Confronting the HIV epidemic in Asia and the Pacific: developing successful strategies to minimize the spread of HIV infection.

PIP: In Asia, the cumulative total of HIV-infected adults will reach 1.22 million by 1995, and, by 2000, the number is estimated to reach 11-45 million. The modes of transmission vary from country to country and include injecting drug users, commercial sex workers and their clients, commercial blood donors, hemophiliacs, and homosexuals. Social, cultural, and health factors also affect transmission, such as rites of passage to adulthood, lack of female autonomy, multiple sex partners, wars and civil unrest, and availability of drugs. The HIV epidemic has economic ramifications and causes, e.g., migrant worker camps, the sex industry, and rapid urbanization luring Burmese girls to Thailand. Governments must create an environment for behavior-change through financial, political, and legislative measures. Community organizations also play a role in prevention, as in programs initiated by a squatter settlement in Bangkok, where 36% of IV drug users were found to be HIV-positive. In Maharashtra State, India, peer-based prevention programs were developed for sex workers. Successful behavior change of individuals is based on redefinition of peer norms, understanding the danger and vulnerability to infection, and building confidence to change behavior. Successful programs require placing priority on HIV issues on the political agenda, negotiation and consensus-building skills, and competent program management. For instance, in Zimbabwe a project enlisted 380,000 people in 4500 education sessions within 2 years, and distributed 2.5 million condoms. Among sex workers, condom use increased from 5% to 50%. Implementation strategies include the provision of information and interpersonal education. In Zaire, mass media and social marketing efforts boosted condom sales from less than half a million in 1987 to over 20 million in 1991. The means to change behavior requires the availability of good quality condoms, disinfectants, and syringes. Furthermore, clinical management of gonorrhea, syphilis, human papillomavirus, and chancroid is vital to lowering the risk of HIV transmission. Continuous epidemiological research and the evaluation of prevention programs improve program effectiveness.^ieng

Molecular characterization of the trypanothione reductase gene from Crithidia fasciculata and Trypanosoma brucei: comparison with other flavoprotein disulphide oxidoreductases with respect to substrate specificity and catalytic mechanism.

Trypanothione reductase belongs to the family of flavoprotein disulphide oxidoreductases that include glutathione reductases, dihydrolipoamide dehydrogenases and mercuric reductases. Trypanothione reductase and its substrate, trypanothione disulphide, are unique to parasitic trypanosomatids responsible for several tropical diseases. The crystal structure of the enzyme from Crithidia fasciculata is currently under investigation as an aid in the design of selective inhibitors with a view to producing new drugs. We report here the cloning and sequencing of the genes for trypanothione reductase from C. fasciculata and Trypanosoma brucei. Alignment of the deduced amino acid sequences with 21 other members of this family provides insight into the role of certain amino acid residues with respect to substrate specificity and catalytic mechanism as well as conservation of certain elements of secondary structure.

Active site of trypanothione reductase. A target for rational drug design.

The X-ray crystal structure of the enzyme trypanothione reductase, isolated from the trypanosomatid organism Crithidia fasciculata, has been solved by molecular replacement. The search model was the crystal structure of human glutathione reductase that shares approximately 40% sequence identity. The trypanosomal enzyme crystallizes in the tetragonal space group P4(1) with unit cell lengths of a = 128.9 A and c = 92.3 A. The asymmetric unit consists of a homodimer of approximate molecular mass 108 kDa. We present the structural detail of the active site as derived from the crystallographic model obtained at an intermediate stage of the analysis using diffraction data to 2.8 A resolution with an R-factor of 23.2%. This model has root-mean-square deviations from ideal geometry of 0.026 A for bond lengths and 4.7 degrees for bond angles. The trypanosomid enzyme assumes a similar biological function to glutathione reductase and, although similar in topology to human glutathione reductase, has an enlarged active site and a number of amino acid differences, steric and electrostatic, which allows it to process only the unique substrate trypanothione and not glutathione. This protein represents a prime target for chemotherapy of several debilitating tropical diseases caused by protozoan parasites belonging to the genera Trypanosoma and Leishmania. The structural differences between the parasite and host enzymes and their substrates thus provides a rational basis for the design of new drugs active against trypanosomes. In addition, our model explains the results of site-directed mutagenesis experiments, carried out on recombinant trypanothione reductase and glutathione reductases, designed by consideration of the crystal structure of human glutathione reductase.

Phosphorylation differences among proteins of bloodstream developmental stages of Trypanosoma brucei brucei.

Early in an infection the bloodstream forms of the African trypanosome Trypanosoma brucei brucei are long, slender and rapidly dividing. Later, non-dividing, short, stumpy forms may be found. In this report we described biochemical differences between the two parasite populations in the phosphorylation of their proteins in vitro. Compared with the slender populations, the non-dividing stumpy forms of the parasites exhibit decreased phosphorylation of an 80 kDa protein and enhanced phosphorylation of 37 kDa and 42 kDa proteins (pp37 and pp42). These changes occurred regardless of whether the stumpy trypanosomes were generated naturally during the course of the infection or induced by difluoromethylornithine treatment. The phosphorylation of pp37 and pp42 occurs on serine and threonine residues and is totally dependent upon the presence of Mn2+ or Mg2+. However, excess Mn2+ or Mg2+ inhibits phosphorylation. Maximal phosphorylation of pp42 occurs with 1 mm-Mn2+ or 10 mm-Mg2+, whereas that of pp37 occurs with 50 mM-Mn2+ or greater than 100 mm-Mg2+. The phosphorylation of pp37 is greatly enhanced by KCl, whereas that of pp42 is only slightly increased by this salt. Ca2+, calmodulin, phospholipids and cyclic AMP have no discernible effect upon the phosphorylation of pp42 or pp37 in vitro, whereas heparin, suramin, polylysine, polyarginine and polyamines all inhibit phosphorylation. Thus the enzymes that phosphorylate pp42 and pp37 have properties similar to, but distinct from, those of mammalian casein kinase II. Since the casein-kinase-like activity is higher in the slender than in the stumpy forms, the enhanced phosphorylation of pp42 and pp37 in the non-dividing parasites is probably a result of the enhanced synthesis of these acidic proteins.