BACKGROUND

Nonadherence with asthma therapy is common and may contribute to poor clinical outcomes.

OBJECTIVE

To examine the effect of dosing frequency and mode of delivery of therapy on adherence and clinical outcomes.

METHODS

We examined adherence in patients with mild persistent asthma (15-85 years) enrolled in a randomized study of montelukast (10 mg once daily) or fluticasone (88 microg, 2 puffs twice daily) during a 12-week double-blind treatment period (DB), followed by a 36-week open-label trial (OL). Adherence was monitored using eDEM for montelukast/placebo and MDILog devices for fluticasone/placebo.

RESULTS

Participants used at least 1 puff of inhaled therapy on 83.3% DB/76.8% OL of days and at least 1 dose of oral therapy on 77.5%/71.4% of days (P < .0001). Subjects used inhaled therapy less than prescribed on 49.5%/57.5% of days, compared with 22.5%/28.6% of days for oral therapy (P < .0001). In the DB, a dose-response relationship was observed with fluticasone and asthma rescue-free days (P = .02) and FEV(1) percent predicted (P < .01) only for patients with FEV(1) < or = 86%. In the OL period, a dose-response relationship was observed with fluticasone and FEV(1) percent predicted (P < .001).

CONCLUSIONS

Whereas subjects were more likely to use inhaled fluticasone/placebo at least once a day, subjects were more likely to take once-daily oral montelukast/placebo as prescribed. Clinical outcomes were inconsistently associated with adherence levels.

CONCLUSIONS

Patients were less likely to be fully adherent with twice-daily therapy than with once-daily therapy, but most still achieved adequate asthma control.

METHODS

We have previously demonstrated that oleuropein (OL) and hydroxytyrosol (HT) reduce 17β-estradiol-mediated proliferation in MCF-7 breast cancer (BC) cells without affecting the classical genomic action of estrogen receptor (ER), but activating instead the ERK1/2 pathway. Here, we hypothesized that this inhibition could be mediated by a G-protein-coupled receptor named GPER/GPR30. Using the ER-negative and GPER-positive SKBR3 BC cells as experimental model, we investigated the effects of OL and HT on GPER-mediated activation of downstream pathways.

RESULTS

Docking simulations and ligand-binding studies evidenced that OL and HT are able to bind GPER. MTT cell proliferation assays revealed that both phenols reduced SKBR3 cell growth; this effect was abolished silencing GPER. Focusing on OL and HT GPER-mediated pathways, using Western blot analysis we showed a sustained ERK1/2 activation triggering an intrinsic apoptotic pathway.

CONCLUSIONS

Showing that OL and HT work as GPER inverse agonists in ER-negative and GPER-positive SKBR3 BC cells, we provide novel insights into the potential of these two molecules as tools in the therapy of this subtype of BC.

Various combinations of six candidate attractants--butanone, carbon dioxide (CO2), honey, octenol, lactic acid and mixed phenols--were tested against natural populations of mosquitoes in Everglades National Park, Florida, U.S.A., using unlighted CDC-baited traps. With few exceptions, the attractancy of these candidate compounds to mosquitoes, when used alone, was less than that of CO2 alone. The exceptions were that octenol and honey extract alone attracted larger numbers of Coquillettidia perturbans (Walker). Addition of lactic acid and/or octenol to CO2 increased trap collections of Aedes taeniorhynchus (Wiedemann), Anopheles atropos D. & K., and An. crucians Wiedemann by 1.4-13.8 times. Culex nigripalpus Theobald collections were increased 2.7 times by the addition of lactic acid, while the addition of octenol produced mixed results. Whereas the addition of lactic acid reduced collections of Cx (Melanoconion) spp., the addition of octenol generally increased collections. The opposite happened for Wyeomyia mitchellii (Theobald). For the biting midge, Culicoides furens (poey), octenol (1.6-23.4 x ) and phenol (2.7 x ) alone attracted larger numbers, and lactic acid alone attracted approximately the same numbers as CO2 alone. The combinations octenol + phenol and octenol + 200 ml/min CO2 increased C. furens collections c. 100 times over CO2 alone. The combination of octenol + CO2 increased (1.6 x ) collections of the tabanid Diachlorus ferrugatus (Fabricius). Butanone appeared to decrease the trap collections of all species when combined with CO2 or octenol + CO2.

Tea (Camellia sinensis (L.) O. Kuntze) leaves are a major source of flavonoids that mainly belong to the flavan 3-ols or catechins. Apart from being responsible for tea quality, these compounds have medicinal properties. Flavanone 3-hydroxylase (F3H) is an abundant enzyme in tea leaves that catalyzes the stereospecific hydroxylation of (2S)-naringenin to form (2R,3R)-dihydrokaempferol. We report a full-length cDNA sequence of F3H from tea (CsF3H Accession no. AY641730). CsF3H comprised 1365 bp with an open reading frame of 1107 nt (from 43 to 1149) encoding a polypeptide of 368 amino acids. Expression of CsF3H in an expression vector in Escherichia coli yielded a functional protein with a specific activity of 32 nmol min(-1) mg protein(-1). There was a positive correlation between the concentration of catechins and CsF3H expression in leaves of different developmental stages. CsF3H expression was down-regulated in response to drought, abscisic acid and gibberellic acid treatment, but up-regulated in response to wounding. The concentration of catechins paralleled the expression data. Exposure of tea shoots to 50-100 microM catechins led to down-regulation of CsF3H expression suggesting substrate mediated feedback regulation of the gene. The strong correlation between the concentration of catechins and CsF3H expression indicates a critical role of F3H in catechin biosynthesis.

BACKGROUND

The structural complexity of taxol dictates continued reliance on biological production methods, which may be improved by a detailed understanding of taxol biosynthesis, especially the rate-limiting steps. The biosynthesis of taxol involves the cyclization of the common isoprenoid intermediate geranylgeranyl diphosphate to taxa-4(5), 11(2)-diene followed by extensive, largely oxidative, modification of this diterpene olefin. We set out to define the first oxygenation step in taxol biosynthesis.

RESULTS

Microsomal enzymes from Taxus stem and cultured cells were used to define the first hydroxylation of taxadiene. We confirmed the structure of the reaction product (taxa-4(20), 11(12)-dien-5alpha-ol) by synthesizing this compound. The responsible biological catalyst was characterized as a cytochrome P450 (heme thiolate protein). In vivo studies confirmed that taxadienol is a biosynthetic intermediate and indicated that the hydroxylation step that produces this product is slow relative to subsequent metabolic transformations.

CONCLUSIONS

The structure of the first oxygenated intermediate on the taxol pathway establishes that the hydroxylation reaction proceeds with an unusual double bond migration that limits the mechanistic possibilities for subsequent elaboration of the oxetane moiety of taxol. The reaction is catalyzed by a cytochrome P450, suggesting that the seven remaining oxygenation steps in taxol biosynthesis may involve similar catalysts. Because the first oxygenation step is slow relative to subsequent metabolic transformations, it may be possible to speed taxol biosynthesis by isolating and manipulating the gene for the taxadiene-5-hydroxylase that catalyzes this reaction.

Sensitization to mechanical stimuli is important in most pain syndromes. We evaluated the populations of nociceptors mediating mechanical hyperalgesia and those mediating mu-opioid receptor (MOR) and delta-opioid receptor (DOR) agonist-induced inhibition of hyperalgesia, in the rat. We found that: (1) intradermal injection of both the endogenous ligand for the Ret receptor, glia-derived growth factor (GDNF), and the ligand for the tropomyosin receptor kinase A (TrkA) receptor, nerve growth factor (NGF)-which are present on distinct populations of nociceptors-both produce mechanical hyperalgesia; (2) DOR agonist 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide (SNC) but not MOR agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) inhibit GDNF-induced hyperalgesia; (3) both DAMGO and SNC inhibit NGF hyperalgesia, even in rats pretreated with isolectin B4 (IB4)-saporin, a toxin that destroys IB4-binding neurons; (4) co-administration of low doses of DAMGO and SNC produce enhanced analgesia, and; (5) repeated administration of DAMGO produces cross-tolerance to the analgesic effect of SNC. These findings demonstrate that, most nociceptors have a role in mechanical hyperalgesia, only the DOR agonist inhibits GDNF hyperalgesia, and MOR and DOR are co-localized on a functionally important population of TrkA-positive nociceptors.

The potential of heptylphysostigmine tartrate (pyrrolo [2,3b] indol-5-ol, 3,3a,8,8a-hexahydro-1,3a,8-trimethylheptylcarbamate [ester, (3aS-cis)]) (MF201), a new second-generation cholinesterase inhibitor, to antagonize scopolamine-induced amnesia in rats was assessed in an 8-arm radial maze. Upon completing the training session, the rats were orally administered increasing doses of MF201 (2, 3, 4, 6 and 8 mg/kg) 60 min prior to a s.c. injection of scopolamine (0.25 mg/kg). 9-Amino-1,2,3,4-tetrahydroamino-acridine hydrochloride hydrate (tacrine) (0.25, 0.37, 0.5, 1 and 2 mg/kg), 1-benzil-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methyl piperidine (E2020) (0.125, 0.18, 0.25 and 0.5 mg/kg) and physostigmine (0.15, 0.25, 0.5 and 1 mg/kg) were orally administered and rats were tested in the same task. As previously described, scopolamine induced an impairment in radial maze performance, measured in terms of total number of errors, total time taken to complete the task and the percentage of amnesic animals. The reversal of scopolamine-induced impairment was characterized by the presence of an inverted U-shaped dose-response curve. A significant antagonistic effect was achieved with a dose (mg/kg) of 0.25 for E2020, 0.5 for tacrine and physostigmine and 3, 4 and 6 for MF201, the latter manifesting a broader spectrum of activity (3-6 mg/kg). While the maximal active doses restored the scopolamine-induced modified pattern of arm entry, they were ineffective in reducing hypermotility, suggesting the drugs have a specific effect on cognitive function.

The beta-3 adrenoceptor protein lacks most of the potential phosphorylation sites for beta adrenoceptor kinase and protein kinase A. In addition, it exhibits a lower affinity toward norepinephrine than beta-1 or beta-2 adrenoceptors. It is thus expected that beta-3 adrenoceptors could be less implicated in desensitization processes than the beta-1 or beta-2 adrenoceptors. An attempt to demonstrate the physiological relevance of this prediction was performed by using fat cells having a beta-3 adrenergic responsiveness or not (hamster and guinea pig). The influence of prolonged in vivo exposure to norepinephrine on the beta adrenoceptor-mediated lipolytic responses was investigated in both species. In control guinea pigs, isoproterenol, norepinephrine and epinephrine were fully lipolytic, whereas BRL 37344 [(R',R')-4-2[2-((2[(3-chlorophenyl)-2-hydroxyethyl]amino] propyl)phenyl]phenoxyacetic acid], CGP 12177](+-)-4-(3-tertiarybutylamino-2-hydroxypropoxy)- benzimidazole-2-on hydrochloride] and other beta-3 agonists were inefficient, whereas hamster adipocytes exhibited maximal response to the beta-3 agonists. Blockade of the lipolytic effect of isoproterenol in the guinea pig gave a rank order of beta antagonists [CGP 20712A (1-[2-(3-carbamoyl-4-hydroxyphenoxy)ethylamino]-3-4-(1-methyl-4-tr i-fluoro-methyl-1H-imidazol-2-yl)phenoxy-2-propanol methanesulfonate]>> bupranolol>> or = propranolol>>) ICI 118551 [erythrodl-1-(7- methylindan-4-yloxy)-3-isopropylaminobutan-2-ol)] in agreement with that of a beta-1 effect. In contrast, the selective beta-1 antagonist CGP 20712A did not counteract the effect of BRL 37344 in hamsters and bupranolol was the best beta antagonist tested; a result arguing for the predominance of a beta-3 component in the adrenergic activation of lipolysis, as in rat fat cells. In treated guinea pigs (6-day treatment with osmotic minipumps delivering norepinephrine at the rate of 5 micrograms/min/kg), the adrenocorticotropic hormone dose-response curve was identical to that of controls, but the curves for isoproterenol, norepinephrine and epinephrine were flattened and shifted to the right. A down-regulation of beta-1 and beta-2 adrenoceptors was evidenced by a reduction in [3H]CGP 12177 high-affinity binding sites. In treated hamsters, compared to the controls, there was no change in the lipolytic response to the beta adrenergic agonists. Other protocols of chronic exposure to norepinephrine (e.g., daily injections) at different doses were also unable to reduce the beta-lipolytic effect in the hamster fat cells.(ABSTRACT TRUNCATED AT 400 WORDS)

We have investigated the coupling between opioid receptors and different types of Ca2+ channels in neurons acutely isolated from the nucleus tractus solitarius (NTS) of the rat. Using fura-2-based imaging we found that Ca2+ transients evoked by depolarization with 50 mM KCl were suppressed by the mu-opioid receptor agonist D-Ala2,N-MePhe4,Gly5-ol-enkephalin (DAMGO) and less effectively by the kappa-receptor agonist U-69,593. The delta-receptor agonist D-Pen2,D-Pen5-enkephalin (DPDPE) was ineffective. In whole-cell voltage-clamp recordings from these neurons, depolarizing voltage steps elicited high-threshold Ca2+ currents that could be distinguished pharmacologically into different components. Part of the current could be blocked by dihydropyridines, part by omega-conotoxin-GVIA and part by omega-agatoxin-IVA. This suggests that the neurons contained L-, N-, and P/Q-type Ca2+ channels. DAMGO and U-69,593 both blocked part of the Ca2+ current but DPDPE was ineffective. Perfusion of GTP-gamma-S into the cells produced a rapid rundown of the Ca2+ current and occluded further effects of the opioid agonists, suggesting the involvement of a G-protein in the coupling mechanism. Inhibition of L-channels did not alter the effect of DAMGO. On the other hand inhibition of N-channels occluded about 80% of the effect of DAMGO. Inhibition of the P/Q-current occluded the remainder of the DAMGO effect. Thus, it appears that activation of opioid receptors can inhibit N- and P/Q-type Ca2+ channels but not L-channels in these cells. It is likely that such effects are important in opioid-mediated inhibition of transmitter release in the brain.

Members of the R7 subfamily of regulators of G-protein signaling (RGS) proteins (RGS6, RGS7, RGS9-2, and RGS11) are found in the mouse CNS. The expression of these proteins was effectively reduced in different neural structures by blocking their mRNA with antisense oligodeoxynucleotides (ODNs). This was achieved without noticeable changes in the binding characteristics of labeled beta-endorphin to opioid receptors. Knockdown of R7 proteins enhanced the potency of antinociception promoted by morphine and [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO)-both agonists at mu-opioid receptors. The duration of morphine analgesia was greatly increased in RGS9-2 and in RGS11 knockdown mice. The impairment of R7 proteins brought about different changes in the analgesic activity of selective delta agonists. Knockdown of RGS11 reduced [D-Ala(2)]deltorphin II analgesic effects. Those of RGS6 and RGS9-2 proteins caused [D-Ala(2)]deltorphin II to produce a smoothened time-course curve-the peak effect blunted and analgesia extended during the declining phase. RGS9-2 impairment also promoted a similar pattern of change for [D-Pen(2,5)]-enkephalin (DPDPE). RGS7-deficient mice showed an increased response to both [D-Ala(2)]deltorphin II and DPDPE analgesic effects. A single intracerebroventricular (i.c.v.) ED(80) analgesic dose of morphine gave rise to acute tolerance in control mice, but did not promote tolerance in RGS6, RGS7, RGS9-2, or RGS11 knockdown animals. Thus, R7 proteins play a critical role in agonist tachyphylaxis and acute tolerance at mu-opioid receptors, and show differences in their modulation of delta-opioid receptors.

Acetylcholine (ACh) is a major neurotransmitter but also an important signaling molecule in neuron-glia interactions. Expression of ACh receptors has been reported in several glial cell populations, including oligodendrocytes (OLs). Nonetheless, the characterization of muscarinic receptors in these cells, as well as the description of the cholinergic effects at different stages of OL development, is still incomplete. In this study, we characterized the pattern of expression of muscarinic receptor subtypes in primary cultures of rat oligodendrocyte progenitor cells (OPC) and mature OLs, at both mRNA and protein levels. We found that muscarinic receptor expression is developmentally regulated. M1, M3, and M4 receptors were the main subtypes expressed in OPC, whereas all receptor subtypes were expressed at low levels in mature OLs. Exposure of OPC to muscarine enhanced cell proliferation, an effect mainly due to M1, M3, and M4 receptor subtypes as demonstrated by pharmacological competition with selective antagonists. Conversely, M2 receptor activation impaired OPC survival. In line with the mitogenic activity, muscarinic receptor activation increased the expression of platelet derived growth factor receptor α. Muscarine stimulation increased CX32 and myelin basic protein expression, left unaffected that of myelin proteolipid protein (PLP), and decreased member of the family of epidermal growth factor receptor (EGFR) ErbB3/ErbB4 receptor expression indicating a predominant role of muscarinic receptors in OPC. These findings suggest that ACh may contribute to the maintenance of an immature proliferating progenitor pool and impair the progression toward mature stage. This hypothesis is further supported by increased expression of Notch-1 in OL on muscarinic activation.

Acacia polyphenol (AP) extracted from the bark of the black wattle tree (Acacia meansii) is rich in unique catechin-like flavan-3-ols, such as robinetinidol and fisetinidol. The present study investigated the anti-obesity/anti-diabetic effects of AP using obese diabetic KKAy mice. KKAy mice received either normal diet, high-fat diet or high-fat diet with additional AP for 7 weeks. After the end of administration, body weight, plasma glucose and insulin were measured. Furthermore, mRNA and protein expression of obesity/diabetic suppression-related genes were measured in skeletal muscle, liver and white adipose tissue. As a result, compared to the high-fat diet group, increases in body weight, plasma glucose and insulin were significantly suppressed for AP groups. Furthermore, compared to the high-fat diet group, mRNA expression of energy expenditure-related genes (PPARα, PPARδ, CPT1, ACO and UCP3) was significantly higher for AP groups in skeletal muscle. Protein expressions of CPT1, ACO and UCP3 for AP groups were also significantly higher when compared to the high-fat diet group. Moreover, AP lowered the expression of fat acid synthesis-related genes (SREBP-1c, ACC and FAS) in the liver. AP also increased mRNA expression of adiponectin and decreased expression of TNF-α in white adipose tissue. In conclusion, the anti-obesity actions of AP are considered attributable to increased expression of energy expenditure-related genes in skeletal muscle, and decreased fatty acid synthesis and fat intake in the liver. These results suggest that AP is expected to be a useful plant extract for alleviating metabolic syndrome.

Several classes of flavonoids (flavones, flavanones, 2'-hydroxychalcones and flavan-4-ols) having a variety of substituents on A ring were investigated for their antiproliferative activity against MCF-7 human breast cancer cells. Structure-activity relationships of these compounds were discussed. 2'-hydroxychalcones and methoxylated flavanones were found to be potent inhibitors of MCF-7 cells growth whereas flavones and flavan-4-ols appeared to be weak inhibitory agents except 7,8-dihydroxyflavone.

BACKGROUND

Although growing evidence from trials and population-based studies has supported a protective role for flavonoids in relation to risk of certain chronic diseases, the underlying mechanisms remain unclear. Several previous studies focused on individual inflammatory biomarkers, but because of the limited specificity of any individual marker, an assessment of a combination of biomarkers may be more informative.

OBJECTIVE

We used an inflammation score (IS) that integrated 12 individual inflammatory biomarkers for the examination of associations with intakes of different flavonoid classes.

METHODS

The study was a cross-sectional analysis of 2375 Framingham Heart Study Offspring Cohort participants. Intakes of total flavonoids and their classes (anthocyanins, flavonols, flavanones, flavan-3-ols, polymers, and flavones) were calculated from validated food-frequency questionnaires. Individual inflammatory biomarkers were ranked, standardized, and summed to derive an overall IS and subgroup scores of functionally related biomarkers.

RESULTS

In multivariate analyses, an inverse association between higher anthocyanin and flavonol intakes and IS was observed with a mean ± SE difference between quintile categories 5 and 1 of -1.48 ± 0.32 (P-trend ≤ 0.001) and -0.72 ± 0.33 (P-trend = 0.01), respectively. Results remained significant after additional adjustment for physical activity and vitamin C and fruit and vegetable intakes. Higher anthocyanin intake was inversely associated with all biomarker subgroups, whereas higher flavonol intake was associated only with lower cytokine and oxidative stress biomarker concentrations. In food-based analyses, higher intakes of apples and pears, red wine, and strawberries were associated with a lower IS with differences between quintiles 5 and 1 of -1.02 ± 0.43 (P = 0.006), -1.73 ± 0.39 (P < 0.001), and -0.44 ± 0.88 (P = 0.02), respectively. Although intakes of other classes were not associated with a reduction in overall IS, higher intakes of flavan-3-ols and their polymers were associated with a significant reduction in oxidative stress biomarkers.

CONCLUSIONS

These findings provide evidence to suggest that an anti-inflammatory effect may be a key component underlying the reduction in risk of certain chronic diseases associated with higher intakes of anthocyanins and flavonols. The Framingham Offspring Study was registered at clinicaltrials.gov as NCT00005121 (Framingham Heart Study).

Structural analysis, a purification scheme and stability information on a fluorescent cholesterol analogue, which has been used as a probe in several model and biological systems, are presented. The proposed structure for the fluorophore, cholestatrien-3 beta-ol, closely resembles that of cholesterol. However, problems of low yield during synthesis and rapid decomposition have impeded its use. This study concerns the synthesis and purification of cholestatrien-3 beta-ol by reverse phase high performance liquid chromatography (HPLC). Unlike cholestatrien-3 beta-ol recrystallized from solvents, the fluorescent sterol purified by HPLC was stable over several months at -70 degrees C either as a white, crystalline powder or in ethanolic solution. In model membranes the fluorescence of cholestatrien-3 beta-ol was stable to ultraviolet (UV) light. A simple spectroscopic assay for purity is presented. Included are detailed absorbance, fluorescence, mass, 1H-NMR, and 13C-NMR spectral analyses. The data confirm the structure of cholestatrien-3 beta-ol proposed, but not proven, over 50 years ago, delta 5,7,9(11)-cholestatrien-3 beta-ol.

The ability of a number of flavonoids belonging to the flavone, flavonol, flavanone, and flavan-3-ol subclasses to protect cellular DNA from H2O2-induced single-strand breaks and the underlying molecular mechanisms were investigated in this work. Formation of single-strand breaks on nuclear DNA, after exposure of Jurkat cells to continuously generated H2O2 in the presence or absence of the flavonoid compounds, was evaluated by the comet assay (single-cell gel electrophoresis). The results indicate the following structural requirements of flavonoids for effective DNA protection: (a) the ortho-dihydroxy structure in either ring A or ring B, (b) the hydroxyl moiety on position 3 in combination with the oxo group at position 4, and (c) the presence of a C2, C3 double bond in ring C. In contrast to free flavonoids, the ability of complexes of [Fe2+]/[flavonoid] to protect nuclear DNA was decreased as the ratio increased, and the complex was completely inactive when the ratio reached a certain value. Moreover, it was observed that several of the flavonoids tested were able to remove iron from calcein loaded into cells and that this property was in excellent correlation with their ability to protect DNA (Spearman's correlation coefficient, rho = 0.9, p = 0.005). The antioxidant (electron donating) capacities of the same flavonoids were also evaluated by a conventional method, but no relation with their DNA-protective ability could be established even when their membrane-penetrating abilities were taken into account (p = 0.64). In conclusion, the results presented in this work strongly support the notion that intracellular binding of iron is responsible for the protection offered by flavonoids against H2O2-induced DNA damage.

Epidemiological studies suggest health-protective effects of flavan-3-ols and their derived compounds on chronic diseases. The present study aimed to estimate dietary flavan-3-ol, proanthocyanidin (PA) and theaflavin intakes, their food sources and potential determinants in the European Prospective Investigation into Cancer and Nutrition (EPIC) calibration cohort. Dietary data were collected using a standardised 24 h dietary recall software administered to 36 037 subjects aged 35-74 years. Dietary data were linked with a flavanoid food composition database compiled from the latest US Department of Agriculture and Phenol-Explorer databases and expanded to include recipes, estimations and retention factors. Total flavan-3-ol intake was the highest in UK Health-conscious men (453·6 mg/d) and women of UK General population (377·6 mg/d), while the intake was the lowest in Greece (men: 160·5 mg/d; women: 124·8 mg/d). Monomer intake was the highest in UK General population (men: 213·5 mg/d; women: 178·6 mg/d) and the lowest in Greece (men: 26·6 mg/d in men; women: 20·7 mg/d). Theaflavin intake was the highest in UK General population (men: 29·3 mg/d; women: 25·3 mg/d) and close to zero in Greece and Spain. PA intake was the highest in Asturias (men: 455·2 mg/d) and San Sebastian (women: 253 mg/d), while being the lowest in Greece (men: 134·6 mg/d; women: 101·0 mg/d). Except for the UK, non-citrus fruits (apples/pears) were the highest contributors to the total flavan-3-ol intake. Tea was the main contributor of total flavan-3-ols in the UK. Flavan-3-ol, PA and theaflavin intakes were significantly different among all assessed groups. This study showed heterogeneity in flavan-3-ol, PA and theaflavin intake throughout the EPIC countries.

Morphine is one of the most potent analgesic drugs. However, the utility of morphine in the management of chronic pain is limited by its rapid development of tolerance. Morphine exerts all of its pharmacological effects via the μ-opioid receptor. In many systems, tolerance is associated with phosphorylation and desensitization of G-protein-coupled receptors (GPCRs). In case of the μ-opioid receptor, phosphorylation occurs in an agonist-selective manner. High-efficacy agonists such as [d-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO), fentanyl, or etonitazene stimulate the phosphorylation of both C-terminal threonine 370 (T370) and serine 375 (S375). In contrast, morphine promotes the phosphorylation of S375 but fails to stimulate T370 phosphorylation. Here, we have assessed the contribution of S375 phosphorylation to the development of antinociceptive tolerance to high- and low-efficacy μ agonists in vivo. We show that S375 phosphorylation of the μ-opioid receptor occurs in intact mouse brain in a dose-dependent manner after administration of morphine, fentanyl, or etonitazene. In knock-in mice expressing the phosphorylation-deficient S375A mutant of the μ-opioid receptor, morphine and fentanyl exhibited greater dose-dependent antinociceptive responses than in wild-type mice. However, acute and chronic tolerance to morphine was retained in S375A mutant mice. In contrast, antinociceptive tolerance after repeated subcutaneous application of etonitazene or repeated intracerebroventricular application of DAMGO was diminished. Thus, tolerance to μ agonists with different efficacies develops through distinct pathways. Whereas tolerance induced by DAMGO or etonitazene requires agonist-driven phosphorylation of S375, the development and maintenance of antinociceptive tolerance to morphine occurs independent of S375 phosphorylation.

The microsomal antiestrogen binding site (AEBS) is a high-affinity target for the antitumor drug tamoxifen and its cognate ligands that mediate breast cancer cell differentiation and apoptosis. The AEBS, a hetero-oligomeric complex composed of 3beta-hydroxysterol-Delta8-Delta7-isomerase (D8D7I) and 3beta-hydroxysterol-Delta7-reductase (DHCR7), binds different structural classes of ligands, including ring B oxysterols. These oxysterols are inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), a microsomal epoxide hydrolase that has yet to be molecularly identified. We hypothesized that the AEBS and ChEH might be related entities. We show that the substrates of ChEH, cholestan-5alpha,6alpha-epoxy-3beta-ol (alpha-CE) and cholestan-5beta,6beta-epoxy-3beta-ol (beta-CE), and its product, cholestane-3beta,5alpha,6beta-triol (CT), are competitive ligands of tamoxifen binding to the AEBS. Conversely, we show that each AEBS ligand is an inhibitor of ChEH activity, and that there is a positive correlation between these ligands' affinity for the AEBS and their potency to inhibit ChEH (r2=0.95; n=39; P<0.0001). The single expression of D8D7I or DHCR7 in COS-7 cells slightly increased ChEH activity (1.8- and 2.6-fold), whereas their coexpression fully reconstituted ChEH, suggesting that the formation of a dimer is required for ChEH activity. Similarly, the single knockdown of D8D7I or DHCR7 using siRNA partially inhibited ChEH in MCF-7 cells, whereas the knockdown of both D8D7I and DHCR7 abolished ChEH activity by 92%. Taken together, our findings strongly suggest that the AEBS carries out ChEH activity and establish that ChEH is a new target for drugs of clinical interest, polyunsaturated fatty acids and ring B oxysterols.

The TRPV1 antagonist A-995662 demonstrates analgesic efficacy in monoiodoacetate-induced osteoarthritic (OA) pain in rat, and repeated dosing results in increased in vivo potency and a prolonged duration of action. To identify possible mechanism(s) underlying these observations, release of neuropeptides and the neurotransmitter glutamate from isolated spinal cord was measured. In OA rats, basal release of glutamate, bradykinin and calcitonin gene-related peptide (CGRP) was significantly elevated compared to naïve levels, whereas substance P (SP) levels were not changed. In vitro studies showed that capsaicin-evoked TRPV1-dependent CGRP release was 54.7+/-7.7% higher in OA, relative to levels measured for naïve rats, suggesting that TRPV1 activity was higher under OA conditions. The efficacy of A-995662 in OA corresponded with its ability to inhibit glutamate and CGRP release from the spinal cord. A single, fully efficacious dose of A-995662, 100 micromol/kg, reduced spinal glutamate and CGRP release, while a single sub-efficacious dose of A-995662 (25 micromol/kg) was ineffective. Multiple dosing with A-995662 increased the potency and duration of efficacy in OA rats. Changes in efficacy did not correlate with plasma concentrations of A-995662, but were accompanied with reductions in spinal glutamate release. These findings suggest that repeated dosing of TRPV1 antagonists enhances therapeutic potency and duration of action against OA pain, at least in part, by the sustained reduction in release of glutamate and CGRP from the spinal cord.

The influence of internal (genetic and developmental) and external (environmental) factors on levels of flavonoid gene transcripts, enzyme activity and metabolites was studied in fruit of six cultivated strawberry (Fragaria x ananassa Duch.) genotypes grown at two Italian locations. Gene expression and enzyme activity showed development- and genotype-associated patterns, revealing gene coordination. Analysis clarified the regulation mechanism of the hydroxylation status of the B-ring of the major flavonoid pools and pointed out examples of genotype-specific post-transcriptional regulation mechanisms and key steps of pathway regulation in strawberry fruits. Metabolite profiles were strongly affected by development and genotype. Flavan-3-ols, their proanthocyanidin (PA) derivatives and anthocyanins were the most abundant metabolites. Flavonol levels and PA-associated traits (epicatechin/catechin ratio and mean degree of polymerization) showed significant environmental effects. Multivariate and correlation analyses determined the relationships among genes, enzymes and metabolites. The combined molecular and biochemical information elucidated more in depth the role of genetic and environmental factors on flavonoid metabolism during strawberry fruit development, highlighting the major impact of developmental processes, and revealing genotype-dependent differences and environmental effects on PA-related traits.

One working hypothesis behind transsexuality is that the normal sex differentiation of certain hypothalamic networks is altered. We tested this hypothesis by investigating the pattern of cerebral activation in 12 nonhomosexual male-to-female transsexuals (MFTRs) when smelling 4,16-androstadien-3-one (AND) and estra-1,3,5(10),16-tetraen-3-ol (EST). These steroids are reported to activate the hypothalamic networks in a sex-differentiated way. Like in female controls the hypothalamus in MFTRs activated with AND, whereas smelling of EST engaged the amygdala and piriform cortex. Male controls, on the other hand, activated the hypothalamus with EST. However, when restricting the volume of interest to the hypothalamus activation was detected in MFTR also with EST, and explorative conjunctional analysis revealed that MFTR shared a hypothalamic cluster with women when smelling AND, and with men when smelling EST. Because the EST effect was limited, MFTR differed significantly only from male controls, and only for EST-AIR and EST-AND. These data suggest a pattern of activation away from the biological sex, occupying an intermediate position with predominantly female-like features. Because our MFTRs were nonhomosexual, the results are unlikely to be an effect of sexual practice. Instead, the data implicate that transsexuality may be associated with sex-atypical physiological responses in specific hypothalamic circuits, possibly as a consequence of a variant neuronal differentiation.

We studied spinal analgesic and antiallodynic effects of endomorphin-1 and endomorphin-2 administered i.t. in comparison with Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO) or morphine, during acute, inflammatory and neuropathic pain in rats chronically implanted with intrathecal cannulas. Endomorphin-1 and endomorphin-2 (2.5, 5, 10 microg i.t.) increased the tail-flick latency and, to the lesser extent, the paw pressure latency. The range of potencies in both those models of acute pain was as follows: DAMGO>> morphine = endomorphin-1>> endomorphin-2. In a model of inflammatory pain, the number of formalin-induced flinching episodes was decreased by endomorphin-1. The effect of endomorphin-2 was much less pronounced. Both DAMGO and morphine significantly inhibited the pain-related behavior evoked by formalin. In a neuropathic pain model (sciatic nerve crushing in rats), endomorphin-1 and -2 (5 microg i.t.) had a statistically significant effect on the tail-flick latency and on the cold-water tail flick latency. Morphine, 5 microg, was found to be ineffective. Endomorphin-1 and -2 (2.5 and 5 microg i.t.) dose-dependently antagonized allodynia. Those effects of endomorphins were antagonized in acute (30 microg), inflammatory (30 microg) and neuropathic pain models (60 microg) by cyprodime, a selective mu-opioid receptor antagonist. In conclusion, our results show a strong analgesic action of endomorphins at the spinal cord level. The most interesting finding is a strong, stronger than in the case of morphine, antiallodynic effect of endomorphins in rats subjected to sciatic nerve crushing, which suggests a possible use of these compounds in a very difficult therapy of neuropathic pain.

Cuprizone [bis(cyclohexylidenehydrazide)]-induced toxic demyelination is an experimental animal model commonly used to study de- and remyelination in the central nervous system. In this model, mice are fed with the copper chelator cuprizone which leads to oligodendrocyte death with subsequent demyelination. The underlying mechanisms of cuprizone-induced oligodendrocyte death are still unknown, and appropriate in vitro investigations to study these mechanisms are not available. Thus, we studied cuprizone effects on rat primary glial cell cultures and on the neuroblastoma cell line SH-SY5Y. Treatment of cells with different concentrations of cuprizone failed to show effects on the proliferation and survival of SH-SY5Y cells, microglia, astrocytes, and oligodendrocyte precursor cells (OPC). In contrast, differentiated mature oligodendrocytes (OL) were found to be significantly affected by cuprizone treatment. This was accompanied by a reduced mitochondrial potential in cuprizone-treated OL. These results demonstrate that the main toxic target for cuprizone is mature OL, whilst other glial cells including OPC are not or only marginally affected. This explains the selective demyelination induced by cuprizone in vivo.