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Y Ashani

Publications and source records attributed to Y Ashani.

At least 37 records · Page 2Linked to original sources
PubMed1993

Prevention of soman-induced cognitive deficits by pretreatment with human butyrylcholinesterase in rats.

This study examined the ability of pretreatment with human serum butyrylcholinesterase (HuBChE) to prevent soman-induced cognitive impairments. Behavioral testing was carried out using the Morris water maze task evaluating learning, memory, and reversal learning processes. Pretreatment with HuBChE significantly prevented the memory and reversal learning impairments induced by soman. A small deficiency in performance was observed only during part of the learning period in HuBChE-treated rats after administration of soman. Results support the contention that pretreatment alone with HuBChE is sufficient to increase survival and to prevent impairment in cognitive functioning following exposure to soman.

Animals↗
PubMed1992

Protection against tabun toxicity in mice by prophylaxis with an enzyme hydrolyzing organophosphate esters.

We demonstrate here the correlation between protection afforded by pretreatment alone with parathion hydrolase purified from Pseudomonas sp. against tabun toxicity in mice and the kinetic parameters which are assumed to determine the in vivo detoxification of tabun by the same enzyme. Results show that 15 and 22 micrograms of parathion hydrolase per animal conferred a protective ratio of 3.94 and 5.65 respectively, against tabun toxicity, without post-exposure treatment.

Animals↗
PubMed1992

Mechanism of inhibition of cholinesterases by huperzine A.

Huperzine A, an alkaloid isolated from Huperzia serrata was found to reversibly inhibit acetylcholinesterases (EC 3.1.1.7) and butyrylcholinesterases (EC 3.1.1.8) with on- and off-rates that depend on both the type and the source of enzyme. Long-term incubation of high concentrations of purified cholinesterases (1-8 microM) with huperzine A did not show any chemical modification of huperzine A. A low dissociation constant KI was obtained for mammalian acetylcholinesterase-huperzine (20-40 nM) compared to mammalian butyrylcholinesterase-huperzine (20-40 microM). This indicates that the thermodynamic stability of huperzine-cholinesterase complex may depend on the number and type of aromatic amino acid residues in the catalytic pocket region of the cholinesterase molecule.

Acetylcholinesterase↗
PubMed1991

Refined crystal structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin.

"Aged" organophosphoryl conjugates of serine hydrolases differ from the corresponding "non-aged" conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin were compared in order to understand the molecular basis for this resistance of "aged" conjugates. "Aged" and "non-aged" crystalline organophosphoryl-gamma-chymotrypsin conjugates were obtained by prolonged soaking of native gamma-chymotrypsin crystals with appropriate organophosphates. Thus, a representative "non-aged" conjugate, diethylphosphoryl-gamma-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl-p-nitrophenyl phosphate), and a closely related "aged" conjugate, monoisopropyl-gamma-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 O gamma. Whereas in the "non-aged" conjugate both ethyl groups can be visualized clearly, in the putative "aged" conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the "aged" and "non-aged" conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the "aged" conjugate, however, close proximity (2.6 A) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 N epsilon 2 indicates the presence of a salt bridge between these two moieties. In contrast, in the "non-aged" conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3.6 A away from His57 N epsilon 2. It is suggested that steric constraints imposed by the salt bridge in the "aged" conjugate lie at the basis of its resistance to reactivation.

Alkylation↗
PubMed1991

Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice.

Human butyrylcholinesterase (BChE, EC 3.1.1.8) or acetylcholinesterase (AChE, EC 3.1.1.7) from fetal bovine serum (FBS), administered i.v. in mice, sequestered at approximately 1:1 stoichiometry the highly toxic anti-ChE organophosphate, 1,2,2-trimethylpropyl methyl-fluorophosphonate (soman). A quantitative linear correlation was demonstrated between blood-ChE levels and the protection conferred by exogeneously administered ChE. Results presented here demonstrate that either human BChE or FBS-AChE is an effective prophylactic measure sufficient to protect mice from multiple LD50S of soman without the administration of post-treatment supportive drugs.

Acetylcholinesterase↗
PubMed1991

Prophylaxis against organophosphate poisoning by an enzyme hydrolysing organophosphorus compounds in mice.

Parathion hydrolase purified from Pseudomonas sp. was injected i.v. into mice to demonstrate the feasibility of using organophosphorus acid anhydride (OPA) hydrolases as pretreatment against organophosphates (OP) poisoning. Results show that exogenous administration of as low as 7 to 26 micrograms of parathion hydrolase conferred protection against challenge with multiple median lethal doses (LD50) of diethyl p-nitrophenyl phosphate (paraoxon; 3.8-7.3 x LD50) and diethylfluorophosphate (DEFP; 2.9 x LD50) without administration of supportive drugs. The extent of protection observed was consistent with blood-parathion hydrolase levels and the kinetic constants of the enzymatic hydrolysis of paraoxon and DEFP by parathion hydrolase. OPA hydrolases not only appear to be potential prophylactic drugs capable of increasing survival ratio following OP intoxication but also to alleviate post-exposure symptoms.

Animals↗
PubMed1991

Enzymes as pretreatment drugs for organophosphate toxicity.

We have successfully demonstrated that exogenously administered acetyl- or butyrylcholinesterase (AChE, BChE respectively) will sequester organophosphates (OPs) before they reach their physiological targets. In addition, a third enzyme, endogenous carboxylesterase is known to be capable of scavenging OPs. In these studies, we have administered AChE and BChE to three different species of animals (mice, marmosets and monkeys) which were challenged with three different OPs (VX, MEPQ and soman). Results obtained from these systematic studies demonstrate that: (a) a quantitative linear correlation exists between blood AChE levels and the protection afforded by exogenously administered ChEs in animals challenged with OP, (b) approximately one mole of either AChE or BChE sequesters one mole of OP, (c) such prophylactic measures are sufficient to protect animals against OPs without the administration of any supportive drugs. Thus the OP dose, the blood-level of esterase, the ratio of the circulating enzyme to OP challenge, and the rate of reaction between them determine the overall efficacy of an enzyme as a pretreatment drug. The biochemical mechanism underlying the sequestration of various OPs by the use of exogenously administered scavenging esterases is the same in all species of animals studied. Therefore, the extrapolation of the results obtained by the use of ChE prophylaxis in animals to humans should be more reliable and effective than extrapolating the results from currently used multidrug antidotal modalities.

Animals↗
PubMed1990

Differences in conformational stability between native and phosphorylated acetylcholinesterase as evidenced by a monoclonal antibody.

Monoclonal antibody 25B1 generated against diisopropyl phosphorofluoridate inhibited fetal bovine serum acetylcholinesterase has been extensively characterized with respect to its anticholinesterase properties. This antibody demonstrated considerably different properties from previously reported inhibitory antibodies raised against acetylcholinesterase in terms of the degree of inhibition (greater than 98%), the high degree of specificity, and the stability of the antigen-antibody complex. Monoclonal antibody 25B1 appears to be directed against a conformational epitope located in close proximity to the catalytic center of the enzyme and was found to be most suitable for studying the stabilization of the active site of acetylcholinesterase against denaturation by heat or guanidine following phosphorylation by organophosphorus anticholinesterase compounds. This approach allowed the determination of stability rank order of various phosphorylated acetylcholinesterases. Among all the organophosphates tested, the combination of a methyl group and a negatively charged oxygen attached to the P atom, CH3P(O)(O-)-AChE, conferred the greatest protection to the active site of aged or nonaged organophosphoryl conjugates of acetylcholinesterase.

Acetylcholinesterase↗
PubMed1989

Aged and non-aged pyrenebutyl-containing organophosphoryl conjugates of chymotrypsin. Preparation and comparison by 31P-NMR spectroscopy.

Homologous pairs of non-aged and aged pyrene-containing phosphoryl conjugates of chymotrypsin were prepared in order to characterize by NMR and optical spectroscopy putative differences in the conformation of non-aged and aged organophosphoryl conjugates of serine hydrolases. Pyrenebutyl-O-P(O)(OC2H5)F and pyrenebutyl-O-P(O)(OC2H5)Cl were used to obtain the non-aged form pyrenebutyl-O-P(O)(OC2H5)-Cht, whereas pyrenebutyl-O-P(O)Cl2, pyrenebutyl-O-P(O)(p-nitrophenoxy)Cl, and pyrenebutyl-O-P(O)(p-nitrophenoxy)2 were used to produce the aged conjugate pyrenebutyl-O-P(O)(O )-Cht. These ligands bind covalently to the active site of serine hydrolases. The absorption spectra of both the non-aged and aged conjugates fitted approximately a 1:1 stoichiometry of bound organophosphate and enzyme in the non-aged and aged conjugates. Pyrenebutyl-O-P(O)(OC2H5)-Cht could be reactivated by pyridine-3-aldoxime methiodide, whereas no reactivation was observed for the similarly treated pyrenebutyl-O-P(O)(O-)-Cht. The 31P-NMR and reactivation data taken together strongly support the hypothesis that the aged form of the OP-Cht conjugate contains a P--O- bond. These results provide a partial interpretation for the known resistance of the aged conjugates of serine hydrolases to reactivation.

Chymotrypsin↗
PubMed1989

Conformational differences between aged and nonaged pyrenebutyl-containing organophosphoryl conjugates of chymotrypsin as detected by optical spectroscopy.

Homologous aged and nonaged fluorescent organophosphorus conjugates of alpha-chymotrypsin (Cht) were used in a comparative spectroscopic study of the conformation of their active sites, employing the pyrene group as the fluorescent probe. Steady-state fluorescence measurements showed that the quantum yield of the pyrene probe which is stoichiometrically attached to the active site is ca. 20% lower in the aged conjugate, pyrenebutyl-O-P(O)(O-)-Cht (PBP-Cht), than in the nonaged conjugate, pyrenebutyl-O-P-(O)(OC2H5)-Cht (PBEP-Cht). Furthermore, fluorescence decay data indicate that quenching is dynamic and is not caused by oxygen. These data, together with collisional quenching data, imply that quenching originates in an internal interaction of the fluorophore with a group within the protein. Thus, interaction of the pyrene moiety with the polypeptide chain is significantly stronger in the aged than in the nonaged conjugate, implying a different orientation of the fluorophore with respect to the protein. Circular dichroism measurements, which reflect the asymmetry of the bound pyrene in the ground state, as well as circularly polarized luminescence studies, which reflect its asymmetry in the excited state, also show that the relative configuration of the pyrene moiety and the polypeptide chain is significantly altered upon aging. Aged conjugates obtained by use of various fluorescenct organophosphates [pyrenebutyl-O-P(O)Cl2, pyrenebutyl-O-P(O)(p-nitrophenoxy)Cl, pyrenebutyl-O-P(O)(p-nitrophenoxy)2] exhibit similar spectroscopic features, thus substantiating the hypothesis that instantaneous aging, by use of pyrenebutyl-O-P(O)Cl2, and dynamic aging, by gradual removal of an aryloxy group, yield a similar product. This finding provides strong support for the formation of a P-O- moiety in the aged conjugates, since the only expected common product of the two processes is PB-O-P(O)(O-)-Cht. Formation of excimers of the pyrene-containing organophosphorylchymotrypsin conjugates at concentrations above 3 X 10(-6) M is also reported.

Chymotrypsin↗
PubMed1989

Acetylcholinesterase prophylaxis against organophosphate poisoning. Quantitative correlation between protection and blood-enzyme level in mice.

Fetal bovine serum acetylcholinesterase (FBS-AChE, EC 3.1.1.7) was titrated, both in vitro and in vivo, with a highly toxic anti-ChE organophosphate, 7-(methylethoxyphosphinyloxy)-1-methyl-quinolinium iodie (MEPQ). Approximately 1:1 stoichiometry was obtained for the sequestration of MEPQ by FBS-AChE in mice. A quantitative, linear correlation was demonstrated between blood-AChE levels and the protection afforded by exogenously administered AChE in mice when challenged with anti-ChE MEPQ. The results presented in this report demonstrate that such prophylactic measures are indeed sufficient to protect animals against poisoning by as high as an 8 x LD50 dose of organophosphate without the administration of any supportive drug. Despite the relatively large toxic dose, most of the mice that survived the challenge did not show any classical clinical signs of severe anti-ChE poisoning. MEPQ may be considered a suitable model compound for studying the quantitative aspects of the scavenger prophylactic approach described here.

Acetylcholinesterase↗
PubMed1988

Tritium labeling of a powerful methylphosphonate inhibitor of cholinesterase: synthesis and biological applications.

7-(Methylethoxy phosphinyloxy)-1-methyl-quinolinium iodide (MEPQ), a powerful anti-cholinesterase methylphosphonate ester, was labeled with tritium (9 Ci/mmol) at the methylphosphonyl moiety (TCH2P(O)(OR)X) by an iodine-tritium replacement reaction. Kinetic measurements of the rate of inhibition of acetylcholinesterase (AChE) by [3H]MEPQ and its rate of hydrolysis in alkaline solution confirmed the identity of [3H]MEPQ with authentic MEPQ, which was prepared by the same reaction sequences. Gel-filtration experiments verified the radiospecificity of [3H]MEPQ. In vitro radiolabeling of both AChE and butyrylcholinesterase along with the whole-body autoradiography of [3H]MEPQ-treated mice suggests that [3H]MEPQ is a convenient marker for studying biological systems containing these esterases.

Acetylcholinesterase↗
PubMed1986

Sugar conjugates of pyridinium aldoximes as antidotes against organophosphate poisoning.

A series of pyridinium aldoximes having a sugar conjugated to the pyridine ring has been prepared as potential antidotes against organophosphate poisoning. The sugar residue was attached either directly through C-1 or C-6 of the pyranose ring or through a C3 bridge between the glycosyl group and the nitrogen atom of the pyridine moiety. Attachment of a sugar group to the oxime derivative seems to increase the bioavailability of the antidote. The clearance rate of the sugar conjugates was significantly lower than that of their non-sugar analogs and thus they were retained longer in the blood circulation. The sugar derivatives were more potent in decreasing paraoxon-induced hypothermia (which is regulated within the central nervous system) than N-methyl-2-pyridiniumaldoxime methanesulfonate, one of the most commonly used mono-oximes. The sugar analogs were also less toxic than the non-sugar analogs; some also displayed higher efficacy. The mechanism underlying the improved features of the sugar oximes, and the structural requirements in relation to the sugar attachment to the oxime function, are discussed.

Animals↗
PubMed1986

Synthesis and in vitro properties of a powerful quaternary methylphosphonate inhibitor of acetylcholinesterase. A new marker in blood-brain barrier research.

To substantiate reported data and improve the properties of anticholinesterase drugs in blood-brain barrier (B-BB) research, 7-(methylethoxyphosphinyloxy) 1-methyl-quinolinium iodide (MEPQ) was prepared and evaluated as an inhibitor of both acetyl- and butyrylcholinesterase (AChE and BuChE, respectively) from various sources. The second-order rate constants for the inhibition of cholinesterase from eel, mice brain and horse serum at 25 degrees were found to be 5.3 X 10(8), 1.3 X 10(8) and 5.4 X 10(7) M-1 min-1 respectively. The inhibited enzyme could be reactivated by 1-methyl-2-hydroxy iminomethylpyridinium iodide (2-PAM). The two enantiomers of the racemic mixture MEPQ inhibited AChE at similar rates. Low concentrations of AChE could be determined by the residual enzyme activity and by fluorescence measurements of the leaving group, thus suggesting the application of MEPQ as a sensitive titrant of cholinesterase, as well as a potential tool in studying B-BB permeability changes.

Acetylcholinesterase↗
PubMed1985

A quaternary anti-cholinesterase probe for determining the integrity of the blood--brain barrier.

7-(Methylethoxyphosphinyloxy)-1-methyl quinolinium iodide (MEPQ), a new quaternary anti-cholinesterase (anti-ChE) compound was prepared and evaluated as a potential probe for assessing changes in the blood-brain barrier (B-BB) permeability. MEPQ was found to be 170 times more potent in its cholinesterase inhibitory activity than phospholine iodide, a previously reported anti-ChE probe in B-BB research. In rats and mice with impaired B-BB induced by osmotic opening, MEPQ readily penetrated through the damaged site as demonstrated by considerable reduction of ChE activity. In controls, brain ChE activity remained unaffected. It is suggested that MEPQ is a useful probe for both qualitative (histological staining) and quantitative (brain homogenated) assessment of permeability changes in the B-BB.

Animals↗
PubMed1985

Distribution of 3H-soman in mice.

3H-soman (specific activity 10 Ci/mMol), a potent irreversible cholinesterase inhibitor, was administered IV to mice in a dose of one LD-50, which corresponds to 0.25 mCi/mouse. Animals were sacrificed at 5 min, 2 h and 24 h, and whole body autoradiography was performed. High levels of radioactivity in lung and skin were observed at all time intervals after injection. The central nervous system showed very low concentrations of radioactivity, which remained so for 24 h post-injection. Considerable accumulation of 3H-soman in the urine and gall-bladder, and in the intestinal lumen, may indicate these as pathways of soman excretion. Quantitative determinations of radioactivity in various tissue samples were consistent with the above-mentioned findings. It is concluded that the nature of the persistent binding of soman to lung and skin is striking, and may indicate the existence of specific sites for soman depots.

Animals↗