Possible roles of protein kinase A in cell motility and excystation of the early diverging eukaryote Giardia lamblia.

Since little is known of how the primitive protozoan parasite, Giardia lamblia, senses and responds to its changing environment, we characterized a giardial protein kinase A (gPKA) catalytic subunit with unusual subcellular localization. Sequence analysis of the 1080-base pair open reading frame shows 48% amino acid identity with the cyclic AMP-dependent kinase from Euglena gracilis. Northern analysis indicated a 1.28- kilobase pair transcript at relatively constant concentrations during growth and encystation. gPKA is autophosphorylated, although amino acid residues corresponding to Thr-197 and Ser-338 of human protein kinase A (PKA) that are important for autophosphorylation are absent. Kinetic analysis of the recombinant PKA showed that ATP and magnesium are preferred over GTP and manganese. Kinase activity of the native PKA has also been detected in crude extracts using kemptide as a substrate. A myristoylated PKA inhibitor, amide 14-22, inhibited excystation with an IC(50) of 3 microm, suggesting an important role of gPKA during differentiation from the dormant cyst form into the active trophozoite. gPKA localizes independently of cell density to the eight flagellar basal bodies between the two nuclei together with centrin, a basal body/centrosome-specific protein. However, localization of gPKA to marginal plates along the intracellular portions of the anterior and caudal pairs of flagella was evident only at low cell density and higher endogenous cAMP concentrations or after refeeding with fresh medium. These data suggest an important role of PKA in trophozoite motility during vegetative growth and the cellular activation of excystation.

Giardia lamblia: stimulation of growth by human intestinal mucus and epithelial cells in serumfree medium.

Giardia lamblia trophozoites specifically colonize the upper human small intestine which is normally serumfree but have been grown in vitro only in medium supplemented with serum or serum fractions. Recently, we demonstrated that biliary lipids will support the growth of G. lamblia without added serum. Now, we report that human duodenal jejunal mucus stimulates growth of Giardia in medium with biliary lipids. Stimulation by mucus was enhanced by inclusion of chymotrypsin or crude pancreatic proteases. Coculture of trophozoites with human intestinal epithelial cells also promoted growth, especially in the presence of mucus and/or biliary lipids. With biliary lipids alone, the mean increase in cell number was 3.2 fold and in the presence of mucus 8 fold (P less than 0.01) in 24 serial subcultures. Our demonstration that human intestinal mucus and epithelial cells promote serumfree growth of G. lamblia may help to explain specific colonization of the small intestine by G. lamblia.

Bruceantin, a potent amoebicide from a plant, Brucea antidysenterica.

Bruceantin, purified from an Ethiopian plant used to treat dysentery, killed Entamoeba histolytica in vitro (IC50 [the concentration of drug which decreased the number of colonies to half that of controls] = 0.018 microgram/ml). Six related quassinoids of 17 tested were also amoebicidal. No relationship between quassinoid structure and amoebicidal activity was apparent.

Control of mutation frequency by bacteriophage T4 DNA polymerase. II. Accuracy of nucleotide selection by the L88 mutator, CB120 antimutator, and wild type phage T4 DNA polymerases.

The accuracy of nucleotide selection by wild type, L88 mutator, and CB120 antimutator T4 DNA polymerases has been compared by measuring both stable incorporation of complementary and noncomplementary nucleotides into polymer and the DNA-dependent conversion of deoxynucleoside triphosphate to monophosphate. The increased accuracy of the CB120 antimutator enzyme is shown by a ratio of utilization of incorrect to correct nucleotides with poly(dA)-poly(dT) as template which is only 10 to 30% of that of the wild type enzyme. In contrast, the ratio of incorrect to correct nucleotide utilized by the L88 mutator enzyme with this template was higher than that of the wild type enzyme, in agreement with the report of Hershfield (Hershfield, M.S. (1973) J. Biol, Chem. 248, 1417-1423). The antimutator, mutator, and wild type enzymes each have a much higher apparent Km for the noncomplementary nucleotides than for complementary nucleotides with this template. The L88 mutator polymerase has a higher "Km" for poly(dA)-poly(dT) than the wild type enzyme in reactions with both complementary and noncomplementary nucleotides. The antimutator polymerase has an elevated "Km" for polymer only for reactions with noncomplementary nucleotides. The wild type and L88 mutator enzymes also utilized both noncomplementary nucleotides much more frequently than the CB120 antimutator enzymes with poly [d(A-T)] as the template-primer. The T4 gene 32DNA unwinding protein appears to facilitate the correct reading of this template since it decreases the ratio of incorrect nucleotides utilized by both the wild type and L88 mutator polymerases.