Substitutions of a cysteine conserved among DNA cytosine methylases result in a variety of phenotypes.

The proposed mechanism for DNA (cytosine-5)-methyltransferases envisions a key role for a cysteine residue. It is expected to form a covalent link with carbon 6 of the target cytosine, activating the normally inactive carbon 5 for methyl transfer. There is a single conserved cysteine among all DNA (cytosine-5)-methyltransferases making it the candidate nucleophile. We have changed this cysteine to other amino acids for the EcoRII methylase; which methylates the second cytosine in the sequence 5'-CCWGG-3'. Mutants were tested for their methyl transferring ability and for their ability to form covalent complexes with DNA. The latter property was tested indirectly with the use of a genetic assay involving sensitivity of cells to 5-azacytidine. Replacement of the conserved cysteine with glycine, valine, tryptophan or serine led to an apparent loss of methyl transferring ability. Interestingly, cells carrying the mutant with serine did show sensitivity to 5-azacytidine, suggesting the ability to link to DNA. Unexpectedly, substitution of the cysteine with glycine results in the inhibition of cell growth and the mutant allele can be maintained in the cells only when it is poorly expressed. These results suggest that the conserved cysteine in the EcoRII methylase is essential for methylase action and it may play more than one role in it.     

Synthesis and biological evaluation of 6,7-disubstituted 4-aminopyrido[2,3-d]pyrimidines as adenosine kinase inhibitors

The synthesis and structure-activity relationship of a series of 6,7-disubstituted 4-aminopyrido[2,3-d]pyrimidines as novel non-nucleoside adenosine kinase inhibitors is described. A variety of substituents, primarily aryl, at the C6 and C7 positions of the pyridopyrimidine core were found to yield analogues that are potent inhibitors of adenosine kinase. In contrast to the 5,7-disubstituted and 5,6,7-trisubstituted pyridopyrimidine series, these analogues exhibited only modest potency to inhibit AK in intact cells.     

Maturation of bacteriophage T4 lagging strand fragments depends on interaction of T4 RNase H with T4 32 protein rather than the T4 gene 45 clamp

In the bacteriophage T4 DNA replication system, T4 RNase H removes the RNA primers and some adjacent DNA before the lagging strand fragments are ligated. This 5'-nuclease has strong structural and functional similarity to the FEN1 nuclease family. We have shown previously that T4 32 protein binds DNA behind the nuclease and increases its processivity. Here we show that T4 RNase H with a C-terminal deletion (residues 278-305) retains its exonuclease activity but is no longer affected by 32 protein. T4 gene 45 replication clamp stimulates T4 RNase H on nicked or gapped substrates, where it can be loaded behind the nuclease, but does not increase its processivity. An N-terminal deletion (residues 2-10) of a conserved clamp interaction motif eliminates stimulation by the clamp. In the crystal structure of T4 RNase H, the binding sites for the clamp at the N terminus and for 32 protein at the C terminus are located close together, away from the catalytic site of the enzyme. By using mutant T4 RNase H with deletions in the binding site for either the clamp or 32 protein, we show that it is the interaction of T4 RNase H with 32 protein, rather than the clamp, that most affects the maturation of lagging strand fragments in the T4 replication system in vitro and T4 phage production in vivo.     

Comparative context analysis of codon pairs on an ORFeome scale

Codon context is an important feature of gene primary structure that modulates mRNA decoding accuracy. We have developed an analytical software package and a graphical interface for comparative codon context analysis of all the open reading frames in a genome (the ORFeome). Using the complete ORFeome sequences of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Escherichia coli, we show that this methodology permits large-scale codon context comparisons and provides new insight on the rules that govern the evolution of codon-pair context.     

Interaction of MutS and Vsr: some dominant-negative mutS mutations that disable methyladenine-directed mismatch repair are active in very-short-patch repair

In Escherichia coli and related bacteria, the very-short-patch (VSP) repair pathway uses an endonuclease, Vsr, to correct T-G mismatches that result from the deamination of 5-methylcytosines in DNA to C-G. The products of mutS and mutL, which are required for adenine methylation-directed mismatch repair (MMR), enhance VSP repair. Multicopy plasmids carrying mutS alleles that are dominant negative for MMR were tested for their effects on VSP repair. Some mutS mutations (class I) did not lower VSP repair in a mutS(+) background, and most class I mutations increased VSP repair in mutS cells more than plasmids containing mutS(+). Other plasmid-borne mutS mutations (class II) and mutS(+) decreased VSP repair in the mutS(+) background. Thus, MutS protein lacking functions required for MMR can still participate in VSP repair, and our results are consistent with a model in which MutS binds transiently to the mispair and then translocates away from the mispair to create a specialized structure that enhances the binding of Vsr.     

Comparison of techniques for the measurement of skin temperature during exercise in a hot,humid environment

Exercising or working in a hot, humid environment can results in the onset of heat-related illness when an individual''s temperature is not carefully monitored. The purpose of the present study was to compare three techniques (data loggers, thermal imaging, and wired electrodes) for the measurement of peripheral (bicep) and central (abdominal) skin temperature. Young men and women (N = 30) were recruited to complete the present study. The three skin temperature measurements were made at 0 and every 10-min during 40-min (60% VO₂max) of cycling in a hot (39±2°C), humid (45±5% RH) environment. Data was statistically analyzed using the Bland-Altman method and correlation analysis. For abdominal skin temperature, the Bland-Altman limits of agreement indicated that data loggers (1.5) were a better index of wired than was thermal imaging (3.5), For the bicep skin temperature the limits of agreement was similar between data loggers (1.9) and thermal (1.9), suggesting the both were suitable measurements. We also found that when skin temperature exceeded 35°C, we observed progressively better prediction between data loggers, thermal imaging, and wired skin sensors. This report describes the potential for the use of data loggers and thermal imaging to be used as alternative measures of skin temperature in exercising, human subjects.     

Soil‐nutrient availability under a global‐change scenario in a Mediterranean mountain ecosystem

Changes in rainfall availability will alter soil‐nutrient availability under a climate‐change scenario. However, studies have usually analyzed the effect of either drier or wetter soil conditions, despite the fact that both possibilities will coexist in many climatic regions of the world. Furthermore, its effect may vary across the different habitats of the ecosystem. We experimentally investigated the effect of three contrasting climatic scenarios on different carbon (C), nitrogen (N), and phosphorus (P) fractions in soil and microbial compartments among three characteristic habitats in a Mediterranean‐type ecosystem: forest, shrubland, and open areas. The climatic scenarios were dry summers, according to the 30% summer rainfall reduction projected in the Mediterranean; wet summer, simulating summer storms to reach the maximum historical records in the study area; and current climatic conditions (control). Sampling was replicated during two seasons (spring and summer) and 2 years. The climatic scenario did not affect the nutrient content in the litter layer. However, soil and microbial nutrients varied among seasons, habitats, and climatic scenarios. Soil‐nutrient fractions increased with lower soil‐moisture conditions (dry scenario and summer), whereas microbial nutrients increased under the wet summer scenario and spring. This pattern was consistent both studied years, although it was modulated by habitat, differences being lower with denser plant cover. Holm oak seedlings, used as live control of the experiment, tended to increase their N and P content (although not significantly) with water availability. Thus, the results support the idea that higher rainfall boosts microbial and plant‐nutrient uptake, and hence nutrient cycling. By contrast, a rainfall reduction leads to an accumulation of nutrients in the soil, increasing the risk of nutrient loss by leaching or erosion. These results show that the projected climate change will have significant effects on nutrient cycles, and therefore will have important implications on the ecosystem functioning.