Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes ^3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl⁺ that has been successfully used in other fluorescence quenching assays ^5,13. Tl⁺ permeates the lipid bilayer slowly ⁸ but passes readily through conducting gramicidin channels ^1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results ¹².Download video file.^{(69M, mov)}     

Differential role of the hippocampus, amygdala, and dorsal raphe nucleus in regulating feeding, memory, and anxiety-like behavioral responses to ghrelin

Ghrelin is a peptide hormone produced and secreted from the stomach. Hypothalamic injection of the peptide increases food intake but it is not known if the peptide affects other brain regions. We measured several behavioral parameters such as anxiety (elevated plus maze), memory retention (step down test), and food intake after injections of different doses of the peptide in the hippocampus, amygdala, and dorsal raphe nucleus (DRN). The injection of ghrelin in the hippocampus and DRN significantly and dose dependently increased food intake in relation to controls rats, while injections into the amygdala did not affect the food intake. We also show for the first time that ghrelin clearly and dose dependently increases memory retention in the hippocampus, amygdala, and DRN. Moreover, ghrelin at different potencies induced anxiogenesis in these brain structures while the highest dose of 3 nmol/microl was effective in all of them. The comparison of sensitivity of each brain structure indicates a specific role of them for each of the behaviors studied. The results provide new insight in to the anatomical substrate and the functional role of extrahypothalamic ghrelin targets in the CNS.     

Activation of central melanocortin-4 receptor suppresses lipopolysaccharide-induced fever in rats

Activation of central melanocortin receptors (MCR) inhibits fever, but the identity of the MCR subtype(s) mediating this antipyretic effect is unknown. To determine whether selective central melanocortin receptor-4 (MC4R) activation produces antipyretic effects, the MC4R selective agonist MRLOB-0001 (CO-His-d-Phe-Arg-Trp-Dab-NH(2)) was administered intracerebroventricularly to rats treated with Escherichia coli lipopolysaccharide (LPS, 30 microg/kg ip). Treatment with MRLOB-0001 (150 ng icv) did not lower core body temperature (T(c)) in afebrile rats but did suppress LPS-induced increases in T(c) and associated decreases in tail skin temperature (T(sk)), an indicator of vasomotor thermoeffector function. In contrast, systemic treatment with MRLOB-0001 (150 ng iv) did not produce similar antipyretic effects. Coadministration of the selective MC4R antagonist HS014 (1 microg icv) blocked the antipyretic effects of MRLOB-0001. HS014 alone (1 microg icv) had no significant effect on LPS-induced increases in T(c) or decreases in T(sk) and in afebrile rats had no significant effects on T(c) or T(sk). We conclude that pharmacological activation of central MC4R suppresses febrile increases in T(c) and that inhibition of heat conservation pathways may contribute to this effect. These findings suggest that the central MC4R may mediate the long-recognized antipyretic effects of centrally administered melanocortins.     

Genomewide scan for hand osteoarthritis: a novel mutation in matrilin-3

Osteoarthritis (OA) is the most common human joint disease, characterized by loss and/or remodeling of joint synovium, cartilage, and bone. Here, we describe a genomewide linkage analysis of patients with idiopathic hand OA who were carefully phenotyped for involvement of either or both the distal interphalangeal (DIP) joints and the first carpometacarpal (CMC1) joints. The best linkage peaks were on chromosomes 4q and 3p and on the short arm of chromosome 2. Genomewide significance was reached for a locus on chromosome 2 for patients with affected CMC1 joints (LOD = 4.97); this locus was also significant for patients with OA in both CMC1 and DIP joints (LOD = 4.44). The peak LOD score at this locus coincides with a gene, MATN3, encoding the noncollagenous cartilage extracellular matrix protein, matrilin-3. Subsequent screening of the genomic sequence revealed a missense mutation, of a conserved amino acid codon, changing threonine to methionine in the epidermal growth factor-like domain in matrilin-3. The missense mutation cosegregates with hand OA in several families. The mutation frequency is slightly more than 2% in patients with hand OA in the Icelandic population and has a relative risk of 2.1.     

Novel human G protein-coupled receptors with long N-terminals containing GPS domains and Ser/Thr-rich regions

We report eight novel members of the superfamily of human G protein-coupled receptors (GPCRs) found by searches in the human genome databases, termed GPR97, GPR110, GPR111, GPR112, GPR113, GPR114, GPR115 and GPR116. Phylogenetic analysis shows that these are additional members of a family of GPCRs with long N-termini, previously termed EGF-7TM, LNB-7TM, B2 or LN-7TM. Five of the receptors form their own phylogenetic cluster, while three others form a cluster with the previously reported HE6 and GPR56 (TM7XN1). All the receptors have a GPS domain in their N-terminus and long Ser/Thr-rich regions forming mucin-like stalks. GPR113 has a hormone binding domain and one EGF domain. GPR112 has over 20 Ser/Thr repeats and a pentraxin domain. GPR116 has two immunoglobulin-like repeats and a SEA box. We found several human EST sequences for most of the receptors showing differential expression patterns, which may indicate that some of these receptors participate in reproductive functions while others are more likely to have a role in the immune system.     

Neuronal differentiation is accompanied by NSP-C expression

Neuroendocrine-specific protein (NSP) reticulons are expressed in neural and neuroendocrine tissues and cell cultures derived therefrom, while most other cell types lack NSP-reticulons. Three major subtypes have been identified so far, designated NSP-A, NSP-B, and NSP-C. We have investigated the correlation between the degree of neuronal differentiation, determined by morphological and biochemical criteria, and NSP-reticulon subtype expression. For this purpose, several human neuroblastoma cell lines, exhibiting different degrees of neuronal differentiation, were examined immuno(cyto) chemically. It became obvious that the expression of NSP-C, as detected by immunofluorescence microscopy and Western blotting, is most prominent in cell lines with a high degree of neuronal differentiation, such as LA-N-5. Such highly differentiated cells also express other neural and neuroendocrine markers, such as neural cell adhesion molecule (NCAM), neurofilament proteins, synaptophysin, and chromogranin. NSP-A was observed in all cell lines to a different extent. However, no clear correlation was observed with the degree of neuronal differentiation as defined by other neuronal and neuroendocrine markers or morphology. NSP-B could not be detected. The induction of neuronal differentiation with nerve growth factor, dbcAMP, and retinoic acid in the rat pheochromocytoma cell line PC12 and the human teratocarcinoma cell line hNT2, respectively, induced the expression of NSP-A and NSP-C in these cell lines parallel to the induction of neurofilament protein expression. It is concluded that NSP-C expression, in particular, is strongly correlated with neuronal differentiation.     

Genetic algorithm for large-scale maximum parsimony phylogenetic analysis of proteins

Inferring phylogeny is a difficult computational problem. For example, for only 13 taxa, there are more then 13 billion possible unrooted phylogenetic trees. Heuristics are necessary to minimize the time spent evaluating non-optimal trees. We describe here an approach for heuristic searching, using a genetic algorithm, that can reduce the time required for weighted maximum parsimony phylogenetic inference, especially for data sets involving a large number of taxa. It is the first implementation of a weighted maximum parsimony criterion using amino acid sequences. To validate the weighted criterion, we used an artificial data set and compared it to a number of other phylogenetic methods. Genetic algorithms mimic the natural selection's ability to solve complex problems. We have identified several parameters affecting the genetic algorithm. Methods were developed to validate these parameters, ensuring optimal performance. This approach allows the construction of phylogenetic trees with over 200 taxa in practical time on a regular PC.     

Neuregulin 1 and susceptibility to schizophrenia

The cause of schizophrenia is unknown, but it has a significant genetic component. Pharmacologic studies, studies of gene expression in man, and studies of mouse mutants suggest involvement of glutamate and dopamine neurotransmitter systems. However, so far, strong association has not been found between schizophrenia and variants of the genes encoding components of these systems. Here, we report the results of a genomewide scan of schizophrenia families in Iceland; these results support previous work, done in five populations, showing that schizophrenia maps to chromosome 8p. Extensive fine-mapping of the 8p locus and haplotype-association analysis, supplemented by a transmission/disequilibrium test, identifies neuregulin 1 (NRG1) as a candidate gene for schizophrenia. NRG1 is expressed at central nervous system synapses and has a clear role in the expression and activation of neurotransmitter receptors, including glutamate receptors. Mutant mice heterozygous for either NRG1 or its receptor, ErbB4, show a behavioral phenotype that overlaps with mouse models for schizophrenia. Furthermore, NRG1 hypomorphs have fewer functional NMDA receptors than wild-type mice. We also demonstrate that the behavioral phenotypes of the NRG1 hypomorphs are partially reversible with clozapine, an atypical antipsychotic drug used to treat schizophrenia.     

Watching TV and Food Intake: The Role of Content

Obesity is a serious and growing health concern worldwide. Watching television (TV) represents a condition during which many habitually eat, irrespective of hunger level. However, as of yet, little is known about how the content of television programs being watched differentially impacts concurrent eating behavior. In this study, eighteen normal-weight female students participated in three counter-balanced experimental conditions, including a ‘Boring’ TV condition (art lecture), an ‘Engaging’ TV condition (Swedish TV comedy series), and a no TV control condition during which participants read (a text on insects living in Sweden). Throughout each condition participants had access to both high-calorie (M&Ms) and low-calorie (grapes) snacks. We found that, relative to the Engaging TV condition, Boring TV encouraged excessive eating (+52% g, P = 0.009). Additionally, the Engaging TV condition actually resulted in significantly less concurrent intake relative to the control ‘Text’ condition (−35% g, P = 0.05). This intake was driven almost entirely by the healthy snack, grapes; however, this interaction did not reach significance (P = 0.07). Finally, there was a significant correlation between how bored participants were across all conditions, and their concurrent food intake (beta = 0.317, P = 0.02). Intake as measured by kcals was similarly patterned but did not reach significance. These results suggest that, for women, different TV programs elicit different levels of concurrent food intake, and that the degree to which a program is engaging (or alternately, boring) is related to that intake. Additionally, they suggest that emotional content (e.g. boring vs. engaging) may be more associated than modality (e.g. TV vs. text) with concurrent intake.