Several 4- or 5-monosubsituted and 4,5-disubstituted 1,2,3-triazole analogues of the anti-HIV-1 lead compound [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D- ribofuranosyl]thymine]-3'-spiro-5"-(4"-amino-1",2"-oxathiole 2",2"-dioxide) (TSAO-T) have been prepared and evaluated as inhibitors of HIV-1-induced cytopathicity. These analogues have been prepared by 1,3-diplar cycloaddition of [2,5-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]- 3-spiro-5'-(4'-amino- and 4'-(N-acetylamino)-1',2'-oxathiole 2',2'-dioxide) (TSAO) azides to various substituted acetylenes. Several 4- and 5-substituted 1,2,3-triazole-TSAO analogues proved superior to the unsubstituted derivative by 1-2 orders of magnitude. In particular the 5-substituted amido-, (methylamido)-, and (dimethylamido)-1,2,3-triazole derivatives of TSAO were endowed with potent anti-HIV-1 activity (50% effective concentration: 0.056-0.52 microM). They show a similar resistance spectrum as previously noted for TSAO-T and related derivatives.

The global regulators agr (accessory gene regulator) and sarA (staphylococcal accessory regulator) have been reported to be both activators and repressors of virulence gene expression in Staphylococcus aureus. How the effector of the agr system, RNAIII, interacts with target gene promoters is unknown. SarA, on the other hand, is a DNA‐binding protein, which binds to conserved DNA motifs immediately upstream of both positively and negatively regulated promoters. Here, we searched for additional regulators that could explain the differential effects of RNAIII and SarA. Four differently regulated genes (hla, alpha‐toxin; hld, RNAIII; spa, protein A; ssp, serine protease) were analysed for binding of potential regulatory proteins to the corresponding promoter DNA fragments, linked to magnetic beads. One protein (29 kDa), with affinity for all four promoters, showed a high degree of similarity to SarA and was named SarH1 (Sar homologue 1). Expression of sarH1 was strongly repressed by sarA and agr. Analysis of hla, hld, ssp and spa mRNAs in sarH1, sarA and agr mutants, and in sarA/sarH1 and agr/sarH1 double mutants, revealed that sarH1 has a strong repressive effect on hla and an activating effect on spa transcription. SDS–PAGE analysis of secreted proteins from the different mutants showed that the production of several other exoproteins was affected by sarH1. In conclusion, we show that both the agr‐dependent suppression of protein A production and the sarA‐dependent stimulation of alpha‐toxin production is mediated via a new regulator, SarH1, which belongs to a family of Sar homologues.

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Uridine-cytidine kinases (UCK) have important roles for the phosphorylation of nucleoside analogs that are being investigated for possible use in chemotherapy of cancer. We have cloned the cDNA of two human UCKs. The Ϸ30-kDa proteins, named UCK1 and UCK2, were expressed in Escherichia coli and shown to catalyze the phosphorylation of Urd and Cyd. The enzymes did not phosphorylate deoxyribonucleosides or purine ribonucleosides. UCK1 mRNA was detected as two isoforms of Ϸ1.8 and Ϸ2.7 kb. The 2.7-kb band was ubiquitously expressed in the investigated tissues. The band of Ϸ1.8 kb was present in skeletal muscle, heart, liver, and kidney. The two isoforms of UCK2 mRNA of 1.2 and 2.0 kb were only detected in placenta among the investigated tissues. The genes encoding UCK1 and UCK2 were mapped to chromosome 9q34.2-9q34.3 and 1q22-1q23.2, respectively. We tested 28 cytidine and uridine nucleoside analogs as possible substrates of the enzymes. The enzymes phosphorylated several of the analogs, such as 6-azauridine, 5-fluorouridine, 4-thiouridine, 5-bromouridine, N 4-acetylcytidine, N 4-benzoylcytidine, 5-fluorocytidine, 2-thiocytidine, 5-methylcytidine, and N 4-anisoylcytidine. The cloning and recombinant expression of the two human UCKs will be important for development of novel pyrimidine ribonucleoside analogs and the characterization of their pharmacological activation.

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We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550 -557). Nm3-H4 was synthesized in escherichia coli as the fulllength protein and as a truncated form missing the Nterminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser 129 has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.

Data have been presented indicating thatStaphylococcus aureus produces several cell surface proteins which bind specifically to different host extracellular matrix proteins and plasma proteins (12,13,32). For many of the cell surface proteins a role in colonization and virulence has been demonstrated in animal models of infection (17,23,27,33). Two highly homologous fibronectin-binding proteins (FnBPA and FnBPB), encoded by fnbA and fnbB, have been characterized (14, 21, 25, 41) and shown to be involved in adherence to damaged heart valves (23) and to promote internalization of S. aureus by epithelial cells (9). Although S. aureus is primarily considered to be an extracellular pathogen, the intracellular niche could promote long-term colonization and maintenance of chronic infections.Protein A (Spa), which binds immunoglobulin G (IgG) by the Fc segment, is a major surface protein present in virtually all strains of S. aureus (10, 11). Strains of S. aureus with a high content of Spa are more resistant to phagocytosis by human neutrophils in vitro than strains with less Spa (34). Reduced virulence of a spa mutant compared to that of the corresponding wild type was demonstrated in a mouse intraperitoneal infection (31).We have recently shown that transcription of the fnbA and fnbB genes is negatively regulated by agr and by an agr-independent mechanism that restricts fnb mRNA synthesis to the early exponential phase of growth (38). A similar temporal control of fnb transcription was also found in another strain of S. aureus (Newman) (43). However, only fnbA appeared to be regulated by agr in this strain. It was also found that fnbA, but not fnbB, was positively regulated by sarA. As for fnbA and fnbB, transcription of spa is negatively regulated by agr (20). However, unlike for fnbA, transcription of spa is negatively controlled by sarA (3,42).Data from recent studies indicate that both FnBPs and protein A may be degraded by extracellular proteases (3, 26, 42). Four major extracellular proteases are produced by S. aureus (1): staphylococcal serine protease (V8 protease) (SspA), a metalloprotease named aureolysin (Aur), a cysteine protease (Scp) named staphopain (18), and a second cysteine protease (SspB) encoded within the same operon as SspA (2, 36).

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