BACKGROUND

The successful development of odour-baited trapping systems for mosquitoes depends on the identification of behaviourally active semiochemicals, besides the design and operating principles of such devices. A large variety of 'attractants' has been identified in laboratory investigations, yet few of these increase trap catches in the field. A contained system, intermediate between the laboratory and open field, is presented and previous reports that human foot odour induces behavioural responses of Anopheles gambiae confirmed.

METHODS

The response of 3-5 day old female An. gambiae towards odour-baited counterflow geometry traps (MM-X model; American Biophysics Corp., RI) was studied in semi-field (screen house) conditions in western Kenya. Traps were baited with human foot odour (collected on socks), carbon dioxide (CO2, 500 ml min(-1)), ammonia (NH3), 1-octen-3-ol, or various combinations thereof. Trap catches were log (x+1) transformed and subjected to Latin square analysis of variance procedures.

RESULTS

Apart from 1-octen-3-ol, all odour baits caused significant (P < 0.05) increases in trap catches over non-baited traps. Foot odour remained behaviourally active for at least 8 days after collection on nylon or cotton sock fabric. A synergistic response (P < 0.001) was observed towards the combination of foot odour and CO2, which increased catches of these odours alone by 3.8 and 2.7 times, respectively.

CONCLUSIONS

These results are the first to report behavioural responses of an African malaria vector to human foot odour outside the laboratory, and further investigation of fractions and/or individual chemical components of this odour complex are called for. Semi-field systems offer the prospect of high-throughput screening of candidate kairomones, which may expedite the development of efficient trap-bait systems for this and other African mosquito species.

Intracranial self-administration of mu- and delta-opioid agonists was demonstrated in male Long-Evans rats. Independent groups were allowed to lever-press for ventral tegmental area (VTA) microinfusions of morphine, the selective mu agonist [D-Ala2,N-Me-Phe4-Gly5-ol]-enkephalin (DAMGO), the selective delta-agonist [D-Pen2,D-Pen5]-enkephalin (DPDPE), or ineffective drug vehicle. Morphine, DAMGO, and DPDPE were each effective in establishing and maintaining lever-pressing habits. Lever-pressing responses were extinguished during a session when vehicle was substituted for drug, and reinstated when drug reinforcement was reestablished. Thus, it appears that VTA mu- and delta-opioid receptors are each involved in reinforcement of opiate self-administration. The effective dose of DAMGO--both for establishing and for maintaining the lever-press habit--was 100 times lower than the effective doses for DPDPE and morphine, suggesting that the major contribution of VTA mechanisms to intravenous heroin self-administration involves an action on mu-opioid receptors.

The pre-Bötzinger complex (PBC) inspiratory center remains active in a transverse brainstem slice. Such slices are studied at high (8-10 mM) superfusate [K+], which could attenuate the sensitivity of the PBC to neuromodulators such as opiates. Findings may also be confounded because slice boundaries, drug injection sites, or location of rhythmogenic interneurons are rarely verified histologically. Thus, we first generated PBC slices with defined boundaries using novel "on-line histology" based on our finding that rostrocaudal extensions of brainstem respiratory marker nuclei are constant in newborn rats between postnatal days 0-4. At physiological superfusate [K+] (3 mM), 500- and 600-microm-thick slices with the PBC in the center and the caudal boundary 0.70 and 0.76 mm caudal to the facial motonucleus generated rhythm for >2 and approximately 4 h, respectively. Rhythm was abolished by low nanomolar concentrations of the mu-opiate receptor agonist DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin). After spontaneous arrest of bursting, rhythm was reactivated at clinically relevant or physiological concentrations by 3,5-dihydroxyphenylglycine, thyrotropin-releasing hormone, or rolipram, each affecting distinct second-messenger pathways. Two-photon/confocal Ca2+ imaging revealed that these agents reactivated the same PBC neurons initially active in 3 mM [K+]. The data show that "calibrated" PBC slices at physiological [K+] generate rhythm with a high sensitivity to neuromodulators for extended time periods, whereas spontaneous "in vitro apnea" is an important tool to study the interaction of signaling pathways that modulate rhythm. Our approaches and findings provide the basis for a pharmacological and structure-function analysis of the isolated respiratory center in a histologically well defined substrate at physiological [K+].

OBJECTIVE

The aim of this study was to test the feasibility of estimating preference weights for all health states defined by the Child Health Utility 9D (CHU9D), a new generic measure of health-related quality of life for children aged 7-11 years. The estimation of preference weights will allow the calculation of QALYs for use in paediatric economic evaluation.

METHODS

Valuation interviews were undertaken with 300 members of the UK adult general population to obtain preference weights for a sample of the health states in the CHU9D descriptive system. Both standard gamble and ranking valuation methods were used. Regression modelling was undertaken to estimate models that could predict a value for every health state defined by the system. A range of models were tested and were evaluated based on their predictive performance.

RESULTS

Models estimated on the standard gamble data performed better than the rank model. All models had a few inconsistencies or insignificant levels and so further modelling was done to estimate a parsimonious consistent regression model using the general-to-specific approach, by combining inconsistent levels and removing non-significant levels. The final preferred model was an ordinary least squares (OLS) model. All the coefficients in this model were significant, there were no inconsistencies and the model had the best predictive performance and a low mean absolute error.

CONCLUSIONS

This research has demonstrated it is feasible to value the CHU9D descriptive system, and preference weights for each health state can be generated to allow the calculation of QALYs. The CHU9D can now be used in the economic evaluation of paediatric healthcare interventions. Further research is needed to investigate the impact of children's preferences for the health states and what methods could be used to obtain these preferences.

The essential oils obtained from the aerial parts of Origanum scabrum and Origaum microphyllum, both endemic species in Greece, were analyzed by means of GC and GC-MS. Forty-eight constituents were identified, representing 98.59 and 98.66% of the oils, respectively. Carvacrol, terpinen-4-ol, linalool, sabinene, alpha-terpinene, and gamma-terpinene were found as the major components. Furthermore, both samples exhibited a very interesting antimicrobial profile after they were tested against six Gram-negative and -positive bacteria and three pathogenic fungi.

Connexins, the family of proteins that form vertebrate gap junctions, have key roles during development and in the adult. Previously, the physiological actions of connexins have been ascribed solely to formation of gap junction channels and thought to be mediated by the transfer of small molecules between neighboring cells. In conflict with this hypothesis here we demonstrate that Cx43 can affect cell growth independently of gap junction formation. This conclusion is based on four findings: (1) There is a lack of correlation between the action of Cx43 mutants Cx43-S255A, Cx43-S279A, and Cx43-S282A on growth and cell coupling in 3T3 A31 fibroblasts. (2) Blockade of gap junction formation, by either heptan-1-ol treatment or culturing cells at low density, had no effect on the ability of the Cx43 mutants to control growth. (3) Wildtype Cx43 inhibited growth of Neuro2a cells under conditions where gap junctions were unable to form. (4) The CT domain of Cx43, which lacks intrinsic gap junction activity, is as effective as the wildtype molecule in suppressing the growth of Neuro2a cells. These observations demonstrate that Cx43 has dual functions: first, its well-accepted role in forming a gap junction channel and, second, a direct action of the connexin protein on growth that is mediated via the cytoplasmic carboxyl domain.

BACKGROUND

Online haemodiafiltration (OL-HDF) is considered to confer clinical benefits over haemodialysis (HD) in terms of solute removal in patients undergoing maintenance HD. The aim of this study was to compare postdilution OL-HDF and high-flux HD in terms of morbidity and mortality.

METHODS

In this prospective, randomized, controlled trial, we enrolled 782 patients undergoing thrice-weekly HD and randomly assigned them in a 1:1 ratio to either postdilution OL-HDF or high-flux HD. The mean age of patients was 56.5 ± 13.9 years, time on HD 57.9 ± 44.6 months with a diabetes incidence of 34.7%. The follow-up period was 2 years, with the mean follow-up of 22.7 ± 10.9 months. The primary outcome was a composite of death from any cause and nonfatal cardiovascular events. The major secondary outcomes were cardiovascular and overall mortality, intradialytic complications, hospitalization rate, changes in several laboratory parameters and medications used.

RESULTS

The filtration volume in OL-HDF was 17.2 ± 1.3 L. Primary outcome was not different between the groups (event-free survival of 77.6% in OL-HDF versus 74.8% in the high-flux group, P = 0.28), as well as cardiovascular and overall survival, hospitalization rate and number of hypotensive episodes. In a post hoc analysis, the subgroup of OL-HDF patients treated with a median substitution volume >17.4 L per session (high-efficiency OL-HDF, n = 195) had better cardiovascular (P = 0.002) and overall survival (P = 0.03) compared with the high-flux HD group. In adjusted Cox-regression analysis, treatment with high-efficiency OL-HDF was associated with a 46% risk reduction for overall mortality {RR = 0.54 [95% confidence interval (95% CI) 0.31-0.93], P = 0.02} and a 71% risk reduction for cardiovascular mortality [RR = 0.29 (95% CI 0.12-0.65), P = 0.003] compared with high-flux HD.

CONCLUSIONS

The composite of all-cause mortality and nonfatal cardiovascular event rate was not different in the OL-HDF and in the high-flux HD groups. In a post hoc analysis, OL-HDF treatment with substitution volumes over 17.4 L was associated with better cardiovascular and overall survival.

This was a multicenter, double-blind (DB), placebo-controlled, randomized discontinuation trial to evaluate the efficacy of pregabalin monotherapy for durability of effect on fibromyalgia (FM) pain. The trial included a 6-week open-label (OL) pregabalin-treatment period followed by 26-week DB treatment with placebo or pregabalin. Adults with FM and 40-mm score on 100-mm pain visual analog scale (VAS) were eligible. During OL weeks 1-3, patients received escalating dosages of pregabalin to determine their optimal dosages. During OL weeks 4-6, patients received their optimal fixed dosages (300, 450, 600mg/d). To be randomized, patients must have had 50% decrease in pain VAS and a self-rating of "much" or "very much" improved on Patient Global Impression of Change (PGIC) at the end of OL. Double-blind treatment was with placebo or the patient's optimal fixed dosage of pregabalin. Primary outcome was time to loss of therapeutic response (LTR), defined as <30% reduction in pain (from OL baseline) or worsening of FM. A total of 1051 patients entered OL; 287 were randomized to placebo, 279 to pregabalin. Time to LTR was longer for pregabalin versus placebo (P<.0001). Kaplan-Meier estimates of time-to-event showed half the placebo group had LTR by Day 19; half the pregabalin group still had not lost response by trial end. At the end of DB, 174 (61%) placebo patients met LTR criteria versus 90 (32%) pregabalin patients. Pregabalin was well tolerated, though 178 (17%) discontinued during OL for treatment-related adverse events (AE), and more pregabalin than placebo patients discontinued for AEs during DB. In those who respond, pregabalin demonstrated durability of effect for relieving FM pain.

The regulation of glutamate (Glu) release from the excitatory input to dopamine cells in the ventral tegmental area (VTA) during acute withdrawal from morphine was studied in slices from animals treated for 6-7 d with morphine. EPSCs were inhibited by opioid agonists acting at micro-subtype receptors but not by selective delta- or kappa-subtype agonists. The opioid inhibition was reduced by 65% with the potassium channel blocker 4-aminopyridine (4-AP; 100 microM) and a 12-lipoxygenase inhibitor, baicalein (5 microM), suggesting that opioids acted via a transduction pathway involving activation of a voltage-dependent potassium conductance by lipoxygenase metabolites as has been shown in the periaqueductal gray (). During withdrawal, neither the potency nor the efficacy of D-Ala-Met-enkephalin-Gly-ol (DAMGO) were changed; however, the blockade of micro-opioid inhibition by both 4-AP and baicalein was reduced. In addition, the potency of baclofen to depress EPSCs by GABA-B receptors and the effects of the GABA-uptake inhibitor NO-711 (10 microM) were increased in withdrawn rats. Finally, group 2 (but not group 4 or 1) metabotropic glutamate receptor-mediated presynaptic inhibition was also enhanced in morphine-withdrawn rats. These results suggest that one of the consequences of withdrawal from chronic morphine is an enhanced presynaptic inhibition of the excitatory inputs to the dopamine cells of the VTA. Inhibition of glutamate release during acute withdrawal would add to the inhibition of dopamine cells that is mediated by an augmented release of GABA ().

An Arabidopsis thaliana cDNA encoding the enzyme beta-carotene hydroxylase was identified by functional complementation in Escherichia coli. The product of this cDNA adds hydroxyl groups to both beta rings of the symmetrical beta-carotene (beta,beta-carotene) to form zeaxanthin (beta,beta-carotene-3,3'-diol) and converts the monocyclic beta-zeacarotene (7',8'-dihydro-beta,psi-carotene) to hydroxy-beta-zeacarotene (7',8'-dihydro-beta,psi-carotene-3-ol). The epsilon rings of delta-carotene (epsilon,psi-carotene) and alpha-zeacarotene (7',8'-dihydro-epsilon,psi-carotene) are poor substrates for the enzyme. The predicted amino acid sequence of the A. thaliana enzyme resembles the four known bacterial beta-carotene hydroxylase enzymes (31-37% identity) but is much longer, with an N-terminal extension of more than 130 amino acids. Truncation of the cDNA to produce a polypeptide lacking the first 69 amino acids does not impair enzyme activity in E. coli. Truncation to yield a polypeptide of a length comparable with the bacterial enzymes (lacking 129 N-terminal amino acids) resulted in the accumulation of the monohydroxy intermediate beta-cryptoxanthin (beta,beta-carotene-3-ol), predominantly, when beta-carotene was provided as the substrate. It is suggested that amino acid residues 70-129 of the A. thaliana enzyme may play a role in formation of a functional homodimer.

A potential drug target for treatment of Chagas disease, sterol 14alpha-demethylase from Trypanosoma cruzi (TCCYP51), was found to be catalytically closely related to animal/fungi-like CYP51. Contrary to the ortholog from Trypanosoma brucei (TB), which like plant CYP51 requires C4-monomethylated sterol substrates, TCCYP51 prefers C4-dimethylsterols. Sixty-six CYP51 sequences are known from bacteria to human, their sequence homology ranging from approximately 25% between phyla to approximately 80% within a phylum. TC versus TB is the first example of two organisms from the same phylum, in which CYP51s (83% amino acid identity) have such profound differences in substrate specificity. Substitution of animal/fungi-like Ile105 in the B' helix to Phe, the residue found in this position in all plant and the other six CYP51 sequences from Trypanosomatidae, dramatically alters substrate preferences of TCCYP51, converting it into a more plant-like enzyme. The rates of 14alpha-demethylation of obtusifoliol and its 24-demethyl analog 4alpha-,4alpha-dimethylcholesta-8,24-dien-3beta-ol(norlanosterol) increase 60- and 150-fold, respectively. Turnover of the three 4,4-dimethylated sterol substrates is reduced approximately 3.5-fold. These catalytic properties correlate with the sterol binding parameters, suggesting that Phe in this position provides necessary interactions with C4-monomethylated substrates, which Ile cannot. The CYP51 substrate preferences imply differences in the post-squalene portion of sterol biosynthesis in TC and TB. The phyla-specific residue can be used to predict preferred substrates of new CYP51 sequences and subsequently for the development of new artificial substrate analogs, which might serve as highly specific inhibitors able to kill human parasites.

Promotion of remyelination is an important therapeutic strategy to facilitate functional recovery after traumatic spinal cord injury (SCI). Transplantation of neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) has been used to enhance remyelination after SCI. However, the microenvironment in the injured spinal cord is inhibitory for oligodendrocyte (OL) differentiation of NSCs or OPCs. Identifying the signaling pathways that inhibit OL differentiation in the injured spinal cord could lead to new therapeutic strategies to enhance remyelination and functional recovery after SCI. In the present study, we show that reactive astrocytes from the injured rat spinal cord or their conditioned media inhibit OL differentiation of adult OPCs with concurrent promotion of astrocyte differentiation. The expression of bone morphogenetic proteins (BMP) is dramatically increased in the reactive astrocytes and their conditioned media. Importantly, blocking BMP activity by BMP receptor antagonist, noggin, reverse the effects of active astrocytes on OPC differentiation by increasing the differentiation of OL from OPCs while decreasing the generation of astrocytes. These data indicate that the upregulated bone morphogenetic proteins in the reactive astrocytes are major factors to inhibit OL differentiation of OPCs and to promote its astrocyte differentiation. These data suggest that manipulation of BMP signaling in the endogenous or grafted NSCs or OPCs may be a useful therapeutic strategy to increase their OL differentiation and remyelination and enhance functional recovery after SCI.

Recent reports have demonstrated that disruption of CB(1) receptor signaling impairs extinction of learned responses in conditioned fear and Morris water maze paradigms. Here, we test the hypothesis that elevating brain levels of the endogenous cannabinoid anandamide through either genetic deletion or pharmacological inhibition of its primary catabolic enzyme fatty-acid amide hydrolase (FAAH) will potentiate extinction in a fixed platform water maze task. FAAH (-/-) mice and mice treated with the FAAH inhibitor OL-135, did not display any memory impairment or motor disruption, but did exhibit a significant increase in the rate of extinction. Unexpectedly, FAAH-compromised mice also exhibited a significant increase in acquisition rate. The CB(1) receptor antagonist SR141716 (rimonabant) when given alone had no effects on acquisition, but disrupted extinction. Additionally, SR141716 blocked the effects of OL-135 on both acquisition and extinction. Collectively, these results indicate that endogenous anandamide plays a facilitatory role in extinction through a CB(1) receptor mechanism of action. In contrast, the primary psychoactive constituent of marijuana, Delta(9)-tetrahydrocannabinol, failed to affect extinction rates, suggesting that FAAH is a more effective target than a direct acting CB(1) receptor agonist in facilitating extinction. More generally, these findings suggest that FAAH inhibition represents a promising pharmacological approach to treat psychopathologies hallmarked by an inability to extinguish maladaptive behaviors, such as post-traumatic stress syndrome and obsessive-compulsive disorder.

To determine the scope of gene expression controlled by the maize transcription factors C1/R and P, which are responsible for activating flavonoid synthesis, we used GeneCalling, an open-ended, gel-based, mRNA-profiling technology, to analyze cell suspension lines of the maize inbred Black Mexican Sweet (BMS) that harbored estradiol-inducible versions of these factors. BMS cells were transformed with a continually expressed estrogen receptor/maize C1 activator domain fusion gene (ER-C1) and either a fusion of C1 and R (CRC), P, or luciferase genes regulated by a promoter containing four repeats of an estrogen receptor binding site. Increasing amounts of luciferase activity, anthocyanins, and flavan-4-ols were detected in the respective cell lines after the addition of estradiol. The expression of both known and novel genes was detected simultaneously in these BMS lines by profiling the mRNA isolated from replicate samples at 0, 6, and 24 hr after estradiol treatment. Numerous cDNA fragments were identified that showed a twofold or greater difference in abundance at 6 and 24 hr than at 0 hr. The cDNA fragments from the known flavonoid genes, except chalcone isomerase (chi1), were induced in the CRC-expressing line after hormone induction, whereas only the chalcone synthase (c2) and flavanone/dihydroflavonol reductase (a1) genes were induced in the P-expressing line, as was expected. Many novel cDNA fragments were also induced or repressed by lines expressing CRC alone, P alone, or both transcription factors in unique temporal patterns. The temporal differences and the evidence of repression indicate a more diverse set of regulatory controls by CRC or P than originally expected. GeneCalling analysis was successful in detecting members of complex metabolic pathways and uncovering novel genes that were either coincidentally regulated or directly involved in such pathways.

The purpose of the present study was to define the optimal loads (OL) for eliciting maximal power-outputs (PO) in the leg and arm modes of the 30s Wingate Anaerobic Test (WAnT). Eighteen female and seventeen male physical education students, respectively 20.6 +/- 1.6 and 24.1 +/- 2.5 years old, volunteered to participate. In each of the total five sessions, the test was administered twice on a convertible, mechanically braked cycle-ergometer, once for the legs and once for the arms. The five randomized, evenly-spaced resistance loads ranged from 2.43 to 5.39 Joule per pedal revolution per kg body weight (B. W.) for the legs, and from 1.96 to 3.92 for the arms. The measured variables were mean (MP x kg-1) and peak PO as well as absolute and relative measures of fatigue. A parabola-fitting technique was employed to define the optimal loads from the MP x kg-1 data. The resulting OL were 5.04 and 5.13 Joule x Rev-1 x kg B.W.-1 in the leg and 2.82 and 3.52 in the arm tests for the women and men, respectively. OL were shown to depend on PO magnitude. However, within a two-load span (0.98 Joule x Rev-1 x kg B.W.-1) about the OL, MP x kg-1 did not vary by more than 1.4% in the leg and 2.2% in the arm tests. It is suggested that although the WAnT is rather insensitive to moderate variation in load assignment, improved results could be obtained by using the stated OL as guidelines that may be modified according to individual body build, composition, and, particularly, anaerobic fitness level.

The mu-opioid receptor mediates the analgesic and addictive properties of morphine. Despite the clinical importance of this G-protein-coupled receptor and many years of pharmacological research, few intracellular signaling mechanisms triggered by morphine and other mu-opioid agonists have been described. We report that mu-opioid agonists stimulate three different effectors of a phosphoinositide 3-kinase (PI3K)-dependent signaling cascade. By using a cell line stably transfected with the mu-opioid receptor cDNA, we show that the specific agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) stimulates the activity of Akt, a serine/threonine protein kinase implicated in protecting neurons from apoptosis. Activation of Akt by DAMGO correlates with its phosphorylation at serine 473. The selective PI3K inhibitors wortmannin and LY294002 blocked phosphorylation of this site, previously shown to be necessary for Akt enzymatic activity. DAMGO also stimulates the phosphorylation of two other downstream effectors of PI3K, the p70 S6 kinase and the repressors of mRNA translation, 4E-BP1 and 4E-BP2. Upon mu-opioid receptor stimulation, p70 S6 kinase is activated and phosphorylated at threonine 389 and at threonine 421/serine 424. Phosphorylation of p70 S6 kinase and 4E-BP1 is also repressed by PI3K inhibitors as well as by rapamycin, the selective inhibitor of FRAP/mTOR. Consistent with these findings, DAMGO-stimulated phosphorylation of 4E-BP1 impairs its ability to bind the translation initiation factor eIF-4E. These results demonstrate that the mu-opioid receptor activates signaling pathways associated with neuronal survival and translational control, two processes implicated in neuronal development and synaptic plasticity.

2-methylpropan-1-ol (isobutanol) is a leading candidate biofuel for the replacement or supplementation of current fossil fuels. Recent work has demonstrated glucose to isobutanol conversion through a modified amino acid pathway in a recombinant organism. Although anaerobic conditions are required for an economically competitive process, only aerobic isobutanol production has been feasible due to an imbalance in cofactor utilization. Two of the pathway enzymes, ketol-acid reductoisomerase and alcohol dehydrogenase, require nicotinamide dinucleotide phosphate (NADPH); glycolysis, however, produces only nicotinamide dinucleotide (NADH). Here, we compare two solutions to this imbalance problem: (1) over-expression of pyridine nucleotide transhydrogenase PntAB and (2) construction of an NADH-dependent pathway, using engineered enzymes. We demonstrate that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH-dependent pathway and transhydrogenase over-expressing strain. Our results show how engineering cofactor dependence can overcome a critical obstacle to next-generation biofuel commercialization.

OBJECTIVE

Animal and clinical studies have suggested that polyphenols in fruits, red wine, and tea may delay the development of atherosclerosis through their antioxidant and anti-inflammatory properties. We investigated whether individual dietary polyphenols representing different polyphenolic classes, namely quercetin (flavonol), (-)-epicatechin (flavan-3-ol), theaflavin (dimeric catechin), sesamin (lignan), or chlorogenic acid (phenolic acid), reduce atherosclerotic lesion formation in the apolipoprotein E (ApoE)(-/-) gene-knockout mouse.

RESULTS

Quercetin and theaflavin (64-mg/kg body mass daily) significantly attenuated atherosclerotic lesion size in the aortic sinus and thoracic aorta (P<0.05 versus ApoE(-/-) control mice). Quercetin significantly reduced aortic F(2)-isoprostane, vascular superoxide, vascular leukotriene B(4), and plasma-sP-selectin concentrations; and augmented vascular endothelial NO synthase activity, heme oxygenase-1 protein, and urinary nitrate excretion (P<0.05 versus control ApoE(-/-) mice). Theaflavin showed similar, although less extensive, significant effects. Although (-)-epicatechin significantly reduced F(2)-isoprostane, superoxide, and endothelin-1 production (P<0.05 versus control ApoE(-/-) mice), it had no significant effect on lesion size. Sesamin and chlorogenic acid treatments exerted no significant effects. Quercetin, but not (-)-epicatechin, significantly increased the expression of heme oxygenase-1 protein in lesions versus ApoE(-/-) controls.

CONCLUSIONS

Specific dietary polyphenols, in particular quercetin and theaflavin, may attenuate atherosclerosis in ApoE(-/-) gene-knockout mice by alleviating inflammation, improving NO bioavailability, and inducing heme oxygenase-1. These data suggest that the cardiovascular protection associated with diets rich in fruits, vegetables, and some beverages may in part be the result of flavonoids, such as quercetin.

In areas where the practise of betel quid chewing is widespread and the chewers also often smoke and drink alcohol, the relation between oral precancerous lesion and condition to the three habits is probably complex. To explore such association and their attributable effect on oral leukoplakia (OL) and oral submucous fibrosis (OSF), a gender-age-matched case-control study was conducted at Kaohsiung, southern Taiwan. This study included 219 patients with newly diagnosed and histologically confirmed OL or OSF, and 876 randomly selected community controls. All information was collected by a structured questionnaire through in-person interviews. A preponderance of younger patients had OSF, while a predominance of older patients had OL. Betel quid chewing was strongly associated with both these oral diseases, the attributable fraction of OL being 73.2% and of OSF 85.4%. While the heterogeneity in risk for areca nut chewing across the two diseases was not apparent, betel quid chewing patients with OSF experienced a higher risk at each exposure level of chewing duration, quantity and cumulative measure than those who had OL. Alcohol intake did not appear to be a risk factor. However, cigarette smoking had a significant contribution to the risk of OL, and modified the effect of chewing based on an additive interaction model. For the two oral premalignant diseases combined, 86.5% was attributable to chewing and smoking. Our results suggested that, although betel quid chewing was a major cause for both OL and OSF, its effect might be difference between the two diseases. Cigarette smoking has a modifying effect in the development of oral leukoplakia.

The rewarding properties of the psychoactive constituents of marijuana, termed "cannabinoids," may reflect actions on synaptic transmission in the nucleus accumbens (NAc). Furthermore, long-term changes in these synapses may support the addictive process. Excitatory and inhibitory synapses are acutely inhibited by cannabinoids in the NAc, and endogenous cannabinoids (endocannabinoids) play a critical role in the expression of long-term depression (LTD) of excitatory cortical afferents in this structure. Because humans often use marijuana for prolonged periods, we examined the impact of long-term cannabinoid exposure on synaptic processes in an animal model. Electrophysiological recordings in rat brain slices containing the NAc were performed after chronic exposure to vehicle solution, Delta9-tetrahydrocannabinol (THC), or the cannabinoid agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)methanone mesylate (WIN55,212-2). Extracellular glutamatergic postsynaptic potentials and whole-cell GABAergic IPSCs were concentration-dependently inhibited by WIN55,212-2 in slices from naive or vehicle-treated animals. However, the sensitivity to WIN55,212-2 was diminished in chronic agonist-treated animals. In addition, cross-tolerance to the inhibitory effect of the mu-opioid agonist Tyr-D-Ala2, N-CH3-Phe4,Gly-ol-enkephalin was observed. Endocannabinoid-mediated LTD was initiated via electrical stimulation (5 min, 10 Hz) of glutamatergic afferents to the NAc and was completely blocked by the cannabinoid receptor antagonist SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide] in vehicle-treated animals. LTD was not observed in brain slices from rats chronically treated with Delta9-THC or WIN55,212-2. These data demonstrate that long-term exposure to the active ingredient of marijuana blocks synaptic plasticity in the NAc and reduces the sensitivity of GABAergic and glutamatergic synapses to both cannabinoids and opioids.

Several analogues of somatostatin were examined in the Mia PaCa-2 human pancreatic cancer cell line for their ability to promote tyrosine phosphatase activity affecting the receptors for the epidermal growth factor. The inhibition of growth of the Mia PaCa-2 cells in culture was also evaluated to determine the mechanism of action of somatostatin analogues and their relative effectiveness in inhibiting cancer growth. Of the analogues tested D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) caused the greatest stimulation of tyrosine phosphatase activity. Analogue D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (RC-121) had less effect but was more potent than somatostatin-14. Analogue D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(ol) (SMS 201-995) produced no significant dephosphorylation. The analogues displayed the same order of activity in assays on growth inhibition of Mia PaCa-2 cells in cultures. Analogue (SMS-201-995) caused virtually no tyrosine phosphatase stimulation or growth inhibition in this cancer cell line, although it possesses a much higher antisecretory activity than somatostatin-14 in normal tissues. These observations indicate that somatostatin and some of its analogues can act as growth inhibitors in cancer cells through the activation of tyrosine phosphatase. These data reinforce the view that somatostatin analogue RC-160 and related compounds could be used for treatment of pancreatic cancer.

Previous studies indicate that the CB1 cannabinoid receptor antagonist, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1H-pyrazole-3-carboxamide HCl (SR141716A), inhibits the anandamide- and delta9-tetrahydrocannabinol- (THC) induced hypotension and bradycardia in anesthetized rats with a potency similar to that observed for SR141716A antagonism of THC-induced neurobehavioral effects. To further test the role of CB1 receptors in the cardiovascular effects of cannabinoids, we examined two additional criteria for receptor-specific interactions: the rank order of potency of agonists and stereoselectivity. A series of cannabinoid analogs including the enantiomeric pair (-)-11-OH-delta9-THC dimethylheptyl (+)-11-OH-delta9-THC dimethylheptyl were evaluated for their effects on arterial blood pressure and heart rate in urethane anesthetized rats. Six analogs elicited pronounced and long lasting hypotension and bradycardia that were blocked by 3 mg/kg of SR141716A. The rank order of potency was (-)-11-OH-delta9-THC dimethylheptyl>> or = (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol>> (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol>> THC>> anandamide>> or = (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol, which correlated well with CB1 receptor affinity or analgesic potency (r = 0.96-0.99). There was no hypotension or bradycardia after palmitoylethanolamine or (+)-11-OH-delta9-THC dimethylheptyl. An initial pressor response was also observed with THC and anandamide, which was not antagonized by SR141716A. We conclude that the similar rank orders of potency, stereoselectivity and sensitivity to blockade by SR141716A indicate the involvement of CB1-like receptors in the hypotensive and bradycardic actions of cannabinoids, whereas the mechanism of the pressor effect of THC and anandamide remains unclear.

The cannabinoid receptor 1 (CB1), a member of the class A G protein-coupled receptor family, is expressed in brain tissue where agonist stimulation primarily activates the pertussis toxin-sensitive inhibitory G protein (G(i)). Ligands such as CP55940 ((1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3- hydroxypropyl)cyclohexan-1-ol) and Δ(9)-tetrahydrocannabinol are orthosteric agonists for the receptor, bind the conventional binding pocket, and trigger G(i)-mediated effects including inhibition of adenylate cyclase. ORG27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide) has been identified as an allosteric modulator that displays positive cooperativity for CP55940 binding to CB1 yet acts as an antagonist of G protein coupling. To examine this apparent conundrum, we used the wild-type CB1 and two mutants, T210A and T210I (D'Antona, A. M., Ahn, K. H., and Kendall, D. A. (2006) Biochemistry 45, 5606-5617), which collectively cover a spectrum of receptor states from inactive to partially active to more fully constitutively active. Using these receptors, we demonstrated that ORG27569 induces a CB1 receptor state that is characterized by enhanced agonist affinity and decreased inverse agonist affinity consistent with an active conformation. Also consistent with this conformation, the impact of ORG27569 binding was most dramatic on the inactive T210A receptor and less pronounced on the already active T210I receptor. Although ORG27569 antagonized CP55940-induced guanosine 5'-3-O-(thio)triphosphate binding, which is indicative of G protein coupling inhibition in a concentration-dependent manner, the ORG27569-induced conformational change of the CB1 receptor led to cellular internalization and downstream activation of ERK signaling, providing the first case of allosteric ligand-biased signaling via CB1. ORG27569-induced ERK phosphorylation persisted even after pertussis toxin treatment to abrogate G(i) and occurs in HEK293 and neuronal cells.

In American football, impacts to the helmet and the resulting head accelerations are the primary cause of concussion injury and potentially chronic brain injury. The purpose of this study was to quantify exposures to impacts to the head (frequency, location and magnitude) for individual collegiate football players and to investigate differences in head impact exposure by player position. A total of 314 players were enrolled at three institutions and 286,636 head impacts were recorded over three seasons. The 95th percentile peak linear and rotational acceleration and HITsp (a composite severity measure) were 62.7g, 4378rad/s(2) and 32.6, respectively. These exposure measures as well as the frequency of impacts varied significantly by player position and by helmet impact location. Running backs (RB) and quarter backs (QB) received the greatest magnitude head impacts, while defensive line (DL), offensive line (OL) and line backers (LB) received the most frequent head impacts (more than twice as many than any other position). Impacts to the top of the helmet had the lowest peak rotational acceleration (2387rad/s(2)), but the greatest peak linear acceleration (72.4g), and were the least frequent of all locations (13.7%) among all positions. OL and QB had the highest (49.2%) and the lowest (23.7%) frequency, respectively, of front impacts. QB received the greatest magnitude (70.8g and 5428rad/s(2)) and the most frequent (44% and 38.9%) impacts to the back of the helmet. This study quantified head impact exposure in collegiate football, providing data that is critical to advancing the understanding of the biomechanics of concussive injuries and sub-concussive head impacts.