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Investigation of organophosphorus pesticide effects on soil bacteriaHinds, Laura January 2000 (has links)
The toxicity of OP insecticides in aqueous solution and soil pore water was assessed using the lux-marked biosensors E. coli HB101 pUCD607 and P. fluorescens 10586r pUCD607. The effect of OP insecticides on soil microbial processes (respiration and nitrification) and the soil microbial community (viable counts and fatty acid analysis of whole soil extract) was investigated. The relative impact of a formulated insecticide (biomalathion) and the corresponding active ingredient (malathion) was assessed. The lux-biosensors successfully reported on the toxicity of OP insecticides in aqueous bioassays. P. fluorescens was the most sensitive biosensor and biomalathion was the most toxic of the OP insecticides tested. OP insecticides generally had stimulatory effects on soil microbial processes. It was not possible to predict effects on soil microbial processes on the basis of lux-biosensor response, due to the difficulties in interpreting the microbial process tests. For example, it is not clear whether a stimulatory effect should be classed as a toxic impact. Plate counts of OP spiked soil showed an increase in the bacterial population density of both total heterotrophic bacteria and Pseudomonas. However, fatty acid analysis of whole soil extracts failed to detect any effect of OP insecticide treatment on the soil microbial community. This may be because the OP insecticides are having a selective effect on the culturable fraction of the soil microbial community. This thesis attempted to assess the impact of an agrochemical on the soil microbial community using an array of techniques. Both the traditional and modern techniques provided useful information about the effect of OP insecticides on the soil microbial community. Changes due to insecticide treatment were evident. However, further research is needed to assess whether these changes will have a long term impact on soil quality and agricultural sustainability.
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The development of biomarkers in freshwater insects for the biological monitoring of pollutionParker, Penelope Judy-Ann Nicole January 1996 (has links)
No description available.
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Pesticide residue analysis using supercritical fluid extractionAhmad, Maqbool January 1993 (has links)
No description available.
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Directed evolution of phosphotriesterase for detoxification of the nerve agent VXGhanem, Eman Mohamed 30 October 2006 (has links)
Phosphotriesterase (PTE) isolated from the soil bacterium Flavobacterium sp. is
a member of the amidohydrolase superfamily. PTE catalyzes the hydrolysis of a broad
spectrum of organophosphate triesters including the insecticide paraoxon, and the
chemical warfare agents; GF, sarin, and soman. In addition, PTE has been shown to
catalytically hydrolyze the lethal nerve agent, VX. However, the rate of VX hydrolysis
is significantly slower. PTE was subjected to directed evolution studies to identify
variants with enhanced activity towards VX hydrolysis. First generation libraries
targeted amino acid residues in the substrate binding site. The H254A mutation
displayed a 4-fold enhancement in kcat and a 2-fold enhancement in kcat/Km over wild
type PTE. The double mutant H254Q/H257F was isolated from the second generation
libraries and displayed a 10-fold enhancement in kcat and a 3-fold enhancement in
kcat/Km. In addition, H254Q/H257F displayed a 9-fold enhancement in kcat/Km for the
hydrolysis of the VX analog, demeton-S.
An in vivo selection approach utilizing organophosphate triesters as the sole
phosphorus source is discussed. The selection is based on co-expressing PTE with the
phosphodiesterase (GpdQ) from E. aerogenes. Substrate specificity of GpdQ was investigated using a small library of structurally diverse organophosphate diesters and
phosphonate monoesters. Results obtained from the in vivo growth assays showed that
GpdQ enabled E. coli to utilize various organophosphate diesters and phosphonate
monoesters as the sole phosphorus source. Cells co-expressing PTE and GpdQ were
tested for their ability to utilize two different organophosphate triesters as the sole
phosphorus source. The results from this experiment indicate that the growth rate is
limited by the phosphotriesterase activity.
Protein translocation to the periplasm was proven advantageous for in vivo
selection since it overcomes the limitation of intercellular delivery of the substrate of
interest. Translocation of PTE to the periplasmic space of E. coli was examined. Two
signal peptides were tested; the native leader peptide from Flavobacterium sp. and the
signal sequence of alkaline phosphatase. The results obtained from cellular fractionation
indicated that neither signal peptides were able to translocate PTE to the periplasm and
that the protein remained in the cytoplasm.
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The toxicity of tricresyl phosphate towards cultured nerve cells and isolated nerveFowler, Maxine Joanne January 2000 (has links)
No description available.
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Reactivation of Acetylcholinesterase Inhibited by Organophosphates in Peripheral Rat TissuesBennett, Joshua Peay 09 May 2015 (has links)
The aim of this study was to determine the ability of twenty novel substituted phenoxyalkyl pyridinium oximes to reactivate phosphylated acetylcholinesterase (AChE) in peripheral rat tissues, in vitro, inhibited by organophosphate anticholinesterase nerve agent surrogates. A sarin surrogate, phthalimidyl isopropyl methylphosphonate (PIMP), and a VX surrogate, 4-nitrophenyl ethyl methylphosphonate (NEMP), were used to inhibit AChE in skeletal muscle and serum samples. Reactivation of the widely used oxime 2-PAM was tested for comparison with the novel oximes. The novel oximes displayed a range of 23-102% reactivation of AChE in vitro across both tissue types. Most of the novel oximes tested in the present study demonstrated a higher percent reactivation of AChE, than 2-PAM. Therefore, these novel oximes have the potential to be effective antidotes used during the treatment of OP toxicity.
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Efficacy of Novel Pyridinium Oximes against Two Organophosphates in Female Sprague Dawley RatsGarcia, Jason Michael 03 May 2019 (has links)
Anticholinesterase organophosphate (OP) compounds were developed as insecticides and are also used as nerve agents in chemical warfare. Treatment against acute OP toxicity includes oximes which reactivate phosphorylated acetylcholinesterase (AChE) restoring enzymatic activity. The oxime currently approved for use in the U.S., pralidoxime (2-PAM), has limited efficacy penetrating the blood-brain barrier. Our laboratory has developed novel substituted phenoxyalkyl pyridinium oximes (US Patent 9,227,937) designed to more effectively penetrate the central nervous system. This research investigated any differences in oxime reactivation among four age/sex groups and also survivability in adult female Sprague Dawley rats challenged with a lethal dose of OP. Initially in in vitro experiments, paraoxon (PXN) and a nerve agent (sarin) surrogate, 4-nitrophenyl isopropyl methylphosphonate (NIMP), were incubated with pooled rat brain homogenate from four sex/age groups: adult male or female, and 12-day old male or female rats. Reactivation was performed utilizing 2-PAM or one of three novel oximes (15, 20, or 55), alone or in combination, and AChE activity was measured in a spectrophotometric assay. Overall, the oximes were more effective reactivating inhibition from PXN than from NIMP. Out of all the oximes tested, 2-PAM showed the greatest reactivation percentages. Of the novel oximes, 15 and 20 displayed the highest reactivation against PXN and NIMP, respectively. No statistical difference was detected in reactivation for any oxime among the age/sex groups. For the in vivo study, female adult Sprague-Dawley rats were treated with LD99 dosages of NIMP or PXN. After development of seizure-like behavior, atropine and one of four oximes, 2-PAM, novel oxime 15, 20, or 55 or Multisol vehicle was administered. Animals were closely monitored for signs of cholinergic toxicity and 24-hour survivability. Against PXN, novel oximes 15 and 55 demonstrated an improved odds ratio of 6.5 and 3.1, respectively, over 2-PAM. The most effective oxime against NIMP was novel oxime 20 demonstrating an odds ratio of 3.2 over treatment with 2-PAM. These data indicate that the novel pyridinium oximes are equally efficacious reactivators in adult and juvenile rats of both sexes and enhance survivability against lethal-level OP toxicity as compared to 2-PAM in adult female rats.
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Improving Reactivity Against Target Organothiophosphates via Active-Site Directed Mutagenisis of a Bacterial PhosphotriesteraseGithens, Tyler 1986- 14 March 2013 (has links)
Phosphotriesters, also known as organophosphates (OP), represent a class of toxic compounds first synthesized in Germany. Enzymatic removal of harmful insecticides and breakdown products is a promising alternative to skimming or dredging. Wild type bacterial phosphotriesterase (PTE) was screened against 7 agricultural organophosphates: coumaphos, chlorpyrifos, fenitrothion, temephos, profenofos, pirimiphosmethyl and diazinon. The initial results laid the groundwork for a mutagenesis study to investigate the determining factors in enzyme reactivity. Coumaphos is hydrolyzed more efficiently than any other target by the wild type cobalt enzyme (kcat/Km = 2 x 10^7 M^-1s^-1). Coumaphos, fenitrothion and chlorpyrifos had the lowest Km values from the initial screen and were targets for steady state kinetic characterization of active site mutants. Site directed mutagenesis of binding sites was conducted and the most reactive point mutants, F132G, F132V and S308G, were used as backgrounds for subsequent mutation. Seven active site double mutants: F132G/S308G, F132G/S308T, F132V/S308G, F132V/S308T, F132G/I106T, F132V/I106T and G308/W309 were purified to homogeneity for kinetic characterization. The double mutant G308/F132V enhanced chlorpyrifos reactivity relative to the wild type enzyme. This enhancement of reactivity is proposed to result from conformational rearrangement following substrate bond hydrolysis.
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Development of enzyme-based biosensors for the detection of organophosphate neurotoxinsPaliwal, Sheetal, Simonian, Aleksandr L., January 2008 (has links) (PDF)
Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 32-46).
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Array biosensor for the detection of organophosphatesRamanathan, Madhumati, Simonian, Aleksandr L. January 2006 (has links) (PDF)
Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.
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