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.     

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.     

Structural and electronic properties of the liver fluke hemoglobin heme cavity by nuclear magnetic resonance: hemin isotope labeling

Reconstitution of liver fluke (Dicrocoelium dendriticum) apo-hemoglobin with hemins selectively deuterated at specific positions has permitted the assignment of several heme resonances in the proton nuclear magnetic resonance spectrum of the Met-aquo and Met-cyano forms of the holoprotein. It was established that in the Met-aquo form the meso protons resonate at positions characteristic of a six-co-ordinated in-plane iron. From this, we deduced that the Met-aquo species retains a bound water molecule at pH values as low as 4.5. The orientation of the proximal histidine imidazole ring with respect to the heme group in the cavity was determined through the identification of the heme methyl signals and the analysis of the hyperfine shift pattern in the Met-cyano hemoglobin proton nuclear magnetic resonance spectrum. Compared to sperm whale myoglobin, the heme appears to be rotated by 180 degrees about the alpha, gamma meso-axis. Protein isomers with the heme group in a reversed orientation were not detected, even shortly after reconstitution. In the Met-cyano form, the resonances most affected by the Bohr transition were shown to arise from the heme propionates.     

NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements

The 1H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid----alkaline transition with pK approximately 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by 1H NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in Aplysia Mb; approximately 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between -16 and -35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shown to be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T-1 Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant S = 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme alpha-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfine shift values for assigned resonances, the NOE connectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotational mobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mnemonic aspects of Escherichia coli DNA polymerase I. Interaction with one template influences the next interaction with another template

When Escherichia coli DNA polymerase I (Pol I) replicates a homopolymer, the excision/polymerization (exo/pol) ratio varies with enzyme and initiator concentration. The study of this effect in the case of poly(dA).oligo(dT) replication led us to propose a mnemonic model for Pol I, in which the 3' to 5' excision activity warms up when the enzyme is actively polymerizing, and cools down when it dissociates from the template. The model predicts that the exo/pol ratio must increase with processivity length and initiator concentration and decrease with enzyme concentration. It predicts also that contact of the enzyme with one template alters its excision efficiency towards another template. The exo/pol ratio and processivities of Pol I and its Klenow fragment were studied on four templates: poly(dA).(dT)10, poly(dT).(dA)10, poly(dC).(dG)10 and poly(dI).(dC)10. We show that the Klenow fragment is usually much less processive than Pol I and when this is the case it has a much lower exo/pol ratio. At equal processivity, the exo/pol ratios are nearly equal. Furthermore, many factors that influence processivity length (e.g. manganese versus magnesium, inorganic pyrophosphate, ionic strength) influence the exo/pol ratio in the same direction. The study of deaminated poly(dC) replication, where we followed incorporation and excision of both G and A residues, allowed us to assign the origin of the dNMP variations to changes in the 3' to 5' proof-reading activity of Pol I. Similarly, the lower dNMP turnover of the Klenow fragment observed with deaminated poly(dC) was specifically assigned to a decreased 3' to 5' exonuclease activity. The exo/pol ratio generally increased with initiator and decreased with enzyme concentration, in agreement with the model, except for poly(dI).oligo(dC), where it decreased with initiator concentration. However, by terminating chain elongation with dideoxy CTP, we showed directly that, even in this system, excision is relatively inefficient at the beginning of synthesis. Interaction of Pol I with poly(dA).(dT) or with poly(dC).(dG) modifies its exo/pol characteristics in the replication of poly(dI).(dC) and poly(dA).(dT), respectively. The Klenow enzyme is not sensitive to such influences and this correlates with its reduced processivity on the influencing templates. Our results reveal the existence of differences between Pol I and its Klenow fragment that are more profound than has been thought previously.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prekallikrein and kininogens in blister fluids in burn patients

Blister fluids removed from benign cutaneous burns contain the components of the prekallikrein-kininogens system in a non activated state. Their occurrence in the fluids is explained by a diffusion from the blood plasma.     

Abnormal electrophoretic mobility of spectrin tetramers in hereditary elliptocytosis

Summary Hereditary elliptocytosis (HE) is a genetically determined disorder of the red cell membrane. The main protein which composes the proteinaceous skeleton of the membrane is an elongated molecule named spectrin which is a heterodimer composed of two chains, and . In the membrane spectrin dimers are associated head-to-head to form tetrameric structures. We and other authors have reported that spectrin studied from many HE patients exhibited a dimer self-association defect (type I HE). A mutation in the head of the spectrin chain was mostly found in type I HE. We have previously described one of the three known spectrin pathological variants shown on mild tryptic digest pattern. This variant was characterized by the appearance of an abnormal 65,000-dalton peptide (Sp ^I/65). Using nondenaturating gel electrophoresis, we describe in this paper a triplicated pattern of the spectrin tetramer bands which is found in heterozygous HE cases displaying the 65,000-dalton variant. Study of a homozygous case allowed us to characterize the electrophoretic mobility of the abnormal symmetrical spectrin tetramer (₂ ^I/65-₂) and to study the correlation between the fraction of this abnormal symmetrical tetramer found in heterozygous patients and the amount of the 65,000-dalton peptide observed in spectrin tryptic digests.     

Selective inactivation of the 3'' to 5'' exonuclease activity of Escherichia coli DNA polymerase I by heat

Heat selectively inactivates the 3' to 5' exonuclease activity of E. coli DNA polymerase I, resulting in reduced dNTP turnover and lower fidelity of replication of homopolymer and natural DNA templates.