Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors

Measurements of affinity and efficacy are fundamental for work on agonists both in drug discovery and in basic studies on receptors. In this review I wish to consider methods for measuring affinity and efficacy at G protein coupled receptors (GPCRs). Agonist affinity may be estimated in terms of the dissociation constant for agonist binding to a receptor using ligand binding or functional assays. It has, however, been suggested that measurements of affinity are always contaminated by efficacy so that it is impossible to separate the two parameters. Here I show that for many GPCRs, if receptor/G protein coupling is suppressed, experimental measurements of agonist affinity using ligand binding (K-obs) provide quite accurate measures of the agonist microscopic dissociation constant (K-A). Also in pharmacological functional studies, good estimates of agonist dissociation constants are possible. Efficacy can be quantitated in several ways based on functional data ( maximal effect of the agonist (E-max), ratio of agonist dissociation constant to concentration of agonist giving half maximal effect in functional assay ( K-obs/ EC50), a combined parameter EmaxKobs/EC50). Here I show that EmaxKobs/EC50 provides the best assessment of efficacy for a range of agonists across the full range of efficacy for full to partial agonists. Considerable evidence now suggests that ligand efficacy may be dependent on the pathway used to assess it. The efficacy of a ligand may, therefore, be multidimensional. It is still, however, necessary to have accurate measures of efficacy in different pathways.

The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

Longitudinal investigation of the faecal microbiota of healthy full-term infants using fluorescence in situ hybridization and denaturing gradient gel electrophoresis.

From birth onwards, the gastrointestinal (GI) tract of infants progressively acquires a complex range of micro-organisms. It is thought that by 2 years of age the GI microbial population has stabilized. Within the developmental period of the infant GI microbiota, weaning is considered to be most critical, as the infant switches from a milk-based diet (breast and/or formula) to a variety of food components. Longitudinal analysis of the biological succession of the infant GI/faecal microbiota is lacking. In this study, faecal samples were obtained regularly from 14 infants from 1 month to 18 months of age. Seven of the infants (including a set of twins) were exclusively breast-fed and seven were exclusively formula-fed prior to weaning, with 175 and 154 faecal samples, respectively, obtained from each group. Diversity and dynamics of the infant faecal microbiota were analysed by using fluorescence in situ hybridization and denaturing gradient gel electrophoresis. Overall, the data demonstrated large inter- and intra-individual differences in the faecal microbiological profiles during the study period. However, the infant faecal microbiota merged with time towards a climax community within and between feeding groups. Data from the twins showed the highest degree of similarity both quantitatively and qualitatively. Inter-individual variation was evident within the infant faecal microbiota and its development, even within exclusively formula-fed infants receiving the same diet. These data can be of help to future clinical trials (e.g. targeted weaning products) to organize protocols and obtain a more accurate outline of the changes and dynamics of the infant GI microbiota.

DC-SIGN increases the affinity of HIV-1 envelope glycoprotein interaction with CD4

Mannose-binding C-type lectin receptors, expressed on Langerhans cells and subepithelial dendritic cells (DCs) of cervico-vaginal tissues, play an important role in HIV-1 capture and subsequent dissemination to lymph nodes. DC-SIGN has been implicated in both productive infection of DCs and the DC-mediated trans infection of CD4(+) T cells that occurs in the absence of replication. However, the molecular events that underlie this efficient transmission have not been fully defined. In this study, we have examined the effect of the extracellular domains of DC-SIGN and Langerin on the stability of the interaction of the HIV-1 envelope glycoprotein with CD4 and also on replication in permissive cells. Surface plasmon resonance analysis showed that DC-SIGN increases the binding affinity of trimeric gp140 envelope glycoproteins to CD4. In contrast, Langerin had no effect on the stability of the gp140:CD4 complex. In vitro infection experiments to compare DC-SIGN enhancement of CD4-dependent and CD4-independent strains demonstrated significantly lower enhancement of the CD4-independent strain. In addition DC-SIGN increased the relative rate of infection of the CD4-dependent strain but had no effect on the CD4-independent strain. DC-SIGN binding to the HIV envelope protein effectively increases exposure of the CD4 binding site, which in turn contributes to enhancement of infection.

Large is fast, small is tight: determinants of speed and affinity in subunit capture by a periplasmic chaperone

The chaperone/usher pathway assembles surface virulence organelles of Gram-negative bacteria, consisting of fibers of linearly polymerized protein subunits. Fiber subunits are connected through 'donor strand complementation': each subunit completes the immunoglobulin (Ig)-like fold of the neighboring subunit by donating the seventh β-strand in trans. Whereas the folding of Ig domains is a fast first-order process, folding of Ig modules into the fiber conformation is a slow second-order process. Periplasmic chaperones separate this process in two parts by forming transient complexes with subunits. Interactions between chaperones and subunits are also based on the principle of donor strand complementation. In this study, we have performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 capsular subunit from Yersinia pestis and analyzed the effect of the mutations on the structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. The results suggest that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enables Caf1M to rapidly bind an early folding intermediate of Caf1 and direct its partial folding. The switch from the Caf1M-Caf1 contact to the less hydrophobic, but considerably tighter and less dynamic Caf1-Caf1 contact occurs via the zip-out-zip-in donor strand exchange pathway with pocket 5 acting as the initiation site. Based on these findings, Caf1M was engineered to bind Caf1 faster, tighter, or both faster and tighter. To our knowledge, this is the first successful attempt to rationally design an assembly chaperone with improved chaperone function.

Affinity purification of 61- and 65-kDa rat brain corticotropin-releasing factor receptors and receptor-associated G proteins.

Corticotropin-releasing factor (CRF) has been shown to have a central role in physiological adaptation to stress. It is recognized for stimulating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonomic and immunological responses to stress. Much confusion has surrounded the characterization of CRF receptors, with proteins of varying molecular weights having been identified but never purified and characterized. Recently, two CRF receptors have been cloned from brain and pituitary gland, but evidence from in-situ hybridization studies suggests that further CRF receptor types exist. We therefore developed two techniques which enable the isolation of CRF receptors from whole rat brain. The use of a solid-phase CRF analogue affinity column and elution using a competing ligand resulted in the purification of a single protein of 61 kDa. A second technique was devised which allowed the co-isolation of associated signalling proteins and the identification of CRF bound species following purification. CRF was covalently cross-linked to receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximately 65 kDa and associated alpha and beta gamma G protein subunits. This study demonstrates the successful isolation of CRF receptors which are of different molecular weights to those previously observed from affinity cross-linking studies or predicted from cloned genes. In addition, we confirm the involvement of G proteins in CRF stimulated cell signalling by demonstrating their association with purified CRF receptor.

Superfast non-linear diffusion: capillary transport in particulate porous media

The migration of liquids in porous media, such as sand, has been commonly considered at high saturation levels with liquid pathways at pore dimensions. In this letter we reveal a low saturation regime observed in our experiments with droplets of extremely low volatility liquids deposited on sand. In this regime the liquid is mostly found within the grain surface roughness and in the capillary bridges formed at the contacts between the grains. The bridges act as variable-volume reservoirs and the flow is driven by the capillary pressure arising at the wetting front according to the roughness length scales. We propose that this migration (spreading) is the result of interplay between the bridge volume adjustment to this pressure distribution and viscous losses of a creeping flow within the roughness. The net macroscopic result is a special case of non-linear diffusion described by a superfast diffusion equation (SFDE) for saturation with distinctive mathematical character. We obtain solutions to a moving boundary problem defined by SFDE that robustly convey a time power law of spreading as seen in our experiments.

Combined use of two genetic fingerprinting methods, pulsed-field gel electrophoresis and ribotyping, for characterization of Escherichia coli O157 isolates from food animals, retail meats, and cases of human disease

Two genetic fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and ribotyping, were used to characterize 207 Escherichia coli O157 isolates from food animals, foods of animal origin, and cases of human disease (206 of the isolates were from the United Kingdom). In addition, 164 of these isolates were also phage typed. The isolates were divided into two general groups: (i) unrelated isolates not known to be epidemiologically linked (n = 154) and originating from food animals, foods and the environment, or humans and (ii) epidemiologically related isolates (n = 53) comprised of four related groups (RGs) originating either from one farm plus the abattoir where cattle from that farm were slaughtered or from one of three different English abattoirs. PFGE was conducted with the restriction endonuclease XbaI. while for ribotyping, two restriction endonucleases (PstI and SphI) were combined to digest genomic DNAs simultaneously. The 207 E. coli O157 isolates produced 97 PFGE profiles and 51 ribotypes. The two genetic fingerprinting methods had similar powers to discriminate the 154 epidemiologically unrelated E. coli O157 isolates in the study (Simpson's index of diversity [D] = 0.98 and 0.94 for PFGE typing and ribotyping, respectively). There was no correlation between the source of an isolate (healthy meat or milk animals, retail meats, or cases of human infection) and either particular PFGE or ribotype profiles or clusters. Combination of the results of both genetic fingerprinting methods produced 146 types, significantly more than when either of the two methods was used individually. Consequently, the superior discriminatory performance of the PFGE-ribotyping combination was proven in two ways: (i) by demonstrating that the majority of the E. coli O157 isolates with unrelated histories were indeed distinguishable types and (ii) by identifying some clonal groups among two of the four RGs of E. coli O157 isolates (comprising PFGE types different by just one or two bands), the relatedness of which would have remained unconfirmed otherwise.

Use of denaturing gradient gel electrophoresis to detect Actinobacteria associated with the human faecal microbiota

With the exceptions of the bifidobacteria, propionibacteria and coriobacteria, the Actinobacteria associated with the human gastrointestinal tract have received little attention. This has been due to the seeming absence of these bacteria from most clone libraries. In addition, many of these bacteria have fastidious growth and atmospheric requirements. A recent cultivation-based study has shown that the Actinobacteria of the human gut may be more diverse than previously thought. The aim of this study was to develop a denaturing gradient gel electrophoresis (DGGE) approach for characterizing Actinobacteria present in faecal samples. Amount of DNA added to the Actinobacteria-specific PCR used to generate strong PCR products of equal intenstity from faecal samples of five infants, nine adults and eight elderly adults was anti-correlated with counts of bacteria obtained using fluorescence in situ hybridization probe HGC69A. A nested PCR using Actinobacteria-specific and universal PCR-DGGE primers was used to generate profiles for the Actinobacteria. Cloning of sequences from the DGGE bands confirmed the specificity of the Actinobacteria-specific primers. In addition to members of the genus Bifidobacterium, species belonging to the genera Propionibacterium, Microbacterium, Brevibacterium, Actinomyces and Corynebacterium were found to be part of the faecal microbiota of healthy humans.

Neurogenic responses mediated by vanilloid receptor-1 (TRPV1) are blocked by the high affinity antagonist, iodo-resiniferatoxin

(1) Stimulation of the vanilloid receptor-1 (TRPV1) results in the activation of nociceptive and neurogenic inflammatory responses. Poor specificity and potency of TRPV1 antagonists has, however, limited the clarification of the physiological role of TRPV1. (2) Recently, iodo-resiniferatoxin (I-RTX) has been reported to bind as a high affinity antagonist at the native and heterologously expressed rat TRPV1. Here we have studied the ability of I-RTX to block a series of TRPV1 mediated nociceptive and neurogenic inflammatory responses in different species (including transfected human TRPV1). (3) We have demonstrated that I-RTX inhibited capsaicin-induced mobilization of intracellular Ca(2+) in rat trigeminal neurons (IC(50) 0.87 nM) and in HEK293 cells transfected with the human TRPV1 (IC(50) 0.071 nM). (4) Furthermore, I-RTX significantly inhibited both capsaicin-induced CGRP release from slices of rat dorsal spinal cord (IC(50) 0.27 nM) and contraction of isolated guinea-pig and rat urinary bladder (pK(B) of 10.68 and 9.63, respectively), whilst I-RTX failed to alter the response to high KCl or SP. (5) Finally, in vivo I-RTX significantly inhibited acetic acid-induced writhing in mice (ED(50) 0.42 micro mol kg(-1)) and plasma extravasation in mouse urinary bladder (ED(50) 0.41 micro mol kg(-1)). (6) In in vitro and in vivo TRPV1 activated responses I-RTX was approximately 3 log units and approximately 20 times more potent than capsazepine, respectively. This high affinity antagonist, I-RTX, may be an important tool for future studies in pain and neurogenic inflammatory models.

Capillary assay device with internal hydrophilic coating

The present invention provides assay devices having a unitary body with an exterior surface, the unitary body being substantially transparent to visible light and formed from a material having a refractive index in the range 1.26 to 1.40, the refractive index being measured at 20 °C with light of wavelength 589 nm, and wherein the unitary body is formed from a hydrophobic material, and at least two capillary bores extending internally along the unitary body, wherein at least a portion of the surface of each capillary bore includes a hydrophilic layer for retaining an assay reagent, and wherein the hydrophilic layer is also substantially transparent to visible light to allow optical interrogation of the capillary bores through the capillary wall. The present invention also provides assay systems including such assay devices, methods of performing an assay using such assay devices and method of method for manufacturing such assay devices.

A new topology of ACBP from Moniliophthora perniciosa

Acyl-CoA binding protein (ACBP) is a housekeeping protein and is an essential protein in human cell lines and in Trypanosoma brucei. The ACBP of Moniliophthora perniciosa is composed of 104 amino acids and is possibly a non-classic isoform exclusively from Basidiomycetes. The M. perniciosa acbp gene was cloned, and the protein was expressed and purified. Acyl-CoA ester binding was analyzed by isoelectric focusing, native gel electrophoresis and isothermal titration calorimetry. Our results suggest an increasing affinity of ACBP for longer acyl-CoA esters, such as myristoyl-CoA to arachidoyl-CoA, and best fit modeling indicates two binding sites. ACBP undergoes a shift from a monomeric to a dimeric state, as shown by dynamic light scattering, fluorescence anisotropy and native gel electrophoresis in the absence and presence of the ligand. The protein`s structure was determined at 1.6 angstrom resolution and revealed a new topology for ACBP, containing five a-helices instead of four. alpha-helices 1, 2, 3 and 4 adopted a bundled arrangement that is unique from the previously determined four-helix folds of ACBP, while alpha-helices 1, 2, 4 and 5 formed a classical four-helix bundle. A MES molecule was found in the CoA binding site, suggesting that the CoA site could be a target for small compound screening. (C) 2009 Elsevier B.V. All rights reserved.

Proinsulin C-peptide induces c-Jun N-terminal kinase 1 expression in LEII mouse lung capillary endothelial cells

To characterize the roles of C-peptide in vascular homeostatic processes, we examined the genes regulated by C-peptide in LEII mouse lung microvascular endothelial cells. Treatment of the cells with C-peptide increased the expression of c-Jun N-terminal kinase 1 (JNK1) mRNA dose-dependently, accompanied by an increase in JNK1 protein content. Prior treatment of the cells with PD98059, an ERK kinase inhibitor or SB203580, a p38MAPK inhibitor, abrogated the C-peptide-elicited JNK1 mRNA expression. These results indicate that C-peptide increases JNK1 protein levels, possibly through ERK- and p38MAPK-dependent activation of JNK. gene transcription.

Structure-Based Approach for the Study of Estrogen Receptor Binding Affinity and Subtype Selectivity

Estrogens exert important physiological effects through the modulation of two human estrogen receptor (hER) subtypes, alpa (hER alpha) and beta (hER beta). Because the levels and relative proportion of hER alpha and hER beta differ significantly in different target cells, selective hER ligands could target specific tissues or pathways regulated by one receptor subtype without affecting the other. To understand the structural and chemical basis by which small molecule modulators are able to discriminate between the two subtypes, we have applied three-dimensional target-based approaches employing a series of potent hER-ligands. Comparative molecular field analysis (CoMFA) studies were applied to a data set of 81 hER modulators, for which binding affinity values were collected for both hER alpha and hER beta. Significant statistical coefficients were obtained (hER alpha, q(2) = 0.76; hER beta, q(2) = 0.70), indicating the internal consistency of the models. The generated models were validated using external test sets, and the predicted values were in good agreement with the experimental results. Five hER crystal structures were used in GRID/PCA investigations to generate molecular interaction fields (MIF) maps. hER alpha and hER beta were separated using one factor. The resulting 3D information was integrated with the aim of revealing the most relevant structural features involved in hER subtype selectivity. The final QSAR and GRID/PCA models and the information gathered from 3D contour maps should be useful for the design or novel hER modulators with improved selectivity.