevant doses, salicylates cause cells to release micromolar concentrations of adenosine, which serves as an endogenous ligand for at least four different types of well-characterized receptors. Previously, we have shown that adenosine mediates the antiinflammatory effects of other potent and widely used antiinflammatory agents, methotrexate and sulfasalazine, both in vitro and in vivo. To determine in vivo whether clinically relevant levels of salicylate act via adenosine, via NFκB, or via the “inflammatory” cyclooxygenase COX-2, we studied acute inflammation in the generic murine air-pouch model by using wild-type mice and mice rendered deficient in either COX-2 or p105, the precursor of p50, one of the components of the multimeric transcription factor NFκB. Here, we show that the antiinflammatory effects of aspirin and sodium salicylate, but not glucocorticoids, are largely mediated by the antiinflammatory autacoid adenosine independently of inhibition of prostaglandin synthesis by COX-1 or COX-2 or of the presence of p105. Indeed, both inflammation and the antiinflammatory effects of aspirin and sodium salicylate were independent of the levels of prostaglandins at the inflammatory site. These experiments also provide in vivo confirmation that the antiinflammatory effects of glucocorticoids depend, in part, on the p105 component of NFκB.

Potentiation of the Oxidative Burst and Isoflavonoid Phytoalexin Accumulation by Serine Protease Inhibitors1

Treatment of soybean (Glycine max L. cv Williams 82) cell-suspension cultures with Pseudomonas syringae pv glycinea (Psg) harboring an avirulence gene (avrA) or with yeast elicitor resulted in an oxidative burst characterized by the accumulation of H2O2. This burst, and the resultant induction of glutathione S-transferase transcripts, occurred more rapidly and was more prolonged if cells were simultaneously treated with serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) or diisopropylfluorophosphate. PMSF and diisopropylfluorophosphate potentiate a large oxidative burst in cells exposed to Psg harboring the avrC avirulence gene, which is not recognized by the soybean cultivar used in this study. The potentiated burst was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and by the protein kinase inhibitor K252a. PMSF treatment of elicited cells or cells exposed to Psg:avrA caused a large increase in the accumulation of the isoflavonoid phytoalexin glyceollin; however, this was not associated with increased levels of transcripts encoding key phytoalexin biosynthetic enzymes. Glyceollin accumulation was inhibited by diphenylene iodonium; however, the oxidative burst in cells treated with Psg:avrC and PMSF was not followed by phytoalexin accumulation. We conclude that active oxygen species from the oxidative burst are necessary but not sufficient for inducing isoflavonoid phytoalexin accumulation in soybean cells.

The Role of EDTA in Lead Transport and Accumulation by Indian Mustard1

Indian mustard (Brassica juncea) plants exposed to Pb and EDTA in hydroponic solution were able to accumulate up to 55 mmol kg−1 Pb in dry shoot tissue (1.1% [w/w]). This represents a 75-fold concentration of Pb in shoot tissue over that in solution. A threshold concentration of EDTA (0.25 mm) was found to be required to stimulate this dramatic accumulation of both Pb and EDTA in shoots. Below this threshold concentration, EDTA also accumulated in shoots but at a reduced rate. Direct measurement of a complex of Pb and EDTA (Pb-EDTA) in xylem exudate of Indian mustard confirmed that the majority of Pb in these plants is transported in coordination with EDTA. The accumulation of EDTA in shoot tissue was also observed to be directly correlated with the accumulation of Pb. Exposure of Indian mustard to high concentrations of Pb and EDTA caused reductions in both the transpiration rate and the shoot water content. The onset of these symptoms was correlated with the presence of free protonated EDTA (H-EDTA) in the hydroponic solution, suggesting that free H-EDTA is more phytotoxic than Pb-EDTA. These studies clearly demonstrate that coordination of Pb transport by EDTA enhances the mobility within the plants of this otherwise insoluble metal ion, allowing plants to accumulate high concentrations of Pb in shoots. The finding that both H-EDTA and Pb-EDTA are mobile within plants also has important implications for the use of metal chelates in plant nutritional research.

Regulation of Tomato Fruit Polygalacturonase mRNA Accumulation by Ethylene: A Re-Examination1

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 μL/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 μL/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 μL/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 μL/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 μL/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.

Anion Channels and the Stimulation of Anthocyanin Accumulation by Blue Light in Arabidopsis Seedlings1

Activation of anion channels by blue light begins within seconds of irradiation in seedlings and is related to the ensuing growth inhibition. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a potent, selective, and reversible blocker of these anion channels in Arabidopsis thaliana. Here we show that 20 μm NPPB blocked 72% of the blue-light-induced accumulation of anthocyanin pigments in seedlings. Feeding biosynthetic intermediates to wild-type and tt5 seedlings provided evidence that NPPB prevented blue light from up-regulating one or more steps between and including phenylalanine ammonia lyase and chalcone isomerase. NPPB was found to have no significant effect on the blue-light-induced increase in transcript levels of PAL1, CHS, CHI, or DFR, which are genes that encode anthocyanin-biosynthetic enzymes. Immunoblots revealed that NPPB also did not inhibit the accumulation of the chalcone synthase, chalcone isomerase, or flavanone-3-hydroxylase proteins. This is in contrast to the reduced anthocyanin accumulation displayed by a mutant lacking the HY4 blue-light receptor, as hy4 displayed reduced expression of the above enzymes. Taken together, the data indicate that blue light acting through HY4 leads to an increase in the amount of biosynthetic enzymes, but blue light must also act through a separate, anion-channel-dependent system to create a fully functional biosynthetic pathway.

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus1

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.

Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-γ ligand troglitazone in patients with liposarcoma

Agonist ligands for the nuclear receptor peroxisome proliferator-activated receptor-γ have been shown to induce terminal differentiation of normal preadipocytes and human liposarcoma cells in vitro. Because the differentiation status of liposarcoma is predictive of clinical outcomes, modulation of the differentiation status of a tumor may favorably impact clinical behavior. We have conducted a clinical trial for treatment of patients with advanced liposarcoma by using the peroxisome proliferator-activated receptor-γ ligand troglitazone, in which extensive correlative laboratory studies of tumor differentiation were performed. We report here the results of three patients with intermediate to high-grade liposarcomas in whom troglitazone administration induced histologic and biochemical differentiation in vivo. Biopsies of tumors from each of these patients while on troglitazone demonstrated histologic evidence of extensive lipid accumulation by tumor cells and substantial increases in NMR-detectable tumor triglycerides compared with pretreatment biopsies. In addition, expression of several mRNA transcripts characteristic of differentiation in the adipocyte lineage was induced. There was also a marked reduction in immunohistochemical expression of Ki-67, a marker of cell proliferation. Together, these data indicate that terminal adipocytic differentiation was induced in these malignant tumors by troglitazone. These results indicate that lineage-appropriate differentiation can be induced pharmacologically in a human solid tumor.

Prevention of cardiovascular and renal pathology of aging by the advanced glycation inhibitor aminoguanidine.

Human aging is impacted severely by cardiovascular disease and significantly but less overtly by renal dysfunction. Advanced glycation endproducts (AGEs) have been linked to tissue damage in diabetes and aging, and the AGE inhibitor aminoguanidine (AG) has been shown to inhibit renal and vascular pathology in diabetic animals. In the present study, the effects of AG on aging-related renal and vascular changes and AGE accumulation were studied in nondiabetic female Sprague-Dawley (S-D) and Fischer 344 (F344) rats treated with AG (0.1% in drinking water) for 18 mo. Significant increases in the AGE content in aged cardiac (P < 0.05), aortic (P < 0.005), and renal (P < 0.05) tissues were prevented by AG treatment (P < 0.05 for each tissue). A marked age-linked vasodilatory impairment in response to acetylcholine and nitroglycerine was prevented by AG treatment (P < 0.005), as was an age-related cardiac hypertrophy evident in both strains (P < 0.05). While creatinine clearance was unaffected by aging in these studies, the AGE/ creatinine clearance ratio declined 3-fold in old rats vs. young rats (S-D, P < 0.05; F344, P < 0.01), while it declined significantly less in AG-treated old rats (P < 0.05). In S-D but not in F344 rats, a significant (P < 0.05) age-linked 24% nephron loss was completely prevented by AG treatment, and glomerular sclerosis was markedly suppressed (P < 0.01). Age-related albuminuria and proteinuria were markedly inhibited by AG in both strains (S-D, P < 0.01; F344, P < 0.01). These data suggest that early interference with AGE accumulation by AG treatment may impart significant protection against the progressive cardiovascular and renal decline afflicting the last decades of life.

Autoregulation at the level of mRNA 3′ end formation of the suppressor of forked gene of Drosophila melanogaster is conserved in Drosophila virilis

The Drosophila melanogaster Suppressor of forked [Su(f)] protein shares homology with the yeast RNA14 protein and the 77-kDa subunit of human cleavage stimulation factor, which are proteins involved in mRNA 3′ end formation. This suggests a role for Su(f) in mRNA 3′ end formation in Drosophila. The su(f) gene produces three transcripts; two of them are polyadenylated at the end of the transcription unit, and one is a truncated transcript, polyadenylated in intron 4. Using temperature-sensitive su(f) mutants, we show that accumulation of the truncated transcript requires wild-type Su(f) protein. This suggests that the Su(f) protein autoregulates negatively its accumulation by stimulating 3′ end formation of the truncated su(f) RNA. Cloning of su(f) from Drosophila virilis and analysis of its RNA profile suggest that su(f) autoregulation is conserved in this species. Sequence comparison between su(f) from both species allows us to point out three conserved regions in intron 4 downstream of the truncated RNA poly(A) site. These conserved regions include the GU-rich downstream sequence involved in poly(A) site definition. Using transgenes truncated within intron 4, we show that sequence up to the conserved GU-rich domain is sufficient for production of the truncated RNA and for regulation of this production by su(f). Our results indicate a role of su(f) in the regulation of poly(A) site utilization and an important role of the GU-rich sequence for this regulation to occur.

The PDE1-encoded Low-Affinity Phosphodiesterase in the Yeast Saccharomyces cerevisiae Has a Specific Function in Controlling Agonist-induced cAMP Signaling

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiesterase. The physiological function of the low-affinity enzyme Pde1 is unclear. We show that deletion of PDE1, but not PDE2, results in a much higher cAMP accumulation upon addition of glucose or upon intracellular acidification. Overexpression of PDE1, but not PDE2, abolished the agonist-induced cAMP increases. These results indicate a specific role for Pde1 in controlling glucose and intracellular acidification-induced cAMP signaling. Elimination of a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine252 into alanine resulted in a Pde1ala252 allele that apparently had reduced activity in vivo. Its presence in a wild-type strain partially enhanced the agonist-induced cAMP increases compared with pde1Δ. The difference between the Pde1ala252 allele and wild-type Pde1 was strongly dependent on PKA activity. In a RAS2val19 pde2Δ background, the Pde1ala252 allele caused nearly the same hyperaccumulation of cAMP as pde1Δ, while its expression in a PKA-attenuated strain caused the same reduction in cAMP hyperaccumulation as wild-type Pde1. These results suggest that serine252 might be the first target site for feedback inhibition of cAMP accumulation by PKA. We show that Pde1 is rapidly phosphorylated in vivo upon addition of glucose to glycerol-grown cells, and this activation is absent in the Pde1ala252 mutant. Pde1 belongs to a separate class of phosphodiesterases and is the first member shown to be phosphorylated. However, in vitro the Pde1ala252 enzyme had the same catalytic activity as wild-type Pde1, both in crude extracts and after extensive purification. This indicates that the effects of the S252A mutation are not caused by simple inactivation of the enzyme. In vitro phosphorylation of Pde1 resulted in a modest and variable increase in activity, but only in crude extracts. This was absent in Pde1ala252, and phosphate incorporation was strongly reduced. Apparently, phosphorylation of Pde1 does not change its intrinsic activity or affinity for cAMP but appears to be important in vivo for protein-protein interaction or for targeting Pde1 to a specific subcellular location. The PKA recognition site is conserved in the corresponding region of the Schizosaccharomyces pombe and Candida albicans Pde1 homologues, possibly indicating a similar control by phosphorylation.

Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation

The human cytomegalovirus UL97 kinase, an important target of antiviral therapy, has an impact on at least two distinct phases of viral replication. Compared with wild-type virus, the UL97 deletion mutant exhibits an early replication defect that reduces DNA accumulation by 4- to 6-fold, as well as a late capsid maturation defect responsible for most of the observed 100- to 1000-fold reduction in replication. Block-release experiments with the antiviral 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)-benzimidazole revealed an important role for UL97 kinase in capsid assembly. Although cleavage of concatemeric DNA intermediates to unit-length genomes remained unaffected, progeny mutant virus maturation was delayed, with accumulation of progeny at significantly reduced levels compared with wild type after release of this block. Transmission electron microscopy confirmed the aberrant accumulation of empty A-like capsids containing neither viral DNA nor an internal scaffold structure, consistent with a failure to stably package DNA in mutant virus-infected cells. The function of UL97 in DNA synthesis as well as capsid assembly suggests that protein phosphorylation mediated by this herpesvirus-conserved kinase increases the efficiency of these two distinct phases of virus replication.

Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.

Metapopulation extinction caused by mutation accumulation

Theory suggests that the risk of extinction by mutation accumulation can be comparable to that by environmental stochasticity for an isolated population smaller than a few thousand individuals. Here we show that metapopulation structure, habitat loss or fragmentation, and environmental stochasticity can be expected to greatly accelerate the accumulation of mildly deleterious mutations, lowering the genetic effective size to such a degree that even large metapopulations may be at risk of extinction. Because of mutation accumulation, viable metapopulations may need to be far larger and better connected than would be required under just stochastic demography.

Rhizosphere Bacteria Enhance Selenium Accumulation and Volatilization by Indian Mustard1

Indian mustard (Brassica juncea L.) accumulates high tissue Se concentrations and volatilizes Se in relatively nontoxic forms, such as dimethylselenide. This study showed that the presence of bacteria in the rhizosphere of Indian mustard was necessary to achieve the best rates of plant Se accumulation and volatilization of selenate. Experiments with the antibiotic ampicillin showed that bacteria facilitated 35% of plant Se volatilization and 70% of plant tissue accumulation. These results were confirmed by inoculating axenic plants with rhizosphere bacteria. Compared with axenic controls, plants inoculated with rhizosphere bacteria had 5-fold higher Se concentrations in roots (the site of volatilization) and 4-fold higher rates of Se volatilization. Plants with bacteria contained a heat-labile compound in their root exudate; when this compound was added to the rhizosphere of axenic plants, Se accumulation in plant tissues increased. Plants with bacteria had an increased root surface area compared with axenic plants; the increased area was unlikely to have caused their increased tissue Se accumulation because they did not accumulate more Se when supplied with selenite or selenomethionine. Rhizosphere bacteria also possibly increased plant Se volatilization because they enabled plants to overcome a rate-limiting step in the Se volatilization pathway, i.e. Se accumulation in plant tissues.