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M E Selkirk

Publications and source records attributed to M E Selkirk.

At least 37 records · Page 2Linked to original sources
PubMed1997

Purification and properties of monomeric (G1) forms of acetylcholinesterase secreted by Nippostrongylus brasiliensis.

Acetylcholinesterase (AChE) activity secreted by Nippostrongylus brasiliensis was resolved by sucrose density centrifugation and gel permeation chromatography in single peaks estimated at 4.3 S and 60-85 kDa, respectively. Sedimentation was unaffected by the inclusion of detergent. AChE was purified by affinity chromatography on 9-[Nbeta-(epsilon-aminocaproyl)-beta-aminopropylamino]-acridinium bromide hydrobromide-coupled sepharose 4B. Three forms of the enzyme (A, B and C) were distinguished by non-denaturating polyacrylamide gel electrophoresis, and displayed apparent masses of 74, 69 and 71 kDa respectively when resolved by SDS-PAGE. All three isoforms showed a preference for acetylthiocholine (ASCh) as substrate. They were highly sensitive to inhibition by the AChE-specific inhibitor bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with inhibitor concentration reducing initial activity by 50% (IC50) between 0.1 and 0.8 microM, but activity was unaffected by tetramonoisopropylpyrophosphortetramide (iso-OMPA) at concentrations up to 10 mM. We conclude that the secreted enzymes are authentic AChEs of hydrophilic monomeric (G1) form and broadly similar properties, but which can be distinguished by molecular mass, inhibitor sensitivities and the degree of excess substrate inhibition.

Acetylcholinesterase↗
PubMed1997

Identification of serine/threonine protein kinases secreted by Trichinella spiralis infective larvae.

Serine/threonine protein kinase activity was identified in excretory/secretory (ES) products of Trichinella spiralis infective larvae, via phosphorylation of exogenous and endogenous substrates. Protein kinase activity was identified as an authentic secretory product via blockade of release into culture medium by brefeldin A. Enzyme activity was reductant-dependent, and the relative resistance to a panel of inhibitors suggested that it could not be readily assigned to any of the major documented subfamilies of serine/threonine protein kinases. There was no evidence for protein tyrosine kinase activity in ES products. The major phosphorylated proteins in this compartment resolved at 50 and 55 kDa by SDS-PAGE, and are therefore designated pp50/55. These proteins contained mainly phosphoserine, and appear to represent differentially glycosylated variants of a 35 kDa polypeptide, modified via the addition of three and four N-linked oligosaccharides, respectively. An autophosphorylation assay following separation by SDS-PAGE identified two protein kinases of 70 and 135 kDa in ES products.

Animals↗
PubMed1997

Cytostatic and cytotoxic effects of activated macrophages and nitric oxide donors on Brugia malayi.

The susceptibility of Brugia malayi microfilariae and adults to injury by the murine macrophage cell line J774 activated with gamma interferon and bacterial lipopolysaccharide has been examined in vitro. Parasites of both stages showed a decline in viability over 48 h of coculture with activated macrophages, assessed by their capacity to reduce the tetrazolium salt 3-[4,5-diethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), although adult parasites were more resistant than microfilariae. Removal of parasites to cell-free medium following exposure to activated macrophages for up to 48 h resulted in partial recovery of their capacity to reduce MTT, suggesting that the effects were primarily cytostatic. However, prolonged exposure to activated J774 cells for 72 h resulted in parasite death. Addition of the nitric oxide synthase inhibitor L-NMMA (N(G)-monomethyl-L-arginine monoacetate) indicated that nitric oxide derivatives were responsible for cytostasis and ultimate toxicity. The toxicity of nitric oxide derivatives was confirmed by coincubation of parasites with chemical donors, although far higher concentrations were required than those generated by activated J774 cells, implying additional complexity in macrophage-mediated cytotoxicity. These experiments further suggested that peroxynitrite or its by-products were more potently damaging to filariae than nitric oxide per se. Examination of ultrastructural changes on exposure of parasites to activated macrophages or donors of nitric oxide indicated that hypodermal mitochondria were highly vacuolated, with less prominent cristae. The data are discussed with reference to immunity to lymphatic filariae and their mechanisms of energy generation.

Animals↗
PubMed1996

Characterization of a platelet-activating factor acetylhydrolase secreted by the nematode parasite Nippostrongylus brasiliensis.

Nippostrongylus brasiliensis, a small nematode parasite of the gastrointestinal tract of rodents, secretes an enzyme that cleaves the proinflammatory molecule platelet-activating factor to its inactive lyso-form. The enzyme activity of Ca(2+)-dependent and does not exhibit interfacial activation. It does not require the addition of reducing agents for maximal activity, and is not inhibited by thiol-active reagents. Sensitivity of inhibitors suggests the involvement of serine and histidine residues in the enzyme activity. As described for other platelet-activating factor acetylhydrolases, it cannot cleave, nor is it inhibited by, long-chain diacyl phospholipids that are typical substrates for phospholipases A2. The purified enzyme was resolved by SDS/PAGE as a heterodimer composed of two protein subunits with apparent molecular masses of 38 and 25 kDa. The properties of the nematode enzyme thus differ from those described for the mammalian enzymes, but are more closely related to those of an acetylhydrolase than a phospholipase.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

Structural organisation and lipid composition of the epicuticular accessory layer of infective larvae of Trichinella spiralis.

The epicuticle of infective larvae of Trichinella spiralis represents the interface between this intracellular nematode parasite and the cytosol of mammalian skeletal muscle. The macromolecular structures that make up the epicuticle were studied by freeze-fracture electron microscopy and compositional analysis. Three fracture planes were observed: one with a typical plasma membrane-type bilayer organisation which was overlaid by two extended layers of lipid in an inverted cylindrical configuration. This overall structure remained unchanged in response to variations in temperature between 20 degrees C and 45 degrees C. The lipid cylinders were on average 6.8 nm in diameter, with randomly-associated particles that were not dissociated by high-salt treatment, indicative of hydrophobically associated proteins. The majority of the lipids were non-polar, consisting of cholesterol, cholesterol esters, mono- and tri-glycerides, and free fatty acids. Three major classes of phospholipids were identified: phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. Total lipid extracts did not adopt an inverted cylindrical or micellar configuration on isolation, but formed flat sheets of lamellae as did the purified polar and non-polar fractions of the lipids. Isolated lipids did not undergo thermally-induced polymorphism between 20 degrees C and 60 degrees C and there was no pH dependency of the structures adopted. The fatty acid saturation levels of the phospholipids were compatible with the observation that they did not form polymorphic structures on isolation. We suggest that this unusual configuration is probably stabilised by the associated (glyco)proteins and may be required for selective permeation of nutrients from the host cell cytosol and/or for maintaining the high curvature of the parasite within the cell.

Animals↗
PubMed1996

Brugia malayi: ultrastructural morphology of the cuticular surface membranes of adult parasites.

We have obtained and examined several fracture planes close to the surface of the cuticle of adult Brugia malayi by freeze-fracture electron microscopy. We observed three exocytoplasmic and two protoplasmic fracture faces exhibiting the periodic annulations characteristic of the outer layers of the cuticle. The outermost exocytoplasmic fracture face we observed contained small randomly distributed particles and appears to originate from within the epicuticle. The second exocytoplasmic face appeared as a featureless surface representing a nonmembranous proteinaceous structural arrangement adjacent to the epicuticle. The innermost fracture plane was represented by an exocytoplasmic face with a morphology dominated by large particles regularly appearing as organised linear arrays. This fracture plane seems to originate from a membrane-like organisation directly below the annulated proteinaceous layer. The two annulated protoplasmic faces were exposed by two discrete planes of fracture and were complementary to the exocytoplasmic planes. The above observations are considered in terms of the overall structural organisation of the cuticle of adult B. malayi.

Animals↗
PubMed1996

Identification and composition of lipid classes in surface and somatic preparations of adult Brugia malayi.

The cuticle of adult Brugia malayi is the organisms's major point of interaction with the mammalian host environment. We therefore undertook an investigation in order to define the lipid composition of this outermost layer of the parasite. The lipid class and fatty acid composition of the cuticle of adult Brugia malayi was examined by surface specific radioiodination, organic extraction, thin layer chromatography and gas chromatography. The data were compared with those derived from similar analyses of somatic preparations of the parasites. The composition of the cuticular lipid fraction was found to be highly unusual and distinct from that of the internal lipids. Cholesterol esters and wax esters were absent from the cuticular lipid fraction, which was however enriched in unesterified fatty acids. The major polar lipids in both cuticular and somatic preparations were phosphatidylcholine and phosphatidylethanolamine, but unusually high levels of lysophosphatidylethanolamine were observed in the cuticular extracts. Analyses of cuticular polar lipids indicated that there is an asymmetric distribution of the fatty acids in phosphatidylethanolamine, assuming that lysophosphatidylethanolamine is derived from deacylation of the former molecule in the cuticle. The major fatty acids in all lipid fractions examined were the 18-carbon, mono- and di-unsaturated type, while significant amounts of palmitic, palmitoleic, stearic and eicosatrienoic acids were also found. A highly unusual feature of the cuticular lipid fraction was that it contained large amounts of a novel polar lipid species which, on exposure to atmospheric oxygen, degraded to a hydrophobic and a hydrophilic moiety. This polar lipid was absent from the somatic preparations. The data are discussed in terms of the possible resistance or susceptibility of the parasite to reactive oxygen species.

Animals↗
PubMed1996

Heterologous expression of the cuticular-glutathione peroxidase of lymphatic filariae in an attenuated vaccine strain of Salmonella typhimurium abrogates H-2 restriction of specific antibody responses.

The major soluble cuticular glycoprotein of lymphatic filariae, gp29, has been expressed in the Salmonella typhimurium aroA aroD live vaccine strain BRD509. Two distinct constructs were generated: a) pgp29, in which gp29 was expressed directly via the inducible promoter nirB, or b) pTetC-gp29, in which it was expressed as a C-terminal fusion to the non-toxic immunogenic fragment C of tetanus toxin, again under the control of nirB. In both cases, plasmid stability in vivo was demonstrated by recovery of recombinant bacteria from livers and spleens of mice immunized via the intravenous route. Negligible gp29-specific antibodies were detected in animals immunized with bacteria expressing the fragment C fusion protein, but bacteria expressing the non-fused protein resulted in gp29-specific antibody production in a proportion of animals immunized. Notably, a number of BALB/c and B10.D2/n (i.e. mice of the H-2d haplotype) responded, in contrast to previously documented nonresponsiveness during infection, or immunization with parasite extracts. Presentation of gp29 by live attenuated S. typhimurium resulted in a broad spectrum of antigen-specific IgG isotypes.

Animals↗
PubMed1995

Heterologous expression and enzymatic properties of a selenium-independent glutathione peroxidase from the parasitic nematode Brugia pahangi.

A full-length cDNA from the parasitic nematode Brugia pahangi encoding a secreted homolog of glutathione peroxidase in which the codon for the active site selenocysteine is substituted naturally by a cysteine codon has been expressed in Spodoptera frugiperda (insect) cells via Autographa californica nuclear polyhedrosis virus (baculovirus). The recombinant protein was glycosylated and secreted from the cells in tetrameric form. The purified protein showed glutathione peroxidase activity with a range of organic hydroperoxides, including L-alpha-phosphatidylcholine hydroperoxide, but no significant activity against hydrogen peroxide. Glutathione was the only thiol tested that served as a substrate for the enzyme, which showed no activity with the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH). No glutathione-conjugating activity was detected against a range of electrophilic compounds that are common substrates for glutathione S-transferases. The apparent (pseudo)m for glutathione was determined as 4.9 mM at a fixed concentration of linolenic acid hydroperoxide (3 microM). The enzyme showed low affinity for hydroperoxide substrates (apparent Km for linolenic acid hydroperoxide and L-alpha-phosphatidylcholine hydroperoxide of 3.8 and 9.7 mM, respectively at a fixed glutathione concentration of 3 mM).

Animals↗
PubMed1995

Brugia malayi: localisation and differential expression of extracellular and cytoplasmic CuZn superoxide dismutases in adults and microfilariae.

We have determined the levels of superoxide dismutase (SOD) in different stages of the lymphatic filarial nematode parasite of man, Brugia malayi. Adult male worm extracts showed the highest levels of enzyme activity at 34.5 U mg-1, and there was no significant difference in the overall levels of SOD in extracts of adult female worms and microfilariae (27.1 and 26.7 U mg-1, respectively). SOD activity was detected in the culture medium of parasites maintained in vitro, with particularly high levels of specific activity in media in which males and females were maintained (357 and 339 U mg-1, respectively), indicative of active secretion. In all cases, this was accounted for predominantly by CuZn SOD, assessed by potassium cyanide inhibition. Northern blots with cDNA probes specific for cytoplasmic and extracellular CuZn SODs indicated that levels of mRNA for the cytoplasmic form were similar between adults and microfilariae, whereas expression of the extracellular form was 10x higher in adult worms. Western blots with an antibody to recombinant CuZn SOD demonstrated that higher levels of the extracellular protein were present in adult male worms, whereas the cytoplasmic form was present in roughly equivalent amounts in males, females, and microfilaria. Iodination and immunoprecipitation experiments indicated that the extracellular enzyme was accessible to surface labeling of both male and female adult worms, but not microfilaria. Immuno-electron microscopy showed that CuZn SOD was localised predominantly in the hypodermis of adult parasites, with an asymmetric distribution in the intercordal regions suggestive of compartmentalisation into several distinct syncytia. No labeling was evident in the cuticle, and thus the accessibility of the extracellular enzyme to extrinsic iodination in adult worms remains unclear. No binding of antibody was demonstrable in the glandular region of the oesophagus or the uterus of females, presumed to be major sites of synthesis for secreted proteins. Dense labeling was observed in the seminal fluid surrounding spermatazoa in the vas deferens of male parasites. These data also suggest that, as observed in mammals, nematode spermatazoa are particularly susceptible to oxidative damage and are protected during storage by secreted anti-oxidant enzymes.

Animals↗
PubMed1995

Brugia malayi: differential susceptibility to and metabolism of hydrogen peroxide in adults and microfilariae.

The sensitivity of microfilariae and adult Brugia malayi to hydrogen peroxide (H2O2) was determined in vitro, and parasite viability assessed by incorporation of 2-deoxy-D-[1-3H]glucose. Both stages were surprisingly resistant to peroxide stress. Microfilariae tolerated the direct addition of H2O2 to medium in which they were incubated at concentrations up to 50 microM, whereas adult worms survived the addition of 100 microM H2O2 and showed slightly impaired viability at 150 microM H2O2. Higher concentrations were lethal in both cases. This observation of differential susceptibility was reproducible when parasites were subjected to continuous generation of H2O2 via glucose/glucose oxidase. Microfilariae remained viable over a 4-hr period when challenged with concentrations which generated 20 microM H2O2 in the absence of parasites. Adults survived higher concentrations of glucose oxidase, which generated 200 microM H2O2 over the same time period. Under these conditions the parasites effectively countered the rate of peroxide generation by metabolising the product. Protein carbonyl formation was detectable at sublethal concentrations of glucose/glucose oxidase, but malonaldehyde formation was only detectable coincident with parasite death. The rate of H2O2 consumption by parasites was determined and showed that adult worms metabolised it at a rate 23x faster than microfilariae, expressed as activity per wet weight. Assessment of enzyme activities in parasite extracts demonstrated that H2O2 metabolism was effected principally by catalase activity, which was elevated in adult worms relative to microfilariae. Cytochrome c peroxidase activity was also detected and was roughly equivalent in both stages. Glutathione peroxidase and NADH/NADPH-dependent consumption of H2O2 were absent, and the rate of nonenzymic reduction of H2O2 coupled to glutathione oxidation did not contribute significantly to metabolism. Glutathione reductase activity and total glutathione content were equivalent in adults and microfilariae. This study illustrates that Brugia malayi are much more resistant to H2O2 than other filarial species examined to date and can effectively metabolise levels in excess of those potentially generated by activated leucocytes.

Animals↗
PubMed1995

T-cell activation and the balance of antibody isotypes in human lymphatic filariasis.

Human filarial infection presents a spectrum of clinical states with two major poles: asymptomatic microfilaraemia and amicrofilaraemic chronic disease. Microfilaremia is associated with a Th1-type tolerance, and maximal IgG4 antibodies, while elephantiasis patients react across a broad range of immune parameters. In this review, Rick Maizels and his colleagues discuss recent advances in the immunology of human filariasis and present a summary of their latest studies in an endemic area of Indonesia.

Journal Article↗
PubMed1995

Differential antibody isotype reactivity to specific antigens in human lymphatic filariasis: gp15/400 preferentially induces immunoglobulin E (IgE), IgG4, and IgG2.

Lymphatic filarial infection in humans is associated with a strong skewing of the immune response towards the TH2 arm, with prominent interleukin 4-producing cells and elevated levels of immunoglobulin G4 (IgG4) and IgE antibodies in peripheral blood. To determine how such a generalized TH2 imbalance governs responses to individual parasite antigens, the profiles of isotypes of antibodies to two recombinant proteins of Brugia spp. were studied. One molecule was the C-terminal portion of the filarial heat shock protein 70 (Bpa-26), representative of a cytoplasmic protein, and the second antigen was a single unit of the tandem repeats of a Brugia polypeptide (BpL-4), a secreted product which is prominently exposed to the immune system. Serum samples from 146 individuals resident in areas in which brugian filariasis is endemic were used, and it was found that whereas the levels of IgG1 and IgG3 responses to both Bpa-26 and BpL-4 were high, IgG4 and IgE antibodies to only BpL-4, not to Bpa-26, were prominent. Thus, an antigen which is chronically exposed to the immune system elicited a TH2-dependent isotype switch, as manifested by increased IgG4 and IgE responses. Moreover, IgG4 and IgE responses to BpL-4 showed a strong negative association, suggesting that mediators other than interleukin 4 must be responsible for such differential regulation of these two isotypes. When the data were analyzed as a function of clinical status, a striking association between elevated levels of IgG3 antibodies to Bpa-26 and manifestation of chronic obstructive disease was found; elephantiasis patients showed significantly higher levels of IgG3 antibodies to Bpa-26 than microfilaremics and asymptomatic amicrofilaremics. This indicates that an imbalance of isotypes of antibodies to particular filarial antigens might play a role in the pathogenesis of chronic disease.

Adolescent↗
PubMed1994

Extracellular and cytoplasmic CuZn superoxide dismutases from Brugia lymphatic filarial nematode parasites.

We have isolated full-length cDNAs encoding two distinct types of CuZn superoxide dismutases (SODs) from the filarial nematode parasite Brugia pahangi. The derived amino acid sequences suggested that one class of cDNAs represented a cytoplasmic form of SOD and the second class represented an extracellular (EC) variant. The predicted proteins were highly homologous to each other, but the sequence of the latter contained an additional 43 residues at the N terminus, the first 16 of which were markedly hydrophobic, and four potential sites for N-linked glycosylation. Western blotting (immunoblotting) with an antiserum to a partial SOD expressed in Escherichia coli revealed two proteins with estimated molecular masses of 19 and 29 kDa. Digestion with N-glycanase indicated that the latter protein corresponded to the EC form, as it possessed N-linked oligosaccharide chains at three sites, leaving a peptide backbone with an estimated molecular mass of 22 kDa, which was consistent with the additional 27 amino acids predicted from the cDNA sequence. Gel filtration indicated that both enzymes were dimeric in their native forms, in contrast to the human EC-SOD, which is tetrameric. Comparison of the primary structure of the parasite EC-SOD with that of the human EC enzyme revealed two major differences: the N-terminal extension of the parasite enzyme was shorter by 25 residues, and it also lacked the C-terminal charged extension which mediates binding to cell surface sulfated proteoglycans. Lavage of Mongolian jirds infected intraperitoneally with Brugia malayi resulted in the recovery of filarial CuZn SODs, principally the EC form, indicating that this form of SOD is secreted in vivo. This EC enzyme may contribute to parasite persistence by neutralizing superoxide generated by activated leukocytes, thus acting as both an antioxidant and an anti-inflammatory factor.

Amino Acid Sequence↗
PubMed1993

Immunological modulation and evasion by helminth parasites in human populations.

Helminth parasites are highly prevalent in human communities in developing countries. In an endemic area an infected individual may harbour parasitic worms for most of his or her life, and the ability of these infections to survive immunological attack has long been a puzzle. But new techniques are starting to expose the diverse mechanisms by which these agents modulate or evade their hosts' defences, creating a dynamic interaction between the human immune system and the parasite population.

Animals↗
PubMed1993

Differential expression of IgE and IgG4 specific antibody responses in asymptomatic and chronic human filariasis.

A population of 164 adult individuals resident in an area endemic for Brugia malayi lymphatic filariasis has been studied for humoral immune responses to filarial parasites. Antibody levels to Ag extracted from adult worms were determined for each of the IgG subclasses, for IgM and for IgE. The dominant isotype of antifilarial antibody was IgG4, which represented 88% of total IgG in asymptomatic microfilaremics, most of whom possessed 100 to 1000 micrograms/ml of specific antibody of this subclass (geometric mean 762 micrograms/ml). Patients with chronic disease (elephantiasis), who were generally amicrofilaremic, had substantially higher levels of IgG1, IgG2, and IgG3, but a 3.4-fold lower geometric mean level of specific IgG4 (222 micrograms/ml) than asymptomatics with or without microfilaremia. In contrast, specific IgE antibody levels in cases of elephantiasis were on average 4.5 times higher than those found in the asymptomatic carrier state. The majority of microfilaremics were therefore typified by extremely high specific IgG4 concentrations and relatively low IgE reactivities, whereas clinical cases tended to show the reverse relationship. The possible roles of these isotypes and the implications of changing IgG4/IgE balances in disease are discussed.

Adolescent↗