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Ruqaiyyah Siddiqui - One of the best experts on this subject based on the ideXlab platform.
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Antiamoebic activity of 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one library against Acanthamoeba castellanii
Parasitology Research, 2020Co-Authors: Muhammad Saquib Shahbaz, Areeba Anwar, Ayaz Anwar, Ruqaiyyah Siddiqui, Khalid Mohammed Khan, Syed Muhammad Saad, Naveed Ahmed KhanAbstract:Acanthamoeba castellanii is a free-living amoeba which can cause a blinding keratitis and fatal granulomatous amoebic encephalitis. The treatment of Acanthamoeba infections is challenging due to formation of cyst. Quinazolinones are medicinally important scaffold against parasitic diseases. A library of nineteen new 3-aryl-6,7-dimethoxyquinazolin-4(3 H )-one derivatives was synthesized to evaluate their antiamoebic activity against Acanthamoeba castellanii . One-pot synthesis of 3-aryl-6,7-dimethoxyquinazolin-4(3 H )-ones ( 1–19 ) was achieved by reaction of 2-amino-4,5-dimethoxybenzoic acid, trimethoxymethane, and different substituted anilines. These compounds were purified and characterized by standard chromatographic and spectroscopic techniques. Antiacanthamoebic activity of these compounds was determined by amoebicidal, encystation, excystation and host cell cytopathogenicity in vitro assays at concentrations of 50 and 100 μg/mL. The IC_50 was found to be between 100 and 50 μg/mL for all the compounds except compound 5 which did not exhibit amoebicidal effects at these concentrations. Furthermore, lactate dehydrogenase assay was also performed to evaluate the in vitro cytotoxicity of these compounds against human keratinocyte (HaCaT) cells. The results revealed that eighteen out of nineteen derivatives of quinazolinones significantly decreased the viability of A. castellanii . Furthermore, eighteen out of nineteen tested compounds inhibited the encystation and excystation, as well as significantly reduced the A. castellanii –mediated cytopathogenicity against human cells. Interestingly, while tested against human normal cell line HaCaT keratinocytes, all compounds did not exhibit any overt cytotoxicity. Furthermore, a detailed structure-activity relationship is also studied to optimize the most potent hit from these synthetic compounds. This report presents several potential lead compounds belonging to 3-aryl-6,7-dimethoxyquinazolin-4(3 H )-one derivatives for drug discovery against infections caused by Acanthamoeba castellanii .
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Antiamoebic activity of synthetic tetrazoles against Acanthamoeba castellanii belonging to T4 genotype and effects of conjugation with silver nanoparticles
Parasitology Research, 2020Co-Authors: Areeba Anwar, Yim Pei Yi, Itrat Fatima, Ruqaiyyah Siddiqui, Naveed Ahmed Khan, Khalid Mohammed Khan, Ayaz AnwarAbstract:Acanthamoeba causes diseases such as Acanthamoeba keratitis (AK) which leads to permanent blindness and granulomatous Acanthamoeba encephalitis (GAE) where there is formation of granulomas in the brain. Current treatments such as chlorhexidine, diamidines, and azoles either exhibit undesirable side effects or require immediate and prolonged treatment for the drug to be effective or prevent relapse. Previously, antifungal drugs amphotericin B, nystatin, and fluconazole-conjugated silver with nanoparticles have shown significantly increased activity against Acanthamoeba castellanii . In this study, two functionally diverse tetrazoles were synthesized, namely 5-(3-4-dimethoxyphenyl)-1 H -tetrazole and 1-(3-methoxyphenyl)-5-phenoxy-1 H -tetrazole, denoted by T1 and T2 respectively. These compounds were evaluated for anti- Acanthamoeba effects at different concentrations ranging from 5 to 50 μM. Furthermore, these compounds were conjugated with silver nanoparticles (AgNPs) to enhance their efficacy. Particle size analysis showed that T1-AgNPs and T2-AgNPs had an average size of 52 and 70 nm respectively. After the successful synthesis and characterization of tetrazoles and tetrazole-conjugated AgNPs, they were subjected to anti- Acanthamoeba studies. Amoebicidal assay showed that at concentration 10 μM and above, T2 showed promising antiamoebic activities between the two compounds while encystation and excystation assays reveal that both T1 and T2 have inhibited differentiation activity against Acanthamoeba castellanii . Conjugation of T1 and T2 to AgNP also increased efficacy of tetrazoles as anti- Acanthamoeba agents. This may be due to the increased bioavailability as AgNP allows better delivery of treatment compounds to A. castellanii . Human cell cytotoxicity assay revealed that tetrazoles and AgNPs are significantly less toxic towards human cells compared with chlorhexidine which is known to cause undesirable side effects. Cytopathogenicity assay also revealed that T2 conjugated with AgNPs significantly reduced cytopathogenicity of A. castellanii compared with T2 alone, suggesting that T2-conjugated AgNP is an effective and safe anti- Acanthamoeba agent. The use of a synthetic azole compound conjugated with AgNPs can be an alternative strategy for drug development against A. castellanii . However, mechanistic and in vivo studies are needed to explore further translational values.
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repositioning of guanabenz in conjugation with gold and silver nanoparticles against pathogenic amoebae Acanthamoeba castellanii and naegleria fowleri
ACS Infectious Diseases, 2019Co-Authors: Areeba Anwar, Ayaz Anwar, Naveed Ahmed Khan, Mohammad Ridwane Mungroo, William Sullivan, Ruqaiyyah SiddiquiAbstract:Brain-eating amoebae cause devastating infections in the central nervous system of humans, resulting in a mortality rate of 95%. There are limited effective therapeutic options available clinically for treating granulomatous amoebic encephalitis and primary amoebic meningoencephalitis caused by Acanthamoeba castellanii (A. castellanii) and Naegleria fowleri (N. fowleri), respectively. Here, we report for the first time that guanabenz conjugated to gold and silver nanoparticles has significant antiamoebic activity against both A. castellanii and N. fowleri. Gold and silver conjugated guanabenz nanoparticles were synthesized by the one-phase reduction method and were characterized by ultraviolet–visible spectrophotometry and atomic force microscopy. Both metals were facilely stabilized by the coating of guanabenz, which was examined by surface plasmon resonance determination. The average size of gold nanoconjugated guanabenz was found to be 60 nm, whereas silver nanoparticles were produced in a larger size ...
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oleic acid conjugated silver nanoparticles as efficient antiamoebic agent against Acanthamoeba castellanii
Parasitology Research, 2019Co-Authors: Ayaz Anwar, Ruqaiyyah Siddiqui, Sumayah Abdelnasir Osman Abdalla, Muhammad Raza Shah, Zara Aslam, Naveed Ahmed KhanAbstract:Acanthamoeba castellanii belonging to the T4 genotype is an opportunistic pathogen which is associated with blinding eye keratitis and rare but fatal central nervous system infection. A. castellanii pose serious challenges in antimicrobial chemotherapy due to its ability to convert into resistant, hardy shell-protected cyst form that leads to infection recurrence. The fatty acid composition of A. castellanii trophozoites is known to be most abundant in oleic acid which chemically is an unsaturated cis-9-Octadecanoic acid and naturally found in animal and vegetable fats and oils. This study was designed to evaluate antiacanthamoebic effects of oleic acid against trophozoites, cysts as well as parasite-mediated host cell cytotoxicity. Moreover, oleic acid–conjugated silver nanoparticles (AgNPs) were also synthesized and tested against A. castellanii. Oleic acid–AgNPs were synthesized by chemical reduction method and characterized by ultraviolet-visible spectrophotometry, atomic force microscopy, dynamic light scattering analysis, and Fourier transform infrared spectroscopy. Viability, growth inhibition, encystation, and excystation assays were performed with 10 and 5 μM concentration of oleic acid alone and oleic acid–conjugated AgNPs. Bioassays revealed that oleic acid alone and oleic acid–conjugated AgNPs exhibited significant antiamoebic properties, whereas nanoparticle conjugation further enhanced the efficacy of oleic acid. Phenotype differentiation assays also showed significant inhibition of encystation and excystation at 5 μM. Furthermore, oleic acid and oleic acid–conjugated AgNPs also inhibited amoebae-mediated host cell cytotoxicity as determined by lactate dehydrogenase release. These findings for the first time suggest that oleic acid–conjugated AgNPs exhibit antiacanthamoebic activity that hold potential for therapeutic applications against A. castellanii.
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Targeting cyst wall is an effective strategy in improving the efficacy of marketed contact lens disinfecting solutions against Acanthamoeba castellanii cysts
Contact Lens and Anterior Eye, 2016Co-Authors: Farhat Abjani, Farzana Abubakar Yousuf, Naveed Ahmed Khan, Ruqaiyyah SiddiquiAbstract:Acanthamoeba cysts are highly resistant to contact lens disinfecting solutions. Acanthamoeba cyst wall is partially made of 1,4 β-glucan (i.e., cellulose) and other complex polysaccharides making it a hardy shell that protects the resident amoeba. Here, we hypothesize that targeting the cyst wall structure in addition to antiamoebic compound would improve the efficacy of marketed contact lens disinfecting solutions. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents abolished viability of Acanthamoeba castellanii cysts and trophozoites. When tested alone, none of the agents nor contact lens disinfecting solutions completely destroyed A. castellanii cysts and trophozoites. The absence of cyst wall-degrading enzymes in marketed contact lens disinfecting solutions render them ineffective against Acanthamoeba cysts. It is concluded that the addition of cyst wall degrading molecules in contact lens disinfecting solutions will enhance their efficacy in decreasing the incidence of Acanthamoeba effectively.
Johnathan A Napier - One of the best experts on this subject based on the ideXlab platform.
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the alternative pathway c20 δ8 desaturase from the non photosynthetic organism Acanthamoeba castellanii is an atypical cytochrome b5 fusion desaturase
FEBS Letters, 2006Co-Authors: Olga Sayanova, Richard P Haslam, Colin M Lazarus, Johnathan A NapierAbstract:Abstract A cDNA encoding a C20 Δ8-desaturase was isolated from the free-living soil amoeba, Acanthamoeba castellanii and functionally characterised by heterologous expression. The open reading frame of the A. castellanii C20 Δ8-desaturase showed similarity to other microsomal front-end desaturases, but the N-terminal domain contained a variant form of the conserved heme-binding motif in which H-P-G-G is replaced by H-P-A-G. Co-expression of the A. castellani Δ8-desaturase with the Isochrysis galbana Δ9-elongase in transgenic Arabidopsis plants confirmed the activity observed in yeast and its role in the alternative pathway for C20 polyunsaturated fatty acid synthesis. Acyl-CoA profiles of these transgenic plants revealed an unexpected accumulation of C20 fatty acids in the acyl-CoA pool. This is the first report of an alternative pathway C20 Δ8-desaturase from a non-photosynthetic organism, and also the first report of a front-end desaturase lacking the canonical cytochrome b5 domain.
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the alternative pathway c20 δ8 desaturase from the non photosynthetic organism Acanthamoeba castellanii is an atypical cytochrome b5 fusion desaturase
FEBS Letters, 2006Co-Authors: Olga Sayanova, Richard P Haslam, Colin M Lazarus, Johnathan A NapierAbstract:A cDNA encoding a C20 Delta8-desaturase was isolated from the free-living soil amoeba, Acanthamoeba castellanii and functionally characterised by heterologous expression. The open reading frame of the A. castellanii C20 Delta8-desaturase showed similarity to other microsomal front-end desaturases, but the N-terminal domain contained a variant form of the conserved heme-binding motif in which H-P-G-G is replaced by H-P-A-G. Co-expression of the A. castellani Delta8-desaturase with the Isochrysis galbana Delta9-elongase in transgenic Arabidopsis plants confirmed the activity observed in yeast and its role in the alternative pathway for C20 polyunsaturated fatty acid synthesis. Acyl-CoA profiles of these transgenic plants revealed an unexpected accumulation of C20 fatty acids in the acyl-CoA pool. This is the first report of an alternative pathway C20 Delta8-desaturase from a non-photosynthetic organism, and also the first report of a front-end desaturase lacking the canonical cytochrome b5 domain.
Naveed Ahmed Khan - One of the best experts on this subject based on the ideXlab platform.
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Antiamoebic activity of 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one library against Acanthamoeba castellanii
Parasitology Research, 2020Co-Authors: Muhammad Saquib Shahbaz, Areeba Anwar, Ayaz Anwar, Ruqaiyyah Siddiqui, Khalid Mohammed Khan, Syed Muhammad Saad, Naveed Ahmed KhanAbstract:Acanthamoeba castellanii is a free-living amoeba which can cause a blinding keratitis and fatal granulomatous amoebic encephalitis. The treatment of Acanthamoeba infections is challenging due to formation of cyst. Quinazolinones are medicinally important scaffold against parasitic diseases. A library of nineteen new 3-aryl-6,7-dimethoxyquinazolin-4(3 H )-one derivatives was synthesized to evaluate their antiamoebic activity against Acanthamoeba castellanii . One-pot synthesis of 3-aryl-6,7-dimethoxyquinazolin-4(3 H )-ones ( 1–19 ) was achieved by reaction of 2-amino-4,5-dimethoxybenzoic acid, trimethoxymethane, and different substituted anilines. These compounds were purified and characterized by standard chromatographic and spectroscopic techniques. Antiacanthamoebic activity of these compounds was determined by amoebicidal, encystation, excystation and host cell cytopathogenicity in vitro assays at concentrations of 50 and 100 μg/mL. The IC_50 was found to be between 100 and 50 μg/mL for all the compounds except compound 5 which did not exhibit amoebicidal effects at these concentrations. Furthermore, lactate dehydrogenase assay was also performed to evaluate the in vitro cytotoxicity of these compounds against human keratinocyte (HaCaT) cells. The results revealed that eighteen out of nineteen derivatives of quinazolinones significantly decreased the viability of A. castellanii . Furthermore, eighteen out of nineteen tested compounds inhibited the encystation and excystation, as well as significantly reduced the A. castellanii –mediated cytopathogenicity against human cells. Interestingly, while tested against human normal cell line HaCaT keratinocytes, all compounds did not exhibit any overt cytotoxicity. Furthermore, a detailed structure-activity relationship is also studied to optimize the most potent hit from these synthetic compounds. This report presents several potential lead compounds belonging to 3-aryl-6,7-dimethoxyquinazolin-4(3 H )-one derivatives for drug discovery against infections caused by Acanthamoeba castellanii .
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Isoniazid Conjugated Magnetic Nanoparticles Loaded with Amphotericin B as a Potent Antiamoebic Agent against Acanthamoeba castellanii.
Antibiotics, 2020Co-Authors: Kawish Iqbal, Areeba Anwar, Ayaz Anwar, Sumayah Abdelnasir Osman Abdalla, Kanwal Muhammad Iqbal, Muhammad Raza Shah, Naveed Ahmed KhanAbstract:The pathogenic free-living amoeba, Acanthamoeba castellanii, is responsible for a rare but deadly central nervous system infection, granulomatous amoebic encephalitis and a blinding eye disease called Acanthamoeba keratitis. Currently, a combination of biguanides, amidine, azoles and antibiotics are used to manage these infections; however, the host cell cytotoxicity of these drugs remains a challenge. Furthermore, Acanthamoeba species are capable of transforming to the cyst form to resist chemotherapy. Herein, we have developed a nano drug delivery system based on iron oxide nanoparticles conjugated with isoniazid, which were further loaded with amphotericin B (ISO-NPs-AMP) to cause potent antiamoebic effects against Acanthamoeba castellanii. The IC50 of isoniazid conjugated with magnetic nanoparticles and loaded with amphotericin B was found to be 45 μg/mL against Acanthamoeba castellanii trophozoites and 50 μg/mL against cysts. The results obtained in this study have promising implications in drug discovery as these nanomaterials exhibited high trophicidal and cysticidal effects, as well as limited cytotoxicity against rat and human cells.
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Antiamoebic activity of synthetic tetrazoles against Acanthamoeba castellanii belonging to T4 genotype and effects of conjugation with silver nanoparticles
Parasitology Research, 2020Co-Authors: Areeba Anwar, Yim Pei Yi, Itrat Fatima, Ruqaiyyah Siddiqui, Naveed Ahmed Khan, Khalid Mohammed Khan, Ayaz AnwarAbstract:Acanthamoeba causes diseases such as Acanthamoeba keratitis (AK) which leads to permanent blindness and granulomatous Acanthamoeba encephalitis (GAE) where there is formation of granulomas in the brain. Current treatments such as chlorhexidine, diamidines, and azoles either exhibit undesirable side effects or require immediate and prolonged treatment for the drug to be effective or prevent relapse. Previously, antifungal drugs amphotericin B, nystatin, and fluconazole-conjugated silver with nanoparticles have shown significantly increased activity against Acanthamoeba castellanii . In this study, two functionally diverse tetrazoles were synthesized, namely 5-(3-4-dimethoxyphenyl)-1 H -tetrazole and 1-(3-methoxyphenyl)-5-phenoxy-1 H -tetrazole, denoted by T1 and T2 respectively. These compounds were evaluated for anti- Acanthamoeba effects at different concentrations ranging from 5 to 50 μM. Furthermore, these compounds were conjugated with silver nanoparticles (AgNPs) to enhance their efficacy. Particle size analysis showed that T1-AgNPs and T2-AgNPs had an average size of 52 and 70 nm respectively. After the successful synthesis and characterization of tetrazoles and tetrazole-conjugated AgNPs, they were subjected to anti- Acanthamoeba studies. Amoebicidal assay showed that at concentration 10 μM and above, T2 showed promising antiamoebic activities between the two compounds while encystation and excystation assays reveal that both T1 and T2 have inhibited differentiation activity against Acanthamoeba castellanii . Conjugation of T1 and T2 to AgNP also increased efficacy of tetrazoles as anti- Acanthamoeba agents. This may be due to the increased bioavailability as AgNP allows better delivery of treatment compounds to A. castellanii . Human cell cytotoxicity assay revealed that tetrazoles and AgNPs are significantly less toxic towards human cells compared with chlorhexidine which is known to cause undesirable side effects. Cytopathogenicity assay also revealed that T2 conjugated with AgNPs significantly reduced cytopathogenicity of A. castellanii compared with T2 alone, suggesting that T2-conjugated AgNP is an effective and safe anti- Acanthamoeba agent. The use of a synthetic azole compound conjugated with AgNPs can be an alternative strategy for drug development against A. castellanii . However, mechanistic and in vivo studies are needed to explore further translational values.
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repositioning of guanabenz in conjugation with gold and silver nanoparticles against pathogenic amoebae Acanthamoeba castellanii and naegleria fowleri
ACS Infectious Diseases, 2019Co-Authors: Areeba Anwar, Ayaz Anwar, Naveed Ahmed Khan, Mohammad Ridwane Mungroo, William Sullivan, Ruqaiyyah SiddiquiAbstract:Brain-eating amoebae cause devastating infections in the central nervous system of humans, resulting in a mortality rate of 95%. There are limited effective therapeutic options available clinically for treating granulomatous amoebic encephalitis and primary amoebic meningoencephalitis caused by Acanthamoeba castellanii (A. castellanii) and Naegleria fowleri (N. fowleri), respectively. Here, we report for the first time that guanabenz conjugated to gold and silver nanoparticles has significant antiamoebic activity against both A. castellanii and N. fowleri. Gold and silver conjugated guanabenz nanoparticles were synthesized by the one-phase reduction method and were characterized by ultraviolet–visible spectrophotometry and atomic force microscopy. Both metals were facilely stabilized by the coating of guanabenz, which was examined by surface plasmon resonance determination. The average size of gold nanoconjugated guanabenz was found to be 60 nm, whereas silver nanoparticles were produced in a larger size ...
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oleic acid conjugated silver nanoparticles as efficient antiamoebic agent against Acanthamoeba castellanii
Parasitology Research, 2019Co-Authors: Ayaz Anwar, Ruqaiyyah Siddiqui, Sumayah Abdelnasir Osman Abdalla, Muhammad Raza Shah, Zara Aslam, Naveed Ahmed KhanAbstract:Acanthamoeba castellanii belonging to the T4 genotype is an opportunistic pathogen which is associated with blinding eye keratitis and rare but fatal central nervous system infection. A. castellanii pose serious challenges in antimicrobial chemotherapy due to its ability to convert into resistant, hardy shell-protected cyst form that leads to infection recurrence. The fatty acid composition of A. castellanii trophozoites is known to be most abundant in oleic acid which chemically is an unsaturated cis-9-Octadecanoic acid and naturally found in animal and vegetable fats and oils. This study was designed to evaluate antiacanthamoebic effects of oleic acid against trophozoites, cysts as well as parasite-mediated host cell cytotoxicity. Moreover, oleic acid–conjugated silver nanoparticles (AgNPs) were also synthesized and tested against A. castellanii. Oleic acid–AgNPs were synthesized by chemical reduction method and characterized by ultraviolet-visible spectrophotometry, atomic force microscopy, dynamic light scattering analysis, and Fourier transform infrared spectroscopy. Viability, growth inhibition, encystation, and excystation assays were performed with 10 and 5 μM concentration of oleic acid alone and oleic acid–conjugated AgNPs. Bioassays revealed that oleic acid alone and oleic acid–conjugated AgNPs exhibited significant antiamoebic properties, whereas nanoparticle conjugation further enhanced the efficacy of oleic acid. Phenotype differentiation assays also showed significant inhibition of encystation and excystation at 5 μM. Furthermore, oleic acid and oleic acid–conjugated AgNPs also inhibited amoebae-mediated host cell cytotoxicity as determined by lactate dehydrogenase release. These findings for the first time suggest that oleic acid–conjugated AgNPs exhibit antiacanthamoebic activity that hold potential for therapeutic applications against A. castellanii.
Olga Sayanova - One of the best experts on this subject based on the ideXlab platform.
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the alternative pathway c20 δ8 desaturase from the non photosynthetic organism Acanthamoeba castellanii is an atypical cytochrome b5 fusion desaturase
FEBS Letters, 2006Co-Authors: Olga Sayanova, Richard P Haslam, Colin M Lazarus, Johnathan A NapierAbstract:Abstract A cDNA encoding a C20 Δ8-desaturase was isolated from the free-living soil amoeba, Acanthamoeba castellanii and functionally characterised by heterologous expression. The open reading frame of the A. castellanii C20 Δ8-desaturase showed similarity to other microsomal front-end desaturases, but the N-terminal domain contained a variant form of the conserved heme-binding motif in which H-P-G-G is replaced by H-P-A-G. Co-expression of the A. castellani Δ8-desaturase with the Isochrysis galbana Δ9-elongase in transgenic Arabidopsis plants confirmed the activity observed in yeast and its role in the alternative pathway for C20 polyunsaturated fatty acid synthesis. Acyl-CoA profiles of these transgenic plants revealed an unexpected accumulation of C20 fatty acids in the acyl-CoA pool. This is the first report of an alternative pathway C20 Δ8-desaturase from a non-photosynthetic organism, and also the first report of a front-end desaturase lacking the canonical cytochrome b5 domain.
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the alternative pathway c20 δ8 desaturase from the non photosynthetic organism Acanthamoeba castellanii is an atypical cytochrome b5 fusion desaturase
FEBS Letters, 2006Co-Authors: Olga Sayanova, Richard P Haslam, Colin M Lazarus, Johnathan A NapierAbstract:A cDNA encoding a C20 Delta8-desaturase was isolated from the free-living soil amoeba, Acanthamoeba castellanii and functionally characterised by heterologous expression. The open reading frame of the A. castellanii C20 Delta8-desaturase showed similarity to other microsomal front-end desaturases, but the N-terminal domain contained a variant form of the conserved heme-binding motif in which H-P-G-G is replaced by H-P-A-G. Co-expression of the A. castellani Delta8-desaturase with the Isochrysis galbana Delta9-elongase in transgenic Arabidopsis plants confirmed the activity observed in yeast and its role in the alternative pathway for C20 polyunsaturated fatty acid synthesis. Acyl-CoA profiles of these transgenic plants revealed an unexpected accumulation of C20 fatty acids in the acyl-CoA pool. This is the first report of an alternative pathway C20 Delta8-desaturase from a non-photosynthetic organism, and also the first report of a front-end desaturase lacking the canonical cytochrome b5 domain.
Peter H.h. Weekers - One of the best experts on this subject based on the ideXlab platform.
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Bacteriolytic activities of the free-living soil amoebae, Acanthamoeba castellanii, Acanthamoeba polyphaga and Hartmannella vermiformis.
Antonie van Leeuwenhoek, 1995Co-Authors: Peter H.h. Weekers, Andreas M. W. Engelberts, Godfried D. VogelsAbstract:Bacteriolytic activities of axenically grown free-living soil amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga and Hartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined. A spectrophotometric assay revealed that the specific bacteriolytic activities of both Acanthamoeba species were higher as those of the three Hartmannella strains. Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus and Pseudomonas fluorescens were more easily lysed than the other bacteria tested. Agrobacterium tumefaciens, Klebsiella aerogenes and Serratia marcescens were hardly affected at all by the amoebal bacteriolytic activities. Among the Gram-negative bacteria we observed differences in lysis sensitivity while the Gram-positive bacteria tested were sensitive to lysis. Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3-10 was performed to separate the bacteriolytic isoenzymes of amoebae. Bacteriolytic patterns were shown by using an activity assay in which lysis bands were formed in the agar/bacteria gel-overlay. The activity assay revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae. Distinct differences between typical pI points of bacteriolytic activities in Acanthamoeba and Hartmannella were shown. Bacteriolytic activities of Hartmannella were more pronounced and observed in the isoelectric points (pI) range of 4.0-9.3 while for Acanthamoeba the range was pI 4.5-8.9.
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nitrogen metabolizing enzyme activities in the free living soil amoebae Acanthamoeba castellanii Acanthamoeba polyphaga and hartmannella vermiformis
Journal of Eukaryotic Microbiology, 1993Co-Authors: Peter H.h. Weekers, Chris Van Der DriftAbstract:. Free-living soil amoebae consume a wide variety of food, including algae, yeast, small protozoa and especially bacteria, which they digest to fulfil their nutritional requirements. Amoebae play an active role in the nitrogen mineralization in soils due to their nitrogen metabolizing capacities. However, little is known about nitrogen metabolizing enzyme activities in these free-living soil amoebae. In this study a number of key enzymes involved in the nitrogen metabolism of the axeaically cultivated free-living soil amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga and two different strains of Hartmannella vermiformis were determined. the specific enzyme activities for exponential growth phase ceils were calculated and it appeared that the species tested possessed urate oxidase, glutamine synthetase, NADH-dependent glutamate dehydrogenase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase activity. Glutamate synthase activity could not be detected in any of these species. the levels of specific activities varied depending on the enzymes tested. For all species the highest activities were detected for the transaminase reactions yielding glutamate, and for glutamate dehydrogenase. A general conclusion is that the pathway of nitrogen assimilation in free-living soil amoebae is similar to the one observed for other eukaryotes. Differences in specific activities were detected between the species.
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Effects of Grazing by the Free-Living Soil Amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis on Various Bacteria.
Applied and environmental microbiology, 1993Co-Authors: Peter H.h. Weekers, Paul L. E. Bodelier, John P. H. Wijen, Godfried D. VogelsAbstract:Cultures of 10 different bacteria were used to serve as food sources for axenically grown Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis. The nonpigmented enterobacteriaceae Escherichia coli K-12 and Klebsiella aerogenes appeared to be excellent feed to all three amoebae. Hardly any growth or ammonium production was observed in tests with Chromatium vinosum and Serratia marcescens, which share the presence of pigmented compounds. Distinct differences in net ammonium production were detected and were correlated to the amoebal growth yield. In general, growth of amoebae and ammonium production increased in the order A. polyphaga, A. castellanii, and H. vermiformis.
