West Nile virus methyltransferase catalyzes two methylations of the viral RNA cap through a substrate-repositioning mechanism

Flaviviruses encode a single methyltransferase domain that sequentially catalyzes two methylations of the viral RNA cap, GpppA-RNA-->m(7)GpppA-RNA-->m(7)GpppAm-RNA, by using S-adenosyl-l-methionine (SAM) as a methyl donor. Crystal structures of flavivirus methyltransferases exhibit distinct binding sites for SAM, GTP, and RNA molecules. Biochemical analysis of West Nile virus methyltransferase shows that the single SAM-binding site donates methyl groups to both N7 and 2'-O positions of the viral RNA cap, the GTP-binding pocket functions only during the 2'-O methylation, and two distinct sets of amino acids in the RNA-binding site are required for the N7 and 2'-O methylations. These results demonstrate that flavivirus methyltransferase catalyzes two cap methylations through a substrate-repositioning mechanism. In this mechanism, guanine N7 of substrate GpppA-RNA is first positioned to SAM to generate m(7)GpppA-RNA, after which the m(7)G moiety is repositioned to the GTP-binding pocket to register the 2'-OH of the adenosine with SAM, generating m(7)GpppAm-RNA. Because N7 cap methylation is essential for viral replication, inhibitors designed to block the pocket identified for the N7 cap methylation could be developed for flavivirus therapy.     

Transgenic mice overexpressing GLUT-1 protein in muscle exhibit increased muscle glycogenesis after exercise

The purpose of the present study was to determine the rates of muscle glycogenolysis and glycogenesis during and after exercise in GLUT-1 transgenic mice and their age-matched littermates. Male transgenic mice (TG) expressing a high level of human GLUT-1 and their nontransgenic (NT) littermates underwent 3 h of swimming. Glycogen concentration was determined in gastrocnemius and extensor digitorum longus (EDL) muscles before exercise and at 0, 5, and 24 h postexercise, during which food (chow) and 10% glucose solution (as drinking water) were provided. Exercise resulted in approximately 90% reduction in muscle glycogen in both NT (from 11.2 +/- 1.4 to 2. 1 +/- 1.3 micromol/g) and TG (from 99.3 +/- 4.7 to 11.8 +/- 4.3 micromol/g) in gastrocnemius muscle. During recovery from exercise, the glycogen concentration increased to 38.2 +/- 7.3 (5 h postexercise) and 40.5 +/- 2.8 micromol/g (24 h postexercise) in NT mice. In TG mice, however, the increase in muscle glycogen concentration during recovery was greater (to 57.5 +/- 7.4 and 152.1 +/- 15.7 micromol/g at 5 and 24 h postexercise, respectively). Similar results were obtained from EDL muscle. The rate of 2-deoxyglucose uptake measured in isolated EDL muscles was 7- to 10-fold higher in TG mice at rest and at 0 and 5 h postexercise. There was no difference in muscle glycogen synthase activation measured in gastrocnemius muscles between NT and TG mice immediately after exercise. These results demonstrate that the rate of muscle glycogen accumulation postexercise exhibits two phases in TG: 1) an early phase (0-5 h), with rapid glycogen accumulation similar to that of NT mice, and 2) a progressive increase in muscle glycogen concentration, which differs from that of NT mice, during the second phase (5-24 h). Our data suggest that the high level of steady-state muscle glycogen in TG mice is due to the increase in muscle glucose transport activity.     

N-Halamine-coated cotton for antimicrobial and detoxification applications

A new N-halamine precursor, 3-(2,3-dihydroxypropyl)-7,7,9,9-tetramethyl-1,3,8-triazaspiro[4.5]decane-2,4-dione (TTDD diol), was synthesized and bonded onto cotton fabrics. Fabrics with variable amounts of chlorine loading were prepared by using several concentrations of TTDD diol. A second N-halamine precursor, 3-(3-triethoxysilylpropyl)-7,7,9,9-tetramethyl-1,3,8-triazaspiro[4.5]decane-2,4-dione (TTDD siloxane), was also synthesized and bound to cotton for comparison purposes. The coated cotton fabrics contained two types of N–Cl moieties after chlorination of the amine and amide groups. Swatches with variable chlorine loadings were challenged with Staphylococcus aureus and Escherichia coli O157:H7 as a function of contact time. The biocidal test results showed that the chlorine loadings and surface hydrophobicities influenced the antimicrobial efficacies. The chlorinated swatches have also been employed to oxidize the simulant of chemical mustard to the less toxic sulfoxide derivative.