Hepatitis C NS3 protease inhibition by peptidyl-alpha-ketoamide inhibitors: kinetic mechanism and structure

A series of novel peptidyl-alpha-ketoamide compounds were evaluated as inhibitors of the deltaNS3-NS4A serine protease from the hepatitis C virus. These peptidyl-alpha-ketoamide inhibitors with Ki values ranging from 0.17 nM to 5.6 microM exhibited slow-binding inhibition. Kinetic studies established one-step kinetic mechanisms and dissociation rate constants in the 3-7 x 10(-5) s(-1) range for these compounds. The association rate constants, which ranged from 10 to 263,000 M(-1) s(-1), were responsible for the greater than four order of magnitude overall binding affinity range exhibited by this series. An X-ray crystal structure of a protease-inhibitor complex revealed an unusual interaction between the oxyanion of the adduct and the protein as well as a significant movement in the S1' region of the protein loop comprising residues 35-42. These results are quite different from peptidyl-alpha-ketoacid inhibition of HCV protease, which reportedly undergoes no notable conformational changes and proceeds with a two-step slow-binding kinetic mechanism.     

Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants reveals strain-specific atypical regulation of the Hrp type III secretion system

The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL::omegaCmr and hrpS::omegaCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative sigma54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE::uidA, pelL::uidA, and hrpN::uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.     

Identification of beta-arrestin2 as a G protein-coupled receptor-stimulated regulator of NF-kappaB pathways

Norepinephrine released by the sympathetic nerve terminals regulates the immune system primarily via its stimulation of beta(2)-adrenergic receptor (beta(2)AR), but the underlying molecular mechanisms remain to be elicited. Beta(2)AR, a well-studied G protein-coupled receptor (GPCR), is functionally regulated by beta-arrestin2, which not only causes receptor desensitization and internalization but also serves as a signaling molecule in GPCR signal transduction. Here we show that beta-arrestin2 directly interacts with IkappaBalpha (inhibitor of NF-kappaB, the key molecule in innate and adaptive immunity) and thus prevents the phosphorylation and degradation of IkappaBalpha. Consequently, beta-arrestin2 effectively modulates activation of NF-kappaB and expression of NF-kappaB target genes. Moreover, stimulation of beta(2)AR significantly enhances beta-arrestin2-IkappaBalpha interaction and greatly promotes beta-arrestin2 stabilization of IkappaBalpha, indicating that beta-arrestin2 mediates a crosstalk between beta(2)AR and NF-kappaB signaling pathways. Taken together, the current study may present a novel mechanism for regulation of the immune system by the sympathetic nervous system.     

A novel phosphatidylinositol 4,5-bisphosphate-binding domain targeting the Phg2 kinase to the membrane in Dictyostelium cells

Phg2 is a ser/thr kinase involved in adhesion, motility, actin cytoskeleton dynamics, and phagocytosis in Dictyostelium cells. In a search for Phg2 domains required for its localization to the plasma membrane, we identified a new domain interacting with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 4-phosphate (PI(4)P) membrane phosphoinositides. Deletion of this domain prevented membrane recruitment of Phg2 and proper function of the protein in the phagocytic process. Moreover, the overexpression of this PI(4,5)P₂-binding domain specifically had a dominant-negative effect by inhibiting phagocytosis. Therefore, plasma membrane recruitment of Phg2 is essential for its function. The PI(4,5)P₂-binding domain fused to GFP (green fluorescent protein) (GFP-Nt-Phg2) was also used to monitor the dynamics of PI(4,5)P₂ during macropinocytosis and phagocytosis. GFP-Nt-Phg2 disappeared from macropinosomes immediately after their closure. During phagocytosis, PI(4,5)P₂ disappeared even before the sealing of phagosomes as it was already observed in mammalian cells. Together these results demonstrate that PI(4,5)P₂ metabolism regulates the dynamics and the function of Phg2.     

Mitochondrial phylogeography and demographic history of the vicuña: implications for conservation

The vicu?a (Vicugna vicugna; Miller, 1924) is a conservation success story, having recovered from near extinction in the 1960s to current population levels estimated at 275,000. However, lack of information about its demographic history and genetic diversity has limited both our understanding of its recovery and the development of science-based conservation measures. To examine the evolution and recent demographic history of the vicu?a across its current range and to assess its genetic variation and population structure, we sequenced mitochondrial DNA from the control region (CR) for 261 individuals from 29 populations across Peru, Chile and Argentina. Our results suggest that populations currently designated as Vicugna vicugna vicugna and Vicugna vicugna mensalis comprise separate mitochondrial lineages. The current population distribution appears to be the result of a recent demographic expansion associated with the last major glacial event of the Pleistocene in the northern (18 to 22 degrees S) dry Andes 14-12,000 years ago and the establishment of an extremely arid belt known as the 'Dry Diagonal' to 29 degrees S. Within the Dry Diagonal, small populations of V. v. vicugna appear to have survived showing the genetic signature of demographic isolation, whereas to the north V. v. mensalis populations underwent a rapid demographic expansion before recent anthropogenic impacts.     

Functional and Structural Characterization of the Antiphagocytic Properties of a Novel Transglutaminase from Streptococcus suis

Streptococcus suis serotype 2 (Ss2) is an important swine and human zoonotic pathogen. In the present study, we identified a novel secreted immunogenic protein, SsTGase, containing a highly conserved eukaryotic-like transglutaminase (TGase) domain at the N terminus. We found that inactivation of SsTGase significantly reduced the virulence of Ss2 in a pig infection model and impaired its antiphagocytosis in human blood. We further solved the crystal structure of the N-terminal portion of the protein in homodimer form at 2.1 Å. Structure-based mutagenesis and biochemical studies suggested that disruption of the homodimer directly resulted in the loss of its TGase activity and antiphagocytic ability. Characterization of SsTGase as a novel virulence factor of Ss2 by acting as a TGase would be beneficial for developing new therapeutic agents against Ss2 infections.     

Body mass of wild bornean orangutans living in human‐dominated landscapes: Implications for understanding their ecology and conservation

Body mass is a key determinant of a species' ecology, including locomotion, foraging strategies, and energetics. Accurate information on the body mass of wild primates allows us to develop explanatory models for relationships among body size, ecology, and behavior and is crucial for reconstructing the ecology and behavior of fossil primates and hominins. Information on body mass can also provide indirect information on health and can be an important tool for conservation in the context of increasingly widespread habitat disturbance. This study reports body mass data recorded for wild Northeast Bornean orangutans (Pongo pygmaeus morio) during relocation efforts in forestry and oil palm plantations in East Kalimantan, Indonesia. The average mass of flanged adult males (n = 12, 74 ± 9.78 kg) and adult females (n = 7, 35.29 ± 7.32 kg) from this study were 13.6% and 9% lower, respectively, than the only other published wild Bornean orangutan body mass measurements, but the range of weights for both males and females was larger for this study. This pattern could be due to sampling error, data collection differences, or the influence of habitat disturbance, specifically a lack of access to resources, on individual health. When necessary relocations present the opportunity, we encourage researchers to prioritize the collection of body size data for the purposes of understanding ecology but also as an indirect means of monitoring population viability. As primate habitat becomes increasingly fragmented and altered by humans such data will become critical to our ability to make informed conservation decisions. Am J Phys Anthropol 157:339–346, 2015. © 2015 Wiley Periodicals, Inc.     

Amputation du gland lors de la circoncision: A propos de 19 cas

Purpose

To assess the clinical features and describe treatment difficulties associated with glandular amputation during circumcision in our daily urological practice in Senegal and to warn about the risks of this procedure, which remain underestimated in this country.

Patients and Methods

The authors report a retrospective series of 19 patients who suffered glandular amputation during circumcision and who were admitted to their urology unit. The medical charts of patients hospitalised during this period were studied.

Results

The mean age of these patients was 7.6 years. 84.2% of patients presented with complete glandular amputation. The mean time to consultation was 17.6 months generally in a context of urinary tract complications. Surgical treatment consisted of meatoplasty in 68.4% of cases. Four cases were treated by glandular reimplantation which unfortunately failed.

Conclusion

The severity of this complication and the difficulties of management in our regions must encourage prevention.