Dihydroxythiophenes are novel potent inhibitors of human immunodeficiency virus integrase with a diketo acid-like pharmacophore.

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.

A developmentally regulated aconitase related to iron-regulatory protein-1 is localized in the cytoplasm and in the mitochondrion of Trypanosoma brucei.

Mitochondrial energy metabolism and Krebs cycle activities are developmentally regulated in the life cycle of the protozoan parasite Trypanosoma brucei. Here we report cloning of a T. brucei aconitase gene that is closely related to mammalian iron-regulatory protein 1 (IRP-1) and plant aconitases. Kinetic analysis of purified recombinant TbACO expressed in Escherichia coli resulted in a K(m) (isocitrate) of 3 +/- 0.4 mM, similar to aconitases of other organisms. This was unexpected since an arginine conserved in the aconitase protein family and crucial for substrate positioning in the catalytic center and for activity of pig mitochondrial aconitase (Zheng, L., Kennedy, M. C., Beinert, H., and Zalkin, H. (1992) J. Biol. Chem. 267, 7895-7903) is substituted by leucine in the TbACO sequence. Expression of the 98-kDa TbACO was shown to be lowest in the slender bloodstream stage of the parasite, 8-fold elevated in the stumpy stage, and increased a further 4-fold in the procyclic stage. The differential expression of TbACO protein contrasted with only minor changes in TbACO mRNA, indicating translational or post-translational mechanisms of regulation. Whereas animal cells express two distinct compartmentalized aconitases, mitochondrial aconitase and cytoplasmic aconitase/IRP-1, TbACO accounts for total aconitase activity in trypanosomes. By cell fractionation and immunofluorescence microscopy, we show that native as well as a transfected epitope-tagged TbACO localizes in both the mitochondrion (30%) and in the cytoplasm (70%). Together with phylogenetic reconstructions of the aconitase family, this suggests that animal IRPs have evolved from a multicompartmentalized ancestral aconitase. The possible functions of a cytoplasmic aconitase in trypanosomes are discussed.

Iron-dependent regulation of transferrin receptor expression in Trypanosoma brucei.

Transferrin is an essential growth factor for African trypanosomes. Here we show that expression of the trypanosomal transferrin receptor, which bears no structural similarity with mammalian transferrin receptors, is regulated by iron availability. Iron depletion of bloodstream forms of Trypanosoma brucei with the iron chelator deferoxamine resulted in a 3-fold up-regulation of the transferrin receptor and a 3-fold increase of the transferrin uptake rate. The abundance of expression site associated gene product 6 (ESAG6) mRNA, which encodes one of the two subunits of the trypanosome transferrin receptor, is regulated 5-fold by a post-transcriptional mechanism. In mammalian cells the stability of transferrin receptor mRNA is controlled by iron regulatory proteins (IRPs) binding to iron-responsive elements (IREs) in the 3'-untranslated region (UTR). Therefore, the role of a T. brucei cytoplasmic aconitase (TbACO) that is highly related to mammalian IRP-1 was investigated. Iron regulation of the transferrin receptor was found to be unaffected in Deltaaco::NEO/Deltaaco::HYG null mutants generated by targeted disruption of the TbACO gene. Thus, the mechanism of post-transcriptional transferrin receptor regulation in trypanosomes appears to be distinct from the IRE/IRP paradigm. The transferrin uptake rate was also increased when trypanosomes were transferred from medium supplemented with foetal bovine serum to medium supplemented with sera from other vertebrates. Due to varying binding affinities of the trypanosomal transferrin receptor for transferrins of different species, serum change can result in iron starvation. Thus, regulation of transferrin receptor expression may be a fast compensatory mechanism upon transmission of the parasite to a new host species.

Cloning, sequencing, and characterization of a gene (narT) encoding a transport protein involved in dissimilatory nitrate reduction in Staphylococcus carnosus.

A Tn917 mutant of Staphylococcus carnosus TM300, nrIII, was isolated and characterized. Mutant nrIII did not take up nitrate or accumulate nitrite when grown in B-medium supplemented with up to 10 mM nitrate under anoxic conditions; however, it displayed wild-type levels of benzyl Delta viologen-linked nitrate reductase activity. Cultivated in B-medium with nitrate under oxic conditions, mutant nrIII accumulated fivefold less nitrite than the wild-type. The mutation in S. carnosus nrIII could be complemented with a 2-kb chromosomal EcoRI-HpaI fragment from the wild-type. The gene affected by transposon insertion in mutant nrIII was cloned and sequenced. Analysis of the deduced amino acid sequence revealed that this gene, designated narT, encodes a highly hydrophobic 42-kDa transmembrane protein of 388 amino acids and shows similarities to transport proteins that play a role in nitrate import or nitrite export. The inability of nrIII to take up nitrate under anoxic conditions and its ability to take up and accumulate nitrite in the presence of benzyl viologen, a nitrate ionophore, under the same conditions suggest that NarT represents a transport protein required for nitrate uptake under anoxic conditions in S. carnosus.

The strengths model in long-term care: linking cost containment and consumer empowerment.

This article outlines how the strengths model of case management can be applied to long-term-care case management. The strengths model was first articulated in the early 1980s as an alternative to brokered case management in mental health. The strengths approach to social service practice focuses on helping people and communities discover and develop their own capacities, talents, skills, and interests, and on connecting them with the resources needed to achieve their goals. This article explicates the components of the strengths model as adapted to long-term care. The authors explore the link between the application of strengths-based strategies at critical intervention points and cost containment, giving examples of how case managers serving older adults through Medicaid waivers have implemented the strengths approach and delayed nursing facility admissions. Finally, the authors describe ways that cost-containment methods can be interfaced with a consumer-driven empowerment orientation. Initial responses gathered from consumers and long-term-care case managers trained in the strengths model support its applicability to older adults. Clear articulation of how the strengths model of case management can be adapted for use with adults in need of long-term care is basic to further implementation and evaluation.