17Beta-estradiol (E2) rapidly (<20 min) attenuates the ability of mu-opioids to hyperpolarize guinea pig hypothalamic neurons. We have used intracellular recordings from female guinea pig hypothalamic slices to characterize the receptor and intracellular pathway(s) mediating E2's rapid effects. E2 acts stereospecifically with physiologically relevant concentration-dependence (EC50 = 8 nM) to cause a fourfold reduction in the potency of the mu-opioid agonist (D-Ala2-N-Me-Phe4-Gly5-ol)-enkephalin and the GABA(B) agonist baclofen to activate an inwardly rectifying K+ conductance in hypothalamic neurons. Both the nonsteroidal estrogen diethylstilbestrol and the anti-estrogen ICI 164,384 blocked E2 actions to uncouple mu-opioid receptors. Using a pharmacological Schild analysis, we found that ICI 164,384 competed for this E2 receptor with a Ke of approximately 0.3 nM. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the mu-opioid receptor from its K+ channel, implying a rapid, nongenomic mechanism of E2 action. The effects of E2 were mimicked by the bath application of the protein kinase A (PKA) activators, forskolin and Sp-cAMP, and the protein kinase C (PKC) activator phorbol-12,13-dibutyrate. Furthermore, the selective PKA antagonists Rp-cAMP and KT5720, which have different chemical structures and modes of action, both blocked the effects of E2. In addition, the actions of E2 were blocked by the selective PKC inhibitor Calphostin C. Therefore, it appears that E2 can activate both PKA and PKC to cause a heterologous desensitization of both mu-opioid and GABA(B) receptors, which has the potential to alter synaptic transmission in many regions of the CNS.

Polyphenolic compounds of apple may play an important role in physiologic functions related to human health. Different polyphenolics may have varied biological activities including antioxidant activity. The objective of this study was to investigate the profiles of polyphenolic compounds in different apple varieties and different parts of an apple. The total and individual polyphenolics differed significantly among the eight apple cultivars grown in Ontario, and the peels had higher concentrations than the flesh. Among the tested cultivars, Red Delicious and Northern Spy had the highest concentrations and Empire the lowest. Five major polyphenolic groups with a total of 16 identified individual compounds were found, among which the dihydroxycinnamic acid esters, phloretin glycosides, and flavan-3-ols were found in both flesh and peel, whereas quercetin glycosides were almost exclusively found in the peel. Cyanidin 3-galactoside was unique to and found only in red apple peels. In both apple peel and flesh, the predominant group of polyphenolics was the procyanidins, followed by quercetin glycosides in the peel and hydroxycinnamic acid esters in the flesh. 3-Hydroxyphloretin 2'-xyloglucoside was newly identified in apple. The results obtained in this study will further the understanding of the polyphenolic composition of apples and their roles in health-promoting physiological functions.

TGR5 (Gpbar-1) is a membrane-bound bile acid receptor in the gastrointestinal tract and immune cells with pleiotropic actions. As shown in the present study, TGR5 is also expressed in astrocytes and neurons. Here, TGR5 may act as a neurosteroid receptor, which is activated by nanomolar concentrations of 5β-pregnan-3α-ol-20-one and micromolar concentrations of 5β-pregnan-3α-17α-21-triol-20-one and 5α-pregnan-3α-ol-20-one (allopregnanolone). TGR5 stimulation in astrocytes and neurons is coupled to adenylate cyclase activation, elevation of intracellular Ca(2+) and the generation of reactive oxygen species. In cultured rat astrocytes, TGR5 mRNA is downregulated in the presence of neurosteroids and ammonia already at concentrations of 0.5 mmol L(-1). Furthermore, TGR5 protein levels are significantly reduced in isolated rat astrocytes after incubation with ammonia. A marked downregulation of TGR5 mRNA is also found in cerebral cortex from cirrhotic patients dying with hepatic encephalopathy (HE) when compared with brains from noncirrhotic control subjects. It is concluded that TGR5 is a novel neurosteroid receptor in brain with implications for the pathogenesis of HE.

It is becoming increasingly recognized that G protein-coupled receptors physically interact. These interactions may provide a mechanism for allosteric modulation of receptor function. In this study, we examined this possibility by using an established model system of a receptor heteromer consisting of μ and δ opioid receptors. We examined the effect of a number of μ receptor ligands on the binding equilibrium and association and dissociation kinetics of a radiolabeled δ receptor agonist, [(3)H]deltorphin II. We also examined the effect of δ receptor ligands on the binding equilibrium and association and dissociation kinetics of a radiolabeled μ receptor agonist, [(3)H][d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin ([(3)H]DAMGO). We show that μ receptor ligands are capable of allosterically enhancing δ receptor radioligand binding and vice versa. Thus, there is strong positive cooperativity between the two receptor units with remarkable consequences for ligand pharmacology. We find that the data can be simulated by adapting an allosteric receptor model previously developed for small molecules, suggesting that the ligand-occupied protomers function as allosteric modulators of the partner receptor's activity.

This paper presents a circular microfluidic compartmentalized co-culture platform that can be used for central nervous system (CNS) axon myelination research. The microfluidic platform is composed of a soma compartment and an axon/glia compartment connected through arrays of axon-guiding microchannels. Myelin-producing glia, oligodendrocytes (OLs), placed in the axon/glia compartment, interact with only axons but not with neuronal somata confined to the soma compartment, reminiscent to in vivo situation where many axon fibres are myelinated by OLs at distance away from neuronal cell bodies. Primary forebrain neurons from embryonic day 16-18 rats were cultured inside the soma compartment for two weeks to allow them to mature and form extensive axon networks. OL progenitors, isolated from postnatal day 1-2 rat brains, were then added to the axon/glia compartment and co-cultured with neurons for an additional two weeks. The microdevice showed fluidic isolation between the two compartments and successfully isolated neuronal cell bodies and dendrites from axons growing through the arrays of axon-guiding microchannels into the axon/glia compartment. The circular co-culture device developed here showed excellent cell loading characteristics where significant numbers of cells were positioned near the axon-guiding microchannels. This significantly increased the probability of axons crossing these microchannels as demonstrated by the more than 51 % of the area of the axon/glia compartment covered with axons two weeks after cell seeding. OL progenitors co-cultured with axons inside the axon/glia compartment successfully differentiated into mature OLs. These results indicate that this device can be used as an excellent in vitro co-culture platform for studying localized axon-glia interaction and signalling.

The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5 alpha-acetoxy-10 beta-ol. Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations. The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences. Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates. This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10 beta-hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10 beta-hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means. The 10 beta-hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway.

(-)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111) is a novel human opiate receptor-like orphan receptor (ORL-1) antagonist that has high affinity for the clonal human ORL-1 receptor (hORL-1 K(i) = 0.33 nM), selectivity versus mu-(174-fold), delta-(6391-fold), and kappa (486-fold)-opioid receptors and is able to inhibit nociceptin signaling via hORL-1 in a whole cell gene reporter assay. SB-612111 has no measurable antinociceptive effects in vivo in the mouse hot-plate test after intravenous administration but is able to antagonize the antimorphine action of nociceptin [ED(50) = 0.69 mg/kg, 95% confidence limit (CL) = 0.34-1.21]. SB-62111 administration can also reverse tolerance to morphine in this model, established via repeated morphine administration. In addition, intravenous SB-612111 can antagonize nociceptin-induced thermal hyperalgesia in a dose-dependent manner (ED(50) = 0.62 mg/kg i.v., 95% CL = 0.22-1.89) and is effective per se at reversing thermal hyperalgesia in the rat carrageenan inflammatory pain model. These data show that an ORL-1 receptor antagonist may be a useful adjunct to chronic pain therapy with opioids and can be used to treat conditions in which thermal hyperalgesia is a significant component of the pain response.

BACKGROUND

Manipulations of nicotinic cholinergic receptors have been shown to influence both alcohol and nicotine intake. Sazetidine-A [6-(5(((S)-azetidine-2-yl)methoxy)pyridine-3-yl)hex-5-yn-1-ol] is a novel compound that potently and selectively desensitizes alpha4beta2 nicotinic receptors with only modest receptor activation.

OBJECTIVE

The goal of the present study was to examine the effects of sazetidine-A on alcohol and nicotine self-administration in alcohol-preferring (P) rats.

METHODS

P rats were given the choice of water or alcohol. Once stable baselines were established, the acute (0, 0.1, 0.3, 1, and 3 mg/kg, s.c.) and chronic (3 mg/kg for 10 days) effects of sazetidine-A on alcohol intake were assessed. Naltrexone (2.5 mg/kg) served as a positive control. The effect of sazetidine-A (3 mg/kg) and naltrexone (4 mg/kg) on saccharin (0.2%) preference was also assessed. In addition, the acute effects of sazetidine-A (3 mg/kg) and naltrexone (4 mg/kg) on alcohol intake after alcohol deprivation were evaluated. In another experiment, the effects of sazetidine-A (0, 1, or 3 mg/kg) on i.v. nicotine self-administration in P and NP rats were assessed.

RESULTS

Sazetidine-A caused a dose-dependent reduction in alcohol intake. Chronic sazetidine-A also effectively reduced alcohol intake until the seventh day of treatment, when partial tolerance appeared to develop. In the post-deprivation study, sazetidine-A significantly reduced alcohol intake and preference. Sazetidine-A at 3 mg/kg significantly reduced nicotine self-administration in both lines.

CONCLUSIONS

Sazetidine-A significantly reduced alcohol and nicotine intake in P rats that self-administer higher levels of both drugs. Sazetidine-A may hold promise for the treatment of alcohol and nicotine addiction.

1. Protoplasts of Saccharomyces cerevisiae N.C.Y.C. 366 were prepared by incubating washed exponential-phase cells in buffered mannitol (0.8m) containing 10mm-magnesium chloride and snail gut juice (about 8mg. of protein/ml. of reaction mixture). Protoplast membranes were obtained by bursting protoplasts in ice-cold phosphate buffer (pH7.0) containing 10mm-magnesium chloride. 2. Protoplast membranes accounted for 13-20% of the dry weight of the yeast cell. They contained on a weight basis about 39% of lipid, 49% of protein, 6% of sterol (assayed spectrophotometrically) and traces of RNA and carbohydrate (glucan+mannan). 3. The principal fatty acids in membrane lipids were C(16:0), C(16:1) and C(18:1) acids. Whole cells contained a slightly greater proportion of C(16:0) and a somewhat smaller proportion of C(18:1) acids. Membrane and whole-cell lipids included monoglycerides, diglycerides, triglycerides, sterols, sterol esters, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine. Phosphorus analyses on phospholipid fractions from membranes and whole cells showed that membranes contained proportionately more phosphatidylethanolamine and phosphatidylinositol+phosphatidylserine than whole cells, which in turn were richer in phosphatidylcholine. Phospholipid fractions from membranes and whole cells had similar fatty acid compositions. 4. Membranes and whole cells contained two major and three minor sterol components. Gas-liquid chromatography, mass spectrometry and u.v. and i.r. spectra indicated that the major components were probably Delta(5,7,22,24(28))-ergostatetraen-3beta-ol and zymosterol. The minor sterol components in whole cells were probably episterol (or fecosterol), ergosterol and a C(29) di-unsaturated sterol. 5. Defatted whole cells contained slightly more glutamate and ornithine and slightly less leucine and isoleucine than membranes. Otherwise, no major differences were detected in the amino acid compositions of defatted whole cells and membranes.

Multiple studies have shown that migration, proliferation, and differentiation of oligodendrocyte (OL) lineage cells are influenced by fibroblast growth factor-2 (FGF-2) signaling through its receptors (FGFR) FGFR-1, FGFR-2, and FGFR-3. We report the effectiveness and specificity of a unique inhibitor, PD173074, for inhibiting FGF receptor signaling in OL-lineage cells. Three FGF-mediated responses of OL progenitors and two of differentiated OLs were examined by immunofluorescence microscopy and immunoblotting. PD173074 effectively antagonized the effect of FGF-2 on proliferation and differentiation of OL progenitors in culture. One dose of PD173074 at nanomolar concentrations was sufficient to inhibit ongoing FGF-2 mediated proliferation for prolonged periods, in a non-toxic, dose-dependent manner. In contrast, platelet-derived growth factor (PDGF)-induced proliferation was unaffected by PD173074. Similarly, mitogen-activated protein kinase (MAPK) activation, a downstream event after activation of either FGFR or PDGFR, was also blocked by PD173074 in OL progenitors stimulated with FGF-2 but not PDGF. A general tyrosine kinase inhibitor (PD166285), however, antagonized both FGF-2- and PDGF-mediated responses. PD173074 also completely antagonized two phenotypic alterations of differentiated OLs, specifically downregulation of myelin proteins, and their re-entry into the cell cycle. We conclude that PD173704 is an effective and specific inhibitor for multiple FGF-2-mediated responses of both OL progenitors and differentiated OLs. This inhibitor provides a direct approach for identifying the importance of FGF signaling, comparable in effect to a knockout of all FGF receptors and all FGF ligands, while leaving other pathways unaffected. Thus, PD173704 is an excellent tool for investigating the role of FGF signaling in vivo in the context of combinatorial interactions of other signals.

Using human gamma-aminobutyric acid type A (GABAA) receptor subunit combinations, expressed in cell lines and Xenopus laevis oocytes, the pharmacology of a number of ligands interacting directly with the GABA recognition site has been studied in [3H]muscimol binding and electrophysiologically. The binding affinity of GABAA agonist and antagonist ligands showed small but statistically significant dependence on the subunit composition of receptors that include gamma 2 and different alpha and beta subunits. The potency of antagonist ligands was largely independent of receptor subunit composition, whereas the composition of receptors expressed in oocytes strongly influenced the EC50 value of agonists. An apparent reciprocal correlation between subunits favoring agonist binding and antagonist binding, respectively, was observed. Whereas antagonists showed comparable potencies in binding and functional studies, the potency of agonists in binding studies was generally two to three orders of magnitude higher than the agonist potencies measured electrophysiologically. 5-(4-Piperidyl)isothiazol-3-ol, which behaves as a low efficacy partial agonist at GABAA receptors in cultured cortical neurons, showed no efficacy in oocytes, but produced pure antagonist effects with a binding/functional affinity ratio between those observed for the agonists and antagonists. It is concluded that the GABAA receptor mechanisms transducing binding into physiological response, but not the binding per se, is dependent on the receptor subunit composition.

Numerous hormones, cytokines and transforming oncogenes activate phosphoinositide 3-kinase (PI-3K), a lipid kinase that initiates signal transduction cascades regulating cellular proliferation, survival, protein synthesis and glucose metabolism. PI-3K catalyses the production of the 3'-phosphoinositides PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3), which recruit downstream effector enzymes to the membrane via their pleckstrin homology (PH) domains. Recent studies have indicated that another signalling lipid, the sphingolipid ceramide, inhibits several PI-3K-dependent events, including insulin-stimulated glucose uptake and growth-factor-stimulated cell survival. Here we show that ceramide analogues specifically prevent the recruitment of the PtdIns(3,4,5)P(3)-binding proteins Akt/protein kinase B (PKB) or the general receptor for phosphoinositides-1 (GRP1). Specifically, the short-chain ceramide derivative C2-ceramide inhibited the platelet-derived growth factor (PDGF)-stimulated translocation of full-length Akt/PKB, as well as truncated proteins encoding only the PH domains of Akt/PKB or GRP1. C2-ceramide did not alter the membrane localization of the PH domain for phospholipase Cdelta, which preferentially binds PtdIns(4,5)P(2), nor did it affect the PDGF-stimulated production of PtdIns(3,4)P(2) or PtdIns(3,4,5)P(3). Interestingly, a glucosylceramide synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol (PDMP), shown previously to increase intracellular ceramide concentrations without affecting PI-3K [Rani, Abe, Chang, Rosenzweig, Saltiel, Radin and Shayman (1995) J. Biol. Chem. 270, 2859-2867], recapitulated the inhibitory effects of C2-ceramide on PDGF-stimulated Akt/PKB phosphorylation. These studies indicate that ceramide prevents the translocation of certain PtdIns(3,4,5)P(3)-binding proteins, despite the presence of a full complement of PtdIns(3,4)P(2) or PtdIns(3,4,5)P(3). Furthermore, these findings suggest a mechanism by which stimuli that induce ceramide synthesis could negate the fundamental signalling pathways initiated by PI-3K.

Leaf alcohol (Z)-3-hexen-1-ol (Z-3-ol) is emitted by green plants upon mechanical damage. Exposure of intact maize plants to Z-3-ol induces the emission of a volatile blend that is typically released after caterpillar feeding and attracts natural enemies of the herbivores [herbivore-induced volatile organic compounds (HI-VOC)]. Thus, it has been suggested that Z-3-ol might have a function in indirect plant defense mediating plant-plant signaling and intraplant information transfer. Here, we demonstrate that HI-VOC induction by Z-3-ol is synergized by the phytohormone ethylene. Exposure to Z-3-ol at doses of 100 and 250 nmol induced HI-VOC emission in intact maize plants. HI-VOC emissions increased by 2.5-fold when ethylene was added. The effect of ethylene was more pronounced (5.1- to 6.6-fold) when only total sesquiterpene release was considered. In contrast, ethylene alone had no inductive effect but rather decreased the emission of the constitutive maize volatile linalool. We suggest that ethylene plays a synergistic role in plant-plant signaling mediated by green leaf volatiles.

Central nervous system (CNS) myelination by oligodendrocytes (OLs) is a highly orchestrated process involving well-defined steps from specification of neural stem cells into proliferative OL precursors followed by terminal differentiation and subsequent maturation of these precursors into myelinating OLs. These specification and differentiation processes are mediated by profound global changes in gene expression, which are in turn subject to control by both extracellular signals and regulatory networks intrinsic to the OL lineage. Recently, basic transcriptional mechanisms that control OL differentiation and myelination have begun to be elucidated at the molecular level and on a genome scale. The interplay between transcription factors activated by differentiation-promoting signals and master regulators likely exerts a crucial role in controlling stage-specific progression of the OL lineage. In this review, we describe the current state of knowledge regarding the transcription factors and the epigenetic programs including histone methylation, acetylation, chromatin remodeling, micro-RNAs, and noncoding RNAs that regulate development of OLs and myelination.

BACKGROUND

Cells of the oligodendrocyte (OL) lineage play a vital role in the production and maintenance of myelin, a multilamellar membrane which allows for saltatory conduction along axons. These cells may provide immense therapeutic potential for lost sensory and motor function in demyelinating conditions, such as spinal cord injury, multiple sclerosis, and transverse myelitis. However, the molecular mechanisms controlling OL differentiation are largely unknown. MicroRNAs (miRNAs) are considered the "micromanagers" of gene expression with suggestive roles in cellular differentiation and maintenance. Although unique patterns of miRNA expression in various cell lineages have been characterized, this is the first report documenting their expression during oligodendrocyte maturation from human embryonic stem (hES) cells. Here, we performed a global miRNA analysis to reveal and identify characteristic patterns in the multiple stages leading to OL maturation from hES cells including those targeting factors involved in myelin production.

RESULTS

We isolated cells from 8 stages of OL differentiation. Total RNA was subjected to miRNA profiling and validations preformed using real-time qRT-PCR. A comparison of miRNAs from our cultured OLs and OL progenitors showed significant similarities with published results from equivalent cells found in the rat and mouse central nervous system. Principal component analysis revealed four main clusters of miRNA expression corresponding to early, mid, and late progenitors, and mature OLs. These results were supported by correlation analyses between adjacent stages. Interestingly, the highest differentially-expressed miRNAs demonstrated a similar pattern of expression throughout all stages of differentiation, suggesting that they potentially regulate a common target or set of targets in this process. The predicted targets of these miRNAs include those with known or suspected roles in oligodendrocyte development and myelination including C11Orf9, CLDN11, MYTL1, MBOP, MPZL2, and DDR1.

CONCLUSIONS

We demonstrate miRNA profiles during distinct stages in oligodendroglial differentiation that may provide key markers of OL maturation. Our results reveal pronounced trends in miRNA expression and their potential mRNA target interactions that could provide valuable insight into the molecular mechanisms of differentiation.

The present study was designed to reexamine the muscarinic acetylcholine receptor subtype mediating carbachol-induced contraction of human urinary bladder and to investigate the underlying signal transduction. Based upon the nonselective tolterodine, the highly M(2)-selective (R)-4-[2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl]piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M(3)-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M(3) receptors. The phospholipase C inhibitor 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122) (1-10 microM) did not significantly affect carbachol-stimulated bladder contraction. The phospholipase D inhibitor butan-1-ol relative to its negative control butan-2-ol (0.3% each) caused small but detectable inhibition of carbachol-induced bladder contraction. The Ca(2+) entry blocker nifedipine (10-100 nM) strongly inhibited carbachol-induced bladder contraction. In contrast, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SK&F 96,365) (1-10 microM), an inhibitor of store-operated Ca(2+) channels, caused little inhibition. The protein kinase C inhibitor bisindolylmaleimide I (1-10 microM) did not significantly affected carbachol-induced bladder contraction. In contrast, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y 27,632) (1-10 microM), an inhibitor of rho-associated kinases, concentration dependently and effectively attenuated the carbachol responses. We conclude that carbachol-induced contraction of human urinary bladder via M(3) receptors largely depends on Ca(2+) entry through nifedipine-sensitive channels and activation of a rho kinase, whereas phospholipase D and store-operated Ca(2+) channels contribute only in a minor way. Surprisingly, phospholipase C or protein kinase C do not seem to be involved to a relevant extent.

BACKGROUND

Amygdala-related circuitry helps translate learned Pavlovian associations into appetitive and aversive motivation, especially upon subsequent encounters with cues.

OBJECTIVE

We asked whether μ-opioid stimulation via microinjections of the specific agonist D-Ala(2), N-MePhe(4), Gly-ol)-enkephalin (DAMGO) in central nucleus of amygdala (CeA), or the adjacent basolateral amygdala (BLA) would magnify sucrose or sex "wanting", guided by available cues.

METHODS

CeA or BLA DAMGO enhancement of cue-triggered "wanting" was assessed using Pavlovian to instrumental transfer (PIT). Unconditioned food "wanting" was measured via intake, and male sexual "wanting" for an estrous female was measured in a sexual approach test. Sucrose hedonic taste "liking" was measured in a taste reactivity test.

RESULTS

CeA (but not BLA) DAMGO increased the intensity of phasic peaks in instrumental sucrose seeking stimulated by Pavlovian cues over precue levels in PIT, while suppressing seeking at other moments. CeA DAMGO also enhanced food intake, as well as sexual approach and investigation of an estrous female by males. DAMGO "wanting" enhancements were localized to CeA, as indicated by "Fos plume"-based anatomical maps for DAMGO causation of behavioral effects. Despite increasing "wanting", CeA DAMGO decreased the hedonic impact or "liking" for sucrose in a taste reactivity paradigm.

CONCLUSIONS

CeA μ-opioid stimulation specifically enhances incentive salience, which is dynamically guided to food or sex by available cues.

Endogenous tri-potential neural stem cells (eNSCs) exist in the adult spinal cord and differentiate primarily into oligodendrocytes (OLs) and astrocytes. Previous in vivo and in vitro studies have shown that during development proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) depend on activity in neighboring axons. However, this activity-dependent development of OPCs has not been examined in the adult CNS. In the present study, we stimulated unilateral corticospinal (CS) axons of the adult rat and investigated proliferation and differentiation of OPCs in dorsal corticospinal tract (dCST). eNSCs were labeled with the mitotic indicator 5-bromo-2'-deoxyuridine (BrdU). Phenotypes of proliferating cells were identified by double-immunolabeling of BrdU with a panel of antibodies to cell markers: NG2, Nkx2.2, APC, GFAP, and Glut-1. Electrical stimulation of CS axons increased BrdU labeled eNSCs and promoted the proliferation and differentiation of OPCs, but not astrocytes and endothelial cells. Our findings demonstrate the importance of neural activity in regulating OPC proliferation/differentiation in the mature CNS. Selective pathway electrical stimulation could be used to promote remyelination and recovery of function in CNS injury and disease.

Oxidative injury to premyelinating oligodendrocytes (preOLs) in developing white matter has been implicated in the pathogenesis of periventricular leukomalacia, the lesion underlying most cases of cerebral palsy in premature infants. In this study, we investigated the pathways of OL death induced by intracellular glutathione (GSH) depletion. We found that the lipoxygenase (LOX) inhibitors AA-861 and BMD-122 (N-benzyl-N-hydroxy-5-phenylpentamide; BHPP), but not the cyclooxygenase (COX) inhibitor indomethacin, fully protected the cells from GSH depletion caused by cystine deprivation. Arachidonic acid (AA), the substrate for 12-LOX, potentiated the toxicity of mild cystine deprivation and at higher concentration was itself toxic. This toxicity was also blocked by 12-LOX inhibitors. Consistent with a role for 12-LOX in the cell death pathway, 12-LOX activity increased following cystine deprivation in OLs. Blocking 12-LOX with AA-861 effectively inhibited the accumulation of reactive oxygen species (ROS) induced by cystine deprivation. These data suggest that, in OLs, intracellular GSH depletion leads to activation of 12-LOX, ROS accumulation and cell death. Mature OLs were more resistant than preOLs to cystine deprivation. The difference in sensitivity was not due to a difference in 12-LOX activity but rather appeared to be related to the presence of stronger antioxidant defense mechanisms in mature OLs. These results suggest that 12-LOX activation plays a key role in oxidative stress-induced OL death.

BACKGROUND

Epidemiologic studies have suggested associations between flavonoid intake and health benefits. Traditional Mediterranean diets consist of a high consumption of plant products rich in flavonoids.

OBJECTIVE

This study estimates dietary flavonoid intake and main food sources in a Mediterranean population (Spanish adults).

METHODS

The study included 40,683 subjects aged 35 to 64 years from northern and southern regions of Spain who were included in the European Prospective Investigation into Cancer and Nutrition study Spanish cohort. Usual food intake was assessed by personal interviews using a computerized version of a validated diet history method. Expanded US Department of Agriculture databases for the flavonoid, isoflavone, and proanthocyanidin content were used.

RESULTS

The median and mean of total flavonoids were 269.17 and 313.26 mg/day, respectively. The most abundant flavonoid subgroup was proanthocyanidins (60.1%), followed by flavanones (16.9%), flavan-3-ols (10.3%), flavonols (5.9%), anthocyanidins (5.8%), flavones (1.1%), and isoflavones (<0.01%). The main sources of total flavonoid intake were apples (23%), red wine (21%), unspecified fruit (12.8%), and oranges (9.3%).

CONCLUSIONS

These results should be very useful for evaluating the relationships between flavonoid intake and several diseases.

Circular dichroism is a powerful tool for establishing the absolute configuration of flavonoids and proanthocyanidin analogues. It has been utilized to study the configuration of flavanones, dihydroflavonols (3-hydroxyflavanones), flavan-3-ols, flavan-4-ols, flavan-3,4-diols, flavans, isoflavans, isoflavanones, pterocarpans, 6a-hydroxypterocarpans, rotenoids, 12a-hydroxyrotenoids, neoflavonoids, 3,4-dihydro-4-arylcoumarins, 4-arylflavan-3-ols, auronols, homoisoflavanones, proanthocyanidins, and various classes of biflavonoids. Results relevant to the correlation of circular dichroic data and the absolute configuration of the diastereoisomers of some of the above classes of compounds will be discussed.

Functional gene clusters, containing two or more genes encoding different enzymes for the same pathway, are sometimes observed in plant genomes, most often when the genes specify the synthesis of specialized defensive metabolites. Here, we show that a cluster of genes in tomato (Solanum lycopersicum; Solanaceae) contains genes for terpene synthases (TPSs) that specify the synthesis of monoterpenes and diterpenes from cis-prenyl diphosphates, substrates that are synthesized by enzymes encoded by cis-prenyl transferase (CPT) genes also located within the same cluster. The monoterpene synthase genes in the cluster likely evolved from a diterpene synthase gene in the cluster by duplication and divergence. In the orthologous cluster in Solanum habrochaites, a new sesquiterpene synthase gene was created by a duplication event of a monoterpene synthase followed by a localized gene conversion event directed by a diterpene synthase gene. The TPS genes in the Solanum cluster encoding cis-prenyl diphosphate-utilizing enzymes are closely related to a tobacco (Nicotiana tabacum; Solanaceae) diterpene synthase encoding Z-abienol synthase (Nt-ABS). Nt-ABS uses the substrate copal-8-ol diphosphate, which is made from the all-trans geranylgeranyl diphosphate by copal-8-ol diphosphate synthase (Nt-CPS2). The Solanum gene cluster also contains an ortholog of Nt-CPS2, but it appears to encode a nonfunctional protein. Thus, the Solanum functional gene cluster evolved by duplication and divergence of TPS genes, together with alterations in substrate specificity to utilize cis-prenyl diphosphates and through the acquisition of CPT genes.

OBJECTIVE

To study the association between dietary flavonoid and lignan intakes, and the risk of development of type 2 diabetes among European populations.

METHODS

The European Prospective Investigation into Cancer and Nutrition-InterAct case-cohort study included 12,403 incident type 2 diabetes cases and a stratified subcohort of 16,154 participants from among 340,234 participants with 3.99 million person-years of follow-up in eight European countries. At baseline, country-specific validated dietary questionnaires were used. A flavonoid and lignan food composition database was developed from the Phenol-Explorer, the U.K. Food Standards Agency, and the U.S. Department of Agriculture databases. Hazard ratios (HRs) from country-specific Prentice-weighted Cox regression models were pooled using random-effects meta-analysis.

RESULTS

In multivariable models, a trend for an inverse association between total flavonoid intake and type 2 diabetes was observed (HR for the highest vs. the lowest quintile, 0.90 [95% CI 0.77-1.04]; P value trend = 0.040), but not with lignans (HR 0.88 [95% CI 0.72-1.07]; P value trend = 0.119). Among flavonoid subclasses, flavonols (HR 0.81 [95% CI 0.69-0.95]; P value trend = 0.020) and flavanols (HR 0.82 [95% CI 0.68-0.99]; P value trend = 0.012), including flavan-3-ol monomers (HR 0.73 [95% CI 0.57-0.93]; P value trend = 0.029), were associated with a significantly reduced hazard of diabetes.

CONCLUSIONS

Prospective findings in this large European cohort demonstrate inverse associations between flavonoids, particularly flavanols and flavonols, and incident type 2 diabetes. This suggests a potential protective role of eating a diet rich in flavonoids, a dietary pattern based on plant-based foods, in the prevention of type 2 diabetes.

The morphogen Sonic Hedgehog (Shh) controls the generation of oligodendrocyte (OLs) during embryonic development and regulates OL production in adulthood in the cortex and corpus callosum. The roles of Shh in CNS repair following lesions associated with demyelinating diseases are still unresolved. Here, we address this issue by using a model of focal demyelination induced by lysolecithin in the corpus callosum of adult mice. Shh transcripts and protein were not detected in control animals but were upregulated in a time-dependent manner in the oligodendroglial lineage within the lesion. We report an increased transcription of Shh target genes suggesting a broad reactivation of the Shh pathway. We show that the adenovirus-mediated transfer of Shh into the lesioned brain results in the attenuation of the lesion extent with the increase of OL progenitor cells (OPCs) and mature myelinating OL numbers due to survival, proliferation, and differentiation activities as well as the decrease of astrogliosis and macrophage infiltration. Furthermore, the blocking of Shh signaling during the lesion, using its physiological antagonist, Hedgehog interacting protein, results in a decrease of OPC proliferation and differentiation, preventing repair. Together, our findings identify Shh as a necessary factor playing a positive role during demyelination and indicate that its signaling activation stands as a potential therapeutic approach for myelin diseases.