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Background and purpose The δ-subunit-containing GABAA receptors, α4 β1 δ and α4 β3 δ, in dentate gyrus granule cells (DGGCs) are known to exhibit both spontaneous channel openings (i.e. constitutive activity) and agonist-induced current. The functional implications of spontaneous gating are unclear. In this study, we tested the hypothesis that constitutively active α4 β1/3 δ receptors limit agonist efficacy. Experimental approach Whole-cell electrophysiological recordings of adult male rat and mouse hippocampal DGGCs were used to characterize known agonists and antagonists at δ-subunit-containing GABAA receptors. To separate constitutive and agonist-induced currents, different recording conditions were employed. Key results Recordings at either 24°C or 34°C, including the PKC autoinhibitory peptide (19-36) intracellularly, removed spontaneous gating by GABAA receptors. In the absence of spontaneous gating, DGGCs responded to the α4 β1/3 δ orthosteric agonist Thio-THIP with a four-fold increased efficacy relative to recording conditions favouring constitutive activity. Surprisingly, the neutral antagonist gabazine was unable to antagonize the current by Thio-THIP. Furthermore, a current was elicited by gabazine alone only when the constitutive current was silenced (EC50 2.1 μM). The gabazine-induced current was inhibited by picrotoxin, potentiated by DS2, completely absent in δ-/- mice and reduced in β1 -/- mice, but could not be replicated in human α4 β1/3 δ receptors expressed heterologously in HEK cells. Conclusion and implications Kinase activity infers spontaneous gating in α4 β1/3 δ receptors in DGGCs. This significantly limits the efficacy of GABAA agonists and has implications in pathologies involving aberrant excitability caused by phosphorylation (e.g. addiction and epilepsy). In such cases, the efficacy of δ-preferring GABAA ligands may be reduced.

This chapter proposes a critical intervention in digital diaspora studies by foregrounding a relational approach that is inspired by feminist and postcolonial theory. This innovative framework allows us to grasp contemporary human mobility as shaped by and constitutive of an unevenly interconnected world. Relational implies taking into account different perspectives and methodologies on diaspora studies which defy ossified notions of ‘here’ and ‘there’, and of ethnic absolutism but sees diaspora as a continuum that needs to be critically scrutinized in its different manifestations. This holds also for the notion of digital diaspora. Recent buzzwords including ‘the connected migrant’, ‘digital diaspora’, ‘online diaspora’ and ‘e-diasporas’ commonly champion agency, particularly of non-white communities hailing from the Global South. This perspective risks glossing over the ways in which everyday offline and online contexts are steeped in intersecting gendered, racial, classed, generational and geo-political power relations. We provide a genealogy of digital diasporas scholarship in order to counter this lack of critical attention for power differences and material, social and emotional contexts. We will do so by combining media and non-media centric paradigm shifts in internet studies with the several turns and takes in critical digital diaspora studies.

Artificial metalloenzymes (AME's) are an interesting class of selective catalysts, where the chiral environment of proteins is used as chiral ligand for a catalytic metal. Commonly, the active site of an enzyme is modified with a catalytically active metal. Here we present an approach, where the commercial proteins lysozyme (LYS) and bovine serum albumin (BSA) can be converted into highly active and enantioselective AME's. This is achieved by acylation of the proteins primary amino groups, which affords the metal salts in the core of the protein. A series of differently acylated LYS and BSA were reacted with K2 OsO2 (OH)4 , RuCl3 , and Ti(OMe)4 , respectively, and the conjugates were tested for their catalytic activity in dihydroxylation and epoxidation of styrene and its derivatives. The best suited system for dihydroxylation is fully acetylated LYS conjugated with K2 OsO2 (OH)4 , which converts styrene to 1,2-phenylethanediol with an enantioselectivity of 95 % ee (S). BSA fully acylated with hexanoic acid and conjugated with three moles RuCl3 per mole protein shows the highest ee values for the conversion of styrene to the respective epoxide with enenatioselectivities of over 80 % ee (R), a TON of more than 2500 and a yield of up to 78 % within 24 h at 40 °C. LYS has two favored selective binding sites for the metal catalyst and BSA has even three. The AME's with titanate in the active center invert the enantioselectivity of styrene epoxidation.

Protein-based pharmaceuticals represent the fastest growing group of drugs in development in the pharmaceutical industry. One of the major challenges in the discovery, development, and distribution of biopharmaceuticals is the assessment of changes in their higher-order structure due to chemical modification. Here, we investigated the interactions of three different biochemical probes (Fab s) generated to detect conformational changes in a therapeutic IgG1 antibody (mAbX) by local hydrogen-deuterium exchange mass spectrometry (HDX-MS). We show that two of the probes target the Fc part of the antibody, whereas the third probe binds to the hinge region. Through HDX-ETD, we could distinguish specific binding patterns of the Fc -binding probes on mAbX at the amino-acid level. Preliminary surface plasmon resonance (SPR) experiments showed that these domain-selective Fab probes are sensitive to conformational changes in distinct regions of a full-length therapeutic antibody upon oxidation.

The transmembrane helix of the amyloid precursor protein is subject to proteolytic cleavages by γ-secretase at different sites resulting in Aβ peptides of different length and toxicity. A number of point mutations within this transmembrane helix alter the cleavage pattern thus enhancing production of toxic Aβ peptide species that are at the root of familial Alzheimer's disease. Here, we investigated how one of the most devastating mutations, the ‘Austrian’ mutation T43I, affects this transmembrane helix. Site-resolved deuterium/hydrogen amide exchange experiments reveal that the mutation destabilizes amide hydrogen bonds in the hinge which connects dimerization and cleavage regions. Weaker intrahelical hydrogen bonds at the hinge may enhance helix bending and thereby affect recognition of the transmembrane substrate by the enzyme and/or presentation of its cleavage sites to the catalytic cleft.

A dorsal-fin photo-identification technique paired with a non-invasive parallel laser photogrammetry technique was used to non-invasively identify individual Sphyrna mokarran over time. Based on the data collected over a duration of 59 days, 16 different S. mokarran (mean ± S.D. pre-caudal length: 220·82 ± 13·66 cm; mean ± S.D. cephalofoil width: 71·38 ± 7·94 cm) were identified using dorsal-fin photo-identification, with a mean ± S.D. shark re-sighting frequency of 4·05 ± 3·06 at-sea days. The results illustrate a high S. mokarran sighting rate and therefore, the utilization of parallel laser photogrammetry and dorsal-fin photo-identification may be a plausible multi-year approach to aid in non-invasively determining the growth rate and inter-annual site fidelity of these animals.

Introduction Quality of life (QOL) is an important dimension to consider in Alzheimer's disease (AD), but few large-scale studies have analyzed self and caregiver reports of patient QOL. Methods Patient QOL was evaluated in a cohort of 574 AD patients with the QOL-AD scale over 2 years. Results Caregiver reports of patient QOL were lower at baseline than self reports. Older patient age was associated with overestimation of QOL by caregivers, whereas neuropsychiatric inventory score and caregiver burden were associated with underestimation. Activities of daily living limitation, depressive symptoms, and caregiver burden were systematically associated with poorer QOL, whereas caregiver relationship and apathy were associated with poorer QOL only for self reports or caregiver reports, respectively. Cognitive function and professional care were not associated with QOL. Self-rated patient QOL did not change over time, whereas disease severity markers and caregiver-rated patient QOL declined. Discussion It is important to assess both self and caregiver ratings when assessing patient QOL.

This study employed a non-lethal measurement tool, which combined an existing photo-identification technique with a surface, parallel laser photogrammetry technique, to accurately estimate the size of free-ranging white sharks Carcharodon carcharias. Findings confirmed the hypothesis that surface laser photogrammetry is more accurate than crew-based estimations that utilized a shark cage of known size as a reference tool. Furthermore, field implementation also revealed that the photographer's angle of reference and the shark's body curvature could greatly influence technique accuracy, exposing two limitations. The findings showed minor inconsistencies with previous studies that examined pre-caudal to total length ratios of dead specimens. This study suggests that surface laser photogrammetry can successfully increase length estimation accuracy and illustrates the potential utility of this technique for growth and stock assessments on free-ranging marine organisms, which will lead to an improvement of the adaptive management of the species.

OBJECTIVE: Gray matter (GM) atrophy occurs in all multiple sclerosis (MS) phenotypes. We investigated whether there is a spatiotemporal pattern of GM atrophy that is associated with faster disability accumulation in MS. METHODS: We analyzed 3,604 brain high-resolution T1-weighted magnetic resonance imaging scans from 1,417 participants: 1,214 MS patients (253 clinically isolated syndrome [CIS], 708 relapsing-remitting [RRMS], 128 secondary-progressive [SPMS], and 125 primary-progressive [PPMS]), over an average follow-up of 2.41 years (standard deviation [SD] = 1.97), and 203 healthy controls (HCs; average follow-up = 1.83 year; SD = 1.77), attending seven European centers. Disability was assessed with the Expanded Disability Status Scale (EDSS). We obtained volumes of the deep GM (DGM), temporal, frontal, parietal, occipital and cerebellar GM, brainstem, and cerebral white matter. Hierarchical mixed models assessed annual percentage rate of regional tissue loss and identified regional volumes associated with time-to-EDSS progression. RESULTS: SPMS showed the lowest baseline volumes of cortical GM and DGM. Of all baseline regional volumes, only that of the DGM predicted time-to-EDSS progression (hazard ratio = 0.73; 95% confidence interval, 0.65, 0.82; p < 0.001): for every standard deviation decrease in baseline DGM volume, the risk of presenting a shorter time to EDSS worsening during follow-up increased by 27%. Of all longitudinal measures, DGM showed the fastest annual rate of atrophy, which was faster in SPMS (-1.45%), PPMS (-1.66%), and RRMS (-1.34%) than CIS (-0.88%) and HCs (-0.94%; p < 0.01). The rate of temporal GM atrophy in SPMS (-1.21%) was significantly faster than RRMS (-0.76%), CIS (-0.75%), and HCs (-0.51%). Similarly, the rate of parietal GM atrophy in SPMS (-1.24-%) was faster than CIS (-0.63%) and HCs (-0.23%; all p values

Conjugates of enzymes and poly(2-methyloxazoline) were used as organosoluble amphiphilic polymer nanocontainers for dissolving osmate, thereby converting the enzymes into organosoluble artificial metalloenzymes. These were shown to catalyze the dihydroxylation of different alkenes with high enantioselectivity. The highest selectivities, found for osmate complexed with laccase polymer-enzyme conjugates (PECs), even exceed those of classical Sharpless catalysts.

Aims The prevention of adverse drug events (ADEs) demands co-ordination of different health care professionals. ADE scorecards are a novel approach to raise the team awareness regarding ADE risks and causes. It makes information on numbers and on possible causes of possible ADE cases available to the clinical team. The aim of the study was to investigate the usage and acceptance of ADE scorecards by healthcare professionals and their impact on rates of possible ADEs. Methods ADE scorecards were introduced in three departments of a French hospital. A controlled time series analysis of ADE data was conducted to assess the impact of the ADE scorecards. In addition, qualitative interviews and a standardized survey with all participating staff members were performed. Results Physicians, nurses and pharmacists found ADE scorecards effective to increase medication safety and recommended future usage. The time-series analysis did not show changes in rates of possible ADEs. Conclusion ADE scorecards appear to be useful to raise awareness of ADE-related issues among professionals. Although the evaluation did not show significant reductions of ADE rates, the participating physicians, nurses and pharmacists believed that the ADE scorecards could contribute to increased patient safety and to a reduction in ADE rates. Strategies need to be designed to integrate ADE scorecards better into the clinical routine and to increase the precision of ADE detection.

Background and Purpose The histamine H4 receptor, originally thought to signal merely through Gαi proteins, has recently been shown to also recruit and signal via β-arrestin2. Following the discovery that the reference antagonist indolecarboxamide JNJ 7777120 appears to be a partial agonist in β-arrestin2 recruitment, we have identified additional biased hH4R ligands that preferentially couple to Gαi or β-arrestin2 proteins. In this study, we explored ligand and receptor regions that are important for biased hH4R signalling. Experimental Approach We evaluated a series of 48 indolecarboxamides with subtle structural differences for their ability to induce hH4R-mediated Gαi protein signalling or β-arrestin2 recruitment. Subsequently, a Fingerprints for Ligands and Proteins three-dimensional quantitative structure–activity relationship analysis correlated intrinsic activity values with structural ligand requirements. Moreover, a hH4R homology model was used to identify receptor regions important for biased hH4R signalling. Key Results One indolecarboxamide (75) with a nitro substituent on position R7 of the aromatic ring displayed an equal preference for the Gαi and β-arrestin2 pathway and was classified as unbiased hH4R ligand. The other 47 indolecarboxamides were β-arrestin2-biased agonists. Intrinsic activities of the unbiased as well as β-arrestin2-biased indolecarboxamides to induce β-arrestin2 recruitment could be correlated with different ligand features and hH4R regions. Conclusion and Implications Small structural modifications resulted in diverse intrinsic activities for unbiased (75) and β-arrestin2-biased indolecarboxamides. Analysis of ligand and receptor features revealed efficacy hotspots responsible for biased-β-arrestin2 recruitment. This knowledge is useful for the design of hH4R ligands with biased intrinsic activities and aids our understanding of the mechanism of H4R activation. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/ 10.1111/bph.2013.170.issue-1

Background and Purpose An alanine to valine exchange at amino acid position 280 (A280V) in the third intracellular loop of the human histamine H3 receptor was first identified in a patient suffering from Shy–Drager syndrome and later reported as a risk factor for migraine. Here, we have compared the pharmacological and signalling properties of wild-type (hH3RWT) and A280V mutant (hH3RA280V) receptors stably expressed in CHO-K1 cells. Experimental Approach The hH3RA280V cDNA was created by overlapping extension PCR amplification. Receptor expression and affinity were assessed by radioligand (N-α-[methyl-3H]-histamine) binding to cell membranes, and receptor function by the inhibition of forskolin-induced cAMP accumulation and stimulation of ERK1/2 phosphorylation in intact cells, as well as stimulation of [35S]-GTPγS binding to cell membranes. Key Results Both receptors were expressed at similar levels with no significant differences in their affinities for H3 receptor ligands. Upon activation the hH3RWT was significantly more efficacious to inhibit forskolin-induced cAMP accumulation and to stimulate [35S]-GTPγS binding, with no difference in pEC50 estimates. The hH3RWT was also more efficacious to stimulate ERK1/2 phosphorylation, but this difference was not significant. The inverse agonist ciproxifan was more efficacious at hH3RWT to reduce [35S]-GTPγS binding but, for both receptors, failed to enhance forskolin-induced cAMP accumulation. Conclusions and Implications The A280V mutation reduces the signalling efficacy of the human H3 receptor. This effect may be relevant to the pathophysiology of disorders associated with the mutation. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/ 10.1111/bph.2013.170.issue-1

Background and Purpose Chemogenomics focuses on the discovery of new connections between chemical and biological space leading to the discovery of new protein targets and biologically active molecules. G-protein coupled receptors (GPCRs) are a particularly interesting protein family for chemogenomics studies because there is an overwhelming amount of ligand binding affinity data available. The increasing number of aminergic GPCR crystal structures now for the first time allows the integration of chemogenomics studies with high-resolution structural analyses of GPCR-ligand complexes. Experimental Approach In this study, we have combined ligand affinity data, receptor mutagenesis studies, and amino acid sequence analyses to high-resolution structural analyses of (hist)aminergic GPCR-ligand interactions. This integrated structural chemogenomics analysis is used to more accurately describe the molecular and structural determinants of ligand affinity and selectivity in different key binding regions of the crystallized aminergic GPCRs, and histamine receptors in particular. Key Results Our investigations highlight interesting correlations and differences between ligand similarity and ligand binding site similarity of different aminergic receptors. Apparent discrepancies can be explained by combining detailed analysis of crystallized or predicted protein-ligand binding modes, receptor mutation studies, and ligand structure-selectivity relationships that identify local differences in essential pharmacophore features in the ligand binding sites of different receptors. Conclusions and Implications We have performed structural chemogenomics studies that identify links between (hist)aminergic receptor ligands and their binding sites and binding modes. This knowledge can be used to identify structure-selectivity relationships that increase our understanding of ligand binding to (hist)aminergic receptors and hence can be used in future GPCR ligand discovery and design. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/ 10.1111/bph.2013.170.issue-1

Background and Purpose The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H4 receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E1825.46 in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C983.36 in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C983.36 by functioning as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide a valuable tool for H4 receptor research. Experimental Approach We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH4 receptor radioligand binding, G protein activation and β-arrestin2 recruitment experiments. The ability of VUF14480 to act as a covalent binder was assessed both chemically and pharmacologically. Key Results VUF14480 was shown to be a partial agonist of hH4 receptor-mediated G protein signalling and β-arrestin2 recruitment. VUF14480 bound covalently to the hH4 receptor with submicromolar affinity. Serine substitution of C983.36 prevented this covalent interaction. Conclusion and Implications VUF14480 is thought to bind covalently to the hH4 receptor-C983.36 residue and partially induce hH4 receptor-mediated G protein activation and β-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH4 receptor. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/ 10.1111/bph.2013.170.issue-1

BACKGROUND AND PURPOSE The C-X-C chemokine receptors 3 (CXCR3) and C-X-C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross-inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers. EXPERIMENTAL APPROACH CXCR3–CXCR4 heteromers were identified in HEK293T cells using co-immunoprecipitation, time-resolved fluorescence resonance energy transfer, saturation BRET and the GPCR-heteromer identification technology (HIT) approach. Equilibrium competition binding and dissociation experiments were performed to detect negative binding cooperativity. KEY RESULTS We provide evidence that chemokine receptors CXCR3 and CXCR4 form heteromeric complexes in HEK293T cells. Chemokine binding was mutually exclusive on membranes co-expressing CXCR3 and CXCR4 as revealed by equilibrium competition binding and dissociation experiments. The small CXCR3 agonist VUF10661 impaired binding of CXCL12 to CXCR4, whereas small antagonists were unable to cross-inhibit chemokine binding to the other chemokine receptor. In contrast, negative binding cooperativity between CXCR3 and CXCR4 chemokines was not observed in intact cells. However, using the GPCR-HIT approach, we have evidence for specific b-arrestin2 recruitment to CXCR3-CXCR4 heteromers in response to agonist stimulation. CONCLUSIONS AND IMPLICATIONS This study indicates that heteromeric CXCR3–CXCR4 complexes may act as functional units in living cells, which potentially open up novel therapeutic opportunities

Background and Purpose Induction of cellular migration is the primary effect of chemokine receptor activation. However, several chemokine receptor-like proteins bind chemokines without subsequent induction of intracellular signalling and chemotaxis. It has been suggested that they act as chemokine scavengers, which may control local chemokine levels and contribute to the function of chemokines during inflammation. This has been verified for the chemokine-like receptor proteins D6 and DARC as well as CCX-CKR. Here, we provide evidence for an additional biological function of human (h)CCX-CKR. Experimental Approach We used transfection strategies in HEK293 and human T cells. Key Results Co-expression of hCCX-CKR completely inhibits hCXCR3-induced chemotaxis. We found that hCCX-CKR forms complexes with hCXCR3, suggesting a relationship between CCX-CKR heteromerization and inhibition of chemotaxis. Moreover, negative binding cooperativity induced by ligands both for hCXCR3 and hCCX-CKR was observed in cells expressing both receptors. This negative cooperativity may also explain the hCCX-CKR-induced inhibition of chemotaxis. Conclusions and Implications These findings suggest that hCCX-CKR prevents hCXCR3-induced chemotaxis by heteromerization thus representing a novel mechanism of regulation of immune cell migration. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

G protein-coupled chemokine receptors and their peptidergic ligands are interesting therapeutic targets due to their involvement in various immune-related diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, HIV-1 infection and cancer. To tackle these diseases, a lot of effort has been focused on discovery and development of small-molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, AMD3100 (CXCR4) and Maraviroc (CCR5) for stem cell mobilization and treatment of HIV-1 infection respectively. The recent GPCR crystal structures together with mutagenesis and pharmacological studies have aided in understanding how small-molecule ligands interact with chemokine receptors. Many of these ligands display behaviour deviating from simple competition and do not interact with the chemokine binding site, providing evidence for an allosteric mode of action. This review aims to give an overview of the evidence supporting modulation of this intriguing receptor family by a range of ligands, including small molecules, peptides and antibodies. Moreover, the computer-assisted modelling of chemokine receptor-ligand interactions is discussed in view of GPCR crystal structures. Finally, the implications of concepts such as functional selectivity and chemokine receptor dimerization are considered.

The effects of the histamine H(4) receptor antagonist JNJ7777120 were evaluated in a model of acute skin inflammation induced by local application of croton oil. The influence of strain on the effect of JNJ7777120 was investigated in four different mouse strains (CD-1, NMRI, BALB/c and C57BL/6J). In CD-1 mice, JNJ777720 (30-100 mg/kg subcutaneously, s.c.) exerted a dose-dependent inhibition of croton oil-induced ear inflammation and polymorphonuclear leucocyte infiltration, as confirmed by histological evaluation of ear tissues. JNJ7777120 (30-100 mg/kg) did not reduce ear oedema in NMRI, BALB/c or C57BL/6J mice. The positive control, dexamethasone (2 mg/kg s.c.) induced significant anti-inflammatory effects only in CD-1 and NMRI mice. In these strains, also the histamine H(1) -receptor blocker pyrilamine (30 mg/kg s.c.) significantly reduced ear oedema at 2 h after croton oil challenge, being as effective as JNJ7777120 in CD-1 mice. Taken together, these data demonstrate that the H(4) receptor antagonist JNJ7777120 may reduce acute croton oil-induced skin inflammation as effectively as H(1) receptor blockade. However, present experiments evidenced for the first time marked strain-related differences in the JNJ7777120 pharmacological activity, which have to be carefully considered when using this ligand to characterize histamine H(4) receptor functions in murine models and translating preclinical data to clinical human settings.

Most cells express a panel of different G protein-coupled receptors (GPCRs) allowing them to respond to at least a corresponding variety of extracellular ligands. In order to come to an integrative well-balanced functional response these ligand-receptor pairs can often cross-regulate each other. Although most GPCRs are fully capable to induce intracellular signalling upon agonist binding on their own, many GPCRs, if not all, appear to exist and function in homomeric and/or heteromeric assemblies for at least some time. Such heteromeric organization offers unique allosteric control of receptor pharmacology and function between the protomers and might even unmask 'new' features. However, it is important to realize that some functional consequences that are proposed to originate from heteromeric receptor interactions may also be observed due to intracellular crosstalk between signalling pathways of non-associated GPCRs. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.

Reactions of 11C recoil atoms with C6H6 in the presence of O2 lead to the formation of high yields of labelled C6H5CHO in the gaseous (6%) and liquid (8-10%) phase, whereas none was detected in the solid phase. For the liquid phase, 63% of the 11C activity in C6H5CHO is in the -CHO group, while 37% is part of the aromatic ring; in the case of gaseous C6H6 with 37% O2, the amount of 11C in the ring is only 11%. The reactions possibly leading to the formation of [11C]C6H5CHO and the 11C distribution over the molecule are discussed.

We report the chain extension of enamines by two carbon atoms through acid catalyzed nucleophilic substitution of the amine function by ene-1,1-diamines 2. By this vinylogous transamination we have prepared a series of diene-1,1-diamines 10a-h, 12a-e and diene-1,1,4-triamines 14a-e.

Herein we describe the scalable diastereoselective and enantioselective syntheses of eight enantiomers of hydroxy metabolites of saperconazole. The in vitro antifungal activity of the eight stereoisomers (compounds 1-8) was compared against a broad panel of Candida spp. (n=93), Aspergillus spp. (n=10), Cryptococcus spp. (n=19), and dermatophytes (n=27). The four 2S isomers 1-4 of the new agent were generally slightly more active than the four 2R isomers 5-8. All eight isomers were tested in a model of experimental A. fumigatus infection in guinea pigs by intravenous inoculation of the fungal conidia. Treatment doses were 1.25 mg kg(-1) and 2.5 mg kg(-1) per day. Infection severity was measured in terms of mean survival time (MST) after infection and mean tissue burdens in brain, liver, spleen, and kidney at postmortem examination. Among the eight isomers, the 2S diastereomers 1-4 showed a generally higher level of activity than the 2R diastereomers 5-8, revealing compounds 1 and 4 as the most potent overall in eradicating tissue burden and MST. Compared with reference compounds itraconazole and saperconazole, the hydroxy isomers 1-8 are less potent inhibitors of the growth of A. fumigatus in vitro and of ergosterol biosynthesis in both A. fumigatus and C. albicans.

Histamine is a well known mediator of allergic skin diseases and, with the discovery of the histamine H4 receptor, the role of histamine is re-evaluated. There are only limited published data elucidating the role of the histamine H4 receptor in dogs. Twelve beagles intradermally injected with histamine (0.25 μmol and 2.5 μmol/site) reacted with a classical wheal and flare reaction. None of the dogs showed signs of pruritus. The dogs reacted with a wheal and flare reaction after intradermal injection of histamine H4 receptor agonist/H3 receptor antagonist clobenpropit (0.1 μmol) and selective histamine H4 receptor agonist VUF 8430 (1.5 μmol). Again, no scratching occurred in any of the dogs. The highly selective histamine H4 receptor antagonist JNJ 7777120 reduced the histamine-induced wheal reaction in nine out of 12 dogs. To determine whether canine mast cells are susceptible to histamine H4 receptor-mediated reactions, effects of clobenpropit and VUF 8430 were tested in canine mastocytoma cells (C2). Incubation with histamine H4 receptor agonists (up to 10 μmol/L) induced a distinct calcium2+ influx. C2 cells also responded with enhanced chemotaxis when stimulated with histamine, VUF 8430 and clobenpropit. Neither VUF 8430, nor clobenpropit (up to 10 μmol/L) led to a modulation of histamine concentration in supernatants of canine mastocytoma cells, whereas mastoparan, used as a positive control, enhanced histamine concentration in supernatants. For treatment of allergic skin diseases in dogs, a combination of H1R and H4R antagonists might be advantageous. Resume L’histamine est un mediateur bien connu des dermatoses allergiques et, depuis la decouverte du recepteur H4, son role est reevalue. Les publications restent limitees sur le role du recepteur H4a l’histamine chez le chien. Douze beagles ont recu des injections intradermiques d’histamine (0.25 μmol et 2.5 μmol/zone) produisant une reaction classique en plaque ortiee erythemateuse. Aucun des chiens n’a montre de prurit. Les chiens ont reagi avec une plaque ortiee erythemateuse apres injection intradermique de clobenpropit (0.1 μmol), un agoniste du recepteur H4/antagoniste du recepteur H3 et de VUF 8430 (1.5 μmol) un agoniste selectif du recepteur H4. La encore, aucun signe de prurit n’a ete observe. L’antagoniste JNJ 7777120 hautement selectif du recepteur H4 diminue la plaque ortiee induite par l’histamine pour 9 des douze chiens. Afin de determiner si les mastocytes canins sont sensibles aux reactions mediees par les recepteurs H4, le clobenpropit et le VUF 8430 ont ete testes sur des cellules de mastocytomes canins (C2). L’incubation avec l’agoniste des recepteurs H4 (jusqu’a 10 μmol/L) induisait un influx de Ca2+. Les cellules C2 repondaient egalement par une augmentation du chimiotactisme a une stimulation par l’histamine, le VUF 8430 et le clobenpropit. Ni le VUF 8430, ni le clobenpropit (jusqu’a 10 μmol/L) ne modifierent des concentrations d’histamine dans les surnageants des mastocytes canins, tandis que le mastoparan, utilise comme controle positif, augmenta la concentration en histamine dans le surnageant. Une association d’antagonistes aux recepteurs H1R et H4R pourraient etre avantageuse dans le traitement des dermatoses allergique du chien. Resumen La histamina es un mediador bien conocido de las enfermedades alergicas de la piel y, con el descubrimiento del receptor de histamina H4, el papel de la histamina es reevaluado. Solo hay unos pocos trabajos publicados que estudian el papel de los receptores de histamina H4 en perros. Doce perros de raza Beagle que se inyectaron con histamina por via intradermica (0,25 μmol y 2,5 μmol/lugar) reaccionaron con el clasico habon y enrojecimiento. Ninguno de los perros mostro signos de prurito. Los perros reaccionaron con habon y enrojecimiento tras la inyeccion intradermica del agonista del receptor de histamina H4/antagonista del receptor H3 clobenpropit (0,1 μmol) y con al agonista selectivo del receptor VUF 8430 (1,5 μmol). De nuevo, no se observoo picor en ninguno de los perros. El antagonista altamente selectivo del receptor JNJ 7777120 redujo el habon producido por histamina en nueve de los 12 perros. Para determinar si los mastocitos caninos son susceptibles a las reacciones mediadas por los receptores de histamina H4, se probaron los efectos de clobenpropit y VUF 8430 en celulas de mastocitoma canino (C2). La incubacion con agonistas del receptor de histamina H4 (hasta 10 μmol/l) indujo un influjo claro de calcio2+. Las celulas C2 tambien respondieron con una quimiotaxis acentuada cuando se estimularon con histamina, VUF 8430 y clobenpropit. Ni VUF 8430, ni el clobenpropit (hasta 10 μmol/L) produjeron una modificacion de la concentracion de histamina en los sobrenadantes de las celulas de mastocitoma, mientras que el mastoparan, utilizado como control positivo, aumento la concentracion de histamina en los sobrenadantes. Para el tratamiento de las enfermedades alergicas en perros puede ser beneficioso utilizar una combinacion de antagonistas H1R y H4R. Zusammenfassung Histamin ist ein bekannter Mediator allergischer Hauterkrankungen und mit der Entdeckung des Histamin H4 Rezeptors wurde die Rolle von Histamin re-evaluiert. Es gibt nur wenig publiziertes Material, welches die Rolle des Histamin H4 Rezeptors bei Hunden beleuchtet. Zwolf Beagles, denen Histamin (0,25μmol und 2,5 μmol/Injektionsstelle) intradermal injiziert worden war, reagierten mit einer klassischen “Wheal und flare” Reaktion. Keiner der Hunde zeigte Anzeichen von Juckreiz. Die Hunde reagierten nach einer intradermalen Injektion des Histamin H4 Rezeptor Agonisten/H3 Rezeptor Antagonisten Clobenpropit (0,1μmol) und dem selektiven Histamin H4 Rezeptor Agonisten VUF 8430 (1,5 μmol) mit einer “Wheal und flare” Reaktion. Wiederum kratzte keiner der Hunde. Der hochselektive Histamin H4 Rezeptor Antagonist JNJ 7777120 reduzierte die durch Histamin induzierte “Wheal Reaktion” in neun von 12 Hunden. Um festzustellen, ob canine Mastzellen fur Histamin H4 Rezeptor-mediierte Reaktionen empfanglich sind, wurden die Auswirkungen von Clobenpropit und VUF 8430 auf Mastozytomzellen (C2) getestet. Die Inkubation mit Histamin H4 Rezeptor Agonisten (bis zu 10 μmol/l) bewirkte ein deutliches Einstromen von Ca2+. Die C2 Zellen reagierten mit zunehmender Chemotaxis, wenn sie durch Histamin, VUF 8430 und Clobenpropit stimuliert wurden. Weder VUF 8430 noch Clobenpropit (bis zu 10 μmol/l) fuhrten zu einer Modulation der Histaminkonzentrationen im Uberstand der caninen Mastozytomzellen, wahrend Mastoparan, welches als Positivkontrolle verwendet wurde, die Histaminkonzentration im Uberstand erhohte. Fur die Behandlung von allergischen Erkrankungen bei Hunden, konnte eine Kombination von H1R und H4R Antagonisten von Vorteil sein.

H(3)R inverse agonists based on an aminopropoxy-phenyloxazoline framework constitute highly valuable druglike lead compounds that display efficacy in a mouse model of recognition memory.

Background and purpose: Functional interactions between the G protein-coupled dopamine D1 and histamine H3 receptors have been described in the brain. In the present study we investigated the existence of D1–H3 receptor heteromers and their biochemical characteristics. Experimental approach: D1–H3 receptor heteromerization was studied in mammalian transfected cells with Bioluminescence Resonance Energy Transfer and binding assays. Furthermore, signalling through mitogen-activated protein kinase (MAPK) and adenylyl cyclase pathways was studied in co-transfected cells and compared with cells transfected with either D1 or H3 receptors. Key results: Bioluminescence Resonance Energy Transfer and binding assays confirmed that D1 and H3 receptors can heteromerize. Activation of histamine H3 receptors did not lead to signalling towards the MAPK pathway unless dopamine D1 receptors were co-expressed. Also, dopamine D1 receptors, usually coupled to Gs proteins and leading to increases in cAMP, did not couple to Gs but to Gi in co-transfected cells. Furthermore, signalling via each receptor was blocked not only by a selective antagonist but also by an antagonist of the partner receptor. Conclusions and implications: D1–H3 receptor heteromers constitute unique devices that can direct dopaminergic and histaminergic signalling towards the MAPK pathway in a Gs-independent and Gi-dependent manner. An antagonist of one of the receptor units in the D1–H3 receptor heteromer can induce conformational changes in the other receptor unit and block specific signals originating in the heteromer. This gives rise to unsuspected therapeutic potentials for G protein-coupled receptor antagonists.

Background and purpose: We compare the pharmacological profiles of a new histamine H4 receptor agonist 2-(2-guanidinoethyl)isothiourea (VUF 8430) with that of a previously described H4 receptor agonist, 4-methylhistamine. Experimental approach: Radioligand binding and functional assays were performed using histamine H4 receptors expressed in mammalian cell lines. Compounds were also evaluated ex vivo in monocyte-derived dendritic cells endogenously expressing H4 receptors and in vivo in anaesthetized rats for gastric acid secretion activity. Key results: Both VUF 8430 and 4-methylhistamine were full agonists at human H4 receptors with lower affinity at rat and mouse H4 receptors. Both compounds induced chemotaxis of monocyte-derived dendritic cells. VUF 8430 also showed reasonable affinity and was a full agonist at the H3 receptor. Agmatine is a metabolite of arginine, structurally related to VUF 8430, and was a H4 receptor agonist with micromolar affinity. At histamine H3 receptors, agmatine was a full agonist, whereas 4-methylhistamine was an agonist only at high concentrations. Both VUF 8430 and agmatine were inactive at H1 and H2 receptors, whereas 4-methylhistamine is as active as histamine at H2 receptors. In vivo, VUF 8430 only caused a weak secretion of gastric acid mediated by H2 receptors, whereas 4-methylhistamine, dimaprit, histamine and amthamine, at equimolar doses, induced 2.5- to 6-fold higher output than VUF 8430. Conclusions and implications: Our results suggest complementary use of 4-methylhistamine and VUF 8430 as H4 receptor agonists. Along with H4 receptor antagonists, both agonists can serve as useful pharmacological tools in studies of histamine H4 receptors.

The elucidation of the human genome has had a major impact on histamine receptor research. The identification of the human H4 receptor by several groups has been instrumental for a new appreciation of the role of histamine in the modulation of immune function. In this review, we summarize the historical developments and the molecular and biochemical pharmacology of the H4 receptor.

The CXCR3 chemokine receptor was first discovered in 1996 and has been shown to play an important role in several diseases, most of which are related to inflammation. This review describes in detail the development of small CXCR3 ligands and their therapeutic potential. Classes of CXCR3 antagonists with strikingly variable core structures have emerged. Some of these compounds have confirmed the beneficial role of CXCR3 antagonism in animal models of disease. One of the compounds, AMG487, progressed to Phase II clinical trials but has been withdrawn because of lack of efficacy. New antagonist classes are being developed to reveal the full therapeutic potential of CXCR3. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.