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.     

Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro

Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases. This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma. The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity. With E. coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites. The addition of E. coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis. Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587. These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.     

Distribution and innervation of short, interdigitated muscle fibers in parallel-fibered muscles of the cat hindlimb

The cat hindlimb contains several long, biarticular strap muscles composed of parallel muscle fascicles that attach to short tendons. Three of these muscles--sartorius, tenuissimus, and semitendinosus--were studied by dissecting individual gold-stained fibers and determining the surface distribution of acetylcholinesterase-stained end-plate zones. In each muscle, fascicles were composed of muscle fibers that ran only part of the fascicle length and tapered to end as fine strands that interdigitated with other tapering fibers within the muscle mass. Most muscle fibers measured 2-3 cm in length. Fascicles of muscle fibers were crossed by short transverse bands of endplates (1 mm wide by 1-5 mm long) that were spaced at fairly regular intervals from the origin to the insertion of the muscle. The endplate pattern suggested that the fiber fascicles were organized into multiple longitudinal strips. In the sartorius, the temporospatial distribution of electromyographic (EMG) activity evoked by stimulating fine, longitudinal branches of the parent nerve confirmed that each strip was selectively innervated by a small subset of the motor axons. These axons appeared to distribute their endings throughout the entire length of the fascicles, providing for synchronous activation of their in-series fibers.     

Response of microbial adhesives and biofilm matrix polymers to chemical treatments as determined by interference reflection microscopy and light section microscopy.

The polymers involved in the adhesion of Pseudomonas fluorescens H2S to solid surfaces were investigated to determine whether differences between cell surface adhesives and biofilm matrix polymers could be detected. Two optical techniques, i.e., interference reflection microscopy (IRM) and light section microscopy (LSM), were used to compare the responses of the two types of polymer to treatment with electrolytes, dimethyl sulfoxide (DMSO), and Tween 20. To evaluate initial adhesive polymers, P. fluorescens H2S cells were allowed to attach to glass cover slip surfaces and were immediately examined with IRM, and their response to chemical solutions was tested. With IRM, changes in cell-substratum separation distance between 0 and ca. 100 nm are detectable as changes in relative light intensity of the image; a contraction of the polymer would be detected as a darkening of the image, whereas expansion would appear as image brightening. To evaluate the intercellular polymer matrix in biofilms, 3-day-old biofilms were exposed to similar solutions, and the resultant change in biofilm thickness was measured with LSM, which measures film thicknesses between 10 and 1,000 microns. The initial adhesive and biofilm polymers were similar in that both appeared to contract when treated with electrolytes and to expand when treated with Tween 20. However, with DMSO treatment, the initial adhesive polymer appeared to contract, whereas there was no change in thickness of the biofilm polymer. These results indicate that both polymers bear acidic groups and thus act electrostatically with cations and are able to enter into hydrophobic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The association of thymidine kinase activity and thymidine transport in Escherichia coli

We have constructed a series of mutants within the putative nucleoside-binding site of the herpes simplex type-1 virus (HSV-1) thymidine kinase (TK)-encoding gene (tk), contained within an expression vector. While most mutations within this sequence produce an inactive protein, we find no absolute requirement for the wild-type Ile166 and Ala167. The uptake of thymidine (dT) into Escherichia coli tdk-, lacking functional endogenous TK activity, is proportional to the amount of TK activity expressed from the heterologous HSV-1 tk gene. In contrast, there is no enhancement in deoxycytidine uptake into E. coli producing (HSV-1) TK. These results imply a specific role for TK in the active transport of dT into E. coli.     

Ease of removal of barnacles from various polymeric materials

The forces required to remove living, fully attached barnacles from the surface of a number of polymeric solids were measured. The forces were related to the surface energy components of the materials. The results indicate a positive correlation between polymer surface energy and force required for barnacle removal. The nonpolar component of the surface energy was more closely related to the removal force than the polar component, although the polar component is significant. Adherence to some composite materials was greater than was consistent with the correlation for noncomposites.     

On the fidelity of DNA replication. Lack of primer position effect on the fidelity of mammalian DNA polymerases

Mechanisms for the fidelity of DNA replication in eucaryotes are not adequately understood. Certain hypotheses can be tested by examining whether the first nucleotide inserted is incorporated with a significantly higher error rate than subsequent nucleotides. Using synthetic oligodeoxynucleotides, we have measured the effect of primer position on single-base misinsertion frequencies at an amber site in phi X174 DNA. Our results show a lack of position effect, indicating that processivity and the most direct "energy relay" proofreading mechanisms are not important determinants in eucaryotic replication fidelity.