ABSTRACT
The oxidation of guanine (G) to 7,8-dihydro-8-oxoguanine (GO) forms one of the major DNA lesions generated by reactive oxygen species (ROS). The GO can be corrected by GO DNA glycosylases (Ogg), enzymes involved in base excision repair (BER). Unrepaired GO induces mismatched base pairing with adenine (A); as a result, the mismatch causes a point mutation, from G paired with cytosine (C) to thymine (T) paired with adenine (A), during DNA replication. Here, we report the characterization of a putative Ogg from the thermoacidophilic archaeon Thermoplasma volcanium. The 204-amino acid sequence of the putative Ogg (TVG_RS00315) shares significant sequence homology with the DNA glycosylases of Methanocaldococcus jannaschii (MjaOgg) and Sulfolobus solfataricus (SsoOgg). The six histidine-tagged recombinant TVG_RS00315 protein gene was expressed in Escherichia coli and purified. The Ogg protein is thermostable, with optimal activity near a pH of 7.5 and a temperature of 60°C. The enzyme displays DNA glycosylase, and apurinic/apyrimidinic (AP) lyase activities on GO/N (where N is A, T, G, or C) mismatch; yet it cannot eliminate U from U/G or T from T/G, as mismatch glycosylase (MIG) can. These results indicate that TvoOgg-encoding TVG_RS00315 is a member of the Ogg2 family of T. volcanium.
Subject(s)
DNA Glycosylases/metabolism , DNA/metabolism , Guanine/analogs & derivatives , Thermoplasma/enzymology , DNA Glycosylases/chemistry , DNA Glycosylases/genetics , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Guanine/metabolism , Hydrogen-Ion Concentration , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Temperature , Thermoplasma/geneticsABSTRACT
We evaluated the biocompatibility of a newly developed vitamin E hemodialyzer (CL-EE; Terumo Co Ltd, Tokyo, Japan) by neutrophil function and oxidant stress in patients with end-stage renal failure in a randomized crossover study. Ten patients underwent hemodialysis using either the CL-EE or a control dialyzer membrane identical to the CL-EE except for vitamin E binding for 12 weeks in a crossover fashion after a 1-month washout period with hemophane membranes. White blood cell counts, serum oxidized low-density lipoprotein (Ox-LDL) levels, and malondialdehyde (MDA) levels during hemodialysis sessions were measured at the initiation and end of the CL-EE and control trials. Superoxide anion production by neutrophils just before and 4 hours after starting the session also was measured. Leukocytopenia at 1 hour after starting the session was detected to a similar extent in both membranes. However, the degree of reduction was less in the CL-EE trial after repeated use. Superoxide anion production by neutrophils just before a hemodialysis session was reduced after repeated use of the CL-EE membrane. Serum Ox-LDL levels increased, whereas serum MDA levels decreased during sessions to a similar extent in both trials. However, these parameters were significantly lower in the CL-EE trial after repeated use. Serum LDL concentrations significantly decreased with repeated use of the CL-EE membrane. These data suggest that repeated use of the CL-EE membrane for 3 months improves neutrophil function, oxidant stress, and LDL concentrations in patients with renal failure. This membrane may be useful to reduce the incidence of cardiovascular events in patients with renal failure.
