Chemical synthesis of 5-azacytidine nucleotides and preparation of tRNAs containing 5-azacytidine in its 3''-terminus

5-azacytidine-5'-triphosphate prepared from 5-azacytidine by chemical phosphorylation is a substrate for AMP (CMP) tRNA nucleotidyl transferase from yeast. tRNAsPhe from yeast containing 5-azacytidine in their 3'-termini were prepared enzymatically. tRNAPhe-Cpn5CpA and tRNAPhe-n5Cpn5CpA can be aminoacylated by phenylalanyl-tRNA synthetase from yeast and they are active in the poly(U)-dependent synthesis of poly(Phe) on E. coli ribosomes. The decomposition of 5-azacytidine via hydrolysis of the triazine ring is significantly accelerated by a phosphate group on the 5'-position of the nucleotide. After the incorporation of 5-azacytidine-5'-phosphate into a polynucleotide chain the rate of hydrolysis of the triazine ring decreases considerably.     

Sterols of the cultured euglenid Eutreptia viridis: A novel Δ23-unsaturated sterol.

The sterol mixture isolated from the marine alga $\text{Eutreptia viridis}$ consists mainly of Δ^5,7-dienes which account for 80% of the free sterols. Eighteen different sterols were detected, including a novel sterol with the rare Δ²³-unsaturation, viz. 24-ethylcholesta-5,7,23Z-trien-3β-ol.     

Distribution of alginate gene sequences in the Pseudomonas rRNA homology group I-Azomonas-Azotobacter lineage of superfamily B procaryotes.

Chromosomal DNA from group I Pseudomonas species, Azotobacter vinelandii, Azomonas macrocytogens, Xanthomonas campestris, Serpens flexibilis, and three enteric bacteria was screened for sequences homologous to four Pseudomonas aeruginosa alginate (alg) genes (algA, pmm, algD, and algR1). All the group I Pseudomonas species tested (including alginate producers and nonproducers) contained sequences homologous to all the P. aeruginosa alg genes used as probes, with the exception of P. stutzeri, which lacked algD. Azotobacter vinelandii also contained sequences homologous to all the alg gene probes tested, while Azomonas macrocytogenes DNA showed homology to all but algD. X. campestris contained sequences homologous to pmm and algR1 but not to algA or algD. The helical bacterium S. flexibilis showed homology to the algR1 gene, suggesting that an environmentally responsive regulatory gene similar to algR1 exists in S. flexibilis. Escherichia coli showed homology to the algD and algR1 genes, while Salmonella typhimurium and Klebsiella pneumoniae failed to show homology with any of the P. aeruginosa alg genes. Since all the organisms tested are superfamily B procaryotes, these results suggest that within superfamily B, the alginate genes are distributed throughout the Pseudomonas group I-Azotobacter-Azomonas lineage, while only some alg genes have been retained in the Pseudomonas group V (Xanthomonas) and enteric lineages.     

Interaction of 5-fluoro-4-thio-2'-deoxyuridine 5'-phosphate with mammalian tumour thymidylate synthase: role of the pyrimidine N(3)-H dissociation

The role of the pyrimidine N(3)-H in binding of dUMP derivatives to thymidylate synthase was evaluated with the aid of a new dUMP analogue, 5-fluoro-4-thio-dUMP, synthesized by an improved thiation and enzymatic phosphorylation. The interaction of this analogue, and of 5-FdUMP, with the enzyme, and the pH-dependence of these interactions, were compared. Both were slow-binding competitive inhibitors of the enzyme from Ehrlich carcinoma, L1210 and CCRF-CEM cells, with Ki an order of magnitude higher for 5-fluoro-4-thio-dUMP than for 5-FdUMP. With both nucleotides, as well as the parent nucleosides, enzyme inactivation increased as the pH was lowered from 8 to 6. Maximum inactivation with 5-FdUrd was at pH 7.0, and with 5-fluoro-4-thio-dUrd at pH 6.0, in agreement with the higher pKa for the N(3)-H dissociation of the former, and pointing to participation of the N(3)-H as a hydrogen donor in binding to the enzyme.     

An in vitro and in vivo investigation of the effects of diesel exhaust on human airway lining fluid antioxidants

Breathing high concentrations of diesel exhaust (DE) induces pulmonary inflammation, bronchoconstriction, increased airway reactivity, and oxidative stress in healthy subjects. To examine if these responses occur at environmentally relevant concentrations of DE, we exposed 25 healthy subjects to DE (PM(10) 100 microg/m(3), 0.6 ppm NO(2) for 2-h) and filtered air on separate occasions. Immediately following DE exposure, subjects displayed an increase in subjective symptoms and a mild bronchoconstriction. Six hours following the cessation of DE exposure neither airway inflammation, nor antioxidant depletion (ascorbate, urate, and reduced glutathione), was seen at any level of the respiratory tract. Instead, an increased flux of reduced glutathione into the bronchial (p < 0.01) and nasal airways (p < 0.05) was observed. In separate, in vitro experiments, DE was found to have comparable oxidative activity to the transition metal rich residual oil fly ash (ROFA) particle, significantly depleting lung lining fluid ascorbic acid and reduced glutathione in a transition metal and superoxide-dependent mechanism. Together, these data indicate that even though DE has marked oxidative activity, this effect is not observed to any great extent in the airways of healthy subjects. We interpret these findings as being indicative that the antioxidant network at the air-lung interface in healthy subjects is capable of dealing with the oxidative challenge posed by DE at ambient concentrations.     

Naive CD4+ T cells from lupus-prone Fas-intact MRL mice display TCR-mediated hyperproliferation due to intrinsic threshold defects in activation

Autoreactive T cell activation is a consistent feature of murine lupus; however, the mechanism of such activation remains unclear. We hypothesized that naive CD4+ T cells in lupus have a lower threshold of activation through their TCR-CD3 complex that renders them more susceptible to stimulation with self-Ags. To test this hypothesis, we compared proliferation, IL-2 production, and single cell calcium signaling of naive CD4+ T cells isolated from Fas-intact MRL/+(Fas-lpr) mice with H-2k-matched B10.BR and CBA/CaJ controls, following anti-CD3 stimulation in the presence or absence of anti-CD28. We also assessed the responsiveness of naive CD4+ T cells isolated from Fas-intact MRL and control mice bearing a rearranged TCR specific for amino acids 88-104 of pigeon cytochrome c to cognate and low affinity peptide Ags presented by bone marrow-matured dendritic cells. TCR transgenic and wild-type CD4+ T cells from MRL mice displayed a lower threshold of activation than control cells, a response that was class II MHC dependent. The rise in intracellular calcium in MRL vs controls was enhanced and prolonged following anti-CD3 triggering, suggestive of proximal defects in TCR-engendered signaling as the mechanism for the observed hyperactivity. These findings were observed as early as 1-2 mo postweaning and, based on analysis of F1 T cells, appeared to be dominantly expressed. This genetically altered threshold for activation of MRL T cells, a consequence of a proximal defect in CD3-mediated signal transduction, may contribute to the abrogation of T cell tolerance to self-Ags in lupus.