Tag E. Mansour

Cloning and characterization of actin depolymerizing factor from Toxoplasma gondii

We determined the predicted amino acid sequence of actin depolymerizing factor (ADF) from Toxoplasma gondii by sequencing the full-length cDNA. T. gondii ADF consists of 118 amino acids (calculated molecular weight 13,400) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially Acanthamoeba actophorin, plant ADFs and yeast and vertebrate cofilin. ADF from T. gondii is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that T. gondii ADF is a single-copy gene. Homogeneous recombinant T. gondii ADF purified from E. coli is active in binding actin monomers and depolymerizing F-actin. Localization of ADF by immunofluorescence and immunoelectron microscopy indicates ADF is scattered throughout the cytoplasm and prominently localized beneath the plasma membrane in T. gondii.

Conserved classes of homeodomains in Schistosoma mansoni, an early bilateral metazoan

Genes containing a homeobox can be divided into classes based on the distinctive peptide sequences of their diverged homeodomains. Many of these classes, including Antennapedia, engrailed and paired, are strongly conserved in higher multicellular animals, but have not previously been found in platyhelminths, the flatworms which represent the most primitive bilateral metazoans. We have screened cDNA libraries of the platyhelminth Schistosoma mansoni using a degenerate oligonucleotide derived from the third helix of the homeodomain, and have identified numerous schistosome homeobox-containing sequences, including members of the Antennapedia, engrailed and paired classes. The schistosome homeodomain sequences are more similar to the higher animals sequences in their respective classes than they are to each other, indicating that the establishment of these three distinctive classes is at least as ancient as the flatworms. Our data suggest that the ancestral functions of the Antennapedia, engrailed and paired classes involve fundamental features of all bilateral metazoan development. The putative full-length coding sequence of the S. mansoni en homologue is presented.

Isolation of a female-specific, highly repeated Schistosoma mansoni DNA probe and its use in an assay of cercarial sex

A 476 bp fragment of female-specific Schistosoma mansoni genomic DNA, clone W1, represents a degenerative repeat present in more than 500 copies per female genome, and may be part of the constitutive heterochromatin of the W chromosome. The cloning method described can be used as a general approach for isolating sex-specific, repeated DNA fragments. Using W1 as a probe, we have developed a rapid and accurate dot-blot assay for determining the sex of S. mansoni cercariae.

The Pharmacology and Biochemistry of Parasitic Helminths

Publisher Summary This chapter reviews some of the recent studies concerning the carbohydrate metabolism and neuromuscular system of some parasitic helminths. The known effects of a relatively small number of chemical agents on these systems have also been discussed. In spite of the early recognition of the differences among members of different phyla, many biologists ignored this fact in their approach to the study of the effect of chemical agents on these organisms. Some of them have used animals of a phylum other than those that include the parasitic helminths. The differences can be established not only by the presence or absence of a certain physiological or biochemical system but also by systems that carry on the same function in different parasites. The biochemical and physiological differences between the host and parasitic helminths has been demonstrated at various levels: on the intact organisms, on cell-free extracts, and on isolated enzyme systems. It has been observed that many of the chemotherapeutic agents that are now in use against helminths owe their effect to a selective action on certain physiological or biochemical processes in the parasite. Advances in the chemotherapy of helminthiasis should depend greatly on more knowledge concerning the physiology, biochemistry, and pharmacology of these animals. The presence of the same metabolic pathway or the same physiological process in the host and parasite does not rule out differences between the two. Such differences have been shown to occur even among enzymes catalyzing the same reactions. Further studies along these lines must lead to the development of more effective and less toxic chemotherapeutic agents.

Glucose as a regulator of glycogen phosphorylase un rat diaphragm: I. Effect of glucose and related compounds of phosphorylase and glycogen levels☆

Abstract 1. 1. Incubation of isolated rat diaphrahm in the presence of glucose resulted in significantly lower levels of phosphorylase a , as compared to control samples incubated without glucose. Total phosphorylase (defined as phosphorylase a plus phosphorylase b) was not significantly affected. The effect of glucose on phosphorylase a was found to be specific, as evidence by the fact that other sugars had little or no effect on the enzyme. 2. 2. Glc-6- P and Glc-1- P also caused a decreased in diaphragm phoshorylase a . The esters were found to be more potent on a molar basis than glucose. In contrast, Fru-1–6- P 2 caused an increase in phosphorylase a levels. None of the hexose phosphates had a significant effect on total phosphorylase. 3. 3. Tissue glycogen was markedly depleted during incubation in the absence of glucose. Normal tissue glycogen levels were maintained or even increased in diaphragms incubated with glucose. Thus, an increased level of phosphorylase a was associated with glycogenolysis, while a decrease in phosphorylase a was associated with a maintenance of normal tissue glycogen. It is suggested that regualtion of glycogen levels by glucose, through an action on phorphorylase a , could be of physicological significance.

Biochemical effects of lysergic acid diethylamide on the liver fluke Fasciola hepatica.

Abstract Serotonin (5-hydroxytryptamine) and lysergic acid diethylamide cause an increase in glycolysis in the liver fluke Fasciola hepatica. The effect of serotonin on glycosis appears to be due to an increase in phosphofructokinase activity and is mediated through cyclic 3′,5′-AMP. The biochemical effects of lysergic acid diethylamide on the liver flukes were examined in the present paper. Like serotonin, lysergic acid diethylamide causes an increase in the activity of phosphofructokinase in the liver flukes. This effect can be obtained either by preincubating the intact flukes with lysergic acid diethylamide or by adding it directly to fluke homogenates. While serotonin activates phosphofructokinase through its effect on a particulate fraction from the flukes, lysergic acid diethylamide did not have an effect on this fraction. The question whether lysergic acid diethylamide effect is mediated through serotonin was investigated. The concentration of serotonin in intact liver flukes was found to be 231 ng/g wet weight. The level of serotonin in the flukes is markedly increased when the flukes are incubated with 5-hydroxytryptophan but is unchanged following incubation with tryptophan. Lysergic acid diethylamide does not change the levels of endogenous serotonin in control flukes. It also does not affect the levels of serotonin synthesized by the organism from 5-hydroxytryptophan. The data does not support the idea that the effect of lysergic acid diethylamide is mediated through an increase in serotonin levels although the effects on carbohydrate metabolism of both agents are similar.

Purification and properties of a pyrophosphate-dependent phosphofructokinase from Toxoplasma gondii

Inorganic pyrophosphate-dependent phosphofructokinase was identified in toxoplasma gondii and purified to near homogeneity from the crude extracts. The purified enzyme displayed one major protein band which coincided with enzyme activity on nondenaturing polyacrylamide gel electrophoresis. This phosphofructokinase had a molecular weight of 100,000 determined by gel filtration and was composed of one type of subunit with the molecular weight of 45,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme is a homodimer. Some kinetic parameters of the purified enzyme were investigated in the forward and the reverse directions. The substrate saturation curves for fructose 6-phosphate and pyrophosphate were all hyperbolic. The apparent Km values for fructose 6-phosphate and for pyrophosphate were 2.7 x 10(-4) M and 3.3 x 10(-5) M respectively. Kinetics for Fru-1,2-P2 and for Pi in the reverse reaction were also hyperbolic. The activity of this enzyme was magnesium-dependent. Nucleoside triphosphates and polyphosphates did not serve as phosphate donor and the enzyme activity was not altered in the presence of any of these nucleotides. As in the case of pyrophosphate-dependent phosphofructokinases from other anaerobic eukaryotes the Toxoplasma enzyme was not activated by fructose 2,6-biphosphate.

Some phosphonic acid analogs as inhibitors of pyrophosphate-dependent phosphofructokinase, a novel target in Toxoplasma gondii

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was identified previously in Toxoplasma gondii as the only kinase that phosphorylates fructose-6-P to fructose-1,6-bisP. Since such an enzyme is not present in mammals, it was considered to be a good target for prospective selective inhibitors of the parasite. We have examined the effects of several phosphonic acid derivatives, analogs of pyrophosphate, on PPi-PFK activity, as well as on the replication of T. gondii in human foreskin fibroblast (HFF) cells. The most active compound in inhibiting PPi-PFK was tetrasodium carbonyldiphosphonate. Several bisphosphonates and related arylhydrazones showed inhibition of the enzyme, but with higher IC50 values. Although several phosphonoacetic acid derivatives also inhibited PPi-PFK, as a group they were less potent than the bisphosphonate derivatives. Comparison among the structures of various inhibitors and their effects against PPi-PFK indicates that a carbonyl (C=O) or amino (C=N) group between two phosphoryl moieties is associated with more potent enzyme inhibiton. Tetrasodium carbonyldiphosphonate did not show a significant effect against replication of T. gondii cells, probably because, as a charged molecule, it could not cross the cell membrane to reach the intracellular parasite. Tetraisopropyl carbonyldiphosphonate 2,4-dinitrophenylhydrazone showed some selective inhibitory effect against replication of the parasite in the HFF cells and protected the mammalian cells from damage by T. gondii. The results indicate that carbonyldiphosphonic acid is a good prototype compound that is amenable to chemical manipulation, which, in turn, may optimize selective inhibition of T. gondii PPi-PFK and increase accessibility to the intracellular parasite.

Measurement of chemotaxis in the slime mold Physarum polycephalum

Abstract A method is described for assaying chemotaxis in the acellular slime mold Physarum polycephalum. It consists of measuring the amount of plasmodium that moves on a strip of nitrocellulose membrane filter Millipore in response to a gradient of an attractant. Time course of chemotactic response of the slime mold is described. Different factors that affect chemotaxis in the slime mold such as: culture care and stage of growth of microplasmodia, substratum used for cell movement, nature of the gradient, effect of salts, pH and temperature are described. From concentration-response curves for different attractants several parameters of the chemotactic effect, such as threshold concentration, half maximal concentration, and maximal effective concentration can be determined. As a group, sugars are more effective chemotactic agents than amino acids. Glucose and galactose, which support the growth of the slime mold, are shown to have high positive chemotactic effect. 3-O-Methyl- d -glucose and 2-deoxy- d -glucose are two sugars that do not support growth but are very effective attractants. Conversely, fructose which supports slime mold growth is at best a weak attractant. The results support the view that the chemotactic effects of different sugars are not dependent on their growth-supporting value.

Glucose as a regulator of glycogen phosphorylase in rat diaphragm: II. Effect of glucose and related compounds on phosphorylase phosphate

Abstract 1. 1. The mechanism by which glucose causes a decrease in phosphorylase a levels in isolated rat diaphragm was investigated. Although glucose is known to be an inhibitor of both phosphorylases a and b , the effect of glucose could not be explained by a direct inhibitory action of the hexose on these enzymes. In contrast, it has been found that glucose markedly activates phosphorylase phosphatase, thus decreasing the level of phosphorylase a . 2. 2. In addition to glucose, glycogen and Glc-6- P also enhanced phosphatase activity, while Fru-1,6- P 2 and P i were inhibitory. 3. 3. Possible mechanismsof glucose activation of diaphragm phosphorylase phosphatase were investigated. Results indicated that the observed effect cannot be explained by a change in the molecular form of the substrate, phosphprylase a . Glucose may alter the susceptibility of the substrate and/or its partially phosphorylated intermediates to phosphatase action. Alternatively, glucose may directly activate the phosphatase itself.