The effect of 2′-fluoro-2′-deoxycytidine on herpes virus growth

The effect of 2′-fluoro-2′-deoxycytidine (dCfl) on the growth of certain viruses of the herpes type was investigated. It is shown that the compound has considerable anti-viral activity against HSV-I, HSV-II, pseudorabies virus and equine abortion virus. It has an effect comparable to that of araC and is more efficient than br⁵dC, but less so than acyclovir. Experiments with thymidine kinase-negative strains of HSV-I indicated that dCfl was phosphorylated by the viral kinase, and its K_m appears to be low and close to that of thymidine. Density gradient centrifugation enabled us to show that dCfl was incorporated into cellular and viral DNA and RNA. The cytotoxic activity of dCfl appears to be about 10-times smaller than that of araC. Removal of the nucleoside analog, washing and replacement with deoxycytidine reversed this effect, indicating rather a cytostatic than cytotoxic effect.     

Antimutagenic effects of cinnamaldehyde on chemical mutagenesis in Escherichia coli

Antimutagenic effects of cinnamaldehyde on mutagenesis by chemical agents were investigated in Escherichia coli WP2 uvrA- trpE-. Cinnamaldehyde, when added to agar medium, greatly reduced the number of Trp+ revertants induced by 4-nitroquinoline 1-oxide (4-NQO) without any decrease of cell viability. This antimutagenic effect could not be explained by inactivation of 4-NQO caused by direct interaction with cinnamaldehyde. Mutagenesis by furylfuramide (AF-2) was also suppressed significantly. Mutations induced by methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) were slightly inhibited. However, cinnamaldehyde was not at all effective on the mutagenesis of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Two derivatives of cinnamaldehyde, cinnamyl alcohol and trans-cinnamic acid, did not have as strong antimutagenic effects on 4-NQO mutagenesis as cinnamaldehyde had. Because cinnamaldehyde showed marked antimutagenic effects against mutations induced by UV-mimic mutagens but not those induced by MNNG or EMS, it seems that cinnamaldehyde might act by interfering with an inducible error-prone DNA repair pathway.     

Genomic distribution and heterogeneity of MocR-like transcriptional factors containing a domain belonging to the superfamily of the pyridoxal-5'-phosphate dependent enzymes of fold type I

Bacterial proteins belonging to the MocR/GabR family are chimeric proteins incorporating a short N-terminal helix-turn-helix containing domain with DNA-binding properties, and a long C-terminal domain belonging to the superfamily of the pyridoxal-5′-phosphate enzymes of fold type I. The first purpose of this report is to give an overview of the distribution of these factors among the different taxonomical bacterial divisions and to determine the degree of conservation of the main structural features of the PLP binding domain. Complete proteomes of bacteria phyla were scanned with a hidden Markov model representative of the MocR family. Results indicate that presence of MocR factors is heterogeneous even within the single bacterial phylum: some species miss completely the factors, while others possess one or even more regulators. Absence of MocR factors is distinctive of some phyla such as Chlamydiae. The genomic distribution of MocR is, as expected, highly correlated to the size of the genome. At variance, phyla missing MocR regulators generally are characterized by compact genomes, of the order of 1.0–2.0 Mb, such as the case of Mollicutes or Chlamydiae. Apparently, the minimum genome size compatible with the presence of MocR genes is around 2.0–2.5 Mb. Conservation of the residues corresponding to those involved in the interaction with the cofactor pyridoxal-5′-phosphate in the homologous 2-aminoadipate aminotransferase, was analyzed in the multiple sequence alignments of MocR within each phyla considered. In the vast majority of cases, residues are conserved or conservatively replaced. This result suggests that, in most cases, MocR factors preserve at least ability to bind the cofactor and very likely some catalytic abilities.     

Measurement of adenine nucleotides in plasma

The goal of this study was to identify the most important variables affecting bioluminescent ATP, ADP and AMP measurements in plasma and to develop an assay that takes these variables into account. Blood samples were drawn from conscious dogs. A ‘stop solution’ containing EDTA was prepared, which greatly retarded plasma ATP degradation by chelating Mg⁺² and Ca⁺² that are co‐factors for many ATPases. Stop solution and blood were mixed using a two‐syringe withdrawal system. Samples were centrifuged twice in order to remove red blood cells, and ATP was measured in the supernatant using the firefly luciferase assay. Sample pH was adjusted to the optimal range (7.75–7.95) and Mg²⁺ (necessary for the luciferase reaction) was added back to the sample within the luminometer 2 s prior to luciferase addition. Four assay tubes were prepared for each plasma sample, containing standard additions of 0–15 pmol added ATP, in order to quantify native plasma ATP content. In separate plasma/stop solution samples ADP + ATP was measured after converting ADP to ATP via the pyruvate kinase reaction, and AMP + ADP + ATP was measured after addition of both myokinase and pyruvate kinase. Addition of forskolin and isobutylmethylxanthine (IBMX) to the stop solution to inhibit platelets resulted in lower ATP concentrations. Measurement of ATP and haemoglobin from lysed erythrocytes revealed that haemolysis exerts a strong influence on plasma ATP concentration that must be taken into account. Copyright © 2003 John Wiley & Sons, Ltd.