The present study was designed to investigate the rewarding effect, G-protein activation and dopamine (DA) release following partial sciatic nerve ligation in the rat. Here we show for the first time that morphine failed to produce a place preference in rats with nerve injury. Various studies provide arguments to support that the mesolimbic dopaminergic system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc), is critical of the motivational effects of opioids. In the present study, there were no significant differences between sham-operated and sciatic nerve-ligated rats in the increases in guanosine-5'-o-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to membranes of the N.Acc stimulated by either DA, the D1 receptor agonist SKF81297, the D2 receptor agonist N-propylnoraporphine or the D3 receptor agonist 7-hydroxy-2-dipropylaminotetralin (7-OH DPAT). In contrast, the increases in [35S]GTPgammaS binding to membranes of the VTA induced by either morphine or a selective micro -opioid receptor agonist [d-Ala2, NMePhe4, Gly(ol)5]enkephalin were significantly attenuated in nerve-ligated rats as compared with sham- operated rats. Furthermore, the enhancement of DA release in the N.Acc stimulated by morphine was significantly suppressed by sciatic nerve ligation. These findings suggest that attenuation of the morphine-induced place preference under neuropathic pain may result from a decrease in the morphine-induced DA release in the N.Acc with reduction in the mu-opioid receptor-mediated G-protein activation in the VTA.

Ips typographus beetles were collected in the field, separated into eight attack phases (from beetles walking on the trunk of a tree under attack to those excavating gallery systems with a mother gallery longer than 4 cm), and immediately frozen in liquid nitrogen. 2-Methyl-3-buten-2-ol,cis- andtrans-verbenol, verbenone, myrtenol, trans-myrtanol, ipsenol, ipsdienol, and 2-phenylethanol were quantified from excised hindguts against an internal standard, heptyl acetate, in the extraction solvent. Methylbutenol, the pinene alcohols, and 2-phenylethanol showed the same pattern of variation between attack phases in males, with the largest amounts present before accepting females and then a fast decline. Ipsenol and ipsdienol were not detected in males before the females were accepted, and the amounts increased when the females start their egg laying. Verbenone occurred only in trace amounts. The beetles were sampled from five Norway spruce trees (Picea abies) of differing resin flow. The correlations between the nine pheromone components and five major host monoterpenes in the gut showed that the variation in the amount of methyl-butenol, ipsenol, and ipsdienol could not be explained by the variation in the amounts of host monoterpenes. In contrast over 80% of the quantitative variation ofcis-verbenol,trans-verbenol, and myrtenol was explained by the amount of α-pinene. The nine pheromone components from 36 individual males were also quantified. Both methylbutenol andcis-verbenol showed a large variation in both amounts and proportions. Females containedtrans-verbenol and traces of most other components found in males. When accepted by the male, they also contained a female-specific compound, β-isophorone. Behavioral and biosynthetic implications of the results are discussed.

OBJECTIVE

We examine socio-economic status (SES) and geographic differences in willingness of respondents to pay for community-based health insurance (CBHI).

METHODS

The study took place in Anambra and Enugu states, south-east Nigeria. It involved a rural, an urban and a semi-urban community in each of the two states. A pre-tested interviewer-administered questionnaire was used to collect information from a total of 3070 households selected by simple random sampling. Contingent valuation was used to elicit willingness to pay (WTP) using the bidding game format. Data were examined for correlation between SES and geographic locations with WTP. Log ordinary least squares (OLS) was used to examine the construct validity of elicited WTP.

RESULTS

Generally, less than 40% of the respondents were willing to pay for CBHI membership for themselves or other household members. The proportions of people who were willing to pay were much lower in the rural communities, at less than 7%. The average that respondents were willing to pay as a monthly premium for themselves ranged from 250 Naira (US$1.7) in a rural community to 343 Naira (US$2.9) in an urban community. The higher the SES group, the higher the stated WTP amount. Similarly, the urbanites stated higher WTP compared with peri-urban and rural dwellers. Males and people with more education stated higher WTP values than females and those with less education. Log OLS also showed that previously paying out-of-pocket for health care was negatively related to WTP. Previously paying for health care using any health insurance mechanism was positively related to WTP.

CONCLUSIONS

Economic status and place of residence amongst other factors matter in peoples' WTP for CBHI membership. Consumer awareness has to be created about the benefits of CBHI, especially in rural areas, and the amount to be paid has to be augmented with other means of financing (e.g. government and/or donor subsidies) to ensure success and sustainability of CBHI schemes.

The human preterm brain is particularly susceptible to cerebral white matter injury (WMI) that disrupts the normal progression of developmental myelination. Advances in the care of preterm infants have resulted in a sustained reduction in the severity of WMI that has shifted from more severe focal necrotic lesions to milder diffuse WMI. Nevertheless, WMI remains a global health problem and the most common cause of chronic neurological morbidity from cerebral palsy and diverse neurobehavioral disabilities. Diffuse WMI involves maturation-dependent vulnerability of the oligodendrocyte (OL) lineage with selective degeneration of late oligodendrocyte progenitors (preOLs) triggered by oxidative stress and other insults. The magnitude and distribution of diffuse WMI are related to both the timing of appearance and regional distribution of susceptible preOLs. Diffuse WMI disrupts the normal progression of OL lineage maturation and myelination through aberrant mechanisms of regeneration and repair. PreOL degeneration is accompanied by early robust proliferation of OL progenitors that regenerate and augment the preOL pool available to generate myelinating OLs. However, newly generated preOLs fail to differentiate and initiate myelination along their normal developmental trajectory despite the presence of numerous intact-appearing axons. Disrupted preOL maturation is accompanied by diffuse gliosis and disturbances in the composition of the extracellular matrix and is mediated in part by inhibitory factors derived from reactive astrocytes. Signaling pathways implicated in disrupted myelination include those mediated by Notch, WNT-beta catenin, and hyaluronan. Hence, there exists a potentially broad but still poorly defined developmental window for interventions to promote white matter repair and myelination and potentially reverses the widespread disturbances in cerebral gray matter growth that accompanies WMI.

The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.

The aim of this study was to characterize the activity of the cannabinoid CB2 receptor selective antagonist, N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazo le-3-carboxamide] (SR144528) in a number of biochemical assays and to look for evidence of cannabinoid CB2 receptors in the rat central nervous system. SR144528 displaced [3H]CP 55,940 ((-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-[3-hydroxyprop yl]cyclohexan-1-ol) from binding sites in CB2- and CB1-transfected cells (Ki = 0.67+/-0.30 and 33.0+/-5.09 nM) and from rat cerebellum and whole brain membrane homogenates (Ki = 54.7+/-9.70 and 54.8+/-7.86 nM). In the GTPgammaS binding assay, SR144528 antagonized a number of cannabinoid receptor agonists (K(B) values ranging from 26.3 to 76.6 nM) in rat cerebellar membranes and in rat whole brain membranes (K(B) = 50.8 nM). SR144528 also antagonized CP 55,940-stimulated GTPgammaS binding in a CB2-expressing cell line (K(B) = 6.34 nM). In Xenopus oocytes co-expressing the CB1 receptor and G-protein coupled inwardly rectifying K+ channels (GIRK 1/4), SR144528 antagonized WIN 55212-2((R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrolo [1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone) -stimulated K+ currents (K(B) = 558 nM). In summary, this report characterizes the cannabinoid CB2 receptor-selective cannabinoid antagonist, SR144528, and additionally suggests an absence of cannabinoid CB2 receptors in the rat central nervous system, an observation confirmed by Northern blot.

The spontaneous, synchronous activity induced by 4-aminopyridine (4AP, 50 microM) in the adult rat entorhinal cortex was analyzed with simultaneous field potential and intracellular recordings in an in vitro slice preparation. Four-AP induced isolated negative-going field potentials (interval of occurrence = 27.6 +/- 9.9 (SD) s; n = 27 slices) that corresponded to intracellular long-lasting depolarizations (LLDs), and ictallike epileptiform discharges (interval of occurrence = 10.4 +/- 5.7 min; n = 27 slices) that were initiated by the negative field potentials. LLDs recorded with K-acetate-filled microelectrodes triggered few action potentials of variable amplitude and had a duration of 1.7 +/- 0.8 s (n = 26 neurons), a peak amplitude of 11.8 +/- 5.0 mV (n = 26 neurons) and a reversal potential of -66.2 +/- 3.9 mV (n = 17 neurons). The ictal discharges studied with K-acetate microelectrodes consisted of prolonged depolarizations (duration = 72.9 +/- 44.3 s; peak amplitude = 29.2 +/- 11.4 mV; n = 25 neurons) with action-potential firing during both the tonic and the clonic phase. These depolarizations had a reversal potential of -45.3 +/- 3.8 mV (n = 4 neurons). Intracellular Cl- diffusion from KCl-filled microelectrodes made both LLDs and ictal depolarizations increase in amplitude (30.5 +/- 8.2 mV, n = 8 and 41.8 +/- 9.8 mV, n = 6 neurons, respectively). LLDs recorded with KCl and 2-(trimethyl-amino)N-(2, 6-dimethylphenyl)-acetamide (QX-314) microelectrodesreached an amplitude of 36.3 +/- 5.2 mV, lasted 12.5 +/- 6.5 s, and had a reversal potential of -31.3 +/- 2.5 mV (n = 4 neurons); under these recording procedures the ictal discharge amplitude was 41.5 +/- 5.0 mV and the reversal potential -24.0 +/- 7.0 mV (n = 4 neurons). The N-methyl-D-aspartate (NMDA) receptor antagonist 3,3-(2-carboxy-piperazine-4-yl)-pro-pyl-l-phosphonate (10 microM, n = 5 neurons) alone or concomitant with the nonNMDA receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (10 microM, n = 4 neurons) abolished ictal discharges, without influencing LLDs. LLDs were blocked by the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (BMI, 10 microM, n = 6 neurons) or the mu-opioid receptor agonist (-Ala2-N-Me-Phe, Gly-ol) enkephalin (DAGO, 10 microM, n = 2 neurons). Application of BMI (n = 4 neurons) or DAGO (n = 2 neurons) to control the medium abolished LLDs and ictal discharges but disclosed a novel type of epileptiform depolarization that lasted 3.5 +/- 1.2 s and occurred every 5.2 +/- 2.6 s (n = 6 neurons). Our data indicate that 4AP induces in the rat entorhinal cortex a synchronous, GABA-mediated potential that is instrumental in initiating NMDA-dependent, ictal discharges. Moreover we present evidence for an active role played by GABAA-mediated potentials in the maintenance and termination of these prolonged epileptiform events.

Dopamine (DA) D3 and D2 receptor mechanisms are implicated in cocaine's abuse-related behavioral effects, but the relative contribution of the two receptor subtypes is only partially characterized. This study investigated the role of D3 and D2 subtype mechanisms by determining the degree to which the D3-preferring antagonist PG01037 [N-{4-[4-(2,3-dichlorophenyl)-piperazin- 1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide HCl] and the D2-preferring antagonist L-741626 [3-[4-(4-chlorophenyl)-4- hydroxypiperidin-1-yl]methyl-1H-indole] attenuated several behavioral effects of cocaine in squirrel monkeys. Quantitative observational studies established doses of each antagonist that did not produce untoward effects, which were used in subsequent comparisons. In addition, the ability of the D3-preferring agonist PD128907 [(R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol)] and the D2-preferring agonist sumanirole [(R)-5,6-dihydro-5-(methylamino)-4H- imidazo[4,5,1-ij]quinolin-2(1H)-one(Z)-2-butenedioate] to reproduce cocaine's discriminative stimulus (DS) and priming effects were compared. In monkeys trained to discriminate cocaine from vehicle, both DA antagonists attenuated and both DA agonists partially reproduced cocaine's DS effects. PG01037 also selectively attenuated the cocaine-like DS effects of PD128907, whereas L-741626 attenuated the cocaine-like DS effects of both agonists. In self-administration studies, L-741626 nonselectively reduced cocaine- and food-maintained responding, whereas PG01037 was ineffective against either reinforcer. In studies involving reinstatement of extinguished cocaine seeking, both antagonists attenuated cocaine-induced reinstatement of responding, and both agonists induced at least partial reinstatement of cocaine seeking. L-741626 also attenuated sumanirole-induced, but not PD128907-induced, reinstatement of responding, whereas PG01037 was ineffective against either DA agonist. The results are consistent with a role for D3 and D2 receptor mechanisms in cocaine's DS effects and cocaine-induced reinstatement of drug seeking, but provide no evidence for a major role of D3 receptors in the direct reinforcing effects of cocaine.

Chronic morphine treatment has been shown to produce constitutive activation of mu-opioid receptors, and this transition might contribute to the development of tolerance and dependence. The apparent ability of chronic morphine to increase the spontaneous, agonist-independent activation of mu-opioid receptors may be unique, due to its distinct partial agonist properties of possessing a relatively high intrinsic activity coupled with a poor ability to produce desensitization and down-regulation. Therefore, the present study tested the hypothesis that prolonged exposure to morphine would produce greater constitutive activity of mu-opioid receptors than exposure to the full agonist [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO). GH(3) cells expressing mu-opioid receptors were exposed to chronic morphine, DAMGO, or no opioid under conditions determined to produce maximal desensitization, down-regulation, and cAMP rebound. After chronic treatment, the mu-opioid antagonists naloxone and beta-chlornaltrexamine (beta-CNA) were evaluated in two assays predictive of inverse agonist activity. Both antagonists produced a concentration-dependent inhibition of [(35)S]GTP gamma S binding only in membranes prepared from cells chronically exposed to opioids. This effect was reversed by the neutral mu-opioid antagonist CTAP. Additionally, conditions known to uncouple G protein-coupled receptors from G proteins produced a leftward shift in the competition curve of beta-CNA for [(3)H]DAMGO binding only in membranes prepared from chronically treated cells. In contrast, these conditions produced no shift in the competition curve by the neutral antagonist CTAP in cells exposed to chronic DAMGO. Therefore, prolonged exposure of GH(3)MOR cells to opioids produced constitutive activation of mu-opioid receptors. Surprisingly, chronic treatment with the more efficacious agonist DAMGO produced greater increases in both measures of inverse agonist activity than did morphine. These observations may lend novel insight into the mechanisms of opioid tolerance and dependence.

Intracellular recordings were obtained from rat neostriatal slices. Bath-applied dopamine (1-10 microM) produced a reversible inhibition of the action potentials evoked by direct stimulation and a decrease in the amplitude of the intrastriatally evoked depolarizing postsynaptic potentials. No change in membrane potential was detected during the application of 1-10 microM dopamine. Dopamine application also produced a decrease in anomalous rectification in the depolarizing direction. This subthreshold inward rectification was abolished by tetrodotoxin, but not by calcium-free and cadmium (0.1-1 mM)-containing solutions. The dopamine-induced decrease in excitatory postsynaptic potential amplitude was evident at resting membrane potential or at more positive levels, but was absent at hyperpolarized values of the membrane potential. Addition of bicuculline (50-500 microM) to the medium did not affect the inhibitory action of dopamine. The inhibitory action of dopamine also persisted in calcium-free and cadmium-containing solutions. The adenosine 3',5'-cyclic monophosphate analogue, 8-bromo-adenosine 3',5'-cyclic monophosphate (0.1-1 mM), mimicked the effects produced by D1 receptor activation. Bath application of 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF 38393) (1-10 microM), a selective D1 dopaminergic agonist, mimicked the effects of micromolar concentrations of dopamine. The D2 dopaminergic agonists, 4,4a,5,6,7,8,8a,9-octahydro-5-n-propyl-2H-pyrazolo-3,4-g-quinoline (LY 171555) and bromocriptine (both at 10 nM-10 microM), had no effects on neostriatal cells. The inhibition induced by micromolar doses of dopamine or SKF 38393 was antagonized by bath applications of R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin++ +-7-ol (SCH 23390; 0.1-10 microM), a D1-selective antagonist, but not by sulpiride (10 nM-10 microM), a D2 antagonist. We conclude that the inhibitory effect of dopamine on rat striatal neurons is postsynaptically mediated by the activation of D1 dopaminergic receptors via the reduction of a voltage-dependent tetrodotoxin-sensitive inward conductance.

The binding of the cyclic peptide (+)-D-Phe1-Cys2-Phe3-D-Trp4-(+)-Lys5-Thr6- Cys7-Thr(ol)8, a somatostatin analogue (SMS 201-995), and the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS) to lipid membranes was investigated with high-sensitivity titration calorimetry. The binding enthalpy of the peptide was found to vary dramatically with the vesicle size. For highly curved vesicles with a diameter of d congruent to 30 nm, the binding reaction was enthalpy-driven with delta H congruent to -7.0 +/- 0.3 kcal/mol; for large vesicles with more tightly packed lipids, the binding reaction became endothermic with delta H congruent to +1.0 +/- 0.3 kcal/mol and was entropy-driven. In contrast, the free energy of binding was almost independent of the vesicle size. The thermodynamic analysis suggests that the observed enthalpy-entropy compensation of about 8 kcal/mol can be related to a change in the internal tension of the bilayer and is brought about by an entropy increase of the lipid matrix. The "entropy potential" of the membrane may have its molecular origin in the excitation of the hydrocarbon chains to a more disordered configuration and may play a more important role in membrane partition equilibria than the classical hydrophobic effect. The binding of the peptide to the membrane surface induced a pK shift of the peptide terminal amino group. Neutral membranes were found to destabilize the NH3+ group, leading to a decrease in pK; negatively charged membranes, generated an apparent increase in pK due to the increase in proton concentration near the membrane surface. No pK shifts were seen for TNS. Titration calorimetry combined with the Gouy-Chapman theory can be used to determine both the reaction enthalpy and the binding constant of the membrane-binding equilibrium.

1. Intracellular recordings were made from neurons of the nucleus raphe magnus (NRM) from rat (n = 128) and guinea-pig (n = 115). Two types of cells were found in each, primary (103 in rat, 27 in guinea-pig) and secondary cells (25 in rat, 88 in guinea-pig). 2. Primary cells had input resistances of 186 +/- 9 M omega (n = 9) in rat and 255 +/- 50 M omega (n = 11) in guinea-pig. The action potential in each was about 1.5 ms in duration. Synaptic potentials were evoked by focal electrical stimulation and consisted of both gamma-aminobutyric acid (GABA) and excitatory amino acid components. 3. Morphine, [Met5]enkephalin (ME) and [D-Ala2,N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) depressed the amplitude of the GABA-mediated synaptic potential by a maximum of 50-65% and had little effect on the excitatory amino acid-mediated synaptic potential. There was no effect of these opioids on the resting membrane potential or input resistance of primary cells in rat or guinea-pig. 4. Secondary cells had short duration action potentials (less than 1 ms) and an input resistance of 354 +/- 47 M omega in rat (n = 6) and 290 +/- 40 M omega in guinea-pig (n = 15). The synaptic potential observed in the cells of this group was mediated by activation of only excitatory amino acid receptors. 5. ME hyperpolarized and/or abolished the spontaneous firing in sixteen out of twenty-four neurons in the secondary group from rat and eight out of eighty-four neurons from guinea-pig. ME induced an outward current at -60 mV that reversed polarity at potentials more negative than -92 +/- 3 mV in rat (n = 6) and -98 +/- 2 mV in guinea-pig (n = 18). The reversal potential of the opioid current was shifted to less negative potentials when the external potassium concentration was increased, as predicted by the Nernst equation. 6. The morphology of the two types of cells were distinguishable in that primary cells were oval (29 x 18 microns in rat; 36 x 19 microns in guinea-pig) with two to four thick tapering dendrites that branched within 50 microns of the cell body. Secondary cells were generally round or oval (about 24 x 13 microns in rat; 27 x 17 microns in guinea-pig) with two to five thin non-tapering dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)

Eighteen Adh-negative mutations were selected with 1-pentyn-3-ol after feeding of formaldehyde. Twelve of the 18 were shown by cytological and genetic analysis to be deletions. Cytological examination of the deletions allowed us to localize the Adh gene to a region including bands 35B3-5 on the left arm of chromosome 2. The deletions were also used to order known visible loci located near Adh.--The vital loci near Adh were also investigated. A total of 109 lethal mutations were generated with EMS and 33 of these, localized within a region defined by the overlap of two of the deletions, were found to belong to 13 complementation groups. If one includes three other loci known to belong there (el, Adh and Sco) a total of 16 complemetation groups have been identified in the region close to Adh.

trans-8, trans-10-Dodecadien-1-ol is a sex attractant of the codling moth, Laspeyresia pomonella. Antennal responses (electroantennograms) to a series of monounsaturated compounds were used in determining the location and configuration of the two double bonds. The synthetic compound is very attractive to male codling moths in the field.

Photobleaching and related photochemical processes are recognized experimental barriers to quantification of fluorescence by microscopy. We have measured the kinetics of photobleaching of fluorophores in living and fixed cells and in microemulsions, and have demonstrated the spatial variability of these processes within individual cells. An inverted fluorescence microscope and a high-sensitivity camera, together with high-speed data acquisition by a computer-controlled image processor, have been used to control precisely exposure time to excitation light and to record images. To improve the signal-to-noise ratio, 32 digital images were integrated. After correction for spatial variations in camera sensitivity and background fluorescence, the images of the relative fluorescence intensities for 0.065 micron2 areas in the object plane were obtained. To evaluate photobleaching objectively, an algorithm was developed to fit a three-parameter exponential equation to 20 images recorded from the same microscope field as a function of illumination time. The results of this analysis demonstrated that the photobleaching process followed first-order reaction kinetics with rate constants that were spatially heterogeneous and varied, within the same cell, between 2- and 65-fold, depending on the fluorophore. The photobleaching rate constants increased proportionally with increasing excitation intensity and, for benzo(a)pyrene, were independent of probe concentration over three orders of magnitude (1.25 microM to 1.25 mM). The propensity to photobleach was different with each fluorophore. Under the cellular conditions used in these studies, the average rates of photobleaching decreased in this order: N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol greater than acridine orange greater than rhodamine-123 greater than benzo(a)pyrene greater than fluorescein greater than tetramethylrhodamine greater than 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine. The photobleaching appears to be an oxidation reaction, in that the addition of saturated solutions of Na2S2O5 to mineral oil microemulsions eliminated photobleaching of N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol or benzo(a)pyrene. We identified experimental conditions to observe, without detectable photobleaching, fluorophores in living cells, which can not be studied anaerobically. Useful images were obtained when excitation light was reduced to eliminate photobleaching, as determined from zero-time images calculated from the exponential fit routine.(ABSTRACT TRUNCATED AT 400 WORDS)

We report a method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites. We used radioligands that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands, sufentanil for mu sites, [D-Ala2, D-Leu5] enkephalin for delta sites, and dynorphin A for kappa sites. After enrichment for mu receptors, [3H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [3H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but we found no evidence for residual mu or delta sites in kappa-enriched membranes. We used this method to identify ligands that are highly selective for each of the three types of sites: Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, sufentanil, and morphiceptin for mu sites; (D- Pen2 , D- Pen5 ]enkephalin and [D- Pen2 ,L- Pen5 ]enkephalin for delta sites; and tifluadom and U50 ,488 for kappa sites.

Growth factors, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected mu-opioid receptor (MOR-1) and kappa-opioid receptor (KOR-1) expression and immunoreactivity in embryonic stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell differentiation and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages.

BACKGROUND

Recent evidence suggests that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that tumorous tissues have higher 5alpha-reductase (5alphaR) and lower 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO activities. The resulting higher levels of 5alpha-reduced progesterone metabolites such as 5alpha-pregnane-3,20-dione (5alphaP) in tumorous tissue promote cell proliferation and detachment, whereas the 4-pregnene metabolites, 4-pregnen-3alpha-ol-20-one (3alphaHP) and 4-pregnen-20alpha-ol-3-one (20alphaDHP), more prominent in normal tissue, have the opposite (anti-cancer-like) effects. The aim of this study was to determine if the differences in enzyme activities between tumorous and nontumorous breast tissues are associated with differences in progesterone metabolizing enzyme gene expression.

METHODS

Semi-quantitative RT-PCR was used to compare relative expression (as a ratio of 18S rRNA) of 5alphaR type 1 (SRD5A1), 5alphaR type 2 (SRD5A2), 3alpha-HSO type 2 (AKR1C3), 3alpha-HSO type 3 (AKR1C2) and 20alpha-HSO (AKR1C1) mRNAs in paired (tumorous and nontumorous) breast tissues from 11 patients, and unpaired tumor tissues from 17 patients and normal tissues from 10 reduction mammoplasty samples.

RESULTS

Expression of 5alphaR1 and 5alphaR2 in 11/11 patients was higher (mean of 4.9- and 3.5-fold, respectively; p < 0.001) in the tumor as compared to the paired normal tissues. Conversely, expression of 3alpha-HSO2, 3alpha-HSO3 and 20alpha-HSO was higher (2.8-, 3.9- and 4.4-fold, respectively; p < 0.001) in normal than in tumor sample. The mean tumor:normal expression ratios for 5alphaR1 and 5alphaR2 were about 35-85-fold higher than the tumor:normal expression ratios for the HSOs. Similarly, in the unmatched samples, the tumor:normal ratios for 5alphaR were significantly higher than the ratios for the HSOs.

CONCLUSIONS

The study shows changes in progesterone metabolizing enzyme gene expression in human breast carcinoma. Expression of SRD5A1 (5alphaR1) and SRD5A2 (5alphaR2) is elevated, and expression of AKR1C1 (20alpha-HSO), AKR1C2 (3alpha-HSO3) and AKR1C3 (3alpha-HSO2) is reduced in tumorous as compared to normal breast tissue. The changes in progesterone metabolizing enzyme expression levels help to explain the increases in mitogen/metastasis inducing 5alphaP and decreases in mitogen/metastasis inhibiting 3alphaHP progesterone metabolites found in breast tumor tissues. Understanding what causes these changes in expression could help in designing protocols to prevent or reverse the changes in progesterone metabolism associated with breast cancer.

R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390) is a widely used, highly selective antagonist of D1 dopamine receptors. While investigating the crosstalk between D1 and D3 dopamine receptor signaling pathways, we discovered that in addition to being a D1 receptor antagonist, SCH23390 and related compounds inhibit G protein-coupled inwardly rectifying potassium (GIRK) channels. We present evidence that SCH23390 blocks endogenous GIRK currents induced by either somatostatin or D3 dopamine receptors in AtT-20 cells (IC50, 268 nM). The inhibition is receptor-independent because constitutive GIRK currents in Chinese hamster ovary cells expressing only GIRK channels are also blocked by SCH23390. The inhibition of GIRK channels is somewhat selective because members of the closely related Kir2.0 family of inwardly rectifying potassium channels, as well as various endogenous cationic currents present in AtT-20 cells, are not affected. In addition, in current clamp recordings, SCH23390 can depolarize the membrane potential and induce AtT-20 cells to fire action potentials, indicating potential physiological significance of the GIRK channel inhibition. To identify the chemical features that contribute to GIRK channel block, we tested several structurally related compounds [SKF38393, R-(+)-7-chloro-8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (nor-methyl-SCH23390), and R-(+)-2,3,4,5-tetrahydro-8-iodo-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrochloride (iodo-SCH23390)], and our results indicate that the halide atom is critical for blocking GIRK channels. Taken together, our results suggest that SCH23390 and related compounds might provide the basis for designing novel GIRK channel-selective blockers. Perhaps more importantly, some studies that have exclusively used SCH23390 to probe D1 receptor function or as a diagnostic of D1 receptor involvement may need to be reevaluated in light of these results.

Interval suction traps were used to study the attractant effect of CO2 and 1-octen-3-ol on trap catches of mosquito populations at 2 different locations in Florida. There was no significant increase in the numbers of mosquitoes caught when the concentration of CO2 was increased from 200 to 1,000 cc/min. One-octen-3-ol used by itself attracted mosquitoes in numbers similar to CO2 released at 200 cc/min. One-octen-3-ol and CO2 acted synergistically in attracting significantly greater numbers of Aedes taeniorhynchus, Anopheles spp. and Wyeomyia mitchellii than either bait used singly, although the response of Culex spp. to this bait combination was less pronounced. Ceratopogonidae (Culicoides furens) and Tabanidae (Diachlorus ferrugatus, Tabanus nigrovittatus and Chrysops spp.) were also attracted to the combined bait.

The midbrain periaqueductal gray (PAG) is a major site of opioid analgesic action, and a significant site of cellular adaptations to chronic morphine treatment (CMT). We examined mu-opioid receptor (MOP) regulation of voltage-gated calcium channel currents (I(Ca)) and G-protein-activated K channel currents (GIRK) in PAG neurons from CMT mice. Mice were injected s.c. with 300 mg kg(-1) of morphine base in a slow release emulsion three times over 5 days, or with emulsion alone (vehicles). This protocol produced significant tolerance to the antinociceptive effects of morphine in a test of thermal nociception. Voltage clamp recordings were made of I(Ca) in acutely isolated PAG neurons and GIRK in PAG slices. The MOP agonist DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) inhibited I(Ca) in neurons from CMT mice (230 nM) with a similar potency to vehicle (150 nM), but with a reduced maximal effectiveness (37% inhibition in vehicle neurons, 27% in CMT neurons). Inhibition of I(Ca) by the GABA(B) agonist baclofen was not altered by CMT. Met-enkephalin-activated GIRK currents recorded in PAG slices were significantly smaller in neurons from CMT mice than vehicles, while GIRK currents activated by baclofen were unaltered. These data demonstrate that CMT-induced antinociceptive tolerance is accompanied by homologous reduction in the effectiveness of MOP agonists to inhibit I(Ca) and activate GIRK. Thus, a reduction in MOP number and/or functional coupling to G proteins accompanies the characteristic cellular adaptations to CMT previously described in PAG neurons.

An HPLC method, using detection after postcolumn derivatization with p-dimethylaminocynnamaldehyde (DMACA), was developed for the quantitative analysis of individual flavanols in food. This method was applied to flavanol determination in 56 different kinds of Spanish food products, including fruit, vegetables, legumes, beverages (cider, coffee, beer, tea, and wine), and chocolate. The determined compounds corresponded to the catechins and proanthocyanidin dimers and trimers usually present in food and, therefore, they were representative of the flavanols of low degree of polymerization consumed with the diet. The data generated could be used for calculation of the dietary intake of either individual or total flavanols, which would allow the further establishment of epidemiological correlations with the incidence of chronic diseases. Similar flavanol profiles were found in the different samples of a similar type of product, even though important variations could exist in the concentrations of total and individual flavanols among them. This was attributed to factors such as sample origin, stage of ripeness, post-harvesting conservation, and processing. Total flavanol contents varied from nondetectable in most of the vegetables to 184 mg/100 g found in a sample of broad bean. Substantial amounts were also found in some fruits, such as plum and apple, as well as in tea and red wine. Epicatechin was the most abundant flavanol, followed by catechin and procyanidin B2. In general, catechins were found in all the flavanol-containing products, but the presence of gallocatechins was only relevant in pomegranate, broad bean, lentil, grape, wine, beer, and tea, and most of the berries. Galloyled flavanols were only detected in strawberry, medlar, grape, and tea.

An extracellular beta-glucosidase from Thermoascus aurantiacus was purified to homogeneity by DEAE-Sepharose, Ultrogel AcA 44 and Mono-P column chromatography. The enzyme was a homotrimer, with a monomer molecular mass of 120 kDa; only the trimer was optimally active at 80 degrees C and at pH 4.5. At 90 degrees C, the enzyme showed 70% of its optimal activity. It was stable at pH 5.2 and at temperatures up to 70 degrees C for 48 h, but stability decreased above 70 degrees C and at pH values above and below 5.0. The enzyme hydrolysed aryl and alkyl beta-d-glucosides and cello-oligosaccharides, and was specific for substrates with a beta-glycosidic linkage. The hydroxy groups at positions 2, 4 and 6 of a glucose residue at the non-reducing end of a disaccharide appeared to be essential for catalysis. The enzyme had the lowest K(m) towards p-nitrophenyl beta-d-glucoside (0.1137 mM) and the highest k(cat) towards cellobiose and beta,beta-trehalose (17052 min(-1)). It released one glucose unit at a time from the non-reducing end of cello-oligosaccharides, and the rate of hydrolysis decreased with an increase in chain length. Glucose and d-delta-gluconolactone inhibited the beta-glucosidase competitively, with K(i) values of 0.29 mM and 8.3 nM respectively, while methanol, ethanol and propan-2-ol activated the enzyme. The enzyme catalysed the synthesis of methyl, ethyl and propyl beta-d-glucosides in the presence of methanol, ethanol and propan-2-ol respectively with either glucose or cellobiose, although cellobiose was preferred. An acidic pH favoured hydrolysis and transglycosylation, but high concentrations of alcohols favoured the latter reaction. The stereochemistry of cellobiose hydrolysis revealed that beta-glucosidase from T. aurantiacus is a retaining glycosidase, while N-terminal amino acid sequence alignment indicated that it is a member of glycoside hydrolase family 3.

Xyloglucan endotransglycosylase (XET) from the core tissue of ripe kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) was purified 3000-fold to homogeneity. The enzyme has a molecular weight of 34 kDa, is N-glycosylated, and is active between pH 5.0 and 8.0, with an optimum between 5.5 and 5.8. The Km was 0.6 mg.mL-1 for kiwifruit xyloglucan and 100 microM for [3H]XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan. Kiwifruit core XET was capable of depolymerising xyloglucan in the absence of [3H]XXXG-ol by hydrolysis, and in the presence of [3H]XXXG-ol by hydrolysis and endotransglycosylation. Six cDNA clones (AdXET1-6) with homology to other reported XETs were isolated from ripe kiwifruit mRNA. The six cDNA clones share 93-99% nucleotide identity and appear to belong to a family of closely related genes. Peptide sequencing indicated that ripe kiwifruit XET was encoded by AdXET6. Northern analysis indicated that expression of the AdXET1-6 gene family was induced in ripening kiwifruit when endogenous ethylene production could first be detected, and peaked in climacteric samples when fruit were soft. A full-length cDNA clone (AdXET5) was overexpressed in E. coli to produce a recombinant protein that showed endotransglycosylase activity when refolded.