Two nrdF genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ribonucleotide reductase (RR) from Mycobacterium tuberculosis have 71% identity at the amino acid level and are both highly homologous with Salmonella typhimurium R2F. The calculated molecular masses of R2-1 and R2-2 are 36,588 (322 amino acids [aa]) and 36,957 (324 aa) Da, respectively. Western blot analysis of crude M. tuberculosis extracts indicates that both R2s are expressed in vivo. Recombinant R2-2 is enzymatically active when assayed with pure recombinant M. tuberculosis R1 subunit. Both ATP and dATP are activators for CDP reduction up to 2 and 1 mM, respectively. The gene encoding M. tuberculosis R2-1, nrdF1, is not linked to nrdF2, nor is either gene linked to the gene encoding the large subunit, M. tuberculosis nrdE. The gene encoding MTP64 was found downstream from nrdF1, and the gene encoding alcohol dehydrogenase was found downstream from nrdF2. A nrdA(Ts) strain of E. coli (E101) could be complemented by simultaneous transformation with M. tuberculosis nrdE and nrdF2. An M. tuberculosis nrdF2 variant in which the codon for the catalytically necessary tyrosine was replaced by the phenylalanine codon did not complement E101 when cotransformed with M. tuberculosis nrdE. Similarly, M. tuberculosis nrdF1 and nrdE did not complement E101. Activity of recombinant M. tuberculosis RR was inhibited by incubating the enzyme with a peptide corresponding to the 7 C-terminal amino acid residues of the R2-2 subunit. M. tuberculosis is a species in which a nrdEF system appears to encode the biologically active species of RR and also the only bacterial species identified so far in which class I RR subunits are not arranged on an operon.Mycobacterium tuberculosis is an aerobic, gram-positive bacterium containing a high-GϩC-content genome of approximately 4 ϫ 10 6 bp with a relatively long mean generation time of 24 h and a chromosomal replication time (C period) of 11 h (13). M. tuberculosis can remain in a dormant, nonreplicating state in the human host for years and can be reactivated to cause disease under certain immunological and/or environmental conditions (38). The molecular mechanisms that govern these growth patterns and the regulation of DNA synthesis during these growth phases are now the focus of several lines of investigation. DNA polymerase I (14), topoisomerase I (42), and gyrA and gyrB (21, 33) from M. tuberculosis have been cloned and sequenced. In addition, the origins of replication of M. tuberculosis, M. smegmatis, and M. leprae have been analyzed and shown to contain a typical gram-positive pattern of genes, rnpA-rpmH-dnaA-dnaN-recF-gyrB-gyrA (28). Recently, ribonucleotide reductase (RR), potentially the rate-limiting step in DNA replication, was purified from M. tuberculosis, and the gene encoding the large subunit (R1) was isolated, sequenced, and expressed in Escherichia coli (41).The enzymatic reduction of ribonucleotides by RR regulates the deoxynucleotide precursor pool for DNA synthesis in a wide variety of bacteria and ...

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Combination antiplatelet therapy using aspirin and ticlopidine has been the standard of care for prevention of subacute thrombosis following coronary stent implantation. However, the use of ticlopidine is associated with a significant risk of adverse hematologic side effects. Clopidogrel is an inhibitor of ADP-induced platelet aggregation that has a better safety profile than ticlopidine. We examined the 30-day clinical outcome following coronary stent implantation in 253 consecutive patients treated with clopidogrel and aspirin. Follow-up was achieved in 99% of patients and four adverse events were documented. Two patients had angiographically confirmed subacute stent thrombosis (0.8%), and both of these patients underwent successful repeat angioplasty at the stent site. There were two patient deaths during follow-up (0. 8%). One was sudden within 1 week of stent placement and the other occurred in a patient with multisystem organ failure after an extensive myocardial infarction that antedated the stent procedure, with no clinical evidence for stent thrombosis. The combined frequency of subacute stent thrombosis and death was 1.6%. This is comparable to prior studies using the combination of ticlopidine and aspirin following stenting. Therefore, clopidogrel in combination with aspirin appears to be a safe and effective therapy in the prevention of subacute thrombosis following coronary stent implantation.