Structural basis of von Willebrand factor activation by the snake toxin botrocetin

The A1 domain of von Willebrand factor (vWF) mediates platelet adhesion to sites of vascular injury by binding to the platelet receptor glycoprotein Ib (GpIb), an interaction that is regulated by hydrodynamic shear forces. The GpIb binding surface of A1 is distinct from a regulatory region, suggesting that ligand binding is controlled allosterically. Here we report the crystal structures of the "gain-of-function" mutant A1 domain (I546V) and its complex with the exogenous activator botrocetin. We show that botrocetin switches the mutant A1 back toward the wild-type conformation, suggesting that affinity is enhanced by augmenting the GpIb binding surface rather than through allosteric control. Functional studies of platelet adhesion under flow further suggest that the activation mechanism is distinct from that of the gain-of-function mutation.     

Protective immunity against Taenia crassiceps murine cysticercosis induced by DNA vaccination with a Taenia saginata tegument antigen

This study investigated the protective capacity of the recombinant Taenia saginata Tso18 antigen administered as a DNA vaccine in the Taenia crassiceps murine model of cysticercosis. This Tso18 DNA sequence, isolated from a T. saginata oncosphere cDNA library, has homologies with Taenia solium and Echinococcus sp. It was cloned in the pcDNA3.1 plasmid and injected once intramuscularly into mice. Compared to saline-vaccinated control mice, immunization reduced the parasite burden by 57.3-81.4%, while lower levels of non-specific protection were induced in control mice injected with the plasmid pcDNA3.1 (18.8-33.1%) or a plasmid with irrelevant construct, pcDNA3.1/3D15 (33.4-38.8%). Importantly, significant levels of protection were observed between the pcDNA3.1/Tso18 plasmid and pcDNA3.1/3D15 plasmid immunized mice. Mice immunized with pTso18 synthesized low levels of, primarily IgG1 sub-class, antibodies. These antibodies were shown to recognize a 66 kDa antigen fraction of T. crassiceps and T. solium. Splenocytes enriched in both CD4+CD8- and CD4-CD8+ T cells from these vaccinated mice proliferated in vitro when exposed to antigens from both T. solium and T. crassiceps cestodes. Immunolocalization studies revealed the Tso18 antigen in oncospheres of T. saginata and T. solium, in the adult tapeworm and in the tegument of T. solium cysticerci. The protective capacity of this antigen and its extensive distribution in different stages, species and genera of cestodes points to the potential of Tso18 antigen for the possible design of a vaccine against cestodes.     

Effect of olive oil mill wastewater on extracellular ligninolytic enzymes produced by Phanerochaete flavido-alba

Four genes, fagA, B, C and D, encoding products with 32-47% identity to proteins involved in bacterial iron uptake systems, were identified immediately downstream of the Corynebacterium pseudotuberculosis phospholipase D gene. beta-Galactosidase assays on a C. pseudotuberculosis strain carrying a fagA-lacZ fusion indicated that the putative fagABC operon was poorly expressed in iron-rich media. However, similar experiments in iron-limited media resulted in an approximately three-fold increase in beta-galactosidase activity, suggesting that this operon is regulated by iron in vitro. Although no defect in iron utilization could be determined for a C. pseudotuberculosis fagB(C) mutant in vitro, this mutant showed reduced virulence compared to wild-type in a goat model of caseous lymphadenitis. Thus, expression of the fag genes in the host appears to contribute to virulence.     

Evolution of ragweed pollen concentrations, sensitization and related allergic clinical symptoms in Parma (northern Italy)

The pollen released in the atmosphere by the ragweed represents a question of public health in several European countries. In Italy, the ragweed is mostly distributed in the North. In our region (Emilia Romagna), the presence of ragweed was not described yet if not occasionally, but this plant is thriving well in the North of the Po river. The aim of our study was to estimate the concentration trends of ragweed pollens in the air of Parma starting from 1992 until 2008 and to describe the clinical related situation. The aerobiological surveillance was made with the methods standardized by the Italian Association of Aerobiology. We analyzed 19,468 outpatients affected by respiratory disease. The patients studied address our clinical Center, mainly with pathologies respiratory, most of them with allergic origin. To detect the existence of significant trends and correlations since, we used the non-parametric tests with SPSS software. Our observations showed that since 1995, the year until when pollens of ragweed were only sporadically observed in the air of Parma, there has been a significant increase in ragweed pollens. Among the patients addressed at our clinical Center, 876 patients had positive SPT (skin prick test) for ragweed pollen with respiratory illnesses, all polysensitized. Besides, we found a significant increase in patients with positive SPT for the ragweed, and among these, the increase in the asthma has been significant. On the basis of our results, we expect, in the absence of intervention from public authorities, a more significant increase in the positive subjects and an aggravation of the symptoms related to the presence of ragweed pollen in the air of Parma.     

Impact of nightshift work on overweight and abdominal obesity among workers of a poultry processing plant in southern Brazil

The authors examined the associations of shiftwork with overweight and abdominal obesity through a cross-sectional study of 1206 employees 18 to 50 yrs of age who were working on a production line in a poultry processing plant. Night-shift workers (n = 800) were considered exposed, whereas day shiftworkers (n = 406) were considered nonexposed. Overweight was defined as a body mass index ≥ 25 kg/m(2) and abdominal obesity as a waist circumference ≥ 88 cm in women and ≥ 102 cm in men. The mean age of the workers was 30.5 yrs (standard deviation = 8.7 yrs), and 65.2% were women. Nightshift workers compared to dayshift workers showed higher prevalences of overweight (42.2% vs. 34.3%; p=?.020) and abdominal obesity (24.9% vs. 19.5%; p =?.037). After adjusting for sociodemographics, parental overweight status, behavioral characteristics, and sleep characteristics, including hours of sleep, the prevalence ratios for overweight and abdominal obesity were 1.27 (95% confidence interval [ CI]: 1.00-1.61) and 1.45 (95% CI: 1.10-1.92), respectively, for the nightshift workers compared to the dayshift workers. A consistent finding in our study was the independent contribution of night shiftwork to overweight and abdominal obesity among Brazilian workers. Further studies are needed to understand the biological mechanisms involved and the complex behavioral and social adaptations experienced by night-shift workers.     

An old enzyme for current needs: adenosine deaminase and a dendritic cell vaccine for HIV

After nearly three decades of searching for a vaccine against HIV, a cure for this pandemic disease still remains elusive. The low immunogenicity of the surface proteins and the huge variability of the virus, together with the immunocompromised status of the host, have made developing an HIV vaccine an uphill battle. Over the past few years, both immunogen design and immunization strategies have improved, providing hope for future, although the anti-HIV responses achieved still remain modest. As developing a prophylactic vaccine seems unlikely nowadays, efforts have focused on alternative therapeutic immunization approaches, although these still need to be further optimized. Using an immunomodulator capable of restoring immune function in the context of infection, thereby boosting cell-mediated and humoral responses, could be critical in effectively improving current therapeutic approaches. Adenosine deaminase, a protein with a pivotal role in T-cell co-stimulation, has been shown to robustly enhance specific T-cell responses against HIV in vitro. Although its role in humoral responses has not yet been assessed, genetic defects in this enzyme are associated with impaired cellular and humoral responses. Importantly, this molecule is already commercially available pharmaceutically and, therefore, it fulfils all the requirements to be assayed as an anti-HIV vaccine adjuvant.     

Contribution of lateral gene transfer to the gene repertoire of a gut-adapted methanogen

Methanobrevibacter smithii is the most abundant archaeon in the human colon. As most of its neighbors are bacterial species, it is expected that lateral gene acquisition from bacteria might have contributed to the evolution and adaptation of this archaeon. We performed a tree-based genome-wide survey of putative lateral gene transfer products in M. smithii, using a phylogenetic pipeline. Over 15% of the coding genes of M. smithii are inferred to be bacterial in origin, based on this analysis. Laterally acquired genes have had the largest contribution to surface functions, and encode glycosyl-transferases and adhesin-like proteins. In addition, several important ABC transporters, especially metal transporters are of bacterial origin. Thus, bacterial genes contributed to the host-adaptation by allowing a larger variety of surface structures and increasing the efficiency of metal ion uptake in the competitive gut niche.     

Development aggravates the severity of skeletal muscle catabolism induced by endotoxemia in neonatal pigs

Accretion rates of muscle protein are elevated in normal neonates, but this anabolic drive decreases with maturation. As this change occurs, it is not known whether development also influences muscle protein catabolism induced by sepsis. We hypothesize that protein degradation in skeletal muscle induced by endotoxemia becomes more severe as the neonate develops. Fasted 7- and 26-day-old pigs were infused for 8 h with LPS (0 and 10 μg·kg(-1)·h(-1)), while plasma amino acids (AA), 3-methylhistidine (3-MH), and α-actin concentrations and muscle protein degradation signal activation were determined (n = 5-7/group/age). Plasma full-length α-actin was greater in 7- than 26-day-old pigs, suggesting a higher baseline protein turnover in neonatal pigs. LPS increased plasma total AA, 3-MH, and full-length and cleaved α-actin in 26- than in 7-day-old pigs. In muscle of both age groups, LPS increased AMPK and NF-κB phosphorylation, the abundances of activated caspase 3 and E-3 ligases MuRF1 and atrogin1, as well as the abundance of cleaved α-actin, suggesting activation of muscle proteolysis by endotoxin in muscle. LPS decreased Forkhead box 01 (Fox01) and Fox04 phosphorylation and increased procaspase 3 abundance in muscle of 26-day-old pigs despite the lack of effect of LPS on PKB phosphorylation. The results suggest that skeletal muscle in healthy neonatal pigs maintains high baseline degradation signal activation that cannot be enhanced by endotoxin, but as maturation advances, the effect of LPS on muscle protein catabolism manifests its severity.     

Phosphodiesterase type-2 and NO-dependent S-nitrosylation mediate the cardioinhibition of the antihypertensive catestatin

The chromogranin A (CHGA)-derived peptide catestatin (CST: hCHGA(352-372)) is a noncompetitive catecholamine-release inhibitor that exerts vasodilator, antihypertensive, and cardiosuppressive actions. We have shown that CST directly influences the basal performance of the vertebrate heart where CST dose dependently induced a nitric oxide-cGMP-dependent cardiosuppression and counteracted the effects of adrenergic stimulation through a noncompetitive antagonism. Here, we sought to determine the specific intracardiac signaling activated by CST in the rat heart. Physiological analyses performed on isolated, Langendorff-perfused cardiac preparations revealed that CST-induced negative inotropism and lusitropism involve β(2)/β(3)-adrenergic receptors (β(2)/β(3)-AR), showing a higher affinity for β(2)-AR. Interaction with β(2)-AR activated phosphatidylinositol 3-kinase/endothelial nitric oxide synthase (eNOS), increased cGMP levels, and induced activation of phosphodiesterases type 2 (PDE2), which was found to be involved in the antiadrenergic action of CST as evidenced by the decreased cAMP levels. CST-dependent negative cardiomodulation was abolished by functional denudation of the endothelium with Triton. CST also increased the eNOS expression in cardiac tissue and human umbilical vein endothelial cells. cells, confirming the involvement of the vascular endothelium. In ventricular extracts, CST increased S-nitrosylation of both phospholamban and β-arrestin, suggesting an additional mechanism for intracellular calcium modulation and β-adrenergic responsiveness. We conclude that PDE2 and S-nitrosylation play crucial roles in the CST regulation of cardiac function. Our results are of importance in relation to the putative application of CST as a cardioprotective agent against stress, including excessive sympathochromaffin overactivation.