Substrate specificity and the effect of calcium on Trypanosomabrucei metacaspase 2

Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca<sup>2+</sup> on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has an unambiguous preference for basic amino acids at the P<sub>1</sub> position of peptide substrates and that this is most probably a result of hydrogen bonding from the P<sub>1</sub> residue to Asp95 and Asp211 in TbMCA2. In addition, TbMCA2 also has a preference for charged residues at the P<sub>2</sub> and P<sub>3</sub>positions and for small residues at the prime side of a peptide substrate. Studies into the effects of Ca<sup>2+</sup> on the enzyme revealed the presence of two Ca<sup>2+</sup> binding sites and a reversible structural modification of the enzyme upon Ca<sup>2+</sup> binding. In addition, the concentration of Ca<sup>2+</sup> used for activation of TbMCA2 was found to produce a differential effect on the activity of TbMCA2, but only when a series of peptides that differed in P<sub>2</sub> were examined, suggesting that Ca<sup>2+</sup>activation of TbMCA2 has a structural effect on the enzyme in the vicinity of the S2 binding pocket. Collectively, these data give new insights into the substrate specificity and Ca<sup>2+</sup> activation of TbMCA2. This provides important functional details and leads to a better understanding of metacaspases, which are known to play an important role in trypanosomes and make attractive drug targets due to their absence in humans

The delivery of therapeutic oligonucleotides

The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology

Geographic variation in adult survival and reproductive tactics of the mosquito Aedes albopictus

Climate differences across latitude can result in seasonal constraints and selection on life history characters. Since Aedes albopictus (Skuse) invaded North America in the mid-1980s, it has spread across a range of approximate to 14 degrees latitude and populations in the north experience complete adult mortality due to cold winter temperatures that are absent in the south. Life table experiments were conducted to test for differences in the adult survival and reproductive schedules of Ae. albopictus females from two populations from the northern (Bloomington, IN [BL] and Manassas, VA [VA]; approximate to 39 degrees N) and southern (Tampa, FL and Fort Myers, FL; approximate to 27-28 degrees N) extremes of the species distribution in North America. Regardless of population origin, age-specific hazard rate increased with reproductive output and decreased with number of bloodmeals. Larger females took fewer bloodmeals, and they had greater hazard rates than did smaller females. There were no consistent differences between northern versus southern populations in resource allocation between reproduction and maintenance, reproduction over time, and reproductive investment among offspring, suggesting that latitudinal variation in climate is probably not a main selective factor impinging on adult mortality and reproductive schedules. One possible effect of climate on geographic differences in life history was detected. BL had lower survivorship, lower lifetime reproductive output, and lower adult reproductive rate than did all other populations. This result may be an indirect result of lower egg survivorship due to the severity of winter in BL compared with other populations, including VA at approximately the same latitude. Such a scenario may make the BL population more prone to extinction, irregularly recolonized from more favorable sites, and thus more susceptible to founder effects, genetic drift, and inbreeding, resulting in lower mean values of fitness-related traits

Nischarin, a Novel Protein That Interacts with the Integrin α5 Subunit and Inhibits Cell Migration

Integrins have been implicated in key cellular functions, including cytoskeletal organization, motility, growth, survival, and control of gene expression. The plethora of integrin α and β subunits suggests that individual integrins have unique biological roles, implying specific molecular connections between integrins and intracellular signaling or regulatory pathways. Here, we have used a yeast two-hybrid screen to identify a novel protein, termed Nischarin, that binds preferentially to the cytoplasmic domain of the integrin α5 subunit, inhibits cell motility, and alters actin filament organization. Nischarin is primarily a cytosolic protein, but clearly associates with α5β1, as demonstrated by coimmunoprecipitation. Overexpression of Nischarin markedly reduces α5β1-dependent cell migration in several cell types. Rat embryo fibroblasts transfected with Nischarin constructs have “basket-like” networks of peripheral actin filaments, rather than typical stress fibers. These observations suggest that Nischarin might affect signaling to the cytoskeleton regulated by Rho-family GTPases. In support of this, Nischarin expression reverses the effect of Rac on lamellipodia formation and selectively inhibits Rac-mediated activation of the c-fos promoter. Thus, Nischarin may play a negative role in cell migration by antagonizing the actions of Rac on cytoskeletal organization and cell movement

Effects of habitat type and drying on Ascogregarina barretti (Eugregarinida : Lecudinidae) infection in Aedes triseritatus (Diptera : Culicidae)

The intensity and prevalence of parasitism by Ascogregarina barretti (Vavra) in aedes triseriatus (Say) did not differ between tires and tree holes in field samples taken in September 1996. There was significant variation in the intensity of parasitism among containers that was not significantly cor-related with the pH, conductivity, or temperature of the container water. In an experiment manipulating habitat drying, treatments had a significant effect on A. barretti infection of Ae. triseriatus, only during midsummer in one of two years. Containers maintained at maximal volume had the lowest prevalence of parasitism, and containers that dried out had the greatest prevalence. In this experiment, there was also a season-dependent difference in the intensity and prevalence of infection between tree holes and tires. The first larvae to reach the fourth instar in tires in the early summer had lower intensity and prevalence of infection than did larvae in tree holes during the same period. The seasonal difference in intensity of parasitism between tires and tree holes was not related to differences in pH, conductivity, and temperature

Integrin Targeted Delivery of Gene Therapeutics

Integrins have become key targets for molecular imaging and for selective delivery of anti-cancer agents. Here we review recent work concerning the targeted delivery of antisense and siRNA oligonucleotides via integrins. A variety of approaches have been used to link oligonucleotides to ligands capable of binding integrins with high specificity and affinity. This includes direct chemical conjugation, incorporating oligonucleotides into lipoplexes, and use of various polymeric nanocarriers including dendrimers. The ligand-oligonucleotide conjugate or complex associates selectively with the integrin, followed by internalization into endosomes and trafficking through subcellular compartments. Escape of antisense or siRNA from the endosome to the cytosol and nucleus may come about through endogenous trafficking mechanisms, or because of membrane disrupting capabilities built into the conjugate or complex. Thus a variety of useful strategies are available for using integrins to enhance the pharmacological efficacy of therapeutic oligonucleotides

Tributação e a Organização dos Prestadores de Serviços no Brasil

The real impact of taxes on the economy can only be assessed when we consider the behavioral responses of economic agents due to these taxes. This paper shows how income taxation in Brazil gives incentives to some classes of taxpayers to incorporate in order to reduce their tax burden. Microdata analysis on these taxpayers characteristics indicate that the probability of incorporation is strongly related to tax ruleTaxation, Organizational Form, Tax Avoidance.