An-Najah Blogs :: Publication http://blogs.najah.edu/author/ashussein An-Najah Blogs :: Publication en-us Wed, 20 Sep 2017 21:16:13 IDT Wed, 20 Sep 2017 21:16:13 IDT webmaster@najah.edu webmaster@najah.edu Properties and characterization of γ-glutamyl transpeptidase from Onchocerca volvulushttp://blogs.najah.edu/staff/ashussein/article/Properties-and-characterization-of-gamma-glutamyl-transpeptidase-from-Onchocerca-volvulusPublished Articles-glutamyl transpeptidase GGT from Onchocerca volvulus is involved in the oxidative stress The enzyme catalyzes the breakdown of glutathione GSH and thus provide the parasite with cysteine for the synthesis of GSH and other amnio acids The enzyme which was highly purified from Onchocerca volvulus is found to be membrane-bound with a specific activity of 100 U mg-1 and a molecular mass of 68 KDa The apparent Km-values for the -glutamyl donor L-glutamic acid -4-nitroanilide is 0023 0013 mM The data presented in this study showed that various amino acids and dipeptides served for the -glutamyl moieties of the enzyme reaction products and showed Km values in the mM range Acivicin was an irreversible inhibitor of the enzyme with a pseudo-first-order kinetics kmax of 034 0004 min1 and KI = 059 004 mM These finding indicate the physiological role of the GGT of this parasite nematode in the catabolism of GSH Further studies are required to investigate the use of this enzyme as a potential target for the development of chemotherapy against O volvulus Keywords: -glutamyl transpeptidase Onchocerca volvulus acivicin GlutathioneAssociation between Factor V Leiden Mutation and Poor Pregnancy Outcomes among Palestinian Womenhttp://blogs.najah.edu/staff/ashussein/article/Association-between-Factor-V-Leiden-Mutation-and-Poor-Pregnancy-Outcomes-among-Palestinian-WomenPublished ArticlesPregnancy is a hypercoagulable state with increased tendency for thrombus formation a condition that is increased when combined with acquired or inherited risk factors that lead to thrombophilia Among the inherited risk factors is Factor V Leiden mutation an autosomal dominant trait with reduced penetrance The mutation seems to be associated with different poor pregnancy outcomes including recurrent miscarriages In the present study we performed a case-control study to investigate the association between the Leiden mutation and poor pregnancy outcome among the Palestinian population in the West bank region of Palestine The study included 145 subjects with recurrent miscarriages and 205 matched control subjects with successful pregnancies who experienced normal delivery and no apparent complications Leiden mutation was detected in 41 of the145 study subjects 282 and in 24 of the 205 control subjects 117 Subjects homozygous for the mutant allele were identified only among the test and not the control group Data analysis indicates a significant association between the mutant allele and recurrent miscarriages p-value 005 Furthermore this association is significant between the mutant haplotype with either early or late miscarriages compared to control group Results show also that a significantly higher frequency of factor V leiden polymorphism among either primary or secondary aborters compared to control groups The odds ratio for the primary aborters was 971and p0001 while the ratio for the secondary aborters was 114 and p-value =0007 In conclusion these results provide evidence for a significant correlation between recurrent miscarriages and Factor V mutation in our populationPrevalence of intestinal parasites among school children in northern districts of West Bank- Palestinehttp://blogs.najah.edu/staff/ashussein/article/Prevalence-of-intestinal-parasites-among-school-children-in-northern-districts-of-West-Bank--PalestinePublished ArticlesParasite infections are amongst the most common infections worldwide The objectives of this study were to assess the prevalence of intestinal parasite infections in northern districts of West Bank Palestine and to determine associated sociodemographic factors The study involved a random sample of 735 school children of mean age 95 years old from rural and urban areas Fecal samples were collected from participants alongside a questionnaire about their demographic and hygiene habits Microscopy and PCR methods were performed to screen for protozoan and helminths parasites The overall prevalence was 222 The rates of infections with ameba Giardia intestinalis Entrobius vermicularis and Ascaris lumbricoides were 97 41 16 and 38 respectively Real-time PCR was performed to differentiate between Entamoeba histolytica and E dispar Results showed that 14 of samples positive with microscopy for ameba were positive for E histolytica There was no significant association between sex and rates of infections P-value005 There were however significant association between parasite infections and parents education place of residence washing hands habits P-value005 No significant assiciation was found with number of family members or eating in school canteens P-value005 The study concludes endemic intestinal parasite infections in West Bank and thus recommends intervention programs including health education and sanitationCryptosporidium parvum causes gastroenteritis epidemics in the Nablus region of Palestinehttp://blogs.najah.edu/staff/ashussein/article/Cryptosporidium-parvum-causes-gastroenteritis-epidemics-in-the-Nablus-region-of-PalestinePublished ArticlesA total of 30 fecal samples collected from individuals admitted to a local hospital in Nablus city in Palestine with gastroenteritis symptoms plus 5 fecal samples from healthy individuals living in the same area were screened for the presence of Cryptosporidium spp by microscopic analysis using malachite green negative staining Molecular techniques were used to confirm the microscopic identification Of these all the 30 samples from individuals with gastroenteritis symptoms were positive by both techniques No other parasites were found in the fecal material of patients or healthy individuals To explore the source of the outbreak water was collected from various reservoirs and springs which supply the city with drinking water Al-Qaryoon water spring was also found to be contaminated with Cryptosporidium using both microscopic and molecular analysis No other water resources was found to be contaminated Genotyping analysis of Cryptosporidium oocysts using PCR-RFLP technique identified the parasite as C parvum This study is the first of its type in the country and it reports the presence of cryptosporidiosis in the regionAyman S. HUSSEIN, Sami K. ABDEL-HAFEZ and Ahmad M. KHALIL. Isolation and characterization of excretory/secretory products from in vitro developmental stages of Echinococcus granulosus. Jpn J. Parasitol., (1994) vol. 43. No. 2, 82-89.http://blogs.najah.edu/staff/ashussein/article/Ayman-S-HUSSEIN-Sami-K-ABDEL-HAFEZ--and-Ahmad-M-KHALIL-Isolation-and-characterization-of-excretorysecretory-products-from-in-vitro-developmental-stages-of-Echinococcus-granulosus-Jpn-J-Parasitol-1994-vol-43-No-2-82-89Published ArticlesExcretorysecretory ES products of Echinococcus granulosus were prepared from presentation stages PS1 PS2 PS3 and PS4 and one segmented S5 stage which corresponded to 1- 5- 12- 26- and 43-day old in vitro cultured parasites respectively Cultures were initiated from protoscolices collected from hydatid cysts of indigenous Jordanian ewes The ES products were characterized using SDS-PAGE with silver Comassie-blue and PAS staining as well as immunobltting with sera from hydatid patients and immunized mice Younger cultured stages yielded small quantities of ES materials compared to the banding PS4 and segmented S5 stages The ES products from various stages of cultured parasites were heterogeneous and contained 15-28 protein bands and 3-6-PAS bands A total of 13 protein bands were shared among the ES products of various cultured stages 11 of which were also shared with crude sheep hydatid fluid CSHF tegumental and somatic antigens The ES products of PS1-S5 stages and CSHF shared 3 PAS bands All the immunoreactive components of ES products except for a band of 144 KDa were detected by sera of unicellular hydatid Jordanian patients and were also reactive to sera of patients infected with E multilocularisHussein AS, Walter RD. Purification and characterization of gamma-glutamylcysteine synthetase from Ascaris suum. Mol Biochem Parasitol. 1995 Jun;72(1-2):57-64.http://blogs.najah.edu/staff/ashussein/article/Hussein-AS-Walter-RD-Purification-and-characterization-of-gamma-glutamylcysteine-synthetase-from-Ascaris-suum-Mol-Biochem-Parasitol-1995-Jun721-257-64Published ArticlesWe have purified and characterized the Ascaris suum gamma-glutamylcysteine synthetase the rate-limiting step in the glutathione biosynthesis The purified enzyme exhibited a specific activity of 18 U mg protein-1 Estimation of the molecular mass of the native enzyme by FPLC on Superdex S-200 revealed the presence of two enzyme activity peaks corresponding to molecular masses of 100 and 70 kDa The higher-molecular-mass component could be dissociated by repeated gel filtration into the 70-kDa protein which is the enzymatically active subunit The apparent Km values of the A suum enzyme for L-aminobutyrate L-cysteine and L-glutamate were 031 041 and 094 mM respectively DL-Buthionine-SR-sulfoximine and cystamine showed time-dependent irreversible inhibitory effects on the A suum enzyme activity with Ki values of 005 and 111 microM respectively The Ki values for the corresponding enzyme from rat kidney with DL-buthionine-SR-sulfoximine and cystamine were 719 and 222 microM respectively The time of half-inactivation of the enzyme at infinite concentration of DL-buthionine-SR-sulfoximine tau 50 was determined to be 31 and 134 min for the parasite and mammalian enzymes respectively For cystamine a tau 50 value of 332 min for the A suum gamma-glutamylcysteine synthetase was determined while a value of 2 min in case of rat kidney enzyme was found The A suum enzyme activity was competitively inhibited by glutathione with a Ki value of 011 mMHussein AS, Walter RD. Inhibition of glutathione synthesis of Ascaris suum by buthionine sulfoximine. Parasitol Res. 1996;82(4):372-4.http://blogs.najah.edu/staff/ashussein/article/Hussein-AS-Walter-RD-Inhibition-of-glutathione-synthesis-of-Ascaris-suum-by-buthionine-sulfoximine-Parasitol-Res-1996824372-4Published ArticlesWe investigated the effect of DL-buthionine-SR-sulfoximine BSO a selective glutathione GSH-depleting agent on the GSH synthesis of Ascaris suum The GSH concentrations of the reproductive and muscle tissues of A suum were determined to be 85 - 03 and 143 - 13 n = 3 nmolmg protein respectively After treatment of the parasites with 10 microM BSO for 24 h the GSH content of the reproductive tissue of A suum was totally depleted as compared with that of untreated controls However the GSH levels of the muscle tissue were reduced to only 50 after treatment of the worms for 24 h with 10 microM BSO Exogenous GSH had no significant effect on the GSH level of the parasites when the worms were incubated for 4 h in RPMI 1640 medium supplemented with 1 mM GSH In the presence of exogenous GSH BSO was less effective in depleting the GSH levels of the parasites which may indicate that the parasites can replenish their GSH levels GSH depletion which has been discussed as being therapeutically effective when normal and tumor cells or parasites have markedly different requirements for GSH may have applications in the development of drugs against nematode infectionsHussein AS, Walter RD. Purification and characterization of gamma-glutamyl transpeptidase from Ascaris suum. Mol Biochem Parasitol. 1996 Apr;77(1):41-7.http://blogs.najah.edu/staff/ashussein/article/Hussein-AS-Walter-RD-Purification-and-characterization-of-gamma-glutamyl-transpeptidase-from-Ascaris-suum-Mol-Biochem-Parasitol-1996-Apr77141-7Published Articlesgamma-Glutamyl transpeptidase which is of central importance in the degradation of glutathione was purified from Ascaris suum to apparent homogeneity The enzyme was found to be a predominantly membrane-bound protein and was solubilized by Triton X-100 The purified enzyme which exhibits a specific activity of 1009 U mg protein-1 showed a molecular mass of 70 kDa and was found to be composed of two non-identical subunits of molecular mass 43 and 30 kDa Concerning the kinetic properties of the enzyme the data presented in this study showed that various amino acids and dipeptides with L-configuration served as acceptors for the gamma-glutamyl moieties of the enzyme reaction products and showed Km-values in the mM range The apparent Km-value for the gamma-glutamyl donor L-glutamyl-gamma-7-amido-4-methylcoumarin of the enzyme was found to be 003 mM L- and D-serine in combination with borate ions were competitive inhibitors of the enzyme activity with Ki-values of 030 and 061 mM respectively Acivicin was an irreversible inhibitor of the enzyme with a Ki-value of 042 mM and with a pseudo-first-order kinetics kinact of 018 min-1 In vitro treatment of the adult A suum with acivicin resulted in a dose-dependent inhibition of the enzyme activity and an increase of the glutathione levels These findings indicate the physiological role of the gamma-glutamyl transpeptidase of this parasitic nematode in the catabolism of glutathioneGrigg ME, Tang L, Hussein AS, Selkirk ME. Purification and properties of monomeric (G1) forms of acetylcholinesterase secreted by Nippostrongylus brasiliensis. Mol Biochem Parasitol. 1997 Dec 15;90(2):513-24.http://blogs.najah.edu/staff/ashussein/article/Grigg-ME-Tang-L-Hussein-AS-Selkirk-ME-Purification-and-properties-of-monomeric-G1-forms-of-acetylcholinesterase-secreted-by-Nippostrongylus-brasiliensis-Mol-Biochem-Parasitol-1997-Dec-15902513-24Published ArticlesAcetylcholinesterase AChE activity secreted by Nippostrongylus brasiliensis was resolved by sucrose density centrifugation and gel permeation chromatography in single peaks estimated at 43 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 bis4-allyldimethylammoniumphenylpentan-3-one dibromide with inhibitor concentration reducing initial activity by 50 IC50 between 01 and 08 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 inhibitionSelkirk, ME, Hussein, AS, Russel, W., Grigg, ME., Chacon, MR., Smith, AM., Henson, S. and Tippins, JR. Secretory of acetylcholinesterases from the nematode parasite Nippostrongylus brasiliensis: properties and implications for mucosal immunity. Structure and function of cholinesterase and related Proteins. Ed Doctor et al., Plenum Press, New York, 1998http://blogs.najah.edu/staff/ashussein/article/Selkirk-ME-Hussein-AS-Russel-W-Grigg-ME-Chacon-MR-Smith-AM-Henson-S-and-Tippins-JR-Secretory-of-acetylcholinesterases-from-the-nematode-parasite-Nippostrongylus-brasiliensis-properties-and-implications-for-mucosal-immunity-Structure-and-function-of-cholinesterase-and-related--Proteins-Ed-Doctor-et-al-Plenum-Press-New-York-1998Published ArticlesNo abstractHussein AS, Grigg ME, Selkirk ME. Nippostrongylus brasiliensis: characterisation of a somatic amphiphilic acetylcholinesterase with properties distinct from the secreted enzymes. Exp Parasitol. 1999 Feb;91(2):144-50http://blogs.najah.edu/staff/ashussein/article/Hussein-AS-Grigg-ME-Selkirk-ME-Nippostrongylus-brasiliensis-characterisation-of-a-somatic-amphiphilic-acetylcholinesterase-with-properties-distinct-from-the-secreted-enzymes-Exp-Parasitol-1999-Feb912144-50Published ArticlesWe have previously determined that Nippostrongylus brasiliensis secretes three monomeric nonamphiphilic G1na variants of acetylcholinesterase AChE with broadly similar properties In this study we have examined AChE expression in somatic extracts of N brasiliensis and report the identification of an additional enzyme which is not secreted The enzyme was resolved by sucrose density gradient centrifugation with a sedimentation coefficient of 102 S which was shifted to 94 S in the presence of Triton X-100 identifying the enzyme as a tetrameric amphiphilic G4a form The amphiphilic properties of this enzyme were confirmed by charge-shift electrophoresis in which migration was accelerated by interaction with sodium deoxycholate The enzyme showed low activity with butyrylthiocholine and a Michaelis constant of 91 - 13 microM for acetylthiocholine was determined It was highly sensitive to the AChE-specific inhibitor bis 4-allyldimethylammoniumphenylpentan-3-one dibromide with an IC50 of 65 - 04 microM but was also inhibited by the butyrylcholinesterase-specific inhibitor tetramonoisopropylpyrophosphortetramide albeit with a higher IC50 of 465 - 61 microM This enzyme can therefore be distinguished from the secreted AChEs by its amphiphilic properties sedimentation in sucrose gradients and sensitivity to cholinesterase inhibitorsHussein AS, Chacón MR, Smith AM, Tosado-Acevedo R, Selkirk ME. Cloning, expression, and properties of a nonneuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis. J Biol Chem. 1999 Apr 2;274(14):9312-9.http://blogs.najah.edu/staff/ashussein/article/Hussein-AS-Chacoacuten-MR-Smith-AM-Tosado-Acevedo-R-Selkirk-ME-Cloning-expression-and-properties-of-a-nonneuronal-secreted-acetylcholinesterase-from-the-parasitic-nematode-Nippostrongylus-brasiliensis-J-Biol-Chem-1999-Apr-2274149312-9-2Published ArticlesWe have isolated a full-length cDNA encoding an acetylcholinesterase secreted by the nematode parasite Nippostrongylus brasiliensis The predicted protein is truncated in comparison with acetylcholinesterases from other organisms such that the carboxyl terminus aligns closely to the end of the catalytic domain of the vertebrate enzymes The residues in the catalytic triad are conserved as are the six cysteines which form the three intramolecular disulfide bonds Three of the fourteen aromatic residues which line the active site gorge in the Torpedo enzyme are substituted by nonaromatic residues corresponding to Tyr-70 Thr Trp-279 Asn and Phe-288 Met High level expression was obtained via secretion from Pichia pastoris The purified enzyme behaved as a monomeric hydrophilic species Although of invertebrate origin and possessing the above substitutions in the active site gorge residues the enzyme efficiently hydrolyzed acetylthiocholine and showed minimal activity against butyrylthiocholine It displayed excess substrate inhibition with acetylthiocholine at concentrations over 2 5 mM and was highly sensitive to both active site and peripheral site inhibitors Northern blot analysis indicated a progressive increase in mRNA for AChE B in parasites isolated from 6 days postinfection Hussein AS, Chacón MR, Smith AM, Tosado-Acevedo R, Selkirk ME. Cloning, expression, and properties of a nonneuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis. J Biol Chem. 1999 Apr 2;274(14):9312-9.http://blogs.najah.edu/staff/ashussein/article/Hussein-AS-Chacoacuten-MR-Smith-AM-Tosado-Acevedo-R-Selkirk-ME-Cloning-expression-and-properties-of-a-nonneuronal-secreted-acetylcholinesterase-from-the-parasitic-nematode-Nippostrongylus-brasiliensis-J-Biol-Chem-1999-Apr-2274149312-9-1Published ArticlesWe have isolated a full-length cDNA encoding an acetylcholinesterase secreted by the nematode parasite Nippostrongylus brasiliensis The predicted protein is truncated in comparison with acetylcholinesterases from other organisms such that the carboxyl terminus aligns closely to the end of the catalytic domain of the vertebrate enzymes The residues in the catalytic triad are conserved as are the six cysteines which form the three intramolecular disulfide bonds Three of the fourteen aromatic residues which line the active site gorge in the Torpedo enzyme are substituted by nonaromatic residues corresponding to Tyr-70 Thr Trp-279 Asn and Phe-288 Met High level expression was obtained via secretion from Pichia pastoris The purified enzyme behaved as a monomeric hydrophilic species Although of invertebrate origin and possessing the above substitutions in the active site gorge residues the enzyme efficiently hydrolyzed acetylthiocholine and showed minimal activity against butyrylthiocholine It displayed excess substrate inhibition with acetylthiocholine at concentrations over 2 5 mM and was highly sensitive to both active site and peripheral site inhibitors Northern blot analysis indicated a progressive increase in mRNA for AChE B in parasites isolated from 6 days postinfection Hussein AS, Chacón MR, Smith AM, Tosado-Acevedo R, Selkirk ME. Cloning, expression, and properties of a nonneuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis. J Biol Chem. 1999 Apr 2;274(14):9312-9.http://blogs.najah.edu/staff/ashussein/article/Hussein-AS-Chacoacuten-MR-Smith-AM-Tosado-Acevedo-R-Selkirk-ME-Cloning-expression-and-properties-of-a-nonneuronal-secreted-acetylcholinesterase-from-the-parasitic-nematode-Nippostrongylus-brasiliensis-J-Biol-Chem-1999-Apr-2274149312-9Published ArticlesWe have isolated a full-length cDNA encoding an acetylcholinesterase secreted by the nematode parasite Nippostrongylus brasiliensis The predicted protein is truncated in comparison with acetylcholinesterases from other organisms such that the carboxyl terminus aligns closely to the end of the catalytic domain of the vertebrate enzymes The residues in the catalytic triad are conserved as are the six cysteines which form the three intramolecular disulfide bonds Three of the fourteen aromatic residues which line the active site gorge in the Torpedo enzyme are substituted by nonaromatic residues corresponding to Tyr-70 Thr Trp-279 Asn and Phe-288 Met High level expression was obtained via secretion from Pichia pastoris The purified enzyme behaved as a monomeric hydrophilic species Although of invertebrate origin and possessing the above substitutions in the active site gorge residues the enzyme efficiently hydrolyzed acetylthiocholine and showed minimal activity against butyrylthiocholine It displayed excess substrate inhibition with acetylthiocholine at concentrations over 2 5 mM and was highly sensitive to both active site and peripheral site inhibitors Northern blot analysis indicated a progressive increase in mRNA for AChE B in parasites isolated from 6 days postinfection Lüersen K, Müller S, Hussein A, Liebau E, Walter RD. The gamma-glutamylcysteine synthetase of Onchocerca volvulus. Mol Biochem Parasitol. 2000 Dec;111(2):243-51.http://blogs.najah.edu/staff/ashussein/article/Luumlersen-K-Muumlller-S-Hussein-A-Liebau-E-Walter-RD-The-gamma-glutamylcysteine-synthetase-of-Onchocerca-volvulus-Mol-Biochem-Parasitol-2000-Dec1112243-51Published ArticlesThe 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 gamma-glutamylcysteine synthetase gamma-GCS The gamma-GCS was partially purified from the filarial parasite Onchocerca volvoulus and preliminary steady state kinetics were performed The Ki-value for L-buthionine-SR-sulphoximine BSO a specific inhibitor of gamma-GCS was determined to be 013 microM which is 54-fold lower than the Ki-value for the mammalian enzyme Filarial gamma-GCS was also inhibited by cystamine with a Ki-value of 39 microM compared with 222 microM determined for the rat enzyme Further the cDNA and the gene of the O volvulus gamma-GCS were cloned and sequenced The gene of 5762 bp is composed of 14 exons and 13 introns Southern blot analysis indicates that the gamma-GCS gene is present as a single-copy gene In accordance with Northern blot analysis the entire cDNA sequence encompasses 2377 bp At its 5 end a nematode-specific spliced leader 130 bp upstream of the first in frame methionine was identified The cDNA encodes a polypeptide of 652 amino acids with 50 and 69 sequence identity to the human and the Caenorhabditis elegans counterparts respectively The filarial gamma-GCS is proposed as a potential drug targetHussein AS, Smith AM, Chacón MR, Selkirk ME. Determinants of substrate specificity of a second non-neuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis. Eur J Biochem. 2000 Apr;267(8):2276-82.http://blogs.najah.edu/staff/ashussein/article/Hussein-AS-Smith-AM-Chacoacuten-MR-Selkirk-ME-Determinants-of-substrate-specificity-of-a-second-non-neuronal-secreted-acetylcholinesterase-from-the-parasitic-nematode-Nippostrongylus-brasiliensis-Eur-J-Biochem-2000-Apr26782276-82Published ArticlesWe 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 A recombinant form of AChE C was highly expressed by Pichia pastoris The enzyme was monomeric and hydrophilic and displayed a marked preference for acetylthiocholine as substrate A double mutation W302FW345F corresponding to positions 290 and 331 in Torpedo rendered the enzyme 10-fold less sensitive to excess substrate inhibition and two times less susceptible to the bis quaternary inhibitor BW284C51 but did not radically affect substrate specificity or sensitivity to the peripheral site inhibitor propidium iodide In contrast a triple mutant M300GW302FW345F efficiently hydrolysed propionylthiocholine and butyrylthiocholine in addition to acetylthiocholine while remaining insensitive to the butyrylcholinesterase-specific inhibitor iso-OMPA and displaying a similar profile of excess substrate inhibition as the double mutant These data highlight a conserved pattern of active site architecture for nematode secreted AChEs characterized to date and provide an explanation for the substrate specificity that might otherwise appear inconsistent with the primary structure in comparison to other invertebrate AChEsRussell WS, Henson SM, Hussein AS, Tippins JR, Selkirk ME.Nippostrongylus brasiliensis: infection induces upregulation of acetylcholinesterase activity on rat intestinal epithelial cells. Exp Parasitol. 2000 Dec;96(4):222-30.http://blogs.najah.edu/staff/ashussein/article/Russell-WS-Henson-SM-Hussein-AS-Tippins-JR-Selkirk-MENippostrongylus-brasiliensis-infection-induces-upregulation-of-acetylcholinesterase-activity-on-rat-intestinal-epithelial-cells-Exp-Parasitol-2000-Dec964222-30Published ArticlesExpression of cholines terases and muscarinic acetylcholine receptors in the jejunal mucosa has been investigated during infection of rats with the nematode parasite Nippostrongylus brasiliensis Selective expression of m3 receptors was observed on epithelial cells from uninfected rats and animals 7 days postinfection and saturation binding with [3H]quinuclidinyl benzilate indicated that receptor expression on cell membranes was unaltered by infection Butyrylcholinesterase was highly expressed in mucosal epithelia but acetylcholinesterase was present at low levels in uninfected animals In contrast discrete foci of intense acetylcholinesterase activity were observed on the basement membrane of intestinal epithelial cells in animals infected with N brasiliensis This was demonstrated to be due to upregulation of expression of endogenous enzyme which peaked at Day 10 postinfection and subsequently declined to preinfection levels It is suggested that this occurs in response to hyper-activation of the enteric nervous system as a result of infection and may benefit the host by limiting excessive fluid secretion due to cholinergic stimulation Copyright 2000 Academic Press Hussein AS, Kichenin K, Selkirk ME. Suppression of secreted acetylcholinesterase expression in Nippostrongylus brasiliensis by RNA interference. Mol Biochem Parasitol. 2002 Jun;122(1):91-4http://blogs.najah.edu/staff/ashussein/article/Hussein-AS-Kichenin-K-Selkirk-ME-Suppression-of-secreted-acetylcholinesterase-expression-in-Nippostrongylus-brasiliensis-by-RNA-interference-Mol-Biochem-Parasitol-2002-Jun122191-4Published ArticlesNo abstractHussein AS, Harel M, Selkirk ME. A distinct family of acetylcholinesterases is secreted by Nippostrongylus brasiliensis. Mol Biochem Parasitol. 2002 Aug 28;123(2):125-34http://blogs.najah.edu/staff/ashussein/article/Hussein-AS-Harel-M-Selkirk-ME-A-distinct-family-of-acetylcholinesterases-is-secreted-by-Nippostrongylus-brasiliensis-Mol-Biochem-Parasitol-2002-Aug-281232125-34Published ArticlesA third variant of acetylcholinesterase AChE A secreted by the parasitic nematode Nippostrongylus brasiliensis has been isolated which shows 63-64 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 Copyright 2002 Elsevier Science BVSelkirk ME, Hussein AS, Chambers AE, Goulding D, Gares MP, Vásquez-Lopez C, Gárate T, Parkhouse RM, Gounaris K. Trichinella spiralis secretes a homologue of prosaposin. Mol Biochem Parasitol. 2004 May;135(1):49-56.http://blogs.najah.edu/staff/ashussein/article/Selkirk-ME-Hussein-AS-Chambers-AE-Goulding-D-Gares-MP-Vaacutesquez-Lopez-C-Gaacuterate-T-Parkhouse-RM-Gounaris-K-Trichinella-spiralis-secretes-a-homologue-of-prosaposin-Mol-Biochem-Parasitol-2004-May135149-56Published ArticlesInfective 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 36-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 discussedSelkirk ME, Lazari O, Hussein AS, Matthews JB. Nematode acetylcholinesterases are encoded by multiple genes and perform non-overlapping functions. Chem Biol Interact. 2005 Dec 15;157-158:263-8.http://blogs.najah.edu/staff/ashussein/article/Selkirk-ME-Lazari-O-Hussein-AS-Matthews-JB--Nematode-acetylcholinesterases-are-encoded-by-multiple-genes-and-perform-non-overlapping-functions-Chem-Biol-Interact-2005-Dec-15157-158263-8Published ArticlesNematodes 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 parasiteHussein AS. Brugia malayi: Depletion of glutathione by buthionine sulfoximine. An-Najah Univ. J. res. (N. Sc.), Vol. 19 (2005); 83-90.http://blogs.najah.edu/staff/ashussein/article/Hussein-AS-Brugia-malayi-Depletion-of-glutathione-by-buthionine-sulfoximine-An-Najah-Univ-J-res-N-Sc-Vol-19-2005-83-90Published ArticlesGlutathione is an intracellular reducing agent It is synthesized by a two step reaction catalyzed by gamma-glutamylcysteine synthetase GCS and glutathione synthetase GCS which is the rate limiting enzyme in the synthesis of glutathione is inhibited specifically by buthionine sulfoximine BSO The enzyme was partially purified from the parasitic nematode Brugia malayi the causative agent of lymphatic filariasis BSO inhibits the enzyme activity in an irreversible manner The Brugia enzyme was found to be 24-fold more sensitive in depleting the glutathione contents of the parasites with low concentration The results may conclude that GCS is a potential target for the development of drugs against filariasisOxidative stress in Caenorhabditis elegans: protective effects of the Omega class glutathione transferase (GSTO-1)http://blogs.najah.edu/staff/ashussein/article/Oxidative-stress-in-Caenorhabditis-elegans-protective-effects-of-the-Omega-class-glutathione-transferase-GSTO-1Published ArticlesTo elucidate the function of Omega class glutathione transferases GSTs EC 25118 in multicellular organisms the GSTO-1 from Caenorhabditis elegans GSTO-1; C29E47 was investigated Disc diffusion assays using Escherichia coli overexpressing GSTO-1 provided a test of resistance to long-term exposure under oxidative stress After affinity purification the recombinant GSTO-1 had minimal catalytic activity toward classic GST substrates but displayed significant thiol oxidoreductase and dehydroascorbate reductase activity Microinjection of the GSTO-1-promoter green fluorescent protein construct and immunolocalization by electron microscopy localized the protein exclusively in the intestine of all postembryonic stages of C elegans Deletion analysis identified an 300-nucleotide sequence upstream of the ATG start site necessary for GSTO-1 expression Site-specific mutagenesis of a GATA transcription factor binding motif in the minimal promoter led to the loss of reporter expression Similarly RNA interference RNAi of Elt-2 indicated the involvement of this gut-specific transcription factor in GSTO-1 expression Transcriptional up-regulation under stress conditions of GSTO-1 was confirmed by analyzing promoter-reporter constructs in transgenic C elegans strains To investigate the function of GSTO-1 in vivo transgenic animals overexpressing GSTO-1 were generated exhibiting an increased resistance to juglone- paraquat- and cumene hydroperoxide-induced oxidative stress Specific silencing of the GSTO-1 by RNAi created worms with an increased sensitivity to several prooxidants arsenite and heat shock We conclude that the stress-responsive GSTO-1 plays a key role in counteracting environmental stressBurmeister C Lersen K Heinick A Hussein A Domagalski M Walter R D Liebau E Oxidative stress in Caenorhabditis elegans: protective effects of the Omega class glutathione transferase GSTO-1 http:wwwfasebjorgcgicontentabstract222343