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.     

Comparative mitogenic and galactopoietic effects of IGF-I, IGF-II and Des-3-IGF-I in bovine mammary gland in vitro.

Insulin-like growth factors (IGFs) I and II (IGF-I, IGF-II) and Des-3-IGF-I at physiological concentrations are potent mitogens of bovine undifferentiated mammary epithelial cells cultured in collagen in a serum-free medium. Des-3-IGF-I was found to be as potent as IGF-I, while IGF-II was significantly less active. All three factors acted either synergistically or additively with epidermal growth factor (EGF), cholera toxin and fetal calf serum (FCS). Indirect evidence indicates that despite its lower mitogenic activity the action of IGF-II is mediated through IGF-I receptors. The galactopoietic activity of Des-3-IGF-I and IGF-II was studied in an organ culture of bovine lactating mammary glands using lactogen-responsive fat synthesis as a test. Neither Des-3-IGF-I nor IGF-II exhibited galactopoietic activity nor did they affect the galactopoietic activity of prolactin.     

Identification of chromosome inheritance modifiers in Drosophila melanogaster

Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a "sensitized" minichromosome substrate (J21A) to screen approximately 3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.     

Swimming from island to island: healing practice in Tonga

The health care system of the Pacific island nation of Tonga serves as an example of enduring medical pluralism which incorporates traditional and Western medical practice and accommodates contemporary political and social change. Biomedicine is represented by the hospital and the community health centers; traditional medicine is practiced in homes by healers. Both types of therapies are popularly utilized for different ailments or for the same problem at different points in the illness. Contemporary healing is described and is also analyzed as an expression of social change occurring in Tonga as a result of a political movement toward democracy.     

Development of adenovirus vectors encoding rat complement regulators for use in therapy in rodent models of inflammatory diseases

C activation has been implicated in the pathogenesis of numerous inflammatory human diseases and disease models. A therapy based on C inhibition might therefore be of benefit to reduce inflammation and ameliorate disease. C inhibition in vivo can be accomplished by the delivery of soluble recombinant C regulators either systemically or directly to a target site, but effects are transitory. We have developed a strategy for the efficient delivery of the membrane-bound rat C inhibitors, CD59, Crry, and decay-accelerating factor (DAF), using replication-deficient adenovirus vectors with the intention of treating rat models of disease in which C is implicated. The adenovirus recombinants(RAd), RAdCD59, RAdCrry, and RAdDAF, respectively, have been tested for expression and function of the transgene in vitro. Infection of human fetal foreskin fibroblasts resulted in high levels of expression of each of the rat inhibitors. The constructs were also tested for inhibition of rat C-mediated cell lysis and C3b deposition. In a cell lysis assay, each inhibited to varying degrees of efficiency in the order RAdCD59 = RAdDAF > RAdCrry. In a C3b deposition assay, RAdDAF caused a greater reduction in C3b deposition than RAdCrry and RAdCD59 was ineffective. These agents, individually or in combination, provide the tools for testing the effects of prolonged inhibition of C at a target site on the progress of experimental models of disease.     

The genetic identity of alien chromosomes in potato breeding lines revealed by sequential GISH and FISH analyses using chromosome-specific cytogenetic DNA markers.

Genomic in situ hybridization (GISH) is one of the most popular and effective techniques for detecting alien chromatin introgressed into breeding lines; however, GISH analysis alone does not reveal the genetic identity of the alien chromosomes. We previously isolated a set of bacterial artificial chromosomes (BACs) specific to each of the 12 potato chromosomes. These BAC clones can be used as chromosome-specific cytogenetic DNA markers (CSCDMs) for potato chromosome identification. Here we demonstrate that GISH and fluorescence in situ hybridization (FISH), using CSCDMs, can be performed sequentially on the same chromosome preparations. Somatic metaphase chromosomes prepared using an enzymatic digestion and "flame-drying" procedure allows repeated probing up to five times without significant damage to chromosome morphology. The sequential GISH and FISH analyses reveal the genomic origin and genetic identity of the alien chromosomes in a single experiment and also determine whether an alien chromosome has been added to the genetic background of potato or is substituting for a homoeologous potato chromosome. The sequential GISH and FISH procedures should be widely applicable for germplasm characterization, especially in plant species with small-sized chromosomes.     

Mechanisms of arsenic hyperaccumulation in Pteris vittata. Uptake kinetics,interactions with phosphate,and arsenic speciation

The mechanisms of arsenic (As) hyperaccumulation in Pteris vittata, the first identified As hyperaccumulator, are unknown. We investigated the interactions of arsenate and phosphate on the uptake and distribution of As and phosphorus (P), and As speciation in P. vittata. In an 18-d hydroponic experiment with varying concentrations of arsenate and phosphate, P. vittata accumulated As in the fronds up to 27,000 mg As kg(-1) dry weight, and the frond As to root As concentration ratio varied between 1.3 and 6.7. Increasing phosphate supply decreased As uptake markedly, with the effect being greater on root As concentration than on shoot As concentration. Increasing arsenate supply decreased the P concentration in the roots, but not in the fronds. Presence of phosphate in the uptake solution decreased arsenate influx markedly, whereas P starvation for 8 d increased the maximum net influx by 2.5-fold. The rate of arsenite uptake was 10% of that for arsenate in the absence of phosphate. Neither P starvation nor the presence of phosphate affected arsenite uptake. Within 8 h, 50% to 78% of the As taken up was distributed to the fronds, with a higher translocation efficiency for arsenite than for arsenate. In fronds, 49% to 94% of the As was extracted with a phosphate buffer (pH 5.6). Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectroscopy showed that >85% of the extracted As was in the form of arsenite, and the remaining mostly as arsenate. We conclude that arsenate is taken up by P. vittata via the phosphate transporters, reduced to arsenite, and sequestered in the fronds primarily as As(III).     

Hydrobenzoin-based rigid chiral polymer

We prepared a rigid, chiral polymer (1) from optically active hydrobenzoin-based subunits. Nonracemic monomer units 6 and 8 were prepared by asymmetric dihydroxylation (AD) methodology and polymerization was carried out under Sonagashira coupling conditions. Polymer 1 was obtained in good yield with a molecular weight M(n) = 5,100 (PDI = 2.3). Modeling suggests that polymer 1 could form a stable helical mainchain conformation in solution or the solid state. The chiroptical data of the polymer and a low-molecular weight model compound (9) are compared.     

Hormone trajectories leading to human birth

The mechanisms regulating human parturition and labor remain unknown. This ignorance is expensive as preterm birth is responsible for 70% of neonatal mortality and 50% of cerebral palsy. Methods for the prediction of preterm birth and treatments for women in preterm labor have poor efficacy reflecting our limited knowledge of the mechanisms involved. Recent research has supported the view that parturition is a cascade of events that commences early in pregnancy and involves the mother, fetus, placenta, membranes, cervix and myometrium. Although a number of the key hormones and proteins involved have been identified, the relationships between these factors in time and tissues remain unclear. Placental production of Corticotropin-releasing hormone (CRH) is proposed as an early event regulating the cascade of events. Central to the onset of parturition will be a mechanism for progesterone withdrawal and estrogen activation in human. Two forms of progesterone receptor with opposing actions exist in the human myometrium. Progesterone receptor A (PR-A) is a dominant negative repressor of progesterone receptor B (PR-B). Preliminary studies strongly support the hypothesis that the onset of human parturition is initiated by rising concentrations of PR-A in the myometrium.