Ayman Hussein - Academia.edu (original) (raw)

Papers by Ayman Hussein

Molecular and Biochemical Parasitology, 1996

T-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from r... more T-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from rat pancreas has been purified to homogeneity and shown to be a glycoprotein of apparent molecular weight 68000, composed of one heavy and one light subunit, with respective molecular weights 43000 and 25000. At the optimum pH 8.0 the specific activity of the purified enzyme is 630 units/mg protein, with L-T-glutamyl-pnitroanilide as substrate (K m : 0.9 raM) and 20 mM glycylgiycine as acceptor. The enzyme is inactivated by the active-site modifying agent and glutamine analogue, 6-diazo-5-oxo-L-norleucine, through a specific and stoichiometric reaction with the light subunit (K i : 1.2 mM); both the inactivation and the modification of the light subunit are accelerated by maleate and prevented by S-methylglutathione. The enzyme is also inactivated by the fluorescent alkylating agent 5-iodoacetamidofluorescein, by specific and stoichiometric incorporation of the fluorescent moiety into the light subunit, which is likewise prevented by S-methylglutathione, but is unaffected by maleate. Antiserum to rat kidney T-glutamyltransferase cross-reacts with the pancreas enzyme in immunodiffusion and inhibits its activity in the p-nitroanilide assay. Despite structural, enzymological and immunological similarities between the pancreas and kidney enzymes, their amino acid compositions are markedly different. The rat pancreas enzyme shows an interesting ontological development, being present in minimal amounts in the fetus, and increasing dramatically on birth and during the following 2 days.

Molecular and Biochemical Parasitology, 1995

T-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from r... more T-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from rat pancreas has been purified to homogeneity and shown to be a glycoprotein of apparent molecular weight 68000, composed of one heavy and one light subunit, with respective molecular weights 43000 and 25000. At the optimum pH 8.0 the specific activity of the purified enzyme is 630 units/mg protein, with L-T-glutamyl-pnitroanilide as substrate (K m : 0.9 raM) and 20 mM glycylgiycine as acceptor. The enzyme is inactivated by the active-site modifying agent and glutamine analogue, 6-diazo-5-oxo-L-norleucine, through a specific and stoichiometric reaction with the light subunit (K i : 1.2 mM); both the inactivation and the modification of the light subunit are accelerated by maleate and prevented by S-methylglutathione. The enzyme is also inactivated by the fluorescent alkylating agent 5-iodoacetamidofluorescein, by specific and stoichiometric incorporation of the fluorescent moiety into the light subunit, which is likewise prevented by S-methylglutathione, but is unaffected by maleate. Antiserum to rat kidney T-glutamyltransferase cross-reacts with the pancreas enzyme in immunodiffusion and inhibits its activity in the p-nitroanilide assay. Despite structural, enzymological and immunological similarities between the pancreas and kidney enzymes, their amino acid compositions are markedly different. The rat pancreas enzyme shows an interesting ontological development, being present in minimal amounts in the fetus, and increasing dramatically on birth and during the following 2 days.

Molecular and Biochemical Parasitology, 2000

The tripeptide glutathione (GSH) plays an important role in the maintenance of the intracellular ... more The tripeptide glutathione (GSH) plays an important role in the maintenance of the intracellular thiol redox state and in detoxification processes. The intracellular GSH level depends on glutathione reductase as well as on GSH synthesis. The first and rate limiting step in the synthetic pathway is catalysed by γ-glutamylcysteine synthetase (γ-GCS). The γ-GCS was partially purified from the filarial parasite

Faseb Journal, 2007

The lifespan of Caenorhabditis elegans can be extended by the administration of synthetic superox... more The lifespan of Caenorhabditis elegans can be extended by the administration of synthetic superoxide dismutase/ catalase mimetics (SCMs) without any effects on development or fertility. Here we demonstrate that the mimetics, Euk-134 and Euk-8, confer resistance to the oxidative stress-inducing agent, paraquat and to thermal stress. The protective effects of the compounds are apparent with treatments either during development or during adulthood and are independent of an insulin/IGF-I-like signalling pathway also known to affect thermal and oxidative stress resistance. Worms exposed to the compounds do not induce a cellular stress response and no detrimental effects are observed.

Journal of Cardiovascular Electrophysiology, 2009

Introduction: The Hansen robotic system has only recently been used in the United States for cath... more Introduction: The Hansen robotic system has only recently been used in the United States for catheter ablation procedures in humans. Atrial fibrillation (AF) ablation may be performed utilizing this system. We report our management of complications with early experience of this system.Methods and Results: All 71 patients in whom the system was utilized were included. In all patients, a 2-operator technique was to be employed; one operator manipulates the ablation catheter via the robot and the other manipulates the circular mapping and intracardiac echocardiogram catheters. There was no procedure-related mortality. All vascular complications occurred in the first 25 procedures performed. There were 6 intraoperative procedural-related complications. These included significant vascular complications (n = 4), one of whom required iliac vein stenting, and 2 cardiac tamponade (one related to a pop-phenomenon)—successfully treated by pericardiocentesis. Early complications (n = 3) were 1 tamponade several hours post-procedure, 1 vascular complication, and 1 pericarditis. Late complications included 5 patients with severe pulmonary vein stenosis (all in first 27 patients) and 1 patient with gastroparesis. All complications were successfully managed without persistent morbidity and occurred earlier in our experience. This led to specific alterations in our vascular access and ablation techniques. These include the use of a longer 14 Fr sheath, through which the robotic sheath is more safely advanced. The choice of ablation catheter and titration of power, particularly when the catheter has a perpendicular orientation to the atrial wall, is also important.Conclusions: The suggested modifications may make the system easier to use with the potential to reduce complications.

Heart Rhythm, 2009

The purpose of this study was to evaluate the feasibility, safety, and outcomes of radiofrequency... more The purpose of this study was to evaluate the feasibility, safety, and outcomes of radiofrequency ablation of atrial fibrillation (AF) in patients with mechanical mitral valve replacement (MVR).

Journal of The American College of Cardiology, 2011

The purpose of this study was to evaluate the feasibility, safety, and outcomes of radiofrequency... more The purpose of this study was to evaluate the feasibility, safety, and outcomes of radiofrequency ablation of atrial fibrillation (AF) in patients with mechanical mitral valve replacement (MVR).

Molecular and Biochemical Parasitology, 2003

We describe the molecular cloning, expression and biochemical characterisation of recombinant for... more We describe the molecular cloning, expression and biochemical characterisation of recombinant forms of two secreted acetylcholinesterases from adult Dictyocaulus viviparus. The two variants (designated Dv-ACE-1 and Dv-ACE-2) were 613 and 615 amino acids long and showed 94.7% identity to one another. The highest level of identity to other cholinesterases was with ACE-2 of Caenorhabditis elegans. Dv-ACE-1 and Dv-ACE-2 showed 48.0 and 47.7% identity to C. elegans ACE-2 over 577 amino acids, respectively. The primary structure of both enzymes showed conservation of the catalytic triad and of a tryptophan residue known to be critical for the choline-binding site, but differed in the number of potential glycosylation sites and at one amino acid in the peripheral anionic site. Southern blotting and PCR experiments indicated that the genes encoding these enzymes are distinct. When expressed in Pichia pastoris, the enzymes were active, but differed subtly in their biochemical characteristics. Both enzymes exhibited a preference for acetylcholine as substrate, but differed in the extent of excess substrate inhibition and in their optimal pH for activity. The lack of an obvious carboxy-terminal membrane anchor and the presence of an insertion at the molecular surface were other features which, thus far, appear to be characteristic of parasite secreted acetylcholinesterases.

Experimental Parasitology, 2000

Experimental Parasitology, 1999

European Journal of Biochemistry, 2000

We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode p... more We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis. Here we describe the primary structure and enzymatic properties of a second secreted variant, termed AChE C after the designation of native AChE isoforms from this parasite. As for the former enzyme, AChE C is truncated at the carboxyl terminus in comparison with the Torpedo AChE, and three of the 14 aromatic residues that line the active site gorge are substituted by nonaromatic residues, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met).

Molecular and Biochemical Parasitology, 2002

Chemico-biological Interactions, 2005

Nematodes are unusual in that diverse molecular forms of acetylcholinesterase are the product of ... more Nematodes are unusual in that diverse molecular forms of acetylcholinesterase are the product of distinct genes. This is best characterised in the free living organism Caenorhabditis elegans, in which 3 genes are known to give rise to distinct enzymes, with a fourth likely to be non-functional. ACE-1 is an amphiphilic tetramer associated with a hydrophobic non-catalytic subunit, analogous to vertebrate T enzymes, whereas ACE-2 and ACE-3 are glycosylphosphatidylinositol-linked amphiphilic dimers. The different ace genes show distinct anatomical patterns of expression in muscles, sensory neurons and motor neurons, with only a few examples of coordinated expression. Clear homologues of ace-1 and ace-2 have now been isolated from a variety of parasitic nematodes, and the predicted proteins have very similar C-terminal amino acid sequences, implying an analogous means of anchorage to membranes. In addition to these membrane-bound enzymes, many parasitic nematodes which colonise mucosal surfaces secrete acetylcholinesterases to the external (host) environment. These hydrophilic enzymes are separately encoded in the genome, so that some parasites may thus have a total complement of six ace genes. The secretory enzymes have been characterised from the intestinal nematode Nippostrongylus brasiliensis and the lungworm Dictyocaulus viviparus. These show a number of common features, including a truncated C-terminus and an insertion at the molecular surface, when compared to other nematode acetylcholinesterases. Although the function of these enzymes has not been determined, they most likely alter host physiological responses to promote survival of the parasite.

Molecular and Biochemical Parasitology, 2002

A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongyl... more A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongylus brasiliensis has been isolated which shows 63 Á/64% identity to AChE B and AChE C, with a truncated carboxyl terminus and a short internal insertion relative to AChEs from other species. Three of the fourteen aromatic residues which line the active site gorge in Torpedo AChE are substituted by non-aromatic residues (Y70T, W279D and F288M). All three enzymes have 8 cysteine residues in conserved positions, including 6 which have been implicated in disulphide bonds in other AChEs. Phylogenetic analysis suggests that these enzymes form a distinct group which evolved after speciation and are most closely related to ACE-2 of Caenorhabditis elegans . Recombinant AChE A secreted by Pichia pastoris was monomeric and hydrophilic, with a substrate preference for acetylthiocholine and negligible activity against butyrylthiocholine. A model structure of AChE A built from the coordinates of the Torpedo californica AChE suggests that W345 (F331 in Torpedo ) limits the docking of butyrylcholine. This model is consistent with mutational analysis of the nematode enzymes. Expression of AChE A is regulated at the transcriptional level independently of the other 2 secreted variants, with maximal expression by fourth stage larvae and young adult worms. These enzymes thus appear to represent an unusual family of AChEs with conserved structural features which operate outside the normal boundaries of known functions in regulation of endogenous neurotransmitter activity. #

Molecular and Biochemical Parasitology, 2004

Infective larvae and adult stage Trichinella spiralis secrete a protein homologous to prosaposin,... more Infective larvae and adult stage Trichinella spiralis secrete a protein homologous to prosaposin, the precursor of sphingolipid activator proteins (saposins) A-D originally defined in vertebrates. The protein contains four saposin domains, with the six cysteine residues which form the three intramolecular disulphide bonds in close register in each case. It differs substantially from vertebrate prosaposins in the N-terminal prodomain, the region separating saposins A and B, and completely lacks the C-terminal domain which has been demonstrated to be essential for lysosomal targetting in these organisms. The protein is secreted in unprocessed form with an estimated mass of 56 kDa, and contains a single N-linked glycan which is bound by the monoclonal antibody NIM-M1, characteristic of the TSL-1 antigens which are capped by tyvelose (3,6-dideoxy-d-arabinohexose). Immuno-electron microscopy localised the protein to membrane-bound vesicles and more complex multi-lamellar organelles in diverse tissues including the hypodermis, intestine and stichosomes, although it was absent from the dense-core secretory granules typical of the latter. Possible functions of a secreted prosaposin are discussed.

Molecular and Biochemical Parasitology, 1997

Acetylcholinesterase (AChE) activity secreted by Nippostrongylus brasiliensis was resolved by suc... more 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.

The quadrupole moments were determined by means of Coulomb excitation experiments. Highly enriche... more The quadrupole moments were determined by means of Coulomb excitation experiments. Highly enriched targets were bombarded with 53 MeV ¹⁶O ions. Elastically and inelastically scattered ions were detected in a split pole spectrograph. The results are in good agreement with muonic x-ray data.

Molecular and Biochemical Parasitology, 1996

T-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from r... more T-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from rat pancreas has been purified to homogeneity and shown to be a glycoprotein of apparent molecular weight 68000, composed of one heavy and one light subunit, with respective molecular weights 43000 and 25000. At the optimum pH 8.0 the specific activity of the purified enzyme is 630 units/mg protein, with L-T-glutamyl-pnitroanilide as substrate (K m : 0.9 raM) and 20 mM glycylgiycine as acceptor. The enzyme is inactivated by the active-site modifying agent and glutamine analogue, 6-diazo-5-oxo-L-norleucine, through a specific and stoichiometric reaction with the light subunit (K i : 1.2 mM); both the inactivation and the modification of the light subunit are accelerated by maleate and prevented by S-methylglutathione. The enzyme is also inactivated by the fluorescent alkylating agent 5-iodoacetamidofluorescein, by specific and stoichiometric incorporation of the fluorescent moiety into the light subunit, which is likewise prevented by S-methylglutathione, but is unaffected by maleate. Antiserum to rat kidney T-glutamyltransferase cross-reacts with the pancreas enzyme in immunodiffusion and inhibits its activity in the p-nitroanilide assay. Despite structural, enzymological and immunological similarities between the pancreas and kidney enzymes, their amino acid compositions are markedly different. The rat pancreas enzyme shows an interesting ontological development, being present in minimal amounts in the fetus, and increasing dramatically on birth and during the following 2 days.

Molecular and Biochemical Parasitology, 1995

T-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from r... more T-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from rat pancreas has been purified to homogeneity and shown to be a glycoprotein of apparent molecular weight 68000, composed of one heavy and one light subunit, with respective molecular weights 43000 and 25000. At the optimum pH 8.0 the specific activity of the purified enzyme is 630 units/mg protein, with L-T-glutamyl-pnitroanilide as substrate (K m : 0.9 raM) and 20 mM glycylgiycine as acceptor. The enzyme is inactivated by the active-site modifying agent and glutamine analogue, 6-diazo-5-oxo-L-norleucine, through a specific and stoichiometric reaction with the light subunit (K i : 1.2 mM); both the inactivation and the modification of the light subunit are accelerated by maleate and prevented by S-methylglutathione. The enzyme is also inactivated by the fluorescent alkylating agent 5-iodoacetamidofluorescein, by specific and stoichiometric incorporation of the fluorescent moiety into the light subunit, which is likewise prevented by S-methylglutathione, but is unaffected by maleate. Antiserum to rat kidney T-glutamyltransferase cross-reacts with the pancreas enzyme in immunodiffusion and inhibits its activity in the p-nitroanilide assay. Despite structural, enzymological and immunological similarities between the pancreas and kidney enzymes, their amino acid compositions are markedly different. The rat pancreas enzyme shows an interesting ontological development, being present in minimal amounts in the fetus, and increasing dramatically on birth and during the following 2 days.

Molecular and Biochemical Parasitology, 2000

The tripeptide glutathione (GSH) plays an important role in the maintenance of the intracellular ... more The tripeptide glutathione (GSH) plays an important role in the maintenance of the intracellular thiol redox state and in detoxification processes. The intracellular GSH level depends on glutathione reductase as well as on GSH synthesis. The first and rate limiting step in the synthetic pathway is catalysed by γ-glutamylcysteine synthetase (γ-GCS). The γ-GCS was partially purified from the filarial parasite

Faseb Journal, 2007

The lifespan of Caenorhabditis elegans can be extended by the administration of synthetic superox... more The lifespan of Caenorhabditis elegans can be extended by the administration of synthetic superoxide dismutase/ catalase mimetics (SCMs) without any effects on development or fertility. Here we demonstrate that the mimetics, Euk-134 and Euk-8, confer resistance to the oxidative stress-inducing agent, paraquat and to thermal stress. The protective effects of the compounds are apparent with treatments either during development or during adulthood and are independent of an insulin/IGF-I-like signalling pathway also known to affect thermal and oxidative stress resistance. Worms exposed to the compounds do not induce a cellular stress response and no detrimental effects are observed.

Journal of Cardiovascular Electrophysiology, 2009

Introduction: The Hansen robotic system has only recently been used in the United States for cath... more Introduction: The Hansen robotic system has only recently been used in the United States for catheter ablation procedures in humans. Atrial fibrillation (AF) ablation may be performed utilizing this system. We report our management of complications with early experience of this system.Methods and Results: All 71 patients in whom the system was utilized were included. In all patients, a 2-operator technique was to be employed; one operator manipulates the ablation catheter via the robot and the other manipulates the circular mapping and intracardiac echocardiogram catheters. There was no procedure-related mortality. All vascular complications occurred in the first 25 procedures performed. There were 6 intraoperative procedural-related complications. These included significant vascular complications (n = 4), one of whom required iliac vein stenting, and 2 cardiac tamponade (one related to a pop-phenomenon)—successfully treated by pericardiocentesis. Early complications (n = 3) were 1 tamponade several hours post-procedure, 1 vascular complication, and 1 pericarditis. Late complications included 5 patients with severe pulmonary vein stenosis (all in first 27 patients) and 1 patient with gastroparesis. All complications were successfully managed without persistent morbidity and occurred earlier in our experience. This led to specific alterations in our vascular access and ablation techniques. These include the use of a longer 14 Fr sheath, through which the robotic sheath is more safely advanced. The choice of ablation catheter and titration of power, particularly when the catheter has a perpendicular orientation to the atrial wall, is also important.Conclusions: The suggested modifications may make the system easier to use with the potential to reduce complications.

Heart Rhythm, 2009

The purpose of this study was to evaluate the feasibility, safety, and outcomes of radiofrequency... more The purpose of this study was to evaluate the feasibility, safety, and outcomes of radiofrequency ablation of atrial fibrillation (AF) in patients with mechanical mitral valve replacement (MVR).

Journal of The American College of Cardiology, 2011

The purpose of this study was to evaluate the feasibility, safety, and outcomes of radiofrequency... more The purpose of this study was to evaluate the feasibility, safety, and outcomes of radiofrequency ablation of atrial fibrillation (AF) in patients with mechanical mitral valve replacement (MVR).

Molecular and Biochemical Parasitology, 2003

We describe the molecular cloning, expression and biochemical characterisation of recombinant for... more We describe the molecular cloning, expression and biochemical characterisation of recombinant forms of two secreted acetylcholinesterases from adult Dictyocaulus viviparus. The two variants (designated Dv-ACE-1 and Dv-ACE-2) were 613 and 615 amino acids long and showed 94.7% identity to one another. The highest level of identity to other cholinesterases was with ACE-2 of Caenorhabditis elegans. Dv-ACE-1 and Dv-ACE-2 showed 48.0 and 47.7% identity to C. elegans ACE-2 over 577 amino acids, respectively. The primary structure of both enzymes showed conservation of the catalytic triad and of a tryptophan residue known to be critical for the choline-binding site, but differed in the number of potential glycosylation sites and at one amino acid in the peripheral anionic site. Southern blotting and PCR experiments indicated that the genes encoding these enzymes are distinct. When expressed in Pichia pastoris, the enzymes were active, but differed subtly in their biochemical characteristics. Both enzymes exhibited a preference for acetylcholine as substrate, but differed in the extent of excess substrate inhibition and in their optimal pH for activity. The lack of an obvious carboxy-terminal membrane anchor and the presence of an insertion at the molecular surface were other features which, thus far, appear to be characteristic of parasite secreted acetylcholinesterases.

Experimental Parasitology, 2000

Experimental Parasitology, 1999

European Journal of Biochemistry, 2000

We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode p... more We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis. Here we describe the primary structure and enzymatic properties of a second secreted variant, termed AChE C after the designation of native AChE isoforms from this parasite. As for the former enzyme, AChE C is truncated at the carboxyl terminus in comparison with the Torpedo AChE, and three of the 14 aromatic residues that line the active site gorge are substituted by nonaromatic residues, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met).

Molecular and Biochemical Parasitology, 2002

Chemico-biological Interactions, 2005

Nematodes are unusual in that diverse molecular forms of acetylcholinesterase are the product of ... more Nematodes are unusual in that diverse molecular forms of acetylcholinesterase are the product of distinct genes. This is best characterised in the free living organism Caenorhabditis elegans, in which 3 genes are known to give rise to distinct enzymes, with a fourth likely to be non-functional. ACE-1 is an amphiphilic tetramer associated with a hydrophobic non-catalytic subunit, analogous to vertebrate T enzymes, whereas ACE-2 and ACE-3 are glycosylphosphatidylinositol-linked amphiphilic dimers. The different ace genes show distinct anatomical patterns of expression in muscles, sensory neurons and motor neurons, with only a few examples of coordinated expression. Clear homologues of ace-1 and ace-2 have now been isolated from a variety of parasitic nematodes, and the predicted proteins have very similar C-terminal amino acid sequences, implying an analogous means of anchorage to membranes. In addition to these membrane-bound enzymes, many parasitic nematodes which colonise mucosal surfaces secrete acetylcholinesterases to the external (host) environment. These hydrophilic enzymes are separately encoded in the genome, so that some parasites may thus have a total complement of six ace genes. The secretory enzymes have been characterised from the intestinal nematode Nippostrongylus brasiliensis and the lungworm Dictyocaulus viviparus. These show a number of common features, including a truncated C-terminus and an insertion at the molecular surface, when compared to other nematode acetylcholinesterases. Although the function of these enzymes has not been determined, they most likely alter host physiological responses to promote survival of the parasite.

Molecular and Biochemical Parasitology, 2002

A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongyl... more A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongylus brasiliensis has been isolated which shows 63 Á/64% identity to AChE B and AChE C, with a truncated carboxyl terminus and a short internal insertion relative to AChEs from other species. Three of the fourteen aromatic residues which line the active site gorge in Torpedo AChE are substituted by non-aromatic residues (Y70T, W279D and F288M). All three enzymes have 8 cysteine residues in conserved positions, including 6 which have been implicated in disulphide bonds in other AChEs. Phylogenetic analysis suggests that these enzymes form a distinct group which evolved after speciation and are most closely related to ACE-2 of Caenorhabditis elegans . Recombinant AChE A secreted by Pichia pastoris was monomeric and hydrophilic, with a substrate preference for acetylthiocholine and negligible activity against butyrylthiocholine. A model structure of AChE A built from the coordinates of the Torpedo californica AChE suggests that W345 (F331 in Torpedo ) limits the docking of butyrylcholine. This model is consistent with mutational analysis of the nematode enzymes. Expression of AChE A is regulated at the transcriptional level independently of the other 2 secreted variants, with maximal expression by fourth stage larvae and young adult worms. These enzymes thus appear to represent an unusual family of AChEs with conserved structural features which operate outside the normal boundaries of known functions in regulation of endogenous neurotransmitter activity. #

Molecular and Biochemical Parasitology, 2004

Infective larvae and adult stage Trichinella spiralis secrete a protein homologous to prosaposin,... more Infective larvae and adult stage Trichinella spiralis secrete a protein homologous to prosaposin, the precursor of sphingolipid activator proteins (saposins) A-D originally defined in vertebrates. The protein contains four saposin domains, with the six cysteine residues which form the three intramolecular disulphide bonds in close register in each case. It differs substantially from vertebrate prosaposins in the N-terminal prodomain, the region separating saposins A and B, and completely lacks the C-terminal domain which has been demonstrated to be essential for lysosomal targetting in these organisms. The protein is secreted in unprocessed form with an estimated mass of 56 kDa, and contains a single N-linked glycan which is bound by the monoclonal antibody NIM-M1, characteristic of the TSL-1 antigens which are capped by tyvelose (3,6-dideoxy-d-arabinohexose). Immuno-electron microscopy localised the protein to membrane-bound vesicles and more complex multi-lamellar organelles in diverse tissues including the hypodermis, intestine and stichosomes, although it was absent from the dense-core secretory granules typical of the latter. Possible functions of a secreted prosaposin are discussed.

Molecular and Biochemical Parasitology, 1997

Acetylcholinesterase (AChE) activity secreted by Nippostrongylus brasiliensis was resolved by suc... more 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.

The quadrupole moments were determined by means of Coulomb excitation experiments. Highly enriche... more The quadrupole moments were determined by means of Coulomb excitation experiments. Highly enriched targets were bombarded with 53 MeV ¹⁶O ions. Elastically and inelastically scattered ions were detected in a split pole spectrograph. The results are in good agreement with muonic x-ray data.