Studies with neutralizing antibodies suggest CXCL8-mediated neutrophil activation is independent of C-C motif chemokine receptor-like 2 (CCRL2) ligand binding function.

C-C motif chemokine receptor-like 2 (CCRL2) is a non-signaling 7 transmembrane receptor that binds chemotactic ligands to shape leukocyte recruitment to sites of inflammation. However, there is a lack of consensus on the ligands that directly bind CCRL2 or their functional impact. Studies with CCRL2 knockout mice have demonstrated that neutrophils have impaired degranulation and migration in response to CXCL8, where the underlying molecular mechanism is proposed to be due to the formation of CCRL2 heterodimers with the chemokine receptor CXCR2. Herein, we characterized the ligands that bind directly to CCRL2 and interrogated the impact of CCRL2 neutralization on CXCL8 signaling in neutrophils using pharmacological antibody tools. Using flow cytometry and Surface Plasmon Resonance microscopy (SPRm) cell binding experiments, we confirmed that chemerin, but not previously reported C-C chemokines, binds CCRL2. Furthermore, we identified human and mouse CCRL2 antibodies that neutralized chemerin binding to CCRL2. Unexpectedly, we found that neutralization of CCRL2 with these antibodies did not attenuate CXCL8-induced human neutrophil degranulation nor CXCL8-induced murine neutrophil recruitment to the peritoneum. Based on the observed differences in modulating CCRL2 function with neutralizing antibodies compared to the reported CCRL2 deficient murine models, we hypothesize that the ligand binding function of CCRL2 is dispensable for CXCL8 signaling in neutrophils. Finally, extensive profiling of CCRL2 expression on peripheral blood leukocytes revealed monocytes, dendritic cells (DC), and subpopulations of natural killer T (NKT) cells as additional targets, highlighting potential roles for CCRL2 in human cell types beyond neutrophils that warrants future investigation.

Demonstration of In Vitro to In Vivo Translation of a TYK2 Inhibitor That Shows Cross Species Potency Differences.

Translation of modulation of drug target activity to therapeutic effect is a critical aspect for all drug discovery programs. In this work we describe the profiling of a non-receptor tyrosine-protein kinase (TYK2) inhibitor which shows a functionally relevant potency shift between human and preclinical species (e.g. murine, dog, macaque) in both biochemical and cellular assays. Comparison of the structure and sequence homology of TYK2 between human and preclinical species within the ATP binding site highlights a single amino acid (I960 → V) responsible for the potency shift. Through TYK2 kinase domain mutants and a TYK2 980I knock-in mouse model, we demonstrate that this single amino acid change drives a functionally relevant potency difference that exists between human and all evaluated preclinical species, for a series of TYK2 inhibitors which target the ATP binding site.

Design and optimization of a series of 4-(3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-2-amines: Dual inhibitors of TYK2 and JAK1.

Herein, we disclose a new series of TYK2/ JAK1 inhibitors based upon a 3.1.0 azabicyclic substituted pyrimidine scaffold. We illustrate the use of structure-based drug design for the initial design and subsequent optimization of this series of compounds. One advanced example 19 met program objectives for potency, selectivity and ADME, and demonstrated oral activity in the adjuvant-induced arthritis rat model.

[INCREMENT]9-Tetrahydrocannabinol discriminative stimulus effects of AM2201 and related aminoalkylindole analogs in rats.

The recent recreational use of synthetic cannabinoid ligands, collectively referred to as 'Spice', has raised concerns about their safety and possible differences in their biological effect(s) from marijuana/Δ-tetrahydrocannabinol (THC). AM2201, a highly efficacious, potent cannabinoid receptor 1 (CB1R) agonist, is a recently detected compound in 'Spice' preparations. Furthermore, structural analogs of AM2201 are now being found in 'Spice'. The present studies were conducted to investigate their Δ-THC-like effects using drug (Δ-THC) discrimination in rats. Results show that the tested compounds were potent cannabinergics that generalized to the response to Δ-THC, with AM2201 being most potent, exhibiting a 14-fold potency difference over Δ-THC. The other analogs were between 2.5-fold and 4-fold more potent than THC. Surmountable antagonism of AM2201 with the selective CB1R antagonist/inverse agonist rimonabant also established that the discrimination is CB1R dependent. Time-course data reveal that AM2201 likely peaks rapidly with an in-vivo functional half-life of only 60 min. The present data confirm and extend previous observations regarding Δ-THC-like effects of 'Spice' components.

Effects of elevated atmospheric CO2 concentrations, clipping regimen and differential day/night atmospheric warming on tissue nitrogen concentrations of a perennial pasture grass.

Forecasting the effects of climate change on nitrogen (N) cycling in pastures requires an understanding of changes in tissue N. We examined the effects of elevated atmospheric CO2 concentration, atmospheric warming and simulated grazing (clipping frequency) on aboveground and belowground tissue N concentrations and C : N ratios of a C3 pasture grass. Phalaris aquatica L. cv. 'Holdfast' was grown in the field in six transparent temperature gradient tunnels (18 × 1.5 × 1.5 m each), three at ambient atmospheric CO2 and three at 759 p.p.m. CO2. Within each tunnel, there were three air temperature treatments: ambient control, +2.2/+4.0 °C above ambient day/night warming and +3.0 °C continuous warming. A frequent and an infrequent clipping treatment were applied to each warming × CO2 combination. Green leaf N concentrations were decreased by elevated CO2 and increased by more frequent clipping. Both warming treatments increased leaf N concentrations under ambient CO2 concentrations, but did not significantly alter leaf N concentrations under elevated CO2 concentrations. Nitrogen resorption from leaves was decreased under elevated CO2 conditions as well as by more frequent clipping. Fine root N concentrations decreased strongly with increasing soil depth and were further decreased at the 10-60 cm soil depths by elevated CO2 concentrations. The interaction between the CO2 and warming treatments showed that leaf N concentration was affected in a non-additive manner. Changes in leaf C : N ratios were driven by changes in N concentration. Overall, the effects of CO2, warming and clipping treatments on aboveground tissue N concentrations were much greater than on belowground tissue.

Probing the carboxyester side chain in controlled deactivation (-)-δ(8)-tetrahydrocannabinols.

We recently reported on a controlled deactivation/detoxification approach for obtaining cannabinoids with improved druggability. Our design incorporates a metabolically labile ester group at strategic positions within the THC structure. We have now synthesized a series of (-)-Δ(8)-THC analogues encompassing a carboxyester group within the 3-alkyl chain in an effort to explore this novel cannabinergic chemotype for CB receptor binding affinity, in vitro and in vivo potency and efficacy, as well as controlled deactivation by plasma esterases. We have also probed the chain's polar characteristics with regard to fast onset and short duration of action. Our lead molecule, namely 2-[(6aR,10aR)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-3-yl]-2-methyl-propanoic acid 3-cyano-propyl ester (AM7438), showed picomolar affinity for CB receptors and is deactivated by plasma esterases while the respective acid metabolite is inactive. In further in vitro and in vivo experiments, the compound was found to be a remarkably potent and efficacious CB1 receptor agonist with relatively fast onset/offset of action.

"Herbal incense": designer drug blends as cannabimimetics and their assessment by drug discrimination and other in vivo bioassays.

Recently, synthetic cannabinoids originally designed for testing in the laboratory only have found use recreationally in designer herbal blends, originally called "Spice". The myriad of compounds found are for the most part potent full agonists of the cannabinoid receptor 1, producing effects similar to tetrahydrocannabinol (THC) and marijuana. Drug discrimination of these compounds offers a specific behavioral test that can help determine whether these new synthetic compounds share a similar "subjective high" with the effects of marijuana/THC. By utilization of drug discrimination and other behavioral techniques, a better understanding of these new "designer" cannabinoids may be reached to assist in treating both the acute and chronic effects of these drugs. The paper provides a brief exposé of modern cannabinoid research as a backdrop to the recreational use of designer herbal blend cannabimimetics.

Controlled-deactivation cannabinergic ligands.

We report an approach for obtaining novel cannabinoid analogues with controllable deactivation and improved druggability. Our design involves the incorporation of a metabolically labile ester group at the 2'-position on a series of (-)-Δ(8)-THC analogues. We have sought to introduce benzylic substituents α to the ester group which affect the half-lives of deactivation through enzymatic activity while enhancing the affinities and efficacies of individual ligands for the CB1 and CB2 receptors. The 1'-(S)-methyl, 1'-gem-dimethyl, and 1'-cyclobutyl analogues exhibit remarkably high affinities for both CB receptors. The novel ligands are susceptible to enzymatic hydrolysis by plasma esterases in a controllable manner, while their metabolites are inactive at the CB receptors. In further in vitro and in vivo experiments key analogues were shown to be potent CB1 receptor agonists and to exhibit CB1-mediated hypothermic and analgesic effects.

Antagonism of ∆9-THC induced behavioral effects by rimonabant: time course studies in rats.

The objective was to examine the time course of the cannabinoid 1 receptor antagonist/inverse agonist rimonabant's ability to antagonize in vivo cannabinergic agonist effects. We used two behavioral procedures sensitive to the effects of ∆9-tetrahydrocannabinol (∆9-THC): rat drug discrimination (EXP-1) and suppression of fixed-ratio responding (FR) for food reinforcement (EXP-2). Two training doses of ∆9-THC (1.8 and 3 mg/kg) served as discriminative cues in two groups discriminating ∆9-THC from vehicle; injections were i.p. 20 min before session onset. Tests assessed the dose-response functions of ∆9-THC and the time course for rimonabant in its ability to block the discriminative stimulus effects of ∆9-THC. For antagonism testing, the training doses of ∆9-THC were used and the rimonabant dose was 1mg/kg. Tests were 20, 60, 120, and 240 min post rimonabant administration; ∆9-THC was always administered 20 min prior to testing. For EXP-2, only one response lever was activated and every 10th (FR-10) press on that lever resulted in food delivery. Once the response rate stabilized, tests occurred with ∆9-THC, rimonabant and combinations of the drugs. The ED(50) estimates for the dose-response functions were 0.38 (±0.28-0.51) and 0.50 (±0.40-0.63) mg/kg for the training doses of 1.8 and 3 mg/kg ∆9-THC, respectively. The time course studies suggested functional half-life estimates of 128.4 (±95.7-172.2) and 98.4 (±64.2-150.7) min by rimonabant for the two groups in EXP-1, respectively. Similarly, the functional half-life of rimonabant was 118.9 (±66.1-213.9) min in EXP-2. Thus, antagonism of ∆9-THC by rimonabant is relatively short lasting.

A comment on the quantitative significance of aerobic methane release by plants.

A recent study by Keppler et al. (2006; Nature 439, 187-191) demonstrated CH4 emission from living and dead plant tissues under aerobic conditions. This work included some calculations to extrapolate the findings from the laboratory to the global scale and led various commentators to question the value of planting trees as a greenhouse mitigation option. The experimental work of Keppler et al. (2006) appears to be largely sound, although some concerns remain about the quantification of emission rates. However, whilst accepting their basic findings, we are critical of the method used for extrapolating results to a global scale. Using the same basic information, we present alternative calculations to estimate global aerobic plant CH4 emissions as 10-60 Mt CH4 year-1. This estimate is much smaller than the 62-236 Mt CH4 year-1 reported in the original study and can be more readily reconciled within the uncertainties in the established sources and sinks in the global CH4 budget. We also assessed their findings in terms of their possible relevance for planting trees as a greenhouse mitigation option. We conclude that consideration of aerobic CH4 emissions from plants would reduce the benefit of planting trees by between 0 and 4.4%. Hence, any offset from CH4 emission is small in comparison to the significant benefit from carbon sequestration. However, much critical information is still lacking about aerobic CH4 emission from plants. For example, we do not yet know the underlying mechanism for aerobic CH4 emission, how CH4 emissions change with light, temperature and the physiological state of leaves, whether emissions change over time under constant conditions, whether they are related to photosynthesis and how they relate to the chemical composition of biomass. Therefore, the present calculations must be seen as a preliminary attempt to assess the global significance from a basis of limited information and are likely to be revised as further information becomes available.

A critical overview of model estimates of net primary productivity for the Australian continent.

Net primary production links the biosphere and the climate system through the global cycling of carbon, water and nutrients. Accurate quantification of net primary productivity (NPP) is therefore critical in understanding the response of the world's ecosystems to global climate change, and how changes in ecosystems might themselves feed back to the climate system.

Plant respiration in productivity models: conceptualisation, representation and issues for global terrestrial carbon-cycle research.

Plant respiratory regulation is too complex for a mechanistic representation in current terrestrial productivity models for carbon accounting and global change research. Accordingly, simpler approaches that attempt to capture the essence of respiration are commonly adopted. Several approaches have been used in the literature: respiration may be embedded implicitly in growth algorithms; assumed values for specific respiration rates may be adopted; respiration may be calculated in terms of growth and maintenance components; conservatism in the ratio of respiration to photosynthesis (R : P) may be assumed; or a more complex process or residual approach may be adopted. Review of this literature suggests that the assumption of conservative R : P ratio is an effective and practicable approach in the context of C-cycle modelling for global change research and documentation, requiring minimal ecosystem-specific data on respiration.Some long-standing controversies in respiration are now becoming resolved. The apparently wasteful process of cyanide-resistant respiration by the alternative oxidase may not be wasteful, as it is thought to be involved in protecting the plant from 'reactive oxygen species'. It is now clear that short-term respiratory response coefficients of plants (e.g. the Q10) do not predict their long-term temperature response. A new experimental approach suggests that leaf respiration is not suppressed by light as previously thought. Careful experiments, taking account of several proposed measurement artefacts, indicate that plant respiration is not suppressed by elevated CO2 concentration in a short-term reversible way.