Determination of fusaric acid in maize using molecularly imprinted SPE clean-up.

A new LC method to detect fusaric acid (FA) in maize is reported based on a molecularly imprinted SPE clean-up using mimic-templated molecularly imprinted polymers. Picolinic acid was used as a toxin analog for imprinting polymers during a thermolytic synthesis. Both acidic and basic functional monomers were predicted to have favorable binding interactions by MP2 ab initio calculations. Imprinted polymers synthesized with methacrylic acid or 2-dimethylaminoethyl methacrylate exhibited imprinting effects in SPE analysis. FA levels were determined using RP ion-pairing chromatography with diode-array UV detection and tetrabutylammonium hydrogen sulfate in the mobile phase. A method was developed to detect FA in maize using molecularly imprinted SPE analysis within the range of 1-100 µg/g with recoveries between 83.9 and 92.1%.

Pharmacological profile of radioligand binding to the norepinephrine transporter: instances of poor indication of functional activity.

Binding assays for the norepinephrine (NE) transporter (NET) with [3H]nisoxetine have generally yielded weak potencies for compounds related to cocaine and 1-(2-(di(4-fluorophenyl)-methoxy)-ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909), as compared with their functional activity in inhibiting NE uptake. In the present work with HEK-293 cells expressing the human NET (hNET), potential underlying causes for this discrepancy have been addressed: ambient temperature of the binding assay, buffer in the assay, preparation used for source of the NET, and radioligand. The results indicate that the standard [3H]nisoxetine binding assay at 0 degrees C with cell membrane preparations in high Na+ buffer underestimates the functional potency of compounds related to cocaine and GBR 12909; in drug development studies it is advisable to either carry out [3H]nisoxetine binding assays with intact cells under uptake conditions, or perform classical [3H]NE uptake studies with intact cells (or with synaptosomes from brain tissue).

Structure-activity relationships for substrate recognition by the human dopamine transporter.

Information is available on the structure-activity relationships for dopamine as a substrate for uptake by the dopamine transporter. However, dopamine transport is a complex process involving substrate binding, translocation, release as well as transporter reorientation. The present study examines only the substrate recognition step by assessment of the potency of various dopamine-related compounds in inhibiting the binding of the cocaine analog [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([3H]WIN 35,428) to human dopamine transporters expressed in HEK-293 cells. alpha-Methylation of the side chain, the presence of the amine, and the 2-carbon-length of the side chain were found to be important for binding affinity, whereas beta-hydroxylation of the side chain and methoxylation at the phenyl ring generated weaker compounds. In addition, the presence of both m- and p-OH at the phenyl ring bestowed an increase in potency but the presence of p-OH alone a decrease. N-alkylation (propylation or methylation) had little or an even slightly beneficial effect on affinity, whereas alpha-carbonylation and alpha-methanoylation reduced affinity. Amino naphthalene compounds with a fused benzenoid ring system retained some potency consonant with the extended (i.e. beta-rotameric) trans (=anti) form of the side chain in dopamine when interacting with the transporter. In a second series of experiments, the interaction between dopamine and structural variants was assessed by monitoring the capability of a compound to shift the dopamine inhibition curve to the right as expected for a competitive inhibitor acting at the same site. Appreciable deviation from competitive interaction was observed by removal of the amine from the side chain, by alpha-carbonylation, and by alpha-methanoylation. Two blocker-type compounds, semi-rigid variants of cocaine, also displayed significant deviation. A substrate-based compound, inhibiting cocaine analog binding without interfering with dopamine recognition, could be a cocaine antagonist allowing conformational changes to occur during dopamine uptake.

Binding of cocaine-like radioligands to the dopamine transporter at 37 degrees C: effect of Na+ and substrates.

Although dopamine (DA) translocation by the DA transporter (DAT) requires Na+, a role for Na+ in the DA recognition step in the translocation cycle has been questioned. Thus, when binding techniques were used to indirectly measure the affinity of DA for DAT via its potency in inhibiting cocaine analog binding, no stimulation of DA binding was observed when assay temperature was at or below room temperature. The present work describes the use of 3H-labeled cocaine analogs for assays at 37 degrees C. When there is sufficient Na+ in the medium (> or =25 mM), [3H]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([3H]CIT) is an excellent radioligand to label human DAT with high affinity in membrane preparations of HEK-293 cells expressing the transporter. However, at 0 and 5 mM of Na+, appreciable binding of [3H]CIT occurs to proteins other than DAT, hampering accurate assessment of DAT-associated binding. No such problems occur with the binding of the 4-fluoro homolog of [3H]CIT, [3H]CFT at 37 degrees C, and this radioligand can be used at low [Na+], provided enough protein is present in the assay. The application of these assays show that, in contrast to the strong Na+ dependency of the binding of CFT, the substrates DA, D-amphetamine, p-tyramine, and DL-octopamine are not stimulated by Na+. This demonstrates that lack of Na+ stimulation of binding of substrates, including DA to DAT, in membrane preparations at room temperature is not caused by the reduced fluidity of the frozen state of the hydrocarbon membrane interior at this temperature as compared with the liquid-expanded state at 37 degrees C.

The role of N-glycosylation in function and surface trafficking of the human dopamine transporter.

The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V(max), and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type.