GPCRs

With our increasing appreciation of the true complexity of diseases and pathophysiologies, it is clear that this knowledge needs to inform the future development of pharmacotherapeutics. For many disorders, the disease mechanism itself is a complex process spanning multiple signaling networks, tissues, and organ systems. Identifying the precise nature and locations of the pathophysiology is crucial for the creation of systemically effective drugs. Diseases once considered constrained to a limited range of organ systems, e.g., central neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington' disease (HD), the role of multiple central and peripheral organ systems in the etiology of such diseases is now widely accepted. With this knowledge, it is increasingly clear that these seemingly distinct neurodegenerative disorders (AD, PD, and HD) possess multiple pathophysiological similarities thereby demonstrating an inter-related con...

Video of my talk at MIT, Center for Bits and Atoms (Andreas Mershin's lab), 9 December 2015: https://www.youtube.com/watch?v=ej45lyl3rzA

Research on olfaction is on the rise. The discovery of the olfactory receptor genes by Linda Buck and Richard Axel in 1991 catapulted olfaction from eccentricity into core neurobiological research. With the identification of the receptors as G-protein coupled receptors olfactory research became affiliated with a wide range of cell signaling studies. While a lot of fundamental questions about olfactory processing are still open to dispute, the consensus is that the olfactory pathway will facilitate a greater neurobiological understanding of signal processing. One of the open questions that attracted greater attention over the past ten years is the longstanding problem of how the brain ‘maps’ smells. My postdoc project centers on olfaction to analyze theoretical and experimental breakthroughs through scientific methodology. Its current dynamics and susceptibility to the revision of its core premises makes olfactory research an excellent example to study the ambiguity of determining what is a reliable research strategy. One of the key reasons for the historical marginalization of olfaction was the experimental difficulty of conducting research into olfaction: How do you ‘materialize’ odors and by what criteria do you make them comparable? Part of the contemporary significance of research in olfaction is its potential for methodological innovations: How do you overcome the persistent difficulties in modeling transmembrane receptors and how should the organization of activation patterns in the cortex be modeled? By tracing the emergence, success, decline and failure of standard lab routines in past and present research on the sense of smell, I want to understand the uncertainties and the ‘missing knowledge’ that drive contemporary (neuro)scientific research.

Talk has three parts: quick general philosophy intro, odorant receptors and GPCRs, olfactory cortex mapping.
(Correction: Linda Buck tried 3 not 8 years finding the receptors.)

Retinal Pigment Epithelial cells (RPE) express both GPR143 and myocilin, which interact in a signal
transduction-dependent manner. In heterologous systems, activation of GPR143 with ligand causes
transient recruitment of myocilin to internalized receptors, which appears to be the entry point of
myocilin to the endocytic pathway. In some but not all cells, myocilin also traffics through the multivesicular
body (MVB) and is released on the surface of exosomes in a signal transduction-dependent
fashion. Little is known regarding the role of exosomes in RPE, but they likely serve as a mode of
communication between the RPE and the outer retina. In this study, we used posterior poles with retina
removed from fresh human donor eyes as a model to test the relationship between GPR143, myocilin,
and exosomes in an endogenous system. We isolated exosomes released by RPE using differential
centrifugation of media conditioned by the RPE for 25 min, and then characterized the exosomes using
nanoparticle tracking to determine the number and size of the exosomes. Next, we tested whether ligand
stimulation of GPR143 using L-DOPA altered RPE exosome release. Finally, we investigated whether
myocilin was present on the exosomes released by RPE and whether L-DOPA stimulation of GPR143
caused recruitment of myocilin to the endocytic pathway, as we have previously observed using cultured
cells. Activation of GPR143 halted RPE exosome release, while simultaneously recruiting myocilin to the
endocytic compartment. Together, our results indicate that GPR143 and myocilin function in a signal
transduction system that can control exosome release from RPE

Intronless genes (IGs) fraction varies between 2.7 and 97.7% in eukaryotic genomes. Although many databases on exons and introns exist, there was no curated database for such genes which allowed their study in a concerted manner. Such a database would be useful to identify the functional features and the distribution of these genes across the genome. Here, a new database of IGs in eukaryotes based on GenBank data was described. This database, called IGD (Intronless Gene Database), is a collection of gene sequences that were annotated and curated. The current version of IGD contains 687 human intronless genes with their protein and CDS sequences. Some features of the entries are given in this paper. Data was extracted from GenBank release 183 using a Perl script. Data extraction was followed by a manual curation step. Intronless genes were then analyzed based on their RefSeq annotation and Gene Ontology functional class. IGD represents a useful resource for retrieval and in silico study of intronless genes. IGD is available at http://www.bioinfocbs.org/igd with comprehensive help and FAQ pages that illustrate the main uses of this resource.

G-protein-coupled receptors (GPCRs) are a large superfamily of membrane receptors that are involved in a wide range of signaling pathways. To fulfill their tasks, GPCRs interact with a variety of partners, including small molecules, lipids and proteins. They are accompanied by different proteins during all phases of their life cycle. Therefore, GPCR interactions with their partners are of great interest in basic cell-signaling research and in drug discovery.

Genomics and proteomics discovery is leading to the identification of all proteins and to the opportunity, and challenge, to reveal the protein recognition networks that drive virtually all biological processes. Over the past decade, biosensors have emerged as a key technology for detection and analysis of biomolecular interactions. An important limitation in developing such biosensors is that the focus has been mainly on sensor platforms, the transducing hardware that converts interaction signals into recorded data, without adequately considering the role of molecular interfaces, the elements of sensors that interact with analytes to produce signals. We have investigated this alternative focus by identifying and, where necessary, designing molecular interfaces that will more effectively drive new biosensor development and utilization in biomedical and biotechnological investigations. Here we describe our recent studies of coiled coil and lipid bilayer interfaces and the potential to use these to expand sensing technologies for multiplexed target detection and analysis in increasingly biologically relevant membrane like environments. Copyright © 2004 John Wiley & Sons, Ltd.

Neurobiology studies mechanisms of cell signalling. A key question is how cells recognise specific signals. In this context, olfaction has become an important experimental system over the past 25 years. The olfactory system responds to an array of structurally diverse stimuli. The discovery of the olfactory receptors (ORs), recognising these stimuli, established the olfactory pathway as part of a greater group of signalling mechanisms mediated by G-protein-coupled receptors (GPCRs). GPCRs are the largest protein family in the mammalian genome and involved in numerous fundamental physiological processes. The OR family exhibits two characteristics that make them an excellent model system to understand GPCRs: its size and the structural diversity of its members. Research on the OR binding site investigates what amino acid sequences determine the receptor binding capacity. This promises a better understanding of how the basic genetic makeup of GPCRs relates to their diversification in ligand binding capacities.

A QSAR (quantitative structureeactivity relationship) analysis of the binding affinities for a series of 43 quinoline derivatives active against the alpha2C adrenergic receptor was performed. Multiple linear regressions (MLR) were obtained using the minimum topological difference (MTD) descriptor and various descriptors which were calculated with Dragon3.0. The variable selection was performed either through the forward stepwise method or backward stepwise combined with forward stepwise methods, providing two satisfactory models. The first one, obtained as a result of the forward stepwise method, contains MTD, Mor24v, MATS5m, MATS7m, G3m, L1s, G_N_N descriptors, while the other one obtained through the combination of backward and forward stepwise methods contains the following descriptors: MTD, ZM2V, X5V, IC5, MATS4v, and E2u. Both models highlight the importance of steric interactions and can be used as tools for predicting the binding affinity of related compounds.

Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT 1 / Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT 1 receptor, a non-functional AT 1 receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas. Cells single-expressing Mas or C18F/K20A did not show any binding for AngII. The co-expression of the wild-type AT 1 receptor and Mas showed a binding profile similar to that observed for the wild-type AT 1 expressed alone. Surprisingly, the co-expression of the double mutant C18F/K20A and Mas evoked a total recovery of the binding affinity for AngII to a level similar to that obtained for the wild-type AT 1 . Functional measurements using inositol phosphate and extracellular acidification rate assays also showed a clear recovery of activity for AngII on cells co-expressing the mutant C18F/ K20A and Mas. In addition, immunofluorescence analysis localized the AT 1 receptor mainly at the plasma membrane and the mutant C18F-K20A exclusively inside the cells. However, the co-expression of C18F-K20A mutant with the Mas changed the distribution pattern of the mutant, with intense signals at the plasma membrane, comparable to those observed in cells expressing the wild-type AT 1 receptor. These results support the hypothesis that Mas is able to rescue binding and functionality of the defective C18F-K20A mutant by dimerization.

Many G-protein coupled receptors are palmitoylated in their C-terminal, intracellular regions. So far no enzymes responsible for this modification have been described. We identified an interaction of the membrane proximal helix 8 of somatostatin receptor 5 (SSTR5) with the N-terminal region of the putative palmitoyltransferase ZDHHC5 using the Ras recruitment interaction screening system. ZDHHC5 and SSTR5 are colocalized at the plasma membrane and can be efficiently coimmunoprecipitated from transfected cells. Coexpression of ZDHHC5 in HEK293 cells increased palmitoylation of SSTR5 whereas knock-down of endogenous ZDHHC5 by siRNAs decreased it. Our data identify the first palmitoyltransferase for a G-protein coupled receptor. Structured summary of protein interactions: SSTR5 physically interacts with ZDHHC5 by ras recruitment system (View interaction) SSTR5 and ZDHHC5 colocalize by fluorescence microscopy (View interaction) SSTR5 physically interacts with ZDHHC5 by pull down (View interaction)

The Staphylococcus aureus genome contains three toxin-antitoxin modules, including one mazEF module, SamazEF. Using an on-column separation protocol we are able to obtain large amounts of wild-type SaMazF toxin. The protein is well-folded and highly resistant against thermal unfolding but aggregates at elevated temperatures. Crystallographic and nuclear magnetic resonance (NMR) solution studies show a well-defined dimer. Differences in structure and dynamics between the X-ray and NMR structural ensembles are found in three loop regions, two of which undergo motions that are of functional relevance. The same segments also show functionally relevant dynamics in the distantly related CcdB family despite divergence of function. NMR chemical shift mapping and analysis of residue conservation in the MazF family suggests a conserved mode for the inhibition of MazF by MazE.

DNA fingerprinting by pulsed-field gel electrophoresis (PFGE) 1 is the methodology used by the PulseNet International network for worldwide surveillance of foodborne diseases [1]. Success relies on the participants' adherence to PulseNet standard working protocols that make ...

CONSPECTUS: Protein function is inextricably linked to protein dynamics. As we move from a static structural picture to a dynamic ensemble view of protein structure and function, novel computational paradigms are required for observing and understanding conformational dynamics of proteins and its functional implications. In principle, molecular dynamics simulations can provide the time evolution of atomistic models of proteins, but the long time scales associated with functional dynamics make it difficult to observe rare dynamical transitions. The issue of extracting essential functional components of protein dynamics from noisy simulation data presents another set of challenges in obtaining an unbiased understanding of protein motions. Therefore, a methodology that provides a statistical framework for efficient sampling and a human-readable view of the key aspects of functional dynamics from data analysis is required. The Markov state model (MSM), which has recently become popular worldwide for studying protein dynamics, is an example of such a framework. In this Account, we review the use of Markov state models for efficient sampling of the hierarchy of time scales associated with protein dynamics, automatic identification of key conformational states, and the degrees of freedom associated with slow dynamical processes. Applications of MSMs for studying long time scale phenomena such as activation mechanisms of cellular signaling proteins has yielded novel insights into protein function. In particular, from MSMs built using large-scale simulations of GPCRs and kinases, we have shown that complex conformational changes in proteins can be described in terms of structural changes in key structural motifs or "molecular switches" within the protein, the transitions between functionally active and inactive states of proteins proceed via multiple pathways, and ligand or substrate binding modulates the flux through these pathways. Finally, MSMs also provide a theoretical toolbox for studying the effect of nonequilibrium perturbations on conformational dynamics. Considering that protein dynamics in vivo occur under nonequilibrium conditions, MSMs coupled with nonequilibrium statistical mechanics provide a way to connect cellular components to their functional environments. Nonequilibrium perturbations of protein folding MSMs reveal the presence of dynamically frozen glass-like states in their conformational landscape. These frozen states are also observed to be rich in β-sheets, which indicates their possible role in the nucleation of β-sheet rich aggregates such as those observed in amyloid-fibril formation. Finally, we describe how MSMs have been used to understand the dynamical behavior of intrinsically disordered proteins such as amyloid-β, human islet amyloid polypeptide, and p53. While certainly not a panacea for studying functional dynamics, MSMs provide a rigorous theoretical foundation for understanding complex entropically dominated processes and a convenient lens for viewing protein motions.

Many G protein-coupled receptor (GPCR) models have been built over the years. The release of the structure of bovine rhodopsin in August 2000 enabled us to analyze models built before that period to learn more about the models we build today. We conclude that the GPCR modelling field is riddled with ‘common knowledge’ similar to Lord Kelvin’s remark in 1895 that “heavier-than-air flying machines are impossible”, and we summarize what we think are the (im)possibilities of modelling GPCRs using the coordinates of bovine rhodopsin as a template. Associated WWW pages: http://www.gpcr.org/articles/2003_mod/.

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Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of b-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.

Anxiety and stress-related disorders, namely posttraumatic stress disorder (PTSD), generalized anxiety disorder (GAD), obsessive-compulsive disorder (ODC), social and specific phobias, and panic disorder, are a major public health issue. A growing body of evidence suggests that glutamatergic neurotransmission may be involved in the biological mechanisms underlying stress response and anxiety-related disorders. The glutamatergic system mediates the acquisition and extinction of fear-conditioning. Thus, new drugs targeting glutamatergic neurotransmission may be promising candidates for new pharmacological treatments. In particular, N-methyl-D-aspartate receptors (NMDAR) antagonists (AP5, AP7, CGP37849, CGP39551, LY235959, NPC17742, and MK-801), NMDAR partial agonists (DCS, ACPC), α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) antagonists (topiramate), and several allosteric modulators targeting metabotropic glutamate receptors (mGluRs) mGluR1, mGluR2/3, and mGluR5, have shown anxiolytic-like effects in several animal and human studies. Several studies have suggested that polyamines (agmatine, putrescine, spermidine, and spermine) may be involved in the neurobiological mechanisms underlying stress-response and anxiety-related disorders. This could mainly be attributed to their ability to modulate ionotropic glutamate receptors, especially NR2B subunits. The aim of this review is to establish that glutamate neurotransmission and polyaminergic system play a fundamental role in the onset of anxiety-related disorders. This may open the way for new drugs that may help to treat these conditions.

The glutamatergic pathway has been consistently involved in the physiopathology of depressive disorder. However a complete dissection and integration of its role in the context of other known mechanisms is lacking. We summarized and integrated the evidence of various levels of interaction between glutamatergic and monoaminergic pathways (see videos). We identified six molecular pathways, some of which with specific regional

Among the structurally similar guanidinonaltrindole (GNTI) compounds 5'-GNTI is an antagonist while 6'-GNTI is an agonist of the KOR opioid receptor. To explore how a subtle alteration of the ligand structure influences the receptor destiny we investigated two concurrent processes: the final steps of ligand binding in the receptor binding site and the initial steps of receptor activation. To trace such early activation steps the membranous part of the receptor was built on an inactive receptor template while the extracellular loops were built using the ab initio CABS method. We used the simulated annealing procedure for ligand docking and all-atom molecular dynamics simulations for finding the immediate changes in the structure of the ligand-receptor complex. Binding of an agonist, contrary to antagonist, induced breaking of the "3-7 lock" between helices TM3 and TM7. We also observed an action of the extended rotamer toggle switch which can suggest interdependence between those two switches.

Fenofibrate is a peroxisome proliferator activated receptor alpha agonist that contains both pro and anti-inflammatory properties, and has been used in the treatment of dyslipidemia and diabetes for decades. Its receptors are expressed in the liver, skeletal muscle, cardiac, enteric, and renal cells, which allow it to provide systemic regulation of lipoprotein metabolism, fatty acid oxidation, and fatty acid transport. Hyperglycemia is a common complication found in the burn population because hepatic glucose production and catecholamine-mediated hepatic glycogenolysis are augmented. Insulin resistance occurs often in these patients and is associated with poor outcomes. In the pediatric burn population, fenofibrate has been found to ameliorate or decrease the number of hypoglycemic episodes when compared to management with insulin alone. Its mechanism of action is thought to involve an improvement in insulin signaling in skeletal muscle, as well as improvements in mitochondrial function, glucose oxidation, and insulin sensitivity. The long term use of fenofibrate in severely burned patients may improve hyperglycemia and insulin resistance, as well as improve wound healing, and reduce apoptosis, and oxidative stress.

Detergent-resistant membrane microdomains in the plasma membrane, known as lipid rafts, have been implicated in various cellular processes. We report here that a low-density Triton X-100-insoluble membrane (detergent-resistant membrane; DRM) fraction is present in bovine rod photoreceptor outer segments (ROS). In dark-adapted ROS, transducin and most of cGMPphosphodiesterase (PDE) were detergent-soluble. When ROS membranes were exposed to light, however, a large portion of transducin localized in the DRM fraction. Furthermore, on addition of guanosine 5-3-O-(thio)triphosphate (GTP␥S) to light-bleached ROS, transducin became detergent-soluble again. PDE was not recruited to the DRM fraction after light stimulus alone, but simultaneous stimulation by light and GTP␥S induced a massive translocation of all PDE subunits to the DRM. A cholesterol-removing reagent, methyl-␤-cyclodextrin, selectively but partially solubilized PDE from the DRM, suggesting that cholesterol contributes, at least in part, to the association of PDE with the DRM. By contrast, transducin was not extracted by the depletion of cholesterol. These data suggest that transducin and PDE are likely to perform their functions in phototransduction by changing their localization between two distinct lipid phases, rafts and surrounding fluid membrane, on disc membranes in an activation-dependent manner.

A series of 5-alkyl pyrazole-3-carboxylic acids were prepared and found to act as potent and selective agonists of the human GPCR, GPR109a, the high affinity nicotinic acid receptor. No activity was observed at the highly homologous low affinity niacin receptor, GPR109b. A further series of 4-fluoro-5-alkyl pyrazole-3-carboxylic acids were shown to display similar potency. One example from the series was shown to have improved properties in vivo compared to niacin.

The interaction between the anaphylatoxin C5a and its receptor involves two distinct sites. One site is formed by acidic residues at the receptor N-terminus and contributes to only ligand binding. The second site, responsible for activation, is less well defined. In this study, we demonstrate that the receptor residue D 282 , near the extracellular face of transmembrane domain VII, is a component of the second ligand-binding site. Mutation of D 282 to A decreases the sensitivity of the receptor to activation by intact C5a but not by its less potent metabolite, C5adR 74 , which lacks the C-terminal arginine 74 . The mutation of the R 74 residue of C5a to A causes a 60-fold decrease in wild-type receptor sensitivity, but only a 2-fold decrease for the receptor mutated at D 282 . In contrast, the mutation of R 74 to D makes C5a completely inactive on both wild-type and A 282 C5a receptors. The mutation of D 282 to R partly restores the response to C5a [D 74 ], which is a more effective ligand than C5a at the mutant receptor. A peptide mimic of the C5a activation domain with a C-terminal R potently activates the wild type but is only a weak agonist at the mutant D 282 R-C5a receptor. Conversely, a peptide with D at the C-terminus is a more effective activator of D 282 R than wild-type C5a receptors. These data indicate that the R 74 side chain of C5a makes an interaction with receptor D 282 that is responsible for the higher potency of intact C5a versus that of C5adR 74 .

A 2A adenosine receptor-mediated signaling affects a variety of important processes in the central nervous system both in physiological and pathological conditions, and has been indicated as possible novel therapeutic target in several nervous system diseases. In the present work, cell death induction was investigated after neuronal PC 12 cell treatment with proinflammatory cytokines and adenosine receptor ligands. Interleukin-1-beta (IL-1-beta, 500 U/mL), tumor necrosis factor-alpha (TNF-alpha, 1000 U/mL) and the non selective adenosine receptor agonist, 5 0-N-ethylcarboxamidoadenosine (NECA), caused a significant reduction of cell viability with a maximal effect within 3-48 hr. Moreover, an addictive effect was detected when the cells were simultaneously treated with Interleukin-1-beta and NECA for 3 hr. To investigate the adenosine receptor subtypes involved in PC 12 cell death, the effects of several adenosine receptor agonists/ antagonists were evaluated. The endogenous nucleoside, adenosine, and the selective A 2A adenosine receptor agonist, 2-(carboxyethylphenylethylamino)adenosine-5 0-carboxamide (CGS21680) reduced PC 12 cell viability. This effect was counteracted by the selective A 2A adenosine receptor antagonist, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3e]-1,2,4-triazolo[1,5c]pyrimidine (SCH58261), but not by selective A 2B adenosine receptor antagonist N-(4-acethylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8yl)phenoxy]acetamide (MRS1706), suggesting the specific involvement of A 2A adenosine receptor subtype in adenosine-mediated cytotoxicity. Moreover, the selective A 1 adenosine receptor agonist, N 6-cyclohexyladenosine (CHA), did not induce any significant effect on cell viability. By ELISA immunoassay cell death detection and transmission electron microscopy (TEM) we demonstrated that A 2A adenosine receptor ligands and cytokines induced cell death through an apoptotic pathway. In conclusion, our results showed that A 2A adenosine receptors are involved in the control of PC 12 cell survival/death and may contribute to modulate cellular activity in response to tissue damage associated with inflammatory mediator production.

The human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) to treat various types of cancer and viral diseases including Hepatitis B/C infections. To improve the pharmacological properties of the drug, a new pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFNalpha(2a). (40)PEG-IFNalpha(2a) is a mixture of mainly six monopegylated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. Here we report the detailed structural and biophysical characterization of (40)PEG-IFNalpha(2a) and its positional isomers, in comparison with IFNalpha(2a), using NMR spectroscopy, analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Our results show that the three-dimensional structure of IFNalpha(2a) is not modified b...

The metabotropic glutamate receptors (mGluRs) are a subset of the Class C G-Protein Coupled Receptors (GPCRs). Recently, an emerging strategy for drug-discovery efforts targeting mGluRs has been to develop compounds acting at the so-called allosteric site in the 7-transmembrane (7TM) domain, common to all GPCRs, rather than the extracellular (EC) domain containing the orthosteric glutamate-binding site. We examine herein some of the intrinsic relative merits of targeting these two domains. Comparisons are made among amino-acid sequences in the two domains and among X-ray structures and homology models of the EC domain. We show that there is greater sequence diversity in the EC domains than in the transmembrane (TM) domains. Thus, contrary to generally accepted descriptions of there being greater evolutionary pressure to preserve the EC domain, it is the 7TM domain that is more highly conserved. Within the EC domain, the glutamate-binding site of the Venus flytrap region has hitherto received the most attention as a target site. Analysis of examples of the three-dimensional structures of the EC domains at the glutamate-binding site reveals differences as well, thereby supporting the viability of targeting the EC domain, even at the glutamate-binding site, for drug discovery. To exemplify this strategy, we present examples of active compounds identified via high-throughput docking in the EC region.

Background: Problem-based learning (PBL) has become the most significant innovation in medical education of the past 40 years. In contrast to exam-centered, lecture-based conventional curricula, PBL is a comprehensive curricular strategy that fosters student-centred learning and the skills desired in physicians. The rapid spread of PBL has produced many variants. One of the most common is 'hybrid PBL' where conventional teaching methods are implemented alongside PBL. This paper contends that the mixing of these two opposing educational philosophies can undermine PBL and nullify its positive benefits. Schools using hybrid PBL and lacking medical education expertise may end up with a dysfunctional curriculum worse off than the traditional approach. Discussion: For hybrid PBL schools with a dysfunctional curriculum, standard PBL is a cost-feasible option that confers the benefits of the PBL approach. This paper describes the signs of a dysfunctional PBL curriculum to aid hybrid PBL schools in recognising curricular breakdown. Next it discusses alternative curricular strategies and costs associated with PBL. It then details the four critical factors for successful conversion to standard PBL: dealing with staff resistance, understanding the role of lectures, adequate time for preparation and support from the administrative leadership. Summary: Hybrid PBL curricula without oversight by staff with medical education expertise can degenerate into dysfunctional curricula inferior even to the traditional approach from which PBL emerged. Such schools should inspect their curriculum periodically for signs of dysfunction to enable timely corrective action. A decision to convert fully to standard PBL is cost feasible but will require time, expertise and commitment which is only sustainable with supportive leadership.

A series of 3-nitro-4-substituted-aminobenzoic acids were prepared and found to act as potent and highly selective agonists of the orphan human GPCR GPR109b, a low affinity receptor for niacin. No activity was observed at the closely homologous high affinity niacin receptor, GPR109a. A second series, comprising 6-amino-substituted nicotinic acids was, also prepared and several analogues showed comparable activity to the nitroaryl series.