Heme Amplifies the Innate Immune Response to Microbial Molecules through Spleen Tyrosine Kinase (Syk)-dependent Reactive Oxygen Species Generation

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.     

Estradiol-17beta-D-glucuronide induces endocytic internalization of Bsep in rats

Endocytic internalization of the multidrug resistance-associated protein 2 (Mrp2) was previously suggested to be involved in estradiol-17beta-D-glucuronide (E217G)-induced cholestasis. Here we evaluated in the rat whether a similar phenomenon occurs with the bile salt export pump (Bsep) and the ability of DBcAMP to prevent it. E217G (15 micromol/kg i.v.) impaired bile salt (BS) output and induced Bsep internalization, as assessed by confocal microscopy and Western blotting. Neither cholestasis nor Bsep internalization occurred in TR- rats lacking Mrp2. DBcAMP (20 micromol/kg i.v.) partially prevented the decrease in bile flow and BS output and substantially prevented E217G-induced Bsep internalization. In hepatocyte couplets, E217G (50 microM) diminished canalicular accumulation of a fluorescent BS and decreased Bsep-associated fluorescence in the canalicular membrane; DBcAMP (10 microM) fully prevented both effects. In conclusion, our results suggest that changes in Bsep localization are involved in E217G-induced impairment of bile flow and BS transport and that DBcAMP prevents this effect by stimulating insertion of canalicular transporter-containing vesicles. Mrp2 is required for E217G to induce its harmful effect.     

Biochemical characterization and molecular cloning of a plasminogen activator proteinase (LV-PA) from bushmaster snake venom

The protein (LV-PA) from bushmaster (Lachesis muta muta) venom is a serine proteinase which specifically activates the inactive proenzyme plasminogen. LV-PA is a single chain glycoprotein with an apparent molecular mass of 33 kDa that fell to 28 kDa after treatment with N-Glycosidase F (PNGase F). Approximately 93% of its protein sequence was determined by automated Edman degradation of various fragments derived from a digestion with trypsin. A cDNA library of L. m. muta was constructed to generate expressed sequence tags (ESTs) and the plasminogen activator precursor cDNA was sequenced. The complete amino acid sequence of the enzyme was deduced from the cDNA sequence. LV-PA is composed of 234 residues and contains a single asparagine-linked glycosylation site, Asn-X-Ser, bearing sugars that account for approximately 10% of the enzyme's total molecular mass of 33 kDa. The sequence of LV-PA is highly similar to the plasminogen activators (PAs) TSV-PA from Trimeresurus stejnegeri venom and Haly-PA from Agkistrodon halys. Furthermore, the mature protein sequence of LV-PA exhibits significant similarity with other viperidae venom serine proteinases which affect many steps of hemostasis, ranging from the blood coagulation cascade to platelet function. The Michaelis constant (Km) and the catalytic rate constant (kcat) of LV-PA on four chromogenic substrates were obtained from Lineweaver-Burk plots. In addition, we used an indirect enzyme-linked immunoabsorbent assay (ELISA) to explore the phylogenetic range of immunological cross-reactivity (using antibodies raised against LV-PA) with analogous serine proteinases from two viperidae venoms and mammals.     

Geographical Variation in Health-Related Physical Fitness and Body Composition among Chilean 8th Graders: A Nationally Representative Cross-Sectional Study

Purpose

In addition to excess adiposity, low cardiorespiratory fitness (CRF) and low musculoskeletal fitness (MSF) are important independent risk factors for future cardio-metabolic disease in adolescents, yet global fitness surveillance in adolescents is poor. The objective of this study was to describe and investigate geographical variation in levels of health-related physical fitness, including CRF, MSF, body mass index (BMI), and waist circumference (WC) in Chilean 8th graders.

Methods

This cross-sectional study was based on a population-based, representative sample of 19,929 8th graders (median age = 14 years) in the 2011 National Physical Education Survey from Chile. CRF was assessed with the 20-meter shuttle run test, MSF with standing broad jump, and body composition with BMI and WC. Data were classified according to health-related standards. Prevalence of levels of health-related physical fitness was mapped for each of the four variables, and geographical variation was explored at the country level by region and in the Santiago Metropolitan Area by municipality.

Results

Girls had significantly higher prevalence of unhealthy CRF, MSF, and BMI than boys (p<0.05). Overall, 26% of boys and 55% of girls had unhealthy CRF, 29% of boys and 35% of girls had unhealthy MSF, 29% of boys and 44% of girls had unhealthy BMI, and 31% of adolescents had unhealthy WC. High prevalence of unhealthy fitness levels concentrates in the northern and middle regions of the country and in the North and Southwest sectors for the Santiago Metropolitan Area.

Conclusion

Prevalence of unhealthy CRF, MSF, and BMI is relatively high among Chilean 8th graders, especially in girls, when compared with global estimates. Identification of geographical regions and municipalities with high prevalence of unhealthy physical fitness presents opportunity for targeted intervention.     

Toxicity,behavior impairment,and repellence of essential oils from pepper‐rosmarin and patchouli to termites

Plant essential oils are potential sources of insecticidal compounds, but have rarely been explored for their effect on termites. In the present study, we assessed the chemical composition of essential oils of Lippia sidoides Cham. (pepper‐rosmarin; Verbenaceae) and Pogostemon cablin (Blanco) Benth. (patchouli; Lamiacaeae) and evaluated their toxicity, behavioral impairment, and repellence to termite species of the genera Amitermes and Microcerotermes (Isoptera: Termitidae: Termitinae). The main components of essential oils of L. sidoides and P. cablin were thymol (44.6%) and patchouli alcohol (36.6%), respectively. The essential oil of P. cablin was most potent against Amitermes cf. amifer Silvestri and had the lowest LD₅₀ (0.63 μg mg^?1). There was no difference in toxicity for Microcerotermes indistinctus Mathews between the essential oils of L. sidoides (LD₅₀ = 1.49 μg mg^?1) and P. cablin (LD₅₀ = 1.67 μg mg^?1). Pogostemon cablin essential oil was the most toxic to M. indistinctus (LC₅₀ = 0.32 μl ml^?1) and A. cf. amifer (LC₅₀ = 0.29 μl ml^?1). The essential oils analyzed exhibited high toxicity and repellence to the termites, in addition to reducing behavioral interactions among individuals, thus constituting potential termiticides.     

Integrated Proteomics Identified Up-Regulated Focal Adhesion-Mediated Proteins in Human Squamous Cell Carcinoma in an Orthotopic Murine Model

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.     

Structure and DNA-binding properties of the Bacillus subtilis SpoIIIE DNA translocase revealed by single-molecule and electron microscopies

SpoIIIE/FtsK are a family of ring-shaped, membrane-anchored, ATP-fuelled motors required to segregate DNA across bacterial membranes. This process is directional and requires that SpoIIIE/FtsK recognize highly skewed octameric sequences (SRS/KOPS for SpoIIIE/FtsK) distributed along the chromosome. Two models have been proposed to explain the mechanism by which SpoIIIE/FtsK interact with DNA. The loading model proposes that SpoIIIE/FtsK oligomerize exclusively on SpoIIIE recognition sequence/orienting polar sequences (SRS/KOPS) to accomplish directional DNA translocation, whereas the target search and activation mechanism proposes that pre-assembled SpoIIIE/FtsK hexamers bind to non-specific DNA, reach SRS/KOPS by diffusion/3d hopping and activate at SRS/KOPS. Here, we employ single-molecule total internal reflection imaging, atomic force and electron microscopies and ensemble biochemical methods to test these predictions and obtain further insight into the SpoIIIE–DNA mechanism of interaction. First, we find that SpoIIIE binds DNA as a homo-hexamer with neither ATP binding nor hydrolysis affecting the binding mechanism or affinity. Second, we show that hexameric SpoIIIE directly binds to double-stranded DNA without requiring the presence of SRS or free DNA ends. Finally, we find that SpoIIIE hexamers can show open and closed conformations in solution, with open-ring conformations most likely resembling a state poised to load to non-specific, double-stranded DNA. These results suggest how SpoIIIE and related ring-shaped motors may be split open to bind topologically closed DNA.     

Humerus development in moles (Talpidae,Mammalia)

The humerus of fossorial moles has a highly derived anatomy, reflecting the ecological specialization of these animals for digging. It is short and broad, with enlarged muscle attachment sites and pronounced articulations compared to non‐fossorial sister taxa and other mammals. Both condyles are rotated in opposite directions, resulting in a torsion which is unique among eutherian mammals. The development of this exceptional bone was studied in embryonic stages of the fossorial Iberian mole (Talpa occidentalis) from mesenchymal condensation to incipient ossification based on histological serial sections using 3D reconstruction methods. For comparison, embryonic stages of the semi‐fossorial Japanese shrew mole (Urotrichus talpoides) as well as a sister taxon of moles, the terrestrial North American least shrew (Cryptotis parva), were studied. Results show that the humerus of Talpa already shows its derived anatomy with broadened muscle attachment sites and distinct articulations at early cartilaginous stages, when ossification has just started in the mid‐diaphyseal region. The torsion takes place simultaneously with the medial rotation of the forelimbs. The supracondylar foramen is closed in all studied Talpa embryos, but patent in Cryptotis and Urotrichus. This is an example of developmental penetrance, suggesting that variation of adult elements can be found at early stages as well.