Determination of the Cannabinoid CB1 Receptor's Positive Allosteric Modulator Binding Site through Mutagenesis Studies.

Positive allosteric modulators (PAMs) of the cannabinoid CB1 receptor (CB1) offer potential therapeutic advantages in the treatment of neuropathic pain and addiction by avoiding the adverse effects associated with orthosteric CB1 activation. Here, molecular modeling and mutagenesis were used to identify residues central to PAM activity at CB1. Six putative allosteric binding sites were identified in silico, including novel sites previously associated with cholesterol binding, and key residues within each site were mutated to alanine. The recently determined ZCZ011 binding site was found to be essential for allosteric agonism, as GAT228, GAT229 and ZCZ011 all increased wild-type G protein dissociation in the absence of an orthosteric ligand; activity that was abolished in mutants F191A3.27 and I169A2.56. PAM activity was demonstrated for ZCZ011 in the presence of the orthosteric ligand CP55940, which was only abolished in I169A2.56. In contrast, the PAM activity of GAT229 was reduced for mutants R220A3.56, L404A8.50, F191A3.27 and I169A2.56. This indicates that allosteric modulation may represent the net effect of binding at multiple sites, and that allosteric agonism is likely to be mediated via the ZCZ011 site. This study underlines the need for detailed understanding of ligand receptor interactions in the search for pure CB1 allosteric modulators.

The Leptospermum scoparium (Manuka)-Specific Nectar and Honey Compound 3,6,7-Trimethyllumazine (LepteridineTM) That Inhibits Matrix Metalloproteinase 9 (MMP-9) Activity.

3,6,7-trimethyllumazine (Lepteridine™) is a newly discovered natural pteridine derivative unique to Manuka (Leptospermum scoparium) nectar and honey, with no previously reported biological activity. Pteridine derivative-based medicines, such as methotrexate, are used to treat auto-immune and inflammatory diseases, and Manuka honey reportedly possesses anti-inflammatory properties and is used topically as a wound dressing. MMP-9 is a potential candidate protein target as it is upregulated in recalcitrant wounds and intestinal inflammation. Using gelatin zymography, 40 µg/mL LepteridineTM inhibited the gelatinase activities of both pro- (22%, p < 0.0001) and activated (59%, p < 0.01) MMP-9 forms. By comparison, LepteridineTM exerted modest (~10%) inhibition against a chromogenic peptide substrate and no effect against a fluorogenic peptide substrate. These findings suggest that LepteridineTM may not interact within the catalytic domain of MMP-9 and exerts a negligible effect on the active site hydrolysis of small soluble peptide substrates. Instead, the findings implicate fibronectin II domain interactions by LepteridineTM which impair gelatinase activity, possibly through perturbed tethering of MMP-9 to the gelatin matrix. Molecular modelling analyses were equivocal over interactions at the S1' pocket versus the fibronectin II domain, while molecular dynamic calculations indicated rapid exchange kinetics. No significant degradation of synthetic or natural LepteridineTM in Manuka honey occurred during simulated gastrointestinal digestion. MMP-9 regulates skin and gastrointestinal inflammatory responses and extracellular matrix remodelling. These results potentially implicate LepteridineTM bioactivity in Manuka honey's reported beneficial effects on wound healing via topical application and anti-inflammatory actions in gastrointestinal disorder models via oral consumption.

Work-Family Enrichment: Influence of Job Autonomy on Job Satisfaction of Knowledge Employees.

In order to explore the specific path of the influence of job autonomy on the satisfaction of knowledge employees, the current study deduced and established a Chain Mediation Model, which was based on the Resource-Gain-Development Model and the Conservation of Resources Theory. Primary data were gathered through questionnaire surveys at several cities in China by using a professional platform named "Wenjuanxing." The target populations were employees with a bachelor's degree or above, who were engaged in higher knowledge content and have mastered certain professional knowledge and skills, including technical R & D personnel, management personnel, professionals (such as accountants, lawyers, and medics) and other personnel generally recognized by the academic community. In order to improve the reliability of the sample and reduce the error caused by regional differences, the questionnaires were disseminated to involve as many cities in China as possible, such as Tianjin, Beijing, Chengdu, Wuhan, and Guangzhou. SPSS24.0 and Aoms24.0 were used as multivariate data analysis tools for statistical analysis. The results showed that job autonomy can significantly improve the satisfaction of knowledge employees; however, it cannot affect the satisfaction of knowledge employees through self-efficacy. The findings of the study also revealed that job autonomy has a positive impact on both resource source domain satisfaction and resource acceptance domain satisfaction through work-family enrichment, especially the positive emotions in the resource source domain. Job autonomy improves the self-efficacy of knowledge employees, which, in turn, improves their overall satisfaction through the work-family enrichment path.

Habituation underpins preference for mates with novel phenotypes in the guppy.

Populations harbour enormous genetic diversity in ecologically important traits. Understanding the processes that maintain this variation is a long-standing challenge in evolutionary biology. Recent evidence indicates that a mating preference for novel sexual signals can be a powerful force maintaining genetic diversity. However, the proximate underpinnings of this preference, and its generality, remain unclear. Here, we test the hypothesis that preference for novel sexual signals is underpinned by habituation, a nearly ubiquitous form of learning whereby individuals become less responsive to repetitive stimuli. We use the Trinidadian guppy ( Poecilia reticulata), in which male colour patterns are diverse yet heritable. We show that repeated exposure to males with a given colour pattern reduces female interest in males with that pattern, and that interest recovers following brief isolation. These results fulfil two core criteria of habituation: responsiveness decline and spontaneous recovery. To distinguish habituation from sensory adaptation and fatigue, we also demonstrate stimulus specificity and dishabituation. These results provide the first evidence that habituation causes a preference for novel sexual signals, addressing the mechanistic underpinnings of this mating preference. Given the pervasiveness of habituation among taxa and sensory contexts, our findings suggest that preference for novelty may play an underappreciated role in mate choice and the maintenance of genetic variation.

Effects of dietary vitamin A restriction during finishing on color display life, lipid oxidation, and sensory traits of longissimus and triceps brachii steaks from early and traditionally weaned steers.

Our objective was to determine the effects of vitamin A restriction during finishing on color display life, lipid oxidation, and sensory traits of longissimus lumborum (LL) and triceps brachii (TB) steaks from early and traditionally weaned steers. Forty-eight steers weaned at either 137±26 days (EW) or 199±26 days (TW) were supplemented with either 15,400IU/kg dry matter of vitamin A (HA) or restricted to no supplemental vitamin A (LA) during finishing for 210 and 150 days, respectively. Both LL and TB steaks from the HA steers had the darkest (P<0.05) color scores after 3 days of retail display in PVC packaging at 2°C, and the highest (P<0.05) thiobarbaturic acid reactive substances (TBARS) values. Instrumental a∗, b∗, and saturation index values were lowest (P<0.05) in LL steaks from the HA steers. Instrumental L∗ values were lower (P<0.05) on days 4-6 in TB steaks from TW steers fed LA than those from EW steers fed HA. No differences were found in Warner-Bratzler shear force values or sensory traits in either muscle. No supplemental vitamin A versus high levels of vitamin A inclusion in finishing diets has potential to increase color display life and reduce lipid oxidation, with no effects on meat palatability.

Effects of vitamin A supplementation and weaning age on serum and liver retinol concentrations, carcass traits, and lipid composition in market beef cattle.

Angus crossbred steers (n=48) were either early-weaned (EW) at 137 days or weaned at a traditional age (TW) of 199 days to determine effects of weaning age and dietary vitamin A on serum and liver retinol, carcass traits, and lipid composition. Steers from both weaning ages were allotted to receive either 42,180IU vitamin A/day (HA) or no supplemental vitamin A (NA). Early-weaned and TW steers consumed vitamin A treatments for 235 and 175 days, respectively. Serum and liver retinol of HA steers were dramatically higher (P<0.01) than those of NA steers at the end of finishing. Steers were harvested in two groups 35 days apart at an average ultrasound 12th rib fat thickness of 1.0cm. Live and HCW were similar (P>0.10) between NA and HA steers, but HA steers had numerically greater (P⩾0.10) fat thickness (1.05 vs. 0.87cm). Marbling score and %IMF fat were numerically (P>0.10) higher for EWNA than EWHA steers. Ratio of marbling score/12th rib fat thickness was greater (P=0.08), and ratios of either marbling or %IMF per unit of 12th rib fat thickness, days on finishing diet, unit of HCW, and tenth of yield grade consistently favored steers fed NA, particularly EW steers. Proportions of serum fatty acids changed (P<0.05) during finishing; proportions of individual fatty acids of the longissimus muscle did not change. Restricting vitamin A during finishing has potential to increase carcass marbling and to decrease waste fat, particularly for EW.

Salmeterol and fluticasone propionate given as a combination. Lack of systemic pharmacodynamic and pharmacokinetic interactions.

OBJECTIVE: To investigate the potential for systemic pharmacodynamic and pharmacokinetic interactions between inhaled salmeterol and fluticasone propionate when repeat doses of the two drugs are given in combination to healthy subjects. METHODS: Twenty-eight healthy subjects received salmeterol 100 microg, salmeterol 100 microg/fluticasone propionate 500 microg and fluticasone propionate 500 microg via a Diskus dry powder inhaler twice daily for 11 days according to a randomised, double-blind, placebo-controlled, crossover design. Subjects in the placebo group also received a single dose of salmeterol 100 microg on the morning of day 10. On day 10, the systemic effects of salmeterol [on pulse rate, blood pressure, corrected QT (QTc) interval and serum potassium and glucose levels] and fluticasone propionate (on 24-h urinary cortisol and morning plasma cortisol levels) were assessed. Maximal number and affinity of lymphocyte beta2-adrenoceptors and beta2-adrenoceptor polymorphism at loci 16 and 27 were also determined. Plasma pharmacokinetics of salmeterol and fluticasone propionate were determined after the morning dose on day 10. Dosing continued on the evening of day 10 and on day 11, and on day 12 the effect of repeat-dose treatment with salmeterol and salmeterol/fluticasone propionate on the systemic effects of cumulative doses of inhaled salbutamol (up to a total dosage of 3,200 microg) was evaluated. RESULTS: All treatments were safe and well tolerated. With the exception of a higher pulse rate after repeat administration of salmeterol [66.2 beats per minute (bpm) versus 63.6 bpm], there were no significant differences between the single-dose and repeat-dose salmeterol groups. The systemic pharmacodynamic effects of inhaled salmeterol were not affected by the co-administration of fluticasone propionate. Eleven days of treatment with salmeterol induced tachyphylaxis to the systemic effects of cumulative doses of salbutamol; however, co-administration of fluticasone propionate did not affect the response to salbutamol. Fluticasone propionate reduced 24-h urinary cortisol excretion (22.4 microg compared with 48.6 microg with placebo), but this was unaffected by the co-administration of salmeterol. Morning plasma cortisol levels were not reduced compared with placebo. There was no significant treatment effect on lymphocyte beta2-adrenoceptors and no correlation of beta2-adrenoceptor polymorphism at loci 16 and 27 with the development of tachyphylaxis. Salmeterol plasma concentrations were measurable only during the first half-hour after dosing. Co-administration of fluticasone propionate did not affect the peak plasma concentration (Cmax) of salmeterol. For fluticasone propionate, there were no statistically significant differences between salmeterol/fluticasone propionate and fluticasone propionate with respect to Cmax, plasma concentration at the end of the dosing interval (Ct), terminal elimination half-life (t1/2) or time to Cmax (tmax). The area under the concentration-time curve within a dosing interval (AUCt) for fluticasone propionate after inhalation of salmeterol/fluticasone propionate was statistically significantly higher (about 8%) than after inhalation of fluticasone propionate alone (P=0.0135). However, the 90% confidence intervals (CIs) for the AUCt and Cmax ratios for the two treatments were within the accepted limits for bioequivalence (1.03, 1.13 and 0.97, 1.12, respectively). CONCLUSION: These results in healthy subjects indicate that there is no systemic pharmacodynamic or pharmacokinetic interaction between inhaled salmeterol and fluticasone propionate when given in combination.

The low potential for drug interactions with zanamivir.

OBJECTIVE: The objective of this study was to assess the potential of zanamivir, a specific inhibitor of influenza A and B virus neuraminidase, to interact with other coadministered therapies in the clinical setting. DESIGN: Potential interactions with zanamivir were examined in a series of in vitro and in vivo model systems. INTERVENTIONS: The expression of microsomal cytochrome P450 (CYP) isoenzymes was examined after daily treatment of rats with intravenous zanamivir. The ability of zanamivir to inhibit the metabolism of CYP probe substrates was studied in human liver microsomes. The binding of zanamivir to human and animal red blood cell fractions and plasma proteins was measured. Finally, the effect of commonly coadministered drugs on the ability of zanamivir to inhibit viral replication in vitro was tested. RESULTS: Zanamivir had no effect on the expression of microsomal CYP isoenzymes after daily intravenous treatment of rats with zanamivir 1, 9 or 90 mg/kg for 5 weeks. Zanamivir at concentrations up to 500 mumol/L (150 mg/L) had no effect on the metabolism of the CYP probe substrates bufuralol, chlorzoxazone, coumarin, ethoxyresorufin, mephenytoin, midazolam, phenacetin and tolbutamide by human liver microsomes. The binding of zanamivir 0.05 to 10 mg/L to human, dog and rat red blood cells and plasma proteins was low. The in vitro potency of zanamivir against influenza virus in Madin Darby canine kidney cells was not adversely affected by aspirin (acetylsalicylic acid) 1.2 mmol/L, paracetamol (acetaminophen) 6.6 mmol/L, ibuprofen 243 mumol/L, phenylephrine 6 mmol/L, oxymetazoline 380 mumol/L, promethazine 35 mumol/L and co-amoxiclav (amoxicillin-clavulanic acid) 1.66 mmol/L. CONCLUSIONS: These data suggest the following: (i) there is no theoretical basis for expecting metabolic interactions between zanamivir and other coadministered compounds; (ii) zanamivir is unlikely to interact with coadministered compounds that are protein bound; and (iii) commonly coadministered drugs will not interfere with the antiviral activity of zanamivir in vivo. Although none of these in vitro or in vivo studies were exhaustive, and although none were performed in humans, all the data are consistent with zanamivir having a very low potential for interactions with coadministered drugs in the clinical setting.

Single dose pharmacokinetics of lamivudine in subjects with impaired renal function and the effect of haemodialysis.

AIMS: The purpose of this study was to investigate the pharmacokinetics of a single oral dose of lamivudine administered to subjects with renal impairment and to determine whether lamivudine was dialysable in subjects with severe renal impairment undergoing haemodialysis. METHODS: Twenty-nine subjects were enrolled, nine with normal renal function (creatinine clearance (CL(CR)) 82-117 ml min(-1)), eight with moderately impaired renal function (CL(CR) 25-49 ml min(-1)), six with severe impairment (CL(CR) 13-19 ml min(-1)) and six with severe impairment who were also receiving haemodialysis. After an overnight fast, nondialysis subjects received a single oral dose of lamivudine. Subjects on haemodialysis were given two doses on separate occasions (intra and interdialysis). Blood was obtained before lamivudine administration and at regular intervals to 48 h post dose. Timed urine collections were performed for subjects able to produce urine. Pharmacokinetic parameters were calculated by using standard non compartmental techniques. RESULTS: Decreasing renal function was associated with reduced lamivudine clearance in a proportional and apparently linear relationship. Lamivudine was well dialysed with an extraction ratio in the order of 50%. However, because lamivudine has a large volume of distribution (approximately 100 1), a haemodialysis session of 4 h did not affect overall exposure to a clinically significant degree in most subjects. CONCLUSIONS: Impaired renal function does require lamivudine dose modification according to the degree of impairment, but no further modification of dose is required for subjects undergoing regular haemodialysis.

Pharmacokinetics of lamivudine and BCH-189 in plasma and cerebrospinal fluid of nonhuman primates.

2'-Deoxy-3'-thiacytidine is a dideoxycytidine analog with a sulfur in place of the 3' carbon of the ribose. There are two enantiomeric forms of the compound, both of which inhibit human immunodeficiency virus type 1 and 2 replication in vitro. However, the (-) enantiomer (lamivudine) appears to be significantly less cytotoxic to uninfected lymphocytes than is the (+) enantiomer. Lamivudine has entered initial clinical trials, and the present study was designed to describe the pharmacokinetic behavior of this compound in both plasma and cerebrospinal fluid (CSF) of primates. Lamivudine was administered as an intravenous bolus dose of 20 mg/kg for 24 h for administration. Urine samples were also obtained from two animals. The same dose of the racemate (BCH-189) was administered to one animal. The drug was quantitated in CSF and plasma with a reverse-phase high-pressure liquid chromatography technique. Elimination of lamivudine from plasma was biexponential, with a mean alpha phase half-life of 5.4 min, a mean beta phase half-life of 84 min, and a total clearance of 6.1 liters/h. The total clearance of the same dose of BCH-189 in a single animal was 11.0 liters/h. In two animals from which urine was obtained for 12 h postadministration, 32 and 59% of the drug was recovered unchanged. The deamination product of lamivudine was not detected. The CSF/plasma ratio of lamivudine was significantly higher when the drug was measured in the lumbar CSF (mean, 0.41) than when it was measured in the ventricular CSF (mean, 0.079). The measured CSF/plasma ratio for ventricular CSF is equivalent to that of other dideoxycytidine analogs, confirming the importance of the nucleobase in determining the degree of CSF penetration. The difference in lamivudine exposure in ventricular and lumbar CSF suggests that there is a transport mechanism for efflux of cytidine analogs from ventricular CSF.

Interspecies scaling and pharmacokinetic parameters of 3TC in humans.

3TC is a dideoxy-nucleoside analogue that has demonstrated in-vitro activity against human immunodeficiency virus (HIV). 3TC concentrations in humans were predicted before the initiation of clinical trials by interspecies scaling of pharmacokinetic parameters observed in animal species. Clearance and volume of distribution were estimated for humans using linear regression on a log-log scale of each parameter versus body weight for rats and dogs. The concentration-time profile and the average serum concentration at steady state after various dosage regimens were estimated as a basis for initial dose selection for clinical trials. The predicted parameters (clearance of 16.3 L/hr and volume of distribution of 40 L for a 70-kg man) were compared with that observed (mean clearance of 24 L/hr and mean volume of distribution of 96 L, mean weight of 74 kg) in 20 asymptomatic, HIV positive, volunteers after single intravenous doses of 3TC. Interspecies scaling was applied prospectively as a rationale for dose selection of 3TC in clinical trials.

Comparison of timed insemination with insemination at estrus following synchronization of estrus with a MGA-prostaglandin system in beef heifers.

Three trials utilizing 231 beef heifers were conducted in 1993 to determine if a timed insemination would result in similar synchronized pregnancy rates as insemination by estrus following synchronization of estrus using the 14-d MGA-prostaglandin system. All heifers were fed 0.5 mg MGA/h/d fof 14 d and given a 25 mg injection of PGF(2)alpha im 17 d after the final day of MGA feeding. Heifers in Group 1 (timed AI treatment) were inseminated at 72 h after the prostaglandin injection independent of whether or not they were observed in estrus. Heifers in Group 2 (AI by estrus) were inseminated 12 to 18 h after the onset of estrus. Since the trial was a significant source of variation for synchronized pregnancy rate, the effect of treatment on pregnancy rate was analyzed for each trial. Synchronized pregnancy rates in Trials 2 and 3 were similar in both treatment groups; 37 vs 35% and 61 vs 58% for the timed AI vs AI by estrus (Groups 1 and 2) in Trials 2 and 3, respectively. In both of these trials the degree of estrous synchrony was high. In Trial 1, the synchronized pregnancy rate in heifers that were time-inseminated was significantly lower than that of heifers that were inseminated by estrus (29 vs 57%). The lower synchronized pregnancy rate of Group 1 (timed AI) heifers in Trial 1 appeared to be due to the low degree of estrous synchrony in this trial. Our results indicate that using timed insemination with the 14-d MGA-prostaglandin system will give similar synchronized pregnancy rates as inseminating by estrus in groups of beef heifers where the degree of synchrony is high. However, in heifers where the degree of estrous synchrony is low, a timed insemination reduces synchronized pregnancy rates.

A molecular link between the bats of New Zealand and South America.

Along with the kiwis (Apteryx), tuatara (Sphenodon) and leiopelmatid frogs, the now rare lesser short-tailed bat (Mystacina tuberculata), one of only two species in the endemic family Mystacinidae, has long been viewed as one of New Zealand's archaic, mystery vertebrates, and has presented taxonomists with a major puzzle since its first description in 1843 (ref. 3). We report here the results of immunological comparisons involving the albumin and transferrin of Mystacina which indicate that its closest phylogenetic affinities are with the New World phyllostomoids--noctilionids, mormoopids and phyllostomoids. We estimate the separation between the Noctilio and Mystacina lineages to have occurred about 35 Myr ago.

Immunization of pigs against the boar taint steroid androstenone.

Tissue concentrations of androstenone were measured in untreated control pigs and pigs immunized against 5 alpha-androst-16-en-3-one. Results confirmed that active immunization of male pigs against androstenone is unlikely to prevent the problem of "boar taint" in the carcass meat.