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Sample records for mammalian receptor repertoire

  1. Specific repertoire of olfactory receptor genes in the male germ cells of several mammalian species

    SciTech Connect

    Vanderhaeghen, P.; Schurmans, S.; Vassart, G.; Parmentier, M.

    1997-02-01

    Olfactory receptors constitute the largest family among G protein-coupled receptors, with up to 1000 members expected. We have previously shown that genes belonging to this family were expressed in the male germ line from both dog and human. We have subsequently demonstrated the presence of one of the corresponding olfactory receptor proteins during dog spermatogenesis and in mature sperm cells. In this study, we investigated whether the unexpected pattern of expression of olfactory receptors in the male germ line was conserved in other mammalian species. Using reverse transcription-PCR with primers specific for the olfactory receptor gene family, about 20 olfactory receptor cDNA fragments were cloned from the testis of each mammalian species tested. As a whole, they displayed no sequence specificity compared to other olfactory receptors, but highly homologous, possibly orthologous, genes were amplified from different species. Finally, their pattern of expression, as determined by RNase protection assay, revealed that many but not all of these receptors were expressed predominantly in testis. The male germ line from each mammalian species tested is thus characterized by a specific repertoire of olfactory receptors, which display a pattern of expression suggestive of their potential implication in the control of sperm maturation, migration, or fertilization. 34 refs., 4 figs., 1 tab.

  2. The human olfactory receptor repertoire

    PubMed Central

    Zozulya, Sergey; Echeverri, Fernando; Nguyen, Trieu

    2001-01-01

    Background The mammalian olfactory apparatus is able to recognize and distinguish thousands of structurally diverse volatile chemicals. This chemosensory function is mediated by a very large family of seven-transmembrane olfactory (odorant) receptors encoded by approximately 1,000 genes, the majority of which are believed to be pseudogenes in humans. Results The strategy of our sequence database mining for full-length, functional candidate odorant receptor genes was based on the high overall sequence similarity and presence of a number of conserved sequence motifs in all known mammalian odorant receptors as well as the absence of introns in their coding sequences. We report here the identification and physical cloning of 347 putative human full-length odorant receptor genes. Comparative sequence analysis of the predicted gene products allowed us to identify and define a number of consensus sequence motifs and structural features of this vast family of receptors. A new nomenclature for human odorant receptors based on their chromosomal localization and phylogenetic analysis is proposed. We believe that these sequences represent the essentially complete repertoire of functional human odorant receptors. Conclusions The identification and cloning of all functional human odorant receptor genes is an important initial step in understanding receptor-ligand specificity and combinatorial encoding of odorant stimuli in human olfaction. PMID:11423007

  3. Mammalian sweet taste receptors.

    PubMed

    Nelson, G; Hoon, M A; Chandrashekar, J; Zhang, Y; Ryba, N J; Zuker, C S

    2001-08-10

    The sense of taste provides animals with valuable information about the quality and nutritional value of food. Previously, we identified a large family of mammalian taste receptors involved in bitter taste perception (the T2Rs). We now report the characterization of mammalian sweet taste receptors. First, transgenic rescue experiments prove that the Sac locus encodes T1R3, a member of the T1R family of candidate taste receptors. Second, using a heterologous expression system, we demonstrate that T1R2 and T1R3 combine to function as a sweet receptor, recognizing sweet-tasting molecules as diverse as sucrose, saccharin, dulcin, and acesulfame-K. Finally, we present a detailed analysis of the patterns of expression of T1Rs and T2Rs, thus providing a view of the representation of sweet and bitter taste at the periphery. PMID:11509186

  4. VDJtools: Unifying Post-analysis of T Cell Receptor Repertoires

    PubMed Central

    Bolotin, Dmitriy A.; Britanova, Olga V.; Putintseva, Ekaterina V.; Pogorelyy, Mikhail V.; Nazarov, Vadim I.; Zvyagin, Ivan V.; Kirgizova, Vitalina I.; Kirgizov, Kirill I.; Skorobogatova, Elena V.; Chudakov, Dmitriy M.

    2015-01-01

    Despite the growing number of immune repertoire sequencing studies, the field still lacks software for analysis and comprehension of this high-dimensional data. Here we report VDJtools, a complementary software suite that solves a wide range of T cell receptor (TCR) repertoires post-analysis tasks, provides a detailed tabular output and publication-ready graphics, and is built on top of a flexible API. Using TCR datasets for a large cohort of unrelated healthy donors, twins, and multiple sclerosis patients we demonstrate that VDJtools greatly facilitates the analysis and leads to sound biological conclusions. VDJtools software and documentation are available at https://github.com/mikessh/vdjtools. PMID:26606115

  5. Sex Hormone Receptor Repertoire in Breast Cancer

    PubMed Central

    Higa, Gerald M.; Fell, Ryan G.

    2013-01-01

    Classification of breast cancer as endocrine sensitive, hormone dependent, or estrogen receptor (ER) positive refers singularly to ERα. One of the oldest recognized tumor targets, disruption of ERα-mediated signaling, is believed to be the mechanistic mode of action for all hormonal interventions used in treating this disease. Whereas ERα is widely accepted as the single most important predictive factor (for response to endocrine therapy), the presence of the receptor in tumor cells is also of prognostic value. Even though the clinical relevance of the two other sex hormone receptors, namely, ERβ and the androgen receptor remains unclear, two discordant phenomena observed in hormone-dependent breast cancers could be causally related to ERβ-mediated effects and androgenic actions. Nonetheless, our understanding of regulatory molecules and resistance mechanisms remains incomplete, further compromising our ability to develop novel therapeutic strategies that could improve disease outcomes. This review focuses on the receptor-mediated actions of the sex hormones in breast cancer. PMID:24324894

  6. The repertoire of olfactory C family G protein-coupled receptors in zebrafish: candidate chemosensory receptors for amino acids

    PubMed Central

    Alioto, Tyler S; Ngai, John

    2006-01-01

    Background Vertebrate odorant receptors comprise at least three types of G protein-coupled receptors (GPCRs): the OR, V1R, and V2R/V2R-like receptors, the latter group belonging to the C family of GPCRs. These receptor families are thought to receive chemosensory information from a wide spectrum of odorant and pheromonal cues that influence critical animal behaviors such as feeding, reproduction and other social interactions. Results Using genome database mining and other informatics approaches, we identified and characterized the repertoire of 54 intact "V2R-like" olfactory C family GPCRs in the zebrafish. Phylogenetic analysis – which also included a set of 34 C family GPCRs from fugu – places the fish olfactory receptors in three major groups, which are related to but clearly distinct from other C family GPCRs, including the calcium sensing receptor, metabotropic glutamate receptors, GABA-B receptor, T1R taste receptors, and the major group of V2R vomeronasal receptor families. Interestingly, an analysis of sequence conservation and selective pressure in the zebrafish receptors revealed the retention of a conserved sequence motif previously shown to be required for ligand binding in other amino acid receptors. Conclusion Based on our findings, we propose that the repertoire of zebrafish olfactory C family GPCRs has evolved to allow the detection and discrimination of a spectrum of amino acid and/or amino acid-based compounds, which are potent olfactory cues in fish. Furthermore, as the major groups of fish receptors and mammalian V2R receptors appear to have diverged significantly from a common ancestral gene(s), these receptors likely mediate chemosensation of different classes of chemical structures by their respective organisms. PMID:17156446

  7. Complement receptors and the shaping of the natural antibody repertoire.

    PubMed

    Holers, V Michael

    2005-03-01

    Complement and complement receptors have been known for several decades to play important roles in immune effector mechanisms related to pathogen elimination and tissue inflammation. In addition, studies over the last 10 years have clearly demonstrated a key role for the complement C3d activation fragment receptor designated CR2 (complement receptor type 2) in the switched-isotype, high-affinity and memory humoral immune responses to T-dependent foreign antigens. More recent studies have extended those observations to include a key role for CR2 and C3d in the humoral immune response to T-independent foreign antigens. Conversely, as these studies have proceeded, a parallel series of analyses have linked defects in expression or function of complement C4 and other classical pathway activation pathway proteins, as well as CR2 and the closely related CR1, to the loss of self tolerance to nuclear antigens such as double-stranded DNA and chromatin in systemic lupus erythematosus. With regard to the topic of this issue, it is now becoming increasingly clear that CR2 also plays a major role in the development of the natural antibody repertoire. Specifically, in the absence of this receptor natural IgM and IgG develop in the naïve animal that demonstrate clearly altered recognition patterns for specific natural antibody targets. This repertoire change is important physiologically in at least one setting because these CR2-dependent natural antibodies are necessary for the recognition of ischemic self tissues. In addition, it is possible that certain of the phenotypes manifest by CR2-deficient mice may be strongly influenced not only by effects on later stages of B cell activation and maturation, as commonly thought, but also by alterations in the pre-existing pool of natural antibodies that are influenced by this receptor. This review will examine the evidence that has accumulated over the last few years supporting these hypotheses. PMID:15614507

  8. Ghrelin Receptors in Non-Mammalian Vertebrates

    PubMed Central

    Kaiya, Hiroyuki; Kangawa, Kenji; Miyazato, Mikiya

    2012-01-01

    The growth hormone secretagogue-receptor (GHS-R) was discovered in humans and pigs in 1996. The endogenous ligand, ghrelin, was discovered 3 years later, in 1999, and our understanding of the physiological significance of the ghrelin system in vertebrates has grown steadily since then. Although the ghrelin system in non-mammalian vertebrates is a subject of great interest, protein sequence data for the receptor in non-mammalian vertebrates has been limited until recently, and related biological information has not been well organized. In this review, we summarize current information related to the ghrelin receptor in non-mammalian vertebrates. PMID:23882259

  9. Strategies for B-Cell Receptor Repertoire Analysis in Primary Immunodeficiencies: From Severe Combined Immunodeficiency to Common Variable Immunodeficiency

    PubMed Central

    IJspeert, Hanna; Wentink, Marjolein; van Zessen, David; Driessen, Gertjan J.; Dalm, Virgil A. S. H.; van Hagen, Martin P.; Pico-Knijnenburg, Ingrid; Simons, Erik J.; van Dongen, Jacques J. M.; Stubbs, Andrew P.; van der Burg, Mirjam

    2015-01-01

    The antigen receptor repertoires of B- and T-cells form the basis of the adaptive immune response. The repertoires should be sufficiently diverse to recognize all possible pathogens. However, careful selection is needed to prevent responses to self or harmless antigens. Limited antigen receptor repertoire diversity leads to immunodeficiency, whereas unselected or misdirected repertoires can result in autoimmunity. The antigen receptor repertoire harbors information about abnormalities in many immunological disorders. Recent developments in next generation sequencing allow the analysis of the antigen receptor repertoire in much greater detail than ever before. Analyzing the antigen receptor repertoire in patients with mutations in genes responsible for the generation of the antigen receptor repertoire will give new insights into repertoire formation and selection. In this perspective, we describe strategies and considerations for analysis of the naive and antigen-selected B-cell repertoires in primary immunodeficiency patients with a focus on severe combined immunodeficiency and common variable immunodeficiency. PMID:25904919

  10. New perspectives for large-scale repertoire analysis of immune receptors.

    PubMed

    Boudinot, Pierre; Marriotti-Ferrandiz, Maria Encarnita; Pasquier, Louis Du; Benmansour, Abdenour; Cazenave, Pierre-André; Six, Adrien

    2008-05-01

    In vertebrates, the world of antigenic motifs is matched to large populations of lymphocytes through specific recognition of an epitope by a given receptor unique to a lymphocyte clone. The concept of immune repertoire was proposed to describe this diversity of lymphocyte receptors - Ig and TCR - required by the network of interactions. The immune repertoires became useful tools to describe lymphocyte and receptor populations through the development of the immune system and in pathological situations. Recently, the development of mass technologies made possible a comprehensive survey of immune repertoires at the genome, transcript and protein levels, and some of these techniques have been already adapted to TCR and Ig repertoire analyses. Such approaches generate very big datasets, which necessitates complex and multi-parametric annotations in dedicated databases. They also require new analysis methods, leading to the integration of structure and dynamics of the immune repertoires, at different time scales (immune response, development of the individual, evolution of the species). Such methods may be extended to the analysis of new classes of adaptive-like receptors, which were recently discovered in different invertebrates and in agnathans. Ultimately, they may allow a parallel monitoring of pathogen and immune repertoires addressing the reciprocal influences that decide for the host survival or death. In this review, we first study the characteristics of Ig and TCR repertoires, and we examine several systematic approaches developed for the analysis of these "classical" immune repertoires at different levels. We then consider examples of the recent developments of modeling and statistical analysis, and we discuss their relevance and their importance for the study of the immune diversity. An extended view of immune repertoires is proposed, integrating the diversity of other receptors involved in immune recognition. Also, we discuss how repertoire studies could link

  11. Sequence Analysis of Bitter Taste Receptor Gene Repertoires in Different Ruminant Species.

    PubMed

    Monteiro Ferreira, Ana; Tomás Marques, Andreia; Bhide, Mangesh; Cubric-Curik, Vlatka; Hollung, Kristin; Knight, Christopher Harold; Raundrup, Katrine; Lippolis, John; Palmer, Mitchell; Sales-Baptista, Elvira; Araújo, Susana Sousa; de Almeida, André Martinho

    2015-01-01

    Bitter taste has been extensively studied in mammalian species and is associated with sensitivity to toxins and with food choices that avoid dangerous substances in the diet. At the molecular level, bitter compounds are sensed by bitter taste receptor proteins (T2R) present at the surface of taste receptor cells in the gustatory papillae. Our work aims at exploring the phylogenetic relationships of T2R gene sequences within different ruminant species. To accomplish this goal, we gathered a collection of ruminant species with different feeding behaviors and for which no genome data is available: American bison, chamois, elk, European bison, fallow deer, goat, moose, mouflon, muskox, red deer, reindeer and white tailed deer. The herbivores chosen for this study belong to different taxonomic families and habitats, and hence, exhibit distinct foraging behaviors and diet preferences. We describe the first partial repertoires of T2R gene sequences for these species obtained by direct sequencing. We then consider the homology and evolutionary history of these receptors within this ruminant group, and whether it relates to feeding type classification, using MEGA software. Our results suggest that phylogenetic proximity of T2R genes corresponds more to the traditional taxonomic groups of the species rather than reflecting a categorization by feeding strategy. PMID:26061084

  12. Sequence Analysis of Bitter Taste Receptor Gene Repertoires in Different Ruminant Species

    PubMed Central

    Monteiro Ferreira, Ana; Tomás Marques, Andreia; Bhide, Mangesh; Cubric-Curik, Vlatka; Hollung, Kristin; Knight, Christopher Harold; Raundrup, Katrine; Lippolis, John; Palmer, Mitchell; Sales-Baptista, Elvira; Araújo, Susana Sousa; de Almeida, André Martinho

    2015-01-01

    Bitter taste has been extensively studied in mammalian species and is associated with sensitivity to toxins and with food choices that avoid dangerous substances in the diet. At the molecular level, bitter compounds are sensed by bitter taste receptor proteins (T2R) present at the surface of taste receptor cells in the gustatory papillae. Our work aims at exploring the phylogenetic relationships of T2R gene sequences within different ruminant species. To accomplish this goal, we gathered a collection of ruminant species with different feeding behaviors and for which no genome data is available: American bison, chamois, elk, European bison, fallow deer, goat, moose, mouflon, muskox, red deer, reindeer and white tailed deer. The herbivores chosen for this study belong to different taxonomic families and habitats, and hence, exhibit distinct foraging behaviors and diet preferences. We describe the first partial repertoires of T2R gene sequences for these species obtained by direct sequencing. We then consider the homology and evolutionary history of these receptors within this ruminant group, and whether it relates to feeding type classification, using MEGA software. Our results suggest that phylogenetic proximity of T2R genes corresponds more to the traditional taxonomic groups of the species rather than reflecting a categorization by feeding strategy. PMID:26061084

  13. Mammalian Gravity Receptors: Structure and Metabolism

    NASA Technical Reports Server (NTRS)

    Ross, M. D.

    1985-01-01

    Calcium metabolism in mammalian gravity receptors is examined. To accomplish this objective it is necessary to study both the mineral deposits of the receptors, the otoconia, and the sensory areas themselves, the saccular and utricular maculas. The main focus was to elucidate the natures of the organic and inorganic phases of the crystalline masses, first in rat otoconia but more recently in otoliths and otoconia of a comparative series of vertebrates. Some of the ultrastructural findings in rat maculas, however, have prompted a more thorough study of the organization of the hair cells and innervation patterns in graviceptors.

  14. Co-regulation of a large and rapidly evolving repertoire of odorant receptor genes

    PubMed Central

    Kambere, Marijo B; Lane, Robert P

    2007-01-01

    The olfactory system meets niche- and species-specific demands by an accelerated evolution of its odorant receptor repertoires. In this review, we describe evolutionary processes that have shaped olfactory and vomeronasal receptor gene families in vertebrate genomes. We emphasize three important periods in the evolution of the olfactory system evident by comparative genomics: the adaptation to land in amphibian ancestors, the decline of olfaction in primates, and the delineation of putative pheromone receptors concurrent with rodent speciation. The rapid evolution of odorant receptor genes, the sheer size of the repertoire, as well as their wide distribution in the genome, presents a developmental challenge: how are these ever-changing odorant receptor repertoires coordinated within the olfactory system? A central organizing principle in olfaction is the specialization of sensory neurons resulting from each sensory neuron expressing only ~one odorant receptor allele. In this review, we also discuss this mutually exclusive expression of odorant receptor genes. We have considered several models to account for co-regulation of odorant receptor repertoires, as well as discussed a new hypothesis that invokes important epigenetic properties of the system. PMID:17903278

  15. bcRep: R Package for Comprehensive Analysis of B Cell Receptor Repertoire Data.

    PubMed

    Bischof, Julia; Ibrahim, Saleh M

    2016-01-01

    Immunoglobulins, as well as T cell receptors, play a key role in adaptive immune responses because of their ability to recognize antigens. Recent advances in next generation sequencing improved also the quality and quantity of individual B cell receptors repertoire sequencing. Unfortunately, appropriate software to exhaustively analyze repertoire data from NGS platforms without limitations of the number of sequences are lacking. Here we introduce a new R package, bcRep, which offers a platform for comprehensive analyses of B cell receptor repertoires, using IMGT/HighV-QUEST formatted data. Methods for gene usage statistics, clonotype classification, as well as diversity measures, are included. Furthermore, functions to filter datasets, to do summary statistics about mutations, as well as visualization methods, are available. To compare samples in respect of gene usage, diversity, amino acid proportions, similar sequences or clones, several functions including also distance measurements, as well as multidimensional scaling methods, are provided. PMID:27551775

  16. bcRep: R Package for Comprehensive Analysis of B Cell Receptor Repertoire Data

    PubMed Central

    Ibrahim, Saleh M.

    2016-01-01

    Immunoglobulins, as well as T cell receptors, play a key role in adaptive immune responses because of their ability to recognize antigens. Recent advances in next generation sequencing improved also the quality and quantity of individual B cell receptors repertoire sequencing. Unfortunately, appropriate software to exhaustively analyze repertoire data from NGS platforms without limitations of the number of sequences are lacking. Here we introduce a new R package, bcRep, which offers a platform for comprehensive analyses of B cell receptor repertoires, using IMGT/HighV-QUEST formatted data. Methods for gene usage statistics, clonotype classification, as well as diversity measures, are included. Furthermore, functions to filter datasets, to do summary statistics about mutations, as well as visualization methods, are available. To compare samples in respect of gene usage, diversity, amino acid proportions, similar sequences or clones, several functions including also distance measurements, as well as multidimensional scaling methods, are provided. PMID:27551775

  17. Diversity and divergence of the glioma-infiltrating T-cell receptor repertoire.

    PubMed

    Sims, Jennifer S; Grinshpun, Boris; Feng, Yaping; Ung, Timothy H; Neira, Justin A; Samanamud, Jorge L; Canoll, Peter; Shen, Yufeng; Sims, Peter A; Bruce, Jeffrey N

    2016-06-21

    Although immune signaling has emerged as a defining feature of the glioma microenvironment, how the underlying structure of the glioma-infiltrating T-cell population differs from that of the blood from which it originates has been difficult to measure directly in patients. High-throughput sequencing of T-cell receptor (TCR) repertoires (TCRseq) provides a population-wide statistical description of how T cells respond to disease. We have defined immunophenotypes of whole repertoires based on TCRseq of the α- and β-chains from glioma tissue, nonneoplastic brain tissue, and peripheral blood from patients. Using information theory, we partitioned the diversity of these TCR repertoires into that from the distribution of VJ cassette combinations and diversity due to VJ-independent factors, such as selection due to antigen binding. Tumor-infiltrating lymphocytes (TILs) possessed higher VJ-independent diversity than nonneoplastic tissue, stratifying patients according to tumor grade. We found that the VJ-independent components of tumor-associated repertoires diverge more from their corresponding peripheral repertoires than T-cell populations in nonneoplastic brain tissue, particularly for low-grade gliomas. Finally, we identified a "signature" set of TCRs whose use in peripheral blood is associated with patients exhibiting low TIL divergence and is depleted in patients with highly divergent TIL repertoires. This signature is detectable in peripheral blood, and therefore accessible noninvasively. We anticipate that these immunophenotypes will be foundational to monitoring and predicting response to antiglioma vaccines and immunotherapy. PMID:27261081

  18. Identification of antigen-specific B cell receptor sequences using public repertoire analysis

    PubMed Central

    Galson, Jacob D.; Rance, Richard; Parkhill, Julian; Lunter, Gerton; Pollard, Andrew J.; Kelly, Dominic F.

    2014-01-01

    High-throughput sequencing allows detailed study of the B cell receptor (BCR) repertoire post-immunization but it remains unclear to what extent the de novo identification of antigen-specific sequences from the total BCR repertoire is possible. A Hib-MenC-TT conjugate vaccine containing H. influenzae type b (Hib) and group C meningococcal (MenC) polysaccharides as well as tetanus toxoid (TT) was used to investigate the BCR repertoire of adult humans following immunization and test the hypothesis that public or convergent repertoire analysis could identify antigen specific sequences. A number of antigen-specific BCR sequences have previously been reported for Hib and TT which made a vaccine containing these 2 antigens an ideal immunological stimulus. Analysis of identical complementarity determining region (CDR)3 amino acid (AA) sequences that were shared by individuals in the post-vaccine repertoire identified a number of known Hib-specific sequences but only one previously described TT sequence. The extension of this analysis to non-identical but highly similar CDR3 AA sequences revealed a number of other TT-related sequences. The anti-Hib avidity index post-vaccination was strongly correlated with the relative frequency of Hib-specific sequences, indicating that the post-vaccination public BCR repertoire may be related to more conventional measures of immunogenicity correlating with disease protection. Analysis of public BCR repertoire provided evidence of convergent BCR evolution in individuals exposed to the same antigens. If this finding is confirmed, the public repertoire could be used for rapid and direct identification of protective antigen-specific BCR sequences from peripheral blood. PMID:25392534

  19. Atlantic salmon possesses two clusters of type I interferon receptor genes on different chromosomes, which allows for a larger repertoire of interferon receptors than in zebrafish and mammals.

    PubMed

    Sun, Baojian; Greiner-Tollersrud, Linn; Koop, Ben F; Robertsen, Børre

    2014-12-01

    Mammalian type I interferons (IFNs) signal through a receptor composed of the IFNAR1 and IFNAR2 chains. In zebrafish two-cysteine IFNs utilize a receptor composed of CRFB1 and CRFB5, while four-cysteine IFNs signal through a receptor formed by CRFB2 and CRFB5. In the present work two CRFB clusters were identified in different chromosomes of Atlantic salmon. Genes of three CRFB5s, one CRFB1, one CRFB2 and the novel CRFB5x were identified, cloned and studied functionally. All CRFBs were expressed in 10 different organs, but the relative expression of CRFBs varied. Mx-reporter assay was used to study which CRFBs might be involved in receptors for salmon IFNa, IFNb and IFNc. The results of Mx-reporter assays suggest that IFNa signals through a receptor composed of CRFB1a as the long chain and either CRFB5a, CRFB5b or CRFB5c as the short chain; IFNc signals through a receptor with CRFB5a or CRFB5c as the short chain while IFNb may signal through a receptor with CRFB5x as a short chain. Taken together, the present work demonstrates that Atlantic salmon has a more diverse repertoire of type I IFN receptors compared to zebrafish or mammals. PMID:25149134

  20. Identification of a Bitter-Taste Receptor Gene Repertoire in Different Lagomorphs Species.

    PubMed

    Ferreira, Ana M; Marques, Andreia T; Fontanesi, Luca; Thulin, Carl-Gustaf; Sales-Baptista, Elvira; Araújo, Susana S; Almeida, André M

    2016-01-01

    The repertoires of bitter-taste receptor (T2R) gene have been described for several animal species, but these data are still scarce for Lagomorphs. The aim of the present work is to identify potential repertoires of T2R in several Lagomorph species, covering a wide geographical distribution. We studied these genes in Lepus timidus, L. europaeus, Oryctolagus cuniculus algirus, Romerolagus diazi, and Sylvilagus floridanus, using O. cuniculus cuniculus as control species for PCR and DNA sequencing. We studied the identities of the DNA sequences and built the corresponding phylogenetic tree. Sequencing was successful for both subspecies of O. cuniculus for all T2R genes studied, for five genes in Lepus, and for three genes in R. diazi and S. floridanus. We describe for the first time the partial repertoires of T2R genes for Lagomorphs species, other than the common rabbit. Our phylogenetic analyses indicate that sequence proximity levels follow the established taxonomic classification. PMID:27092177

  1. Identification of a Bitter-Taste Receptor Gene Repertoire in Different Lagomorphs Species

    PubMed Central

    Ferreira, Ana M.; Marques, Andreia T.; Fontanesi, Luca; Thulin, Carl-Gustaf; Sales-Baptista, Elvira; Araújo, Susana S.; Almeida, André M.

    2016-01-01

    The repertoires of bitter-taste receptor (T2R) gene have been described for several animal species, but these data are still scarce for Lagomorphs. The aim of the present work is to identify potential repertoires of T2R in several Lagomorph species, covering a wide geographical distribution. We studied these genes in Lepus timidus, L. europaeus, Oryctolagus cuniculus algirus, Romerolagus diazi, and Sylvilagus floridanus, using O. cuniculus cuniculus as control species for PCR and DNA sequencing. We studied the identities of the DNA sequences and built the corresponding phylogenetic tree. Sequencing was successful for both subspecies of O. cuniculus for all T2R genes studied, for five genes in Lepus, and for three genes in R. diazi and S. floridanus. We describe for the first time the partial repertoires of T2R genes for Lagomorphs species, other than the common rabbit. Our phylogenetic analyses indicate that sequence proximity levels follow the established taxonomic classification. PMID:27092177

  2. Contrasted evolution of the vomeronasal receptor repertoires in mammals and squamate reptiles.

    PubMed

    Brykczynska, Urszula; Tzika, Athanasia C; Rodriguez, Ivan; Milinkovitch, Michel C

    2013-01-01

    The vomeronasal organ (VNO) is an olfactory structure that detects pheromones and environmental cues. It consists of sensory neurons that express evolutionary unrelated groups of transmembrane chemoreceptors. The predominant V1R and V2R receptor repertoires are believed to detect airborne and water-soluble molecules, respectively. It has been suggested that the shift in habitat of early tetrapods from water to land is reflected by an increase in the ratio of V1R/V2R genes. Snakes, which have a very large VNO associated with a sophisticated tongue delivery system, are missing from this analysis. Here, we use RNA-seq and RNA in situ hybridization to study the diversity, evolution, and expression pattern of the corn snake vomeronasal receptor repertoires. Our analyses indicate that snakes and lizards retain an extremely limited number of V1R genes but exhibit a large number of V2R genes, including multiple lineages of reptile-specific and snake-specific expansions. We finally show that the peculiar bigenic pattern of V2R vomeronasal receptor gene transcription observed in mammals is conserved in squamate reptiles, hinting at an important but unknown functional role played by this expression strategy. Our results do not support the hypothesis that the shift to a vomeronasal receptor repertoire dominated by V1Rs in mammals reflects the evolutionary transition of early tetrapods from water to land. This study sheds light on the evolutionary dynamics of the vomeronasal receptor families in vertebrates and reveals how mammals and squamates differentially adapted the same ancestral vomeronasal repertoire to succeed in a terrestrial environment. PMID:23348039

  3. Repertoire of Chemokine Receptor Expression in the Female Genital Tract

    PubMed Central

    Patterson, Bruce K.; Landay, Alan; Andersson, Jan; Brown, Clark; Behbahani, Homira; Jiyamapa, Dan; Burki, Zareefa; Stanislawski, Donna; Czerniewski, Mary Ann; Garcia, Patricia

    1998-01-01

    Sexually transmitted diseases, genital ulcer disease, and progesterone therapy increase susceptibility to lentivirus transmission. Infection of cells by human immunodeficiency virus (HIV) is dependent on expression of specific chemokine receptors known to function as HIV co-receptors. Quantitative kinetic reverse transcription-polymerase chain reaction was developed to determine the in vivo expression levels of CCR5, CXCR4, CCR3, CCR2b, and the cytomegalovirus-encoded US28 in peripheral blood mononuclear cells and cervical biopsies from 12 women with and without sexually transmitted diseases, genital ulcer disease, and progesterone-predominant conditions. Our data indicate that CCR5 is the major HIV co-receptor expressed in the female genital tract, and CXCR4 is the predominantly expressed HIV co-receptor in peripheral blood. CCR5 mRNA expression in the ectocervix was 10-fold greater than CXCR4, 20-fold greater than CCR2b, and 100-fold greater than CCR3. In peripheral blood, CXCR4 expression was 1.5-fold greater than CCR5, 10-fold greater than CCR2b, and 15-fold greater than CCR3. US28 was not expressed in cervical tissue despite expression in peripheral blood mononuclear cells from five individuals. CCR5 was significantly increased (p < 0.02) in biopsies from women with sexually transmitted diseases and others who were progesterone predominant. In vitro studies demonstrate that progesterone increases CCR5, CXCR4, and CCR3 expression and decreases CCR2b expression in lymphocytes and monocytes/macrophages. Characterization of chemokine receptors at the tissue level provides important information in identifying host determinants of HIV-1 transmission. PMID:9708808

  4. Probability model for molecular recognition in biological receptor repertoires: significance to the olfactory system.

    PubMed

    Lancet, D; Sadovsky, E; Seidemann, E

    1993-04-15

    A generalized phenomenological model is presented for stereospecific recognition between biological receptors and their ligands. We ask what is the distribution of binding constants psi(K) between an arbitrary ligand and members of a large receptor repertoire, such as immunoglobulins or olfactory receptors. For binding surfaces with B potential subsite and S different types of subsite configurations, the number of successful elementary interactions obeys a binomial distribution. The discrete probability function psi(K) is then derived with assumptions on alpha, the free energy contribution per elementary interaction. The functional form of psi(K) may be universal, although the parameter values could vary for different ligand types. An estimate of the parameter values of psi(K) for iodovanillin, an analog of odorants and immunological haptens, is obtained by equilibrium dialysis experiments with nonimmune antibodies. Based on a simple relationship, predicted by the model, between the size of a receptor repertoire and its average maximal affinity toward an arbitrary ligand, the size of the olfactory receptor repertoire (Nolf) is calculated as 300-1000, in very good agreement with recent molecular biological studies. A very similar estimate, Nolf = 500, is independently derived by relating a theoretical distribution of maxima for psi(K) with published human olfactory threshold variations. The present model also has implications to the question of olfactory coding and to the analysis of specific anosmias, genetic deficits in perceiving particular odorants. More generally, the proposed model provides a better understanding of ligand specificity in biological receptors and could help in understanding their evolution. PMID:8475121

  5. Probability model for molecular recognition in biological receptor repertoires: significance to the olfactory system.

    PubMed Central

    Lancet, D; Sadovsky, E; Seidemann, E

    1993-01-01

    A generalized phenomenological model is presented for stereospecific recognition between biological receptors and their ligands. We ask what is the distribution of binding constants psi(K) between an arbitrary ligand and members of a large receptor repertoire, such as immunoglobulins or olfactory receptors. For binding surfaces with B potential subsite and S different types of subsite configurations, the number of successful elementary interactions obeys a binomial distribution. The discrete probability function psi(K) is then derived with assumptions on alpha, the free energy contribution per elementary interaction. The functional form of psi(K) may be universal, although the parameter values could vary for different ligand types. An estimate of the parameter values of psi(K) for iodovanillin, an analog of odorants and immunological haptens, is obtained by equilibrium dialysis experiments with nonimmune antibodies. Based on a simple relationship, predicted by the model, between the size of a receptor repertoire and its average maximal affinity toward an arbitrary ligand, the size of the olfactory receptor repertoire (Nolf) is calculated as 300-1000, in very good agreement with recent molecular biological studies. A very similar estimate, Nolf = 500, is independently derived by relating a theoretical distribution of maxima for psi(K) with published human olfactory threshold variations. The present model also has implications to the question of olfactory coding and to the analysis of specific anosmias, genetic deficits in perceiving particular odorants. More generally, the proposed model provides a better understanding of ligand specificity in biological receptors and could help in understanding their evolution. PMID:8475121

  6. Diet shapes the evolution of the vertebrate bitter taste receptor gene repertoire.

    PubMed

    Li, Diyan; Zhang, Jianzhi

    2014-02-01

    Vertebrate Tas2r taste receptors bind to bitter compounds, which are typically poisonous, to elicit bitter sensation to prevent the ingestion of toxins. Previous studies noted a marked variation in the number of Tas2r genes among species, but the underlying cause is unclear. To address this question, we compile the Tas2r gene repertoires from 41 mammals, 4 birds, 2 reptiles, 1 amphibian, and 6 fishes. The number of intact Tas2r genes varies from 0 in the bottlenose dolphin to 51 in the Western clawed frog, with numerous expansions and contractions of the gene family throughout vertebrates, especially among tetrapods. The Tas2r gene number in a species correlates with the fraction of plants in its diet. Because plant tissues contain more toxic compounds than animal tissues do, our observation supports the hypothesis that dietary toxins are a major selective force shaping the diversity of the Tas2r repertoire. PMID:24202612

  7. Diet Shapes the Evolution of the Vertebrate Bitter Taste Receptor Gene Repertoire

    PubMed Central

    Li, Diyan; Zhang, Jianzhi

    2014-01-01

    Vertebrate Tas2r taste receptors bind to bitter compounds, which are typically poisonous, to elicit bitter sensation to prevent the ingestion of toxins. Previous studies noted a marked variation in the number of Tas2r genes among species, but the underlying cause is unclear. To address this question, we compile the Tas2r gene repertoires from 41 mammals, 4 birds, 2 reptiles, 1 amphibian, and 6 fishes. The number of intact Tas2r genes varies from 0 in the bottlenose dolphin to 51 in the Western clawed frog, with numerous expansions and contractions of the gene family throughout vertebrates, especially among tetrapods. The Tas2r gene number in a species correlates with the fraction of plants in its diet. Because plant tissues contain more toxic compounds than animal tissues do, our observation supports the hypothesis that dietary toxins are a major selective force shaping the diversity of the Tas2r repertoire. PMID:24202612

  8. Diversity-oriented approaches for interrogating T-cell receptor repertoire, ligand recognition, and function

    PubMed Central

    Birnbaum, Michael E.; Dong, Shen; Garcia, K. Christopher

    2012-01-01

    Summary Molecular diversity lies at the heart of adaptive immunity. T-cell receptors and peptide-major histocompatibility complex molecules utilize and rely upon an enormous degree of diversity at the levels of genetics, chemistry, and structure to engage one another and carry out their functions. This high level of diversity complicates the systematic study of important aspects of T-cell biology, but recent technical advances have allowed for the ability to study diversity in a comprehensive manner. In this review, we assess insights gained into T-cell receptor function and biology from our increasingly precise ability to assess the T-cell repertoire as a whole or to perturb individual receptors with engineered reagents. We conclude with a perspective on a new class of high-affinity, non-stimulatory peptide ligands we have recently discovered using diversity-oriented techniques that challenges notions for how we think about T-cell receptor signaling. PMID:23046124

  9. In-Depth Assessment of Within-Individual and Inter-Individual Variation in the B Cell Receptor Repertoire

    PubMed Central

    Galson, Jacob D.; Trück, Johannes; Fowler, Anna; Münz, Márton; Cerundolo, Vincenzo; Pollard, Andrew J.; Lunter, Gerton; Kelly, Dominic F.

    2015-01-01

    High-throughput sequencing of the B cell receptor (BCR) repertoire can provide rapid characterization of the B cell response in a wide variety of applications in health, after vaccination and in infectious, inflammatory and immune-driven disease, and is starting to yield clinical applications. However, the interpretation of repertoire data is compromised by a lack of studies to assess the intra and inter-individual variation in the BCR repertoire over time in healthy individuals. We applied a standardized isotype-specific BCR repertoire deep sequencing protocol to a single highly sampled participant, and then evaluated the method in 9 further participants to comprehensively describe such variation. We assessed total repertoire metrics of mutation, diversity, VJ gene usage and isotype subclass usage as well as tracking specific BCR sequence clusters. There was good assay reproducibility (both in PCR amplification and biological replicates), but we detected striking fluctuations in the repertoire over time that we hypothesize may be due to subclinical immune activation. Repertoire properties were unique for each individual, which could partly be explained by a decrease in IgG2 with age, and genetic differences at the immunoglobulin locus. There was a small repertoire of public clusters (0.5, 0.3, and 1.4% of total IgA, IgG, and IgM clusters, respectively), which was enriched for expanded clusters containing sequences with suspected specificity toward antigens that should have been historically encountered by all participants through prior immunization or infection. We thus provide baseline BCR repertoire information that can be used to inform future study design, and aid in interpretation of results from these studies. Furthermore, our results indicate that BCR repertoire studies could be used to track changes in the public repertoire in and between populations that might relate to population immunity against infectious diseases, and identify the characteristics of

  10. Taste and odorant receptors of the coelacanth--a gene repertoire in transition.

    PubMed

    Picone, Barbara; Hesse, Uljana; Panji, Sumir; Van Heusden, Peter; Jonas, Mario; Christoffels, Alan

    2014-09-01

    G-protein coupled chemosensory receptors (GPCR-CRs) aid in the perception of odors and tastes in vertebrates. So far, six GPCR-CR families have been identified that are conserved in most vertebrate species. Phylogenetic analyses indicate differing evolutionary dynamics between teleost fish and tetrapods. The coelacanth Latimeria chalumnae belongs to the lobe-finned fishes, which represent a phylogenetic link between these two groups. We searched the genome of L. chalumnae for GPCR-CRs and found that coelacanth taste receptors are more similar to those in tetrapods than in teleost fish: two coelacanth T1R2s co-segregate with the tetrapod T1R2s that recognize sweet substances, and our phylogenetic analyses indicate that the teleost T1R2s are closer related to T1R1s (umami taste receptors) than to tetrapod T1R2s. Furthermore, coelacanths are the first fish with a large repertoire of bitter taste receptors (58 T2Rs). Considering current knowledge on feeding habits of coelacanths the question arises if perception of bitter taste is the only function of these receptors. Similar to teleost fish, coelacanths have a variety of olfactory receptors (ORs) necessary for perception of water-soluble substances. However, they also have seven genes in the two tetrapod OR subfamilies predicted to recognize airborne molecules. The two coelacanth vomeronasal receptor families are larger than those in teleost fish, and similar to tetrapods and form V1R and V2R monophyletic clades. This may point to an advanced development of the vomeronasal organ as reported for lungfish. Our results show that the intermediate position of Latimeria in the phylogeny is reflected in its GPCR-CR repertoire. PMID:24106203

  11. Origin of basal activity in mammalian olfactory receptor neurons

    PubMed Central

    2010-01-01

    Mammalian odorant receptors form a large, diverse group of G protein–coupled receptors that determine the sensitivity and response profile of olfactory receptor neurons. But little is known if odorant receptors control basal and also stimulus-induced cellular properties of olfactory receptor neurons other than ligand specificity. This study demonstrates that different odorant receptors have varying degrees of basal activity, which drives concomitant receptor current fluctuations and basal action potential firing. This basal activity can be suppressed by odorants functioning as inverse agonists. Furthermore, odorant-stimulated olfactory receptor neurons expressing different odorant receptors can have strikingly different response patterns in the later phases of prolonged stimulation. Thus, the influence of odorant receptor choice on response characteristics is much more complex than previously thought, which has important consequences on odor coding and odor information transfer to the brain. PMID:20974772

  12. Sequence analysis of a bitter taste receptor gene repertoires in different ruminant species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bitter taste has been extensively studied in mammalian species and is associated with sensitivity to toxins and with food choices that avoid dangerous substances in the diet. At the molecular level, bitter compounds are sensed by bitter taste receptor proteins (T2R) present at the surface of taste r...

  13. Discrimination of T-cell subsets and T-cell receptor repertoire distribution.

    PubMed

    Bretschneider, Isabell; Clemente, Michael J; Meisel, Christian; Guerreiro, Manuel; Streitz, Mathias; Hopfenmüller, Werner; Maciejewski, Jaroslav P; Wlodarski, Marcin W; Volk, Hans-Dieter

    2014-01-01

    Flow cytometry-based analysis of T-cell receptor (TCR) repertoires is an essential tool for the detection of clonal T-cell expansions in physiologic and pathologic conditions. Individual T-cell subsets can be investigated based on their surface properties. The aims of our study were to provide reference values for various disease settings and delineate the contribution of individual TCR repertoires to the human T-cell differentiation pathway. We analyzed blood of 66 healthy subjects aged 0 (cord blood) to 72 years. Lymphocyte subpopulations and TCR repertoires were simultaneously explored using antibodies specific to CD3, CD4, CD8, CD45RA, CCR7, CD27, CD57 and a set of 25 antibodies detecting human TCR-Vβ chains. Statistical analysis included Wilcoxon, paired t and ANOVA tests. Initially, TCR expansion values were calculated based on the analysis of TCR-Vβ distribution on CD4+ and CD8+ T cells. We then established gating strategies and an algorithm for data analysis allowing for discrimination of T-cell subsets and TCR distribution. Dominant TCR expansions were present within effector as opposed to central/effector memory or naive cells, e.g., median TCR-Vβ expansion rate was highest on CD45RA+/CCR7- effector CD4+/8+ cells (eight and 11-fold, respectively). Remarkably, TCR expansions were missing (0) or very low (0.5) on CD4+ and CD8+ central memory population, respectively. No significant gender-related variability of TCR repertoires was identified, and significant impact of chronic cytomegalovirus infection was shown. Our results serve as reference for future studies elucidating clonal TCR dominance of T-cell subsets. PMID:24272857

  14. Ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes.

    PubMed

    McDaniel, Jonathan R; DeKosky, Brandon J; Tanno, Hidetaka; Ellington, Andrew D; Georgiou, George

    2016-03-01

    High-throughput sequencing of the variable domains of immune receptors (antibodies and T cell receptors (TCRs)) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH+VL or TCRβ/δ+TCRα/γ) at the single-cell level for typical samples containing >10(4) lymphocytes is problematic, because immune receptors comprise two polypeptide chains that are encoded by separate mRNAs. Here we present a technology that allows rapid and low-cost determination of a paired immune receptor repertoire from millions of cells with high precision (>97%). Flow focusing is used to encapsulate single cells in emulsions containing magnetic beads for mRNA capture. The mRNA transcripts are then reverse-transcribed, physically linked to their partners by overlap extension PCR, and interrogated by high-throughput paired-end Illumina sequencing. This protocol describes the construction and operation of the flow-focusing device in detail, as well as the bioinformatics pipeline used to interpret the data. The entire procedure can be performed by a single researcher in under 12 h of effort per sample. PMID:26844430

  15. The receptors and cells for mammalian taste.

    PubMed

    Chandrashekar, Jayaram; Hoon, Mark A; Ryba, Nicholas J P; Zuker, Charles S

    2006-11-16

    The emerging picture of taste coding at the periphery is one of elegant simplicity. Contrary to what was generally believed, it is now clear that distinct cell types expressing unique receptors are tuned to detect each of the five basic tastes: sweet, sour, bitter, salty and umami. Importantly, receptor cells for each taste quality function as dedicated sensors wired to elicit stereotypic responses. PMID:17108952

  16. Birds Generally Carry a Small Repertoire of Bitter Taste Receptor Genes

    PubMed Central

    Wang, Kai; Zhao, Huabin

    2015-01-01

    As they belong to the most species-rich class of tetrapod vertebrates, birds have long been believed to possess an inferior taste system. However, the bitter taste is fundamental in birds to recognize dietary toxins (which are typically bitter) in potential food sources. To characterize the evolution of avian bitter taste receptor genes (Tas2rs) and to test whether dietary toxins have shaped the repertoire size of avian Tas2rs, we examined 48 genomes representing all but 3 avian orders. The total number of Tas2r genes was found to range from 1 in the domestic pigeon to 12 in the bar-tailed trogon, with an average of 4, which suggested that a much smaller Tas2r gene repertoire exists in birds than in other vertebrates. Furthermore, we uncovered a positive correlation between the number of putatively functional Tas2rs and the abundance of potential toxins in avian diets. Because plant products contain more toxins than animal tissues and insects release poisonous defensive secretions, we hypothesized that herbivorous and insectivorous birds may demand more functional Tas2rs than carnivorous birds feeding on noninsect animals. Our analyses appear to support this hypothesis and highlight the critical role of taste perception in birds. PMID:26342138

  17. Birds Generally Carry a Small Repertoire of Bitter Taste Receptor Genes.

    PubMed

    Wang, Kai; Zhao, Huabin

    2015-09-01

    As they belong to the most species-rich class of tetrapod vertebrates, birds have long been believed to possess an inferior taste system. However, the bitter taste is fundamental in birds to recognize dietary toxins (which are typically bitter) in potential food sources. To characterize the evolution of avian bitter taste receptor genes (Tas2rs) and to test whether dietary toxins have shaped the repertoire size of avian Tas2rs, we examined 48 genomes representing all but 3 avian orders. The total number of Tas2r genes was found to range from 1 in the domestic pigeon to 12 in the bar-tailed trogon, with an average of 4, which suggested that a much smaller Tas2r gene repertoire exists in birds than in other vertebrates. Furthermore, we uncovered a positive correlation between the number of putatively functional Tas2rs and the abundance of potential toxins in avian diets. Because plant products contain more toxins than animal tissues and insects release poisonous defensive secretions, we hypothesized that herbivorous and insectivorous birds may demand more functional Tas2rs than carnivorous birds feeding on noninsect animals. Our analyses appear to support this hypothesis and highlight the critical role of taste perception in birds. PMID:26342138

  18. Glycosaminoglycan receptors facilitate infection of mammalian cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A growing list of viruses has been reported to use more than one receptor for binding and internalization during infection of the host cell. Sialic acid residues or glycosaminoglycans, such as heparin sulfate, frequently function in this scenario, as a first contact, charge based, low affinity bindi...

  19. Targeting of cancer neoantigens with donor-derived T cell receptor repertoires.

    PubMed

    Strønen, Erlend; Toebes, Mireille; Kelderman, Sander; van Buuren, Marit M; Yang, Weiwen; van Rooij, Nienke; Donia, Marco; Böschen, Maxi-Lu; Lund-Johansen, Fridtjof; Olweus, Johanna; Schumacher, Ton N

    2016-06-10

    Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation-derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies to broaden neoantigen-specific T cell responses are therefore attractive. We found that naïve T cell repertoires of healthy blood donors provide a source of neoantigen-specific T cells, responding to 11 of 57 predicted human leukocyte antigen (HLA)-A*02:01-binding epitopes from three patients. Many of the T cell reactivities involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes. Finally, T cells redirected with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such "outsourced" immune responses in cancer immunotherapy. PMID:27198675

  20. Smallest bitter taste receptor (T2Rs) gene repertoire in carnivores.

    PubMed

    Hu, Ling-Ling; Shi, Peng

    2013-06-01

    Bitter taste reception is presumably associated with dietary selection, preventing animals from ingesting potentially harmful compounds. Accordingly, carnivores, who encounter these toxic substances less often, should have fewer genes associated with bitter taste reception compared with herbivores and omnivores. To investigate the genetic basis of bitter taste reception, we confirmed bitter taste receptor (T2R) genes previously found in the genome sequences of two herbivores (cow and horse), two omnivores (mouse and rat) and one carnivore (dog). We also identified, for the first time, the T2R repertoire from the genome of other four carnivore species (ferret, giant panda, polar bear and cat) and detected 17-20 bitter receptor genes from the five carnivore genomes, including 12-16 intact genes, 0-1 partial but putatively functional genes, and 3-8 pseudogenes. Both the intact T2R genes and the total T2R gene number among carnivores were the smallest among the tested species, supporting earlier speculations that carnivores have fewer T2R genes, herbivores an intermediate number, and omnivores the largest T2R gene repertoire. To further explain the genetic basis for this disparity, we constructed a phylogenetic tree, which showed most of the T2R genes from the five carnivores were one-to-one orthologs across the tree, suggesting that carnivore T2Rs were conserved among mammals. Similarly, the small carnivore T2R family size was likely due to rare duplication events. Collectively, these results strengthen arguments for the connection between T2R gene family size, diet and habit. PMID:23776004

  1. Mammalian EGF receptor activation by the rhomboid protease RHBDL2.

    PubMed

    Adrain, Colin; Strisovsky, Kvido; Zettl, Markus; Hu, Landian; Lemberg, Marius K; Freeman, Matthew

    2011-05-01

    The epidermal growth factor receptor (EGFR) has several functions in mammalian development and disease, particularly cancer. Most EGF ligands are synthesized as membrane-tethered precursors, and their proteolytic release activates signalling. In Drosophila, rhomboid intramembrane proteases catalyse the release of EGF-family ligands; however, in mammals this seems to be primarily achieved by ADAM-family metalloproteases. We report here that EGF is an efficient substrate of the mammalian rhomboid RHBDL2. RHBDL2 cleaves EGF just outside its transmembrane domain, thereby facilitating its secretion and triggering activation of the EGFR. We have identified endogenous RHBDL2 activity in several tumour cell lines. PMID:21494248

  2. Mammalian EGF receptor activation by the rhomboid protease RHBDL2

    PubMed Central

    Adrain, Colin; Strisovsky, Kvido; Zettl, Markus; Hu, Landian; Lemberg, Marius K; Freeman, Matthew

    2011-01-01

    The epidermal growth factor receptor (EGFR) has several functions in mammalian development and disease, particularly cancer. Most EGF ligands are synthesized as membrane-tethered precursors, and their proteolytic release activates signalling. In Drosophila, rhomboid intramembrane proteases catalyse the release of EGF-family ligands; however, in mammals this seems to be primarily achieved by ADAM-family metalloproteases. We report here that EGF is an efficient substrate of the mammalian rhomboid RHBDL2. RHBDL2 cleaves EGF just outside its transmembrane domain, thereby facilitating its secretion and triggering activation of the EGFR. We have identified endogenous RHBDL2 activity in several tumour cell lines. PMID:21494248

  3. Sequential recovery of NK cell receptor repertoire after allogeneic hematopoietic SCT.

    PubMed

    Giebel, S; Dziaczkowska, J; Czerw, T; Wojnar, J; Krawczyk-Kulis, M; Nowak, I; Holowiecka, A; Segatti, A; Kyrcz-Krzemien, S; Kusnierczyk, P; Holowiecki, J

    2010-06-01

    Alloreactivity of natural killer (NK) cells contributes to the GVL reaction after allogeneic hematopoietic SCT (allo-HSCT). However, various procedure-related factors may affect NK cell maturation and their ability to recognize and kill leukemic cells. In this study, we prospectively evaluated expression of NK cell inhibitory receptors in 83 adults treated with myeloablative, killer cell Ig-like receptor (KIR)-ligand-matched allo-HSCT. NK cell maturation was evaluated by comparing the phenotypic patterns after allo-HSCT with the donor ones. The frequencies of KIR3DL1 were comparable to the donor ones on day +28, while they decreased significantly starting from day +100. The expression of KIR2DL2/3 was significantly lower in patients compared with donors up to day +100. The expression of KIR2DL1, despite continues growth, remained significantly decreased for 1 year after allo-HSCT. NKG2A was over-expressed up to day +180. Within 1 year after allo-HSCT, the NK cell phenotypic pattern tended to recapitulate the donor type. The process was disturbed by the use of steroids with significant differences observed on days +56 (P=0.01) and +100 (P=0.04). Up to day +100, reconstitution of NK cell receptor repertoire correlated with the absolute numbers of circulating CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells. Our observations should be taken into account when trying to predict potential benefit from NK cell alloreactivity. PMID:20118994

  4. The gentle touch receptors of mammalian skin.

    PubMed

    Zimmerman, Amanda; Bai, Ling; Ginty, David D

    2014-11-21

    The skin is our largest sensory organ, transmitting pain, temperature, itch, and touch information to the central nervous system. Touch sensations are conveyed by distinct combinations of mechanosensory end organs and the low-threshold mechanoreceptors (LTMRs) that innervate them. Here we explore the various structures underlying the diverse functions of cutaneous LTMR end organs. Beyond anchoring of LTMRs to the surrounding dermis and epidermis, recent evidence suggests that the non-neuronal components of end organs play an active role in signaling to LTMRs and may physically gate force-sensitive channels in these receptors. Combined with LTMR intrinsic properties, the balance of these factors comprises the response properties of mechanosensory neurons and, thus, the neural encoding of touch. PMID:25414303

  5. The gentle touch receptors of mammalian skin

    PubMed Central

    Zimmerman, Amanda; Bai, Ling; Ginty, David D.

    2015-01-01

    The skin is our largest sensory organ, transmitting pain, temperature, itch, and touch information to the central nervous system. Touch sensations are conveyed by distinct combinations of mechanosensory end organs and the low-threshold mechanoreceptors (LTMRs) that innervate them. Here we explore the various structures underlying the diverse functions of cutaneous LTMR end organs. Beyond anchoring of LTMRs to the surrounding dermis and epidermis, recent evidence suggests that the non-neuronal components of end organs play an active role in signaling to LTMRs and may physically gate force-sensitive channels in these receptors. Combined with LTMR intrinsic properties, the balance of these factors comprises the response properties of mechanosensory neurons and, thus, the neural encoding of touch. PMID:25414303

  6. Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation.

    PubMed

    Link, C S; Eugster, A; Heidenreich, F; Rücker-Braun, E; Schmiedgen, M; Oelschlägel, U; Kühn, D; Dietz, S; Fuchs, Y; Dahl, A; Domingues, A M J; Klesse, C; Schmitz, M; Ehninger, G; Bornhäuser, M; Schetelig, J; Bonifacio, E

    2016-06-01

    Allogeneic stem cell transplantation is potentially curative, but associated with post-transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR-α repertoire and its clinical relevance in patients following stem cell transplantation. Using next-generation sequencing we examined the TCR-α repertoire of CD8(+) T cells and CMV-specific CD8(+) T cells in four patients. Additionally, we performed single-cell TCR-αβ sequencing of CMV-specific CD8(+) T cells. The TCR-α composition of human leucocyte antigen (HLA)-A*0201 CMVpp65- and CMVIE -specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8(+) T cells and half of the total CD8(+) T cell repertoire was dominated by few CMV-reactive clonotypes. Some TCR-α clonotypes were shared between patients. Gene expression of the circulating CMV-specific CD8(+) T cells was consistent with chronically activated effector memory T cells. The CD8(+) T cell response to CMV reactivation resulted in an expansion of a few TCR-α clonotypes to dominate the CD8(+) repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti-viral T cell response in this setting. PMID:26800118

  7. Structure of a mammalian ryanodine receptor

    PubMed Central

    Zalk, Ran; Clarke, Oliver B.; des Georges, Amédée; Grassucci, Robert A.; Reiken, Steven; Mancia, Filippo; Hendrickson, Wayne A.; Frank, Joachim; Marks, Andrew R.

    2014-01-01

    Ryanodine receptors (RyRs) mediate rapid release of calcium (Ca2+) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here, we report the closed-state structure of the 2.3 MDa complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle cryo-electron microscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3939 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane (6TM) ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca2+. PMID:25470061

  8. Functional analysis of a mammalian odorant receptor subfamily

    PubMed Central

    Abaffy, Tatjana; Matsunami, Hiroaki; Luetje, Charles W.

    2014-01-01

    Phylogenetic analysis groups mammalian odorant receptors into two broad classes and numerous subfamilies. These subfamilies are proposed to reflect functional organization. Testing this idea requires an assay allowing detailed functional characterization of odorant receptors. Here we show that a variety of Class I and Class II mouse odorant receptors can be functionally expressed in Xenopus laevis oocytes. Receptor constructs included the N-terminal 20 residues of human rhodopsin and were coexpressed with Gαolf and the cystic fibrosis transmembrane regulator to allow electrophysiological measurement of receptor responses. For most mouse odorant receptors tested, these conditions were sufficient for functional expression. Co-expression of accessory proteins was required to allow functional surface expression of some mouse odorant receptors. We used this assay to examine the receptive ranges of all members of the MOR42 subfamily. MOR42-1 responded to dicarboxylic acids, preferring a 10–12 carbon chain length. MOR42-2 responded to monocarboxylic acids (7–10 carbons). MOR42-3 responded to dicarboxylic acids (8–10 carbons) and monocarboxylic acids (10–12 carbons). Thus, the receptive range of each receptor was unique. However, overlap between the individual receptive ranges suggests that the members of this subfamily form one contiguous subfamily receptive range, suggesting that odorant receptor subfamilies do constitute functional units. PMID:16606354

  9. Analysis of the vomeronasal receptor repertoire, expression and allelic diversity in swine.

    PubMed

    Dinka, Hunduma; Le, Minh Thong; Ha, Heekyun; Cho, Hyesun; Choi, Min-Kyeung; Choi, Hojun; Kim, Jin-Hoi; Soundarajan, Nagasundarapandian; Park, Jin-Ki; Park, Chankyu

    2016-05-01

    Here we report a comprehensive analysis of the vomeronasal receptor repertoire in pigs. We identified a total of 25 V1R sequences consisting of 10 functional genes, 3 pseudogenes, and 12 partial genes, while functional V2R and FPR genes were not present in the pig genome. Pig V1Rs were classified into three subfamilies, D, F, and J. Using direct high resolution sequencing-based typing of all functional V1Rs from 10 individuals of 5 different breeds, a total of 24 SNPs were identified, indicating that the allelic diversity of V1Rs is much lower than that of the olfactory receptors. A high expression level of V1Rs was detected in the vomeronasal organ (VNO) and testes, while a low expression level of V1Rs was observed in all other tissues examined. Our results showed that pigs could serve as an interesting large animal model system to study pheromone-related neurobiology because of their genetic simplicity. PMID:26482471

  10. An altered repertoire of T cell receptor V gene expression by rheumatoid synovial fluid T lymphocytes.

    PubMed

    Lunardi, C; Marguerie, C; So, A K

    1992-12-01

    The pattern of T cell receptor V gene expression by lymphocytes from rheumatoid synovial fluid and paired peripheral blood samples was compared using a polymerase chain reaction (PCR)-based assay. Eight rheumatoid arthritis (RA) patients who had varying durations of disease (from 2 to 20 years) were studied. In all patients there was evidence of a different pattern of V gene expression between the two compartments. Significantly increased expression of at least one V alpha or V beta gene family by synovial fluid T cells was observed in all the patients studied. Three different V alpha (V alpha 10, 15 and 18) and three V beta (V beta 4, 5 and 13) families were commonly elevated. Sequencing of synovial V beta transcripts demonstrated that the basis of increased expression of selected V gene families in the synovial fluid was due to the presence of dominant clonotypes within those families, which constituted up to 53% of the sequences isolated from one particular synovial V gene family. There were considerable differences in the NDJ sequences found in synovial and peripheral blood T cell receptor (TCR) transcripts of the same V beta gene family. These data suggest that the TCR repertoire in the two compartments differs, and that antigen-driven expansion of particular synovial T cell populations is a component of rheumatoid synovitis, and is present in all stages of the disease. PMID:1458680

  11. The repertoire of G-protein-coupled receptors in fully sequenced genomes.

    PubMed

    Fredriksson, Robert; Schiöth, Helgi B

    2005-05-01

    The superfamily of G-protein-coupled receptors (GPCRs) is one of the largest and most studied families of proteins. We created Hidden Markov Models derived from sorted groups of GPCRs from our previous detailed phylogenetic classification of human GPCRs and added several other models derived from receptors not found in mammals. We used these models to search entire Genscan data sets from 13 species whose genomes are nearly completely sequenced. We found more than 5000 unique GPCRs that were divided into 15 main groups, and the largest one, the Rhodopsin family, was subdivided into 13 subclasses. The results show that the main families in the human genome, Glutamate, Rhodopsin, Adhesion, Frizzled, and Secretin, arose before the split of nematodes from the chordate lineage. Moreover, several of the subgroups of the Rhodopsin family arose before the split of the linage leading to vertebrates. We also searched expressed sequence tag (EST) databases and identified more than 20,000 sequences that match GPCRs. Although the GPCRs represent typically 1 to 2% of the Genscan predictions, the ESTs that match GPCRs are typically only 0.01 to 0.001%, indicating that GPCRs in most of the groups are expressed at low levels. We also provide searchable data sets that may be used for annotation and further detailed analysis of the GPCR family. This study provides an extensive overview of the expansion of the gene repertoire for families and subgroups of GPCRs. PMID:15687224

  12. Intracellular trafficking of a tagged and functional mammalian olfactory receptor.

    PubMed

    Ivic, Lidija; Zhang, Cen; Zhang, Xinmin; Yoon, Sung Ok; Firestein, Stuart

    2002-01-01

    Tagged G-protein-coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N-terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C-terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N-terminus, or EGFP located at the C-terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells. PMID:11748633

  13. Next Generation Sequencing Reveals Skewing of the T and B Cell Receptor Repertoires in Patients with Wiskott–Aldrich Syndrome

    PubMed Central

    O’Connell, Amy E.; Volpi, Stefano; Dobbs, Kerry; Fiorini, Claudia; Tsitsikov, Erdyni; de Boer, Helen; Barlan, Isil B.; Despotovic, Jenny M.; Espinosa-Rosales, Francisco J.; Hanson, I. Celine; Kanariou, Maria G.; Martínez-Beckerat, Roxana; Mayorga-Sirera, Alvaro; Mejia-Carvajal, Carmen; Radwan, Nesrine; Weiss, Aaron R.; Pai, Sung-Yun; Lee, Yu Nee; Notarangelo, Luigi D.

    2014-01-01

    The Wiskott–Aldrich syndrome (WAS) is due to mutations of the WAS gene encoding for the cytoskeletal WAS protein, leading to abnormal downstream signaling from the T cell and B cell antigen receptors (TCR and BCR). We hypothesized that the impaired signaling through the TCR and BCR in WAS would subsequently lead to aberrations in the immune repertoire of WAS patients. Using next generation sequencing (NGS), the T cell receptor β and B cell immunoglobulin heavy chain (IGH) repertoires of eight patients with WAS and six controls were sequenced. Clonal expansions were identified within memory CD4+ cells as well as in total, naïve and memory CD8+ cells from WAS patients. In the B cell compartment, WAS patient IGH repertoires were also clonally expanded and showed skewed usage of IGHV and IGHJ genes, and increased usage of IGHG constant genes, compared with controls. To our knowledge, this is the first study that demonstrates significant abnormalities of the immune repertoire in WAS patients using NGS. PMID:25101082

  14. Rejection of cardiac allografts by T cells expressing a restricted repertoire of T-cell receptor V beta genes.

    PubMed Central

    Shirwan, H; Barwari, L; Cramer, D V

    1997-01-01

    We have recently shown that T cells infiltrating cardiac allografts early in graft rejection use a limited T-cell receptor (TCR) V beta repertoire. In this study we tested whether this limited repertoire of V beta genes is important for graft rejection. A cell line, AL2-L3, was established from LEW lymphocytes infiltrating ACI heart allografts 2 days after transplantation. This cell line is composed of CD4+ T cells that primarily recognize the class II RTI.B major histocompatibility complex (MHC) molecule expressed by the donor graft. This cell line precipitated acute rejection of donor hearts with a median survival time (MST) of 10.5 days following adoptive transfer to sublethally irradiated LEW recipients. This rate of graft rejection was significantly (P < 0.0007) accelerated when compared with a MST of 60 days for allografts in irradiated control recipients. The AL2-L3-mediated acceleration of graft rejection was donor specific as WF third-party heart allografts were rejected with a delayed tempo (MST = 28.5 days). The V beta repertoire of this cell line was primarily restricted to the expression of V beta 4, 15 and 19 genes. The nucleotide sequence analysis of the beta-chain cDNAs from this cell line demonstrated that the restricted use of the V gene repertoire was not shared with the N, D and J regions. A wide variety of CDR3 loops and J beta genes were used in association with selected V beta genes. These data provide evidence for the role a restricted repertoire of V beta genes plays in cardiac allograft rejection in this model. The restricted usage of the V beta repertoire in an early T-cell response to allografts may provide the opportunity to therapeutically disrupt the rejection reaction by targeting selected T-cell populations for elimination at the time of organ transplantation. Images Figure 2 PMID:9176111

  15. Recognition of Bacterial Signal Peptides by Mammalian Formyl Peptide Receptors

    PubMed Central

    Bufe, Bernd; Schumann, Timo; Kappl, Reinhard; Bogeski, Ivan; Kummerow, Carsten; Podgórska, Marta; Smola, Sigrun; Hoth, Markus; Zufall, Frank

    2015-01-01

    Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system. PMID:25605714

  16. An Endogenous Mammalian Retinoid X Receptor Ligand, At Last!

    PubMed

    de Lera, Ángel R; Krezel, Wojciech; Rühl, Ralph

    2016-05-19

    9-cis-Retinoic acid was identified and claimed to be the endogenous ligand of the retinoid X receptors (RXRs) in 1992. Since then, the endogenous presence of this compound has never been rigorously confirmed. Instead, concerns have been raised by other groups that have reported that 9-cis-retinoic acid is undetectable or that its presence occurs at very low levels. Furthermore, these low levels could not satisfactorily explain the physiological activation of RXR. Alternative ligands, among them various lipids, have also been identified, but also did not fulfill criteria for rigorous endogenous relevance, and their consideration as bona fide endogenous mammalian RXR ligand has likewise been questioned. Recently, novel studies claim that the saturated analogue 9-cis-13,14-dihydroretinoic acid functions as an endogenous physiologically relevant mammalian RXR ligand. PMID:27151148

  17. Genomic architecture of MHC-linked odorant receptor gene repertoires among 16 vertebrate species.

    PubMed

    Santos, Pablo Sandro Carvalho; Kellermann, Thomas; Uchanska-Ziegler, Barbara; Ziegler, Andreas

    2010-09-01

    The recent sequencing and assembly of the genomes of different organisms have shown that almost all vertebrates studied in detail so far have one or more clusters of genes encoding odorant receptors (OR) in close physical linkage to the major histocompatibility complex (MHC). It has been postulated that MHC-linked OR genes could be involved in MHC-influenced mate choice, comprising both pre- as well as post-copulatory mechanisms. We have therefore carried out a systematic comparison of protein sequences of these receptors from the genomes of man, chimpanzee, gorilla, orangutan, rhesus macaque, mouse, rat, dog, cat, cow, pig, horse, elephant, opossum, frog and zebra fish (amounting to a total of 559 protein sequences) in order to identify OR families exhibiting evolutionarily conserved MHC linkage. In addition, we compared the genomic structure of this region within these 16 species, accounting for presence or absence of OR gene families, gene order, transcriptional orientation and linkage to the MHC or framework genes. The results are presented in the form of gene maps and phylogenetic analyses that reveal largely concordant repertoires of gene families, at least among tetrapods, although each of the eight taxa studied (primates, rodents, ungulates, carnivores, proboscids, marsupials, amphibians and teleosts) exhibits a typical architecture of MHC (or MHC framework loci)-linked OR genes. Furthermore, the comparison of the genomic organization of this region has implications for phylogenetic relationships between closely related taxa, especially in disputed cases such as the evolutionary history of even- and odd-toed ungulates and carnivores. Finally, the largely conserved linkage between distinct OR genes and the MHC supports the concept that particular alleles within a given haplotype function in a concerted fashion during self-/non-self-discrimination processes in reproduction. PMID:20680261

  18. Whole Genome Duplications Shaped the Receptor Tyrosine Kinase Repertoire of Jawed Vertebrates

    PubMed Central

    Brunet, Frédéric G.; Volff, Jean-Nicolas; Schartl, Manfred

    2016-01-01

    The receptor tyrosine kinase (RTK) gene family, involved primarily in cell growth and differentiation, comprises proteins with a common enzymatic tyrosine kinase intracellular domain adjacent to a transmembrane region. The amino-terminal portion of RTKs is extracellular and made of different domains, the combination of which characterizes each of the 20 RTK subfamilies among mammals. We analyzed a total of 7,376 RTK sequences among 143 vertebrate species to provide here the first comprehensive census of the jawed vertebrate repertoire. We ascertained the 58 genes previously described in the human and mouse genomes and established their phylogenetic relationships. We also identified five additional RTKs amounting to a total of 63 genes in jawed vertebrates. We found that the vertebrate RTK gene family has been shaped by the two successive rounds of whole genome duplications (WGD) called 1R and 2R (1R/2R) that occurred at the base of the vertebrates. In addition, the Vegfr and Ephrin receptor subfamilies were expanded by single gene duplications. In teleost fish, 23 additional RTK genes have been retained after another expansion through the fish-specific third round (3R) of WGD. Several lineage-specific gene losses were observed. For instance, birds have lost three RTKs, and different genes are missing in several fish sublineages. The RTK gene family presents an unusual high gene retention rate from the vertebrate WGDs (58.75% after 1R/2R, 64.4% after 3R), resulting in an expansion that might be correlated with the evolution of complexity of vertebrate cellular communication and intracellular signaling. PMID:27260203

  19. Whole Genome Duplications Shaped the Receptor Tyrosine Kinase Repertoire of Jawed Vertebrates.

    PubMed

    Brunet, Frédéric G; Volff, Jean-Nicolas; Schartl, Manfred

    2016-01-01

    The receptor tyrosine kinase (RTK) gene family, involved primarily in cell growth and differentiation, comprises proteins with a common enzymatic tyrosine kinase intracellular domain adjacent to a transmembrane region. The amino-terminal portion of RTKs is extracellular and made of different domains, the combination of which characterizes each of the 20 RTK subfamilies among mammals. We analyzed a total of 7,376 RTK sequences among 143 vertebrate species to provide here the first comprehensive census of the jawed vertebrate repertoire. We ascertained the 58 genes previously described in the human and mouse genomes and established their phylogenetic relationships. We also identified five additional RTKs amounting to a total of 63 genes in jawed vertebrates. We found that the vertebrate RTK gene family has been shaped by the two successive rounds of whole genome duplications (WGD) called 1R and 2R (1R/2R) that occurred at the base of the vertebrates. In addition, the Vegfr and Ephrin receptor subfamilies were expanded by single gene duplications. In teleost fish, 23 additional RTK genes have been retained after another expansion through the fish-specific third round (3R) of WGD. Several lineage-specific gene losses were observed. For instance, birds have lost three RTKs, and different genes are missing in several fish sublineages. The RTK gene family presents an unusual high gene retention rate from the vertebrate WGDs (58.75% after 1R/2R, 64.4% after 3R), resulting in an expansion that might be correlated with the evolution of complexity of vertebrate cellular communication and intracellular signaling. PMID:27260203

  20. The complex NOD-like receptor repertoire of the coral Acropora digitifera includes novel domain combinations.

    PubMed

    Hamada, Mayuko; Shoguchi, Eiichi; Shinzato, Chuya; Kawashima, Takeshi; Miller, David J; Satoh, Nori

    2013-01-01

    Innate immunity in corals is of special interest not only in the context of self-defense but also in relation to the establishment and collapse of their obligate symbiosis with dinoflagellates of the genus Symbiodinium. In innate immunity system of vertebrates, approximately 20 tripartite nucleotide oligomerization domain (NOD)-like receptor proteins that are defined by the presence of a NAIP, CIIA, HET-E and TP1 (NACHT) domain, a C-terminal leucine-rich repeat (LRR) domain, and one of three types of N-terminal effector domain, are known to function as the primary intracellular pattern recognition molecules. Surveying the coral genome revealed not only a larger number of NACHT- and related domain nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)-encoding loci (~500) than in other metazoans but also surprising diversity of domain combinations among the coral NACHT/NB-ARC-containing proteins; N-terminal effector domains included the apoptosis-related domains caspase recruitment domain (CARD), death effector domain (DED), and Death, and C-terminal repeat domains included LRRs, tetratricopeptide repeats, ankyrin repeats, and WD40 repeats. Many of the predicted coral proteins that contain a NACHT/NB-ARC domain also contain a glycosyl transferase group 1 domain, a novel domain combination first found in metazoans. Phylogenetic analyses suggest that the NACHT/NB-ARC domain inventories of various metazoan lineages, including corals, are largely products of lineage-specific expansions. Many of the NACHT/NB-ARC loci are organized in pairs or triplets in the Acropora genome, suggesting that the large coral NACHT/NB-ARC repertoire has been generated at least in part by tandem duplication. In addition, shuffling of N-terminal effector domains may have occurred after expansions of specific NACHT/NB-ARC-repeat domain types. These results illustrate the extraordinary complexity of the innate immune repertoire of corals, which may in part reflect adaptive

  1. Deciphering the Receptor Repertoire Encoding Specific Odorants by Time-Lapse Single-Cell Array Cytometry

    PubMed Central

    Suzuki, Masato; Yoshimoto, Nobuo; Shimono, Ken; Kuroda, Shun’ichi

    2016-01-01

    Mammals can recognize a vast number of odorants by using olfactory receptors (ORs) known as G protein-coupled receptors. The OR gene family is one of the most diverse gene families in mammalian genomes. Because of the vast combinations of ORs and odorants, few ORs have thus far been linked to specific odorants. Here, we established a functional screening method for OR genes by using a microchamber array containing >5,400 single olfactory epithelium-derived cells from mice applied to time-lapse single-cell array cytometry. This method facilitated the prompt isolation of single olfactory sensory neurons (OSNs) responding to the odorant of interest. Subsequent single-cell RT-PCR allowed us to isolate the genes encoding respective ORs. By using volatile molecules recognized as biomarkers for lung cancers, this method could deorphanize ORs and thereby reconstitute the OR-mediated signaling cascade in HEK293T cells. Thus, our system could be applied to identify any receptor by using specific ligands in the fields of physiopathology and pharmacology. PMID:26832639

  2. Deciphering the Receptor Repertoire Encoding Specific Odorants by Time-Lapse Single-Cell Array Cytometry.

    PubMed

    Suzuki, Masato; Yoshimoto, Nobuo; Shimono, Ken; Kuroda, Shun'ichi

    2016-01-01

    Mammals can recognize a vast number of odorants by using olfactory receptors (ORs) known as G protein-coupled receptors. The OR gene family is one of the most diverse gene families in mammalian genomes. Because of the vast combinations of ORs and odorants, few ORs have thus far been linked to specific odorants. Here, we established a functional screening method for OR genes by using a microchamber array containing >5,400 single olfactory epithelium-derived cells from mice applied to time-lapse single-cell array cytometry. This method facilitated the prompt isolation of single olfactory sensory neurons (OSNs) responding to the odorant of interest. Subsequent single-cell RT-PCR allowed us to isolate the genes encoding respective ORs. By using volatile molecules recognized as biomarkers for lung cancers, this method could deorphanize ORs and thereby reconstitute the OR-mediated signaling cascade in HEK293T cells. Thus, our system could be applied to identify any receptor by using specific ligands in the fields of physiopathology and pharmacology. PMID:26832639

  3. Receptor-mediated mitophagy in yeast and mammalian systems.

    PubMed

    Liu, Lei; Sakakibara, Kaori; Chen, Quan; Okamoto, Koji

    2014-07-01

    Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease. PMID:24903109

  4. Complex T-Cell Receptor Repertoire Dynamics Underlie the CD8+ T-Cell Response to HIV-1

    PubMed Central

    Costa, Ana I.; Koning, Dan; Ladell, Kristin; McLaren, James E.; Grady, Bart P. X.; Schellens, Ingrid M. M.; van Ham, Petra; Nijhuis, Monique; Borghans, José A. M.; Keşmir, Can; Price, David A.

    2014-01-01

    ABSTRACT Although CD8+ T cells are important for the control of HIV-1 in vivo, the precise correlates of immune efficacy remain unclear. In this study, we conducted a comprehensive analysis of viral sequence variation and T-cell receptor (TCR) repertoire composition across multiple epitope specificities in a group of antiretroviral treatment-naive individuals chronically infected with HIV-1. A negative correlation was detected between changes in antigen-specific TCR repertoire diversity and CD8+ T-cell response magnitude, reflecting clonotypic expansions and contractions related to alterations in cognate viral epitope sequences. These patterns were independent of the individual, as evidenced by discordant clonotype-specific transitions directed against different epitopes in single subjects. Moreover, long-term asymptomatic HIV-1 infection was characterized by evolution of the TCR repertoire in parallel with viral replication. Collectively, these data suggest a continuous bidirectional process of adaptation between HIV-1 and virus-specific CD8+ T-cell clonotypes orchestrated at the TCR-antigen interface. IMPORTANCE We describe a relation between viral epitope mutation, antigen-specific T-cell expansion, and the repertoire of responding clonotypes in chronic HIV-1 infection. This work provides insights into the process of coadaptation between the human immune system and a rapidly evolving lentivirus. PMID:25320304

  5. Network properties derived from deep sequencing of human B-cell receptor repertoires delineate B-cell populations

    PubMed Central

    Bashford-Rogers, Rachael J.M.; Palser, Anne L.; Huntly, Brian J.; Rance, Richard; Vassiliou, George S.; Follows, George A.; Kellam, Paul

    2013-01-01

    The adaptive immune response selectively expands B- and T-cell clones following antigen recognition by B- and T-cell receptors (BCR and TCR), respectively. Next-generation sequencing is a powerful tool for dissecting the BCR and TCR populations at high resolution, but robust computational analyses are required to interpret such sequencing. Here, we develop a novel computational approach for BCR repertoire analysis using established next-generation sequencing methods coupled with network construction and population analysis. BCR sequences organize into networks based on sequence diversity, with differences in network connectivity clearly distinguishing between diverse repertoires of healthy individuals and clonally expanded repertoires from individuals with chronic lymphocytic leukemia (CLL) and other clonal blood disorders. Network population measures defined by the Gini Index and cluster sizes quantify the BCR clonality status and are robust to sampling and sequencing depths. BCR network analysis therefore allows the direct and quantifiable comparison of BCR repertoires between samples and intra-individual population changes between temporal or spatially separated samples and over the course of therapy. PMID:23742949

  6. The mammalian mineralocorticoid receptor: tying down a promiscuous receptor.

    PubMed

    Gomez-Sanchez, Elise P

    2010-01-01

    The mineralocorticoid receptor (MR) has been called a promiscuous receptor because its intrinsic affinity for aldosterone, cortisol and corticosterone is similar. Since glucocorticoids circulate in concentrations 100- to 1000-fold those of aldosterone, stoichiometry dictates that MR should be activated by glucocorticoids, not aldosterone, yet MRs are expressed in many tissues and regulate diverse functions, many of them under the regulation of the renin-angiotensin-aldosterone system. A relatively small number of brain MRs are aldosterone selective and modulate blood pressure. Evidence for possible mechanisms conferring ligand specificity in the context of mineralocorticoid-induced hypertension and the brain are discussed. These include factors (or mechanisms) intrinsic to the receptor, such as alternative splice variants and translation start sites, and extrinsic to the MR, including differential access through the blood-brain barrier, differential recruitment of co-regulators and scaffolding proteins, 11beta-steroid dehydrogenase activity, synthesis of potent acylated aldosterone derivatives and the synthesis of relevant amounts of aldosterone in areas of the brain that modulate blood pressure. PMID:19648477

  7. Evolutionary redesign of the Atlantic cod (Gadus morhua L.) Toll-like receptor repertoire by gene losses and expansions

    PubMed Central

    Solbakken, Monica H.; Tørresen, Ole K.; Nederbragt, Alexander J.; Seppola, Marit; Gregers, Tone F.; Jakobsen, Kjetill S.; Jentoft, Sissel

    2016-01-01

    Genome sequencing of the teleost Atlantic cod demonstrated loss of the Major Histocompatibility Complex (MHC) class II, an extreme gene expansion of MHC class I and gene expansions and losses in the innate pattern recognition receptor (PRR) family of Toll-like receptors (TLR). In a comparative genomic setting, using an improved version of the genome, we characterize PRRs in Atlantic cod with emphasis on TLRs demonstrating the loss of TLR1/6, TLR2 and TLR5 and expansion of TLR7, TLR8, TLR9, TLR22 and TLR25. We find that Atlantic cod TLR expansions are strongly influenced by diversifying selection likely to increase the detectable ligand repertoire through neo- and subfunctionalization. Using RNAseq we find that Atlantic cod TLRs display likely tissue or developmental stage-specific expression patterns. In a broader perspective, a comprehensive vertebrate TLR phylogeny reveals that the Atlantic cod TLR repertoire is extreme with regards to losses and expansions compared to other teleosts. In addition we identify a substantial shift in TLR repertoires following the evolutionary transition from an aquatic vertebrate (fish) to a terrestrial (tetrapod) life style. Collectively, our findings provide new insight into the function and evolution of TLRs in Atlantic cod as well as the evolutionary history of vertebrate innate immunity. PMID:27126702

  8. Evolutionary redesign of the Atlantic cod (Gadus morhua L.) Toll-like receptor repertoire by gene losses and expansions.

    PubMed

    Solbakken, Monica H; Tørresen, Ole K; Nederbragt, Alexander J; Seppola, Marit; Gregers, Tone F; Jakobsen, Kjetill S; Jentoft, Sissel

    2016-01-01

    Genome sequencing of the teleost Atlantic cod demonstrated loss of the Major Histocompatibility Complex (MHC) class II, an extreme gene expansion of MHC class I and gene expansions and losses in the innate pattern recognition receptor (PRR) family of Toll-like receptors (TLR). In a comparative genomic setting, using an improved version of the genome, we characterize PRRs in Atlantic cod with emphasis on TLRs demonstrating the loss of TLR1/6, TLR2 and TLR5 and expansion of TLR7, TLR8, TLR9, TLR22 and TLR25. We find that Atlantic cod TLR expansions are strongly influenced by diversifying selection likely to increase the detectable ligand repertoire through neo- and subfunctionalization. Using RNAseq we find that Atlantic cod TLRs display likely tissue or developmental stage-specific expression patterns. In a broader perspective, a comprehensive vertebrate TLR phylogeny reveals that the Atlantic cod TLR repertoire is extreme with regards to losses and expansions compared to other teleosts. In addition we identify a substantial shift in TLR repertoires following the evolutionary transition from an aquatic vertebrate (fish) to a terrestrial (tetrapod) life style. Collectively, our findings provide new insight into the function and evolution of TLRs in Atlantic cod as well as the evolutionary history of vertebrate innate immunity. PMID:27126702

  9. Repertoire of virus-derived small RNAs produced by mosquito and mammalian cells in response to dengue virus infection.

    PubMed

    Schirtzinger, Erin E; Andrade, Christy C; Devitt, Nicholas; Ramaraj, Thiruvarangan; Jacobi, Jennifer L; Schilkey, Faye; Hanley, Kathryn A

    2015-02-01

    RNA interference (RNAi) is the major defense of many arthropods against arthropod-borne RNA viruses (arboviruses), but the role of RNAi in vertebrate immunity to arboviruses is not clear. RNA viruses can trigger RNAi in vertebrate cells, but the vertebrate interferon response may obscure this interaction. We quantified virus-derived small RNAs (vRNAs) generated by mosquito (U4.4) cells and interferon-deficient (Vero) and interferon-competent (HuH-7) mammalian cells infected with a single isolate of mosquito-borne dengue virus. Mosquito cells produced significantly more vRNAs than mammalian cells, and mosquito cell vRNAs were derived from both the positive- and negative-sense dengue genomes whereas mammalian cell vRNAs were derived primarily from positive-sense genome. Mosquito cell vRNAs were predominantly 21 nucleotides in length whereas mammalian cell vRNAs were between 12 and 36 nucleotides with a modest peak at 24 nucleotides. Hot-spots, regions of the virus genome that generated a disproportionate number of vRNAs, overlapped among the cell lines. PMID:25528416

  10. IMGT/HighV QUEST paradigm for T cell receptor IMGT clonotype diversity and next generation repertoire immunoprofiling.

    PubMed

    Li, Shuo; Lefranc, Marie-Paule; Miles, John J; Alamyar, Eltaf; Giudicelli, Véronique; Duroux, Patrice; Freeman, J Douglas; Corbin, Vincent D A; Scheerlinck, Jean-Pierre; Frohman, Michael A; Cameron, Paul U; Plebanski, Magdalena; Loveland, Bruce; Burrows, Scott R; Papenfuss, Anthony T; Gowans, Eric J

    2013-01-01

    T cell repertoire diversity and clonotype follow-up in vaccination, cancer, infectious and immune diseases represent a major challenge owing to the enormous complexity of the data generated. Here we describe a next generation methodology, which combines 5'RACE PCR, 454 sequencing and, for analysis, IMGT, the international ImMunoGeneTics information system (IMGT), IMGT/HighV-QUEST web portal and IMGT-ONTOLOGY concepts. The approach is validated in a human case study of T cell receptor beta (TRB) repertoire, by chronologically tracking the effects of influenza vaccination on conventional and regulatory T cell subpopulations. The IMGT/HighV-QUEST paradigm defines standards for genotype/haplotype analysis and characterization of IMGT clonotypes for clonal diversity and expression and achieves a degree of resolution for next generation sequencing verifiable by the user at the sequence level, while providing a normalized reference immunoprofile for human TRB. PMID:23995877

  11. Individual heritable differences result in unique cell lymphocyte receptor repertoires of naïve and antigen-experienced cells.

    PubMed

    Rubelt, Florian; Bolen, Christopher R; McGuire, Helen M; Vander Heiden, Jason A; Gadala-Maria, Daniel; Levin, Mikhail; Euskirchen, Ghia M; Mamedov, Murad R; Swan, Gary E; Dekker, Cornelia L; Cowell, Lindsay G; Kleinstein, Steven H; Davis, Mark M

    2016-01-01

    The adaptive immune system's capability to protect the body requires a highly diverse lymphocyte antigen receptor repertoire. However, the influence of individual genetic and epigenetic differences on these repertoires is not typically measured. By leveraging the unique characteristics of B, CD4(+) T and CD8(+) T-lymphocyte subsets from monozygotic twins, we quantify the impact of heritable factors on both the V(D)J recombination process and on thymic selection. We show that the resulting biases in both V(D)J usage and N/P addition lengths, which are found in naïve and antigen experienced cells, contribute to significant variation in the CDR3 region. Moreover, we show that the relative usage of V and J gene segments is chromosomally biased, with ∼1.5 times as many rearrangements originating from a single chromosome. These data refine our understanding of the heritable mechanisms affecting the repertoire, and show that biases are evident on a chromosome-wide level. PMID:27005435

  12. Broad T-Cell Receptor Repertoire in T-Lymphocytes Derived from Human Induced Pluripotent Stem Cells

    PubMed Central

    Chang, Chia-Wei; Lai, Yi-Shin; Lamb, Lawrence S.; Townes, Tim M.

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) have enormous potential for the treatment of inherited and acquired disorders. Recently, antigen-specific T lymphocytes derived from hiPSCs have been reported. However, T lymphocyte populations with broad T cell receptor (TCR) diversity have not been generated. We report that hiPSCs derived from skin biopsy are capable of producing T lymphocyte populations with a broad TCR repertoire. In vitro T cell differentiation follows a similar developmental program as observed in vivo, indicated by sequential expression of CD7, intracellular CD3 and surface CD3. The γδ TCR locus is rearranged first and is followed by rearrangement of the αβ locus. Both γδ and αβ T cells display a diverse TCR repertoire. Upon activation, the cells express CD25, CD69, cytokines (TNF-α, IFN-γ, IL-2) and cytolytic proteins (Perforin and Granzyme-B). These results suggest that most, if not all, mechanisms required to generate functional T cells with a broad TCR repertoire are intact in our in vitro differentiation protocol. These data provide a foundation for production of patient-specific T cells for the treatment of acquired or inherited immune disorders and for cancer immunotherapy. PMID:24828440

  13. Avian olfactory receptor gene repertoires: evidence for a well-developed sense of smell in birds?

    PubMed

    Steiger, Silke S; Fidler, Andrew E; Valcu, Mihai; Kempenaers, Bart

    2008-10-22

    Among vertebrates, the sense of smell is mediated by olfactory receptors (ORs) expressed in sensory neurons within the olfactory epithelium. Comparative genomic studies suggest that the olfactory acuity of mammalian species correlates positively with both the total number and the proportion of functional OR genes encoded in their genomes. In contrast to mammals, avian olfaction is poorly understood, with birds widely regarded as relying primarily on visual and auditory inputs. Here, we show that in nine bird species from seven orders (blue tit, Cyanistes caeruleus; black coucal, Centropus grillii; brown kiwi, Apteryx australis; canary, Serinus canaria; galah, Eolophus roseicapillus; red jungle fowl, Gallus gallus; kakapo, Strigops habroptilus; mallard, Anas platyrhynchos; snow petrel, Pagodroma nivea), the majority of amplified OR sequences are predicted to be from potentially functional genes. This finding is somewhat surprising as one previous report suggested that the majority of OR genes in an avian (red jungle fowl) genomic sequence are non-functional pseudogenes. We also show that it is not the estimated proportion of potentially functional OR genes, but rather the estimated total number of OR genes that correlates positively with relative olfactory bulb size, an anatomical correlate of olfactory capability. We further demonstrate that all the nine bird genomes examined encode OR genes belonging to a large gene clade, termed gamma-c, the expansion of which appears to be a shared characteristic of class Aves. In summary, our findings suggest that olfaction in birds may be a more important sense than generally believed. PMID:18628122

  14. Short latency compound action potentials from mammalian gravity receptor organs

    NASA Technical Reports Server (NTRS)

    Jones, T. A.; Jones, S. M.

    1999-01-01

    Gravity receptor function was characterized in four mammalian species using far-field vestibular evoked potentials (VsEPs). VsEPs are compound action potentials of the vestibular nerve and central relays that are elicited by linear acceleration ramps applied to the cranium. Rats, mice, guinea pigs, and gerbils were studied. In all species, response onset occurred within 1.5 ms of the stimulus onset. Responses persisted during intense (116 dBSPL) wide-band (50 to 50 inverted question mark omitted inverted question mark000 Hz) forward masking, whereas auditory responses to intense clicks (112 dBpeSPL) were eliminated under the same conditions. VsEPs remained after cochlear extirpation but were eliminated following bilateral labyrinthectomy. Responses included a series of positive and negative peaks that occurred within 8 ms of stimulus onset (range of means at +6 dBre: 1.0 g/ms: P1=908 to 1062 micros, N1=1342 to 1475 micros, P2=1632 to 1952 micros, N2=2038 to 2387 micros). Mean response amplitudes at +6 dBre: 1.0 g/ms ranged from 0.14 to 0.99 microV. VsEP input/output functions revealed latency slopes that varied across peaks and species ranging from -19 to -51 micros/dB. Amplitude-intensity slopes also varied ranging from 0.04 to 0.08 microV/dB for rats and mice. Latency values were comparable to those of birds although amplitudes were substantially smaller in mammals. VsEP threshold values were considerably higher in mammals compared to birds and ranged from -8.1 to -10.5 dBre 1.0 g/ms across species. These results support the hypothesis that mammalian gravity receptors are less sensitive to dynamic stimuli than are those of birds.

  15. Functional promiscuity in a mammalian chemosensory system: extensive expression of vomeronasal receptors in the main olfactory epithelium of mouse lemurs.

    PubMed

    Hohenbrink, Philipp; Dempewolf, Silke; Zimmermann, Elke; Mundy, Nicholas I; Radespiel, Ute

    2014-01-01

    The vomeronasal organ (VNO) is functional in most terrestrial mammals, though progressively reduced in the primate lineage, and is used for intraspecific communication and predator recognition. Vomeronasal receptor (VR) genes comprise two families of chemosensory genes (V1R and V2R) that have been considered to be specific for the VNO. However, recently a large number of VRs were reported to be expressed in the main olfactory epithelium (MOE) of mice, but there is little knowledge of the expression of these genes outside of rodents. To explore the function of VR genes in mammalian evolution, we analyzed and compared the expression of 64 V1R and 2 V2R genes in the VNO and the MOE of the gray mouse lemur (Microcebus murinus), the primate with the largest known VR repertoire. We furthermore compared expression patterns in adults of both sexes and seasons, and in an infant. A large proportion (83-97%) of the VR loci was expressed in the VNO of all individuals. The repertoire in the infant was as rich as in adults, indicating reliance on olfactory communication from early postnatal development onwards. In concordance with mice, we also detected extensive expression of VRs in the MOE, with proportions of expressed loci in individuals ranging from 29 to 45%. TRPC2, which encodes a channel protein crucial for signal transduction via VRs, was co-expressed in the MOE in all individuals indicating likely functionality of expressed VR genes in the MOE. In summary, the large VR repertoire in mouse lemurs seems to be highly functional. Given the differences in the neural pathways of MOE and VNO signals, which project to higher cortical brain centers or the limbic system, respectively, this raises the intriguing possibility that the evolution of MOE-expression of VRs enabled mouse lemurs to adaptively diversify the processing of VR-encoded olfactory information. PMID:25309343

  16. Functional promiscuity in a mammalian chemosensory system: extensive expression of vomeronasal receptors in the main olfactory epithelium of mouse lemurs

    PubMed Central

    Hohenbrink, Philipp; Dempewolf, Silke; Zimmermann, Elke; Mundy, Nicholas I.; Radespiel, Ute

    2014-01-01

    The vomeronasal organ (VNO) is functional in most terrestrial mammals, though progressively reduced in the primate lineage, and is used for intraspecific communication and predator recognition. Vomeronasal receptor (VR) genes comprise two families of chemosensory genes (V1R and V2R) that have been considered to be specific for the VNO. However, recently a large number of VRs were reported to be expressed in the main olfactory epithelium (MOE) of mice, but there is little knowledge of the expression of these genes outside of rodents. To explore the function of VR genes in mammalian evolution, we analyzed and compared the expression of 64 V1R and 2 V2R genes in the VNO and the MOE of the gray mouse lemur (Microcebus murinus), the primate with the largest known VR repertoire. We furthermore compared expression patterns in adults of both sexes and seasons, and in an infant. A large proportion (83–97%) of the VR loci was expressed in the VNO of all individuals. The repertoire in the infant was as rich as in adults, indicating reliance on olfactory communication from early postnatal development onwards. In concordance with mice, we also detected extensive expression of VRs in the MOE, with proportions of expressed loci in individuals ranging from 29 to 45%. TRPC2, which encodes a channel protein crucial for signal transduction via VRs, was co-expressed in the MOE in all individuals indicating likely functionality of expressed VR genes in the MOE. In summary, the large VR repertoire in mouse lemurs seems to be highly functional. Given the differences in the neural pathways of MOE and VNO signals, which project to higher cortical brain centers or the limbic system, respectively, this raises the intriguing possibility that the evolution of MOE-expression of VRs enabled mouse lemurs to adaptively diversify the processing of VR-encoded olfactory information. PMID:25309343

  17. NON-MAMMALIAN ESTROGENICITY SCREEN: RAINBOW TROUT ESTROGEN RECEPTOR BINDING

    EPA Science Inventory

    The U.S. EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. Current assays for measuring endocrine activity are primarily mammalian-based. The appropriateness of extrapolating mammalian results to non-mammalian species is uncert...

  18. Evidence for increased olfactory receptor gene repertoire size in two nocturnal bird species with well-developed olfactory ability

    PubMed Central

    Steiger, Silke S; Fidler, Andrew E; Kempenaers, Bart

    2009-01-01

    Background In vertebrates, the molecular basis of the sense of smell is encoded by members of a large gene family, namely olfactory receptor (OR) genes. Both the total number of OR genes and the proportion of intact OR genes in a genome may indicate the importance of the sense of smell for an animal. There is behavioral, physiological, and anatomical evidence that some bird species, in particular nocturnal birds, have a well developed sense of smell. Therefore, we hypothesized that nocturnal birds with good olfactory abilities have evolved (i) more OR genes and (ii) more intact OR genes than closely related and presumably less 'olfaction-dependent' day-active avian taxa. Results We used both non-radioactive Southern hybridization and PCR with degenerate primers to investigate whether two nocturnal bird species that are known to rely on olfactory cues, the brown kiwi (Apteryx australis) and the kakapo (Strigops habroptilus), have evolved a larger OR gene repertoire than their day-active, closest living relatives (for kiwi the emu Dromaius novaehollandiae, rhea Rhea americana, and ostrich Struthio camelus and for kakapo the kaka Nestor meridionalis and kea Nestor notabilis). We show that the nocturnal birds did not have a significantly higher proportion of intact OR genes. However, the estimated total number of OR genes was larger in the two nocturnal birds than in their relatives. Conclusion Our results suggest that ecological niche adaptations such as daily activity patterns may have shaped avian OR gene repertoires. PMID:19467156

  19. Unravelling the Complexity of Human Olfactory Receptor Repertoire by Copy Number Analysis across Population Using High Resolution Arrays

    PubMed Central

    Veerappa, Avinash M.; Vishweswaraiah, Sangeetha; Lingaiah, Kusuma; Murthy, Megha; Manjegowda, Dinesh S.; Nayaka, Radhika; Ramachandra, Nallur B.

    2013-01-01

    Olfactory receptors (OR), responsible for detection of odor molecules, belong to the largest family of genes and are highly polymorphic in nature having distinct polymorphisms associated with specific regions around the globe. Since there are no reports on the presence of copy number variations in OR repertoire of Indian population, the present investigation in 43 Indians along with 270 HapMap and 31 Tibetan samples was undertaken to study genome variability and evolution. Analysis was performed using Affymetrix Genome-Wide Human SNP Array 6.0 chip, Affymterix CytoScan® High-Density array, HD-CNV, and MAFFT program. We observed a total of 1527 OR genes in 503 CNV events from 81.3% of the study group, which includes 67.6% duplications and 32.4% deletions encompassing more of genes than pseudogenes. We report human genotypic variation in functional OR repertoire size across populations and it was found that the combinatorial effect of both “orthologous obtained from closely related species” and “paralogous derived sequences” provide the complexity to the continuously occurring OR CNVs. PMID:23843967

  20. High-throughput sequencing of T cell receptors reveals a homogeneous repertoire of tumor-infiltrating lymphocytes in ovarian cancer

    PubMed Central

    Rieder, Mark J.; Guenthoer, Jamie; Williamson, David W.; Carlson, Christopher S.; Drescher, Charles W.; Tewari, Muneesh; Bielas, Jason H.; Robins, Harlan S.

    2014-01-01

    The cellular adaptive immune system mounts a response to many solid tumors mediated by tumor infiltrating T lymphocytes (TILs). Basic measurements of these TILs, including total count, show promise as prognostic markers for a variety of cancers, including ovarian and colorectal. In addition, recent therapeutic advances are thought to exploit this immune response to effectively fight melanoma with promising studies showing efficacy in additional cancers. However, many of the basic properties of TILs are poorly understood including specificity, clonality, and spatial heterogeneity of the T cell response. We utilize deep sequencing of rearranged T-cell receptor beta (TCRB) genes to characterize the basic properties of TILs in ovarian carcinoma. Due to somatic rearrangement during T cell development, the TCR beta chain sequence serves as a molecular tag for each T cell clone. Using these sequence tags, we assess similarities and differences between infiltrating T cells in discretely sampled sections of large tumors and compare to T cells from peripheral blood. Within the limits of sensitivity of our assay, the TIL repertoires show strong similarity throughout each tumor and are distinct from the circulating T cell repertoire. We conclude that the cellular adaptive immune response within ovarian carcinomas is spatially homogeneous and distinct from the T cell compartment of peripheral blood. PMID:24027095

  1. Insights into the Molecular Mechanisms Underlying Mammalian P2X7 Receptor Functions and Contributions in Diseases, Revealed by Structural Modeling and Single Nucleotide Polymorphisms

    PubMed Central

    Jiang, Lin-Hua; Baldwin, Jocelyn M.; Roger, Sebastien; Baldwin, Stephen A.

    2013-01-01

    The mammalian P2X7 receptors (P2X7Rs), a member of the ionotropic P2X receptor family with distinctive functional properties, play an important part in mediating extracellular ATP signaling in health and disease. A clear delineation of the molecular mechanisms underlying the key receptor properties, such as ATP-binding, ion permeation, and large pore formation of the mammalian P2X7Rs, is still lacking, but such knowledge is crucial for a better understanding of their physiological functions and contributions in diseases and for development of therapeutics. The recent breakthroughs in determining the atomic structures of the zebrafish P2X4.1R in the closed and ATP-bound open states have provided the long-awaited structural information. The human P2RX7 gene is abundant with non-synonymous single nucleotide polymorphisms (NS-SNPs), which generate a repertoire of human P2X7Rs with point mutations. Characterizations of the NS-SNPs identified in patients of various disease conditions and the resulting mutations have informed previously unknown molecular mechanisms determining the mammalian P2X7R functions and diseases. In this review, we will discuss the new insights into such mechanisms provided by structural modeling and recent functional and genetic linkage studies of NS-SNPs. PMID:23675347

  2. Transcriptional and Functional Characterization of the G Protein-Coupled Receptor Repertoire of Gastric Somatostatin Cells.

    PubMed

    Egerod, Kristoffer L; Engelstoft, Maja S; Lund, Mari L; Grunddal, Kaare V; Zhao, Mirabella; Barir-Jensen, Dominique; Nygaard, Eva B; Petersen, Natalia; Holst, Jens J; Schwartz, Thue W

    2015-11-01

    In the stomach, somatostatin (SST) acts as a general paracrine negative regulator of exocrine secretion of gastric acid and pepsinogen and endocrine secretion of gastrin, ghrelin, and histamine. Using reporter mice expressing red fluorescent protein (RFP) under control of the SST promotor, we have characterized the G protein-coupled receptors expressed in gastric Sst-RFP-positive cells and probed their effects on SST secretion in primary cell cultures. Surprisingly, besides SST, amylin and PYY were also highly enriched in the SST cells. Several receptors found to regulate SST secretion were highly expressed and/or enriched. 1) The metabolite receptors calcium-sensing receptor and free fatty acid receptor 4 (GPR120) functioned as positive and negative regulators, respectively. 2) Among the neurotransmitter receptors, adrenergic receptors α1a, α2a, α2b, and β1 were all highly expressed, with norepinephrine and isoproterenol acting as positive regulators. The muscarinic receptor M3 acted as a positive regulator, whereas M4 was conceivably a negative regulator. 3) Of the hormone receptors, the GLP-1 and GIP receptors, CCKb (stimulated by both CCK and gastrin) and surprisingly the melanocortin MC1 receptor were all positive regulators. 4) The neuropeptide receptors for calcitonin gene-related peptide, adrenomedullin, and vasoactive intestinal peptide acted as positive regulators, no effect was observed using galanin and nociceptin although transcripts for the corresponding receptors appeared highly expressed. 5) The SST receptors 1 and 2 functioned in an autocrine negative feedback loop. Thus, the article provides a comprehensive map of receptors through which SST secretion is regulated by hormones, neurotransmitters, neuropeptides and metabolites that act directly on the SST cells in the gastric mucosa. PMID:26181106

  3. A comparison of reptilian and avian olfactory receptor gene repertoires: Species-specific expansion of group γ genes in birds

    PubMed Central

    Steiger, Silke S; Kuryshev, Vladimir Y; Stensmyr, Marcus C; Kempenaers, Bart; Mueller, Jakob C

    2009-01-01

    Background The detection of odorants is mediated by olfactory receptors (ORs). ORs are G-protein coupled receptors that form a remarkably large protein superfamily in vertebrate genomes. We used data that became available through recent sequencing efforts of reptilian and avian genomes to identify the complete OR gene repertoires in a lizard, the green anole (Anolis carolinensis), and in two birds, the chicken (Gallus gallus) and the zebra finch (Taeniopygia guttata). Results We identified 156 green anole OR genes, including 42 pseudogenes. The OR gene repertoire of the two bird species was substantially larger with 479 and 553 OR gene homologs in the chicken and zebra finch, respectively (including 111 and 221 pseudogenes, respectively). We show that the green anole has a higher fraction of intact OR genes (~72%) compared with the chicken (~66%) and the zebra finch (~38%). We identified a larger number and a substantially higher proportion of intact OR gene homologs in the chicken genome than previously reported (214 versus 82 genes and 66% versus 15%, respectively). Phylogenetic analysis showed that lizard and bird OR gene repertoires consist of group α, θ and γ genes. Interestingly, the vast majority of the avian OR genes are confined to a large expansion of a single branch (the so called γ-c clade). An analysis of the selective pressure on the paralogous genes of each γ-c clade revealed that they have been subjected to adaptive evolution. This expansion appears to be bird-specific and not sauropsid-specific, as it is lacking from the lizard genome. The γ-c expansions of the two birds do not intermix, i.e., they are lineage-specific. Almost all (group γ-c) OR genes mapped to the unknown chromosome. The remaining OR genes mapped to six homologous chromosomes plus three to four additional chromosomes in the zebra finch and chicken. Conclusion We identified a surprisingly large number of potentially functional avian OR genes. Our data supports recent

  4. Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells★

    PubMed Central

    Engelstoft, Maja S.; Park, Won-mee; Sakata, Ichiro; Kristensen, Line V.; Husted, Anna Sofie; Osborne-Lawrence, Sherri; Piper, Paul K.; Walker, Angela K.; Pedersen, Maria H.; Nøhr, Mark K.; Pan, Jie; Sinz, Christopher J.; Carrington, Paul E.; Akiyama, Taro E.; Jones, Robert M.; Tang, Cong; Ahmed, Kashan; Offermanns, Stefan; Egerod, Kristoffer L.; Zigman, Jeffrey M.; Schwartz, Thue W.

    2013-01-01

    The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell. PMID:24327954

  5. Pervasive and ongoing positive selection in the vomeronasal-1 receptor (V1R) repertoire of mouse lemurs.

    PubMed

    Hohenbrink, Philipp; Radespiel, Ute; Mundy, Nicholas I

    2012-12-01

    Chemosensory genes are frequently the target of positive selection and are often present in large gene families, but little is known about heterogeneity of selection in these cases and its relation to function. Here, we use the vomeronasal-1 receptor (V1R) repertoire of mouse lemurs (Microcebus spp.) as a model system to study patterns of selection of chemosensory genes at several different levels. Mouse lemurs are small nocturnal strepsirrhine primates and have a large (~200 loci) repertoire of V1R loci that are likely important for intraspecific pheromonal communication and interspecific interactions, for example, recognition of predator cues. We investigated signals and patterns of positive selection among the 105 identified full length V1R loci in the gray mouse lemur and within 7 V1R loci amplified across multiple mouse lemur species. Phylogenetic reconstructions of published sequences revealed at least nine monophyletic clusters of V1Rs in gray mouse lemurs that have diversified since the split between lemurs and lorisoid primates. A large majority of clusters evolved under significant positive selection. Similar results were found in V1Rs of closely related greater galagos. Comparison with function of related V1R clusters in mice suggested a potential relationship between receptor function and strength of selection. Interestingly, most codons identified as being under positive selection are located in the extracellular domains of the receptors and hence likely indicate the position of residues involved in ligand binding. Positive selection was also detected within five V1R loci (=71% of analyzed loci) sequenced from 6 to 10 mouse lemur species, indicating ongoing selection within the genus, which may be related to sexual selection and, potentially, speciation processes. Variation in strength of positive selection on V1Rs showed no simple relationship to cluster size. The diversity of V1R loci in mouse lemurs reflects their adaptive evolution and is most

  6. Structural and Functional Similarity of Amphibian Constitutive Androstane Receptor with Mammalian Pregnane X Receptor

    PubMed Central

    Mathäs, Marianne; Burk, Oliver; Gödtel-Armbrust, Ute; Herlyn, Holger; Wojnowski, Leszek; Windshügel, Björn

    2014-01-01

    The nuclear receptors and xenosensors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2) induce the expression of xenobiotic metabolizing enzymes and transporters, which also affects various endobiotics. While human and mouse CAR feature a high basal activity and low induction upon ligand exposure, we recently identified two constitutive androstane receptors in Xenopus laevis (xlCARα and β) that possess PXR-like characteristics such as low basal activity and activation in response to structurally diverse compounds. Using a set of complementary computational and biochemical approaches we provide evidence for xlCARα being the structural and functional counterpart of mammalian PXR. A three-dimensional model of the xlCARα ligand-binding domain (LBD) reveals a human PXR-like L-shaped ligand binding pocket with a larger volume than the binding pockets in human and murine CAR. The shape and amino acid composition of the ligand-binding pocket of xlCAR suggests PXR-like binding of chemically diverse ligands which was confirmed by biochemical methods. Similarly to PXR, xlCARα possesses a flexible helix 11’. Modest increase in the recruitment of coactivator PGC-1α may contribute to the enhanced basal activity of three gain-of-function xlCARα mutants humanizing key LBD amino acid residues. xlCARα and PXR appear to constitute an example of convergent evolution. PMID:24797902

  7. Contrasting Patterns of Evolutionary Diversification in the Olfactory Repertoires of Reptile and Bird Genomes

    PubMed Central

    Vandewege, Michael W.; Mangum, Sarah F.; Gabaldón, Toni; Castoe, Todd A.; Ray, David A.; Hoffmann, Federico G.

    2016-01-01

    Olfactory receptors (ORs) are membrane proteins that mediate the detection of odorants in the environment, and are the largest vertebrate gene family. Comparative studies of mammalian genomes indicate that OR repertoires vary widely, even between closely related lineages, as a consequence of frequent OR gains and losses. Several studies also suggest that mammalian OR repertoires are influenced by life history traits. Sauropsida is a diverse group of vertebrates group that is the sister group to mammals, and includes birds, testudines, squamates, and crocodilians, and represents a natural system to explore predictions derived from mammalian studies. In this study, we analyzed olfactory receptor (OR) repertoire variation among several representative species and found that the number of intact OR genes in sauropsid genomes analyzed ranged over an order of magnitude, from 108 in the green anole to over 1,000 in turtles. Our results suggest that different sauropsid lineages have highly divergent OR repertoire composition that derive from lineage-specific combinations of gene expansions, losses, and retentions of ancestral OR genes. These differences also suggest that varying degrees of adaption related to life history have shaped the unique OR repertoires observed across sauropsid lineages. PMID:26865070

  8. Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

    PubMed Central

    Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

    2016-01-01

    Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface. PMID:27353000

  9. The full repertoire of Drosophila gustatory receptors for detecting an aversive compound

    PubMed Central

    Shim, Jaewon; Lee, Youngseok; Jeong, Yong Taek; Kim, Yonjung; Lee, Min Goo; Montell, Craig; Moon, Seok Jun

    2015-01-01

    The ability to detect toxic compounds in foods is essential for animal survival. However, the minimal subunit composition of gustatory receptors required for sensing aversive chemicals in Drosophila is unknown. Here we report that three gustatory receptors, GR8a, GR66a and GR98b function together in the detection of L-canavanine, a plant-derived insecticide. Ectopic co-expression of Gr8a and Gr98b in Gr66a-expressing, bitter-sensing gustatory receptor neurons (GRNs) confers responsiveness to L-canavanine. Furthermore, misexpression of all three Grs enables salt- or sweet-sensing GRNs to respond to L-canavanine. Introduction of these Grs in sweet-sensing GRNs switches L-canavanine from an aversive to an attractive compound. Co-expression of GR8a, GR66a and GR98b in Drosophila S2 cells induces an L-canavanine-activated nonselective cation conductance. We conclude that three GRs collaborate to produce a functional L-canavanine receptor. Thus, our results clarify the full set of GRs underlying the detection of a toxic tastant that drives avoidance behaviour in an insect. PMID:26568264

  10. A tetrapod-like repertoire of innate immune receptors and effectors for coelacanths

    USGS Publications Warehouse

    Boudinot, Pierre; Zou, Jun; Ota, Tatsuya; Buonocore, Francesco; Scapigliati, Giuseppe; Canapa, Adriana; Cannon, John; Litman, Gary; Hansen, John D.

    2014-01-01

    The recent availability of both robust transcriptome and genome resources for coelacanth (Latimeria chalumnae) has led to unique discoveries for coelacanth immunity such as the lack of IgM, a central component of adaptive immunity. This study was designed to more precisely address the origins and evolution of gene families involved in the initial recognition and response to microbial pathogens, which effect innate immunity. Several multigene families involved in innate immunity are addressed, including: Toll-like receptors (TLRs), retinoic acid inducible gene 1 (RIG1)-like receptors (RLRs), the nucleotide-binding domain and leucine-rich repeat containing proteins (NLRs), diverse immunoglobulin domain-containing proteins (DICP) and modular domain immune-type receptors (MDIRs). Our analyses also include the tripartite motif-containing proteins (TRIM), which are involved in pathogen recognition as well as the positive regulation of antiviral immunity. Finally, this study addressed some of the downstream effectors of the antimicrobial response including IL-1 family members, type I and II interferons (IFN) and IFN-stimulated effectors (ISGs). Collectively, the genes and gene families in coelacanth that effect innate immune functions share characteristics both in content, structure and arrangement with those found in tetrapods but not in teleosts. The findings support the sister group relationship of coelacanth fish with tetrapods.

  11. A tetrapod-like repertoire of innate immune receptors and effectors for coelacanths.

    PubMed

    Boudinot, Pierre; Zou, Jun; Ota, Tatsuya; Buonocore, Francesco; Scapigliati, Giuseppe; Canapa, Adriana; Cannon, John; Litman, Gary; Hansen, John D

    2014-09-01

    The recent availability of both robust transcriptome and genome resources for coelacanth (Latimeria chalumnae) has led to unique discoveries for coelacanth immunity such as the lack of IgM, a central component of adaptive immunity. This study was designed to more precisely address the origins and evolution of gene families involved in the initial recognition and response to microbial pathogens, which effect innate immunity. Several multigene families involved in innate immunity are addressed, including: Toll-like receptors (TLRs), retinoic acid inducible gene 1 (RIG1)-like receptors (RLRs), the nucleotide-binding domain and leucine-rich repeat containing proteins (NLRs), diverse immunoglobulin domain-containing proteins (DICP) and modular domain immune-type receptors (MDIRs). Our analyses also include the tripartite motif-containing proteins (TRIM), which are involved in pathogen recognition as well as the positive regulation of antiviral immunity. Finally, this study addressed some of the downstream effectors of the antimicrobial response including IL-1 family members, type I and II interferons (IFN) and IFN-stimulated effectors (ISGs). Collectively, the genes and gene families in coelacanth that effect innate immune functions share characteristics both in content, structure and arrangement with those found in tetrapods but not in teleosts. The findings support the sister group relationship of coelacanth fish with tetrapods. PMID:24482296

  12. Picrotoxin dramatically speeds the mammalian circadian clock independent of Cys-loop receptors

    PubMed Central

    Freeman, G. Mark; Nakajima, Masato; Ueda, Hiroki R.

    2013-01-01

    Picrotoxin is extensively and specifically used to inhibit GABAA receptors and other members of the Cys-loop receptor superfamily. We find that picrotoxin acts independently of known Cys-loop receptors to shorten the period of the circadian clock markedly by specifically advancing the accumulation of PERIOD2 protein. We show that this mechanism is surprisingly tetrodotoxin-insensitive, and the effect is larger than any known chemical or genetic manipulation. Notably, our results indicate that the circadian target of picrotoxin is common to a variety of human and rodent cell types but not Drosophila, thereby ruling out all conserved Cys-loop receptors and known regulators of mammalian PERIOD protein stability. Given that the circadian clock modulates significant aspects of cell physiology including synaptic plasticity, these results have immediate and broad experimental implications. Furthermore, our data point to the existence of an important and novel target within the mammalian circadian timing system. PMID:23576702

  13. Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity.

    PubMed Central

    Ip, N Y; Ibáñez, C F; Nye, S H; McClain, J; Jones, P F; Gies, D R; Belluscio, L; Le Beau, M M; Espinosa, R; Squinto, S P

    1992-01-01

    Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 (NT-3) are the three members of the neurotrophin family known to exist in mammals. Recently, a fourth neurotrophin (designated neurotrophin-4 or NT-4), which shares all of the features found in the mammalian neurotrophins, has been identified in Xenopus and viper. We used sequences specific to the Xenopus/viper NT-4 to isolate a neurotrophin from both human and rat genomic DNA that appears to represent the mammalian counterpart of Xenopus/viper NT-4. Human NT-4 as well as a human NT-4 pseudogene colocalize to chromosome 19 band q13.3. Mammalian NT-4 has many unusual features compared to the previously identified neurotrophins and is less conserved evolutionarily than the other neurotrophins. However, mammalian NT-4 displays bioactivity and trk receptor specificity similar to that of Xenopus NT-4. Images PMID:1313578

  14. Diversity of native nicotinic receptor subtypes in mammalian brain.

    PubMed

    Zoli, Michele; Pistillo, Francesco; Gotti, Cecilia

    2015-09-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are a heterogeneous family of pentameric ligand-gated cation channels that are expressed throughout the brain and involved in a wide range of physiological and pathophysiological processes. The nAChR subtypes share a common basic structure, but their biophysical and pharmacological properties depend on their subunit composition, which is therefore central to understanding their function in the nervous system and discovering new subtype selective drugs. The development of new technologies and the generation of mice carrying deletions or the expression of gain-of-function nAChR subunits, or GFP-tagged receptor genes has allowed the in vivo identification of complex subtypes and to study the role of individual subtypes in specific cells and complex neurobiological systems but much less is known about which native nAChR subtypes are involved in specific physiological functions and pathophysiological conditions in human brain. We briefly review some recent findings concerning the structure and function of native nAChRs, focussing on the subtypes identified in the rodent habenulo-interpeduncular pathway, a pathway involved in nicotine reinforcement and withdrawal. We also discuss recent findings concerning the expression of native subtypes in primate brain. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25460185

  15. Dynamic Perturbations of the T-Cell Receptor Repertoire in Chronic HIV Infection and following Antiretroviral Therapy

    PubMed Central

    Heather, James M.; Best, Katharine; Oakes, Theres; Gray, Eleanor R.; Roe, Jennifer K.; Thomas, Niclas; Friedman, Nir; Noursadeghi, Mahdad; Chain, Benjamin

    2016-01-01

    HIV infection profoundly affects many parameters of the immune system and ultimately leads to AIDS, yet which factors are most important for determining resistance, pathology, and response to antiretroviral treatment – and how best to monitor them – remain unclear. We develop a quantitative high-throughput sequencing pipeline to characterize the TCR repertoires of HIV-infected individuals before and after antiretroviral therapy, working from small, unfractionated samples of peripheral blood. This reveals the TCR repertoires of HIV+ individuals to be highly perturbed, with considerably reduced diversity as a small proportion of sequences are highly overrepresented. HIV also causes specific qualitative changes to the repertoire including an altered distribution of V gene usage, depletion of public TCR sequences, and disruption of TCR networks. Short-term antiretroviral therapy has little impact on most of the global damage to repertoire structure, but is accompanied by rapid changes in the abundance of many individual TCR sequences, decreases in abundance of the most common sequences, and decreases in the majority of HIV-associated CDR3 sequences. Thus, high-throughput repertoire sequencing of small blood samples that are easy to take, store, and process can shed light on various aspects of the T-cell immune compartment and stands to offer insights into patient stratification and immune reconstitution. PMID:26793190

  16. Deep sequencing of the T-cell receptor repertoire in CD8+ T-large granular lymphocyte leukemia identifies signature landscapes

    PubMed Central

    Clemente, Michael J.; Przychodzen, Bartlomiej; Jerez, Andres; Dienes, Brittney E.; Afable, Manuel G.; Husseinzadeh, Holleh; Rajala, Hanna L. M.; Wlodarski, Marcin W.; Mustjoki, Satu

    2013-01-01

    New massively parallel sequencing technology enables, through deep sequencing of rearranged T-cell receptor (TCR) Vβ complementarity-determining region 3 (CDR3) regions, a previously inaccessible level of TCR repertoire analysis. The CDR3 repertoire diversity reflects clonal composition, the potential antigenic recognition spectrum, and the quantity of available T-cell responses. In this context, T-large granular lymphocyte (T-LGL) leukemia is a chronic clonal lymphoproliferation of cytotoxic T cells often associated with autoimmune diseases and various cytopenias. Using CD8+ T-LGL leukemia as a model disease, we set out to evaluate and compare the TCR deep-sequencing spectra of both patients and healthy controls to better understand how TCR deep sequencing could be used in the diagnosis and monitoring of not only T-LGL leukemia but also reactive processes such as autoimmune disease and infection. Our data demonstrate, with high resolution, significantly decreased diversity of the T-cell repertoire in CD8+ T-LGL leukemia and suggest that many T-LGL clonotypes may be private to the disease and may not be present in the general public, even at the basal level. PMID:24149287

  17. Repertoire comparison of the B-cell receptor-encoding loci in humans and rhesus macaques by next-generation sequencing

    PubMed Central

    Vigdorovich, Vladimir; Oliver, Brian G; Carbonetti, Sara; Dambrauskas, Nicholas; Lange, Miles D; Yacoob, Christina; Leahy, Will; Callahan, Jonathan; Stamatatos, Leonidas; Sather, D Noah

    2016-01-01

    Rhesus macaques (RMs) are a widely used model system for the study of vaccines, infectious diseases and microbial pathogenesis. Their value as a model lies in their close evolutionary relationship to humans, which, in theory, allows them to serve as a close approximation of the human immune system. However, despite their prominence as a human surrogate model system, many aspects of the RM immune system remain ill characterized. In particular, B cell-mediated immunity in macaques has not been sufficiently characterized, and the B-cell receptor-encoding loci have not been thoroughly annotated. To address these gaps, we analyzed the circulating heavy- and light-chain repertoires in humans and RMs by next-generation sequencing. By comparing V gene segment usage, J-segment usage and CDR3 lengths between the two species, we identified several important similarities and differences. These differences were especially notable in the IgM+ B-cell repertoire. However, the class-switched, antigen-educated B-cell populations converged on a set of similar characteristics, implying similarities in how each species responds to antigen. Our study provides the first comprehensive overview of the circulating repertoires of the heavy- and light-chain sequences in RMs, and provides insight into how they may perform as a model system for B cell-mediated immunity in humans. PMID:27525066

  18. Genetic dissection of the signaling domain of a mammalian steroid receptor in yeast.

    PubMed Central

    Garabedian, M J; Yamamoto, K R

    1992-01-01

    The mechanism of signal transduction by steroid receptor proteins is complex and not yet understood. We describe here a facile genetic strategy for dissection of the rat glucocorticoid receptor "signaling domain," a region of the protein that binds and transduces the hormonal signal. We found that the characteristics of signal transduction by the receptor expressed in yeast were similar to those of endogenous receptors in mammalian cells. Interestingly, the rank order of particular ligands differed between species with respect to receptor binding and biological efficacy. This suggests that factors in addition to the receptor alone must determine or influence ligand efficacy in vivo. To obtain a collection of receptors with distinct defects in signal transduction, we screened in yeast an extensive series of random point mutations introduced in that region in vitro. Three phenotypic classes were obtained: one group failed to bind hormone, a second displayed altered ligand specificity, and a third bound hormone but lacked regulatory activity. Our results demonstrate that analysis of glucocorticoid receptor action in yeast provides a general approach for analyzing the mechanism of signaling by the nuclear receptor family and may facilitate identification of non-receptor factors that participate in this process. Images PMID:1457829

  19. β-Adrenergic receptor signaling and modulation of long-term potentiation in the mammalian hippocampus

    PubMed Central

    O'Dell, Thomas J.; Connor, Steven A.; Guglietta, Ryan

    2015-01-01

    Encoding new information in the brain requires changes in synaptic strength. Neuromodulatory transmitters can facilitate synaptic plasticity by modifying the actions and expression of specific signaling cascades, transmitter receptors and their associated signaling complexes, genes, and effector proteins. One critical neuromodulator in the mammalian brain is norepinephrine (NE), which regulates multiple brain functions such as attention, perception, arousal, sleep, learning, and memory. The mammalian hippocampus receives noradrenergic innervation and hippocampal neurons express β-adrenergic receptors, which are known to play important roles in gating the induction of long-lasting forms of synaptic potentiation. These forms of long-term potentiation (LTP) are believed to importantly contribute to long-term storage of spatial and contextual memories in the brain. In this review, we highlight the contributions of noradrenergic signaling in general and β-adrenergic receptors in particular, toward modulating hippocampal LTP. We focus on the roles of NE and β-adrenergic receptors in altering the efficacies of specific signaling molecules such as NMDA and AMPA receptors, protein phosphatases, and translation initiation factors. Also, the roles of β-adrenergic receptors in regulating synaptic “tagging” and “capture” of LTP within synaptic networks of the hippocampus are reviewed. Understanding the molecular and cellular bases of noradrenergic signaling will enrich our grasp of how the brain makes new, enduring memories, and may shed light on credible strategies for improving mental health through treatment of specific disorders linked to perturbed memory processing and dysfunctional noradrenergic synaptic transmission. PMID:26286656

  20. Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

    SciTech Connect

    Gu, Y.

    1989-01-01

    The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the {alpha}-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced {sup 22}Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the {gamma}, {delta}, and {epsilon} subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four ({alpha}, {beta}, {gamma}, and {delta}) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the {alpha}, {beta}, and {delta} subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the {gamma}- and {epsilon}- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the {gamma} is replaced by {epsilon}.

  1. A high density of tertiary lymphoid structure B cells in lung tumors is associated with increased CD4+ T cell receptor repertoire clonality

    PubMed Central

    Zhu, Wei; Germain, Claire; Liu, Zheng; Sebastian, Yinong; Devi, Priyanka; Knockaert, Samantha; Brohawn, Philip; Lehmann, Kim; Damotte, Diane; Validire, Pierre; Yao, Yihong; Valge-Archer, Viia; Hammond, Scott A; Dieu-Nosjean, Marie-Caroline; Higgs, Brandon W

    2015-01-01

    T and B cell receptor (TCR and BCR, respectively) Vβ or immunoglobulin heavy chain complementarity-determining region 3 sequencing allows monitoring of repertoire changes through recognition, clonal expansion, affinity maturation, and T or B cell activation in response to antigen. TCR and BCR repertoire analysis can advance understanding of antitumor immune responses in the tumor microenvironment. TCR and BCR repertoires of sorted CD4+, CD8+ or CD19+ cells in tumor, non-tumoral distant tissue (NT), and peripheral compartments (blood/draining lymph node [P]) from 47 non-small cell lung cancer (NSCLC) patients (agemedian = 68 y) were sequenced. The clonotype spectra were assessed among different tissues and correlated with clinical and immunological parameters. In all tissues, CD4+ and CD8+ TCR repertoires had greater clonality relative to CD19+ BCR. CD4+ T cells exhibited greater clonality in NT compared to tumor (p = 0.002) and P (p < 0.001), concentrated among older patients (age > 68). Younger patients exhibited greater CD4+ T cell diversity in P compared to older patients (p = 0.05), and greater CD4+ T cell clonality in tumor relative to P (p < 0.001), with fewer shared clonotypes between tumor and P than older patients (p = 0.04). More interestingly, greater CD4+ and CD8+ T cell clonality in tumor and P, respectively (both p = 0.05), correlated with high density of tumor-associated tertiary lymphoid structure (TLS) B cells, a biomarker of higher overall survival in NSCLC. Results indicate distinct adaptive immune responses in NSCLC, where peripheral T cell diversity is modulated by age, and tumor T cell clonal expansion is favored by the presence of TLSs in the tumor microenvironment. PMID:26587322

  2. Siglec receptors impact mammalian lifespan by modulating oxidative stress.

    PubMed

    Schwarz, Flavio; Pearce, Oliver M T; Wang, Xiaoxia; Samraj, Annie N; Läubli, Heinz; Garcia, Javier O; Lin, Hongqiao; Fu, Xiaoming; Garcia-Bingman, Andrea; Secrest, Patrick; Romanoski, Casey E; Heyser, Charles; Glass, Christopher K; Hazen, Stanley L; Varki, Nissi; Varki, Ajit; Gagneux, Pascal

    2015-01-01

    Aging is a multifactorial process that includes the lifelong accumulation of molecular damage, leading to age-related frailty, disability and disease, and eventually death. In this study, we report evidence of a significant correlation between the number of genes encoding the immunomodulatory CD33-related sialic acid-binding immunoglobulin-like receptors (CD33rSiglecs) and maximum lifespan in mammals. In keeping with this, we show that mice lacking Siglec-E, the main member of the CD33rSiglec family, exhibit reduced survival. Removal of Siglec-E causes the development of exaggerated signs of aging at the molecular, structural, and cognitive level. We found that accelerated aging was related both to an unbalanced ROS metabolism, and to a secondary impairment in detoxification of reactive molecules, ultimately leading to increased damage to cellular DNA, proteins, and lipids. Taken together, our data suggest that CD33rSiglecs co-evolved in mammals to achieve a better management of oxidative stress during inflammation, which in turn reduces molecular damage and extends lifespan. PMID:25846707

  3. Siglec receptors impact mammalian lifespan by modulating oxidative stress

    PubMed Central

    Schwarz, Flavio; Pearce, Oliver MT; Wang, Xiaoxia; Samraj, Annie N; Läubli, Heinz; Garcia, Javier O; Lin, Hongqiao; Fu, Xiaoming; Garcia-Bingman, Andrea; Secrest, Patrick; Romanoski, Casey E; Heyser, Charles; Glass, Christopher K; Hazen, Stanley L; Varki, Nissi; Varki, Ajit; Gagneux, Pascal

    2015-01-01

    Aging is a multifactorial process that includes the lifelong accumulation of molecular damage, leading to age-related frailty, disability and disease, and eventually death. In this study, we report evidence of a significant correlation between the number of genes encoding the immunomodulatory CD33-related sialic acid-binding immunoglobulin-like receptors (CD33rSiglecs) and maximum lifespan in mammals. In keeping with this, we show that mice lacking Siglec-E, the main member of the CD33rSiglec family, exhibit reduced survival. Removal of Siglec-E causes the development of exaggerated signs of aging at the molecular, structural, and cognitive level. We found that accelerated aging was related both to an unbalanced ROS metabolism, and to a secondary impairment in detoxification of reactive molecules, ultimately leading to increased damage to cellular DNA, proteins, and lipids. Taken together, our data suggest that CD33rSiglecs co-evolved in mammals to achieve a better management of oxidative stress during inflammation, which in turn reduces molecular damage and extends lifespan. DOI: http://dx.doi.org/10.7554/eLife.06184.001 PMID:25846707

  4. Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

    PubMed Central

    Zhuang, Hanyi; Matsunami, Hiroaki

    2009-01-01

    A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigate odorant-OR relationship in mammals has been an inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein (RTP) family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput “deorphanization” of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently-expressed mammalian odorant receptors in HEK293T cells. The stages of odorant receptor cell-surface expression include cell culture preparation, transfer of cells, transfection, and immunocytochemistry/flow cytometry, odorant stimulation, and luciferase assay. This protocol can be completed in a period of 3 days from transfer of cells to cell-surface expression detection and/or measurement of functional activation. PMID:18772867

  5. Quantitative autoradiography of TRH receptors in discrete brain regions of different mammalian species

    SciTech Connect

    Sharif, N.A.

    1989-01-01

    The results clearly show marked heterogeneity and ubiquity of the CNS distribution of TRH receptors across several mammalian species including man. The use of high resolution autoradiography coupled with image analysis has permitted the visualization and quantification of TRH receptor density in even very small regions and nuclei of the CNS. This technique will undoubtedly help elucidate the other areas of TRH receptor localization that have thus far escaped detection in mammals and that are yet to be studied in lower vertebrates. Although an attempt has been made to correlate the presence of the peptide, its receptors, and its possible physiological functions, only further detailed physiological/behavioral investigations will ultimately unravel and support the diverse neurotransmitter and trophic roles of TRH in CNS and endocrine function. 130 references.

  6. A Highly Focused Antigen Receptor Repertoire Characterizes γδ T Cells That are Poised to Make IL-17 Rapidly in Naive Animals

    PubMed Central

    Wei, Yu-Ling; Han, Arnold; Glanville, Jacob; Fang, Fengqin; Zuniga, Luis Alejandro; Lee, Jacob S.; Cua, Daniel J.; Chien, Yueh-hsiu

    2015-01-01

    Interleukin (IL)-17 plays a key role in immunity. In acute infections, a rapid IL-17 response must be induced without prior antigen exposure, and γδ T cells are the major initial IL-17 producers. In fact, some γδ T cells make IL-17 within hours after an immune challenge. These cells appear to acquire the ability to respond to IL-1 and IL-23 and to make IL-17 naturally in naïve animals. They are known as the natural Tγδ17 (nTγδ17) cells. The rapidity of the nTγδ17 response, and the apparent lack of explicit T cell receptor (TCR) engagement for its induction have led to the view that this is a cytokine (IL-1, IL-23)-mediated response. However, pharmacological inhibition or genetic defects in TCR signaling drastically reduce the nTγδ17 response and/or their presence. To better understand antigen recognition in this rapid IL-17 response, we analyzed the antigen receptor repertoire of IL-1R+/IL-23R+ γδ T cells, a proxy for nTγδ17 cells in naïve animals directly ex vivo, using a barcode-enabled high throughput single-cell TCR sequence analysis. We found that regardless of their anatomical origin, these cells have a highly focused TCR repertoire. In particular, the TCR sequences have limited V gene combinations, little or no junctional diversity and much reduced or no N region diversity. In contrast, IL-23R− cells at mucosal sites similar to most of the splenic γδ T cells and small intestine epithelial γδ lymphocytes expressed diverse TCRs. This remarkable commonality and restricted repertoire of IL-1R+/IL-23R+ γδ T cells underscores the importance of antigen recognition in their establishment/function. PMID:25852688

  7. Amphibian ryanodine receptor isoforms are related to those of mammalian skeletal or cardiac muscle.

    PubMed

    Lai, F A; Liu, Q Y; Xu, L; el-Hashem, A; Kramarcy, N R; Sealock, R; Meissner, G

    1992-08-01

    The ryanodine receptor (RyR)-Ca2+ release channels of frog skeletal muscle have been purified as 30S protein complexes comprised of two high molecular weight polypeptides. The upper and lower bands of the frog doublet comigrated on sodium dodecyl sulfate polyacylamide gels with the mammalian skeletal and cardiac RyR polypeptides, respectively. Immunoblot analysis showed that a polyclonal antiserum to the rat skeletal RyR preferentially cross-reacted with the upper band, whereas monoclonal antibodies to the canine cardiac RyR preferentially cross-reacted with the lower band of the frog receptor doublet. Immunoprecipitation studies indicated the presence of two homooligomer 30S RyR complexes comprised of either the lower or upper polypeptide band of the frog doublet, and immunocytochemical staining revealed their colocalization in frog gastrocnemius muscle. After planar lipid bilayer reconstitution of the 30S frog RyR, single-channel currents were observed that exhibited a Na+ and Ca2+ conductance and pharmacological characteristics similar to those of the mammalian skeletal and cardiac Ca2+ release channels. These results suggest that amphibian skeletal muscle expresses two distinct RyR isoforms that share epitopes in common with the mammalian skeletal or cardiac RyR. PMID:1325114

  8. T cell diversity and TcR repertoires in teleost fish.

    PubMed

    Castro, R; Bernard, D; Lefranc, M P; Six, A; Benmansour, A; Boudinot, P

    2011-11-01

    In vertebrates, the diverse and extended range of antigenic motifs is matched to large populations of lymphocytes. The concept of immune repertoire was proposed to describe this diversity of lymphocyte receptors--IG and TR--required for the recognition specificity. Immune repertoires have become useful tools to describe lymphocyte and receptor populations during the immune system development and in pathological situations. In teleosts, the presence of conventional T cells was first proposed to explain graft rejection and optimized specific antibody production. The discovery of TR genes definitely established the reality of conventional T cells in fish. The development of genomic and EST databases recently led to the description of several key T cell markers including CD4, CD8, CD3, CD28, CTLA4, as well as important cytokines, suggesting the existence of different T helper (Th) subtypes, similar to the mammalian Th1, Th2 and Th17. Over the last decade, repertoire studies have demonstrated that both public and private responses occur in fish as they do in mammals, and in vitro specific cytotoxicity assays have been established. While such typical features of T cells are similar in both fish and mammals, the structure of particular repertoires such as the one of gut intra-epithelial lymphocytes seems to be very different. Future studies will further reveal the particular characteristics of teleost T cell repertoires and adaptive responses. PMID:20804845

  9. Recognition of bacterial signal peptides by mammalian formyl peptide receptors: a new mechanism for sensing pathogens.

    PubMed

    Bufe, Bernd; Schumann, Timo; Kappl, Reinhard; Bogeski, Ivan; Kummerow, Carsten; Podgórska, Marta; Smola, Sigrun; Hoth, Markus; Zufall, Frank

    2015-03-20

    Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system. PMID:25605714

  10. Expression of the avian-specific toll-like receptor 15 in chicken heterophils is mediated by Gram-negative and Gram-postive bacteria, but not TLR agonists

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pattern recognition receptors (PRRs) are essential for recognition of conserved molecular constituents found on infectious microbes. Toll-like receptors (TLRs) are a critical component of the PRR repertoire in both mammalian and avian species. While most mammalian TLRs have been well characterized...

  11. Chicken TLR21 is an innate CpG DNA receptor distinct from mammalian TLR9.

    PubMed

    Keestra, A Marijke; de Zoete, Marcel R; Bouwman, Lieneke I; van Putten, Jos P M

    2010-07-01

    TLRs comprise a family of evolutionary conserved sensory receptors that respond to distinct classes of ligands. For one major evolutionary branch of TLRs, the ligands are still largely unknown. Here we report the cloning and function of one member of this group, chicken TLR21 (chTLR21). This TLR is absent in the human species but has homologs in fish and frog and displays similarity with mouse TLR13. Expression of chTLR21 in HEK293 cells resulted in activation of NF-kappaB in response to unmethylated CpG DNA, typically recognized by mammalian TLR9. Silencing of chTLR21 (but not chTLR4) in chicken macrophages inhibited the response to CpG-DNA (but not to LPS), indicating similar functionality of the endogenous receptor. ChTLR21 responded to human- and murine-specific TLR9 ligands, as well as to bacterial genomic DNA isolated from Salmonella enterica serovar Enteritidis. Confocal microscopy located chTLR21 in the same intracellular compartments as human TLR9. Inhibition of the chTLR21 response by the endosomal maturation inhibitor chloroquine suggested that the receptor is functional in endolysosomes, as known for TLR9. The analogous localization and function of the phylogenetically only distantly related chTLR21 and mammalian TLR9 suggest that during evolution different classes of TLRs have emerged that recognize the same type of ligands. PMID:20498358

  12. Generation of Novel Traj18-Deficient Mice Lacking Vα14 Natural Killer T Cells with an Undisturbed T Cell Receptor α-Chain Repertoire

    PubMed Central

    Dashtsoodol, Nyambayar; Shigeura, Tomokuni; Ozawa, Ritsuko; Harada, Michishige; Kojo, Satoshi; Watanabe, Takashi; Koseki, Haruhiko; Nakayama, Manabu; Ohara, Osamu; Taniguchi, Masaru

    2016-01-01

    Invariant Vα14 natural killer T (NKT) cells, characterized by the expression of a single invariant T cell receptor (TCR) α chain encoded by rearranged Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice, and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans, mediate adjuvant effects to activate various effector cell types in both innate and adaptive immune systems that facilitates the potent antitumor effects. It was recently reported that the Jα18-deficient mouse described by our group in 1997 harbors perturbed TCRα repertoire, which raised concerns regarding the validity of some of the experimental conclusions that have been made using this mouse line. To resolve this concern, we generated a novel Traj18-deficient mouse line by specifically targeting the Traj18 gene segment using Cre-Lox approach. Here we showed the newly generated Traj18-deficient mouse has, apart from the absence of Traj18, an undisturbed TCRα chain repertoire by using next generation sequencing and by detecting normal generation of Vα19Jα33 expressing mucosal associated invariant T cells, whose development was abrogated in the originally described Jα18-KO mice. We also demonstrated here the definitive requirement for NKT cells in the protection against tumors and their potent adjuvant effects on antigen-specific CD8 T cells. PMID:27064277

  13. Extensive analysis of D-J-C arrangements allows the identification of different mechanisms enhancing the diversity in sheep T cell receptor β-chain repertoire

    PubMed Central

    2010-01-01

    Background In most species of mammals, the TRB locus has the common feature of a library of TRBV genes positioned at the 5'- end of two in tandem aligned D-J-C gene clusters, each composed of a single TRBD gene, 6-7 TRBJ genes and one TRBC gene. An enhancer located at the 3'end of the last TRBC and a well-defined promoter situated at the 5'end of the TRBD gene and/or a undefined promoter situated at the 5'end of the TRBD2 are sufficient to generate the full recombinase accessibility at the locus. In ruminant species, the 3'end of the TRB locus is characterized by the presence of three D-J-C clusters, each constituted by a single TRBD, 5-7 TRBJ and one TRBC genes with the center cluster showing a structure combined with the clusters upstream and downstream, suggesting that a unequal crossover occurred in the duplication. An enhancer downstream the last TRBC, and a promoter at the 5'-end of each TRBD gene are also present. Results In this paper we focused our attention on the analysis of a large number of sheep TR β-chain transcripts derived from four different lymphoid tissues of three diverse sheep breed animals to certify the use and frequency of the three gene clusters in the β-chain repertoire. As the sheep TRB locus genomic organization is known, the exact interpretation of the V-D-J rearrangements was fully determined. Our results clearly demonstrate that sheep β-chain constitutes a level of variability that is substantially larger than that described in other mammalian species. This is due not only to the increase of the number of D and J genes available to the somatic recombination, but also to the presence of the trans-rearrangement process. Moreover, the functional complexity of β-chain repertoire is resolved by other mechanisms such as alternative cis- and trans-splicing and recombinational diversification that seems to affect the variety of the constant region. Conclusion All together our data demonstrate that a disparate set of molecular mechanisms

  14. Juno is the egg Izumo receptor and is essential for mammalian fertilization.

    PubMed

    Bianchi, Enrica; Doe, Brendan; Goulding, David; Wright, Gavin J

    2014-04-24

    Fertilization occurs when sperm and egg recognize each other and fuse to form a new, genetically distinct organism. The molecular basis of sperm-egg recognition is unknown, but is likely to require interactions between receptor proteins displayed on their surface. Izumo1 is an essential sperm cell-surface protein, but its receptor on the egg has not been described. Here we identify folate receptor 4 (Folr4) as the receptor for Izumo1 on the mouse egg, and propose to rename it Juno. We show that the Izumo1-Juno interaction is conserved within several mammalian species, including humans. Female mice lacking Juno are infertile and Juno-deficient eggs do not fuse with normal sperm. Rapid shedding of Juno from the oolemma after fertilization suggests a mechanism for the membrane block to polyspermy, ensuring eggs normally fuse with just a single sperm. Our discovery of an essential receptor pair at the nexus of conception provides opportunities for the rational development of new fertility treatments and contraceptives. PMID:24739963

  15. Cross-signaling in metabotropic glutamate 2 and serotonin 2A receptor heteromers in mammalian cells.

    PubMed

    Baki, Lia; Fribourg, Miguel; Younkin, Jason; Eltit, Jose Miguel; Moreno, Jose L; Park, Gyu; Vysotskaya, Zhanna; Narahari, Adishesh; Sealfon, Stuart C; Gonzalez-Maeso, Javier; Logothetis, Diomedes E

    2016-05-01

    We previously reported that co-expression of the Gi-coupled metabotropic glutamate receptor 2 (mGlu2R) and the Gq-coupled serotonin (5-HT) 2A receptor (2AR) in Xenopus oocytes (Fribourg et al. Cell 147:1011-1023, 2011) results in inverse cross-signaling, where for either receptor, strong agonists suppress and inverse agonists potentiate the signaling of the partner receptor. Importantly, through this cross-signaling, the mGlu2R/2AR heteromer integrates the actions of psychedelic and antipsychotic drugs. To investigate whether mGlu2R and 2AR can cross-signal in mammalian cells, we stably co-expressed them in HEK293 cells along with the GIRK1/GIRK4 channel, a reporter of Gi and Gq signaling activity. Crosstalk-positive clones were identified by Fura-2 calcium imaging, based on potentiation of 5-HT-induced Ca(2+) responses by the inverse mGlu2/3R agonist LY341495. Cross-signaling from both sides of the complex was confirmed in representative clones by using the GIRK channel reporter, both in whole-cell patch-clamp and in fluorescence assays using potentiometric dyes, and further established by competition binding assays. Notably, only 25-30 % of the clones were crosstalk-positive. The crosstalk-positive phenotype correlated with (a) increased colocalization of the two receptors at the cell surface, (b) lower density of mGlu2R binding sites and higher density of 2AR binding sites in total membrane preparations, and (c) higher ratios of mGlu2R/2AR normalized surface protein expression. Consistent with our results in Xenopus oocytes, a combination of ligands targeting both receptors could elicit functional crosstalk in a crosstalk-negative clone. Crosstalk-positive clones can be used in high-throughput assays for identification of antipsychotic drugs targeting this receptor heterocomplex. PMID:26780666

  16. TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

    PubMed Central

    Paredes, Alfonso; Romero, Carmen; Dissen, Gregory A.; DeChiara, Tom M.; Reichardt, Louis; Cornea, Anda; Ojeda, Sergio R.; Xu, Baoji

    2009-01-01

    Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells— required for this transition—and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative

  17. Insulin receptor/IGF-I receptor hybrids are widely distributed in mammalian tissues: quantification of individual receptor species by selective immunoprecipitation and immunoblotting.

    PubMed

    Bailyes, E M; Navé, B T; Soos, M A; Orr, S R; Hayward, A C; Siddle, K

    1997-10-01

    The insulin receptor (IR) and type 1 insulin-like growth factor (IGF-I) receptor (IGFR) are both widely expressed in mammalian tissues, and are known to be capable of heteromeric assembly as insulin/IGF hybrid receptors, in addition to the classically described receptors. By selective immunoadsorption of radioligand/receptor complexes and by immunoblotting we have determined the fraction of insulin receptors and IGF receptors occurring as hybrids in different tissues. Microsomal membranes were isolated from tissue homogenates and solubilized with Triton X-100. Solubilized receptors were incubated with 125I-IGF-I, and radioligand/receptor complexes bound by IR-specific and IGFR-specific monoclonal antibodies were quantified. The fraction of IGF-I binding sites behaving as hybrids (anti-IR-bound/anti-IGFR-bound) was approx. 40% in liver and spleen, 70% in placenta, and 85-90% in skeletal muscle and heart, similar results being obtained in rabbit and human tissues. There was no correlation between the proportion of hybrids and the ratio of 125I-insulin/125I-IGF-I binding in different tissues. The fraction of 125I-insulin bound to hybrids was too low for accurate quantification, because of the relatively low affinity of hybrids for insulin. The fraction of insulin receptors present in hybrids was therefore determined by immunoblotting. Receptors in solubilized human placental microsomal membranes were precipitated with IR-specific or IGFR-specific monoclonal antibodies, and after SDS/PAGE, blots were prepared and probed with IR-specific and IGFR-specific antisera. It was found that 15% of IR and 80% of IGFR were present in hybrids. Consistent with these figures, the overall level of IR was estimated, by blotting with the respective antibodies at concentrations shown to give equal signals with equal amounts of receptor, to be 4-fold greater than IGFR. Overall it was concluded that a significant fraction of both IR and IGFR occurs as hybrids in most mammalian tissues

  18. Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells

    PubMed Central

    Rangel, Roberto; Guzman-Rojas, Liliana; le Roux, Lucia G.; Staquicini, Fernanda I.; Hosoya, Hitomi; Barbu, E. Magda; Ozawa, Michael G.; Nie, Jing; Jr, Kenneth Dunner; Langley, Robert R.; Sage, E. Helene; Koivunen, Erkki; Gelovani, Juri G.; Lobb, Roy R.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2012-01-01

    Phage display screening allows the study of functional protein–protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development. PMID:22510693

  19. Ecological adaptation determines functional mammalian olfactory subgenomes

    PubMed Central

    Hayden, Sara; Bekaert, Michaël; Crider, Tess A.; Mariani, Stefano; Murphy, William J.; Teeling, Emma C.

    2010-01-01

    The ability to smell is governed by the largest gene family in mammalian genomes, the olfactory receptor (OR) genes. Although these genes are well annotated in the finished human and mouse genomes, we still do not understand which receptors bind specific odorants or how they fully function. Previous comparative studies have been taxonomically limited and mostly focused on the percentage of OR pseudogenes within species. No study has investigated the adaptive changes of functional OR gene families across phylogenetically and ecologically diverse mammals. To determine the extent to which OR gene repertoires have been influenced by habitat, sensory specialization, and other ecological traits, to better understand the functional importance of specific OR gene families and thus the odorants they bind, we compared the functional OR gene repertoires from 50 mammalian genomes. We amplified more than 2000 OR genes in aquatic, semi-aquatic, and flying mammals and coupled these data with 48,000 OR genes from mostly terrestrial mammals, extracted from genomic projects. Phylogenomic, Bayesian assignment, and principle component analyses partitioned species by ecotype (aquatic, semi-aquatic, terrestrial, flying) rather than phylogenetic relatedness, and identified OR families important for each habitat. Functional OR gene repertoires were reduced independently in the multiple origins of aquatic mammals and were significantly divergent in bats. We reject recent neutralist views of olfactory subgenome evolution and correlate specific OR gene families with physiological requirements, a preliminary step toward unraveling the relationship between specific odors and respective OR gene families. PMID:19952139

  20. T cell receptor β-chain repertoire analysis reveals intratumour heterogeneity of tumour-infiltrating lymphocytes in oesophageal squamous cell carcinoma.

    PubMed

    Chen, Zengchao; Zhang, Chaoting; Pan, Yaqi; Xu, Ruiping; Xu, Changqing; Chen, Ziping; Lu, Zheming; Ke, Yang

    2016-08-01

    Oesophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, due to the lack of effective treatment methods. Immunotherapeutic approaches based on tumour-infiltrating lymphocytes (TILs) have demonstrated that durable responses are produced in some patients with solid tumours, which suggests the potential feasibility of clinical application of immunotherapy for ESCC. However, many of the basic characteristics of TILs in ESCC are poorly understood, including clonality, specificity and spatial heterogeneity of the response of TILs, which depends on the interaction between antigens and T cell receptors (TCRs). We used ultra-deep sequencing of rearranged genes in TCR β-chain (TCRβ) to profile the basic characteristics of T cells in tumour tissues (four to six regions from each tumour) as well as matched adjacent normal tissue and peripheral blood from seven patients diagnosed with primary ESCC. We found that T cell clones within ESCCs were quite different from those of the peripheral blood and even the adjacent normal tissues in general. Although there was a relatively higher degree of overlap of intratumoural TCRβ repertoires than those between the tumour and other tissues, intratumoural TCRβ repertoires were spatially heterogeneous. Due to the restricted sampling, high-throughput TCRβ sequencing could characterize the diversity and composition of a limited (compartment-dependent) fraction of the respective T cell clones in any individual ESCC, expanding our understanding of immune behaviour and immune response and shedding more light on ESCC immunotherapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:27171315

  1. Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization.

    PubMed

    Zimmerman, Shawn W; Manandhar, Gaurishankar; Yi, Young-Joo; Gupta, Satish K; Sutovsky, Miriam; Odhiambo, John F; Powell, Michael D; Miller, David J; Sutovsky, Peter

    2011-01-01

    Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced

  2. Aldehyde Recognition and Discrimination by Mammalian Odorant Receptors via Functional Group-Specific Hydration Chemistry

    PubMed Central

    2015-01-01

    The mammalian odorant receptors (ORs) form a chemical-detecting interface between the atmosphere and the nervous system. This large gene family is composed of hundreds of membrane proteins predicted to form as many unique small molecule binding niches within their G-protein coupled receptor (GPCR) framework, but very little is known about the molecular recognition strategies they use to bind and discriminate between small molecule odorants. Using rationally designed synthetic analogs of a typical aliphatic aldehyde, we report evidence that among the ORs showing specificity for the aldehyde functional group, a significant percentage detect the aldehyde through its ability to react with water to form a 1,1-geminal (gem)-diol. Evidence is presented indicating that the rat OR-I7, an often-studied and modeled OR known to require the aldehyde function of octanal for activation, is likely one of the gem-diol activated receptors. A homology model based on an activated GPCR X-ray structure provides a structural hypothesis for activation of OR-I7 by the gem-diol of octanal. PMID:25181321

  3. Functional characterization of bitter-taste receptors expressed in mammalian testis.

    PubMed

    Xu, Jiang; Cao, Jie; Iguchi, Naoko; Riethmacher, Dieter; Huang, Liquan

    2013-01-01

    Mammalian spermatogenesis and sperm maturation are susceptible to the effects of internal and external factors. However, how male germ cells interact with and respond to these elements including those potentially toxic substances is poorly understood. Here, we show that many bitter-taste receptors (T2rs), which are believed to function as gatekeepers in the oral cavity to detect and innately prevent the ingestion of poisonous bitter-tasting compounds, are expressed in mouse seminiferous tubules. Our in situ hybridization results indicate that Tas2r transcripts are expressed postmeiotically. Functional analysis showed that mouse spermatids and spermatozoa responded to both naturally occurring and synthetic bitter-tasting compounds by increasing intracellular free calcium concentrations, and individual male germ cells exhibited different ligand-activation profiles, indicating that each cell may express a unique subset of T2r receptors. These calcium responses could be suppressed by a specific bitter-tastant blocker or abolished by the knockout of the gene for the G protein subunit α-gustducin. Taken together, our data strongly suggest that male germ cells, like taste bud cells in the oral cavity and solitary chemosensory cells in the airway, utilize T2r receptors to sense chemicals in the milieu that may affect sperm behavior and fertilization. PMID:22983952

  4. Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor

    PubMed Central

    Arighi, Cecilia N.; Hartnell, Lisa M.; Aguilar, Ruben C.; Haft, Carol R.; Bonifacino, Juan S.

    2004-01-01

    The cation-independent mannose 6-phosphate receptor (CI-MPR) mediates sorting of lysosomal hydrolase precursors from the TGN to endosomes. After releasing the hydrolase precursors into the endosomal lumen, the unoccupied receptor returns to the TGN for further rounds of sorting. Here, we show that the mammalian retromer complex participates in this retrieval pathway. The hVps35 subunit of retromer interacts with the cytosolic domain of the CI-MPR. This interaction probably occurs in an endosomal compartment, where most of the retromer is localized. In particular, retromer is associated with tubular–vesicular profiles that emanate from early endosomes or from intermediates in the maturation from early to late endosomes. Depletion of retromer by RNA interference increases the lysosomal turnover of the CI-MPR, decreases cellular levels of lysosomal hydrolases, and causes swelling of lysosomes. These observations indicate that retromer prevents the delivery of the CI-MPR to lysosomes, probably by sequestration into endosome-derived tubules from where the receptor returns to the TGN. PMID:15078903

  5. Sirtuins and the Estrogen Receptor as Regulators of the Mammalian Mitochondrial UPR in Cancer and Aging.

    PubMed

    Germain, D

    2016-01-01

    By being both the source of ATP and the mediator of apoptosis, the mitochondria are key regulators of cellular life and death. Not surprisingly alterations in the biology of the mitochondria have implications in a wide array of diseases including cancer and age-related diseases such as neurodegeneration. To protect the mitochondria against damage the mitochondrial unfolded protein response (UPR(mt)) orchestrates several pathways, including the protein quality controls, the antioxidant machinery, oxidative phosphorylation, mitophagy, and mitochondrial biogenesis. While several reports have implicated an array of transcription factors in the UPR(mt), most of the focus has been on studies of Caenorhabditis elegans, which led to the identification of ATFS-1, for which the mammalian homolog remains unknown. Meanwhile, there are studies which link the UPR(mt) to sirtuins and transcription factors of the Foxo family in both C. elegans and mammalian cells but those have been largely overlooked. This review aims at emphasizing the potential importance of these studies by building on the large body of literature supporting the key role of the sirtuins in the maintenance of the integrity of the mitochondria in both cancer and aging. Further, the estrogen receptor alpha (ERα) and beta (ERβ) are known to confer protection against mitochondrial stress, and at least ERα has been linked to the UPR(mt). Considering the difference in gender longevity, this chapter also includes a discussion of the link between the ERα and ERβ and the mitochondria in cancer and aging. PMID:27037754

  6. Identification of Farnesoid X Receptor β as a Novel Mammalian Nuclear Receptor Sensing Lanosterol

    PubMed Central

    Otte, Kerstin; Kranz, Harald; Kober, Ingo; Thompson, Paul; Hoefer, Michael; Haubold, Bernhard; Remmel, Bettina; Voss, Hartmut; Kaiser, Carmen; Albers, Michael; Cheruvallath, Zaccharias; Jackson, David; Casari, Georg; Koegl, Manfred; Pääbo, Svante; Mous, Jan; Kremoser, Claus; Deuschle, Ulrich

    2003-01-01

    Nuclear receptors are ligand-modulated transcription factors. On the basis of the completed human genome sequence, this family was thought to contain 48 functional members. However, by mining human and mouse genomic sequences, we identified FXRβ as a novel family member. It is a functional receptor in mice, rats, rabbits, and dogs but constitutes a pseudogene in humans and primates. Murine FXRβ is widely coexpressed with FXR in embryonic and adult tissues. It heterodimerizes with RXRα and stimulates transcription through specific DNA response elements upon addition of 9-cis-retinoic acid. Finally, we identified lanosterol as a candidate endogenous ligand that induces coactivator recruitment and transcriptional activation by mFXRβ. Lanosterol is an intermediate of cholesterol biosynthesis, which suggests a direct role in the control of cholesterol biosynthesis in nonprimates. The identification of FXRβ as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs. PMID:12529392

  7. Counting Bungarotoxin Binding Sites of Nicotinic Acetylcholine Receptors in Mammalian Cells with High Signal/Noise Ratios

    PubMed Central

    Simonson, Paul D.; DeBerg, Hannah A.; Ge, Pinghua; Alexander, John K.; Jeyifous, Okunola; Green, William N.; Selvin, Paul R.

    2010-01-01

    Nicotinic acetylcholine receptors are some of the most studied synaptic proteins; however, many questions remain that can only be answered using single molecule approaches. Here we report our results from single α7 and neuromuscular junction type nicotinic acetylcholine receptors in mammalian cell membranes. By labeling the receptors with fluorophore-labeled bungarotoxin, we can image individual receptors and count the number of bungarotoxin-binding sites in receptors expressed in HEK 293 cells. Our results indicate that there are two bungarotoxin-binding sites in neuromuscular junction receptors, as expected, and five in α7 receptors, clarifying previous uncertainty. This demonstrates a valuable technique for counting subunits in membrane-bound proteins at the single molecule level, with nonspecialized optics and with higher signal/noise ratios than previous fluorescent protein-based techniques. PMID:21081055

  8. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

    PubMed

    Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

    2014-10-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. PMID:25149566

  9. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor

    PubMed Central

    Ukhanov, K.; Bobkov, Y.; Corey, E.A.; Ache, B.W.

    2014-01-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca2+ release and/or Ca2+ influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5 mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. PMID:25149566

  10. A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types.

    PubMed Central

    McMahan, C J; Slack, J L; Mosley, B; Cosman, D; Lupton, S D; Brunton, L L; Grubin, C E; Wignall, J M; Jenkins, N A; Brannan, C I

    1991-01-01

    cDNA clones corresponding to an Mr approximately 80,000 receptor (type I receptor) for interleukin-1 (IL-1) have been isolated previously by mammalian expression. Here, we report the use of an improved expression cloning method to isolate human and murine cDNA clones encoding a second type (Mr approximately 60,000) of IL-1 receptor (type II receptor). The mature type II IL-1 receptor consists of (i) a ligand binding portion comprised of three immunoglobulin-like domains; (ii) a single transmembrane region; and (iii) a short cytoplasmic domain of 29 amino acids. This last contrasts with the approximately 215 amino acid cytoplasmic domain of the type I receptor, and suggests that the two IL-1 receptors may interact with different signal transduction pathways. The type II receptor is expressed in a number of different tissues, including both B and T lymphocytes, and can be induced in several cell types by treatment with phorbol ester. Both IL-1 receptors appear to be well conserved in evolution, and map to the same chromosomal location. Like the type I receptor, the human type II IL-1 receptor can bind all three forms of IL-1 (IL-1 alpha, IL-1 beta and IL-1ra). Vaccinia virus contains an open reading frame bearing strong resemblance to the type II IL-1 receptor. Images PMID:1833184

  11. Extreme expansion of the olfactory receptor gene repertoire in African elephants and evolutionary dynamics of orthologous gene groups in 13 placental mammals.

    PubMed

    Niimura, Yoshihito; Matsui, Atsushi; Touhara, Kazushige

    2014-09-01

    Olfactory receptors (ORs) detect odors in the environment, and OR genes constitute the largest multigene family in mammals. Numbers of OR genes vary greatly among species--reflecting the respective species' lifestyles--and this variation is caused by frequent gene gains and losses during evolution. However, whether the extent of gene gains/losses varies among individual gene lineages and what might generate such variation is unknown. To answer these questions, we used a newly developed phylogeny-based method to classify >10,000 intact OR genes from 13 placental mammal species into 781 orthologous gene groups (OGGs); we then compared the OGGs. Interestingly, African elephants had a surprisingly large repertoire (∼ 2000) of functional OR genes encoded in enlarged gene clusters. Additionally, OR gene lineages that experienced more gene duplication had weaker purifying selection, and Class II OR genes have evolved more dynamically than those in Class I. Some OGGs were highly expanded in a lineage-specific manner, while only three OGGs showed complete one-to-one orthology among the 13 species without any gene gains/losses. These three OGGs also exhibited highly conserved amino acid sequences; therefore, ORs in these OGGs may have physiologically important functions common to every placental mammal. This study provides a basis for inferring OR functions from evolutionary trajectory. PMID:25053675

  12. Extreme expansion of the olfactory receptor gene repertoire in African elephants and evolutionary dynamics of orthologous gene groups in 13 placental mammals

    PubMed Central

    Matsui, Atsushi; Touhara, Kazushige

    2014-01-01

    Olfactory receptors (ORs) detect odors in the environment, and OR genes constitute the largest multigene family in mammals. Numbers of OR genes vary greatly among species—reflecting the respective species' lifestyles—and this variation is caused by frequent gene gains and losses during evolution. However, whether the extent of gene gains/losses varies among individual gene lineages and what might generate such variation is unknown. To answer these questions, we used a newly developed phylogeny-based method to classify >10,000 intact OR genes from 13 placental mammal species into 781 orthologous gene groups (OGGs); we then compared the OGGs. Interestingly, African elephants had a surprisingly large repertoire (∼2000) of functional OR genes encoded in enlarged gene clusters. Additionally, OR gene lineages that experienced more gene duplication had weaker purifying selection, and Class II OR genes have evolved more dynamically than those in Class I. Some OGGs were highly expanded in a lineage-specific manner, while only three OGGs showed complete one-to-one orthology among the 13 species without any gene gains/losses. These three OGGs also exhibited highly conserved amino acid sequences; therefore, ORs in these OGGs may have physiologically important functions common to every placental mammal. This study provides a basis for inferring OR functions from evolutionary trajectory. PMID:25053675

  13. Evidence of positive selection at codon sites localized in extracellular domains of mammalian CC motif chemokine receptor proteins

    PubMed Central

    2010-01-01

    Background CC chemokine receptor proteins (CCR1 through CCR10) are seven-transmembrane G-protein coupled receptors whose signaling pathways are known for their important roles coordinating immune system responses through targeted trafficking of white blood cells. In addition, some of these receptors have been identified as fusion proteins for viral pathogens: for example, HIV-1 strains utilize CCR5, CCR2 and CCR3 proteins to obtain cellular entry in humans. The extracellular domains of these receptor proteins are involved in ligand-binding specificity as well as pathogen recognition interactions. In mammals, the majority of chemokine receptor genes are clustered together; in humans, seven of the ten genes are clustered in the 3p21-24 chromosome region. Gene conversion events, or exchange of DNA sequence between genes, have been reported in chemokine receptor paralogs in various mammalian lineages, especially between the cytogenetically closely located pairs CCR2/5 and CCR1/3. Datasets of mammalian orthologs for each gene were analyzed separately to minimize the potential confounding impact of analyzing highly similar sequences resulting from gene conversion events. Molecular evolution approaches and the software package Phylogenetic Analyses by Maximum Likelihood (PAML) were utilized to investigate the signature of selection that has acted on the mammalian CC chemokine receptor (CCR) gene family. The results of neutral vs. adaptive evolution (positive selection) hypothesis testing using Site Models are reported. In general, positive selection is defined by a ratio of nonsynonymous/synonymous nucleotide changes (dN/dS, or ω) >1. Results Of the ten mammalian CC motif chemokine receptor sequence datasets analyzed, only CCR2 and CCR3 contain amino acid codon sites that exhibit evidence of positive selection using site based hypothesis testing in PAML. Nineteen of the twenty codon sites putatively indentified as likely to be under positive selection code for amino acid

  14. Involvement of Mammalian RF-Amide Peptides and Their Receptors in the Modulation of Nociception in Rodents

    PubMed Central

    Ayachi, Safia; Simonin, Frédéric

    2014-01-01

    Mammalian RF-amide peptides, which all share a conserved carboxyl-terminal Arg–Phe–NH2 sequence, constitute a family of five groups of neuropeptides that are encoded by five different genes. They act through five G-protein-coupled receptors and each group of peptide binds to and activates mostly one receptor: RF-amide related peptide group binds to NPFFR1, neuropeptide FF group to NPFFR2, pyroglutamylated RF-amide peptide group to QRFPR, prolactin-releasing peptide group to prolactin-releasing peptide receptor, and kisspeptin group to Kiss1R. These peptides and their receptors have been involved in the modulation of several functions including reproduction, feeding, and cardiovascular regulation. Data from the literature now provide emerging evidence that all RF-amide peptides and their receptors are also involved in the modulation of nociception. This review will present the current knowledge on the involvement in rodents of the different mammalian RF-amide peptides and their receptors in the modulation of nociception in basal and chronic pain conditions as well as their modulatory effects on the analgesic effects of opiates. PMID:25324831

  15. Involvement of Mammalian RF-Amide Peptides and Their Receptors in the Modulation of Nociception in Rodents.

    PubMed

    Ayachi, Safia; Simonin, Frédéric

    2014-01-01

    Mammalian RF-amide peptides, which all share a conserved carboxyl-terminal Arg-Phe-NH2 sequence, constitute a family of five groups of neuropeptides that are encoded by five different genes. They act through five G-protein-coupled receptors and each group of peptide binds to and activates mostly one receptor: RF-amide related peptide group binds to NPFFR1, neuropeptide FF group to NPFFR2, pyroglutamylated RF-amide peptide group to QRFPR, prolactin-releasing peptide group to prolactin-releasing peptide receptor, and kisspeptin group to Kiss1R. These peptides and their receptors have been involved in the modulation of several functions including reproduction, feeding, and cardiovascular regulation. Data from the literature now provide emerging evidence that all RF-amide peptides and their receptors are also involved in the modulation of nociception. This review will present the current knowledge on the involvement in rodents of the different mammalian RF-amide peptides and their receptors in the modulation of nociception in basal and chronic pain conditions as well as their modulatory effects on the analgesic effects of opiates. PMID:25324831

  16. Purification and high-sensitivity membrane photoaffinity labeling of mammalian beta/sub 2/-adrenergic receptor

    SciTech Connect

    Drake, F.H. III

    1986-01-01

    The Beta/sub 2/-Adrenergic receptor (BAR) from guinea pig lung has been purified to near homogeneity. The purified BAR, detected by silver staining or by total radioiodination and autoradiography, migrates on SDS-PAGE as a broad band centered at 66 kilodaltons (kD). This band can be specifically labeled with the adrenergic photoaffinity ligand, /sup 125/I-azidobenzylpindolol. The purified BAR displays the same beta/sub 2/-subtype pharmacology and mobility on SDS-PAGE as the membrane-bound BAR. Microsequenator analysis of the purified BAR suggests that the amino terminus of the receptor is blocked. Several site-specific agents were used to fragment the purified BAR; some of the fragments may be useful for obtaining amino acid sequence of the BAR. Conditions also have developed for photoaffinity labeling the BAR in membranes of mammalian tissue culture cells (human astrocytoma, 1321N1) which contain very low levels of BAR. The BAR from these cells migrates as a broad band of about 66 kD on SDS-PAGE. Endoglycosidase F, which cleaves N-linked oligosaccharides, reduces the apparent molecular weight of the BAR from these cells to 45 kD. Recovery from agonist-induced down-regulation in post-confluent cultures of 1321N1 cells in the presence of tunicamycin (an inhibitor of N-linked glycosylation) results in the appearance of a 41 kD form of the BAR. Despite the apparent absence of N-linked oligosaccharides, this 41 kD form of the BAR retains adrenergic binding activity.

  17. Immunoglobulin heavy chain variable region gene repertoire and B-cell receptor stereotypes in Indian patients with chronic lymphocytic leukemia.

    PubMed

    Rani, Lata; Mathur, Nitin; Gogia, Ajay; Vishnubhatla, Sreenivas; Kumar, Lalit; Sharma, Atul; Dube, Divya; Kaur, Punit; Gupta, Ritu

    2016-10-01

    In chronic lymphocytic leukemia (CLL), the geographical bias in immunoglobulin heavy-chain variable (IGHV) gene usage lead us to analyze IGHV gene usage and B-cell receptor stereotypy in 195 patients from India. IGHV3, IGHV4, and IGHV1 families were the most frequently used. 20.5% sequences had stereotyped BCR and were clustered in 12 pre-defined and 6 novel subsets. Unmutated IGHV was significantly associated with reduced time to first treatment (p < 0.033) and poor overall survival (OS; p = 0.01). We observed a significant difference in OS between IGHV1, IGHV3, and IGHV4 family cases (p = 0.045) in early stage patients. Regarding subfamily usage, only IGHV1-69 expression was found to have statistically significant poor outcome (p = 0.017). Our results from the analysis of various molecular and clinical features suggest that the expression of specific IGHV gene influences the outcome in early stage CLL, and hence its assessment may be added to the clinical leukemia laboratory armamentarium. PMID:26942309

  18. Characterization of the T cell repertoire by deep T cell receptor sequencing in tissues and blood from patients with advanced colorectal cancer

    PubMed Central

    TAMURA, KENJI; HAZAMA, SHOICHI; YAMAGUCHI, RUI; IMOTO, SEIYA; TAKENOUCHI, HIROKO; INOUE, YUKA; KANEKIYO, SHINSUKE; SHINDO, YOSHITARO; MIYANO, SATORU; NAKAMURA, YUSUKE; KIYOTANI, KAZUMA

    2016-01-01

    The aim of the present study was to characterize infiltrated T cell clones that define the tumor immune environment and are important in the response to treatment in patients with advanced colorectal cancer (CRC). In order to explore predictive biomarkers for the efficacy of immunochemotherapies, T cell receptor (TCR) repertoire analysis was performed using blood samples and tumor tissues obtained from patients with advanced CRC that had been treated with a combination of five-cancer peptide vaccines and oxaliplatin-based chemotherapy. The TCR-α/β complementary DNAs (cDNAs), prepared from the messenger RNAs (mRNAs) obtained from 17 tumor tissues and 39 peripheral blood mononuclear cells of 9 CRC patients at various time points, were sequenced. The oligoclonal enrichment of certain TCR sequences was identified in tumor tissues and blood samples; however, only a few TCR sequences with a frequency of >0.1% were commonly detected in pre- and post-treatment tumor tissues, or in post-treatment blood and tissue samples. The average correlation coefficients of the TCR-α and TCR-β clonotype frequencies between the post-treatment tumor tissues and blood samples were 0.023 and 0.035, respectively, and were much lower compared with the correlation coefficients of the TCR-α and TCR-β clonotype frequencies between pre- and post-treatment blood samples (0.430 and 0.370, respectively), suggesting that T cell populations in tumor tissues vary from those in blood. Although the sample size was small, a tendency for the TCR diversity in tumor tissues to drastically decrease during the treatment was indicated in two patients, who exhibited a longer progression-free survival time. The results of the present study suggest that TCR diversity scores in tissues may be a useful predictive biomarker for the therapeutic effect of immunochemotherapy for patients with advanced CRC. PMID:27284367

  19. Influence of the route of infection on development of T-cell receptor beta-chain repertoires of reovirus-specific cytotoxic T lymphocytes.

    PubMed

    Fulton, Jonathan R; Smith, Jeremy; Cunningham, Cynthia; Cuff, Christopher F

    2004-02-01

    It is well established that the route of infection affects the nature of the adaptive immune response. However, little is known about the effects of the route of exposure on development of cytotoxic T-lymphocyte (CTL) responses. Alternative antigen-presenting cell populations, tissue-restricted expression of class I major histocompatibility complex-encoded molecules, and unique T-cell receptor (TCR)-bearing cells in mucosal tissues could influence the selection and expansion of responder T cells. This study addresses the question of whether the route of virus infection affects the selection and expansion of subpopulations of virus-specific CTLs. Mice were infected orally or in the hind footpads with reovirus, and the repertoires of TCR beta-chains expressed on virus-specific CD8(+) T cells in Peyer's patches or lymph nodes and spleens were examined. CD8(+) cells expressing the variable gene segment of the TCR beta-chain 6 (Vbeta6) expanded in the spleens of mice infected by either route and in CTL lines established from the spleens and draining lymphoid tissues. Adoptively transferred Vbeta6(+) CD8(+) T cells from orally or parenterally infected donors expanded in reovirus-infected severe combined immunodeficient recipient mice and mediated cytotoxicity ex vivo. Furthermore, recovered Vbeta6(+) cells were enriched for clones utilizing uniform complementarity-determining region 3 (CDR3) lengths. However, sequencing of CDR3beta regions from Vbeta6(+) CD8(+) cells indicated that Jbeta gene segment usage is significantly more restricted in CTLs from orally infected mice, suggesting that the route of infection affects selection and/or subsequent expansion of virus-specific CTLs. PMID:14722312

  20. A Teaching Repertoire

    ERIC Educational Resources Information Center

    Garrett, Joyce Lynn

    2007-01-01

    Building a repertoire of teaching skills means organizing instructional strategies in some meaningful way. In this brief essay, the author recommends that beginning teachers develop a repertoire comprised of a mix of strategies from each of Joyce, Weil, and Calhoun's (2004) categories: three strategies from the Cognitive Family (Concept…

  1. The Linguistic Repertoire Revisited

    ERIC Educational Resources Information Center

    Busch, Brigitta

    2012-01-01

    This article argues for the relevance of poststructuralist approaches to the notion of a linguistic repertoire and introduces the notion of language portraits as a basis for empirical study of the way in which speakers conceive and represent their heteroglossic repertoires. The first part of the article revisits Gumperz's notion of a linguistic…

  2. Distribution of neurotransmitter receptors and zinc in the pigeon (Columba livia) hippocampal formation: A basis for further comparison with the mammalian hippocampus.

    PubMed

    Herold, Christina; Bingman, Verner P; Ströckens, Felix; Letzner, Sara; Sauvage, Magdalena; Palomero-Gallagher, Nicola; Zilles, Karl; Güntürkün, Onur

    2014-08-01

    The avian hippocampal formation (HF) and mammalian hippocampus share a similar functional role in spatial cognition, but the underlying neuronal mechanisms allowing the functional similarity are incompletely understood. To understand better the organization of the avian HF and its transmitter receptors, we analyzed binding site densities for glutamatergic AMPA, NMDA, and kainate receptors; GABAA receptors; muscarinic M1 , M2 and nicotinic (nACh) acetylcholine receptors; noradrenergic α1 and α2 receptors; serotonergic 5-HT1A receptors; dopaminergic D1/5 receptors by using quantitative in vitro receptor autoradiography. Additionally, we performed a modified Timm staining procedure to label zinc. The regionally different receptor densities mapped well onto seven HF subdivisions previously described. Several differences in receptor expression highlighted distinct HF subdivisions. Notable examples include 1) high GABAA and α1 receptor expression, which rendered distinctive ventral subdivisions; 2) high α2 receptor expression, which rendered distinctive a dorsomedial subdivision; 3) distinct kainate, α2 , and muscarinic receptor densities that rendered distinctive the two dorsolateral subdivisions; and 4) a dorsomedial region characterized by high kainate receptor density. We further observed similarities in receptor binding densities between subdivisions of the avian and mammalian HF. Despite the similarities, we propose that 300 hundred million years of independent evolution has led to a mosaic of similarities and differences in the organization of the avian HF and mammalian hippocampus and that thinking about the avian HF in terms of the strict organization of the mammalian hippocampus is likely insufficient to understand the HF of birds. PMID:24477871

  3. Phosphoinositide-3-Kinase Is the Primary Mediator of Phosphoinositide-Dependent Inhibition in Mammalian Olfactory Receptor Neurons

    PubMed Central

    Ukhanov, Kirill; Corey, Elizabeth; Ache, Barry W.

    2016-01-01

    Odorants inhibit as well as excite primary olfactory receptor neurons (ORNs) in many animal species. Growing evidence suggests that inhibition of mammalian ORNs is mediated by phosphoinositide (PI) signaling through activation of phosphoinositide 3-kinase (PI3K), and that canonical adenylyl cyclase III signaling and PI3K signaling interact to provide the basis for ligand-induced selective signaling. As PI3K is known to act in concert with phospholipase C (PLC) in some cellular systems, the question arises as to whether they work together to mediate inhibitory transduction in mammalian ORNs. The present study is designed to test this hypothesis. While we establish that multiple PLC isoforms are expressed in the transduction zone of rat ORNs, that odorants can activate PLC in ORNs in situ, and that pharmacological blockade of PLC enhances the excitatory response to an odorant mixture in some ORNs in conjunction with PI3K blockade, we find that by itself PLC does not account for an inhibitory response. We conclude that PLC does not make a measurable independent contribution to odor-evoked inhibition, and that PI3K is the primary mediator of PI-dependent inhibition in mammalian ORNs. PMID:27147969

  4. Mammalian non-classical major histocompatibility complex I and its receptors: Important contexts of gene, evolution, and immunity

    PubMed Central

    Pratheek, B. M.; Nayak, Tapas K.; Sahoo, Subhransu S.; Mohanty, Prafulla K.; Chattopadhyay, Soma; Chakraborty, Ntiya G.; Chattopadhyay, Subhasis

    2014-01-01

    The evolutionary conserved, less-polymorphic, nonclassical major histocompatibility complex (MHC) class I molecules: Qa-1 and its human homologue human leukocyte antigen-E (HLA-E) along with HLA-F, G and H cross-talk with the T-cell receptors and also interact with natural killer T-cells and other lymphocytes. Moreover, these nonclassical MHC molecules are known to interact with CD94/NKG2 heterodimeric receptors to induce immune responses and immune regulations. This dual role of Qa-1/HLA-E in terms of innate and adaptive immunity makes them more interesting. This review highlights the new updates of the mammalian nonclassical MHC-I molecules in terms of their gene organization, evolutionary perspective and their role in immunity. PMID:25400340

  5. Control of yeast mating signal transduction by a mammalian. beta. sub 2 -adrenergic receptor and G sub s. alpha. subunit

    SciTech Connect

    King, K.; Caron, M.G.; Lefkowitz, R.J. ); Dohlman, H.G.; Thorner, J. )

    1990-10-05

    To facilitate functional and mechanistic studies of receptor-G protein interactions by expression of the human {beta}{sub 2}-adrenergic receptor (h{beta}-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h{beta}-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h{beta}-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h{beta}-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by {beta}-adrenergic receptor agonists was achieved in cells coexpressing h{beta}-AR and a mammalian G protein (G{sub s}) {alpha} subunit - demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.

  6. Quantitative autoradiographic characterization of GA-BA sub B receptors in mammalian central nervous system

    SciTech Connect

    Chu, D.Chin-Mei.

    1989-01-01

    The inhibitory effects of the amino acid neurotransmitter {gamma}-aminobutyric acid (GABA) within the nervous system appear to be mediated through two distinct classes of receptors: GABA{sub A} and GABA{sub B} receptors. A quantitative autoradiographic method with {sup 3}H-GABA was developed to examine the hypotheses that GABA{sub A} and GABA{sub B} sites have distinct anatomical distributions, pharmacologic properties, and synaptic localizations within the rodent nervous system. The method was also applied to a comparative study of these receptors in postmortem human brain from individuals afflicted with Alzheimer's disease and those without neurologic disease. The results indicated that GABA{sub B} receptors occur in fewer numbers and have a lower affinity for GABA than GABA{sub A} receptors in both rodent and human brain. Within rodent brain, the distribution of these two receptor populations were clearly distinct. GABA{sub B} receptors were enriched in the medial habenula, interpeduncular nucleus, cerebellar molecular layer and olfactory glomerular layer. After selective lesions of postsynaptic neurons of the corticostriatal and perforant pathway, both GABA{sub B} and GABA{sub A} receptors were significantly decreased in number. Lesions of the presynaptic limbs of the perforant but not the corticostriatal pathway resulted in upregulation of both GABA receptors in the area of innervation. GABA{sub B} receptors were also upregulated in CA3 dendritic regions after destruction of dentate granule neurons.

  7. Study of a synthetic human olfactory receptor 17-4: expression and purification from an inducible mammalian cell line.

    PubMed

    Cook, Brian L; Ernberg, Karin E; Chung, Hyeyoun; Zhang, Shuguang

    2008-01-01

    In order to begin to study the structural and functional mechanisms of olfactory receptors, methods for milligram-scale purification are required. Here we demonstrate the production and expression of a synthetically engineered human olfactory receptor hOR17-4 gene in a stable tetracycline-inducible mammalian cell line (HEK293S). The olfactory receptor gene was fabricated from scratch using PCR-based gene-assembly, which facilitated codon optimization and attachment of a 9-residue bovine rhodopsin affinity tag for detection and purification. Induction of adherent cultures with tetracycline together with sodium butyrate led to hOR17-4 expression levels of approximately 30 microg per 150 mm tissue culture plate. Fos-choline-based detergents proved highly capable of extracting the receptors, and fos-choline-14 (N-tetradecylphosphocholine) was selected for optimal solubilization and subsequent purification. Analysis by SDS-PAGE revealed both monomeric and dimeric receptor forms, as well as higher MW oligomeric species. A two-step purification method of immunoaffinity and size exclusion chromatography was optimized which enabled 0.13 milligrams of hOR17-4 monomer to be obtained at >90% purity. This high purity of hOR17-4 is not only suitable for secondary structural and functional analyses but also for subsequent crystallization trials. Thus, this system demonstrates the feasibility of purifying milligram quantities of the GPCR membrane protein hOR17-4 for fabrication of olfactory receptor-based bionic sensing device. PMID:18682799

  8. The chicken yolk sac IgY receptor, a mammalian mannose receptor family member, transcytoses IgY across polarized epithelial cells.

    PubMed

    Tesar, Devin B; Cheung, Evelyn J; Bjorkman, Pamela J

    2008-04-01

    In mammals the transfer of passive immunity from mother to young is mediated by the MHC-related receptor FcRn, which transports maternal IgG across epithelial cell barriers. In birds, maternal IgY in egg yolk is transferred across the yolk sac to passively immunize chicks during gestation and early independent life. The chicken yolk sac IgY receptor (FcRY) is the ortholog of the mammalian phospholipase A2 receptor, a mannose receptor family member, rather than an FcRn or MHC homolog. FcRn and FcRY both exhibit ligand binding at the acidic pH of endosomes and ligand release at the slightly basic pH of blood. Here we show that FcRY expressed in polarized mammalian epithelial cells functioned in endocytosis, bidirectional transcytosis, and recycling of chicken FcY/IgY. Confocal immunofluorescence studies demonstrated that IgY binding and endocytosis occurred at acidic but not basic pH, mimicking pH-dependent uptake of IgG by FcRn. Colocalization studies showed FcRY-mediated internalization via clathrin-coated pits and transport involving early and recycling endosomes. Disruption of microtubules partially inhibited apical-to-basolateral and basolateral-to-apical transcytosis, but not recycling, suggesting the use of different trafficking machinery. Our results represent the first cell biological evidence of functional equivalence between FcRY and FcRn and provide an intriguing example of how evolution can give rise to systems in which similar biological requirements in different species are satisfied utilizing distinct protein folds. PMID:18256279

  9. Skewed T cell receptor repertoire of Vδ1+ γδ T lymphocytes after human allogeneic haematopoietic stem cell transplantation and the potential role for Epstein–Barr virus-infected B cells in clonal restriction

    PubMed Central

    Fujishima, N; Hirokawa, M; Fujishima, M; Yamashita, J; Saitoh, H; Ichikawa, Y; Horiuchi, T; Kawabata, Y; Sawada, K-I

    2007-01-01

    The proliferation of Vδ1+ γδ T lymphocytes has been described in various infections including human immunodeficiency virus (HIV), cytomegalovirus (CMV) and malaria. However, the antigen specificity and functions of the human Vδ1+ T cells remain obscure. We sought to explore the biological role for this T cell subset by investigating the reconstitution of T cell receptor (TCR) repertoires of Vδ1+ γδ T lymphocytes after human allogeneic haematopoietic stem cell transplantation (HSCT). We observed skewed TCR repertoires of the Vδ1+ T cells in 27 of 44 post-transplant patients. Only one patient developed EBV-associated post-transplant lymphoproliferative disorder in the present patient cohort. The -WGI- amino acid motif was observed in CDR3 of clonally expanded Vδ1+ T cells in half the patients. A skew was also detected in certain healthy donors, and the Vδ1+ T cell clone derived from the donor mature T cell pool persisted in the recipient's blood even 10 years after transplant. This T cell clone expanded in vitro against stimulation with autologous EBV–lymphoblastoid cell lines (LCL), and the Vδ1+ T cell line expanded in vitro from the same patient showed cytotoxicity against autologous EBV–LCL. EBV-infected cells could also induce in vitro oligoclonal expansions of autologous Vδ1+ T cells from healthy EBV-seropositive individuals. These results suggest that human Vδ1+ T cells have a TCR repertoire against EBV-infected B cells and may play a role in protecting recipients of allogeneic HSCT from EBV-associated disease. PMID:17425654

  10. Ligand-dependent interactions of the Ah receptor with coactivators in a mammalian two-hybrid assay

    SciTech Connect

    Zhang Shu; Rowlands, Craig; Safe, Stephen

    2008-03-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a high affinity ligand for the aryl hydrocarbon receptor (AhR). In this study, we investigated structure-dependent differences in activation of the AhR by a series of halogenated aromatic hydrocarbons. TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB126) induced CYP1A1-dependent activities in HEK293 human embryonic kidney, Panc1 pancreatic cancer, and Hepa1c1c7 mouse hepatoma cell lines. There was a structure-dependent difference in the efficacy of TCDF and PCB126 in HEK293 and Panc1 cells since induced CYP1A1 mRNA levels were lower than observed for the other congeners. A mammalian two-hybrid assay in cells transfected with GAL4-coactivator and AhR-VP16 chimeras was used to investigate structure-dependent interactions of these chimeras in Panc1, HEK293, and Hepa1c1c7 cells. The reporter construct pGAL4-luc contains five tandem GAL4 response elements linked to the luciferase gene and the GAL4-coactivator chimeras express several coactivators including steroid receptor coactivator 1 (SRC-1), SRC-2 and SRC-3, the mediator coactivator TRAP220, coactivator associated arginine methyl transferase 1 (CARM-1), and peroxisome proliferator-activated receptor {gamma} coactivator 1 (PGC-1). Results of the mammalian two-hybrid studies clearly demonstrate that activation of pGAL4-luc in cells transfected with VP-AhR and GAL4-coactivator chimeras is dependent on the structure of the HAH congener, cell context, and coactivator, suggesting that the prototypical HAH congeners used in this study exhibit selective AhR modulator activity.

  11. Expression of mammalian beta-adrenergic receptors in Xenopus laevis oocytes

    SciTech Connect

    Bahouth, S.W.; Malbon, C.C.

    1987-05-01

    Xenopus laevis oocytes are a useful transcription and expression system for DNA and RNA, respectively. Total cellular RNA was extracted from mouse lymphoma S49 cells and poly(A)/sup +/mRNA prepared by affinity chromatography of RNA on oligo(dT) cellulose. The membranes of S49 cells contain beta-adrenergic receptors that display pharmacological characteristics of beta/sub 2/-subtype. Xenopus laevis oocytes were injected with 50 ng of mRNA/oocyte. Expression of beta-adrenergic receptors in oocytes incubated for 30 hr after microinjection was assessed in membranes by radioligand binding using (/sup 3/H) dihydroalprenolol. The injected oocytes displayed 0.34 fmol receptor/oocyte as compared to 0.02 fmol receptor/oocyte in the control oocytes. The affinity of beta-adrenergic receptors in injected oocytes for this radioligand was 2 nM, a value similar to the affinity of beta-adrenergic receptors for DHA in S49 cell membranes. The potency of beta-adrenergic agonists in competing for DHA binding to oocytes membranes was isoproterenol > epinephrine > norepineprine, indicating that the expressed beta-adrenergic receptors were of the beta/sub 2/-subtype. The K/sub I/ of these agonists for the beta-adrenergic receptor in oocyte membranes was 0.03, 0.15 and 1.2 ..mu..M, respectively. The role of post-translational modification in dictating receptor subtype is analyzed using mRNA of beta/sub 1/- as well as beta/sub 2/-adrenergic receptors.

  12. Nanoscale organization of {beta}{sub 2}-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

    SciTech Connect

    Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer; Bani-Yaghoub, Mahmud; Taylor, Rod; Johnston, Linda J.; Pezacki, John Paul

    2009-04-24

    Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize {beta}{sub 2}-adrenergic receptors ({beta}{sub 2}AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the {beta}{sub 2}AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the {beta}{sub 2}AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of {beta}{sub 2}AR are observed in the membrane of the HEK293 cells that stably overexpress {beta}{sub 2}AR-GFP and {beta}{sub 2}AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use {beta}{sub 2}AR-Venus fusion proteins as models for {beta}{sub 2}AR function. These observations are critical for designing future cell models and assays based on {beta}{sub 2}AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

  13. Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system

    PubMed Central

    Cheng, Hao; Fan, Chen; Zhang, Si-wei; Wu, Zhong-shan; Cui, Zhi-cheng; Melcher, Karsten; Zhang, Cheng-hai; Jiang, Yi; Cong, Yao; Xu, H Eric

    2015-01-01

    Aim: To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target. Methods: α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis. Results: Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel. Conclusion: We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis. PMID:26073323

  14. Characterizing immune repertoires by high throughput sequencing: strategies and applications

    PubMed Central

    Calis, Jorg J.A.; Rosenberg, Brad R.

    2014-01-01

    As the key cellular effectors of adaptive immunity, T and B lymphocytes utilize specialized receptors to recognize, respond to, and neutralize a diverse array of extrinsic threats. These receptors (immunoglobulins in B lymphocytes, T cell receptors in T lymphocytes) are incredibly variable, the products of specialized genetic diversification mechanisms that generate complex lymphocyte repertoires with extensive collections of antigen specificities. Recent advances in high throughput sequencing (HTS) technologies have transformed our ability to examine antigen receptor repertoires at single nucleotide, and more recently, single cell, resolution. Here we review current approaches to examining antigen receptor repertoires by HTS, and discuss inherent biological and technical challenges. We further describe emerging applications of this powerful methodology for exploring the adaptive immune system. PMID:25306219

  15. Phosphoinositide 3-kinase dependent inhibition as a broad basis for opponent coding in Mammalian olfactory receptor neurons.

    PubMed

    Ukhanov, Kirill; Corey, Elizabeth A; Ache, Barry W

    2013-01-01

    Phosphoinositide 3-kinase (PI3K) signaling has been implicated in mediating inhibitory odorant input to mammalian olfactory receptor neurons (ORNs). To better understand the breadth of such inhibition in odor coding, we screened a panel of odorants representing different chemical classes, as well as odorants known to occur in a natural odor object (tomato), for their ability to rapidly activate PI3K-dependent inhibitory signaling. Odorants were screened on dissociated native rat ORNs before and after pre-incubation with the PI3K-isoform specific blockers AS252424 and TGX221. Many different odorants increased their excitatory strength for particular ORNs following PI3K blockade in a manner consistent with activating PI3K-dependent inhibitory signaling in those cells. The PI3K-dependent inhibitory odorants overlapped with conventional excitatory odorants, but did not share the same bias, indicating partial partitioning of the odor space. Finding that PI3K-dependent inhibition can be activated by a wide range of otherwise conventional excitatory odorants strongly implies PI3K-dependent inhibition provides a broad basis for opponent coding in mammalian ORNs. PMID:23585911

  16. The mammalian tachykinin ligand-receptor system: an emerging target for central neurological disorders

    PubMed Central

    Pantaleo, Nick; Chadwick, Wayne; Park, Sung-Soo; Wang, Liyun; Zhou, Yu; Martin, Bronwen; Maudsley, Stuart

    2010-01-01

    Our understanding of the complex signaling neurophysiology of the central nervous system has facilitated the exploration of potential novel receptor-ligand system targets for disorders of this most complex organ. In recent years, many relatively neglected receptor-ligand systems have been re-evaluated with respect to their ability to potently modulate discrete tracts in the central nervous system. One such system is the tachykinin (previously neurokinin) system. The multiple heptahelical G protein-coupled receptors and neuropeptide ligands that comprise this system may be significantly involved in more central nervous systems actions than previously thought, including sleep disorders, amyotrophic lateral sclerosis, Alzheimer’s and Machado-Joseph disease. The development of our understanding of the role of the tachykinin receptor-ligand system in higher order central functions is likely to allow the creation of more specific and selective tachykinin-related neurotherapeutics. PMID:20632965

  17. Juno is the egg Izumo receptor and is essential for mammalian fertilisation

    PubMed Central

    Bianchi, Enrica; Doe, Brendan; Goulding, David; Wright, Gavin J.

    2014-01-01

    Fertilisation occurs when sperm and egg recognise each other and fuse to form a new, genetically distinct organism. The molecular basis of sperm-egg recognition is unknown, but is likely to require interactions between receptor proteins displayed on their surface. Izumo1 is an essential sperm cell surface protein, but its egg receptor has remained a mystery. Here, we identify Juno as the receptor for Izumo1 on mouse eggs, and show this interaction is conserved within mammals. Female mice lacking Juno are infertile and Juno-deficient eggs do not fuse with normal sperm. Rapid shedding of Juno from the oolemma after fertilisation suggests a mechanism for the membrane block to polyspermy, ensuring eggs normally fuse with just a single sperm. Our discovery of an essential receptor pair at the nexus of conception provides opportunities for the rational development of new fertility treatments and contraceptives. PMID:24739963

  18. Natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts.

    PubMed

    Rose, Patrick P; Hanna, Sheri L; Spiridigliozzi, Anna; Wannissorn, Nattha; Beiting, Daniel P; Ross, Susan R; Hardy, Richard W; Bambina, Shelly A; Heise, Mark T; Cherry, Sara

    2011-08-18

    Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter natural resistance-associated macrophage protein (NRAMP) as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were nonpermissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multipass membrane proteins for infection. PMID:21843867

  19. Molecular size of the gamma-aminobutyric acidA receptor purified from mammalian cerebral cortex.

    PubMed

    Mamalaki, C; Barnard, E A; Stephenson, F A

    1989-01-01

    The hydrodynamic behaviour of both the soluble and purified gamma-aminobutyric acidA (GABAA) receptor of bovine or rat cerebral cortex has been investigated in solution in Triton X-100 or in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS). In all the hydrodynamic separations made, it was found that the binding activities for GABA, benzodiazepine, and (where detectable) t-butylbicyclophosphorothionate comigrated. Conditions were established for gel exclusion chromatography and for sucrose density gradient velocity sedimentation that maintain the GABAA receptor in a nonaggregated form. Using these conditions, the molecular weight of the bovine GABAA receptor in the above-mentioned detergents was calculated using the H2O/2H2O method. A value of Mr 230,000-240,000 was calculated for the bovine pure GABAA receptor purified in sodium deoxycholate/Triton X-100 media. A value of Mr 284,000-290,000 was calculated for the nonaggregated bovine or rat cortex receptor in CHAPS, but the Stokes radius is smaller in the latter than in the former medium and the detergent binding in CHAPS is underestimated. Thus the deduced Mr, 240,000, is the best estimate by this method. PMID:2535707

  20. ß-Adrenergic Receptor Signaling and Modulation of Long-Term Potentiation in the Mammalian Hippocampus

    ERIC Educational Resources Information Center

    O'Dell, Thomas J.; Connor, Steven A.; Guglietta, Ryan; Nguyen, Peter V.

    2015-01-01

    Encoding new information in the brain requires changes in synaptic strength. Neuromodulatory transmitters can facilitate synaptic plasticity by modifying the actions and expression of specific signaling cascades, transmitter receptors and their associated signaling complexes, genes, and effector proteins. One critical neuromodulator in the…

  1. Receptor-Mediated Entry of Pristine Octahedral DNA Nanocages in Mammalian Cells.

    PubMed

    Vindigni, Giulia; Raniolo, Sofia; Ottaviani, Alessio; Falconi, Mattia; Franch, Oskar; Knudsen, Birgitta R; Desideri, Alessandro; Biocca, Silvia

    2016-06-28

    DNA offers excellent programming properties for the generation of nanometer-scaled polyhedral structures with a broad variety of potential applications. Translation to biomedical applications requires improving stability in biological fluids, efficient and selective cell binding, and/or internalization of the assembled DNA nanostructures. Here, we report an investigation on the selective mechanism of cellular uptake of pristine DNA nanocages in cells expressing the receptor "oxidized low-density lipoprotein receptor-1" (LOX-1), a scavenger receptor associated with cardiovascular diseases and, more recently, identified as a tumor marker. For this purpose a truncated octahedral DNA nanocage functionalized with a single biotin molecule, which allows DNA cage detection through the biotin-streptavidin assays, was constructed. The results indicate that DNA nanocages are stable in biological fluids, including human serum, and are selectively bound and very efficiently internalized in vesicles only in LOX-1-expressing cells. The amount of internalized cages is 30 times higher in LOX-1-expressing cells than in normal fibroblasts, indicating that the receptor-mediated uptake of pristine DNA nanocages can be pursued for a selective cellular internalization. These results open the route for a therapeutic use of pristine DNA cages targeting LOX-1-overexpressing tumor cells. PMID:27214742

  2. REACTIVITY PROFILE OF LIGANDS OF MAMMALIAN RETINOIC ACID RECEPTORS: A PRELIMINARY COREPA ANALYSIS

    EPA Science Inventory

    Retinoic acid and associated derivatives comprise a class of endogenous hormones that bind to and activate different families of retinoic acid receptors (RARs, RXRs), and control many aspects of vertebrate development. Identification of potential RAR and RXR ligands is of interes...

  3. Functional differences between junctional and extrajunctional adrenergic receptor activation in mammalian ventricle

    PubMed Central

    Ajijola, Olujimi A.; Vaseghi, Marmar; Zhou, Wei; Yamakawa, Kentaro; Benharash, Peyman; Hadaya, Joseph; Lux, Robert L.; Mahajan, Aman

    2013-01-01

    Increased cardiac sympathetic activation worsens dispersion of repolarization and is proarrhythmic. The functional differences between intrinsic nerve stimulation and adrenergic receptor activation remain incompletely understood. This study was undertaken to determine the functional differences between efferent cardiac sympathetic nerve stimulation and direct adrenergic receptor activation in porcine ventricles. Female Yorkshire pigs (n = 13) underwent surgical exposure of the heart and stellate ganglia. A 56-electrode sock was placed over the ventricles to record epicardial electrograms. Animals underwent bilateral sympathetic stimulation (BSS) (n = 8) or norepinephrine (NE) administration (n = 5). Activation recovery intervals (ARIs) were measured at each electrode before and during BSS or NE. The degree of ARI shortening during BSS or NE administration was used as a measure of functional nerve or adrenergic receptor density. During BSS, ARI shortening was nonuniform across the epicardium (F value 9.62, P = 0.003), with ARI shortening greatest in the mid-basal lateral right ventricle and least in the midposterior left ventricle (LV) (mean normalized values: 0.9 ± 0.08 vs. 0.56 ± 0.08; P = 0.03). NE administration resulted in greater ARI shortening in the LV apex than basal segments [0.91 ± 0.04 vs. 0.63 ± 0.05 (averaged basal segments); P = 0.003]. Dispersion of ARIs increased in 50% and 60% of the subjects undergoing BSS and NE, respectively, but decreased in the others. There is nonuniform response to cardiac sympathetic activation of both porcine ventricles, which is not fully explained by adrenergic receptor density. Different pools of adrenergic receptors may mediate the cardiac electrophysiological effects of efferent sympathetic nerve activity and circulating catecholamines. PMID:23241324

  4. Extracellular ATP inhibits chloride channels in mature mammalian skeletal muscle by activating P2Y1 receptors.

    PubMed

    Voss, Andrew A

    2009-12-01

    ATP is released from skeletal muscle during exercise, a discovery dating back to 1969. Surprisingly, few studies have examined the effects of extracellular ATP on mature mammalian skeletal muscle. This electrophysiological study examined the effects of extracellular ATP on fully innervated rat levator auris longus using two intracellular microelectrodes. The effects of ATP were determined by measuring the relative changes of miniature endplate potentials (mEPPs) and voltage responses to step current pulses in individual muscle fibres. Exposure to ATP (20 microm) prolonged the mEPP falling phase by 31 +/- 7.5% (values +/- s.d., n = 3 fibres). Concurrently, the input resistance increased by 31 +/- 2.0% and the time course of the voltage responses increased by 59 +/- 3.0%. Analogous effects were observed using 2 and 5 microm ATP, and on regions distal from the neuromuscular junction, indicating that physiologically relevant levels of ATP enhanced electrical signalling over the entire muscle fibre. The effects of extracellular ATP were blocked by 200 microm anthracene-9-carboxylic acid, a chloride channel inhibitor, and reduced concentrations of extracellular chloride, indicating that ATP inhibited chloride channels. A high affinity agonist for P2Y receptors, 2-methylthioadenosine-5-O-diphosphate (2MeSADP), induced similar effects to ATP with an EC(50) of 160 +/- 30 nm. The effects of 250 nm2MeSADP were blocked by 500 nmMRS2179, a specific P2Y(1) receptor inhibitor, suggesting that ATP acts on P2Y(1) receptors to inhibit chloride channels. The inhibition of chloride channels by extracellular ATP has implications for muscle excitability and fatigue, and the pathophysiology of myotonias. PMID:19805741

  5. G protein-coupled odorant receptors underlie mechanosensitivity in mammalian olfactory sensory neurons

    PubMed Central

    Connelly, Timothy; Yu, Yiqun; Grosmaitre, Xavier; Wang, Jue; Santarelli, Lindsey C.; Savigner, Agnes; Qiao, Xin; Wang, Zhenshan; Storm, Daniel R.; Ma, Minghong

    2015-01-01

    Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR–G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical responses of OSNs. Furthermore, ectopic expression of an OR restores mechanosensitivity in loss-of-function OSNs. Lastly, heterologous expression of an OR confers mechanosensitivity to its host cells. These results indicate that certain ORs are both necessary and sufficient to cause mechanical responses, revealing a previously unidentified mechanism for mechanotransduction. PMID:25550517

  6. Subunit composition of mammalian transient receptor potential channels in living cells.

    PubMed

    Hofmann, Thomas; Schaefer, Michael; Schultz, Günter; Gudermann, Thomas

    2002-05-28

    Hormones, neurotransmitters, and growth factors give rise to calcium entry via receptor-activated cation channels that are activated downstream of phospholipase C activity. Members of the transient receptor potential channel (TRPC) family have been characterized as molecular substrates mediating receptor-activated cation influx. TRPC channels are assumed to be composed of multiple TRPC proteins. However, the cellular principles governing the assembly of TRPC proteins into homo- or heteromeric ion channels still remain elusive. By pursuing four independent experimental approaches--i.e., subcellular cotrafficking of TRPC subunits, differential functional suppression by dominant-negative subunits, fluorescence resonance energy transfer between labeled TRPC subunits, and coimmunoprecipitation--we investigate the combinatorial rules of TRPC assembly. Our data show that (i) TRPC2 does not interact with any known TRPC protein and (ii) TRPC1 has the ability to form channel complexes together with TRPC4 and TRPC5. (iii) All other TRPCs exclusively assemble into homo- or heterotetramers within the confines of TRPC subfamilies--e.g., TRPC4/5 or TRPC3/6/7. The principles of TRPC channel formation offer the conceptual framework to assess the physiological role of distinct TRPC proteins in living cells. PMID:12032305

  7. Mammalian pheromones.

    PubMed

    Liberles, Stephen D

    2014-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors. PMID:23988175

  8. Mammalian Pheromones

    PubMed Central

    Liberles, Stephen D.

    2015-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d ) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors. PMID:23988175

  9. Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors.

    PubMed

    Maksimovic, Srdjan; Nakatani, Masashi; Baba, Yoshichika; Nelson, Aislyn M; Marshall, Kara L; Wellnitz, Scott A; Firozi, Pervez; Woo, Seung-Hyun; Ranade, Sanjeev; Patapoutian, Ardem; Lumpkin, Ellen A

    2014-05-29

    Touch submodalities, such as flutter and pressure, are mediated by somatosensory afferents whose terminal specializations extract tactile features and encode them as action potential trains with unique activity patterns. Whether non-neuronal cells tune touch receptors through active or passive mechanisms is debated. Terminal specializations are thought to function as passive mechanical filters analogous to the cochlea's basilar membrane, which deconstructs complex sounds into tones that are transduced by mechanosensory hair cells. The model that cutaneous specializations are merely passive has been recently challenged because epidermal cells express sensory ion channels and neurotransmitters; however, direct evidence that epidermal cells excite tactile afferents is lacking. Epidermal Merkel cells display features of sensory receptor cells and make 'synapse-like' contacts with slowly adapting type I (SAI) afferents. These complexes, which encode spatial features such as edges and texture, localize to skin regions with high tactile acuity, including whisker follicles, fingertips and touch domes. Here we show that Merkel cells actively participate in touch reception in mice. Merkel cells display fast, touch-evoked mechanotransduction currents. Optogenetic approaches in intact skin show that Merkel cells are both necessary and sufficient for sustained action-potential firing in tactile afferents. Recordings from touch-dome afferents lacking Merkel cells demonstrate that Merkel cells confer high-frequency responses to dynamic stimuli and enable sustained firing. These data are the first, to our knowledge, to directly demonstrate a functional, excitatory connection between epidermal cells and sensory neurons. Together, these findings indicate that Merkel cells actively tune mechanosensory responses to facilitate high spatio-temporal acuity. Moreover, our results indicate a division of labour in the Merkel cell-neurite complex: Merkel cells signal static stimuli, such as

  10. Muscarinic receptors mediate negative and positive inotropic effects in mammalian ventricular myocardium: differentiation by agonists.

    PubMed Central

    Korth, M.; Kühlkamp, V.

    1987-01-01

    , and did not cause an increase in intracellular Na+ activity in the quiescent papillary muscle. The results show that muscarinic receptors of the ventricular myocardium mediate two inotropic effects, which are opposite in direction and differ in their concentration-dependence by a factor of 100. Although agonists differentiate between both inotropic effects, it is unknown whether the receptors involved represent receptor states or separate receptor subpopulations. The negative inotropic effect of choline esters and of oxotremorine can be best explained by adenylate cyclase inhibition.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2434180

  11. Inferring processes underlying B-cell repertoire diversity.

    PubMed

    Elhanati, Yuval; Sethna, Zachary; Marcou, Quentin; Callan, Curtis G; Mora, Thierry; Walczak, Aleksandra M

    2015-09-01

    We quantify the VDJ recombination and somatic hypermutation processes in human B cells using probabilistic inference methods on high-throughput DNA sequence repertoires of human B-cell receptor heavy chains. Our analysis captures the statistical properties of the naive repertoire, first after its initial generation via VDJ recombination and then after selection for functionality. We also infer statistical properties of the somatic hypermutation machinery (exclusive of subsequent effects of selection). Our main results are the following: the B-cell repertoire is substantially more diverse than T-cell repertoires, owing to longer junctional insertions; sequences that pass initial selection are distinguished by having a higher probability of being generated in a VDJ recombination event; somatic hypermutations have a non-uniform distribution along the V gene that is well explained by an independent site model for the sequence context around the hypermutation site. PMID:26194757

  12. Inferring processes underlying B-cell repertoire diversity

    PubMed Central

    Elhanati, Yuval; Sethna, Zachary; Marcou, Quentin; Callan, Curtis G.; Mora, Thierry; Walczak, Aleksandra M.

    2015-01-01

    We quantify the VDJ recombination and somatic hypermutation processes in human B cells using probabilistic inference methods on high-throughput DNA sequence repertoires of human B-cell receptor heavy chains. Our analysis captures the statistical properties of the naive repertoire, first after its initial generation via VDJ recombination and then after selection for functionality. We also infer statistical properties of the somatic hypermutation machinery (exclusive of subsequent effects of selection). Our main results are the following: the B-cell repertoire is substantially more diverse than T-cell repertoires, owing to longer junctional insertions; sequences that pass initial selection are distinguished by having a higher probability of being generated in a VDJ recombination event; somatic hypermutations have a non-uniform distribution along the V gene that is well explained by an independent site model for the sequence context around the hypermutation site. PMID:26194757

  13. Differential properties of type I and type II benzodiazepine receptors in mammalian CNS neurones.

    PubMed Central

    Yakushiji, T.; Shirasaki, T.; Munakata, M.; Hirata, A.; Akaike, N.

    1993-01-01

    1. The effects of benzodiazepine receptor (BZR) partial agonists, Y-23684 and CL218,872, were compared with its full agonist, diazepam, on gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) in acutely dissociated rat cerebral cortex (CTX), cerebellar Purkinje (CPJ) and spinal ventral horn (SVH) neurones, by the whole-cell mode patch-clamp technique. 2. The GABA-induced responses were essentially the same in both SVH and CPJ neurones, but the KD value of the GABA response in CTX neurone was lower than those in the other two brain regions. 3. Enhancement of the GABA response by the two partial agonists was about one-third of that by diazepam in the SVH neurones (where type II subtype of BZR, BZ2, is predominant), whereas these partial agonists potentiated the GABA response as much as diazepam in CPJ neurones (where the type I subtype of BZR, BZ1, is predominant). In CTX neurones where both type I and II variants are expressed, the augmentation ratio of the GABA response by diazepam was between the values in CPJ and SVH neurones. 4. In concentration-response relationships of BZR partial agonists, the threshold concentrations, KD values and maximal augmentation ratio of the GABA response were similar in all CTX, CPJ and SVH neurones. Also, in all preparations, the threshold concentration and KD values of diazepam action were 10 fold less than those induced by partial agonists. 5. All BZR agonists shifted the concentration-response relationship for GABA to the left without changing the maximum current amplitude, indicating that activation of both BZ1 and BZ2 increase the affinity of the GABAA receptor for GABA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8395299

  14. A Role for the Chemokine Receptor CCR6 in Mammalian Sperm Motility and Chemotaxis

    PubMed Central

    Caballero-Campo, Pedro; Buffone, Mariano G.; Benencia, Fabian; Conejo-García, José R.; Rinaudo, Paolo F.; Gerton, George L.

    2013-01-01

    Although recent evidence indicates that several chemokines and defensins, well-known as inflammatory mediators, are expressed in the male and female reproductive tracts, the location and functional significance of chemokine networks in sperm physiology and sperm reproductive tract interactions are poorly understood. To address this deficiency in our knowledge, we examined the expression and function in sperm of CCR6, a receptor common to several chemoattractant peptides, and screened several reproductive tract fluids for the presence of specific ligands. CCR6 protein is present in mouse and human sperm and mainly localized in the sperm tail with other minor patterns in sperm from mice (neck and acrosomal region) and men (neck and midpiece regions). As expected from the protein immunoblotting and immunofluorescence results, mouse Ccr6 mRNA is expressed in the testis. Furthermore, the Defb29 mRNA encoding the CCR6 ligand, β-defensin DEFB29, is expressed at high levels in the epididymis. As determined by protein chip analysis, several chemokines (including some that act through CCR6, such as CCL20/MIP-3α (formerly Macrophage Inflammatory Protein 3α) and protein hormones were present in human follicular fluid, endometrial secretions, and seminal plasma. In functional chemotaxis assays, capacitated human sperm exhibited a directional movement towards CCL20, and displayed modifications in motility parameters. Our data indicate that chemokine ligand/receptor interactions in the male and female genital tracts promote sperm motility and chemotaxis under non-inflammatory conditions. Therefore, some of the physiological reactions mediated by CCR6 ligands in male reproduction extend beyond a pro-inflammatory response and might find application in clinical reproduction and/or contraception. PMID:23765988

  15. The gene repertoire and the common evolutionary history of glutamate, pheromone (V2R), taste(1) and other related G protein-coupled receptors.

    PubMed

    Bjarnadóttir, Thóra K; Fredriksson, Robert; Schiöth, Helgi B

    2005-12-01

    Glutamate receptors (also known as clan C) are one of the main groups of GPCRs with many subgroup linked through complex evolutionary relationships. We performed thorough searches for genes coding for proteins belonging to this family in the human, mouse, Fugu, and zebrafish genomes, as well as in four invertebrate species. We assembled over 70 new full-length sequences from protein predictions excluding pseudogenes. This resulted in a total of 22 full-length sequences from the human genome, 79 from the mouse genome, 30 from the Fugu genome, and 32 from the zebrafish genome (pseudogenes are not included in these numbers). We show that the vertebrate Glutamate GPCRs form four main phylogenetic groups with a total of eight subgroups (Group I: V2R, TAS1R, GPRC6A, and CASR, Group II: GRM, Group III: GABA together with previously unpublished GPR158 and GPR158L and Group IV: GPRC5). All eight receptor subgroups are present both in mammals and fish, except for GPRC5 and GPR158. The pheromone (V2R), GPRC6, and sweet taste (TAS1) receptors were not found in invertebrates while GRM, GABA, and CASR were found in both C. elegans and C. intestinalis. The pheromone receptors are found in high numbers in mouse, zebrafish and Fugu but are only found as pseudogenes in the human genome. This report provides a comprehensive overview of the expansion/deletions of the groups within the Glutamate receptor family. PMID:16229975

  16. Deep mutational scanning of an antibody against epidermal growth factor receptor using mammalian cell display and massively parallel pyrosequencing

    PubMed Central

    Forsyth, Charles M.; Juan, Veronica; Akamatsu, Yoshiko; DuBridge, Robert B.; Doan, Minhtam; Ivanov, Alexander V.; Ma, Zhiyuan; Polakoff, Dixie; Razo, Jennifer; Wilson, Keith; Powers, David B.

    2013-01-01

    We developed a method for deep mutational scanning of antibody complementarity-determining regions (CDRs) that can determine in parallel the effect of every possible single amino acid CDR substitution on antigen binding. The method uses libraries of full length IgGs containing more than 1000 CDR point mutations displayed on mammalian cells, sorted by flow cytometry into subpopulations based on antigen affinity and analyzed by massively parallel pyrosequencing. Higher, lower and neutral affinity mutations are identified by their enrichment or depletion in the FACS subpopulations. We applied this method to a humanized version of the anti-epidermal growth factor receptor antibody cetuximab, generated a near comprehensive data set for 1060 point mutations that recapitulates previously determined structural and mutational data for these CDRs and identified 67 point mutations that increase affinity. The large-scale, comprehensive sequence-function data sets generated by this method should have broad utility for engineering properties such as antibody affinity and specificity and may advance theoretical understanding of antibody-antigen recognition. PMID:23765106

  17. Natural Resistance-associated Macrophage Protein (NRAMP) is a cellular receptor for Sindbis virus in both insect and mammalian hosts

    PubMed Central

    Rose, Patrick P.; Hanna, Sheri L.; Spiridigliozzi, Anna; Wannissorn, Nattha; Beiting, Daniel P.; Ross, Susan R.; Hardy, Richard W.; Bambina, Shelly A.; Heise, Mark T.; Cherry, Sara

    2011-01-01

    Summary Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of for this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter Natural Resistance-Associated Macrophage Protein (NRAMP), as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were non-permissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multi-pass membrane proteins for infection. PMID:21843867

  18. BMP receptor IA is required in mammalian neural crest cells for development of the cardiac outflow tract and ventricular myocardium

    PubMed Central

    Stottmann, Rolf W.; Choi, Murim; Mishina, Yuji; Meyers, Erik N.; Klingensmith, John

    2010-01-01

    Summary The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. We have ablated Bmpr1a specifically in the neural crest, beginning at the five-somite stage. We find that most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium. PMID:15073157

  19. Current Techniques for Studying Oligomer Formations of G-Protein-Coupled Receptors Using Mammalian and Yeast Cells.

    PubMed

    Nakamura, Yasuyuki; Ishii, Jun; Kondo, Akihiko

    2016-05-27

    G-protein-coupled receptors (GPCRs) are physiologically important transmembrane proteins that sense signaling molecules such as hormones, neurotransmitters, and various sensory stimuli; GPCRs represent major molecular targets for drug discovery. Although GPCRs traditionally have been thought to function as monomers or homomers, in the recent years these proteins have also been shown to function as heteromers. Heteromerization among GPCRs is expected to generate potentially large functional and physiological diversity and to provide new opportunities for drug discovery. However, due to the existence of numerous combinations, the larger universe of possible GPCR heteromers is unknown, and thus its functional significance is still poorly understood. The oligomerization of GPCRs in living cells now has been demonstrated in mammalian cells and in native tissues by using genetic, biochemical, and physiological approaches, as well as various resonance energy transfer (RET) technologies. In addition, the yeast Saccharomyces cerevisiae, which can serve as a biosensor for monitoring eukaryotic biological processes, can also be used for the identification of functionally significant heteromer pairs of GPCRs. In this review, we focus on studies of GPCR oligomers, and summarize the technologies used to evaluate GPCR oligomerization. We additionally consider the potential limitations of these methods at present, and envision the possible future applications of these techniques. PMID:27052183

  20. Antisense Oligonucleotides Targeting Parasite Inositol 1,4,5-Trisphosphate Receptor Inhibits Mammalian Host Cell Invasion by Trypanosoma cruzi

    NASA Astrophysics Data System (ADS)

    Hashimoto, Muneaki; Nara, Takeshi; Hirawake, Hiroko; Morales, Jorge; Enomoto, Masahiro; Mikoshiba, Katsuhiko

    2014-02-01

    Chagas disease is caused by an intracellular parasitic protist, Trypanosoma cruzi. As there are no highly effective drugs against this agent that also demonstrate low toxicity, there is an urgent need for development of new drugs to treat Chagas disease. We have previously demonstrated that the parasite inositol 1,4,5-trisphosphate receptor (TcIP3R) is crucial for invasion of the mammalian host cell by T. cruzi. Here, we report that TcIP3R is a short-lived protein and that its expression is significantly suppressed in trypomastigotes. Treatment of trypomastigotes, an infective stage of T. cruzi, with antisense oligonucleotides specific to TcIP3R deceased TcIP3R protein levels and impaired trypomastigote invasion of host cells. Due to the resulting instability and very low expression level of TcIP3R in trypomastigotes indicates that TcIP3R is a promising target for antisense therapy in Chagas disease.

  1. Interaction of Dictyostelium discoideum lysosomal enzymes with the mammalian phosphomannosyl receptor. The importance of oligosaccharides which contain phosphodiesters

    SciTech Connect

    Freeze, H.H.

    1985-07-25

    Mammalian cell lysosomal enzymes or phosphorylated oligosaccharides derived from them are endocytosed by a phosphomannosyl receptor (PMR) found on the surface of fibroblasts. Various studies suggest that 2 residues of Man-6-P in phosphomonoester linkage but not diester linkage (PDE) are essential for a high rate of uptake. The lysosomal enzymes of the slime mold Dictyostelium discoideum are also recognized by the PMR on these cells; however, none of the oligosaccharides from these enzymes contain 2 phosphomonoesters. Instead, most contain multiple sulfate esters and 2 residues of Man-6-P in an unusual PDE linkage. In this study the authors have tried to account for the unexpected highly efficient uptake of the slime mold enzymes. The results show that nearly all of the alpha-mannosidase molecules contain the oligosaccharides required for uptake. Competition of SVI-beta-glucosidase uptake by various carbohydrate-containing fractions indicates that the best inhibitors are those with 2 PDE, either with or without sulfate esters. Complete denaturation of SVI-labeled wild-type beta-glucosidase in sodium dodecyl sulfate/dithiothreitol also reduces its uptake by about 10-fold. Taken together, these results suggest that the interactions of multiple, weakly binding oligosaccharides, especially those with 2 PDE, are important for the high rate of uptake of the slime mold enzymes.

  2. Blocking mammalian target of rapamycin alleviates bone cancer pain and morphine tolerance via µ-opioid receptor.

    PubMed

    Jiang, Zongming; Wu, Shaoyong; Wu, Xiujuan; Zhong, Junfeng; Lv, Anqing; Jiao, Jing; Chen, Zhonghua

    2016-04-15

    The current study was to examine the underlying mechanisms responsible for the role of mammalian target of rapamycin (mTOR) in regulating bone cancer-evoked pain and the tolerance of systemic morphine. Breast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats and this evoked significant mechanical and thermal hyperalgesia. Our results showed that the protein expression of p-mTOR, mTOR-mediated phosphorylation of 4E-binding protein 4 (4E-BP1), p70 ribosomal S6 protein kinase 1 (S6K1) as well as phosphatidylinositide 3-kinase (p-PI3K) pathways were amplified in the superficial dorsal horn of the spinal cord of bone cancer rats compared with control rats. Blocking spinal mTOR by using rapamycin significantly attenuated activities of PI3K signaling pathways as well as mechanical and thermal hyperalgesia. Additionally, rapamycin enhanced attenuations of protein kinase Cɛ (PKCɛ)/protein kinase A (PKA) induced by morphine and further extended analgesia of morphine via µ-opioid receptor (MOR). Our data for the first time revealed specific signaling pathways leading to bone cancer pain, including the activation of mTOR and PI3K and downstream PKCɛ/PKA, and resultant sensitization of MOR. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of bone cancer pain often observed in clinics. PMID:26566757

  3. Expression of Mammalian G Protein-Coupled Receptors in Caenorhabditis elegans

    PubMed Central

    Jastrzebska, Beata; Salom, David; Jin, Hui; Cao, Pengxiu; Sun, Wenyu; Palczewski, Krzysztof; Feng, Zhaoyang

    2014-01-01

    Constituting the largest group of membrane proteins identified in the human genome, G protein-coupled receptors (GPCRs) help control many physiological processes by responding to various stimuli. As targets for more than 40% of all prescribed pharmaceuticals, detailed understanding of GPCR structures is vital for the design and development of more specific medications and improved patient therapies. But structural information for membrane proteins and GPCRs, in particular, is limited despite considerable interest. The major impediment to obtaining sufficient quantities of highly purified GPCRs in their native form for crystallization lies in their low tissue levels, poor yields, and stability. The only exception is rhodopsin, which is abundantly expressed in the eye and stabilized by its covalently bound chromophore, 11-cis-retinal. Expression systems and purification protocols have yet to be developed for all other GPCRs. Here, we present a novel expression system for human GPCRs in Caenorhabditis elegans that produces sufficient amounts of recombinant proteins to allow their biochemical and structural characterization. PMID:23332703

  4. Isotopic labeling of mammalian G protein-coupled receptors heterologously expressed in Caenorhabditis elegans.

    PubMed

    Salom, David; Cao, Pengxiu; Yuan, Yiyuan; Miyagi, Masaru; Feng, Zhaoyang; Palczewski, Krzysztof

    2015-03-01

    High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack post-translational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work, we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with (15)N,(13)C by providing them with isotopically labeled bacteria. (2)H labeling also was achieved by growing C. elegans in the presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the "test" GPCR to demonstrate the viability of this approach. Although the worms' cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization. PMID:25461480

  5. Genetics, Receptor Binding, Replication, and Mammalian Transmission of H4 Avian Influenza Viruses Isolated from Live Poultry Markets in China

    PubMed Central

    Liang, Libin; Deng, Guohua; Shi, Jianzhong; Wang, Shuai; Zhang, Qianyi; Kong, Huihui; Gu, Chunyang; Guan, Yuntao; Suzuki, Yasuo; Li, Yanbing; Jiang, Yongping; Tian, Guobin; Liu, Liling

    2015-01-01

    ABSTRACT H4 avian influenza virus (AIV) is one of the most prevalent influenza virus subtypes in the world. However, whether H4 AIVs pose a threat to public health remains largely unclear. Here, we analyzed the phylogenetic relationships, receptor binding properties, replication, and transmissibility in mammals of H4 AIVs isolated from live poultry markets in China between 2009 and 2012. Genomic sequence analysis of 36 representative H4 viruses revealed 32 different genotypes, indicating that these viruses are undergoing complex and frequent reassortment events. All 32 viruses tested could replicate in the respiratory organs of infected mice without prior adaptation. Receptor binding analysis demonstrated that the H4 AIVs bound to α-2,6-linked glycans, although they retained the binding preference for α-2,3-linked glycans. When we tested the direct-contact transmission of 10 H4 viruses in guinea pigs, we found that three viruses did not transmit to any of the contact animals, one virus transmitted to one of three contact animals, and six viruses transmitted to all three contact animals. When we further tested the respiratory droplet transmissibility of four of the viruses that transmitted efficiently via direct contact, we found that three of them could transmit to one or two of the five exposed animals. Our study demonstrates that the current circulating H4 AIVs can infect, replicate in, and transmit to mammalian hosts, thereby posing a potential threat to human health. These findings emphasize the continual need for enhanced surveillance of H4 AIVs. IMPORTANCE Numerous surveillance studies have documented the wide distribution of H4 AIVs throughout the world, yet the biological properties of H4 viruses have not been well studied. In this study, we found that multiple genotypes of H4 viruses are cocirculating in the live poultry markets of China and that H4 viruses can replicate in mice, possess human-type receptor binding specificity, and transmit between

  6. NMDA and AMPA receptors contribute similarly to temporal processing in mammalian retinal ganglion cells

    PubMed Central

    Stafford, Benjamin K; Manookin, Michael B; Singer, Joshua H; Demb, Jonathan B

    2014-01-01

    Postsynaptic AMPA- and NMDA-type glutamate receptors (AMPARs, NMDARs) are commonly expressed at the same synapses. AMPARs are thought to mediate the majority of fast excitatory neurotransmission whereas NMDARs, with their relatively slower kinetics and higher Ca2+ permeability, are thought to mediate synaptic plasticity, especially in neural circuits devoted to learning and memory. In sensory neurons, however, the roles of AMPARs and NMDARs are less well understood. Here, we tested in the in vitro guinea pig retina whether AMPARs and NMDARs differentially support temporal contrast encoding by two ganglion cell types. In both OFF Alpha and Delta ganglion cells, contrast stimulation evoked an NMDAR-mediated response with a characteristic J-shaped I–V relationship. In OFF Delta cells, AMPAR- and NMDAR-mediated responses could be modulated at low frequencies but were suppressed during 10 Hz stimulation, when responses were instead shaped by synaptic inhibition. With inhibition blocked, both AMPAR- and NMDAR-mediated responses could be modulated at 10 Hz, indicating that NMDAR kinetics do not limit temporal encoding. In OFF Alpha cells, NMDAR-mediated responses followed stimuli at frequencies up to ∼18 Hz. In both cell types, NMDAR-mediated responses to contrast modulation at 9–18 Hz showed delays of <10 ms relative to AMPAR-mediated responses. Thus, NMDARs combine with AMPARs to encode rapidly modulated glutamate release, and NMDAR kinetics do not limit temporal coding by OFF Alpha and Delta ganglion cells substantially. Furthermore, glutamatergic transmission is differentially regulated across bipolar cell pathways: in some, release is suppressed at high temporal frequencies by presynaptic inhibition. PMID:25217374

  7. ORA1, a Zebrafish Olfactory Receptor Ancestral to All Mammalian V1R Genes, Recognizes 4-Hydroxyphenylacetic Acid, a Putative Reproductive Pheromone

    PubMed Central

    Behrens, Maik; Frank, Oliver; Rawel, Harshadrai; Ahuja, Gaurav; Potting, Christoph; Hofmann, Thomas; Meyerhof, Wolfgang; Korsching, Sigrun

    2014-01-01

    The teleost v1r-related ora genes are a small, highly conserved olfactory receptor gene family of only six genes, whose direct orthologues can be identified in lineages as far as that of cartilaginous fish. However, no ligands for fish olfactory receptor class A related genes (ORA) had been uncovered so far. Here we have deorphanized the ORA1 receptor using heterologous expression and calcium imaging. We report that zebrafish ORA1 recognizes with high specificity and sensitivity 4-hydroxyphenylacetic acid. The carboxyl group of this compound is required in a particular distance from the aromatic ring, whereas the hydroxyl group in the para-position is not essential, but strongly enhances the binding efficacy. Low concentrations of 4-hydroxyphenylacetic acid elicit increases in oviposition frequency in zebrafish mating pairs. This effect is abolished by naris closure. We hypothesize that 4-hydroxyphenylacetic acid might function as a pheromone for reproductive behavior in zebrafish. ORA1 is ancestral to mammalian V1Rs, and its putative function as pheromone receptor is reminiscent of the role of several mammalian V1Rs as pheromone receptors. PMID:24831010

  8. CCDI: a new ligand that modulates mammalian type 1 ryanodine receptor (RyR1)

    PubMed Central

    Tian, Chengju; Shao, Chun Hong; Padanilam, Christina; Ezell, Edward; Singh, Jaipaul; Kutty, Shelby; Bidasee, Keshore R

    2014-01-01

    Background and Purpose Ryanodine receptors (RyRs) are Ca2+-release channels on the sarco(endo)plasmic reticulum that modulate a wide array of physiological functions. Three RyR isoforms are present in cells: RyR1, RyR2 and RyR3. To date, there are no reports on ligands that modulate RyR in an isoform-selective manner. Such ligands are not only valuable research tools, but could serve as intermediates for development of therapeutics. Experimental approach Pyrrole-2-carboxylic acid and 1,3-dicyclohexylcarbodiimide were allowed to react in carbon tetrachloride for 24 h at low temperatures and pressures. The chemical structures of the two products isolated were elucidated using NMR spectrometry, mass spectrometry and elemental analyses. [3H]-ryanodine binding, lipid bilayer and time-lapsed confocal imaging were used to determine their effects on RyR isoforms. Key results The major product, 2-cyclohexyl-3-cyclohexylimino-2, 3, dihydro–pyrrolo[1,2-c]imidazol-1-one (CCDI) dose-dependently potentiated Ca2+-dependent binding of [3H]-ryanodine to RyR1, with no significant effects on [3H]-ryanodine binding to RyR2 or RyR3. CCDI also reversibly increased the open probability (Po) of RyR1 with minimal effects on RyR2 and RyR3. CCDI induced Ca2+ transients in C2C12 skeletal myotubes, but not in rat ventricular myocytes. This effect was blocked by pretreating cells with ryanodine. The minor product 2-cyclohexyl-pyrrolo[1,2-c]imidazole-1,3-dione had no effect on either [3H]-ryanodine binding or Po of RyR1, RyR2 and RyR3. Conclusions and implications A new ligand that preferentially modulates RyR1 was identified. In addition to being an important research tool, the pharmacophore of this small molecule could serve as a template for the synthesis of other isoform-selective modulators of RyRs. PMID:24819467

  9. High-throughput sequencing of immune repertoires in multiple sclerosis.

    PubMed

    Lossius, Andreas; Johansen, Jorunn N; Vartdal, Frode; Holmøy, Trygve

    2016-04-01

    T cells and B cells are crucial in the initiation and maintenance of multiple sclerosis (MS), and the activation of these cells is believed to be mediated through specific recognition of antigens by the T- and B-cell receptors. The antigen receptors are highly polymorphic due to recombination (T- and B-cell receptors) and mutation (B-cell receptors) of the encoding genes, which can therefore be used as fingerprints to track individual T- and B-cell clones. Such studies can shed light on mechanisms driving the immune responses and provide new insights into the pathogenesis. Here, we summarize studies that have explored the T- and B-cell receptor repertoires using earlier methodological approaches, and we focus on how high-throughput sequencing has provided new knowledge by surveying the immune repertoires in MS in even greater detail and with unprecedented depth. PMID:27081660

  10. Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

    PubMed Central

    Lee, Terry H. Y.; Mitchell, Ainslie; Liu Lau, Sydney; An, Hongyan; Rajeaskariah, Poornima; Wasinger, Valerie; Raftery, Mark; Bryant, Katherine; Tedla, Nicodemus

    2013-01-01

    The leukocyte immunoglobulin-like receptor (LILR) A3 is a member of the highly homologous activating and inhibitory receptors expressed on leukocytes. LILRA3 is a soluble receptor of unknown functions but is predicted to act as a broad antagonist to other membrane-bound LILRs. Functions of LILRA3 are unclear primarily because of the lack of high quality functional recombinant protein and insufficient knowledge regarding its ligand(s). Here, we expressed and characterized recombinant LILRA3 (rLILRA3) proteins produced in 293T cells, Escherichia coli, and Pichia pastoris. We found that the purified rLILRA3 produced in the mammalian system was the same size as a 70-kDa native macrophage LILRA3. This is 20 kDa larger than the calculated size, suggesting significant post-translational modifications. In contrast, rLILRA3 produced in E. coli was similar in size to the unprocessed protein, but yeast-produced protein was 2–4 times larger than the unprocessed protein. Treatment with peptide-N-glycosidase F reduced the size of the mammalian cell- and yeast-produced rLILRA3 to 50 kDa, suggesting that most modifications are due to glycosylation. Consistent with this, mass spectrometric analysis of the mammalian rLILRA3 revealed canonical N-glycosylation at the predicted Asn140, Asn281, Asn302, Asn341, and Asn431 sites. Functionally, only mammalian cell-expressed rLILRA3 bound onto the surface of monocytes with high affinity, and importantly, only this significantly abrogated LPS-induced TNFα production by monocytes. Binding to monocytes was partially blocked by β-lactose, indicating that optimally glycosylated LILRA3 might be critical for ligand binding and function. Overall, our data demonstrated for the first time that LILRA3 is a potential new anti-inflammatory protein, and optimal glycosylation is required for its functions. PMID:24085305

  11. Regulation of insulin receptor substrate-1 by mTORC2 (mammalian target of rapamycin complex 2)

    PubMed Central

    DeStefano, Michael A.; Jacinto, Estela

    2016-01-01

    mTOR (mammalian target of rapamycin) responds to the presence of nutrients, energy and growth factors to link cellular metabolism, growth and proliferation. The rapamycin-sensitive mTORC (mTOR complex) 1 activates the translational regulator S6K (S6 kinase), leading to increased protein synthesis in the presence of nutrients. On the other hand, the rapamycin-insensitive mTORC2 responds to the presence of growth factors such as insulin by phosphorylating Akt to promote its maturation and allosteric activation. We recently found that mTORC2 can also regulate insulin signalling at the level of IRS-1 (insulin receptor substrate-1). Whereas mTORC1 promotes IRS-1 serine phosphorylation that is linked to IRS-1 down-regulation, we uncovered that mTORC2 mediates its degradation. In mTORC2-disrupted cells, inactive IRS-1 accumulated despite undergoing phosphorylation at the mTORC1-mediated serine sites. Defective IRS-1 degradation was due to attenuated expression of the CUL7 (Cullin 7) ubiquitin ligase substrate-targeting subunit Fbw8. mTORC2 and Fbw8 co-localize at the membrane where mTORC2 phosphorylates Ser86 to stabilize Fbw8 and promotes its cytosolic localization upon insulin stimulation. Under conditions of chronic insulin exposure, inactive serine-phosphorylated IRS-1 and Fbw8 co-localize to the cytosol where the former becomes ubiquitylated via CUL7/Fbw8. Thus mTORC2 negatively feeds back to IRS-1 via control of Fbw8 stability and localization. Our findings reveal that, in addition to persistent mTORC1 signalling, increased mTORC2 signals can promote insulin resistance due to mTORC2-mediated degradation of IRS-1. PMID:23863152

  12. Comparative Expression Study of the Endo–G Protein Coupled Receptor (GPCR) Repertoire in Human Glioblastoma Cancer Stem-like Cells, U87-MG Cells and Non Malignant Cells of Neural Origin Unveils New Potential Therapeutic Targets

    PubMed Central

    Lennon, Sarah; Carapito, Christine; Dong, Jihu; Van Dorsselaer, Alain; Junier, Marie-Pierre; Chneiweiss, Hervé; Cianférani, Sarah; Haiech, Jacques; Kilhoffer, Marie-Claude

    2014-01-01

    Glioblastomas (GBMs) are highly aggressive, invasive brain tumors with bad prognosis and unmet medical need. These tumors are heterogeneous being constituted by a variety of cells in different states of differentiation. Among these, cells endowed with stem properties, tumor initiating/propagating properties and particularly resistant to chemo- and radiotherapies are designed as the real culprits for tumor maintenance and relapse after treatment. These cells, termed cancer stem-like cells, have been designed as prominent targets for new and more efficient cancer therapies. G-protein coupled receptors (GPCRs), a family of membrane receptors, play a prominent role in cell signaling, cell communication and crosstalk with the microenvironment. Their role in cancer has been highlighted but remains largely unexplored. Here, we report a descriptive study of the differential expression of the endo-GPCR repertoire in human glioblastoma cancer stem-like cells (GSCs), U-87 MG cells, human astrocytes and fetal neural stem cells (f-NSCs). The endo-GPCR transcriptome has been studied using Taqman Low Density Arrays. Of the 356 GPCRs investigated, 138 were retained for comparative studies between the different cell types. At the transcriptomic level, eight GPCRs were specifically expressed/overexpressed in GSCs. Seventeen GPCRs appeared specifically expressed in cells with stem properties (GSCs and f-NSCs). Results of GPCR expression at the protein level using mass spectrometry and proteomic analysis are also presented. The comparative GPCR expression study presented here gives clues for new pathways specifically used by GSCs and unveils novel potential therapeutic targets. PMID:24662753

  13. Autoradiographic distribution of 5-HT7 receptors in the human brain using [3H]mesulergine: comparison to other mammalian species

    PubMed Central

    Martín-Cora, Francisco J; Pazos, Angel

    2003-01-01

    The main aim of this investigation was to delineate the distribution of the 5-HT7 receptor in human brain. Autoradiographic studies in guinea-pig and rat brain were also carried out in order to revisit and compare the anatomical distribution of 5-HT7 receptors in different mammalian species.Binding studies were performed in rat frontal cortex membranes using 10 nM [3H]mesulergine in the presence of raclopride (10 μM) and DOI (0.8 μM). Under these conditions, a binding site with pharmacological characteristics consistent with those of the 5-HT7 receptors was identified (rank order of binding affinity values: 5-CT>5-HT>5-MeOT>mesulergine ≈methiothepin>8-OH-DPAT=spiperone ≈(+)-butaclamol≫imipramine ≈(±)-pindolol≫ondansetron ≈clonidine ≈prazosin).The autoradiographic studies revealed that the anatomical distribution of 5-HT7 receptors throughout the human brain was heterogenous. High densities were found over the caudate and putamen nuclei, the pyramidal layer of the CA2 field of the hippocampus, the centromedial thalamic nucleus, and the dorsal raphe nucleus. The inner layer of the frontal cortex, the dentate gyrus of the hippocampus, the subthalamic nucleus and superior colliculus, among others, presented intermediate concentrations of 5-HT7 receptors. A similar brain anatomical distribution of 5-HT7 receptors was observed in all three mammalian species studied.By using [3H]mesulergine, we have mapped for the first time the anatomical distribution of 5-HT7 receptors in the human brain, overcoming the limitations previously found in radiometric studies with other radioligands, and also revisiting the distribution in guinea-pig and rat brain. PMID:14656806

  14. Human KIR repertoires: shaped by genetic diversity and evolution.

    PubMed

    Manser, Angela R; Weinhold, Sandra; Uhrberg, Markus

    2015-09-01

    Killer cell immunoglobulin-like receptors (KIRs) on natural killer (NK) cells are crucially involved in the control of cancer development and virus infection by probing cells for proper expression of HLA class I. The clonally distributed expression of KIRs leads to great combinatorial diversity that develops in the presence of the evolutionary older CD94/NKG2A receptor to create highly stochastic but tolerant repertoires of NK cells. These repertoires are present at birth and are subsequently shaped by an individuals' immunological history toward recognition of self. The single most important factor that shapes functional NK cell repertoires is the genetic diversity of KIR, which is characterized by the presence of group A and B haplotypes with complementary gene content that are present in all human populations. Group A haplotypes constitute the minimal genetic entity that provides high affinity recognition of all major human leukocyte antigen class I-encoded ligands, whereas group B haplotypes contribute to the diversification of NK cell repertoires by providing sets of stimulatory KIR genes that modify NK cell responses. We suggest a cooperative model for the balancing selection of A and B haplotypes, which is driven by the need to provide a suitable corridor of repertoire complexity in which A/A individuals with only 16 different KIR combinations coexist with A/B and B/B donors expressing up to 2048 different clone types. PMID:26284478

  15. Evaluation of TCR repertoire diversity in patients after hematopoietic stem cell transplantation

    PubMed Central

    Xu, Ling

    2015-01-01

    T-cell receptor (TCR) repertoire analyses have been widely used to identify T cell populations of interest in cancer and autoimmunity and for characterizing immune repertoire reconstitution after hematopoietic stem cell transplantation (HSCT). Several decades of development and progress have led to the use of techniques for evaluating TCR repertoires in a more comprehensive, unbiased and fast manner, and the mechanisms of T cell immune reconstitution after HSCT and the new approaches used for recovering T cell repertoire diversity post HSCT have been more exhaustively documented to some degree. To better understand and characterize this progress, here we review recent studies on TCR repertoire diversity recovery in patients with leukemia and autoimmune disease who have received HSCT, impact factors and improvements in approaches for TCR repertoire recovery after HSCT.

  16. Biomonitoring of Non-Dioxin-Like Polychlorinated Biphenyls in Transgenic Arabidopsis Using the Mammalian Pregnane X Receptor System: A Role of Pectin in Pollutant Uptake

    PubMed Central

    Bao, Lieming; Gao, Chen; Li, Miaomiao; Chen, Yong; Lin, Weiqiang; Yang, Yanjun; Han, Ning; Bian, Hongwu; Zhu, Muyuan; Wang, Junhui

    2013-01-01

    Polychlorinated biphenyls (PCBs) are persistent organic pollutants damaging to human health and the environment. Techniques to indicate PCB contamination in planta are of great interest to phytoremediation. Monitoring of dioxin-like PCBs in transgenic plants carrying the mammalian aryl hydrocarbon receptor (AHR) has been reported previously. Herein, we report the biomonitoring of non-dioxin-like PCBs (NDL-PCBs) using the mammalian pregnane X receptor (PXR). In the transgenic Arabidopsis designated NDL-PCB Reporter, the EGFP-GUS reporter gene was driven by a promoter containing 18 repeats of the xenobiotic response elements, while PXR and its binding partner retinoid X receptor (RXR) were coexpressed. Results showed that, in live cells, the expression of reporter gene was insensitive to endogenous lignans, carotenoids and flavonoids, but responded to all tested NDL-PCBs in a dose- and time- dependent manner. Two types of putative PCB metabolites, hydroxy- PCBs and methoxy- PCBs, displayed different activation properties. The vascular tissues seemed unable to transport NDL-PCBs, whereas mutation in QUASIMODO1 encoding a 1,4-galacturonosyltransferase led to reduced PCB accumulation in Arabidopsis, revealing a role for pectin in the control of PCB translocation. Taken together, the reporter system may serve as a useful tool to biomonitor the uptake and metabolism of NDL-PCBs in plants. PMID:24236133

  17. Mutant Thyroid Hormone Receptors (TRs) Isolated from Distinct Cancer Types Display Distinct Target Gene Specificities: a Unique Regulatory Repertoire Associated with Two Renal Clear Cell Carcinomas

    PubMed Central

    Rosen, Meghan D.; Chan, Ivan H.

    2011-01-01

    Thyroid hormone receptors (TRs) are hormone-regulated transcription factors that regulate a diverse array of biological activities, including metabolism, homeostasis, and development. TRs also serve as tumor suppressors, and aberrant TR function (via mutation, deletion, or altered expression) is associated with a spectrum of both neoplastic and endocrine diseases. A particularly high frequency of TR mutations has been reported in renal clear cell carcinoma (RCCC) and in hepatocellular carcinoma (HCC). We have shown that HCC-TR mutants regulate only a fraction of the genes targeted by wild-type TRs but have gained the ability to regulate other, unique, targets. We have suggested that this altered gene recognition may contribute to the neoplastic phenotype. Here, to determine the generality of this phenomenon, we examined a distinct set of TR mutants associated with RCCC. We report that two different TR mutants, isolated from independent RCCC tumors, possess greatly expanded target gene specificities that extensively overlap one another, but only minimally overlap that of the wild-type TRs, or those of two HCC-TR mutants. Many of the genes targeted by either or both RCCC-TR mutants have been previously implicated in RCCC and include a series of metallothioneins, solute carriers, and genes involved in glycolysis and energy metabolism. We propose as a hypothesis that TR mutations from RCCC and HCC may play tissue-specific roles in carcinogenesis, and that the divergent target gene recognition patterns of TR mutants isolated from the two different types of tumors may arise from different selective pressures during development of RCCC vs. HCC. PMID:21622534

  18. Hacking the genetic code of mammalian cells.

    PubMed

    Schwarzer, Dirk

    2009-07-01

    A genetic shuttle: The highlighted article, which was recently published by Schultz, Geierstanger and co-workers, describes a straightforward scheme for enlarging the genetic code of mammalian cells. An orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for a new amino acid can be evolved in E. coli and subsequently transferred into mammalian cells. The feasibility of this approach was demonstrated by adding a photocaged lysine derivative to the genetic repertoire of a human cell line. PMID:19533721

  19. Analysis of the T-cell receptor repertoire of human T-cell leukemia virus type 1 (HTLV-1) Tax-specific CD8+ cytotoxic T lymphocytes from patients with HTLV-1-associated disease: evidence for oligoclonal expansion.

    PubMed

    Utz, U; Banks, D; Jacobson, S; Biddison, W E

    1996-02-01

    Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurological disease characterized by marked degeneration of the spinal cord and the presence of antibodies against HTLV-1. Patients with HAM/TSP, but not asymptomatic carriers, show very high precursor frequencies of HTLV-1-specific CD8+ T cells in peripheral blood and cerebrospinal fluid, suggestive of a role of these T cells in the pathogenesis of the disease. In HLA-A2+ HAM/TSP patients, HTLV-1-specific T cells were demonstrated to be directed predominantly against one HTLV-1 epitope, namely, Tax11-19. In the present study, we analyzed HLA-A2-restricted HTLV-1 Tax11-19-specific cytotoxic T cells from three patients with HAM/TSP. An analysis of the T-cell receptor (TCR) repertoire of these cells revealed an absence of restricted variable (V) region usage. Different combinations of TCR V alpha and V beta genes were utilized between, but also within, the individual patients for the recognition of Tax11-19. Sequence analysis of the TCR showed evidence for an oligoclonal expansion of few founder T cells in each patient. Apparent structural motifs were identified for the CDR3 regions of the TCR beta chains. One T-cell clone could be detected within the same patient over a period of 3 years. We suggest that these in vivo clonally expanded T cells might play a role in the pathogenesis of HAM/TSP and provide information on HTLV-1-specific TCR which may elucidate the nature of the T cells that infiltrate the central nervous system in HAM/TSP patients. PMID:8551623

  20. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors

    PubMed Central

    Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands. PMID:26030739

  1. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

    PubMed

    Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands. PMID:26030739

  2. The Brain-Uterus Connection: Brain Derived Neurotrophic Factor (BDNF) and Its Receptor (Ntrk2) Are Conserved in the Mammalian Uterus

    PubMed Central

    Wessels, Jocelyn M.; Wu, Liang; Leyland, Nicholas A.; Wang, Hongmei; Foster, Warren G.

    2014-01-01

    The neurotrophins are neuropeptides that are potent regulators of neurite growth and survival. Although mainly studied in the brain and nervous system, recent reports have shown that neurotrophins are expressed in multiple target tissues and cell types throughout the body. Additionally, dysregulation of neurotrophins has been linked to several disease conditions including Alzheimer's, Parkinson's, Huntington's, psychiatric disorders, and cancer. Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family that elicits its actions through the neurotrophic tyrosine receptor kinase type 2 (Ntrk2). Together BDNF and Ntrk2 are capable of activating the adhesion, angiogenesis, apoptosis, and proliferation pathways. These pathways are prominently involved in reproductive physiology, yet a cross-species examination of BDNF and Ntrk2 expression in the mammalian uterus is lacking. Herein we demonstrated the conserved nature of BDNF and Ntrk2 across several mammalian species by mRNA and protein sequence alignment, isolated BDNF and Ntrk2 transcripts in the uterus by Real-Time PCR, localized both proteins to the glandular and luminal epithelium, vascular smooth muscle, and myometrium of the uterus, determined that the major isoforms expressed in the human endometrium were pro-BDNF, and truncated Ntrk2, and finally demonstrated antibody specificity. Our findings suggest that BDNF and Ntrk2 are transcribed, translated, and conserved across mammalian species including human, mouse, rat, pig, horse, and the bat. PMID:24714156

  3. A cluster of olfactory receptor genes linked to frugivory in bats.

    PubMed

    Hayden, Sara; Bekaert, Michaël; Goodbla, Alisha; Murphy, William J; Dávalos, Liliana M; Teeling, Emma C

    2014-04-01

    Diversity of the mammalian olfactory receptor (OR) repertoire has been globally reshaped by niche specialization. However, little is known about the variability of the OR repertoire at a shallower evolutionary timeframe. The vast bat radiation exhibits an extraordinary variety of trophic and sensory specializations. Unlike other mammals, bats possess a unique and diverse OR gene repertoire. We elucidated whether the evolution of the OR gene repertoire can be linked to ecological niche specializations, such as sensory modalities and diet. The OR gene repertoires of 27 bat species spanning the chiropteran radiation were amplified and sequenced. For each species, intact and nonfunctional genes were assessed, and the OR gene abundances in each gene family were analyzed and compared. We identified a unique OR pattern linked to the frugivorous diet of New World fruit-eating bats and a similar convergent pattern in the Old World fruit-eating bats. Our results show a strong association between niche specialization and OR repertoire diversity even at a shallow evolutionary timeframe. PMID:24441035

  4. Functional NMDA receptors are expressed by both AII and A17 amacrine cells in the rod pathway of the mammalian retina.

    PubMed

    Zhou, Yifan; Tencerová, Barbora; Hartveit, Espen; Veruki, Margaret L

    2016-01-01

    At many glutamatergic synapses, non-N-methyl-d-aspartate (NMDA) and NMDA receptors are coexpressed postsynaptically. In the mammalian retina, glutamatergic rod bipolar cells are presynaptic to two rod amacrine cells (AII and A17) that constitute dyad postsynaptic partners opposite each presynaptic active zone. Whereas there is strong evidence for expression of non-NMDA receptors by both AII and A17 amacrines, the expression of NMDA receptors by the pre- and postsynaptic neurons in this microcircuit has not been resolved. In this study, using patch-clamp recording from visually identified cells in rat retinal slices, we investigated the expression and functional properties of NMDA receptors in these cells with a combination of pharmacological and biophysical methods. Pressure application of NMDA did not evoke a response in rod bipolar cells, but for both AII and A17 amacrines, NMDA evoked responses that were blocked by a competitive antagonist (CPP) applied extracellularly and an open channel blocker (MK-801) applied intracellularly. NMDA-evoked responses also displayed strong Mg(2+)-dependent voltage block and were independent of gap junction coupling. With low-frequency application (60-s intervals), NMDA-evoked responses remained stable for up to 50 min, but with higher-frequency stimulation (10- to 20-s intervals), NMDA responses were strongly and reversibly suppressed. We observed strong potentiation when NMDA was applied in nominally Ca(2+)-free extracellular solution, potentially reflecting Ca(2+)-dependent NMDA receptor inactivation. These results indicate that expression of functional (i.e., conductance-increasing) NMDA receptors is common to both AII and A17 amacrine cells and suggest that these receptors could play an important role for synaptic signaling, integration, or plasticity in the rod pathway. PMID:26561610

  5. Cytotoxic activity of Bacillus thuringiensis Cry proteins on mammalian cells transfected with cadherin-like Cry receptor gene of Bombyx mori (silkworm).

    PubMed Central

    Tsuda, Yoko; Nakatani, Fumiki; Hashimoto, Keiko; Ikawa, Satoshi; Matsuura, Chikako; Fukada, Takashi; Sugimoto, Kenji; Himeno, Michio

    2003-01-01

    Cry1Aa, an insecticidal protein produced by Bacillus thuringiensis, has been shown to bind to cadherin-like protein, BtR175, in Bombyx mori (silkworm) midgut. We previously reported three variant alleles of BtR175 (BtR175a, b and c). When transiently expressed in COS7 cells, all the three BtR175 variants bound to Cry1Aa. We stably expressed BtR175b in HEK293 cells. These BtR175b-expressing cells swelled and died in the presence of activated Cry1Aa in a dose- and time-dependent manner, showing that BtR175b itself can impart Cry1Aa-susceptibility to mammalian cells. These cells were more susceptible to Cry1Aa than to Cry1Ab and Cry1Ac. Since dispersed B. mori midgut cells were reported to be highly susceptible to Cry1Ac, this result suggested that other Cry1Ac-specific receptor(s) were simultaneously working with BtR175 in the midgut cells. Advantages are also discussed of applying these transfected mammalian cells to toxicity assays of mutant Cry proteins. PMID:12403648

  6. Measurement of Bluetongue Virus Binding to a Mammalian Cell Surface Receptor by an In Situ Immune Fluorescent Staining Technique

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A quantifiable in situ immune fluorescent assay (IFA) was developed to measure bluetongue virus (BTV) binding to mammalian cells. The utility of the assay was demonstrated with both Chinese hamster ovary (CHO) and bovine pulmonary artery endothelial (CPAE) cells. Since heparin sulfate (HS) has been ...

  7. Preschoolers' Prosocial Repertoires: Parents' Perspectives.

    ERIC Educational Resources Information Center

    Bergin, Christi A. C.; And Others

    1995-01-01

    Two studies had parents describe the characteristics of the most prosocial two- and five-year olds they knew and rate the relative importance of each attribute in defining a child as prosocial. Results indicated more similarities than differences between the two age groups and suggested that research has underrepresented the rich repertoire of…

  8. Reconstructing and mining the B cell repertoire with ImmunediveRsity.

    PubMed

    Cortina-Ceballos, Bernardo; Godoy-Lozano, Elizabeth Ernestina; Sámano-Sánchez, Hugo; Aguilar-Salgado, Andrés; Velasco-Herrera, Martín Del Castillo; Vargas-Chávez, Carlos; Velázquez-Ramírez, Daniel; Romero, Guillermo; Moreno, José; Téllez-Sosa, Juan; Martínez-Barnetche, Jesús

    2015-01-01

    The B cell antigen receptor repertoire is highly diverse and constantly modified by clonal selection. High-throughput DNA sequencing (HTS) of the lymphocyte repertoire (Rep-Seq) represents a promising technology to explore such diversity ex-vivo and assist in the identification of antigen-specific antibodies based on molecular signatures of clonal selection. Therefore, integrative tools for repertoire reconstruction and analysis from antibody sequences are needed. We developed ImmunediveRity, a stand-alone pipeline primarily based in R programming for the integral analysis of B cell repertoire data generated by HTS. The pipeline integrates GNU software and in house scripts to perform quality filtering, sequencing noise correction and repertoire reconstruction based on V, D and J segment assignment, clonal origin and unique heavy chain identification. Post-analysis scripts generate a wealth of repertoire metrics that in conjunction with a rich graphical output facilitates sample comparison and repertoire mining. Its performance was tested with raw and curated human and mouse 454-Roche sequencing benchmarks providing good approximations of repertoire structure. Furthermore, ImmunediveRsity was used to mine the B cell repertoire of immunized mice with a model antigen, allowing the identification of previously validated antigen-specific antibodies, and revealing different and unexpected clonal diversity patterns in the post-immunization IgM and IgG compartments. Although ImmunediveRsity is similar to other recently developed tools, it offers significant advantages that facilitate repertoire analysis and repertoire mining. ImmunediveRsity is open source and free for academic purposes and it runs on 64 bit GNU/Linux and MacOS. Available at: https://bitbucket.org/ImmunediveRsity/immunediversity/. PMID:25875140

  9. Reconstructing and mining the B cell repertoire with ImmunediveRsity

    PubMed Central

    Cortina-Ceballos, Bernardo; Godoy-Lozano, Elizabeth Ernestina; Sámano-Sánchez, Hugo; Aguilar-Salgado, Andrés; Velasco-Herrera, Martín Del Castillo; Vargas-Chávez, Carlos; Velázquez-Ramírez, Daniel; Romero, Guillermo; Moreno, José; Téllez-Sosa, Juan; Martínez-Barnetche, Jesús

    2015-01-01

    The B cell antigen receptor repertoire is highly diverse and constantly modified by clonal selection. High-throughput DNA sequencing (HTS) of the lymphocyte repertoire (Rep-Seq) represents a promising technology to explore such diversity ex-vivo and assist in the identification of antigen-specific antibodies based on molecular signatures of clonal selection. Therefore, integrative tools for repertoire reconstruction and analysis from antibody sequences are needed. We developed ImmunediveRity, a stand-alone pipeline primarily based in R programming for the integral analysis of B cell repertoire data generated by HTS. The pipeline integrates GNU software and in house scripts to perform quality filtering, sequencing noise correction and repertoire reconstruction based on V, D and J segment assignment, clonal origin and unique heavy chain identification. Post-analysis scripts generate a wealth of repertoire metrics that in conjunction with a rich graphical output facilitates sample comparison and repertoire mining. Its performance was tested with raw and curated human and mouse 454-Roche sequencing benchmarks providing good approximations of repertoire structure. Furthermore, ImmunediveRsity was used to mine the B cell repertoire of immunized mice with a model antigen, allowing the identification of previously validated antigen-specific antibodies, and revealing different and unexpected clonal diversity patterns in the post-immunization IgM and IgG compartments. Although ImmunediveRsity is similar to other recently developed tools, it offers significant advantages that facilitate repertoire analysis and repertoire mining. ImmunediveRsity is open source and free for academic purposes and it runs on 64 bit GNU/Linux and MacOS. Available at: https://bitbucket.org/ImmunediveRsity/immunediversity/ PMID:25875140

  10. Requirement of full TCR repertoire for regulatory T cells to maintain intestinal homeostasis.

    PubMed

    Nishio, Junko; Baba, Minato; Atarashi, Koji; Tanoue, Takeshi; Negishi, Hideo; Yanai, Hideyuki; Habu, Sonoko; Hori, Shohei; Honda, Kenya; Taniguchi, Tadatsugu

    2015-10-13

    The regulation of intestinal homeostasis by the immune system involves the dynamic interplay between gut commensal microbiota and resident immune cells. It is well known that a large and diverse lymphocyte antigen receptor repertoire enables the immune system to recognize and respond to a wide range of invading pathogens. There is also an emerging appreciation for a critical role the T-cell receptor (TCR) repertoire serves in the maintenance of peripheral tolerance by regulatory T cells (Tregs). Nevertheless, how the diversity of the TCR repertoire in Tregs affects intestinal homeostasis remains unknown. To address this question, we studied mice whose T cells express a restricted TCR repertoire. We observed the development of spontaneous colitis, accompanied by the induction of T-helper type 17 cells in the colon that is driven by gut commensal microbiota. We provide further evidence that a restricted TCR repertoire causes a loss of tolerogenicity to microbiota, accompanied by a paucity of peripherally derived, Helios(-) Tregs and hyperactivation of migratory dendritic cells. These results thus reveal a new facet of the TCR repertoire in which Tregs require a diverse TCR repitoire for intestinal homeostasis, suggesting an additional driving force in the evolutional significance of the TCR repertoire. PMID:26420876

  11. Origin and evolution of the T cell repertoire after posttransplantation cyclophosphamide

    PubMed Central

    Kanakry, Christopher G.; Coffey, David G.; Towlerton, Andrea M.H.; Vulic, Ante; Storer, Barry E.; Chou, Jeffrey; Yeung, Cecilia C.S.; Gocke, Christopher D.; Robins, Harlan S.; O’Donnell, Paul V.; Luznik, Leo; Warren, Edus H.

    2016-01-01

    Posttransplantation cyclophosphamide (PTCy) effectively prevents graft-versus-host disease (GVHD), but its immunologic impact is poorly understood. We assessed lymphocyte reconstitution via flow cytometry (n = 74) and antigen receptor sequencing (n = 35) in recipients of myeloablative, HLA-matched allogeneic BM transplantation using PTCy. Recovering T cells were primarily phenotypically effector memory with lower T cell receptor β (TRB) repertoire diversity than input donor repertoires. Recovering B cells were predominantly naive with immunoglobulin heavy chain locus (IGH) repertoire diversity similar to donors. Numerical T cell reconstitution and TRB diversity were strongly associated with recipient cytomegalovirus seropositivity. Global similarity between input donor and recipient posttransplant repertoires was uniformly low at 1–2 months after transplant but increased over the balance of the first posttransplant year. Blood TRB repertoires at ≥3 months after transplant were often dominated by clones present in the donor blood/marrow memory CD8+ compartment. Limited overlap was observed between the TRB repertoires of T cells infiltrating the skin or gastrointestinal tract versus the blood. Although public TRB sequences associated with herpesvirus- or alloantigen-specific CD8+ T cells were detected in some patients, posttransplant TRB and IGH repertoires were unique to each individual. These data define the immune dynamics occurring after PTCy and establish a benchmark against which immune recovery after other transplantation approaches can be compared. PMID:27213183

  12. Requirement of full TCR repertoire for regulatory T cells to maintain intestinal homeostasis

    PubMed Central

    Nishio, Junko; Baba, Minato; Atarashi, Koji; Tanoue, Takeshi; Negishi, Hideo; Yanai, Hideyuki; Habu, Sonoko; Hori, Shohei; Honda, Kenya; Taniguchi, Tadatsugu

    2015-01-01

    The regulation of intestinal homeostasis by the immune system involves the dynamic interplay between gut commensal microbiota and resident immune cells. It is well known that a large and diverse lymphocyte antigen receptor repertoire enables the immune system to recognize and respond to a wide range of invading pathogens. There is also an emerging appreciation for a critical role the T-cell receptor (TCR) repertoire serves in the maintenance of peripheral tolerance by regulatory T cells (Tregs). Nevertheless, how the diversity of the TCR repertoire in Tregs affects intestinal homeostasis remains unknown. To address this question, we studied mice whose T cells express a restricted TCR repertoire. We observed the development of spontaneous colitis, accompanied by the induction of T-helper type 17 cells in the colon that is driven by gut commensal microbiota. We provide further evidence that a restricted TCR repertoire causes a loss of tolerogenicity to microbiota, accompanied by a paucity of peripherally derived, Helios− Tregs and hyperactivation of migratory dendritic cells. These results thus reveal a new facet of the TCR repertoire in which Tregs require a diverse TCR repitoire for intestinal homeostasis, suggesting an additional driving force in the evolutional significance of the TCR repertoire. PMID:26420876

  13. Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway

    PubMed Central

    Varghese, Binny V.; Koohestani, Faezeh; McWilliams, Michelle; Colvin, Arlene; Gunewardena, Sumedha; Kinsey, William H.; Nowak, Romana A.; Nothnick, Warren B.; Chennathukuzhi, Vargheese M.

    2013-01-01

    Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase–protein kinase B/AKT–mammalian target of rapamycin (PI3K/AKT–mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K–AKT–mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids. PMID:23284171

  14. Membrane glucocorticoid receptors are localised in the extracellular matrix and signal through the MAPK pathway in mammalian skeletal muscle fibres

    PubMed Central

    Boncompagni, Simona; Arthurton, Lewis; Akujuru, Eugene; Pearson, Timothy; Steverding, Dietmar; Protasi, Feliciano; Mutungi, Gabriel

    2015-01-01

    A number of studies have previously proposed the existence of glucocorticoid receptors on the plasma membrane of many cell types, including skeletal muscle fibres. However, their exact localisation and the cellular signalling pathway(s) they utilise to communicate with the rest of the cell are still poorly understood. In this study, we investigated the localisation and the mechanism(s) underlying the non-genomic physiological functions of these receptors in mouse skeletal muscle cells. The results show that the receptors were localised in the cytoplasm in myoblasts, in the nucleus in myotubes, in the extracellular matrix, in satellite cells and in the proximity of mitochondria in adult muscle fibres. Also, they bound laminin in a glucocorticoid-dependent manner. Treating small skeletal muscle fibre bundles with the synthetic glucocorticoid beclomethasone dipropionate increased the phosphorylation (= activation) of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. This occurred within 5 min and depended on the fibre type and the duration of the treatment. It was also abolished by the glucocorticoid receptor inhibitor, mifepristone, and a monoclonal antibody against the receptor. From these results we conclude that the non-genomic/non-canonical physiological functions of glucocorticoids, in adult skeletal muscle fibres, are mediated by a glucocorticoid receptor localised in the extracellular matrix, in satellite cells and close to mitochondria, and involve activation of the mitogen-activated protein kinase pathway. PMID:25846902

  15. The past, present, and future of immune repertoire biology - the rise of next-generation repertoire analysis.

    PubMed

    Six, Adrien; Mariotti-Ferrandiz, Maria Encarnita; Chaara, Wahiba; Magadan, Susana; Pham, Hang-Phuong; Lefranc, Marie-Paule; Mora, Thierry; Thomas-Vaslin, Véronique; Walczak, Aleksandra M; Boudinot, Pierre

    2013-01-01

    T and B cell repertoires are collections of lymphocytes, each characterized by its antigen-specific receptor. We review here classical technologies and analysis strategies developed to assess immunoglobulin (IG) and T cell receptor (TR) repertoire diversity, and describe recent advances in the field. First, we describe the broad range of available methodological tools developed in the past decades, each of which answering different questions and showing complementarity for progressive identification of the level of repertoire alterations: global overview of the diversity by flow cytometry, IG repertoire descriptions at the protein level for the identification of IG reactivities, IG/TR CDR3 spectratyping strategies, and related molecular quantification or dynamics of T/B cell differentiation. Additionally, we introduce the recent technological advances in molecular biology tools allowing deeper analysis of IG/TR diversity by next-generation sequencing (NGS), offering systematic and comprehensive sequencing of IG/TR transcripts in a short amount of time. NGS provides several angles of analysis such as clonotype frequency, CDR3 diversity, CDR3 sequence analysis, V allele identification with a quantitative dimension, therefore requiring high-throughput analysis tools development. In this line, we discuss the recent efforts made for nomenclature standardization and ontology development. We then present the variety of available statistical analysis and modeling approaches developed with regards to the various levels of diversity analysis, and reveal the increasing sophistication of those modeling approaches. To conclude, we provide some examples of recent mathematical modeling strategies and perspectives that illustrate the active rise of a "next-generation" of repertoire analysis. PMID:24348479

  16. The Past, Present, and Future of Immune Repertoire Biology – The Rise of Next-Generation Repertoire Analysis

    PubMed Central

    Six, Adrien; Mariotti-Ferrandiz, Maria Encarnita; Chaara, Wahiba; Magadan, Susana; Pham, Hang-Phuong; Lefranc, Marie-Paule; Mora, Thierry; Thomas-Vaslin, Véronique; Walczak, Aleksandra M.; Boudinot, Pierre

    2013-01-01

    T and B cell repertoires are collections of lymphocytes, each characterized by its antigen-specific receptor. We review here classical technologies and analysis strategies developed to assess immunoglobulin (IG) and T cell receptor (TR) repertoire diversity, and describe recent advances in the field. First, we describe the broad range of available methodological tools developed in the past decades, each of which answering different questions and showing complementarity for progressive identification of the level of repertoire alterations: global overview of the diversity by flow cytometry, IG repertoire descriptions at the protein level for the identification of IG reactivities, IG/TR CDR3 spectratyping strategies, and related molecular quantification or dynamics of T/B cell differentiation. Additionally, we introduce the recent technological advances in molecular biology tools allowing deeper analysis of IG/TR diversity by next-generation sequencing (NGS), offering systematic and comprehensive sequencing of IG/TR transcripts in a short amount of time. NGS provides several angles of analysis such as clonotype frequency, CDR3 diversity, CDR3 sequence analysis, V allele identification with a quantitative dimension, therefore requiring high-throughput analysis tools development. In this line, we discuss the recent efforts made for nomenclature standardization and ontology development. We then present the variety of available statistical analysis and modeling approaches developed with regards to the various levels of diversity analysis, and reveal the increasing sophistication of those modeling approaches. To conclude, we provide some examples of recent mathematical modeling strategies and perspectives that illustrate the active rise of a “next-generation” of repertoire analysis. PMID:24348479

  17. Expression of Tas1 Taste Receptors in Mammalian Spermatozoa: Functional Role of Tas1r1 in Regulating Basal Ca2+ and cAMP Concentrations in Spermatozoa

    PubMed Central

    Meyer, Dorke; Voigt, Anja; Widmayer, Patricia; Borth, Heike; Huebner, Sandra; Breit, Andreas; Marschall, Susan; de Angelis, Martin Hrabé; Boehm, Ulrich; Meyerhof, Wolfgang; Gudermann, Thomas; Boekhoff, Ingrid

    2012-01-01

    Background During their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined. Methodology/Principal Findings The present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami) in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 null mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca2+ ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca2+ levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes. Conclusions/Significance Since Ca2+ and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and/or by tonic receptor activation by

  18. Expression of major guidance receptors is differentially regulated in spinal commissural neurons transfated by mammalian Barh genes.

    PubMed

    Kawauchi, Daisuke; Muroyama, Yuko; Sato, Tatsuya; Saito, Tetsuichiro

    2010-08-15

    During development, commissural neurons in the spinal cord project their axons across the ventral midline, floor plate, via multiple interactions among temporally controlled molecular guidance cues and receptors. The transcriptional regulation of commissural axon-associated receptors, however, is not well characterized. Spinal dorsal cells are transfated into commissural neurons by misexpression of Mbh1, a Bar-class homeobox gene. We examined the function of another Bar-class homeobox gene, Mbh2, and how Mbh1 and Mbh2 modulate expression of the receptors, leading to midline crossing of axons. Misexpression of Mbh1 and Mbh2 showed the same effects in the spinal cord. The competence of spinal dorsal cells to become commissural neurons was dependent on the embryonic stage, during which misexpression of the Mbh genes was able to activate guidance receptor genes such as Rig1 and Nrp2. Misexpression of Lhx2, which has been recently shown to be involved in Rig1 expression, activated Rig1 but not Nrp2, and was less effective in generating commissural neurons. Moreover, expression of Lhx2 was activated by and required the Mbh genes. These findings have revealed a transcriptional cascade, in which Lhx2-dependent and -independent pathways leading to expression of guidance receptors branch downstream of the Mbh genes. PMID:20599893

  19. Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization.

    PubMed

    Chalbi, Myriam; Barraud-Lange, Virginie; Ravaux, Benjamin; Howan, Kevin; Rodriguez, Nicolas; Soule, Pierre; Ndzoudi, Arnaud; Boucheix, Claude; Rubinstein, Eric; Wolf, Jean Philippe; Ziyyat, Ahmed; Perez, Eric; Pincet, Frédéric; Gourier, Christine

    2014-10-01

    Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery. PMID:25209248

  20. Photoaffinity labeling of mammalian. cap alpha. /sub 1/-adrenergic receptors: identification of the ligand binding subunit with a high affinity radioiodinated probe. [Rats, guinea pigs, rabbits

    SciTech Connect

    Leeb-Lundberg, L.M.F.; Dickinson, K.E.J.; Heald, S.L.; Wikberg, J.E.S.; Hagen, P.O.; DeBernardis, J.F.; Winn, M.; Arendsen, D.L.; Lefkowitz, R.J.; Caron, M.G.

    1984-02-01

    A description is given of the synthesised and characterization of a novel high affinity radioiodinated ..cap alpha../sub 1/-adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-(4-(5-(4-azido-3-(/sup 125/I)iodophenyl)pentanoyl)-1-piperazinyl) quinazoline. In the absence of light, this ligand binds with high affinity (K/sub d/ = 130 pm) in a reverisble and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an ..cap alpha../sub 1/-adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical ..cap alpha../sub 1/-adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at M/sub 4/ = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the M/sub r/ = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the ..cap alpha../sub 1/-adrenergic receptor.

  1. Unifying model for molecular determinants of the preselection Vβ repertoire.

    PubMed

    Gopalakrishnan, Suhasni; Majumder, Kinjal; Predeus, Alexander; Huang, Yue; Koues, Olivia I; Verma-Gaur, Jiyoti; Loguercio, Salvatore; Su, Andrew I; Feeney, Ann J; Artyomov, Maxim N; Oltz, Eugene M

    2013-08-20

    The primary antigen receptor repertoire is sculpted by the process of V(D)J recombination, which must strike a balance between diversification and favoring gene segments with specialized functions. The precise determinants of how often gene segments are chosen to complete variable region coding exons remain elusive. We quantified Vβ use in the preselection Tcrb repertoire and report relative contributions of 13 distinct features that may shape their recombination efficiencies, including transcription, chromatin environment, spatial proximity to their DβJβ targets, and predicted quality of recombination signal sequences (RSSs). We show that, in contrast to functional Vβ gene segments, all pseudo-Vβ segments are sequestered in transcriptionally silent chromatin, which effectively suppresses wasteful recombination. Importantly, computational analyses provide a unifying model, revealing a minimum set of five parameters that are predictive of Vβ use, dominated by chromatin modifications associated with transcription, but largely independent of precise spatial proximity to DβJβ clusters. This learned model-building strategy may be useful in predicting the relative contributions of epigenetic, spatial, and RSS features in shaping preselection V repertoires at other antigen receptor loci. Ultimately, such models may also predict how designed or naturally occurring alterations of these loci perturb the preselection use of variable gene segments. PMID:23918392

  2. Trade-offs in antibody repertoires to complex antigens

    PubMed Central

    Childs, Lauren M.; Baskerville, Edward B.; Cobey, Sarah

    2015-01-01

    Pathogens vary in their antigenic complexity. While some pathogens such as measles present a few relatively invariant targets to the immune system, others such as malaria display considerable antigenic diversity. How the immune response copes in the presence of multiple antigens, and whether a trade-off exists between the breadth and efficacy of antibody (Ab)-mediated immune responses, are unsolved problems. We present a theoretical model of affinity maturation of B-cell receptors (BCRs) during a primary infection and examine how variation in the number of accessible antigenic sites alters the Ab repertoire. Naive B cells with randomly generated receptor sequences initiate the germinal centre (GC) reaction. The binding affinity of a BCR to an antigen is quantified via a genotype–phenotype map, based on a random energy landscape, that combines local and distant interactions between residues. In the presence of numerous antigens or epitopes, B-cell clones with different specificities compete for stimulation during rounds of mutation within GCs. We find that the availability of many epitopes reduces the affinity and relative breadth of the Ab repertoire. Despite the stochasticity of somatic hypermutation, patterns of immunodominance are strongly shaped by chance selection of naive B cells with specificities for particular epitopes. Our model provides a mechanistic basis for the diversity of Ab repertoires and the evolutionary advantage of antigenically complex pathogens. PMID:26194759

  3. The Herbicide Atrazine Activates Endocrine Gene Networks via Non-Steroidal NR5A Nuclear Receptors in Fish and Mammalian Cells

    PubMed Central

    Suzawa, Miyuki; Ingraham, Holly A.

    2008-01-01

    Atrazine (ATR) remains a widely used broadleaf herbicide in the United States despite the fact that this s-chlorotriazine has been linked to reproductive abnormalities in fish and amphibians. Here, using zebrafish we report that environmentally relevant ATR concentrations elevated zcyp19a1 expression encoding aromatase (2.2 µg/L), and increased the ratio of female to male fish (22 µg/L). ATR selectively increased zcyp19a1, a known gene target of the nuclear receptor SF-1 (NR5A1), whereas zcyp19a2, which is estrogen responsive, remained unchanged. Remarkably, in mammalian cells ATR functions in a cell-specific manner to upregulate SF-1 targets and other genes critical for steroid synthesis and reproduction, including Cyp19A1, StAR, Cyp11A1, hCG, FSTL3, LHß, INHα, αGSU, and 11ß-HSD2. Our data appear to eliminate the possibility that ATR directly affects SF-1 DNA- or ligand-binding. Instead, we suggest that the stimulatory effects of ATR on the NR5A receptor subfamily (SF-1, LRH-1, and zff1d) are likely mediated by receptor phosphorylation, amplification of cAMP and PI3K signaling, and possibly an increase in the cAMP-responsive cellular kinase SGK-1, which is known to be upregulated in infertile women. Taken together, we propose that this pervasive and persistent environmental chemical alters hormone networks via convergence of NR5A activity and cAMP signaling, to potentially disrupt normal endocrine development and function in lower and higher vertebrates. PMID:18461179

  4. The origin of GPCRs: identification of mammalian like Rhodopsin, Adhesion, Glutamate and Frizzled GPCRs in fungi.

    PubMed

    Krishnan, Arunkumar; Almén, Markus Sällman; Fredriksson, Robert; Schiöth, Helgi B

    2012-01-01

    G protein-coupled receptors (GPCRs) in humans are classified into the five main families named Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin according to the GRAFS classification. Previous results show that these mammalian GRAFS families are well represented in the Metazoan lineages, but they have not been shown to be present in Fungi. Here, we systematically mined 79 fungal genomes and provide the first evidence that four of the five main mammalian families of GPCRs, namely Rhodopsin, Adhesion, Glutamate and Frizzled, are present in Fungi and found 142 novel sequences between them. Significantly, we provide strong evidence that the Rhodopsin family emerged from the cAMP receptor family in an event close to the split of Opisthokonts and not in Placozoa, as earlier assumed. The Rhodopsin family then expanded greatly in Metazoans while the cAMP receptor family is found in 3 invertebrate species and lost in the vertebrates. We estimate that the Adhesion and Frizzled families evolved before the split of Unikonts from a common ancestor of all major eukaryotic lineages. Also, the study highlights that the fungal Adhesion receptors do not have N-terminal domains whereas the fungal Glutamate receptors have a broad repertoire of mammalian-like N-terminal domains. Further, mining of the close unicellular relatives of the Metazoan lineage, Salpingoeca rosetta and Capsaspora owczarzaki, obtained a rich group of both the Adhesion and Glutamate families, which in particular provided insight to the early emergence of the N-terminal domains of the Adhesion family. We identified 619 Fungi specific GPCRs across 79 genomes and revealed that Blastocladiomycota and Chytridiomycota phylum have Metazoan-like GPCRs rather than the GPCRs specific for Fungi. Overall, this study provides the first evidence of the presence of four of the five main GRAFS families in Fungi and clarifies the early evolutionary history of the GPCR superfamily. PMID:22238661

  5. The Origin of GPCRs: Identification of Mammalian like Rhodopsin, Adhesion, Glutamate and Frizzled GPCRs in Fungi

    PubMed Central

    Fredriksson, Robert; Schiöth, Helgi B.

    2012-01-01

    G protein-coupled receptors (GPCRs) in humans are classified into the five main families named Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin according to the GRAFS classification. Previous results show that these mammalian GRAFS families are well represented in the Metazoan lineages, but they have not been shown to be present in Fungi. Here, we systematically mined 79 fungal genomes and provide the first evidence that four of the five main mammalian families of GPCRs, namely Rhodopsin, Adhesion, Glutamate and Frizzled, are present in Fungi and found 142 novel sequences between them. Significantly, we provide strong evidence that the Rhodopsin family emerged from the cAMP receptor family in an event close to the split of Opisthokonts and not in Placozoa, as earlier assumed. The Rhodopsin family then expanded greatly in Metazoans while the cAMP receptor family is found in 3 invertebrate species and lost in the vertebrates. We estimate that the Adhesion and Frizzled families evolved before the split of Unikonts from a common ancestor of all major eukaryotic lineages. Also, the study highlights that the fungal Adhesion receptors do not have N-terminal domains whereas the fungal Glutamate receptors have a broad repertoire of mammalian-like N-terminal domains. Further, mining of the close unicellular relatives of the Metazoan lineage, Salpingoeca rosetta and Capsaspora owczarzaki, obtained a rich group of both the Adhesion and Glutamate families, which in particular provided insight to the early emergence of the N-terminal domains of the Adhesion family. We identified 619 Fungi specific GPCRs across 79 genomes and revealed that Blastocladiomycota and Chytridiomycota phylum have Metazoan-like GPCRs rather than the GPCRs specific for Fungi. Overall, this study provides the first evidence of the presence of four of the five main GRAFS families in Fungi and clarifies the early evolutionary history of the GPCR superfamily. PMID:22238661

  6. scAAV-Mediated Gene Transfer of Interleukin 1-Receptor Antagonist to Synovium and Articular Cartilage in Large Mammalian Joints

    PubMed Central

    Watson, Rachael S.; Broome, Ted A.; Levings, Padraic P.; Rice, Bret L.; Kay, Jesse D.; Smith, Andrew D.; Gouze, Elvire; Gouze, Jean-Noel; Dacanay, E. Anthony; Hauswirth, William W.; Nickerson, David M.; Dark, Michael J.; Colahan, Patrick T.; Ghivizzani, Steven C.

    2012-01-01

    With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for AAV-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1 receptor antagonist (hIL-1Ra) or green fluorescent protein (GFP) was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 1011 vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA. PMID:23151520

  7. Natural killer group 2D and CD28 receptors differentially activate mammalian/mechanistic target of rapamycin to alter murine effector CD8+ T-cell differentiation.

    PubMed

    McQueen, Bryan; Trace, Kelsey; Whitman, Emily; Bedsworth, Taylor; Barber, Amorette

    2016-03-01

    Memory CD8+ T cells are an essential component of anti-tumour and anti-viral immunity. Activation of the mammalian/mechanistic target of rapamycin (mTOR) pathway has been implicated in regulating the differentiation of effector and memory T cells. However, the mechanisms that control mTOR activity during immunity to tumours and infections are not well known. Activation of co-stimulatory receptors, including CD28 and natural killer group 2D (NKG2D), activate phosphatidylinositol-3 kinase and subsequently may activate the mTOR pathway in CD8+ T cells. This study compared the activation of the mTOR signalling pathway after co-stimulation through CD28 or NKG2D receptors in murine effector CD8+ T cells. Compared with CD28 co-stimulation, activation through CD3 and NKG2D receptors had weaker activation of mTORc1, as shown by decreased phosphorylation of mTORc1 targets S6K1, ribosomal protein S6 and eukaryotic initiation factor 4E binding protein 1. NKG2D co-stimulation also showed increased gene expression of tuberous sclerosis protein 2, a negative regulator of mTORc1, whereas CD28 co-stimulation increased gene expression of Ras homologue enriched in brain, an activator of mTORc1, and hypoxia-inducible factor-1α and vascular endothelial growth factor-α, pro-angiogenic factors downstream of mTORc1. Strong mTORc1 activation in CD28-co-stimulated cells also increased expression of transcription factors that support effector cell differentiation, namely T-bet, B lymphocyte-induced maturation protein (BLIMP-1), interferon regulatory factor 4, and inhibitor of DNA binding 2, whereas low levels of mTORc1 activation allowed for the expression of Eomes, B-cell lymphoma 6 (BCL6), and inhibitor of DNA binding 3 during NKG2D stimulation, and increased expression of memory markers CD62 ligand and CD127. These data show that compared with CD28, co-stimulation through the NKG2D receptor leads to the differential activation of the mTOR signalling pathway and potentially supports

  8. Activation of Relaxin Family Receptor 1 from Different Mammalian Species by Relaxin Peptide and Small-Molecule Agonist ML290

    PubMed Central

    Huang, Zaohua; Myhr, Courtney; Bathgate, Ross A. D.; Ho, Brian A.; Bueno, Amaya; Hu, Xin; Xiao, Jingbo; Southall, Noel; Barnaeva, Elena; Agoulnik, Irina U.; Marugan, Juan J.; Ferrer, Marc; Agoulnik, Alexander I.

    2015-01-01

    Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit–human and guinea pig–human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing. PMID:26347712

  9. Activation of Relaxin Family Receptor 1 from Different Mammalian Species by Relaxin Peptide and Small-Molecule Agonist ML290.

    PubMed

    Huang, Zaohua; Myhr, Courtney; Bathgate, Ross A D; Ho, Brian A; Bueno, Amaya; Hu, Xin; Xiao, Jingbo; Southall, Noel; Barnaeva, Elena; Agoulnik, Irina U; Marugan, Juan J; Ferrer, Marc; Agoulnik, Alexander I

    2015-01-01

    Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit-human and guinea pig-human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing. PMID:26347712

  10. Praxis and Poiesis in Piano Repertoire Preparation

    ERIC Educational Resources Information Center

    Dos Santos, Regina Antunes Teixeira; Hentschke, Liane

    2011-01-01

    The piano repertoire preparation of three undergraduate students at three different academic levels--the first, fifth and eighth semesters--was followed during an academic semester. A phenomenological approach was used to collect data in three stages: an introductory interview, observations of the repertoire under preparation and a final…

  11. Evaluation of the Role of G Protein-Coupled Receptor Kinase 3 in Desensitization of Mouse Odorant Receptors in a Mammalian Cell Line and in Olfactory Sensory Neurons

    PubMed Central

    Kato, Aya; Reisert, Johannes; Ihara, Sayoko; Yoshikawa, Keiichi

    2014-01-01

    Thousands of odors are sensed and discriminated by G protein-coupled odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs). G protein-coupled receptor kinases (GRKs) may have a role in desensitization of ORs. However, whether ORs are susceptible to agonist-dependent desensitization and whether GRKs affect odorant responsiveness of OSNs are currently unknown. Here we show that GRK3 attenuated the agonist responsiveness of a specific mouse odorant receptor for eugenol (mOR-EG) upon agonist pretreatment in HEK293 cells, but GRK3 did not affect the response amplitude or the recovery kinetics upon repeated agonist stimulation. We performed electrophysiological recordings of single OSNs which expressed mOR-EG and green fluorescent protein (GFP) in the presence or absence of GRK3. The kinetics and amplitude of agonist responsiveness of individual GFP-labeled mOR-EG neurons were not significantly affected by the absence of GRK3. These results indicate that the role of GRK3 in attenuating ORs responsiveness in OSNs may have been overestimated. PMID:25313015

  12. Pilocarpine modulates the cellular electrical properties of mammalian hearts by activating a cardiac M3 receptor and a K+ current

    PubMed Central

    Wang, Huizhen; Shi, Hong; Lu, Yanjie; Yang, Baofeng; Wang, Zhiguo

    1999-01-01

    Pilocarpine, a muscarinic acetylcholine receptor (mAChR) agonist, is widely used for treatment of xerostomia and glaucoma. It can also cause many other cellular responses by activating different subtypes of mAChRs in different tissues. However, the potential role of pilocarpine in modulating cardiac function remained unstudied.We found that pilocarpine produced concentration-dependent (0.1–10 μM) decrease in sinus rhythm and action potential duration, and hyperpolarization of membrane potential in guinea-pig hearts. The effects were nearly completely reversed by 1 μM atropine or 2 nM 4DAMP methiodide (an M3-selective antagonist).Patch-clamp recordings in dispersed myocytes from guinea-pig and canine atria revealed that pilocarpine induces a novel K+ current with delayed rectifying properties. The current was suppressed by low concentrations of M3-selective antagonists 4DAMP methiodide (2–10 nM), 4DAMP mustard (4–20 nM, an ackylating agent) and p-F-HHSiD (20–200 nM). Antagonists towards other subtypes (M1, M2 or M4) all failed to alter the current.The affinity of pilocarpine (KD) at mAChRs derived from displacement binding of [3H]-NMS in the homogenates from dog atria was 2.2 μM (65% of the total binding) and that of 4DAMP methiodide was 2.8 nM (70% of total binding), consistent with the concentration of pilocarpine needed for the current induction and for the modulation of the cardiac electrical activity and the concentration of 4DAMP to block pilocarpine effects.Our data indicate, for the first time, that pilocarpine modulates the cellular electrical properties of the hearts, likely by activating a K+ current mediated by M3 receptors. PMID:10372814

  13. Mammalian 43-kD acetylcholine receptor-associated protein (RAPsyn) is expressed in some nonmuscle cells

    SciTech Connect

    Musil, L.S.; Frail, D.E.; Merlie, J.P. )

    1989-05-01

    Torpedo electric organ and vertebrate neuromuscular junctions contain the receptor-associated protein of the synapse (RAPsyn) (previously referred to as the 43K protein), a nonactin, 43,000-Mr peripheral membrane protein associated with the cytoplasmic face of postsynaptic membranes at areas of high nicotinic acetylcholine receptor (AChR) density. Although not directly demonstrated, several lines of evidence suggest that RAPsyn is involved in the synthesis and/or maintenance of such AChR clusters. Microscopic and biochemical studies had previously indicated that RAPsyn expression is restricted to differentiated, AChR-synthesizing cells. Our recent finding that RAPsyn is also produced in undifferentiated myocytes led to to examine whether RAPsyn is synthesized in cell types that never express AChR (i.e., cells of other than skeletal muscle origin). Various primary and established rodent cell lines were metabolically labeled with (35S)methionine, and extracts were immunoprecipitated with a monospecific anti-RAPsyn serum. Analysis of these immunoprecipitates by SDS-PAGE revealed detectable RAPsyn synthesis in some (notably fibroblast and Leydig tumor cell lines and primary cardiac cells) but not all (hepatocyte- and lymphocyte-derived) cell types. These results were further substantiated by peptide mapping studies of RAPsyn immunoprecipitated from different cells and quantitation of RAPsyn-encoding mRNA levels in mouse tissues. RAPsyn synthesized in both muscle and nonmuscle cells was shown to be tightly associated with membranes. These findings demonstrate that RAPsyn is not specific to skeletal muscle-derived cells and imply that it may function in a capacity either in addition to or instead of AChR clustering.

  14. Identification of mRNAs coding for mammalian-type melanin-concentrating hormone and its receptors in the scalloped hammerhead shark Sphyrna lewini.

    PubMed

    Mizusawa, Kanta; Amiya, Noriko; Yamaguchi, Yoko; Takabe, Souichirou; Amano, Masafumi; Breves, Jason P; Fox, Bradley K; Grau, E Gordon; Hyodo, Susumu; Takahashi, Akiyoshi

    2012-10-01

    Melanin-concentrating hormone (MCH) is a neuromodulator, synthesized in the hypothalamus, that regulates both appetite and energy homeostasis in mammals. MCH was initially identified in teleost fishes as a pituitary gland hormone that induced melanin aggregation in chromatophores in the skin; however, this function of MCH has not been observed in other vertebrates. Recent studies suggest that MCH is involved in teleost feeding behavior, spurring the hypothesis that the original function of MCH in early vertebrates was appetite regulation. The present study reports the results of cDNAs cloning encoding preproMCH and two MCH receptors from an elasmobranch fish, Sphyrna lewini, a member of Chondrichthyes, the earliest diverged class in gnathostomes. The putative MCH peptide is composed of 19 amino acids, similar in length to the mammalian MCH. Reverse-transcription polymerase chain reaction revealed that MCH is expressed in the hypothalamus in S. lewini MCH cell bodies and fibers were identified by immunochemistry in the hypothalamus, but not in the pituitary gland, suggesting that MCH is not released via the pituitary gland into general circulation. MCH receptor genes mch-r1 and mch-r2 were expressed in the S. lewini hypothalamus, but were not found in the skin. These results indicate that MCH does not have a peripheral function, such as a melanin-concentrating effect, in the skin of S. lewini hypothalamic MCH mRNA levels were not affected by fasting, suggesting that feeding conditions might not affect the expression of MCH in the hypothalamus. PMID:22884735

  15. Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

    PubMed

    Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

    2016-07-01

    Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

  16. Highly sensitive and unbiased approach for elucidating antibody repertoires.

    PubMed

    Lin, Sherry G; Ba, Zhaoqing; Du, Zhou; Zhang, Yu; Hu, Jiazhi; Alt, Frederick W

    2016-07-12

    Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes. PMID:27354528

  17. The ancestral gene repertoire of animal stem cells.

    PubMed

    Alié, Alexandre; Hayashi, Tetsutaro; Sugimura, Itsuro; Manuel, Michaël; Sugano, Wakana; Mano, Akira; Satoh, Nori; Agata, Kiyokazu; Funayama, Noriko

    2015-12-22

    Stem cells are pivotal for development and tissue homeostasis of multicellular animals, and the quest for a gene toolkit associated with the emergence of stem cells in a common ancestor of all metazoans remains a major challenge for evolutionary biology. We reconstructed the conserved gene repertoire of animal stem cells by transcriptomic profiling of totipotent archeocytes in the demosponge Ephydatia fluviatilis and by tracing shared molecular signatures with flatworm and Hydra stem cells. Phylostratigraphy analyses indicated that most of these stem-cell genes predate animal origin, with only few metazoan innovations, notably including several partners of the Piwi machinery known to promote genome stability. The ancestral stem-cell transcriptome is strikingly poor in transcription factors. Instead, it is rich in RNA regulatory actors, including components of the "germ-line multipotency program" and many RNA-binding proteins known as critical regulators of mammalian embryonic stem cells. PMID:26644562

  18. The ancestral gene repertoire of animal stem cells

    PubMed Central

    Alié, Alexandre; Hayashi, Tetsutaro; Sugimura, Itsuro; Manuel, Michaël; Sugano, Wakana; Mano, Akira; Satoh, Nori; Agata, Kiyokazu; Funayama, Noriko

    2015-01-01

    Stem cells are pivotal for development and tissue homeostasis of multicellular animals, and the quest for a gene toolkit associated with the emergence of stem cells in a common ancestor of all metazoans remains a major challenge for evolutionary biology. We reconstructed the conserved gene repertoire of animal stem cells by transcriptomic profiling of totipotent archeocytes in the demosponge Ephydatia fluviatilis and by tracing shared molecular signatures with flatworm and Hydra stem cells. Phylostratigraphy analyses indicated that most of these stem-cell genes predate animal origin, with only few metazoan innovations, notably including several partners of the Piwi machinery known to promote genome stability. The ancestral stem-cell transcriptome is strikingly poor in transcription factors. Instead, it is rich in RNA regulatory actors, including components of the “germ-line multipotency program” and many RNA-binding proteins known as critical regulators of mammalian embryonic stem cells. PMID:26644562

  19. Peroxisome proliferator-activated receptor-γ agonist inhibits the mammalian target of rapamycin signaling pathway and has a protective effect in a rat model of status epilepticus.

    PubMed

    San, Yong-Zhi; Liu, Yu; Zhang, Yu; Shi, Ping-Ping; Zhu, Yu-Lan

    2015-08-01

    Peroxisome proliferator-activated receptor γ (PPAR-γ) has a protective role in several neurological diseases. The present study investigated the effect of the PPAR-γ agonist, pioglitazone, on the mammalian target of rapamycin (mTOR) signaling pathway in a rat model of pentylenetetrazol (PTZ)-induced status epilepticus (SE). The investigation proceeded in two stages. First, the course of activation of the mTOR signaling pathway in PTZ-induced SE was examined to determine the time-point of peak activity, as reflected by phopshorylated (p)-mTOR/mTOR and p-S6/S6 ratios. Subsequently, pioglitazone was administrated intragastrically to investigate its effect on the mTOR signaling pathway, through western blot and immunochemical analyses. The levels of the interleukin (IL)-1β and IL-6 inflammatory cytokines were detected using ELISA, and neuronal loss was observed via Nissl staining. In the first stage of experimentation, the mTOR signaling pathway was activated, and the p-mTOR/mTOR and p-S6/S6 ratios peaked on the third day. Compared with the vehicle treated-SE group, pretreatment with pioglitazone was associated with the loss of fewer neurons, lower levels of IL-1β and IL-6, and inhibition of the activation of the mTOR signaling pathway. Therefore, the mTOR signaling pathway was activated in the PTZ-induced SE rat model, and the PPAR-γ agonist, pioglitazone, had a neuroprotective effect, by inhibiting activation of the mTOR pathway and preventing the increase in the levels of IL-1β and IL-6. PMID:25891824

  20. Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires.

    PubMed Central

    Goodnow, C C

    1996-01-01

    Immunological self-tolerance is ensured by eliminating or inhibiting self-reactive lymphocyte clones, creating physical or functional holes in the B- and T-lymphocyte antigen receptor repertoires. The nature and size of these gaps in our immune defenses must be balanced against the necessity of mounting rapid immune responses to an everchanging array of foreign pathogens. To achieve this balance, only a fraction of particularly hazardous self-reactive clones appears to be physically eliminated from the repertoire in a manner that fully prevents their recruitment into an antimicrobial immune response. Many self-reactive cells are retained with a variety of conditional and potentially flexible restraints: (i) their ability to be triggered by antigen is diminished by mechanisms that tune down signaling by their antigen receptors, (ii) their ability to carry out inflammatory effector functions can be inhibited, and (iii) their capacity to migrate and persist is constrained. This balance between tolerance and immunity can be shifted, altering susceptibility to autoimmune disease and to infection by genetic or environmental differences either in the way antigens are presented, in the tuning molecules that adjust triggering set points for lymphocyte responses to antigen, or in the effector molecules that eliminate, retain, or expand particular clones. Images Fig. 1 Fig. 2 PMID:8637861

  1. In silico prediction of the G-protein coupled receptors expressed during the metamorphic molt of Sagmariasus verreauxi (Crustacea: Decapoda) by mining transcriptomic data: RNA-seq to repertoire.

    PubMed

    Buckley, Sean J; Fitzgibbon, Quinn P; Smith, Gregory G; Ventura, Tomer

    2016-03-01

    Against a backdrop of food insecurity, the farming of decapod crustaceans is a rapidly expanding and globally significant source of food protein. Sagmariasus verreauxi spiny lobster, the subject of this study, are decapods of underdeveloped aquaculture potential. Crustacean neuropeptide G-protein coupled receptors (GPCRs) mediate endocrine pathways that are integral to animal fecundity, growth and survival. The potential use of novel biotechnologies to enhance GPCR-mediated physiology may assist in improving the health and productivity of farmed decapod populations. This study catalogues the GPCRs expressed in the early developmental stages, as well as adult tissues, with a view to illuminating key neuropeptide receptors. De novo assembled contiguous sequences generated from transcriptomic reads of metamorphic and post metamorphic S. verreauxi were filtered for seven transmembrane domains, and used as a reference for iterative re-mapping. Subsequent putative GPCR open reading frames (ORFs) were BLAST annotated, categorised, and compared to published orthologues based on phylogenetic analysis. A total of 85 GPCRs were digitally predicted, that represented each of the four arthropod subfamilies. They generally displayed low-level and non-differential metamorphic expression with few exceptions that we examined using RT-PCR and qPCR. Two putative CHH-like neuropeptide receptors were annotated. Three dimensional structural modelling suggests that these receptors exhibit a conserved extracellular ligand binding pocket, providing support to the notion that these receptors co-evolved with their ligands across Decapoda. This perhaps narrows the search for means to increase productivity of farmed decapod populations. PMID:26850661

  2. Bias in the αβ T-cell repertoire: implications for disease pathogenesis and vaccination.

    PubMed

    Miles, John J; Douek, Daniel C; Price, David A

    2011-03-01

    The naïve T-cell repertoire is vast, containing millions of unique T-cell receptor (TCR) structures. Faced with such diversity, the mobilization of TCR structures from this enormous pool was once thought to be a stochastic, even chaotic, process. However, steady and systematic dissection over the last 20 years has revealed that this is not the case. Instead, the TCR repertoire deployed against individual antigens is routinely ordered and biased. Often, identical and near-identical TCR repertoires can be observed across different individuals, suggesting that the system encompasses an element of predictability. This review provides a catalog of αβ TCR bias by disease and by species, and discusses the mechanisms that govern this inherent and widespread phenomenon. PMID:21301479

  3. B-cell repertoire responses to varicella-zoster vaccination in human identical twins.

    PubMed

    Wang, Chen; Liu, Yi; Cavanagh, Mary M; Le Saux, Sabine; Qi, Qian; Roskin, Krishna M; Looney, Timothy J; Lee, Ji-Yeun; Dixit, Vaishali; Dekker, Cornelia L; Swan, Gary E; Goronzy, Jörg J; Boyd, Scott D

    2015-01-13

    Adaptive immune responses in humans rely on somatic genetic rearrangements of Ig and T-cell receptor loci to generate diverse antigen receptors. It is unclear to what extent an individual's genetic background affects the characteristics of the antibody repertoire used in responding to vaccination or infection. We studied the B-cell repertoires and clonal expansions in response to attenuated varicella-zoster vaccination in four pairs of adult identical twins and found that the global antibody repertoires of twin pair members showed high similarity in antibody heavy chain V, D, and J gene segment use, and in the length and features of the complementarity-determining region 3, a major determinant of antigen binding. These twin similarities were most pronounced in the IgM-expressing B-cell pools, but were seen to a lesser extent in IgG-expressing B cells. In addition, the degree of antibody somatic mutation accumulated in the B-cell repertoire was highly correlated within twin pair members. Twin pair members had greater numbers of shared convergent antibody sequences, including mutated sequences, suggesting similarity among memory B-cell clonal lineages. Despite these similarities in the memory repertoire, the B-cell clones used in acute responses to ZOSTAVAX vaccination were largely unique to each individual. Taken together, these results suggest that the overall B-cell repertoire is significantly shaped by the underlying germ-line genome, but that stochastic or individual-specific effects dominate the selection of clones in response to an acute antigenic stimulus. PMID:25535378

  4. Sequence analysis of T-cell repertoires in health and disease

    PubMed Central

    2013-01-01

    T-cell antigen receptor (TCR) variability enables the cellular immune system to discriminate between self and non-self. High-throughput TCR sequencing (TCR-seq) involves the use of next generation sequencing platforms to generate large numbers of short DNA sequences covering key regions of the TCR coding sequence, which enables quantification of T-cell diversity at unprecedented resolution. TCR-seq studies have provided new insights into the healthy human T-cell repertoire, such as revised estimates of repertoire size and the understanding that TCR specificities are shared among individuals more frequently than previously anticipated. In the context of disease, TCR-seq has been instrumental in characterizing the recovery of the immune repertoire after hematopoietic stem cell transplantation, and the method has been used to develop biomarkers and diagnostics for various infectious and neoplastic diseases. However, T-cell repertoire sequencing is still in its infancy. It is expected that maturation of the field will involve the introduction of improved, standardized tools for data handling, deposition and statistical analysis, as well as the emergence of new and equivalently large-scale technologies for T-cell functional analysis and antigen discovery. In this review, we introduce this nascent field and TCR-seq methodology, we discuss recent insights into healthy and diseased TCR repertoires, and we examine the applications and challenges for TCR-seq in the clinic. PMID:24172704

  5. Laboratory and Data Analysis Methods for Characterization of Human B Cell Repertoires by High-Throughput DNA Sequencing.

    PubMed

    Wang, Chen; Liu, Yi; Roskin, Krishna M; Jackson, Katherine J L; Boyd, Scott D

    2015-01-01

    High-throughput DNA sequencing techniques have greatly accelerated the pace of research into the repertoires of antibody and T cell receptor gene rearrangements that confer antigen specificity to adaptive immune responses. Studies of aging-related changes in human B cell repertoires have benefited from the ability to detect and quantify thousands to millions of B cell clones in human samples, and study the mutational lineages and isotype switching relationships within each clonal lineage. Correlation of repertoire analysis with antibody gene data from antigen-specific B cells is poised to give much greater insight into clinically relevant B cell responses and memory storage. Here, we describe strategies for preparing and analyzing human antibody gene libraries for studying B cell repertoires. PMID:26420720

  6. Characterization of the Sortase Repertoire in Bacillus anthracis

    PubMed Central

    Fouet, Agnès

    2011-01-01

    LPXTG proteins, present in most if not all Gram-positive bacteria, are known to be anchored by sortases to the bacterial peptidoglycan. More than one sortase gene is often encoded in a bacterial species, and each sortase is supposed to specifically anchor given LPXTG proteins, depending of the sequence of the C-terminal cell wall sorting signal (cwss), bearing an LPXTG motif or another recognition sequence. B. anthracis possesses three sortase genes. B. anthracis sortase deleted mutant strains are not affected in their virulence. To determine the sortase repertoires, we developed a genetic screen using the property of the gamma phage to lyse bacteria only when its receptor, GamR, an LPXTG protein, is exposed at the surface. We identified 10 proteins that contain a cell wall sorting signal and are covalently anchored to the peptidoglycan. Some chimeric proteins yielded phage lysis in all sortase mutant strains, suggesting that cwss proteins remained surface accessible in absence of their anchoring sortase, probably as a consequence of membrane localization of yet uncleaved precursor proteins. For definite assignment of the sortase repertoires, we consequently relied on a complementary test, using a biochemical approach, namely immunoblot experiments. The sortase anchoring nine of these proteins has thus been determined. The absence of virulence defect of the sortase mutants could be a consequence of the membrane localization of the cwss proteins. PMID:22076158

  7. A special repertoire of alpha:beta T cells in neonatal mice.

    PubMed Central

    Bogue, M; Candéias, S; Benoist, C; Mathis, D

    1991-01-01

    According to several functional criteria, the mature thymocytes of neonatal and adult mice are distinctly different. We wondered whether these differences in function might have a structural correlate: do neonates have a distinct repertoire of alpha:beta T cells? In this study, we have exploited the power of polymerase chain reaction technology to generate large numbers of T cell receptor sequences from sorted thymocyte populations from newborn and adult mice. The newborn-derived sequences show very few N nucleotide additions, usually the major source of diversity in T cell receptors. Most interestingly, the paucity of N insertions appears to be exaggerated by selection events that operate during T cell differentiation in the thymus. The significance of these results is largely: (i) that they parallel recent findings on the B cell repertoire in neonates, raising questions about the reactivities specified by such a special repertoire; and (ii) that they suggest a means to 'tag' T cells exported perinatally, allowing one to test the premise that autoreactive T cells derive preferentially from the newborn repertoire. Images PMID:1834457

  8. Age-related changes in natural killer cell repertoires: impact on NK cell function and immune surveillance.

    PubMed

    Manser, Angela R; Uhrberg, Markus

    2016-04-01

    A key feature of human natural killer (NK) cells, which enables efficient recognition of infected and malignant target cells, is the expression of HLA class I-specific receptors of the KIR and NKG2 gene families. Cell-to-cell variability in receptor expression leads to the formation of complex NK cell repertoires. As outlined here, NK cells go through major changes from newborns to adults characterized by downregulation of the inhibitory NKG2A receptor and concomitant upregulation of KIR family members. This process is completed in young adults, and in the majority of individuals, KIR/NKG2A repertoires remain remarkably stable until old age. Nonetheless, age-related factors have the potential to majorly influence the complexity of NK cell repertoires: Firstly infection with HCMV is associated with major clonal expansions of terminally differentiated NKG2C- and KIR-expressing NK cells in certain individuals. Secondly, ineffective hematopoiesis can lead to immature and less diversified NK cell repertoires as observed in myelodysplastic syndrome (MDS), a malignant disease of the elderly. Thus, whereas in the majority of elderly the NK cell compartment appears to be highly stable in terms of function and phenotype, in a minority of subjects a breakdown of NK cell repertoire diversity is observed that might influence immune surveillance and healthy aging. PMID:26288343

  9. Englishized Style Repertoire in Modern Japanese Literature.

    ERIC Educational Resources Information Center

    Ono, Reiko

    1992-01-01

    The roles played by English borrowings in modern Japanese literary works are examined. After a brief summary of previous studies, this paper describes the style repertoire and the kinds of stylistic effects produced in Japanese literature by English borrowings, such as attention attractors and in-group-identity markers. (23 references) (Author/LB)

  10. Mammalian sleep

    NASA Astrophysics Data System (ADS)

    Staunton, Hugh

    2005-05-01

    This review examines the biological background to the development of ideas on rapid eye movement sleep (REM sleep), so-called paradoxical sleep (PS), and its relation to dreaming. Aspects of the phenomenon which are discussed include physiological changes and their anatomical location, the effects of total and selective sleep deprivation in the human and animal, and REM sleep behavior disorder, the latter with its clinical manifestations in the human. Although dreaming also occurs in other sleep phases (non-REM or NREM sleep), in the human, there is a contingent relation between REM sleep and dreaming. Thus, REM is taken as a marker for dreaming and as REM is distributed ubiquitously throughout the mammalian class, it is suggested that other mammals also dream. It is suggested that the overall function of REM sleep/dreaming is more important than the content of the individual dream; its function is to place the dreamer protagonist/observer on the topographical world. This has importance for the developing infant who needs to develop a sense of self and separateness from the world which it requires to navigate and from which it is separated for long periods in sleep. Dreaming may also serve to maintain a sense of ‘I’ness or “self” in the adult, in whom a fragility of this faculty is revealed in neurological disorders.