Darrel E. Menking

Pharmacological specificity of a nicotinic acetylcholine receptor optical sensor

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.

Comparison of lindane, bicyclophosphate and picrotoxin binding to the putative chloride channel sites in rat brain and Torpedo electric organ

The relative potencies of lindane, picrotoxin and several bicyclophosphate derivatives were compared in their ability to compete with 35S-t-butylbicyclophosphorothionate (35S-TBPS) binding sites in membranes derived from Torpedo electric organ and rat brain. Lindane proved to be ten times more potent in competing with 35S-TBPS binding in electric organ than rat brain, while the bicyclophosphate analogs displayed up to three orders of magnitude greater affinity for rat brain over electric organ. GABA inhibited 35S-TBPS binding in rat brain with moderate potency (IC50 = 30 microM), while unlabelled TBPS inhibited the binding of 3H-muscimol to the GABA receptor with an IC50 greater than 100 microM. The GABA receptor antagonist bicuculline increased 35S-TBPS binding in rat brain both in the presence and absence of 30 microM GABA. The results of the study are discussed in the context of a pharmacological discrimination between voltage-sensitive and receptor-gated Cl- channels in nervous tissue, with lindane and the i-propylbicyclophosphate derivative being the most selective compounds for discriminating between them.

Inhibition of calcium channel dihydropyridine receptor binding by purified Mojave toxin

Mojave toxin, the principal toxic component of the venom of the Mojave rattlesnake Crotalus scutulatus scutulatus, is a protein complex of about 22,000 mol. wt. The mechanism of action of this potent (LD50 = 0.039 micrograms/g, mouse, IV) neurotoxin is a matter of conjecture, but physiologic data suggest a presynaptic site of action with disruption of stimulus-secretion coupling and neurotransmitter release. The selectivity of Mojave toxins effect on several ion channels involved in neurotransmission was assessed in the present study using competitive radioisotopic binding procedures. Synaptic membranes from rat brain were used to assess the toxins interaction with Ca++ and Cl- channels while membrane fragments from the Torpedo fish electric organ were used to determine toxin interaction with the nicotinic acetylcholine receptor-coupled Na+ channel. Mojave toxin was found to irreversibly inhibit 3H-nitrendipine binding to dihydropyridine receptors associated with Ca++ channels in rat brain, but had no effect on radioligand binding in the Na+ and Cl- channel assays. Saturation analysis of the binding further showed that the effects of MoTX on dihydropyridine binding were noncompetitive, with MoTX producing a decrease in both the affinity and density of 3H-nitrendipine sites. These results are consistent with the hypothesis that MoTX acts selectively on Ca++ channel function and that this interaction occurs via an allosteric mechanism in which MoTX binds to a membrane site that is topologically distinct from the dihydropyridine receptor.

Rapid cleanup of bacterial DNA from field samples

Abstract Polymerase chain reaction (PCR) is an in vitro enzymatic, synthetic method used to amplify specific DNA sequences from organisms. Detection of target DNA using gene probes allows for absolute identification not only of specific organisms, but also of genetic material in recombinant organisms. A major challenge facing detection of DNA from field samples is that they are almost sure to contain impurities, especially impurities that inhibit amplification through PCR. For example, humic material even in quantities as small as 1 ng have been shown to inhibit PCR. DNA has been extracted from sewage/stool samples, food, sputum, water and sediment, and human DNA likewise is being extracted from sources as varied as forensic samples of blood, cigarette butts and human remains. However, multi-step, time consuming methods are required to isolate the DNA from the surrounding contamination. This research focuses on developing a method for rapid cleanup of DNA which combines extraction and purification of DNA while, at the same time, removing inhibitors from ‘dirty samples’ to produce purified, PCR-ready DNA. GeneReleaser™ produces PCR-ready DNA in a rapid 5-min protocol. We report here the rapid extraction/purification of plasmid DNA from recombinant Escherichia coli . GeneReleaser™, inhibitor and ∼10 3 cfu recombinant E. coli containing a plasmid insert were added to PCR tubes, vortexed for 30 s and microwaved for 5 min. DNA was PCR-amplified and identified by gel electrophoresis. GeneReleaser™ (GR) resin was able to cleanup samples containing typical aerosol and water/soil contaminants (dust—60 μg, pollen—100 μg, soot—250 μg, humic acid—75 ng). While these inhibitors were easily removed prior to PCR amplification, other complex inhibitors found in soil and food samples remain a major challenge and detection of DNA in these materials typically requires multi-step procedures that may take up to a day. The advantages of using GR are that it is rapid and inexpensive.

Regional sensitivity of neuroleptic receptors to sub-acute soman intoxication

Sub-acute exposure to anti-cholinesterase organophosphorous compounds induces, in humans, cognitive and emotional deficits which include depression, anxiety, emotional lability, and schizophrenic-like symptoms. Neuroleptic drugs used to treat similar clinical conditions bind to serotonergic (S2) and dopaminergic (D2) receptors, suggesting that these sites are involved in the psychiatric consequences of organophosphate exposure. Rats were given saline or soman (50 micrograms/kg, SC) on a sub-acute regimen (three times weekly for four weeks) and killed 1 hr, 1 day or 3 days after the final injection. Response of regional neuroleptic receptors to soman intoxication was assessed using 3H-spiperone as ligand. Initial high affinity binding experiments using mianserine, haloperidol, or both to identify specific cortical binding revealed that mianserine displaceable binding sites showed the greatest down-regulation in response to soman. Subsequent kinetic analyses of mianserine displaceable 3H-spiperone binding indicated a dramatic decrease in the number of hippocampal binding sites and a decrease in the affinity of cortical binding sites. These S2 sites, considered to be involved with neural excitation, have the ability to self-regulate and appear to be involved in the expression of soman neurotoxicity.