The rhoptry neck protein RON4 re-localizes at the moving junction during Toxoplasma gondii invasion

Host cell invasion in the Apicomplexa is unique in its dependency on a parasite actin-driven machinery and in the exclusion of most host cell membrane proteins during parasitophorous vacuole (PV) formation. This exclusion occurs at a junction between host cell and parasite plasma membranes that has been called the moving junction, a circumferential zone which forms at the apical tip of the parasite, moves backward and eventually pinches the PV from the host cell membrane. Despite having been described by electron microscopic studies 30 years ago, the molecular nature of this singular structure is still enigmatic. We have obtained a monoclonal antibody that recognizes the moving junction of invading tachyzoites of Toxoplasma gondii, in a pattern clearly distinct from those described so far for microneme and rhoptry proteins. The protein recognized by this antibody has been affinity purified. Mass spectrometry analysis showed that it is a rhoptry neck protein (RON4), a hypothetical protein with homologues restricted to Apicomplexa. Our findings reveals for the first time the participation of rhoptry neck proteins in moving junction formation and strongly suggest the conservation of this structure at the molecular level among Apicomplexa.     

Inhibition of matrix metalloproteinase-1 activity by the soybean Bowman–Birk inhibitor

Inductively coupled plasma analysis of soybean Bowman-Birk inhibitor (BBI) indicated that BBI was a metalloprotein which contained magnesium, calcium, and zinc at 0.40, 0.43 and 0.008 atom/mol BBI, respectively. Heparin-enhanced gelatin zymography, quenched fluorescence substrate hydrolysis analysis, and the Biotrak assay of the interaction of BBI with the matrix metalloproteinase-1 (MMP-1) demonstrated that demineralized BBI at 30 nM inhibited MMP-1 activity whereas mineralized BBI was inhibitory at 115 nM.     

Effect of vitamin A deficiency on thymosin-β4 and CD4 concentrations

Vitamins are evaluated for their role in immunity. Recently, vitamin A received a particular attention as a critical micronutrient for regulating immune system. Therefore, the present study aimed to search for new about vitamin A. Forty-eight Egyptian adults aged from 18 to 42?years old from both sexes were subjected to clinical examination and nutrition questionnaire and were screened for vitamin A by using ELISA method. Forty subjects were selected and subdivided into two groups. Group 1 with vitamin A at level >200?µg/dl consists of 10 healthy subjects. Group 2 with vitamin A deficiency at level <50?µg/dl consists of 30 subjects. Tβ4 and CD4 levels were also determined by a commercial ELISA kit. Results showed a significant decrease in serum levels of Tβ4 and CD4 in group 2 than group 1 at P < .003 and P < .019 respectively. Both of Tβ4 and CD4 had positive correlation with vitamin A level at P < .000 and P < .003 respectively as well as with each other at p < .000. We concluded that vitamin A deficiency may be influence the levels of Tβ4 and CD4.     

Distribution and breeding ecology of the Ferruginous Duck Aythya nyroca in Algeria

We conducted a survey of the distribution of Ferruginous Duck Aythya nyroca across Algeria and analysed the influence of nest-site characteristics on nesting success at a Ramsar site, Lake Tonga. The species was found to occupy different wetlands (freshwater lakes, brackish marshes and salt lakes) across three major climatic belts (subhumid coastal strip, semi-arid Hauts Plateaux and arid Sahara). Mean clutch size of successful nests was 13.3 ± 6.0 eggs (N = 26) with a hatching rate of 74% for successful clutches. Nesting success was recorded for 44% of nests with clutch desertion (72.7%) accounting for the majority of failed clutches. Egg size of the study population, which breeds at the southern limit of the species’ range, was significantly smaller than that of its northerly counterparts. Clutch size was negatively related to egg size and positively associated with depth of water below the nest, suggesting that older, more experienced or high-quality birds monopolised the safest sites. Successful clutches were significantly associated with tall vegetation, suggesting that when nesting is carried out mainly on offshore floating islets, protection against aerial predators and heat stress determine nesting outcome. Conspecific brood parasitism was significantly and positively associated with deferred egg-laying and smaller egg size, suggesting an age-dependent mechanism.     

The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny,duplication rate and parasite virulence

Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora‐related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In Toxoplasma gondii, we show that TgArk2 and TgArk3 concentrate at specific sub‐cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock‐down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3‐depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites and highlights Aurora kinase 3 as potential drug target.     

The photo-transferred thermoluminescence phenomenon in case of emergency dose assessment

Radiation and Environmental Biophysics - A major disadvantage of dose reconstruction by means of thermoluminescence (TL) is the fact that during readout of any TL material exposed to ionizing...     

Mutational approaches to improve the biophysical properties of human single-domain antibodies

Monoclonal antibodies are a remarkably successful class of therapeutics used to treat a wide range of indications. There has been growing interest in smaller antibody fragments such as Fabs, scFvs and domain antibodies in recent years. In particular, the development of human V_H and V_L single-domain antibody therapeutics, as stand-alone affinity reagents or as “warheads” for larger molecules, are favored over other sources of antibodies due to their perceived lack of immunogenicity in humans. However, unlike camelid heavy-chain antibody variable domains (V_HHs) which almost unanimously resist aggregation and are highly stable, human V_Hs and V_Ls are prone to aggregation and exhibit poor solubility. Approaches to reduce V_H and V_L aggregation and increase solubility are therefore very active areas of research within the antibody engineering community. Here we extensively chronicle the various mutational approaches that have been applied to human V_Hs and V_Ls to improve their biophysical properties such as expression yield, thermal stability, reversible unfolding and aggregation resistance. In addition, we describe stages of the V_H and V_L development process where these mutations could best be implemented. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

The ROP2 family of Toxoplasma gondii rhoptry proteins: proteomic and genomic characterization and molecular modeling

Four rhoptry proteins (ROP) of Toxoplasma gondii previously identified with mAb have been affinity purified and analyzed by MS; the data obtained allowed the genomic sequences to be assigned to these proteins. As previously suggested for some of them by antibody crossreactivity, these proteins were shown to belong to a family, the prototype of which being ROP2. We describe here the proteins ROP2, 4, 5, and 7. These four proteins correspond to the most abundant products of a gene family that comprises several members which we have identified in genomic and EST libraries. Eight additional sequences were found and we have cloned four of them. All members of the ROP2 family contain a protein-kinase-like domain, but only some of them possess a bona fide kinase catalytic site. Molecular modeling of the kinase domain demonstrates the conservation of residues critical for the stabilization of the protein-kinase fold, especially within a hydrophobic segment described so far as transmembrane and which appears as an helix buried inside the protein. The concomitant synthesis of these ROPs by T. gondii tachyzoites suggests a specific role for each of these proteins, especially in the early interaction with the host cell upon invasion.