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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.

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.

One common way to examine toxicity in vitro is to measure the effect on cell viability. In one such assay, the 3-(4,5- dimethylthiazol-2-yl)-5-(3 -carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt MTS reagent is bioreduced to a formazan product in living cells. Various cell lines may have differing abilities to reduce MTS; therefore, standardization should be carried out for each one. To optimize the assay, the toxicity of MTS, the linear range for signal versus cell number, and a method of background noise reduction were determined. The MTS reagent decreased cell number after 25 hr. The linear range for the neuronal SK-N-Sll, esophageal KYSE-30, breast MCF-7, and cervical HeLa cell lines were established. Finally, media containing no phenol red significantly reduced background noise compared to media with phenol red.

A large semi-synthetic recombinant antibody library was constructed based on a human anti-tetanus library. The library was used to isolate a human anti-cholera toxin B antibody cidne, which is expressed in XL-1 Blue E. Coli bacteria. The clone, B-27 cholera Fab, was expressed in small scale and purified by a two column procedure, which uses the histidine affinity tag, which was engineered into the protein. The purified cholera Fab was tested by ELISA to confirm specificity and by surface plasmon resonance to determine affinity binding constants.

An antibody-based fiber optic biosensor is described. This biosensor will serve as a model for future development of antibody-based detection of biological toxins. It is sensitive, specific, and new assays will be based solely on the antibody recognition element of the sensor.

A Fiber optic evanescent fluorosensor was used to detect the presence of toxins of biological origin. A direct competition assay was used for cholera toxin and staphylococcus entertoxin B, and an indirect competition assay for cholera, botulinum toxoid A and ricin. Detection of toxins for both methods was in the nanomolar range.

Biodegradation of electronic components was investigated from historical and experimental standpoints. A literature review was conducted on new materials. Twisted wire pairs were tested for degradation by decontamination solution (DS-2). Capacitance readings were used to determine degradation. Initial readings of the immersed wires dropped to zero, indicating shorting of the wire, followed by reading oscillation and return to approximately the initial reading. This phenomenon may be due to pinhole sealing. Further testing is necessary but will require the fabrication of a bridge capable of meeting ASTM/MIL STD specifications. (Author)

Synaptic plasma membranes isolated from bovine brain exhibited a low and high affinity calcium (Ca++) + magnesium (Mg++)-dependent ATPase as evidenced by kinetic constants for ATP. One activity that hydrolyzed ATP maximally at pH 7.4 and 7.8 exhibited an eight-fold higher affinity when compared to the second or lower affinity activity that hydrolyzed ATP maximally at pH 7.0. Only slight inhibition was observed in the presence of rotenone and oligomycin. Although both activities were observed to be trifluoperazine sensitive, they differed significantly with regard to other parameters. Thermal denaturization studies indicated that the high affinity activity was stable for 2 min at 45C aft 50% of the activity was lost at 2.5 min. Although kinetic data are consistent with other findings that indicate the presence of different kinetic conformations of a single synaptic membrane protein, we can not rule out the presence of two different proteins maximally operational at two different ATP concentrations.

Efforts to increase the safety margin of opiate-type drugs led to the discovery of the fentanyl series of compounds. These compounds are sufficiently potent to produce surgical anesthesia with few of the negative side effects associated with morphine, except for respiratory depression. Previous research demonstrated a pharmacological dissociation between the analgesic and respiratory effects of narcotics that is based on different types of the opioid receptor. This study assessed the relative potency and selectivity of one fentanyl derivative (carfentanil) for the putative MU, KAPPA, and DELTA, types of opioid receptor. Rat brain tissue homogenates were labeled at the MU, DELTA, and KAPPA receptor sites. Carfentanil's apparent affinity for each receptor was estimated from the concentration required to displace 50% of the specifically bound radioligands. Biphasic displacement curves were observed for each radioligand, which suggest high and low affinity binding sites. Carfentanil appeared equipotent in displacing the MU and KAPPA radioligands with IC5Os of 0. 7 and 100 pM, while displacing the DELTA radioligand with IC5Os of 0.8 and 40 NM. The results are discussed in terms of their significance for explaining the persistence of respiratory depression of the pharmacologic profile of the fentanyl series of compounds.