Resequencing microarray method for molecular diagnosis of human arboviral diseases.

BACKGROUND: Resequencing DNA microarray (RMA) technology uses probes designed to identify a panel of viral sequences. It can be used for detecting emerging viruses by revealing the nucleotide polymorphisms within the target of interest. OBJECTIVES/STUDY DESIGN: As a new tool for molecular diagnosis of arbovirus infection, high density PathogenID v2.0 RMA (PID2-RMA) was assessed for the detection and genetic analysis of dengue, West Nile, and Chikungunya viruses in spiked blood samples or sera from individuals infected with dengue virus. Viral RNAs extracted from biological samples were retrotranscribed into cDNA and amplified using the Phi 29 polymerase-based method. This amplified cDNA was used for hybridization on PID2-RMA. RESULTS: A good specificity of RMA-based detection was demonstrated using a panel of arboviruses including Dengue, West Nile and Chikungunya viruses. This technology was also efficient for the detection and genetic analysis of the different serotypes of dengue virus in sera of infected patients. Furthermore, the mixing of dengue, West Nile and Chikungunya prototype viruses within a single sample of human blood did not interfere with the sensitivity of PID2-RMA. CONCLUSIONS: Our data show that high density PID2-RMA was suitable for the identification of medically important arboviruses. It appears to be particularly adapted to the genetic analysis of dengue, West Nile, and Chikungunya viruses in urgent clinical situations where the rapid identification and characterization of the pathogen is essential.

Potent and selective ligands for the dopamine transporter (DAT): structure-activity relationship studies of novel 4-[2-(diphenylmethoxy)ethyl]-1-(3-phenylpropyl)piperidine analogues.

Molecular structural modifications of 4-[2-(diphenylmethoxy)ethyl]-1-(3-phenylpropyl)piperidine (1a), a dopamine transporter (DAT)-specific ligand, generated several novel analogues. Biological activities of these new molecules for their binding to the DAT and serotonin transporter (SERT) were evaluated in rat striatal membranes. Some of these new analogues were more potent and selective than GBR 12909 when their binding to the DAT relative to SERT was compared. Thus compounds 9 and 19a were among the most potent (IC50 = 6.6 and 6.0 nM, respectively) and selective (DAT/SERT = 33.8 and 30.0, respectively) compounds in this series, and they were more active than GBR 12909 (IC50 = 14 nM, DAT/SERT = 6.1). Introduction of a double bond in the N-propyl side chain of these molecules did not influence their activities to a great extent. Bioisosteric replacement of the aromatic phenyl group by a thiophene moiety produced some of the most potent compounds in this series.

Cations affect [3H]mazindol and [3H]WIN 35,428 binding to the human dopamine transporter in a similar fashion.

The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21 degrees C. Zn2+ (30-100 microM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1-100 microM) had no effect; Hg2+ at approximately 3 microM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0 degrees C, and at 30-100 microM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30-100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21 degrees C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21 degrees C and Hg2+ at 0 degrees C.

Regulation of the functional activity of the human dopamine transporter by protein kinase C.

The role of protein kinase C (PKC) was examined in the regulation of dopamine transport in C6 glioma cells stably expressing the human dopamine transporter. The PKC activating phorbol esters phorbol 12-myristate 13-acetate (PMA) and 4 beta-12,13-dibutyrate phorbol-ester (PDBu) inhibited [3H]dopamine uptake concentration dependently. These effects were attenuated by the PKC inhibitor staurosporine but were unaltered by another inhibitor, chelerythrine, or the phosphatase inhibitor okadaic acid. The potency of PMA in inhibiting [3H]dopamine uptake was similar to that in inhibiting the binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane ([3H]WIN 35,428), and again staurosporine, but not chelerythrine, weakened the effect of PMA. The reduction in dopamine transporter activity by PMA was caused by a decrease in the Vmax value of [3H]dopamine uptake, opposed by a smaller reduction in the Km value, whereas the effect of PMA on [3H]WIN 35,428 binding was caused by a reduction in the Bmax value without a change in the Kd value. The lower Km value in the presence of PMA was accompanied by a higher IC50 of dopamine in inhibiting [3H]WIN 35,428 binding; the latter effect was attenuated by the co-presence of staurosporine. The results are discussed in the context of transporter loss from the cell surface, or a model with phosphorylation affecting the shared dopamine and WIN 35,428 binding domain on the transporter as well as affecting a part of the dopamine binding domain lying outside that for WIN 35,428.

Highly selective, novel analogs of 4-[2-(diphenylmethoxy)ethyl]- 1-benzylpiperidine for the dopamine transporter: effect of different aromatic substitutions on their affinity and selectivity.

Several analogs of the potent and selective dopamine transporter (DAT) ligand 4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine, 1a, were prepared and biologically evaluated at the dopamine and serotonin transporter (SERT) sites. Several substituents were introduced in the aromatic rings to evaluate the influences of electronic and steric interactions in their binding to the DAT. All the novel analogs showed preferential interaction at the DAT compared with the SERT. Different aromatic substitutions in the phenyl ring of the N-benzyl part of the molecule played a key role in the selectivity. In general, compounds with strong electron-withdrawing substituents were most active and selective at the DAT. Thus, compounds 5a (R = F) and 11b (R = NO2) were among the most potent (IC50 = 17.2 and 16.4 nM, respectively) and most selective (SERT/DAT = 112 and 108, respectively) and were far more selective than GBR 12909 (SERT/DAT = 6). Bioisosteric replacement of one of the phenyl rings of the diphenylmethoxy moiety by a thiophene ring was tolerated well and produced the most potent compound 13b (IC50 = 13.8 nM) in the series. Our current structure-activity studies of these piperidine analogs resulted in the generation of second generation of GBR-type compounds, and all of these new compounds reported here were more selective than GBR 12909 in interacting with the DAT over the SERT.

Binding domains for blockers and substrates on the dopamine transporter in rat striatal membranes studied by protection against N-ethylmaleimide-induced reduction of [3H]WIN 35,428 binding.

Binding sites for 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane ([3H]WIN 35,428) on rat striatal membranes were alkylated with N-ethylmaleimide (NEM), and the protective potency was measured of the blockers cocaine, N[1-(2-benzo[b]thiophenyl) cyclohexyl]piperidine (BTCP), benztropine, WIN 35,428; and nomifensine, and of the substrates dopamine, norepinephrine, S(+)-amphetamine, tyramine, and metaraminol. In general, the protective potency was lower (at least 3 times) than the potency in inhibiting [3H]WIN 35,428 binding with the compounds present under the same experimental conditions used for the NEM-induced alkylation. However, the disparity was substantially greater for all substrates tested (10- to 93-fold) than for the blockers (2- to 6-fold), especially cocaine and BTCP (3-fold). [3H]WIN 35,428 binding was best described by a l-site model under the present conditions. The results are discussed in terms of models involving blocker-induced conformational changes and overlapping nonidentical binding domains for blockers and substrates.

Binding domains for blockers and substrates on the cloned human dopamine transporter studied by protection against N-ethylmaleimide-induced reduction of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane ([3H]WIN 35,428) binding.

Binding sites for 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane ([3H]WIN 35,428) on the human dopamine transporter expressed in C6 glioma cells were alkylated with N-ethylmaleimide (NEM), and the protective potency of the blockers cocaine, N[1-(2-benzo[b]thiophenyl) cyclohexyl]piperidine (BTCP), and benztropine, and of the substrates dopamine, d-amphetamine, and norepinephrine was measured. In general, the protective potency was lower (at least 4-5 times) than the potency in inhibiting [3H]WIN 35,428 binding with the compounds present under the same experimental conditions used for the NEM alkylation. However, the disparity was substantially greater for all substrates tested (23- to 44-fold) than for the blockers (4- to 11-fold), especially cocaine (5-fold) and BTCP (4-fold). Benztropine took an intermediate place (11-fold) between cocaine (5-fold) and BTCP (4-fold), on the one hand, and dopamine (23-fold), on the other hand. [3H]WIN 35,428 binding was best described by a one-site model under the present conditions. The results are discussed in terms of models involving blocker-induced conformational changes and overlapping nonidentical binding domains for blockers and substrates.

Translocation of dopamine and binding of WIN 35,428 measured under identical conditions in cells expressing the cloned human dopamine transporter.

Translocation of [3H]dopamine and binding of [3H]WIN 35,428 were measured in intact C6 glioma cells expressing the cloned human dopamine transporter (hDAT) under identical conditions of assay buffer (phosphate-Krebs) and temperature (25 degrees C) with uptake at initial velocity and binding at equilibrium. In the intact cells, [3H]dopamine uptake was a one-component process; in contrast, [3H]WIN 35,428 binding included both a high-affinity component, inhibitable by micromolar concentrations of dopamine, and a low-affinity component only partially inhibited by millimolar concentrations of dopamine. Binding (high-affinity) over uptake Ki ratios were on the average 2.3 for the inhibitors WIN 35,428, cocaine, GBR 12909, and BTCP. The potency of dopamine in inhibiting its own translocation was close to that in inhibiting [3H]WIN 35,428 binding consonant with a more rapid reorientation step of the DAT in the C6-hDAT system than in rat striatal synaptosomes. The similarity in turnover values of the DAT estimated in the current experiments with the C6-hDAT system and in our previous study on rat striatal synaptosomes, performed under comparable conditions, suggest that all DAT's inserted into the C6 cell membrane are functionally active.

Extracellular dopamine, norepinephrine, and serotonin in the nucleus accumbens of freely moving rats during intracerebral dialysis with cocaine and other monoamine uptake blockers.

Monoamine-uptake blockers were applied focally (0.1-1,000 microM) through a dialysis probe in the nucleus accumbens of freely moving rats, and the extracellular concentrations of dopamine, norepinephrine, and serotonin were measured. The selective dopamine-uptake blocker GBR 12935 increased dopamine preferentially with only a small effect on norepinephrine, whereas the selective serotonin-uptake blocker fluoxetine increased serotonin output preferentially. In contrast, the selective norepinephrine-uptake blockers desipramine and nisoxetine enhanced not only norepinephrine, but also serotonin and dopamine appreciably. Cocaine increased all three amines with the greatest effects on dopamine and serotonin. As in our previous study on the ventral tegmental area, there was a positive association between dopamine and norepinephrine output when all blocker data were taken together. The present results suggest a contribution of the increase in norepinephrine, but not serotonin, to the enhancement of dopamine after cocaine applied focally in the nucleus accumbens.

[3H]WIN 35,428 [2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane] binding to rat brain membranes. Comparing dopamine cell body areas with nerve terminal regions.

Potential differences between somatodendritic acid and axonal dopamine transporters were examined by comparing the binding constants of [3H]WIN 35, 428 [2 beta-carbomethoxy- 3 beta-(4-fluorophenyl)tropane] binding to membranes prepared from the rat ventral mesencephalon, containing A9 and A10 dopamine cell bodies, and from the nucleus accumbens. Saturation analysis of [3H]WIN 35,428 binding, in the presence of compounds to occlude norepinephrine and serotonin transporters, was performed by both the "unlabeled" method (varying unlabeled ligand) and "labeled" method (varying radioligand). The density of binding was substantially lower in the ventral mesencephalon than in the nucleus accumbens, but the binding affinity was only slightly different. Likewise, the differences between the two regions in the inhibitory potency of cocaine and GBR 12909 [1-(2-di(4-fluorophenyl)-methoxy-ethyl)4-(3-phenylpropyl)piperazine] were not substantial. The results suggest that somatodendritic and axonal dopamine transporters in the ventral mesencephalon and nucleus accumbens are not very different as far as their binding domains for uptake blockers such as cocaine and GBR 12909 are concerned.

Amygdala kindling of forebrain seizures and the occurrence of brainstem seizures in genetically epilepsy-prone rats.

Forebrain seizures were kindled in rats by daily electrical stimulation of the amygdala. Genetically epilepsy-prone rats scoring 9 (GEPR-9s) on the seizure severity scale during audiogenic seizure (AGS) screening ("brainstem seizure-experienced") required fewer stimulations to achieve fully kindled seizures (forelimb clonus with rearing and falling) than control rats. AGS-naive GEPR-9s required an intermediate number of stimulations, indicating a role for both genetic predisposition and previous acoustically evoked brainstem seizure experience. Other forebrain kindling indices such as afterdischarge threshold/duration and seizure latency/duration also involved genetic as well as phenotypic (previous seizure experience) factors. In most GEPR-9s in both groups, severe brainstem seizures occurred after forebrain stimulation. The occurrence of brainstem seizures had a random nature and was not related to the sequence of kindling-dependent forebrain seizure progression. The lack of a difference in the occurrence of brainstem seizures between seizure-experienced and AGS-naive GEPR-9s suggest that genetic predisposition is the major factor in forebrain seizure-induced activation of brainstem seizure circuitry. This brainstem seizure activity appears to model pertinent aspects of secondary generalization observed in human partial seizures.

Translocation of dopamine and binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl) tropane (WIN 35,428) measured under identical conditions in rat striatal synaptosomal preparations. Inhibition by various blockers.

Translocation of [3H]dopamine and binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]-tropane ([3H]WIN 35,428) were measured in crude synaptosomal preparations from rat striatum under identical conditions of assay buffer (phosphate-Krebs) and temperature (25 degrees). [3H]Dopamine uptake as a function of time was close to linear for at least 8 min, whereas [3H]WIN 35,428 binding had reached equilibrium within 1 min and remained at its plateau value for at least 20 min. The following inhibitors were tested in uptake and binding assays run in parallel with the same synaptosomal preparation: cocaine, WIN 35,428, benztropine, nomifensine, mazindol, methylphenidate, N-[1-(2-benzo[b]-thiophenyl)cyclohexyl]piperidine (BTCP), Lu 19-005 (Indatraline), 1-(2-(di(4-fluorophenyl)-methoxy)-ethyl)-4-(3-phenyl-2-propyl)piperazine (GBR 12909), 1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenyl-2-propyl)piperazine (GBR 12935) and 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo [1,2-a]quinoxaline (CGS 12066B). When present together with [3H]dopamine or [3H]WIN 35,428 for 8 min, the observed binding IC50 values were generally higher (average 1.4-fold) than the uptake IC50 values, with a significant y-axis intercept in linear regression analysis of binding on uptake IC50. For slowly equilibrating inhibitors, estimates of uptake IC50 values were overestimates, and relatively lower values were obtained by monitoring [3H]dopamine uptake for 1 min only during the last minute of the 8-min presence of inhibitor; under these conditions, binding over uptake IC50 ratios were on the average 2.3. Kinetic calculations, taking into account both radioligand and inhibitor equilibration kinetics, indicated that the latter comparison between binding and uptake measurements was most relevant, and suggested the involvement of complexities beyond simple competitive inhibition of dopamine transport, such as different binding domains for substrate and blocker recognition, or spare receptors for blockers. The present data indicate that binding over uptake IC50 ratios should be interpreted with caution, depending on the experimental conditions used to measure these ratios.

Structure-activity relationships for cocaine congeners in inhibiting dopamine uptake into rat brain synaptic vesicles and bovine chromaffin granule ghosts.

Structure-activity relationships of cocaine congeners were determined in inhibiting reserpine-sensitive, Mg++/ATP-dependent uptake of [3H]dopamine into rat brain synaptic vesicles under initial velocity conditions. The C2 carbomethoxy group could be deleted without loss of activity, whereas movement of this group from axial to equatorial orientation increased the potency. There was no need for the ester linkage between the tropane and phenyl rings, nor was there need for the ethylene bridge between C1 and C5 that makes cocaine a tropane instead of a piperidine structure. The structure-activity relationships were different from those for inhibiting neuronal amine transport or blocking voltage-dependent sodium channels. There was no correlation between block of uptake and degree of lipophilicity. The equally lipophilic compounds cocaine and pseudococaine, and WIN 35,065-3 and WIN 35,140, differed in uptake-blocking potency by an order of magnitude (137 vs. 22 microM and 65 vs. 4 microM, respectively). In bovine chromaffin granules, used as a less complex model system for the vesicular uptake system, the rank order of d-amphetamine, cocaine and pseudococaine in perturbing the proton gradient and in changing the membrane potential was different from that in inhibiting uptake. The inhibition of uptake is discussed in terms of the compounds acting as weak bases, transporter substrates or transporter blockers.

GBR 12909 and 12935 block dopamine uptake into brain synaptic vesicles as well as nerve endings.

GBR 12909 and 12935, commonly used as potent neuronal dopamine uptake blockers, also inhibit dopamine uptake into brain synaptic vesicles. The concentrations required for the latter activity (34-45 nM) are one order of magnitude higher than those required for inhibiting neuronal uptake of dopamine (1-6 nM). In contrast, the two activities differ by three orders of magnitude for cocaine (137 microM versus 0.35 microM). We propose that the vesicular effect of GBR-type dopamine uptake blockers should be taken into account when interpreting in vivo experiments.

[3H]WIN 35,428 binding to the dopamine uptake carrier. I. Effect of tonicity and buffer composition.

In the present study, the dopamine transporter on rat striatal membranes was labeled with [3H]WIN 35,428 (2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane), and its binding state or form was manipulated by changing tonicity and buffer composition. Binding to P2 membranes was enhanced by the presence of sucrose in the assay. This effect was not due solely to factors relating to tonicity because creation of isotonicity by dextrose or N-methyl-D-glucamine was less effective, and an increase in binding by sucrose was also observed in assays that were already isotonic by a mixture of sodium phosphate and NaCl. Under the latter conditions, fructose and mannose were equally effective as sucrose. Other important factors were the presence of sodium phosphate in the homogenizing buffer and the presence of sucrose during resuspension of the membranes. When P2 membranes were prepared from homogenates in 0.32 M sucrose, the effect of sucrose in the polytronning step or in the binding assay was restricted to a decrease in the Kd of the main binding component.

[3H]WIN 35,428 binding to the dopamine uptake carrier. II. Effect of membrane fractionation procedure and freezing.

In the present study the dopamine transporter on rat striatal membranes was labeled with [3H]WIN 35,428 (2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane) and its binding state or form was compared between fresh and frozen tissue fractionated by various protocols. Freezing striatal tissue resulted in a decrease in binding to crude or P2 membranes, but did not consistently generate upward concave Scatchard plots. Among various conditions, sodium phosphate buffers containing sucrose, employed for homogenization and for measurement of binding to freshly prepared P2 membranes, were most likely to give multiple binding component resolutions. These results allow us to reconcile the existing literature on [3H]WIN 35,428 binding and to identify (1) the 'universal' binding component common to all studies thus far, (2) a high-affinity component measured with sufficiently low ligand concentration, and (3) a low-affinity component observed with sufficiently high ligand concentration.

Cationic and anionic requirements for the binding of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane to the dopamine uptake carrier.

The present study reports the ion dependency of 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)[3H]tropane ([3H]CFT) binding to the dopamine transporter in the rat striatum. The results indicate that [3H]CFT binding to synaptosomal P2 membranes requires low concentrations of Na+ (peak binding between 20 and 50 mM Na+), is stimulated by phosphate anion or I-, but is unaffected or only slightly affected by F-, Cl-, Br-, NO3-, or SO(4)2-. Concentrations of Na+ of > 50 mM become inhibitory except in the presence of I-, which shifts peak binding levels toward higher Na+ concentrations and also elevates the peak binding level. K+ strongly decreased [3H]CFT binding with a shallow inhibition curve, and Na+ could not overcome this effect. Saturation analysis of [3H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 mM; 1 mM plus 49 mM NaCl versus 10 mM plus 40 mM NaCl; or 1 mM plus 129 mM NaI versus 10 mM plus 120 mM NaI). No differences were observed in the density of CFT binding sites between any of the conditions examined.