Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.     

Amino terminal tyrosine phosphorylation of human MIXL1

Seven members of the Mix family of paired-type homeoproteins regulate mesoderm/endoderm differentiation in amphibians. In mammals, the MIXL1 (Mix. 1 homeobox [Xenopus laevis]-like gene 1) gene is the sole representative of this family. Unlike the amphibian Mix genes that encode an open reading frame of >300 amino acids, mammalian MIXL1 encodes a smaller protein (~230aa). However, mammalian MIXL1 contains a unique proline-rich domain (PRD) with a potential to interact with signal transducing Src homolgy 3 (SH3) domains. Notably, human MIXL1 also contains a unique tyrosine residue Tyr20 that is amino-terminal to the PRD. Here we report that mammalian MIXL1 protein is phosphorylated at Tyr20 and the phosphorylation is dramatically reduced in the absence of PRD. Our findings are consistent with Tyr20 phosphorylation of MIXL1 being a potential regulatory mechanism that governs its activity.     

Tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor-kappaB and degradation of MyoD protein

In this study we have investigated the effect and the mechanisms by which tumor necrosis factor-like weak inducer of apoptosis (TWEAK) modulates myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK inhibited their differentiation evident by a decrease in the expression of creatine kinase, myosin heavy chain-fast twitch, myogenin, and the formation of multinucleated myotubes. TWEAK also inhibited the differentiation of mouse primary myoblasts. Conversely, the proliferation of C2C12 myoblasts and the expression of a cell-cycle regulator cyclin D1 were increased in response to TWEAK treatment. Inhibition of cellular proliferation using hydroxyurea only partially reversed the inhibitory effect of TWEAK on myogenic differentiation. Treatment of C2C12 myoblasts with TWEAK resulted in the activation of nuclear factor-kappaB (NF-kappaB), the (IkappaB) IkappaB kinase (IKK) complex, and the phosphorylation and degradation of IkappaBalpha protein. Inhibition of NF-kappaB activity by overexpression of a dominant negative mutant of IkappaBalpha (IkappaBalphaDeltaN) significantly increased the myogenic differentiation in TWEAK-treated C2C12 cultures. Furthermore, overexpression of a dominant negative mutant of IKKbeta (IKKbetaK44A) but not IKKalpha (IKKalphaK44M) reversed the inhibitory effect of TWEAK on myogenesis. TWEAK inhibited the expression of myogenic regulatory factors MyoD and myogenin and also induced the degradation of MyoD protein. Finally, inhibition of NF-kappaB activation through overexpression of IKKbetaK44A prevented the degradation of MyoD protein. Overall, our data suggest that TWEAK inhibits myogenesis through the activation of NF-kappaB signaling pathway and degradation of MyoD protein.     

Solution structure of human von Willebrand factor studied using small angle neutron scattering

von Willebrand factor (VWF) binding to platelets under high fluid shear is an important step regulating atherothrombosis. We applied light and small angle neutron scattering to study the solution structure of human VWF multimers and protomer. Results suggest that these proteins resemble prolate ellipsoids with radius of gyration (R(g)) of approximately 75 and approximately 30 nm for multimer and protomer, respectively. The ellipsoid dimensions/radii are 175 x 28 nm for multimers and 70 x 9.1 nm for protomers. Substructural repeat domains are evident within multimeric VWF that are indicative of elements of the protomer quarternary structure (16 nm) and individual functional domains (4.5 nm). Amino acids occupy only approximately 2% of the multimer and protomer volume, compared with 98% for serum albumin and 35% for fibrinogen. VWF treatment with guanidine.HCl, which increases VWF susceptibility to proteolysis by ADAMTS-13, causes local structural changes at length scales <10 nm without altering protein R(g). Treatment of multimer but not protomer VWF with random homobifunctional linker BS(3) prior to reduction of intermonomer disulfide linkages and Western blotting reveals a pattern of dimer and trimer units that indicate the presence of stable intermonomer non-covalent interactions within the multimer. Overall, multimeric VWF appears to be a loosely packed ellipsoidal protein with non-covalent interactions between different monomer units stabilizing its solution structure. Local, and not large scale, changes in multimer conformation are sufficient for ADAMTS-13-mediated proteolysis.     

Biomechanics of P-selectin PSGL-1 bonds: shear threshold and integrin-independent cell adhesion

Platelet-leukocyte adhesion may contribute to thrombosis and inflammation. We examined the heterotypic interaction between unactivated neutrophils and either thrombin receptor activating peptide (TRAP)-stimulated platelets or P-selectin-bearing beads (Ps-beads) in suspension. Cone-plate viscometers were used to apply controlled shear rates from 14 to 3000/s. Platelet-neutrophil and bead-neutrophil adhesion analysis was performed using both flow cytometry and high-speed videomicroscopy. We observed that although blocking antibodies against either P-selectin or P-selectin glycoprotein ligand-1 (PSGL-1) alone inhibited platelet-neutrophil adhesion by approximately 60% at 140/s, these reagents completely blocked adhesion at 3000/s. Anti-Mac-1 alone did not alter platelet-neutrophil adhesion rates at any shear rate, though in synergy with selectin antagonists it abrogated cell binding. Unstimulated neutrophils avidly bound Ps-beads and activated platelets in an integrin-independent manner, suggesting that purely selectin-dependent cell adhesion is possible. In support of this, antagonists against P-selectin or PSGL-1 caused dissociation of previously formed platelet-neutrophil and Ps-bead neutrophil aggregates under shear in a variety of experimental systems, including in assays performed with whole blood. In studies where medium viscosity and shear rate were varied, a shear threshold for P-selectin PSGL-1 binding was also noted at shear rates <100/s when Ps-beads collided with isolated neutrophils. Results are discussed in light of biophysical computations that characterize the collision between unequal-size particles in linear shear flow. Overall, our studies reveal an integrin-independent regime for cell adhesion and weak shear threshold for P-selectin PSGL-1 interactions that may be physiologically relevant.     

The effects of temperature, pressure and peptide incorporation on ternary model raft mixtures--a Laurdan fluorescence spectroscopy study

Recently, an increasing evidence accumulated for the existence of lipid microdomains, called lipid rafts, in cell membranes, which may play an important role in many important membrane-associated biological processes. Suitable model systems for studying biophysical properties of lipid rafts are lipid vesicles composed of three-component lipid mixtures, such as POPC/SM/cholesterol, which exhibit a rich phase diagram, including raft-like liquid-ordered/liquid-disordered phase coexistence regions. We explored the temperature, pressure and concentration-dependent phase behavior of such canonical model raft mixtures using the Laurdan fluorescence spectroscopic technique. Hydrostatic pressure has not only been used as a physical parameter for studying the stability and energetics of these systems, but also because high pressure is an important feature of certain natural membrane environments. We show that the liquid-disordered/liquid-ordered phase coexistence regions of POPC/SM/cholesterol model raft mixtures extends over a very wide temperature range of about 50 degrees C. Upon pressurization, an overall ordered membrane state is reached at pressures of approximately 1,000 bar at 20 degrees C, and of approximately 2,000 bar at 40 degrees C. Incorporation of 5 mol% gramicidin as a model ion channel slightly increases the overall order parameter profile in the l(o)+l(d) two-phase coexistence region, probably by selectively partitioning into l(d) domains, does not change the overall phase behavior, however. This behavior is in contrast to the effect of the peptide incorporation into simple, one-component phospholipid bilayer systems.     

Intake rates and the functional response in shorebirds (Charadriiformes) eating macro-invertebrates

As field determinations take much effort, it would be useful to be able to predict easily the coefficients describing the functional response of free-living predators, the function relating food intake rate to the abundance of food organisms in the environment. As a means easily to parameterise an individual-based model of shorebird Charadriiformes populations, we attempted this for shorebirds eating macro-invertebrates. Intake rate is measured as the ash-free dry mass (AFDM) per second of active foraging; i.e. excluding time spent on digestive pauses and other activities, such as preening. The present and previous studies show that the general shape of the functional response in shorebirds eating approximately the same size of prey across the full range of prey density is a decelerating rise to a plateau, thus approximating the Holling type II ('disc equation') formulation. But field studies confirmed that the asymptote was not set by handling time, as assumed by the disc equation, because only about half the foraging time was spent in successfully or unsuccessfully attacking and handling prey, the rest being devoted to searching.A review of 30 functional responses showed that intake rate in free-living shorebirds varied independently of prey density over a wide range, with the asymptote being reached at very low prey densities (<150/m-2). Accordingly, most of the many studies of shorebird intake rate have probably been conducted at or near the asymptote of the functional response, suggesting that equations that predict intake rate should also predict the asymptote.A multivariate analysis of 468 'spot' estimates of intake rates from 26 shorebirds identified ten variables, representing prey and shorebird characteristics, that accounted for 81% of the variance in logarithm-transformed intake rate. But four-variables accounted for almost as much (77.3%), these being bird size, prey size, whether the bird was an oystercatcher Haematopus ostralegus eating mussels Mytilus edulis, or breeding. The four variable equation under-predicted, on average, the observed 30 estimates of the asymptote by 11.6%, but this discrepancy was reduced to 0.2% when two suspect estimates from one early study in the 1960s were removed. The equation therefore predicted the observed asymptote very successfully in 93% of cases. We conclude that the asymptote can be reliably predicted from just four easily measured variables. Indeed, if the birds are not breeding and are not oystercatchers eating mussels, reliable predictions can be obtained using just two variables, bird and prey sizes. A multivariate analysis of 23 estimates of the half-asymptote constant suggested they were smaller when prey were small but greater when the birds were large, especially in oystercatchers. The resulting equation could be used to predict the half-asymptote constant, but its predictive power has yet to be tested. As well as predicting the asymptote of the functional response, the equations will enable research workers engaged in many areas of shorebird ecology and behaviour to estimate intake rate without the need for conventional time-consuming field studies, including species for which it has not yet proved possible to measure intake rate in the field.     

Cytokine responses of bovine macrophages to diverse clinical Mycobacterium avium subspecies paratuberculosis strains

Background  

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease (JD) persistently infects and survives within the host macrophages. While it is established that substantial genotypic variation exists among MAP, evidence for the correlates that associate specific MAP genotypes with clinical or sub-clinical disease phenotypes is presently unknown. Thus we studied strain differences in intracellular MAP survival and host responses in a bovine monocyte derived macrophage (MDM) system.     

Isolation and characterization of bioactive compounds of Clematis gouriana Roxb. ex DC against snake venom phospholipase A2 (PLA2) computational and in vitro insights

Bioactive compounds were isolated from Clematis gouriana Roxb. ex DC. The compounds were separated, characterized, the structures elucidated and submitted to the PubChem Database. The PubChem Ids SID 249494134 and SID 249494135 were tested against phospholipases A₂ (PLA₂) of Naja naja (Indian cobra) venom for PLA₂ activity. Both the compounds showed promising inhibitory activity; computational data also substantiated the results. The two compounds underwent density functional theory calculation to observe the chemical stability and electrostatic potential profile. Molecular interactions between the compounds and PLA₂ were observed at the binding pocket of the PLA₂ protein. Further, this protein–ligand complexes were simulated for a timescale of 100 ns of molecular dynamics simulation. Experimental and computational results showed significant PLA₂ inhibition activity.