Nematode pyruvate dehydrogenase kinases: role of the C-terminus in binding to the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex.
Pyruvate dehydrogenase kinases (PDKs) from the anaerobic parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans were functionally expressed with hexahistidine tags at their N-termini and purified to apparent homogeneity. Both recombinant PDKs (rPDKs) were dimers, were not autophosphorylated and exhibited similar specific activities with the A. suum pyruvate dehydrogenase (E1) as substrate. In addition, the activities of both PDKs were activated by incubation with PDK-depleted A. suum muscle pyruvate dehydrogenase complex (PDC) and were stimulated by NADH and acetyl-CoA. However, the recombinant A. suum PDK (rAPDK) required higher NADH/NAD+ ratios for half-maximal stimulation than the recombinant C. elegans PDK (rCPDK) or values reported for mammalian PDKs, as might be predicted by the more reduced microaerobic mitochondrial environment of the APDK. Limited tryptic digestion of both rPDKs yielded stable fragments truncated at the C-termini (trPDKs). The trPDKs retained their dimeric structure and exhibited substantial PDK activity with the A. suum E1 as substrate, but PDK activity was not activated by incubation with PDK-depleted A. suum PDC or stimulated by elevated NADH/NAD+ or acetyl-CoA/CoA ratios. Direct-binding assays demonstrated that increasing amounts of rCPDK bound to the A. suum PDK-depleted PDC. No additional rCPDK binding was observed at ratios greater than 20 mol of rCPDK/mol of PDC. In contrast, the truncated rCPDK (trCPDK) did not exhibit significant binding to the PDC. Similarly, a truncated form of rCPDK, rCPDK1-334, generated by mutagenesis, exhibited properties similar to those observed for trCPDK. These results suggest that the C-terminus of the PDK is not required for subunit association of the homodimer or catalysis, but instead seems to be involved in the binding of the PDKs to the dihydrolipoyl transacetylase core of the complex. (+info)
Developmentally regulated telomerase activity is correlated with chromosomal healing during chromatin diminution in Ascaris suum.
Telomerase is the ribonucleoprotein complex responsible for the maintenance of the physical ends, or telomeres, of most eukaryotic chromosomes. In this study, telomerase activity has been identified in cell extracts from the nematode Ascaris suum. This parasitic nematode is particularly suited as a model system for the study of telomerase, because it shows the phenomenon of chromatin diminution, consisting of developmentally programmed chromosomal breakage, DNA elimination, and new telomere formation. In vitro, the A. suum telomerase is capable of efficiently recognizing and elongating nontelomeric primers with nematode-specific telomere repeats by using limited homology at the 3' end of the DNA to anneal with the putative telomerase RNA template. The activity of this enzyme is developmentally regulated, and it correlates temporally with the phenomenon of chromatin diminution. It is up-regulated during the first two rounds of embryonic cell divisions, to reach a peak in 4-cell-stage embryos, when three presomatic blastomeres prepare for chromatin diminution. The activity remains high until the beginning of gastrulation, when the last of the presomatic cells undergoes chromatin diminution, and then constantly decreases during further development. In summary, our data strongly argue for a role of this enzyme in chromosome healing during the process of chromatin diminution. (+info)
Sequence-divergent units of the ABA-1 polyprotein array of the nematode Ascaris suum have similar fatty-acid- and retinol-binding properties but different binding-site environments.
Polyproteins comprise long polypeptides that are post-translationally cleaved into proteins of different function, or tandemly repetitive polypeptides which are processed into multiple versions of proteins which are presumed to have the same function. In the latter case the individual units of the polyprotein can differ substantially in sequence. Identity of function between the different units therefore cannot be assumed. Here we have examined the ABA-1 polyprotein allergen of the parasitic nematode Ascaris suum and found it to contain units which show a 50% difference in amino acid sequence. The parasite therefore produces at least two radically different forms of the allergen encoded within the polyprotein array. In fluorescence-based ligand-binding assays, recombinant polypeptides representing the two forms (designated ABA-1A1 and ABA-1B1) showed similar binding affinities for a range of fluorescent active-site probes [retinol, dansylundecanoic acid, dansyl-DL-alpha-amino-octanoic acid, cis-parinaric acid (cPnA)] and for the non-specific hydrophobic surface probe 8-anilinonaphthalene-1-sulphonic acid. However, the molecular environments in the active sites are markedly different, as indicated by disparate fluorescence emission peaks and intensities of bound probes. CD showed that the proteins have similar secondary structures but differ in susceptibility to chemical denaturation/unfolding by guanidinium chloride. Both retain a single conserved tryptophan residue in a characteristic non-polar environment, as revealed by extreme fluorescence blue shift. Thus the gross differences in sequence of the two proteins are not reflected in their ligand-binding specificities but in their binding-site environments. (+info)
Effect of an orally active Th1/Th2 balance modulator, M50367, on IgE production, eosinophilia, and airway hyperresponsiveness in mice.
We have found a novel anti-allergic agent, M50367, which suppresses IgE biosynthesis and eosinophil accumulation in vivo. In this study, we evaluated the ability of M50367 to modulate Th1/Th2 balance in Th2-background BALB/c mice and to inhibit airway hyperresponsiveness in a murine model of atopic asthma. Oral M50367 at 3-30 mg/kg/day exhibited 51 to 73% reduction of IL-4/IL-5 production and 2- to 5-fold augmentation of IFN-gamma production by Ag-stimulated cultured splenocytes of the mice sensitized with DNP-Ascaris. These alterations in Th1/Th2 cytokine production were accompanied by 55-85% suppression of plasma IgE level. Oral M50367 at a dose of 10 mg/kg/day significantly inhibited Ig-independent peritoneal eosinophilia by 54%, which was induced by repeated i.p. injections of Ascaris suum extract. To develop airway hyperresponsiveness caused by allergic airway inflammation, BALB/c mice were sensitized with i.p. OVA injections, followed three times by OVA inhalation. Oral M50367 significantly inhibited the increase in airway reactivity to acetylcholine, together with the elevation of plasma IgE level and pulmonary eosinophilia, which were observed in vehicle-treated mice 1 day after the last inhalation. Moreover, M50367 treatment reduced IL-4 and IL-5 production and tended to enhance IFN-gamma production, not only by cultured splenocytes, but also in bronchoalveolar lavage fluid. These results suggest that M50367 has a modulating ability of Th1/Th2 balance to down-regulate Th2 response in the circulating system as well as at the sites of inflammation, and may be beneficial for the treatment of allergic disorders such as atopic asthma. (+info)
Localized depolymerization of the major sperm protein cytoskeleton correlates with the forward movement of the cell body in the amoeboid movement of nematode sperm.
The major sperm protein (MSP)-based amoeboid motility of Ascaris suum sperm requires coordinated lamellipodial protrusion and cell body retraction. In these cells, protrusion and retraction are tightly coupled to the assembly and disassembly of the cytoskeleton at opposite ends of the lamellipodium. Although polymerization along the leading edge appears to drive protrusion, the behavior of sperm tethered to the substrate showed that an additional force is required to pull the cell body forward. To examine the mechanism of cell body movement, we used pH to uncouple cytoskeletal polymerization and depolymerization. In sperm treated with pH 6.75 buffer, protrusion of the leading edge slowed dramatically while both cytoskeletal disassembly at the base of the lamellipodium and cell body retraction continued. At pH 6.35, the cytoskeleton pulled away from the leading edge and receded through the lamellipodium as its disassembly at the cell body continued. The cytoskeleton disassembled rapidly and completely in cells treated at pH 5.5, but reformed when the cells were washed with physiological buffer. Cytoskeletal reassembly occurred at the lamellipodial margin and caused membrane protrusion, but the cell body did not move until the cytoskeleton was rebuilt and depolymerization resumed. These results indicate that cell body retraction is mediated by tension in the cytoskeleton, correlated with MSP depolymerization at the base of the lamellipodium. (+info)
1H NMR investigation of the distal hydrogen bonding network and ligand tilt in the cyanomet complex of oxygen-avid Ascaris suum hemoglobin.
The O(2)-avid hemoglobin from the parasitic nematode Ascaris suum exhibits one of the slowest known O(2) off rates. Solution (1)H NMR has been used to investigate the electronic and molecular structural properties of the active site for the cyano-met derivative of the recombinant first domain of this protein. Assignment of the heme, axial His, and majority of the residues in contact with the heme reveals a molecular structure that is the same as reported in the A. suum HbO(2) crystal structure (Yang, J., Kloek, A., Goldberg, D. E., and Mathews, F. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4224-4228) with the exception that the heme in solution is rotated by 180 degrees about the alpha,gamma-meso axis relative to that in the crystal. The observed dipolar shifts, together with the crystal coordinates of HbO(2), provide the orientation of the magnetic axes in the molecular framework. The major magnetic axis, which correlates with the Fe-CN vector, is found oriented approximately 30 degrees away from the heme normal and indicates significant steric tilt because of interaction with Tyr(30)(B10). The three side chain labile protons for the distal residues Tyr(30)(B10) and Gln(64)(E7) were identified, and their relaxation, dipolar shifts, and nuclear Overhauser effects to adjacent residues used to place them in the distal pocket. It is shown that these two distal residues exhibit the same orientations ideal for H bonding to the ligand and to each other, as found in the A. suum HbO(2) crystal. It is concluded that the ligated cyanide participates in the same distal H bonding network as ligated O(2). The combination of the strong steric tilt of the bound cyanide and slow ring reorientation of the Tyr(30)(B10) side chain supports a crowded and constrained distal pocket. (+info)
Effect of the injection of an extract of Ascaris suum on macrophage activation during the early phase of Mycobacterium bovis BCG infection in C57Bl/6 mice.
Injection of an Ascaris suum extract (Asc) affects both the humoral and cellular immune responses to unrelated antigens when it is co-administered with these antigens. In the present study we evaluated the effect of Asc on macrophage activation in the early phase of Mycobacterium bovis BCG (Pasteur strain TMCC 1173) infection in C57Bl/6 mice. C57Bl/6 mice were injected intraperitoneally (ip) with 0.1 mg BCG (BCG group) or BCG plus 1 mg Asc (BCG + Asc group). The peritoneal exudates were obtained at 2, 7 and 14 days after infection. The numbers of IFN-gamma-secreting cells were assessed by the ELISPOT assay. Nitric oxide (NO) production was measured by the Griess method and by the evaluation of NADPH diaphorase activity in the peritoneal exudates. The administration of Asc extract increased NADPH diaphorase activity (2 days: control = 0, BCG = 7%, BCG + Asc = 13%, and Asc = 4%; 7 days: control = 4, BCG = 13%, BCG + Asc = 21%, and Asc = 4.5%) and TNF-alpha levels (mean +/- SD; 2 days: control = 0, BCG = 169 +/- 13, BCG + Asc = 202 +/- 37, and Asc = 0; 7 days: control = 0, BCG = 545 +/- 15.5, BCG + Asc = 2206 +/- 160.6, and Asc = 126 +/- 26; 14 days: control = 10 +/- 1.45, BCG = 9 +/- 1.15, BCG + Asc = 126 +/- 18, and Asc = 880 +/- 47.67 pg/ml) in the early phase of BCG infection. Low levels of NO production were detected at 2 and 7 days after BCG infection, increasing at 14 days (mean +/- SD; 2 days: control = 0, BCG = 3.7 +/- 1.59, BCG + Asc = 0.82 +/- 0.005, Asc = 0.48 +/- 0.33; 7 days: control = 0, BCG = 2.78 +/- 1.54, BCG + Asc = 3.07 +/- 1.05, Asc = 0; 14 days: control = 0, BCG = 9.05 +/- 0.53, BCG + Asc = 9.61 +/- 0.81, Asc = 10.5 +/- 0.2 (2 x 10(6)) cells/ml). Furthermore, we also observed that Asc co-injection induced a decrease of BCG-colony-forming units (CFU) in the spleens of BCG-infected mice during the first week of infection (mean +/- SD; 2 days: BCG = 1.13 +/- 0.07 and BCG + Asc = 0.798 +/- 0.305; 7 days: BCG = 1.375 +/- 0. 194 and BCG + Asc = 0.548 +/- 0.0226; 14 days: BCG = 0.473 +/- 0.184 and BCG + Asc = 0.675 +/- 0.065 (x 10(2)) CFU). The present data suggest that Asc induces the enhancement of the immune response in the early phase of BCG infection. (+info)
Phosphocholine-containing, zwitterionic glycosphingolipids of adult Onchocerca volvulus as highly conserved antigenic structures of parasitic nematodes.
Human Onchocerca volvulus infection sera were found to recognize zwitterionic glycolipids of O. volvulus and to cross-react with those of other parasitic nematodes (Ascaris suum, Setaria digitata and Litomosoides sigmodontis). By the use of an epitope-specific monoclonal antibody, zwitterionic glycolipids of all these nematode species were observed to contain the antigenic determinant phosphocholine. A hyperimmune serum specific for arthro-series glycolipid structures reacted with the various neutral glycolipids of all these nematodes, which demonstrated that their oligosaccharide moieties belonged to the arthro-series of protostomial glycolipids. These results indicated that arthro-series glycosphingolipids carrying, in part, phosphocholine substituents, represent highly conserved, antigenic glycolipid markers of parasitic nematodes. Three glycolipid components of the O. volvulus zwitterionic fraction were structurally characterized by matrix-assisted laser-desorption/ionization time-of-flight MS, methylation analysis and exoglycosidase treatment. Their chemical structures were elucidated to be phosphocholine-6GlcNAc(beta1-3)Man(beta1-4)Glc(1-1)ceramide, GalNAc(beta1-4)[phosphocholine-6]GlcNAc(beta1-3)Man(beta1-4)Glc(1-1) ceramide and Gal(alpha1-3)GalNAc(beta1-4)[phosphocholine-6]GlcNAc(beta1-3)Man(beta 1-4)Glc(1-1)ceramide for the zwitterionic ceramide tri-, tetra- and penta-hexosides respectively. The ceramide composition was found to be dominated by 2-hydroxylated docosanoic (C(22h:0)), tricosanoic (C(23h:0)) and tetracosanoic (C(24h:0)) acids, and C(17) sphingosine (C(d17:1)) (where (h) is hydroxylated and (d) is dihydroxylated). (+info)