Discovery of novel inhibitors of Trypanosoma cruzi trans-sialidase from in silico screening

trans-Sialidase from Trypanosoma cruzi (TcTS) has emerged as a potential drug target for treatment of Chagas disease. Here, we report the results of virtual screening for the discovery of novel TcTS inhibitors, which targeted both the sialic acid and sialic acid acceptor sites of this enzyme. A library prepared from the Evotec database of commercially available compounds was screened using the molecular docking program GOLD, following the application of drug-likeness filters. Twenty-three compounds selected from the top-scoring ligands were purchased and assayed using a fluorimetric assay. Novel inhibitor scaffolds, with IC₅₀ values in the submillimolar range were discovered. The 3-benzothiazol-2-yl-4-phenyl-but-3-enoic acid scaffold was studied in more detail, and TcTS inhibition was confirmed by an alternative sialic acid transfer assay. Attempts to obtain crystal structures of these compounds with TcTS proved unsuccessful but provided evidence of ligand binding at the active site.     

Thrombopoietin complements G(i)- but not G(q)-dependent pathways for integrin {alpha}(IIb){beta}(3) activation and platelet aggregation

Binding of thrombopoietin (TPO) to the cMpl receptor on human platelets potentiates aggregation induced by a number of agonists, including ADP. In this work, we found that TPO was able to restore ADP-induced platelet aggregation upon blockade of the G(q)-coupled P2Y1 purinergic receptor but not upon inhibition of the G(i)-coupled P2Y12 receptor. Moreover, TPO triggered platelet aggregation upon co-stimulation of G(z) by epinephrine but not upon co-stimulation of G(q) by the thromboxane analogue U46619. Platelet aggregation induced by TPO and G(i) stimulation was biphasic, and cyclooxygenase inhibitors prevented the second but not the first phase. In contrast to ADP, TPO was unable to induce integrin alpha(IIb)beta(3) activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of integrin alpha(IIb)beta(3) was blocked by antagonists of the G(q)-coupled P2Y1 receptor but was completely restored by the simultaneous co-stimulation of cMpl receptor by TPO. Inside-out activation of integrin alpha(IIb)beta(3) induced by TPO and G(i) stimulation occurred independently of thromboxane A(2) production and was not mediated by protein kinase C, MAP kinases, or Rho-dependent kinase. Importantly, TPO and G(i) activation of integrin alpha(IIb)beta(3) was suppressed by wortmannin and Ly294002, suggesting a critical regulation by phosphatidylinositol 3-kinase. We found that TPO did not activate phospholipase C in human platelets and was unable to restore ADP-induced phospholipase C activation upon blockade of the G(q)-coupled P2Y1 receptor. TPO induced a rapid and sustained activation of the small GTPase Rap1B through a pathway dependent on phosphatidylinositol 3-kinase. In ADP-stimulated platelets, Rap1B activation was reduced, although not abolished, upon blockade of the P2Y1 receptor. However, accumulation of GTP-bound Rap1B in platelets activated by co-stimulation of cMpl and P2Y12 receptor was identical to that induced by the simultaneous ligation of P2Y1 and P2Y12 receptor by ADP. These results indicate that TPO can integrate G(i), but not G(q), stimulation and can efficiently support integrin alpha(IIb)beta(3) activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol 3-kinase and the small GTPase Rap1B.     

Mutational analysis of the RecA protein L1 region identifies this area as a probable part of the co-protease substrate binding site

Previous mutational analysis of the L1 region of the RecA protein suggested that Gly-157 and Glu-158 are 'hot-spots' for the occurrence of constitutive LexA co-protease mutants (coprt^c). In the present study, we clearly establish that position 157 is a hot-spot for the occurrence of such mutants, as 12 of 14 and 10 of 14 substitutions result in this phenotype for UmuD and LexA cleavage respectively. The frequency of such mutations at position 158 is somewhat lower, 8 of 13 and 5 of 13 for UmuD and LexA respectively. Comparison of the UmuD vs. LexA co-protease activity for all single mutants with substitutions at positions 154, 155, 156, 157 and 158 (47 in total) reveals that, although there is good agreement among most mutants regarding their ability to cleave both LexA and UmuD, there are two in particular (Glu-154→Asp and Glu-154→Gln) that show a clear preference for cleavage of UmuD. We also show that three second-site mutations that completely suppress coprt^c activity toward LexA have little or no effect on the coprt^c activity of the primary mutant toward UmuD. In addition, we observe a high frequency of second-site suppressor mutations, suggesting a functional interaction among side-chains in this region. Together, these results support the idea that the L1 region of RecA makes up part of the co-protease substrate-binding site.     

Origin of human immunodeficiency virus type 1 quasispecies emerging after antiretroviral treatment interruption in patients with therapeutic failure

The emergence of antiretroviral (ARV) drug-resistant human immunodeficiency virus type 1 (HIV-1) quasispecies is a major cause of treatment failure. These variants are usually replaced by drug-sensitive ones when the selective pressure of the drugs is removed, as the former have reduced fitness in a drug-free environment. This was the rationale for the design of structured ARV treatment interruption (STI) studies for the management of HIV-1 patients with treatment failure. We have studied the origin of drug-sensitive HIV-1 quasispecies emerging after STI in patients with treatment failure due to ARV drug resistance. Plasma and peripheral blood mononuclear cell samples were obtained the day of treatment interruption (day 0) and 30 and 60 days afterwards. HIV-1 pol and env were partially amplified, cloned, and sequenced. At day 60 drug-resistant variants were replaced by completely or partially sensitive quasispecies. Phylogenetic analyses of pol revealed that drug-sensitive variants emerging after STI were not related to their immediate temporal ancestors but formed a separate cluster, demonstrating that STI leads to the recrudescence and reemergence of a sequestrated viral population rather than leading to the back mutation of drug-resistant forms. No evidence for concomitant changes in viral tropism was seen, as deduced from env sequences. This study demonstrates the important role that the reemergence of quasispecies plays in HIV-1 population dynamics and points out the difficulties that may be found when recycling ARV therapies with patients with treatment failure.     

Morphological and physiological differentiation of seedlings between dry and wet habitats in a tropical dry forest

A common observation in tropical dry forests is the habitat preference of tree species along spatial soil water gradients. This pattern of habitat partitioning might be a result of species differentiation in their strategy for using water, along with competing functions such as maximizing water exploitation and tolerating soil water stress. We tested whether species from drier soil conditions exhibited a tolerance strategy compared with that of wet-habitat species. In a comparison of 12 morphophysiological traits in seedlings of 10 closely related dry and wet-habitat species pairs, we explored what trade-offs guide differentiation between habitats and species. Contrary to our expectations, dry-habitat species showed mostly traits associated with an exploitation strategy (higher carbon assimilation capacity, specific leaf area and leaf-specific conductivity and lower water-use efficiency). Strikingly, dry-habitat species tended to retain their leaves longer during drought. Additionally, we detected multiple strategies to live within each habitat, in part due to variation of strategies among lineages, as well as functional differentiation along the water storage capacity-stem density (xylem safety) trade-off. Our results suggest that fundamental trade-offs guide functional niche differentiation among tree species expressed both within and between soil water habitats in a tropical dry forest.     

Presence of Bartonella species in wild carnivores of northern Spain

The genus Bartonella was detected by PCR in 5.7% (12/212) of wild carnivores from Northern Spain. Based on hybridization and sequence analyses, Bartonella henselae was identified in a wildcat (Felis silvestris), Bartonella rochalimae in a red fox (Vulpes vulpes) and in a wolf (Canis lupus), and Bartonella sp. in badgers (Meles meles).     

On the relative roles of faster-X evolution and dominance in the establishment of intrinsic postzygotic isolating barriers

The modern theory of speciation assigns a prominent role to the recessivity of genetic incompatibilities in the two rules of speciation, namely Haldane's rule and the large X effect, and considers that the contribution of faster evolution of the X versus the autosomes to those patterns is generally of relatively minor importance. By extending Turelli and Orr's previous analysis of the model of two-locus Dobzhansky–Muller incompatibilities, I first show that when the X and the autosomes evolve at the same rate, the two dominance parameters involved in that model are not equally important for the declaration of a large X effect, but that the degree of recessivity of homozygous–homozygous incompatibilities is the major determinant for such a declaration. When the X evolves faster than the autosomes, the model obviously predicts that the importance of both dominance parameters will progressively vanish. It is then of importance to obtain estimates of the relative evolutionary rate of X-linked incompatibility loci. Several different procedures to obtain such estimates from the perspective of the large X effect are suggested. The application of the appropriate test to the only suitable data from Drosophila hybridizations so far available leads to the conclusion that the X actually evolves at least 2.5 times faster than the autosomes, as far as hybrid male sterility determinants are concerned, thus making dominance considerations absolutely irrelevant. Notwithstanding the necessity of further tests, the relative roles currently assigned to faster-X evolution and dominance in the theory of speciation should be revised, giving due prominence to faster-X evolution, at least for hybrid male sterility in the genus Drosophila.     

Polycystin-1 activates and stabilizes the polycystin-2 channel

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent genetic disorder largely caused by mutations in the PKD1 and PKD2 genes that encode the transmembrane proteins polycystin-1 and -2, respectively. Both proteins appear to be involved in the regulation of cell growth and maturation, but the precise mechanisms are not yet well defined. Polycystin-2 has recently been shown to function as a Ca(2+)-permeable, non-selective cation channel. Polycystin-2 interacts through its cytoplasmic carboxyl-terminal region with a coiled-coil motif in the cytoplasmic tail of polycystin-1 (P1CC). The functional consequences of this interaction on its channel activity, however, are unknown. In this report, we show that P1CC enhanced the channel activity of polycystin-2. R742X, a disease-causing polycystin-2 mutant lacking the polycystin-1 interacting region, fails to respond to P1CC. Also, P1CC containing a disease-causing mutation in its coiled-coil motif loses its stimulatory effect on wild-type polycystin-2 channel activity. The modulation of polycystin-2 channel activity by polycystin-1 may be important for the various biological processes mediated by this molecular complex.     

Estrogen receptor,cyclic adenosine monophosphate,and protein kinase A are involved in the nongenomic pathway by which estradiol accelerates oviductal oocyte transport in cyclic rats

This investigation examined the role of estrogen receptor (ER) on the stimulatory effect of estradiol (E2) on protein phosphorylation in the oviduct as well as on E2-induced acceleration of oviductal oocyte transport in cyclic rats. Estrous rats were injected with E2 s.c. and with the ER antagonist ICI 182 780 intrabursally (i.b.), and 6 h later, oviducts were excised and protein phosphorylation was determined by Western blot analysis. ICI 182 780 inhibited the E2-induced phosphorylation of some oviductal proteins. Other estrous rats were treated with E2 s.c. and ICI 182 780 i.b. The number of eggs in the oviduct, assessed 24 h later, showed that ICI 182 780 blocked the E2-induced egg transport acceleration. The possible involvement of adenylyl cyclase, protein kinase A (PK-A), protein kinase C (PK-C), or tyrosine kinases on egg transport acceleration induced by E2 was then examined. Selective inhibitors of adenylyl cyclase or PK-A inhibited the E2-induced egg transport acceleration, whereas PK-C or tyrosine kinase inhibitors had no effect. Furthermore, forskolin, an adenylyl cyclase activator, mimicked the effect of E2 on ovum transport and E2 increased the level of cAMP in the oviduct of cycling rats. Finally, we measured PK-A activity in vitro in the presence of E2 or E2-ER complex. Activity of PK-A in the presence of E2 or E2-ER was similar to PK-A alone, showing that E2 or E2-ER did not directly activate PK-A. We conclude that the nongenomic pathway by which E2 accelerates oviductal egg transport in the rat requires absolute participation of ER and cAMP and partial participation of PK-A signaling pathways in the oviduct.