A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell

Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37 kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.     

Genotype-specific interactions and the trade-off between host and parasite fitness

Background  

Evolution of parasite traits is inextricably linked to their hosts. For instance one common definition of parasite virulence is the reduction in host fitness due to infection. Thus, traits of infection must be viewed in both protagonists and may be under shared genetic and physiological control. We investigated these questions on the oomycete Hyaloperonospora arabidopsis (= parasitica), a natural pathogen of the Brassicaceae Arabidopsis thaliana.     

Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3��-Terminal Exon

Alternative splicing of 3′-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3′-untranslated regions that regulate their fate and their expression. The Xenopus α-tropomyosin pre-mRNA possesses a composite internal/3′-terminal exon (exon 9A9′) that is differentially processed depending on the embryonic tissue. Exon 9A9′ is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3′-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9′ as a 3′-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9′ in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9′ as a 3′-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3′-end processing of the α-tropomyosin pre-mRNA.     

The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

Purification and properties of factor XIII from human placenta.

1. FXIII was isolated and purified over 4000 fold from human placenta to apparent electrophoretic homogeneity by a new procedure including ethanol precipitation. DEAE-Cellulose, molecular sieving on Sephacryl S-300 and Phenyl-Sepharose chromatography. 2. Its pI was about 5.1. Under appropriate conditions, the incubation of FXIII in the presence of thrombin did not lead to inactivation cut in the polypeptidic chain. 3. FXIII was also activated by CaCl2 and, in a lesser extent, by other divalent cations like SrCl2, BaCl2 or MgCl2. 4. The binding of calcium to FXIII exhibited a negative cooperativity. 5. The activity-pH curve of the calcium-activated enzyme did not appear very different from that of the thrombin-activated enzyme.     

RGD Surface Functionalization of the Hydrophilic Acrylic Intraocular Lens Material to Control Posterior Capsular Opacification

Posterior Capsular Opacification (PCO) is the capsule fibrosis developed on implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LECs) undergoing Epithelial Mesenchymal Transition (EMT). Literature has shown that the incidence of PCO is multifactorial including the patient''s age or disease, surgical technique, and IOL design and material. Reports comparing hydrophilic and hydrophobic acrylic IOLs have shown that the former has more severe PCO. On the other hand, we have previously demonstrated that the adhesion of LECs is favored on hydrophobic compared to hydrophilic materials. By combining these two facts and contemporary knowledge in PCO development via the EMT pathway, we propose a biomimetically inspired strategy to promote LEC adhesion without de-differentiation to reduce the risk of PCO development. By surface grafting of a cell adhesion molecule (RGD peptide) onto the conventional hydrophilic acrylic IOL material, the surface-functionalized IOL can be used to reconstitute a capsule-LEC-IOL sandwich structure, which has been considered to prevent PCO formation in literature. Our results show that the innovative biomaterial improves LEC adhesion, while also exhibiting similar optical (light transmittance, optical bench) and mechanical (haptic compression force, IOL injection force) properties compared to the starting material. In addition, compared to the hydrophobic IOL material, our bioactive biomaterial exhibits similar abilities in LEC adhesion, morphology maintenance, and EMT biomarker expression, which is the crucial pathway to induce PCO. The in vitro assays suggest that this biomaterial has the potential to reduce the risk factor of PCO development.