Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Schistosoma mansoni. A potential target for chemotherapy

Hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities are essential for the supply of guanine nucleotides in Schistosoma mansoni schistosomules. In crude extracts of adult S. mansoni, these two activities co-elute in size exclusion, ion exchange, and chromatofocusing chromatography and exhibit similar stabilities to heat treatment, suggesting that they are associated in one enzyme protein hypoxanthine-guanine phosphoribosyltransferase. This enzyme has been purified by a combination of heat treatment at 85 degrees C and chromatofocusing chromatography with elution at an apparent pI of 5.27 +/- 0.15. Pore gradient electrophoresis of the native enzyme followed by subsequent activity staining demonstrate an enzyme molecular weight of 105,000. The activity staining pattern remains the same whether hypoxanthine or guanine is used as the substrate, further supporting the existence of a single protein, hypoxanthine-guanine phosphoribosyltransferase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein results in a single protein band with a subunit molecular weight estimate of 64,000, suggesting that the native enzyme is a dimer. Preliminary kinetic studies showed that the purified hypoxanthine-guanine phosphoribosyltransferase reacted with guanine at a rate twice as fast as it did with hypoxanthine, but it did not act on xanthine at all. A full-length mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase cDNA clone pHPT5 and a plasmid pSV2-gpt containing the xanthine-guanine phosphoribosyltransferase gene for Escherichia coli were utilized as probes on Southern blots of S. mansoni DNA digests, and no significant hybridization was found under relatively relaxed conditions. Polyclonal antibodies made against human erythrocyte hypoxanthine-guanine phosphoribosyltransferase and E. coli xanthine-guanine phosphoribosyltransferase were tested in enzyme-linked immunosorbent assays of S. mansoni protein extracts, and no detectable cross-reacting protein was found. S. mansoni hypoxanthine-guanine phosphoribosyltransferase thus may bear rather limited homology to mammalian hypoxanthine-guanine phosphoribosyltransferase or bacterial xanthine-guanine phosphoribosyltransferase and could be an attractive target for antischistosomal chemotherapeutic drug design.     

Detecting non-neutral heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence

Natural selection, in the form of balancing selection or selective sweeps, can result in a decoupling of the amounts of molecular polymorphism and divergence. Thus natural selection can cause some areas of DNA sequence to have greater silent polymorphism, relative to divergence between species, than other areas. It would be useful to have a statistical test for heterogeneity in the polymorphism to divergence ratio across a region of DNA sequence, one that could identify heterogeneity greater than that expected from the neutral processes of mutation, drift, and recombination. The only currently available test requires that a region be arbitrarily divided into sections that are compared with each other, and the subjectivity of this division could be problematic. Here a test is proposed in which runs of polymorphic and fixed sites are counted, where a "run" is a set of one or more sites of one type preceded and followed by the other type. The number of runs is smaller than otherwise expected if polymorphisms are clumped together. By simulating neutral evolution and comparing the observed number of runs to the simulations, a statistical test is possible which does not require any a priori decisions about subdivision.      

Strongyloides stercoralis: identification of a protease that facilitates penetration of skin by the infective larvae

Host invasion and tissue migration of several helminths have been linked to the expression and release of parasite-derived proteases. One of the most remarkable examples of tissue migration is that of larvae of the nematode parasite Strongyloides stercoralis, which can move through tissue at speeds of up to 10 cm per hour. We have shown the Strongyloides L3 larvae secrete a potent histolytic metalloprotease to facilitate their rapid migration. This protease has elastase activity and catalyzes the degradation of a model of dermal extracellular matrix. The importance of this enzyme in the pathogenesis of strongyloidiasis is underscored by the observation that invasion by larvae of skin in vitro is prevented by metalloprotease inhibitors. These results substantiate the role of proteases as virulence factors in strongyloidiasis, as well as other related parasitic infections, and suggest new approaches to therapy.     

Schistosoma mansoni: Cercarial degradation of a radioactively labeled collagen gel

The apparent penetration activity of Schistosoma mansoni cercariae was quantified by means of an in vitro assay with a radioactively labeled Type I collagen gel. Both live cercariae and cercarial preacetabular gland secretions degraded the collagen. The addition of skin lipid or linoleic acid to the gel surface enhanced the degradation by live cercariae.     

The Influence of Antimicrobial Peptides and Mucolytics on the Integrity of Biofilms Consisting of Bacteria and Yeasts as Affecting Voice Prosthetic Air Flow Resistances

The integrity of biofilms on voice prostheses used to rehabilitate speech in laryngectomized patients causes unwanted increases in airflow resistance, impeding speech. Biofilm integrity is ensured by extracellular polymeric substances (EPS). This study aimed to determine whether synthetic salivary peptides or mucolytics, including N-acetylcysteine and ascorbic acid, influence the integrity of voice prosthetic biofilms. Biofilms were grown on voice prostheses in an artificial throat model and exposed to synthetic salivary peptides, mucolytics and two different antiseptics (chlorhexidine and Triclosan). Synthetic salivary peptides did not reduce the air flow resistance of voice prostheses after biofilm formation. Although both chlorhexidine and Triclosan reduced microbial numbers on the prostheses, only the Triclosan-containing positive control reduced the air flow resistance. Unlike ascorbic acid, the mucolytic N-acetylcysteine removed most EPS from the biofilms and induced a decrease in air flow resistance.     

The cysteine protease of Trypanosoma cruzi as a model for antiparasite drug design

By combining new technology in molecular biology, X-ray crystallography, computer graphics and biochemistry, structure-based drug design provides a parallel and cost-effective strategy for identification of new antiparasite chemotherapy. James McKerrow, Mary McGrath and Juan Engel here discuss an example of the amplication of this strategy is its use in targeting the major cysteine protease in Trypanosoma cruzi. Tools from molecular biology helped overcome the obstacle of limited parasite material to allow production of reagent quantities of enzyme for inhibitor screening. Computer graphics analysis and X-ray crystallography are allowing rapid identification of new inhibitors based on either leads already identified or compounds selected by computer graphics screening of chemical databases.