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.     

Cytokeratin provides a specific marker for human arachnoid cells grown in vitro

Cells from cranial and spinal arachnoid membranes of humans were grown in culture. Their growth characteristics, morphology and details of their cytoskeletal composition are described. Arachnoid membranes, obtained at autopsy, were finely minced and incubated in tissue culture medium. Monolayers of cells of homogeneous morphology grew from these tissue fragments. The cells were flat and polygonal. They divided slowly to form non-overlapping monolayers of low cell density. Electron microscopic examination of cultured arachnoid cells revealed numerous desmosome-like tight junctions and abundant intermediate filaments (tonofilaments). Both morphological features are characteristic of arachnoid cells in situ, but not of cells in the fibroblast-rich dura mater. Immunofluorescence microscopy with monoclonal antibodies demonstrated cytokeratin in the cytoplasm of primary cultures of arachnoid cells. Thus we demonstrated that these cultured cells retained certain of the specific differentiated properties of arachnoid cells in situ and that they are not fibroblasts (which lack tight junctions and cytokeratins). To our knowledge, there have been no previous reports of in vitro growth of arachnoid cells. This in vitro model should be useful in studying the response of arachnoid cells to a variety of substances thought to be involved in the chronic inflammatory condition of the meninges known as arachnoiditis.     

Metabolism and excretion of exogenous [3H]-LTC4 in primates

Four novel omega- and beta-oxidation (from the omega end) products of peptide leukotrienes, 20-hydroxy and 20-carboxy-LTE4, 18-carboxy-19, 20-dinor-LTE4 and 16-carboxy-17,18,19,20-tetranor-14,15-dihydro-LTE4 were prepared by total synthesis and used as standards for identification of biliary and urinary metabolites in the cynomolgus monkey. After intravenous administration 14, 15-[3H] leukotriene C4 (10 microCi kg-1) was partially metabolized in and rapidly cleared from the vascular circulation. This resulted, within 24 hours, in significant urinary excretion (14.8 +/- 2.1%, n = 4), consisting largely of material more polar than LTE4 (61% of urinary excretion) as shown by reverse phase HPLC. The polar fraction demonstrated two predominant metabolites which coeluted in several HPLC solvent systems with synthetic 16-carboxytetranordihydro-LTE4 (major component) and 18-carboxydinor-LTE4 (minor component). Characterization of the major polar metabolite as 16-carboxytetranordihydro-LTE4 was substantiated by conversion to its N-acetylated derivative. The absence of the 14, 15 double bond was confirmed by product analysis of oxidative ozonolysis. In a single animal, the bile duct was cannulated, with significant biliary excretion of radioactivity demonstrated over 4 hours (58.6% recovery). The predominant polar biliary metabolites were also identified as the 18-carboxydinor and 16-carboxytetranordihydro derivatives of LTE4 mentioned above. These data suggest that beta-oxidation products generated from the omega-carboxyl end of the 20-carboxy-LTE4 are important products of [3H] LTC4 metabolism in the monkey. Quantitation of these urinary metabolites may be an important index of in vivo leukotriene production.     

Transformation of Saccharomyces cerevisiae by electroporation

A method for introducing heterologous DNA into Saccharomyces cerevisiae rapidly and efficiently by electroporation was developed. Transformant colonies appeared somewhat sooner than by the LiCl or spheroplast transformation method, and the time spent in manipulation was much less than for these two methods. The pores in the cell membrane formed by the high voltage of electroporation were resealed within 6 to 7 min after electroporation. At a capacitance of 25 microF, the optimum voltage was 2.0 to 2.25 kV/cm. Log-phase cells concentrated to 10 to 20 units at an optical density of 600 nm in 200 microliters of fresh rich medium and electroporated at 2.25 kV/cm in the presence of 0.1 microgram of supercoiled plasmid DNA will yield 1,000 to 4,500 colonies per microgram of DNA.     

Transformation of Saccharomyces cerevisiae by electroporation.

A method for introducing heterologous DNA into Saccharomyces cerevisiae rapidly and efficiently by electroporation was developed. Transformant colonies appeared somewhat sooner than by the LiCl or spheroplast transformation method, and the time spent in manipulation was much less than for these two methods. The pores in the cell membrane formed by the high voltage of electroporation were resealed within 6 to 7 min after electroporation. At a capacitance of 25 microF, the optimum voltage was 2.0 to 2.25 kV/cm. Log-phase cells concentrated to 10 to 20 units at an optical density of 600 nm in 200 microliters of fresh rich medium and electroporated at 2.25 kV/cm in the presence of 0.1 microgram of supercoiled plasmid DNA will yield 1,000 to 4,500 colonies per microgram of DNA.     

Effects of cyclic AMP and butyrate on cell cycle,DNA, RNA,and purine synthesis of cultured astrocytes

Dibutyryl cyclic monophosphate (dBcAMP) has been shown to inhibit growth, and alter the morphology of astrocytes. However, the potential contribution of its hydrolytic product, butyrate, in inducing some of the changes that have been attributed to dBcAMP, is not clear. DNA, RNA, and purine synthesis were therefore studied in primary astrocyte cultures after 24 hours of exposure to varying concentrations of butyrate, dBcAMP, and agents that increase intracellular cAMP levels. Progression of cells through cell cycle was also studied by flow cytometry. Dibutyryl cAMP partially arrested cells in Go/G1 phase of cell cycle while sodium butyrate increased the percentage population of cells in G2/M phase. DNA synthesis and de novo purine synthesis were inhibited after treatment with dBcAMP, sodium butyrate, and various drugs that increase intracellular cAMP levels. RNA synthesis was increased with cAMP but was not affected by sodium butyrate. Our study shows that at millimolar concentrations, butyrate is capable of altering the cell cycle and inhibiting DNA synthesis in primary astrocyte cultures, in a manner that is similar although not identical to the effects of dBcAMP.     

Molecular and symbiotic characterization of exopolysaccharide-deficient mutants of Rhizobium tropici strain CIAT899

We studied the symbiotic behaviour of 20 independent Tn5 mutants of Rhizobium tropici strain CIAT899 that were deficient in exopolysaccharide (EPS) production. The mutants produced non-mucoid colonies, were motile, grew in broth cultures at rates similar to those of the parent, and produced significantly less EPS than did CIAT899 in broth culture. A genomic library of strain CIAT899, constructed in pLA2917, was mobilized into all of the mutants, and cosmids that restored EPS production were identified. EcoRI restriction digests of the cosmids revealed nine unique inserts. Mutant complementation and hybridization analysis showed that the mutations affecting EPS production fell into six functional and physical linkage groups. On bean, the mutants were as efficient in nodulation and as effective in acetylene reduction as strain CIAT899, induced a severe interveinal chlorosis, and all but one were less competitive than CIAT899. On siratro, CIAT899 induced nodules that were ineffective in acetylene reduction, whereas the EPS-deficient mutants induced effective nodules. Microscopic examination of thin sections showed that nodules from both siratro and bean plants inoculated with either CIAT899 or an EPS-deficient mutant contained infected cells. These data indicate that EPS is not required for normal nodulation of bean by R. tropici, that it may contribute to competitiveness of R. tropici on bean, and that the loss of EPS production is accompanied by acquisition of the ability to reduce acetylene on siratro.     

Protein Synthesis Linked to Respiration and Phosphorylation in Intact Euglena Mitochondria

Highly purified condensed mitochondria obtained from bleachedmutant. W₁₀BSmL of Euglena gracilis Klebs var bacillaris Coriincorporate [³⁵S]methionine into protein when fortified withmalate, ADP, Mg²⁺, phosphate and a sucrose osmoticum. Twentyto twenty-five polypeptide bands were found to be labeled inorganello when the labeled protein was subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis. Methionine incorporation,but not respiration or oxidative phosphorylation, was blockedby chloramphenicol and other 70S ribosomal translation inhibitorsbut cycloheximide and ribonuclease were without effect. Inhibitorsof electron transport and uncouplers of oxidative phosphorylationwere excellent inhibitors of protein synthesis. Thus, thesemitochondrial preparations carry out protein synthesis in organellothat is linked to respiration and oxidative phosphorylation. ¹Present address: VA Hospital Outpatient Clinic, 17 Court St.,Boston, MA 02115, U.S.A. ²Present address: Laboratories de Microbiologia e Inmunologia,Universidad Catolica de Chile, Casilla 114-D, Santiago, Chile. ³Present address: Botany Department, University of Massachusetts,Amherst, MA 01003, U.S.A. (Received June 17, 1985; Accepted October 28, 1985)