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The mating capacity of the male of Spodoptera was slightly decreased by male or female treatment with the chemosterilant metepa, but the decrease was much more when both adults were treated. There was no significant difference neither in male preference between treated and untreated females nor in competition between treated and untreated males. Only young sterilized females mated; 4-days old females lost their mating ability. However adult age may not be an important factor limiting the successful eradication of adults by chemosterilants since they live for about 90 days. On the other hand sterilized moths lived only for about 7 days. The average of time of copula in the different crosses was found to be 65, 69, 75 and 77 minutes for the normal, treated-female, treated-male and treated-treated crosses respectively. The differences of the first three of them are Significant. Abnormal copulations in the treated crosses were observed in which the males could not seperate from females. Zusammenfassung Wirkungen von Metepa auf das Paarungs-Verhalten von Spodoptera littoralis (Boisd.) Die Begattungs-Kapazitat der Mannchen von Spodoptera wurde nach Behandlung der Mannchen oder Weibchen mit dem Chemosterilant Metepa etwas verringert, doch war die Verringerung groser, wenn beide Geschlechter behandelt wurden. Es waren weder die Annahme von Mannchen zwischen behandelten und unbehandelten Weibchen noch die Konkurrenz zwischen behandelten und unbehandelten Mannchen signifikant verschieden. Nur junge sterilisierte Weibchen kopulierten; 4 Tage alte Weibchen verloren ihre Kopulationsfahigkeit. Jedoch braucht das Alter kein wesentlicher Begrenzungsfaktor fur eine erfolgreiche Bekampfung der Falter mit Chemosterilantien sein, da sie etwa 90 Tage lang leben. Demgegenuber betrug die Lebenszeit der sterilisierten Falter nur 7 Tage. Die mittlere Kopulationsdauer bei den verschiedenen Paarungen betrug 65, 69, 75 unci 77 min fur Normalfalter, Sterilisation des Weibchens, des Mannchens sowie beider Geschlechter. Die Unterschiede zwischen den drei ersten Formen sind signifikant. Die Paarungen behandelter Tiere waren z. T. abnorm, indem sich die Mannchen nicht mehr von den Weibchen losen konnten.

An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K D ) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/mL, with an IC50 of 2 ng/mL. Other opiates and heroin metabolites that showed >50% crossreactivity when present at 1 µg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (

Organophosphate (OP) anticholinesterases were found to modulate metabolic activities of human neuroblastoma cells and hepatocytes, which was detectable by the Cytosensor microphysiometer. The nerve gas ethyl-S-2-diisopropylaminoethyl methylphosphorothiolate (VX), at 10 microM, produced significant reduction in cell metabolism within 2 min, as measured by changes in the acidification rate of the medium. The reduction was dose- and time-dependent and irreversible after 4 h of exposure. Two alkaline degradation products of VX produced no cytotoxicity. Exposure for 24 h to 3 microM VX caused 36% and 94% irreversible loss of metabolism in hepatocytes and neuroblastoma cells, respectively. The insecticides parathion and chlorpyrifos stimulated hepatocyte metabolism but inhibited neuroblastoma cells. Their oxons were more active. Exposure of neuroblastoma cells for 4 h to VX, parathion, paraoxon, diisopropylfluorophosphate or chlorpyrifos gave an LC50 of 65, 775, 640, 340, or 672 microM, respectively, whereas 24 h gave an LC50 of 0.7, 3.7, 2.5, 29, and 31 microM, respectively. Preincubation of hepatocytes with phenobarbital enhanced their response to parathion and VX due to metabolic bioactivation. Atropine partially blocked the effects of VX and paraoxon on both cell types, which suggests the involvement of a muscarinic receptor as the target for cytotoxicity. There was no correlation between OP in vivo neurotoxicity and in vitro cytotoxicity. It is suggested that the former results from their cholinesterase inhibition, while the latter results from action on different targets and requires much higher concentrations.

A new solid phase fluoroimmunoassay using a fully automated flow fluorometer adapted for urinalysis of drug metabolites is described. Fluorescein-conjugated benzoylecgonine (FL-BE) and monoclonal antibodies (mAb) against benzoylecgonine (BE) were the reagents used for demonstration. The solid phase consisted of anti-BE mAbs immobilized on the surface of polymethyl methacrylate (PMMA) beads. Free BE in solution competed with FL-BE and reduced bead-bound fluorescence in a concentration-dependent manner. The binding of FL-BE to the anti-BE mAb reached steady-state within minutes. FL-BE was not bound by uncoated beads nor beads coated with non-specific proteins or IgG. The signal-to-noise ratio was 33, and the sensitivity of the assay was 2 ng ml(-1) for BE. The effective concentration of BE was 1 to 100 ng ml(-1), with an IC50 value of 12 ng ml(-1). The mAb showed equal affinities for BE, cocaine, and cocaethylene, but a five order-of-magnitude lower affinity for ecgonine and ecgonine methylester. In a double-blind comparison using clinical urine samples, the data from this single-step competitive assay had excellent agreement with results obtained using a fiber-optic biosensor (FOB), and the EMIT assay performed commercially. The assay provided kinetic data rapidly and can be used to detect small analytes for which antibodies and fluorescein conjugates are available. The affinity of the mAb for FL-BE, calculated from kinetic analysis of the time course of the on and off reaction, was 2.25 x 10(-9) M.

The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443–473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: ( i ) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and ( ii ) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC 3 H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.

Both enantiomers of a 2-(4-pentenyl)pyrrolidine derivative 4 (65–90% ee ), prepared via the asymmetric dihydroxylation (AD) of terminal olefin 2 , underwent a second AD to provide all of the four stereoisomers of indolizidine 209D 1 with enantiomeric enhancement (92–98% ee ). The affinity of 1 for nicotinic acetylcholine receptor was evaluated.

The sea nettle jellyfish toxin (SNTX), which contains several polypeptides, was highly toxic to human hepatocytes. The Cytosensor microphysiometer was used continuously to monitor cell media acidification rate as an index of cellular metabolic activity. Cells exposed to > 1 microg SNTX protein/ml media exhibited a transient increase in metabolic activity, followed by a sharp decrease and cell death within minutes. The kinetics of the transient increase and subsequent decline increased with higher concentrations of SNTX. The biphasic and time-dependent response of hepatocytes to SNTX suggests that more than one mechanism may be involved in the toxicity of its different polypeptides. SNTX-induced cytotoxicity of hepatocytes was reduced by the presence of high titer antibodies against a heterologous jellyfish. Phenobarbital-induced cells became more vulnerable to SNTX, suggesting that some toxin component(s) require(s) bioactivation. Short-term exposure (1-2 h) to 10 microg/ml of the calcium ionophore calcimycin, or the non-selective monovalent cation ionophore gramicidin, had no effect on metabolic activity. However, 165 microg/ml gramicidin or 53 microg/ml calcimycin produced slight transient activation followed by steady decline in metabolic activity, while 20 h exposure to either ionophore produced total cell death. Exposure to even a 10-fold lower concentration of either ionophore killed 88% and 75%, respectively. This contrasts with the toxicity of SNTX which is detectable in minutes with as little as 3 microg/ml. Since pre-exposure to the organophosphate anticholinesterases VX and paraoxon, or the chemotherapeutic alkylating agents cyclophosphamide and mechlorethamine reduced the cytotoxic effects of SNTX, it suggests that phosphorylation or alkylation of cell protein(s) interferes with SNTX toxicity.

Philanthotoxins are noncompetitive inhibitors of the nicotinic acetylcholine receptor and the various glutamate receptors. Analogues carrying photoaffinity labels, fluorine atoms for solid-state NMR studies of ligand/receptor interaction, and large head groups such as porphyrins and planar bulky aromatic rings (BIG analogues) for clarifying mode of entry and orientation of analogues in receptors have been synthesized, assayed against the nicotinic acetylcholine receptor, and brief comments are given for the assay results.

The nicotinic acetylcholine receptor (nAChR) of the electric organ of the electric ray. Torpedo sp., the richest source of nAChR, with similar structure and pharmacology to the mammalian skeletal muscle nAChR, carries several binding sites for different ligands. Incubation of Torpedo membrane-bound nAChRs with the agonist carbamylcholine (Carb) stimulated the binding of [3H]thienyl-cyclohexylpiperidine ([3H]TCP), which binds to the receptor's noncompetitive antagonist binding site in its ionic channel, with high affinity (Kd of 196 nM). The agonist-stimulated binding of [3H]TCP (i.e., binding to activated nAChRs) was inhibited in a concentration-dependent manner by four organophosphate (OP) anticholinesterases, chlorpyrifos oxon (CPO), chlorpyrifos (CPS), parathion (PS), and paraoxon (PO) with IC50 (concentration that inhibits 50% of the effect) values of 5, 150, 200, and 300 microM, respectively. The binding of CPO was totally reversible. The OPs had no effect on equilibrium binding of [alpha-125I]bungarotoxin ([alpha-125I]BGT) to the receptor's acetylcholine (ACh)-binding site, but preincubation of the membranes with the OPs increased this site's affinity for Carb. In absence of agonist, 100 microM of the OPs increased the binding of [3H]TCP by two- to fivefold with the following order of decreasing potency: PS > CPO > CPS > PO. The data suggest that in addition to inhibition of acetylcholinesterase, these OPs bind to a site on the nAChR that is different from the sites that bind ACh or TCP and that this binding induces nAChR desensitization. The relevance of this direct action of OPs on nAChRs on their acute toxicities is discussed.

Chlorendic caproic acid (CCA) was used to synthesize hexachlorocyclopentadienefluorescein (FL) and bovine serum albumin (BSA) conjugates. Anti-CCA antibodies (CCA−Abs), which were raised against BSA−CCA and immobilized on quartz fibers, bound FL−CCA selectively and reversibly. Fluorescence generated by evanescent excitation of the bound FL−CCA was used to monitor the binding event. The affinity of CCA−Abs for FL−CCA (KD = 1.9 nM) was calculated from the time courses of association and dissociation of FL−CCA. The cyclodiene insecticides chlordane, heptachlor, dieldrin, endrin, aldrin, and endosulfan competed with FL−CCA for binding to CCA−Abs and reduced fluorescence in a concentration-dependent manner with the following rank order: chlordane > heptachlor > dieldrin > aldrin > endosulfan. This fiber optic fluoroimmunosensor detects cyclodiene insecticides at the ppb level, has low cross-reactivity with γ-hexachlorocyclohexane, and does not detect (p,p'-dichlorodiphenyl)trichloroethane (DDT). Keywords: Cyc...

The replacement of the quartz halogen lamp of an ORD fluorometer with a high brightness blue-light-emitting diode enhanced evanescent excitation of benzoylecgonine-fluorescein bound to the mAbs immobilized on quartz fibers and gave stronger optical signals.

A fiber optic immunosensor, consisting of a quartz fiber coated with partially purified polyclonal anti-PCB antibodies (Abs), was used to detect polychlorinated biphenyls (PCBs). The optical signal was generated by the binding and subsequent fluorescein (FL) conjugate of 2,4,5-trichlorophenoxybutyrate (TCPB) to the Ab-coated fiber. The Abs, immobilized on the fiber, bound TCPB-FL with high affinity, saturably and reversibly. TCPB and PCBs competed with TCPB-FL for binding and decreased fluorescene in a concentration-dependent manner, with a detection limit of 10 ppb TCPB. The biosensor detected 1 ppm Aroclors, but not polychlorinated pesticides, polychlorinated phenols, or trichlorobenzene. It was more selective for TCPB at 1 ppm than the four tested Aroclors (i.e. 1016, 1232, 1250, and 1262) by 1.4-, 2.2-, 5.5-, and 3-fold, respectively. It detected Aroclor 1242 added to river waters and standard reference soil samples with reasonable accuracy. Advantages of this biosensor are its fast detection time (seconds to minutes), high sensitivity, and reversibility. 37 refs., 11 figs., 3 tabs.

A fiber-optic biosensor was developed for detection of cocaine, its metabolites, and other coca alkaloids, using a monoclonal antibody (mAb) against a derivatized benzoylecgonine (BE). The mAb was immobilized noncovalently on quartz fibers and a flow fluorometer was used to detect changes in evanescent wave fluorescence. A fluorescein (FL) conjugate of BE bound to the mAb specifically in a saturable manner and with high affinity (Kd = 7.6 nM). Cocaine or other test compounds competed with FL-BE for binding to the mAb in a concentration-dependent manner, thereby reducing the initial rate or steady-state fluorescence. Addition of cocaine to the flow buffer after reaching steady-state fluorescence enhanced the dissociation of bound FL-BE, and cocaine removal allowed fiber regeneration for multiple measurements. The detection limits for cocaine, cocaethylene, norcocaine, and BE were 5, 5, 29, and 30 ng/ml, respectively, but for ecgonine it was 4600 ng/ml and for methylecgonine it was 2000 ng/ml. Tropacocaine was detected at 10 ng/ml, but atropine was detected at 2900 ng/ml. The biosensor discriminated by 833-fold between cocaine and its stereoisomer pseudococaine. Structural features necessary for high-affinity recognition by this mAb are benzoate and 3 beta configuration, both of which are found in BE, cocaine, norcocaine, and cocaethylene.

Using radioreceptor assay with [ 3 H]α-bungarotoxin (α-BGT) and [ 3 H]phencyclidine (PCP) as probes for the nicotinic acetylcholine receptor (nAChR) in membranes obtained from honeybee heads, the effects of various nAChR ligands, nicotinoids and neonicotinoids were studied. The data indicated differences in pharmacological characteristics between Torpedo electric organ and honeybee brain nAChRs. [ 3 H]α-BGT binds to the acetylcholine (ACh) recognition site of the nAChRs of vertebrate skeletal muscle, Torpedo electric organ and honeybee brain. In vertebrates, [ 3 H]PCP binds to an allosteric site on the receptor's ion channel and its binding is stimulated by receptor activation with agonists. However, the tested vertebrate cholinergic agonists inhibited [ 3 H]α-BGT binding, but did not activate [ 3 H]PCP binding to the honeybee nAChR. Saturation isotherms of the binding of [ 3 H]α-BGT with or without PCP indicated that PCP interacted with the ACh recognition site on the nAChR. Furthermore, nicotine inhibited not only [ 3 H]α-BGT binding but also [ 3 H]PCP binding. Detailed study of [ 3 HjPCP binding indicated that [ 3 H]PCP bound to the honeybee brain membranes both at high and low affinity sites. The former corresponded to the vertebrate allosteric site on the nAChR and the latter to the ACh recognition site. Nicotine, anabasine and nitenpyram bound to both sites, while imidacloprid, 6-Cl-PMN1 and acetamiprid bound selectively to the ACh recognition site. In houseflies, nicotine and imidacloprid produced excitation followed by paralysis, while PCP was anesthetic, even though PCP was as insecticidal as nicotine.

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diisopropylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.

Thin Layer Scintillation Counting (TLSC) is a new approach to ligand binding and transport assays. It uses a thin layer of scintillant to coat the radioactivity trapped on the filter, instead of placing the filter in bulk scintillant as is done in conventional Liquid Scintillation Counting (LSC). It saves on time, labor and cost and addresses better the problem of radioactive waste disposal. The methodology is described and compared to conventional LSC using examples of binding of drugs to brain membranes and uptake into synaptosomes. 1Current address: School of Pharmacy, Cairo University, Cairo, Egypt.

We previously reported (J. Med. Chem. 1993, 36, 1188-1193) that changes to the ring size of the piperidine and cyclohexyl rings of the high-affinity and selective dopamine (DA)-uptake inhibitor 1-[1-(2-benzo[b]thienyl)cyclohexyl]piperidine (BTCP, 2) caused different, and in some cases opposite, changes in affinity for sites on the DA transporter labeled by [3H]BTCP and [3H]-cocaine. These results suggested that the radioligands label different sites on the transporter. In the present study, we extend the structure-activity relationships (SAR) of BTCP by studying the binding characteristics of a series of N,N-disubstituted 1-(2-benzo[b]thienyl)cyclohexylamines 7-32 at the DA transporter. Cyclohexyl was selected as opposed to other ring sizes since it corresponds to BTCP. The binding results indicate that a considerable degree of structural variation is permitted for the N-substituents, while still retaining nanomolar affinity for sites on the transporter (studied in rat forebrain homogenates). As observed in our earlier study, the differential effects of structural change on binding to sites on the DA transporter labeled by these radioligands suggests that they are different and distinct binding sites. In general, and up to a point, increasing the size and lipophilicity of the N substituents resulted in improvements in binding but appeared to have less predictable effects on DA-uptake inhibition (as measured in rat brain synaptosomes). The binding of these compounds to sites labeled by [3H]BTCP appeared to correlate best with IC50 for DA-uptake inhibition. To our surprise, the monoalkyl N-substituted BTCP derivatives displayed the highest affinity for the DA transporter of all the compounds in this series. For example, the N-(cyclopropylmethyl) derivative 14 displayed IC50's = 23 nM ([3H]cocaine) and 1 nM ([3H]-BTCP), and the N-butyl derivative 10 showed IC50's = 60 nM ([3H]cocaine) and 0.3 nM ([3H]-BTCP). BTCP exhibited IC50's of 39 nM ([3H]cocaine) and 5 nM ([3H]BTCP) in this assay. The observation that N,N-dibutyl derivative 31 exhibited low ratios of IC50 [3H]cocaine/IC50 DA reuptake and IC50 [3H]BTCP/IC50 DA reuptake suggests that it may be a potential candidate for cocaine antagonism studies. The effect of additional amino, amide, and aromatic groups on the N-substituents was examined, and the results are discussed. The failure of all of the compounds in this series to bind phenycyclidine receptors coupled with their high affinity and range of selectivities at the DA transporter identifies many of them as useful tools for probing the mode of action of BTCP at this site.

Imazethapyr is the active ingredient of PURSUIT herbicide. This herbicide belongs to the imidazolinone class of compounds. Polyclonal antibodies have been prepared in rabbits and sheep which specifically recognize this class of compounds. Using immune sera, ELISAs have been developed for imazaquin, imazapyr, imazethapyr, and imazmethabenz methyl (the active ingredient for SCEPTER, ARSENAL, PURSUIT, and ASSERT herbicides, respectively) with sensitivity at low nanogram per milliliter levels. The quantitation of imidazolinones in soil requires a certain amount of sample pretreatment, thus, the throughput is not ideal. A simpler immunoassay method for screening large amounts of soil samples economically would be useful

A light addressable potentiometric sensor (LAPS) was used to detect organophosphate and carbamate anticholinesterases (anti-ChEs), using eel acetylcholinesterase (AChE) as the biological sensing element. Biotinylated AChE was preincubated with inhibitor or buffer alone and then captured on biotinylated nitrocellulose membrane via streptavidin cross-linking, or AChE was preimmobilized on the capture membrane and then a sample containing the anti-ChE was filtered through the capture membrane. Hydrolysis of acetylcholine (ACh) by the captured AChE resulted in a strong potentiometric signal, and the immobilized AChE retained its affinity for ACh and anti-ChEs. IC50 values for inhibition of captured AChE obtained by the LAPS agreed with those obtained by a spectrophotometric method or a fiber optic evanescent fluorosensor. Paraoxon and bendiocarb were detected at 10 nM, while higher concentrations were required for monocrotophos, dicrotophos, dichlorvos, phosdrin, diazinon, tetraethyl pyrophosphate, aldicarb, and methomyl. Important features of the LAPS for detection of anti-ChEs are speed (eight samples assayed simultaneously in minutes), precision, and reusability.

Both enantiomers of a 2-(4-pentenyl)pyrrolidine derivative 4 (65–90% ee ), prepared via the asymmetric dihydroxylation (AD) of terminal olefin 2 , underwent a second AD to provide all of the four stereoisomers of indolizidine 209D 1 with enantiomeric enhancement (92–98% ee ). The affinity of 1 for nicotinic acetylcholine receptor was evaluated.

Casein-parathion conjugates, immobilized by adsorption on quartz fibers, selectively adsorbed antiparathion rabbit antibodies raised against bovine serum albumin (BSA)-parathion conjugates from polyclonal immune sera. The presence of free parathion interfered with binding of the rabbit's antiparathion Abs. Fluorescein isothiocyanate(FITC) goat antirabbit IgG were used to generate an optical signal, which was measured by a signal transducer based on total internal reflection fluorescence. Inhibition of binding of rabbit Abs by free parathion, reduced the optical signal and provided the basis for detection of parathion with this optical immununosensor. It could detect 0.3 ppb parathion and was highly specific since its detection of the oxygen analog paraoxon was possible at 100-fold higher concentration.

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M3 subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [3H]quinuclidinyl benzilate ([3H]QNB) was most sensitive to atropine and the M3-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M2 antagonist AFDX116. This, and the binding characteristics of [3H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M3 subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 microM) reduced Bmax of [3H]4-DAMP binding from 27 +/- 4 to 13 +/- 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolin-induced cAMP formation in SMG cells with an EC50 of 200 nM, but paraoxon was greater than 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 microM atropine, that by paraoxon was unaffected by up to 100 microM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect beta-adrenoreceptor function in the preparation, since it did not affect the 10 microM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 microM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells.(ABSTRACT TRUNCATED AT 250 WORDS)

An optical sensor for anticholinesterases (AntiChEs) was constructed by immobilizing fluorescein isothiocyanate (FITC)-tagged eel electric organ acetylcholinesterase (AChE) on quartz fibers and monitoring enzyme activity. The pH-dependent fluorescent signal generated by FITC-AChE, present in the evanescent zone on the fiber surface, was quenched by the protons produced during acetylcholine (ACh) hydrolysis. Analysis of the fluorescence response showed Michaelis-Menten kinetics with a Kapp value of 420 microM for ACh hydrolysis. The reversible inhibitor edrophonium (0.1 mM) inhibited AChE and consequently reduced fluorescence quenching. The biosensor response immediately recovered upon its removal. The carbamate neostigmine (0.1 mM) also inhibited the biosensor response but recovery was much slower. In the presence of ACh, the organophosphate (OP) diisopropylfluorophosphate (DFP) at 0.1 mM did not interfere with the ACh-dependent fluorescent signal quenching, but preexposure of the biosensor to DFP in absence of ACh inhibited totally and irreversibly the biosensor response. However, the DFP-treated AChE biosensor recovered fully after a 10-min perfusion with pralidoxime (2-PAM). Echothiophate, a quaternary ammonium OP, inhibited the ACh-induced fluorescence quenching in the presence of ACh and the phosphorylated biosensor was reactivated with 2-PAM. These effects reflected the mechanism of action of the inhibitors with AChE and the inhibition constants obtained were comparable to those from colorimetric methods. The biosensor detected concentrations of the carbamate insecticides bendiocarb and methomyl and the OPs echothiophate and paraoxon in the nanomolar to micromolar range. Malathion, parathion, and dicrotophos were not detected even at millimolar concentrations; however, longer exposure or prior modification of these compounds (i.e., to malaoxon, paraoxon) may increase the biosensor detection limits. This AChE biosensor is fast, sensitive, reusable, and relatively easy to operate. Since the instrument is portable and can be self-contained, it shows potential adaptability to field use.

Biotinylated acetylcholinesterase (AChE) from Eel was immobilized to a biotinylated cellulose nitrate membrane via a streptavidin bridge. Using a light addressable potentiometric sensor (LAPS), enzyme activity was determined as a function of substrate concentration (i.e. acetylcholine) and amount of enzyme immobilized to the membrane. This biosensor was also evaluated for its potential to detect organophosphate (OP) anticholinesterases (antiChEs).

Five (1R-2-exo-3-exo)-3-(N-phenylcarbamoyl)ecgonine methyl ester analogues were synthesized and characterized by 1H and 13C NMR, IR, and thermospray MS. The compounds were synthesized in two or three steps as (-)-stereoisomers from (-)-ecgonine in good yield (56% overall). These cocaine derivatives were assessed for their ability to inhibit [3H]cocaine binding to rat striatal tissue and to inhibit [3H]dopamine uptake into synaptosomes prepared from the same tissue. The most potent of the analogues was (1R-2-exo-3-exo)-2-(carbomethoxy)-8-methyl-8-azabicyclo[3.2.1]octyl 3-N-(3'-nitrophenyl)carbamate. IC50 values for inhibition of cocaine binding and dopamine uptake were 37 and 178 nM, respectively. Amino derivatives were less active than the nitro and (1R-2-exo-3-exo)-2-(carbomethoxy)-8-methyl-8-azabicyclo [3.2.1]octyl 3-N-(4'-aminophenyl)carbamate had the lowest affinity for the receptor with IC50 values of 63 and greater than 100 microM in the aforementioned assays, respectively.

The mechanism of action of the anticholinesterase paraoxon on the function of a muscarinic receptor subtype in rat brain striatum was investigated. Paraoxon inhibited binding of the muscarinic agonist cis -[ 3 H]methyldioxolane, which binds to a high-affinity population of muscarinic receptors, with K 0.5 of 80 n M , compared to a K 0.5 of 7 μ M for parathion. The inhibition was competitive, suggesting that paraoxon and CD bind to a common site. When this muscarinic receptor (possibly an M 4 subtype) is activated it inhibits cAMP synthesis. Thus, function of the paraoxon-sensitive receptor was assayed by the inhibition of the forskolin-activated [ 3 H]cAMP synthesis. Paraoxon inhibited cAMP synthesis in a dose-dependent manner as did the muscarinic agonist carbachol, and this inhibition was completely blocked by the muscarinic antagonist atropine. When low concentrations of carbachol and paraoxon were used together, there was additive inhibition of cAMP synthesis. However, there was no further increase when both paraoxon and carbachol were present in concentrations that individually produced maximal inhibition. The data suggest that paraoxon acts like carbachol, causing activation of the muscarinic receptor subtype in brain striatum and leading to inhibition of cAMP synthesis. Considering the high affinity that this muscarinic receptor has for paraoxon, it is suggested that this direct reversible action of paraoxon on the muscarinic receptor could affects its toxicity, expecially early on before most of the acetylcholinesterase is phosphorylated.

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype-selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains. In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype-selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist, 11-[2-[dimethylamino)-methyl)1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepin-6 one (AF-DX 116), the M2-selective antagonist, and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) the M3-selective antagonist, was also the same as that of mammalian brains, i.e., 4-DAMP greater than PZ greater than AF-DX 116. The three insect brain receptors had 27-50-fold lower affinity for PZ (Ki 484-900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4-DAMP (Ki 18.9-56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.

The optical signal generated by a nicotinic acetylcholine receptor (nAChR)-based optical biosensor was dependent on the density of toxin binding sites (i.e. receptors) immobilized on the surface of the fiber. The maximum density of nAChR receptors absorbed on the optic fibers, measured using [125I]-alpha-bungarotoxin (the alpha-neurotoxin of the Bungarus snake venom) binding, was 6.2 pmol of receptor sites per fiber. Incubation time, that was required for maximal noncovalent immobilization of the receptor protein on the fiber, was less than 10 min. Immobilization of the nAChR protein on the quartz fiber was affected by pH of the medium, with pH 3.5 as the optimal. Stored in phosphate buffered saline (PBS) or in a dry nitrogen atmosphere, the nAChR optical sensor showed no loss of activity over the first 3 days, then showed a slow but gradual loss in activity (45% over the next 30 days).

The pharmacological specificity of a nicotinic acetylcholine receptor (nAChR) optical biosensor was investigated using three fluorescein isothiocyanate (FITC)-tagged neurotoxic peptides that vary in the reversibility of their receptor inhibition: alpha-bungarotoxin (alpha-BGT), alpha-Naja toxin (alpha-NT), and alpha-conotoxin (GI) (alpha-CNTX). Kinetic analysis of the time course of binding of FITC-neurotoxins to the nAChR-coated fiber gave association rate constants (k+1) of 8.4 x 10(6) M-1 min-1 for FITC-alpha-BGT, 6.0 x 10(6) M-1 min-1 for FITC-alpha-NT and 1.4 x 10(6) M-1 min-1 for FITC-alpha-CNTX. The dissociation rate constants (k-1) for the three neurotoxins were 7.9 x 10(-3) min-1. 4.8 x 10(-2) min-1 and 8.0 x 10(-1) min-1 for FITC-alpha-BGT. FITC-alpha-NT and FITC-alpha-CNTX, respectively. The equilibrium dissociation constant (Kd) values for the three toxins. calculated from these rare constants, were similar to published values obtained from tissue responses or ligand binding assays. The optical signal generated by FITC-alpha-NT binding to the nAChR-coated fiber was effectively quenched by agonists and antagonists of the nAChR but not by most of the tested agonists and antagonists of muscarinic cholinergic, adrenergic, glutamatergic, serotonergic, dopaminergic or GABAergic receptors. Interestingly, 5-hydroxy-tryptamine, haloperidol and (+)cis-methyldioxolane gave significant inhibition of FITC-alpha-NT binding to the immobilized receptor. Equilibrium constants of inhibition (Ki) for d-tubocurarine (d-TC) and carbamylcholine (carb) were determined from competition studies using FITC-alpha-CNTX. FITC-alpha-NT or FITC-alpha-BGT as probes for receptor occupancy. When the more reversible probe FITC-alpha-CNTX was used, the Ki value for d-TC was an order of magnitude lower than those determined using the less reversible probes. Ki values for carb however, were independent of the FITC-toxin probe used.

3-Arylecgonine analogues were synthesized and characterized by 1H and 13C NMR, IR, and MS. The compounds were synthesized as racemates from cycloheptatriene-7-carboxylic acid or enantiomerically from (-)-cocaine. These analogues were tested for their ability to inhibit [3H]cocaine binding to bovine striatal tissue and to inhibit [3H]dopamine uptake into striatal synaptosomes. Methyl (1RS-2-exo-3-exo)-8-methyl-3-phenyl-8-azabicyclo[3.2.1]octane-2-ca rboxylate was the most potent analogue. IC50 values for inhibition of cocaine binding and dopamine uptake were 20 and 100 nM, respectively. The racemates and the 1R isomers were equally potent inhibitors of binding and uptake. Methyl (1RS-2-endo-3-exo)-3-(2,4-dinitrophenyl)-8-methyl-8-azabicyclo[3.2 .1]octane- 2-carboxylate was the least potent. IC50 for inhibition of both binding and uptake was 40 microM.

The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[3H]cis-methyldioxolane ([3H]CD), which has been used to label a high affinity population of M2 receptors. A single population of sites (KD 2.74 nM; Bmax of 82 fmol/mg protein) was detected and [3H]CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [3H]CD and considered to be of the M2 subtype. Up to 70% of the [3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of [3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM [3H]CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic receptors have for these toxicants point to their extra vulnerability. It is suggested that the success of iv administration of the muscarinic receptor inhibitor atropine in initial therapy of poisoning by OP anticholinesterases may be related in part to the extra sensitivity of M2 receptors to certain OPs.

The synaptic action of biogenic amines, in the central and peripheral nervous systems, is terminated by reuptake of the amine transmitters into the presynaptic nerve terminals (1). This neuronal uptake is temperature-dependent and exhibits saturation kinetics and stereospecificity for substrates and inhibitors (2). Striking properties of neuronal amine uptake are its complete dependency on Na+ (1–3) and its sensitivity to the cardiac glycoside ouabain (3).Thus, it was hypothesized that neuronal amine uptake was a Na+-dependent active transport process like uptake of sugars and amino acids (1) and that Na+,K+-ATPase provided the energy to drive amine transport. This model prevailed for the last 20 years despite serious challenges early on (4, 5), and ATP-dependent uptake was never observed. Neuronal amine transporters are the pharmacological receptors for tricyclic antidepressants and central stimulants (e.g. cocaine). Although cocaine inhibits uptake of norepinephrine and 5-hydroxytryptamine as well as dopamine (DA), it is the last effect which correlates best with cocaine's behavioral actions (6–8). In this study, we provide evidence that the cocaine-sensitive uptake of DA into striatal synaptosomes is regulated by ATP.

[3H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [3H]-cocaine with a high affinity site of 170 fmole/mg protein (Kd 16.7 nM). The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [3H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear.

The total synthesis of pyrrolizidines 223H', 239K', 265H', and 267H' has been achieved starting from 1,5-hexadiene via a common synthetic intermediate 5. The affinity of 1-4 for nicotinic acetylcholine receptor was evaluated.

A comparison of the toxinological properties of nematocyst venoms from Old and New World Cassiopea and Aurelia species was undertaken. The cnidom of venomous Cassiopea andromeda (Ca) and Aurelia (Aa(RS)) from the Red Sea was identical to that of nonvenomous Bahamian Cassiopea xamancha (Cx) and Chesapeake Bay Aurelia aurita (Aa(CB)), respectively. A clean nematocyst preparation of Ca and both Aurelias could be obtained but algal particles could not be separated completely from the Cx nematocysts. Further purification of all four nematocyst preparations showed significant differences in the action of their protein. Only the Cassiopea had coexisting dermonecrotic and vasopermeability producing properties and Ca's hemolytic activity was associated with mouse lethality. The protein, hemolysin and phospholipase gel filtration eluant curves of Ca venom were similar. Venomous Aa(RS) actively stung lips and contained more potent mouse lethal, demonecrotic, vasopermeability plus hemolytic factors than Aa(CB). Cross reactivity of convalescent human serum obtained from patients stung by Ca and venomous Cx collected in Central America occurred. This was also observed between sera of bathers stung by Aa(RS) and stinging Aurelia which appeared in Florida during the recent El Niño year. IgG was stimulated by several nematocyst proteins since many venom subfractions tested positive at high titers against convalescent sera. T-cell proliferation of mice primed with either Aurelia venom was positive against the homologous preparation with cross reactivity to the heterologous venom. Crude venoms of both Red Sea jellyfish metabolically stimulated cultured human hepatocytes more than their New World counterparts. This data shows that considerable similarities and differences exist in the venoms of these Old and New World Cassiopea and Aurelia medusae with the Eastern species being more potent.

There is increasing need in industry and government for toxicity assessment of the growing numbers of new chemicals added to the environment such as pesticides, industrial products, hazardous waste, food additives, cosmetics and therapeutics. This is necessary for regulatory and other purposes. Hundreds of thousands of laboratory animals are used in toxicity testing so as to obtain statistically significant results. Because of these and ethical circumstances such as undue pain and suffering, there is a growing demand for use of alternative testing methods. Most importantly is the sometime (inherent) inaccuracy of extrapolation of toxicity data from animal studies to humans. An example is the high potency of demyelinating agents (e.g. tri-orthocresylphosphate) to chicken, cat and human but not to rodents (the choice animals for toxicity studies). Another is dioxin, which is a most potent carcinogen to rodents but much less so to humans. Thus, the only way to reliably predict the human toxicity of a chemical is to test it on humans. This is not feasible, but the availability of many human cell lines make possible the collection of human in vitro toxicity data.

Analyte 2000, a four-channel fiber optic biosensor (FOB), was used for analysis of cocaine and its metabolites (COC) in human urine using a competitive fluorescence immunoassay. Binding of antibenzoylecgonine monoclonal antibody (mAb) to the casein-benzoylecgonine Ag-coated, tapered optical fibers was inhibited by COC. Bound mAb, which inversely correlated with COC concentration, was quantitated by fluorescence produced by evanescent excitation of bound cyanine dye-tagged antimouse antibody (CY5-Ab). The effective concentration range of benzoylecgonine (BE) for inhibiting the fluorescent signals was 0.75-50 ng/mL, with IC50 of 9.0 ng/mL. This FOB had similar affinities for BE, cocaine, and cocaethylene, but very low affinities for ecgonine and ecgonine methyl ester. A sensitivity of 100% and a specificity of 96% were achieved when 54 human urine specimens were analyzed by FOB (cutoff, 300 ng/mL COC) and GC-MS (cutoff, 150 ng/mL BE). All results were in agreement except for one positive FOB result with a GC-MS BE concentration of 148 ng/mL. In addition, regeneration and reuse of the fiber for multiple analyses were performed successfully with no carryover from specimens containing high COC concentrations to specimens containing low COC concentrations.

Ten 3 beta-ecgonine analogues were synthesized and characterized by 1H and 13C NMR, MS, and elemental analysis. The compounds were synthesized as (-)-stereoisomers from (-)-cocaine. These compounds were assessed for their ability to inhibit [3H]cocaine binding to rat striatal tissue and to inhibit [3H]DA uptake into rat striatal synaptosomes. In this series of compounds, the length of the spacer between the aryl group and the tropane skeleton ranged from 1 to 4 bond distances, and conformational flexibility of the linkage and orientation of the aryl ring system were controlled by various types of linkages. The most potent of the analogues was methyl-(1R-2-exo-3-exo)-8-methyl-3-(beta-styrenyl)-8-azabicyclo[3. 2.1] octane-2-carboxylate. One of the less potent compounds was found to inhibit [3H]cocaine binding and [3H]DA uptake with significantly different IC50 values, in contrast to 14 other 3 beta-substituted analogues. Molecular modeling and CoMFA analysis were used to obtain a rigorous structure-function relationship for the studied compounds. The results showed that the potencies of these 3 beta-substituted ecgonine methyl esters were dominated by steric effects and were acutely sensitive to the distance between the aryl ring and the tropane skeleton and to the orientation of the aryl ring system relative to the tropane skeleton. The current study provides a clearer picture of the shape and size of the putative hydrophobic binding pocket for the 3 beta substituent at the cocaine receptor as well as emphasizing the importance of a drug's free energy of solvation in obtaining structure-activity relationships.

A fiber optic evanescent fluoroimmunosensor was used to rapidly detect and quantitate coca alkaloids as cocaine equivalents in leaf extracts of five Erythroxylum species. A monoclonal antibody (mAb) made against benzoylecgonine (BE), a major metabolite of cocaine, was immobilized covalently on quartz fibers and used as the biological sensing element in the portable fluorometer. Benzoylecgonine-fluorescein (BE-FL) was used as the optical signal generator when it bound to the fiber. If present, cocaine competed for the mAb and interfered with the binding of BE-FL, thereby reducing the fluorescence transmitted by the fiber. Calibration curves were prepared by measuring (over 30 s) the rates of fluorescence increase in the absence, or presence of cocaine. Ethanol or acid extracts of dry coca leaves were assayed by this fiber optic biosensor, gas chromatography and a fluorescent polarization immune assay. Biosensor values of cocaine content of leaves from five Erythroxylum species were not significantly different from gas chromatography values, but had higher variance. The biosensor assay was rapid and did not require cleanup of the crude leaf extracts. Cocaine in acid extracts was reduced significantly after 4 weeks at 23 degrees C and after 3 weeks at 37 degrees C. Fibers (mAb-coated), stored at 37 degrees C in phosphate-buffered solution (0.02% NaN3), gave stable responses for 14 days.

The Cytosensor™ microphysiometer was used to continuously monitor perturbations in metabolic rates of the human liver cell line ATCC-CCL-13 when exposed to each of 10 drugs. The effects of exposure to one concentration for 24 hr or to sequential increasing concentrations for 4 hr, and recovery after drug removal, were compared. Paracetamol (acetaminophen) and ethanol were used to establish the assay protocols and determine reversibility of drug effect. All drugs produced concentration-and time-dependent reduction in acidification rate following 24 hr exposure, which may be due to decreased number of viable cells and/or lowered metabolic rates of the live cells. The degree of irreversible inhibition of acidification rate was used as an index of cell death and the IC50 values for the 10 drugs were comparable to those produced in the same cell line by a fluorescence assay using Calcein AM stain (r = 0.991), that fluoresces only in live cells, as well as the [3H]thymidine uptake assay (r = 0.976). There was also excellent correlation (r = 0.958) between IC50 values of 24 hr exposure obtained from the Cytosensor with the 10 drugs and their published human lethal blood concentrations. An advantage of this new methodology over other in vitro assays is that it allows the determination of time points at which reversible change becomes irreversible.

The production of antibodies towards antigens with low immunogenicity is enhanced by the intrinsic efficiency of screening combinatorial libraries of immunoglobulins. The need to isolate clones with rare binding specificities has dictated a highly efficient method of screening and isolating antibody clones. The production of recombinant immunoglobulin libraries in bacteria allows for a more controlled selection of antibody specificity and can be used in circumstances where hybridoma fusions are unable to isolate rare clones with the desired epitope specificity. Botulinum neurotoxin (NT) with associated non neurotoxin proteins (non-NT) as a complex was used to immunize mice to obtain mRNA for the production of a recombinant antibody library with a repertoire of specificities. Initial screens of the combinatorial library revealed clones which recognized the non-neurotoxin proteins of the toxin complex similar to monoclonal antibodies produced by conventional hybridoma fusions. The combinatorial library was re-screened in order to isolate antibodies that specifically recognized the neurotoxin component of the toxin complex. The ability to alter the biopanning selection process affords the researcher a measure of control in the selection process not available with traditional hybridoma fusions.