Schistosoma japonicum: a method for transformation by electroporation

Despite of our knowledge of genetic make up of schistosomes, a number of genes have not been characterized largely due to lack of effective transformation protocols. Here we present electroporation as a strategy for effective introduction of plasmids DNA into schistosomula and adults. Using plasmids of pEGFP-C1 as an expression vector, we first verified that the CMV promoter could direct EGFP to express in primary culture cells from Schistosoma japonicum. Subsequently, the plasmids were introduced into schistosomula and adults by electroporation and EGFP expression was demonstrated using molecular and microscopical methods. Our findings indicate that electroporation is an effective method for transformation of S. japonicum.     

Proteasome inhibition and Tau proteolysis: an unexpected regulation

Increasing evidence suggests that an inhibition of the proteasome, as demonstrated in Parkinson's disease, might be involved in Alzheimer's disease. In this disease and other Tauopathies, Tau proteins are hyperphosphorylated and aggregated within degenerating neurons. In this state, Tau is also ubiquitinated, suggesting that the proteasome might be involved in Tau proteolysis. Thus, to investigate if proteasome inhibition leads to accumulation, hyperphosphorylation and aggregation of Tau, we used neuroblastoma cells overexpressing Tau proteins. Surprisingly, we showed that the inhibition of the proteasome led to a bidirectional degradation of Tau. Following this result, the cellular mechanisms that may degrade Tau were investigated.     

Proteomic analysis of Gal/GalNAc lectin-associated proteins in Entamoeba histolytica

Contact-dependent killing and phagocytosis of target cells by Entamoeba histolytica trophozoites is mediated by the galactose (Gal) and N-acetyl-d-galactosamine (GalNAc)-inhibitable lectin. Previous work has suggested that this lectin functions as part of a signal transduction complex. To identify proteins that might be part of this complex, amebic trophozoites were bound to GalNAc-BSA-labeled magnetic beads and lysed. Bound proteins were eluted from the beads and analyzed by tandem mass spectrometry. Along with the Gal/GalNAc lectin subunits, several cytoskeletal proteins, potential signaling proteins, and a novel transmembrane protein, consistently purified with the GalNAc-BSA beads.     

Influence of hydrophobic matching on association of model transmembrane fragments containing a minimised glycophorin A dimerisation motif

The principles that govern the folding and packing of membrane proteins are still not completely understood. In the present work, we have revisited the glycophorin A (GpA) dimerisation motif that mediates transmembrane (TM) helix association, one of the best-suited models of membrane protein oligomerisation. By using artificial polyleucine TM segments we have demonstrated in this study that a pattern of only five amino acids (GVxxGVxxT) promotes specific dimerisation. Further, we have used this minimised GpA motif to assess the influence of hydrophobic matching on the TM helix packing process in detergent micelles and found that this factor modulates helix-helix association and/or dissociation between TM fragments.     

The cell surface receptor G6b, a member of the immunoglobulin superfamily, binds heparin

The G6b gene, located in the human Major Histocompatibility Complex, encodes a receptor of the immunoglobulin (Ig) superfamily. In this study, we show using a variety of techniques that the extracellular domain of the G6b protein, containing a single Ig-like domain, binds to heparin with high affinity. In an ELISA assay, this binding was displaceable with soluble heparin with an IC50 value of approximately 0.5 microg/ml. Other sulfated glycans showed weaker or no competition. The observed interaction between G6b and heparin is strongly salt dependent suggesting a mainly electrostatic interaction. Heparin might modulate the interaction of G6b with its as yet unidentified protein ligand.     

Dissection of Escherichia coli glutamate 5-kinase: functional impact of the deletion of the PUA domain

Glutamate 5-kinase (G5K) catalyzes the controlling first step of the synthesis of the osmoprotective amino acid proline, which feed-back inhibits G5K. Microbial G5K generally consists of one amino acid kinase (AAK) and one PUA (named after pseudo uridine synthases and archaeosine-specific transglycosylases) domain. To investigate the role of the PUA domain, we have deleted it from Escherichia coli G5K. We show that wild-type G5K requires free Mg for activity, it is tetrameric, and it aggregates to higher forms in a proline-dependent way. G5K lacking the PUA domain remains tetrameric, active, and proline-inhibitable, but the Mg requirement and the proline-triggered aggregation are greatly diminished and abolished, respectively, and more proline is needed for inhibition. We propose that the PUA domain modulates the function of the AAK domain, opening the way to potential PUA domain-mediated regulation of G5K; and that this domain moves, exposing new surfaces upon proline binding.     

Blastocladiella emersonii expresses a centrin similar to Chlamydomonas reinhardtii isoform not found in late-diverging fungi

Centrins are members of the calcium-binding EF-hand protein superfamily which can be divided into two subfamilies, probably associated with different functions: one related to Chlamydomonas reinhardtii centrin, CrCenp, and the other, represented by Saccharomyces cerevisiae isoform, ScCdc31p. ESTs encoding the two isoforms (BeCen1 and BeCen3) from the chytridiomycete Blastocladiella emersonii were isolated, and expression of the CrCenp-type centrin, BeCen1, was analyzed throughout the fungus life cycle. Becen1 mRNA levels increase transiently during sporulation and protein levels present a similar pattern. Immunolocalization studies seem to localize BeCen1 at the basal body zone and in the cytoplasm surrounding the nuclear cap, a zoospore organelle.     

PrP(106-126) activates neuronal intracellular kinases and Egr1 synthesis through activation of NADPH-oxidase independently of PrPc

Prion diseases are characterised by severe neural lesions linked to the presence of an abnormal protease-resistant isoform of cellular prion protein (PrPc). The peptide PrP(106-126) is widely used as a model of neurotoxicity in prion diseases. Here, we examine in detail the intracellular signalling cascades induced by PrP(106-126) in cortical neurons and the participation of PrPc. We show that PrP(106-126) induces the activation of subsets of intracellular kinases (e.g., ERK1/2), early growth response 1 synthesis and induces caspase-3 activity, all of which are mediated by nicotinamide adenine dinucleotide phosphate hydrogen-oxidase activity and oxidative stress. However, cells lacking PrPc are similarly affected after peptide exposure, and this questions the involvement of PrPc in these effects.     

Formation and destabilization of actin filaments with tetramethylrhodamine-modified actin

Actin labeling at Cys(374) with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics.     

Identification of Eggshell Membrane Proteins and Purification of Ovotransferrin and β-NAGase from Hen Egg White

Exposure of selected Gram-positive and Gram-negative bacterial pathogens to egg shell membranes (ESM) significantly reduced their thermal resistance and/or inactivated cells. Although the components responsible for this antibacterial activity have not been conclusively identified, several proteins associated with the ESM activity have been identified including β-N-acetylglucosaminidase, lysozyme and ovotransferrin, with each displaying varying degrees of antibacterial activity. Numerous attempts to purify active fractions of β-N-acetylglucosaminidase, lysozyme and ovotransferrin from the ESM proved somewhat limited; however, hen egg white (HEW) β-N-acetylglucosaminidase was purified using a two-step chromatographic procedure, isoelectric focusing followed by cation exchange chromatography. Pure fractions of ovotransferrin were also obtained in the process. SDS-PAGE electrophoresis and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry were then used to partially characterize the individual protein components. Purified protein fractions such as these will be required in order to fully elucidate the mechanism responsible for the antimicrobial properties associated with the ESM.