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Sample records for mammalian receptor repertoire

  1. Pharmacology of mammalian olfactory receptors.

    PubMed

    Smith, Richard S; Peterlin, Zita; Araneda, Ricardo C

    2013-01-01

    Mammalian species have evolved a large and diverse number of odorant receptors (ORs). These proteins comprise the largest family of G-protein-coupled receptors (GPCRs) known, amounting to ~1,000-different receptors in the rodent. From the perspective of olfactory coding, the availability of such a vast number of chemosensory receptors poses several fascinating questions; in addition, such a large repertoire provides an attractive biological model to study ligand-receptor interactions. The limited functional expression of these receptors in heterologous systems, however, has greatly hampered attempts to deorphanize them. We have employed a successful approach that combines electrophysiological and imaging techniques to analyze the response profiles of single sensory neurons. Our approach has enabled us to characterize the "odor space" of a population of native aldehyde receptors and the molecular range of a genetically engineered receptor, OR-I7.

  2. Mammalian odorant receptors: functional evolution and variation

    PubMed Central

    Jiang, Yue; Matsunami, Hiroaki

    2015-01-01

    In mammals, the perception of smell starts with the activation of odorant receptors (ORs) by volatile molecules in the environment. The mammalian OR repertoire has been subject to rapid evolution, and is highly diverse within the human population. Recent advances in the functional expression and ligand identification of ORs allow for functional analysis of OR evolution, and reveal that changes in OR protein sequences translate into high degrees of functional variations. Moreover, in several cases the functional variation of a single OR affects the perception of its cognate odor ligand, providing clues as to how an odor is coded at the receptor level. PMID:25660959

  3. Comparison of the canine and human olfactory receptor gene repertoires

    PubMed Central

    Quignon, Pascale; Kirkness, Ewen; Cadieu, Edouard; Touleimat, Nizar; Guyon, Richard; Renier, Corinne; Hitte, Christophe; André, Catherine; Fraser, Claire; Galibert, Francis

    2003-01-01

    Background Olfactory receptors (ORs), the first dedicated molecules with which odorants physically interact to arouse an olfactory sensation, constitute the largest gene family in vertebrates, including around 900 genes in human and 1,500 in the mouse. Whereas dogs, like many other mammals, have a much keener olfactory potential than humans, only 21 canine OR genes have been described to date. Results In this study, 817 novel canine OR sequences were identified, and 640 have been characterized. Of the 661 characterized OR sequences, representing half of the canine repertoire, 18% are predicted to be pseudogenes, compared with 63% in human and 20% in mouse. Phylogenetic analysis of 403 canine OR sequences identified 51 families, and radiation-hybrid mapping of 562 showed that they are distributed on 24 dog chromosomes, in 37 distinct regions. Most of these regions constitute clusters of 2 to 124 closely linked genes. The two largest clusters (124 and 109 OR genes) are located on canine chromosomes 18 and 21. They are orthologous to human clusters located on human chromosomes 11q11-q13 and HSA11p15, containing 174 and 115 ORs respectively. Conclusions This study shows a strongly conserved genomic distribution of OR genes between dog and human, suggesting that OR genes evolved from a common mammalian ancestral repertoire by successive duplications. In addition, the dog repertoire appears to have expanded relative to that of humans, leading to the emergence of specific canine OR genes. PMID:14659017

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

    PubMed

    Shugay, Mikhail; Bagaev, Dmitriy V; Turchaninova, Maria A; 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

    2015-11-01

    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.

  5. 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

  6. The repertoire of bitter taste receptor genes in canids.

    PubMed

    Shang, Shuai; Wu, Xiaoyang; Chen, Jun; Zhang, Huanxin; Zhong, Huaming; Wei, Qinguo; Yan, Jiakuo; Li, Haotian; Liu, Guangshuai; Sha, Weilai; Zhang, Honghai

    2017-07-01

    Bitter taste receptors (Tas2rs) play important roles in mammalian defense mechanisms by helping animals detect and avoid toxins in food. Although Tas2r genes have been widely studied in several mammals, minimal research has been performed in canids. To analyze the genetic basis of Tas2r genes in canids, we first identified Tas2r genes in the wolf, maned wolf, red fox, corsac fox, Tibetan fox, fennec fox, dhole and African hunting dog. A total of 183 Tas2r genes, consisting of 118 intact genes, 6 partial genes and 59 pseudogenes, were detected. Differences in the pseudogenes were observed among nine canid species. For example, Tas2r4 was a pseudogene in the dog but might play a functional role in other canid species. The Tas2r42 and Tas2r10 genes were pseudogenes in the maned wolf and dhole, respectively, and the Tas2r5 and Tas2r34 genes were pseudogenes in the African hunting dog; however, these genes were intact genes in other canid species. The differences in Tas2r pseudogenes among canids might suggest that the loss of intact Tas2r genes in canid species is species-dependent. We further compared the 183 Tas2r genes identified in this study with Tas2r genes from ten additional carnivorous species to evaluate the potential influence of diet on the evolution of the Tas2r gene repertoire. Phylogenetic analysis revealed that most of the Tas2r genes from the 18 species intermingled across the tree, suggesting that Tas2r genes are conserved among carnivores. Within canids, we found that some Tas2r genes corresponded to the traditional taxonomic groupings, while some did not. PIC analysis showed that the number of Tas2r genes in carnivores exhibited no positive correlation with diet composition, which might be due to the limited number of carnivores included in our study.

  7. Functional Evolution of Mammalian Odorant Receptors

    PubMed Central

    Adipietro, Kaylin A.; Mainland, Joel D.; Matsunami, Hiroaki

    2012-01-01

    The mammalian odorant receptor (OR) repertoire is an attractive model to study evolution, because ORs have been subjected to rapid evolution between species, presumably caused by changes of the olfactory system to adapt to the environment. However, functional assessment of ORs in related species remains largely untested. Here we investigated the functional properties of primate and rodent ORs to determine how well evolutionary distance predicts functional characteristics. Using human and mouse ORs with previously identified ligands, we cloned 18 OR orthologs from chimpanzee and rhesus macaque and 17 mouse-rat orthologous pairs that are broadly representative of the OR repertoire. We functionally characterized the in vitro responses of ORs to a wide panel of odors and found similar ligand selectivity but dramatic differences in response magnitude. 87% of human-primate orthologs and 94% of mouse-rat orthologs showed differences in receptor potency (EC50) and/or efficacy (dynamic range) to an individual ligand. Notably dN/dS ratio, an indication of selective pressure during evolution, does not predict functional similarities between orthologs. Additionally, we found that orthologs responded to a common ligand 82% of the time, while human OR paralogs of the same subfamily responded to the common ligand only 33% of the time. Our results suggest that, while OR orthologs tend to show conserved ligand selectivity, their potency and/or efficacy dynamically change during evolution, even in closely related species. These functional changes in orthologs provide a platform for examining how the evolution of ORs can meet species-specific demands. PMID:22807691

  8. Practical guidelines for B-cell receptor repertoire sequencing analysis.

    PubMed

    Yaari, Gur; Kleinstein, Steven H

    2015-11-20

    High-throughput sequencing of B-cell immunoglobulin repertoires is increasingly being applied to gain insights into the adaptive immune response in healthy individuals and in those with a wide range of diseases. Recent applications include the study of autoimmunity, infection, allergy, cancer and aging. As sequencing technologies continue to improve, these repertoire sequencing experiments are producing ever larger datasets, with tens- to hundreds-of-millions of sequences. These data require specialized bioinformatics pipelines to be analyzed effectively. Numerous methods and tools have been developed to handle different steps of the analysis, and integrated software suites have recently been made available. However, the field has yet to converge on a standard pipeline for data processing and analysis. Common file formats for data sharing are also lacking. Here we provide a set of practical guidelines for B-cell receptor repertoire sequencing analysis, starting from raw sequencing reads and proceeding through pre-processing, determination of population structure, and analysis of repertoire properties. These include methods for unique molecular identifiers and sequencing error correction, V(D)J assignment and detection of novel alleles, clonal assignment, lineage tree construction, somatic hypermutation modeling, selection analysis, and analysis of stereotyped or convergent responses. The guidelines presented here highlight the major steps involved in the analysis of B-cell repertoire sequencing data, along with recommendations on how to avoid common pitfalls.

  9. Selection of the lamprey VLRC antigen receptor repertoire.

    PubMed

    Holland, Stephen J; Gao, Mingming; Hirano, Masayuki; Iyer, Lakshminarayan M; Luo, Ming; Schorpp, Michael; Cooper, Max D; Aravind, L; Mariuzza, Roy A; Boehm, Thomas

    2014-10-14

    The alternative adaptive immune system of jawless vertebrates is based on different isotypes of variable lymphocyte receptors (VLRs) that are composed of leucine-rich repeats (LRRs) and expressed by distinct B- and T-like lymphocyte lineages. VLRB is expressed by B-like cells, whereas VLRA and VLRC are expressed by two T-like lineages that develop in the thymoid, a thymus-like structure in lamprey larvae. In each case, stepwise combinatorial insertions of different types of short donor LRR cassettes into incomplete germ-line genes are required to generate functional VLR gene assemblies. It is unknown, however, whether the diverse repertoires of VLRs that are expressed by peripheral blood lymphocytes are shaped by selection after their assembly. Here, we identify signatures of selection in the peripheral repertoire of VLRC antigen receptors that are clonally expressed by one of the T-like cell types in lampreys. Selection strongly favors VLRC molecules containing four internal variable leucine-rich repeat (LRRV) modules, although VLRC assemblies encoding five internal modules are initially equally frequent. In addition to the length selection, VLRC molecules in VLRC(+) peripheral lymphocytes exhibit a distinct pattern of high entropy sites in the N-terminal LRR1 module, which is inserted next to the germ-line-encoded LRRNT module. This is evident in comparisons to VLRC gene assemblies found in the thymoid and to VLRC gene assemblies found in some VLRA(+) cells. Our findings are the first indication to our knowledge that selection operates on a VLR repertoire and provide a framework to establish the mechanism by which this selection occurs during development of the VLRC(+) lymphocyte lineage.

  10. Selection of the lamprey VLRC antigen receptor repertoire

    PubMed Central

    Holland, Stephen J.; Gao, Mingming; Hirano, Masayuki; Iyer, Lakshminarayan M.; Luo, Ming; Schorpp, Michael; Cooper, Max D.; Aravind, L.; Mariuzza, Roy A.; Boehm, Thomas

    2014-01-01

    The alternative adaptive immune system of jawless vertebrates is based on different isotypes of variable lymphocyte receptors (VLRs) that are composed of leucine-rich repeats (LRRs) and expressed by distinct B- and T-like lymphocyte lineages. VLRB is expressed by B-like cells, whereas VLRA and VLRC are expressed by two T-like lineages that develop in the thymoid, a thymus-like structure in lamprey larvae. In each case, stepwise combinatorial insertions of different types of short donor LRR cassettes into incomplete germ-line genes are required to generate functional VLR gene assemblies. It is unknown, however, whether the diverse repertoires of VLRs that are expressed by peripheral blood lymphocytes are shaped by selection after their assembly. Here, we identify signatures of selection in the peripheral repertoire of VLRC antigen receptors that are clonally expressed by one of the T-like cell types in lampreys. Selection strongly favors VLRC molecules containing four internal variable leucine-rich repeat (LRRV) modules, although VLRC assemblies encoding five internal modules are initially equally frequent. In addition to the length selection, VLRC molecules in VLRC+ peripheral lymphocytes exhibit a distinct pattern of high entropy sites in the N-terminal LRR1 module, which is inserted next to the germ-line–encoded LRRNT module. This is evident in comparisons to VLRC gene assemblies found in the thymoid and to VLRC gene assemblies found in some VLRA+ cells. Our findings are the first indication to our knowledge that selection operates on a VLR repertoire and provide a framework to establish the mechanism by which this selection occurs during development of the VLRC+ lymphocyte lineage. PMID:25228760

  11. 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

  12. 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

  13. Model for Comparative Analysis of Antigen Receptor Repertoires

    PubMed Central

    Rempala, Grzegorz A.; Seweryn, Michał; Ignatowicz, Leszek

    2010-01-01

    In modern molecular biology one of the standard ways of analyzing a vertebrate immune system is to sequence and compare the counts of specific antigen receptor clones (either immunoglobulins or T-cell receptors) derived from various tissues under different experimental or clinical conditions. The resulting statistical challenges are difficult and do not fit readily into the standard statistical framework of contingency tables primarily due to the serious under-sampling of the receptor populations. This under-sampling is caused, on one hand, by the extreme diversity of antigen receptor repertoires maintained by the immune system and, on the other, by the high cost and labor intensity of the receptor data collection process. In most of the recent immunological literature the differences across antigen receptor populations are examined via non-parametric statistical measures of the species overlap and diversity borrowed from ecological studies. While this approach is robust in a wide range of situations, it seems to provide little insight into the underlying clonal size distribution and the overall mechanism differentiating the receptor populations. As a possible alternative, the current paper presents a parametric method that adjusts for the data under-sampling as well as provides a unifying approach to a simultaneous comparison of multiple receptor groups by means of the modern statistical tools of unsupervised learning. The parametric model is based on a flexible multivariate Poisson-lognormal distribution and is seen to be a natural generalization of the univariate Poisson-lognormal models used in the ecological studies of biodiversity patterns. The procedure for evaluating a model’s fit is described along with the public domain software developed to perform the necessary diagnostics. The model-driven analysis is seen to compare favorably vis a vis traditional methods when applied to the data from T-cell receptors in transgenic mice populations. PMID:20955715

  14. Dramatic variation of the vomeronasal pheromone receptor gene repertoire among five orders of placental and marsupial mammals

    PubMed Central

    Grus, Wendy E.; Shi, Peng; Zhang, Ya-ping; Zhang, Jianzhi

    2005-01-01

    Pheromones are chemicals emitted and sensed by conspecifics to elicit social and sexual responses and are perceived in terrestrial vertebrates primarily by the vomeronasal organ (VNO). Pheromone receptors in the mammalian VNO are encoded by the V1R and V2R gene superfamilies. The V1R superfamily contains 187 and 102 putatively functional genes in the mouse and rat, respectively. To investigate whether this large repertoire size is typical among mammals with functional VNOs, we here describe the V1R repertoires of dog, cow, and opossum based on their draft genome sequences. The dog and cow have only 8 and 32 intact V1R genes, respectively. Thus, the intact V1R repertoire size varies by at least 23-fold among placental mammals with functional VNOs. To our knowledge, this size ratio represents the greatest among-species variation in gene family size of all mammalian gene families. Phylogenetic analysis of placental V1R genes suggests multiple losses of ancestral genes in carnivores and artiodactyls and gains of many new genes by gene duplication in rodents, manifesting massive gene births and deaths. We also identify 49 intact opossum V1R genes and discover independent expansions of the repertoire in placentals and marsupials. We further show a concordance between the V1R repertoire size and the complexity of VNO morphology, suggesting that the latter could indicate the sophistication of pheromone communications within species. In sum, our results demonstrate tremendous diversity and rapid evolution of mammalian V1R gene inventories and caution the generalization of VNO biology from rodents to all mammals. PMID:15790682

  15. Dramatic variation of the vomeronasal pheromone receptor gene repertoire among five orders of placental and marsupial mammals.

    PubMed

    Grus, Wendy E; Shi, Peng; Zhang, Ya-ping; Zhang, Jianzhi

    2005-04-19

    Pheromones are chemicals emitted and sensed by conspecifics to elicit social and sexual responses and are perceived in terrestrial vertebrates primarily by the vomeronasal organ (VNO). Pheromone receptors in the mammalian VNO are encoded by the V1R and V2R gene superfamilies. The V1R superfamily contains 187 and 102 putatively functional genes in the mouse and rat, respectively. To investigate whether this large repertoire size is typical among mammals with functional VNOs, we here describe the V1R repertoires of dog, cow, and opossum based on their draft genome sequences. The dog and cow have only 8 and 32 intact V1R genes, respectively. Thus, the intact V1R repertoire size varies by at least 23-fold among placental mammals with functional VNOs. To our knowledge, this size ratio represents the greatest among-species variation in gene family size of all mammalian gene families. Phylogenetic analysis of placental V1R genes suggests multiple losses of ancestral genes in carnivores and artiodactyls and gains of many new genes by gene duplication in rodents, manifesting massive gene births and deaths. We also identify 49 intact opossum V1R genes and discover independent expansions of the repertoire in placentals and marsupials. We further show a concordance between the V1R repertoire size and the complexity of VNO morphology, suggesting that the latter could indicate the sophistication of pheromone communications within species. In sum, our results demonstrate tremendous diversity and rapid evolution of mammalian V1R gene inventories and caution the generalization of VNO biology from rodents to all mammals.

  16. 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

  17. Persisting fetal clonotypes influence the structure and overlap of adult human T cell receptor repertoires.

    PubMed

    Pogorelyy, Mikhail V; Elhanati, Yuval; Marcou, Quentin; Sycheva, Anastasiia L; Komech, Ekaterina A; Nazarov, Vadim I; Britanova, Olga V; Chudakov, Dmitriy M; Mamedov, Ilgar Z; Lebedev, Yury B; Mora, Thierry; Walczak, Aleksandra M

    2017-07-01

    The diversity of T-cell receptors recognizing foreign pathogens is generated through a highly stochastic recombination process, making the independent production of the same sequence rare. Yet unrelated individuals do share receptors, which together constitute a "public" repertoire of abundant clonotypes. The TCR repertoire is initially formed prenatally, when the enzyme inserting random nucleotides is downregulated, producing a limited diversity subset. By statistically analyzing deep sequencing T-cell repertoire data from twins, unrelated individuals of various ages, and cord blood, we show that T-cell clones generated before birth persist and maintain high abundances in adult organisms for decades, slowly decaying with age. Our results suggest that large, low-diversity public clones are created during pre-natal life, and survive over long periods, providing the basis of the public repertoire.

  18. Persisting fetal clonotypes influence the structure and overlap of adult human T cell receptor repertoires

    PubMed Central

    Pogorelyy, Mikhail V.; Elhanati, Yuval; Sycheva, Anastasiia L.; Nazarov, Vadim I.; Britanova, Olga V.; Mamedov, Ilgar Z.

    2017-01-01

    The diversity of T-cell receptors recognizing foreign pathogens is generated through a highly stochastic recombination process, making the independent production of the same sequence rare. Yet unrelated individuals do share receptors, which together constitute a “public” repertoire of abundant clonotypes. The TCR repertoire is initially formed prenatally, when the enzyme inserting random nucleotides is downregulated, producing a limited diversity subset. By statistically analyzing deep sequencing T-cell repertoire data from twins, unrelated individuals of various ages, and cord blood, we show that T-cell clones generated before birth persist and maintain high abundances in adult organisms for decades, slowly decaying with age. Our results suggest that large, low-diversity public clones are created during pre-natal life, and survive over long periods, providing the basis of the public repertoire. PMID:28683116

  19. 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.

  20. 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.

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

    PubMed Central

    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.

    2016-01-01

    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

  2. Largest vertebrate vomeronasal type 1 receptor gene repertoire in the semiaquatic platypus.

    PubMed

    Grus, Wendy E; Shi, Peng; Zhang, Jianzhi

    2007-10-01

    Vertebrate vomeronasal chemoreception plays important roles in many aspects of an organism's daily life, such as mating, territoriality, and foraging. Vomeronasal type 1 receptors (V1Rs) and vomeronasal type 2 receptors (V2Rs), 2 large families of G protein-coupled receptors, serve as vomeronasal receptors to bind to various pheromones and odorants. Contrary to the previous observations of reduced olfaction in aquatic and semiaquatic mammals, we here report the surprising finding that the platypus, a semiaquatic monotreme, has the largest V1R repertoire and nearly largest combined repertoire of V1Rs and V2Rs of all vertebrates surveyed, with 270 intact genes and 579 pseudogenes in the V1R family and 15 intact genes, 55 potentially intact genes, and 57 pseudogenes in the V2R family. Phylogenetic analysis shows a remarkable expansion of the V1R repertoire and a moderate expansion of the V2R repertoire in platypus since the separation of monotremes from placentals and marsupials. Our results challenge the view that olfaction is unimportant to aquatic mammals and call for further study into the role of vomeronasal reception in platypus physiology and behavior.

  3. 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.

  4. Selective manipulation of the human T-cell receptor repertoire expressed by thymocytes in organ culture.

    PubMed Central

    Merkenschlager, M; Fisher, A G

    1992-01-01

    A recently described organ culture system for human thymocytes is shown to support the generation of a diverse T-cell receptor repertoire in vitro: thymocytes of the alpha beta lineage, including representatives of the V beta families 5.2/5.3, 6.7, and 8, accounted for the majority of T-cell receptor-positive cells throughout a 3-week culture period. Thymocytes bearing gamma delta receptors were also identified, particularly among the CD4 CD8 double-negative subset. The T-cell receptor repertoire expressed in organ culture responded to experimental manipulation with staphylococcal enterotoxins. Staphylococcal enterotoxin D (a powerful activator of human peripheral T cells expressing V beta 5.2/5.3 receptors) caused a marked reduction of V beta 5.2/5.3 expression, as determined with the V beta-specific antibody 42/1C1. Evidence is presented that this loss of V beta 5.2/5.3 expression resulted from the selective deletion of activated thymocytes by apoptosis, in concert with T-cell receptor modulation. These effects of staphylococcal enterotoxin D were specific (since staphylococcal enterotoxin E did not influence V beta 5.2/5.3 expression) and V beta-selective (since expression of V beta 6.7 remained unaffected by staphylococcal enterotoxin D). On the basis of these observations, we suggest that thymic organ culture provides a powerful approach to study the generation of the human T-cell repertoire. Images PMID:1584760

  5. The Evolution of Mammalian Olfactory Receptor Genes

    PubMed Central

    Issel-Tarver, L.; Rine, J.

    1997-01-01

    We performed a comparative study of four subfamilies of olfactory receptor genes first identified in the dog to assess changes in the gene family during mammalian evolution, and to begin linking the dog genetic map to that of humans. The human subfamilies were localized to chromosomes 7, 11, and 19. The two subfamilies that were tightly linked in the dog genome were also tightly linked in the human genome. The four subfamilies were compared in human (primate), horse (perissodactyl), and a variety of artiodactyls and carnivores. Some changes in gene number were detected, but overall subfamily size appeared to have been established before the divergence of these mammals 60-100 million years ago. PMID:9017400

  6. Analysis of cattle olfactory subgenome: the first detail study on the characteristics of the complete olfactory receptor repertoire of a ruminant

    PubMed Central

    2013-01-01

    Background Mammalian olfactory receptors (ORs) are encoded by the largest mammalian multigene family. Understanding the OR gene repertoire in the cattle genome could lead to link the effects of genetic differences in these genes to variations in olfaction in cattle. Results We report here a whole genome analysis of the olfactory receptor genes of Bos taurus using conserved OR gene-specific motifs and known OR protein sequences from diverse species. Our analysis, using the current cattle genome assembly UMD 3.1 covering 99.9% of the cattle genome, shows that the cattle genome contains 1,071 OR-related sequences including 881 functional, 190 pseudo, and 352 partial OR sequences. The OR genes are located in 49 clusters on 26 cattle chromosomes. We classified them into 18 families consisting of 4 Class I and 14 Class II families and these were further grouped into 272 subfamilies. Comparative analyses of the OR genes of cattle, pigs, humans, mice, and dogs showed that 6.0% (n = 53) of functional OR cattle genes were species-specific. We also showed that significant copy number variations are present in the OR repertoire of the cattle from the analysis of 10 selected OR genes. Conclusion Our analysis revealed the almost complete OR gene repertoire from an individual cattle genome. Though the number of OR genes were lower than in pigs, the analysis of the genetic system of cattle ORs showed close similarities to that of the pig. PMID:24004971

  7. 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

  8. Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination

    PubMed Central

    Qi, Qian; Cavanagh, Mary M.; Le Saux, Sabine; NamKoong, Hong; Kim, Chulwoo; Turgano, Emerson; Liu, Yi; Wang, Chen; Mackey, Sally; Swan, Gary E.; Dekker, Cornelia L.; Olshen, Richard A.; Boyd, Scott D.; Weyand, Cornelia M.; Tian, Lu; Goronzy, Jörg J.

    2016-01-01

    Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood is able to escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. While all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells, but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen-reactive TCRs including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important read-out to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection. PMID:27030598

  9. tcR: an R package for T cell receptor repertoire advanced data analysis.

    PubMed

    Nazarov, Vadim I; Pogorelyy, Mikhail V; Komech, Ekaterina A; Zvyagin, Ivan V; Bolotin, Dmitry A; Shugay, Mikhail; Chudakov, Dmitry M; Lebedev, Yury B; Mamedov, Ilgar Z

    2015-05-28

    The Immunoglobulins (IG) and the T cell receptors (TR) play the key role in antigen recognition during the adaptive immune response. Recent progress in next-generation sequencing technologies has provided an opportunity for the deep T cell receptor repertoire profiling. However, a specialised software is required for the rational analysis of massive data generated by next-generation sequencing. Here we introduce tcR, a new R package, representing a platform for the advanced analysis of T cell receptor repertoires, which includes diversity measures, shared T cell receptor sequences identification, gene usage statistics computation and other widely used methods. The tool has proven its utility in recent research studies. tcR is an R package for the advanced analysis of T cell receptor repertoires after primary TR sequences extraction from raw sequencing reads. The stable version can be directly installed from The Comprehensive R Archive Network ( http://cran.r-project.org/mirrors.html ). The source code and development version are available at tcR GitHub ( http://imminfo.github.io/tcr/ ) along with the full documentation and typical usage examples.

  10. High-throughput sequencing of the T-cell receptor repertoire: pitfalls and opportunities.

    PubMed

    Heather, James M; Ismail, Mazlina; Oakes, Theres; Chain, Benny

    2017-01-10

    T-cell specificity is determined by the T-cell receptor, a heterodimeric protein coded for by an extremely diverse set of genes produced by imprecise somatic gene recombination. Massively parallel high-throughput sequencing allows millions of different T-cell receptor genes to be characterized from a single sample of blood or tissue. However, the extraordinary heterogeneity of the immune repertoire poses significant challenges for subsequent analysis of the data. We outline the major steps in processing of repertoire data, considering low-level processing of raw sequence files and high-level algorithms, which seek to extract biological or pathological information. The latest generation of bioinformatics tools allows millions of DNA sequences to be accurately and rapidly assigned to their respective variable V and J gene segments, and to reconstruct an almost error-free representation of the non-templated additions and deletions that occur. High-level processing can measure the diversity of the repertoire in different samples, quantify V and J usage and identify private and public T-cell receptors. Finally, we discuss the major challenge of linking T-cell receptor sequence to function, and specifically to antigen recognition. Sophisticated machine learning algorithms are being developed that can combine the paradoxical degeneracy and cross-reactivity of individual T-cell receptors with the specificity of the overall T-cell immune response. Computational analysis will provide the key to unlock the potential of the T-cell receptor repertoire to give insight into the fundamental biology of the adaptive immune system and to provide powerful biomarkers of disease. © The Author 2017. Published by Oxford University Press.

  11. Contrasted Evolution of the Vomeronasal Receptor Repertoires in Mammals and Squamate Reptiles

    PubMed Central

    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

  12. 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.

  13. Specificity, Privacy, and Degeneracy in the CD4 T Cell Receptor Repertoire Following Immunization

    PubMed Central

    Sun, Yuxin; Best, Katharine; Cinelli, Mattia; Heather, James M.; Reich-Zeliger, Shlomit; Shifrut, Eric; Friedman, Nir; Shawe-Taylor, John; Chain, Benny

    2017-01-01

    T cells recognize antigen using a large and diverse set of antigen-specific receptors created by a complex process of imprecise somatic cell gene rearrangements. In response to antigen-/receptor-binding-specific T cells then divide to form memory and effector populations. We apply high-throughput sequencing to investigate the global changes in T cell receptor sequences following immunization with ovalbumin (OVA) and adjuvant, to understand how adaptive immunity achieves specificity. Each immunized mouse contained a predominantly private but related set of expanded CDR3β sequences. We used machine learning to identify common patterns which distinguished repertoires from mice immunized with adjuvant with and without OVA. The CDR3β sequences were deconstructed into sets of overlapping contiguous amino acid triplets. The frequencies of these motifs were used to train the linear programming boosting (LPBoost) algorithm LPBoost to classify between TCR repertoires. LPBoost could distinguish between the two classes of repertoire with accuracies above 80%, using a small subset of triplet sequences present at defined positions along the CDR3. The results suggest a model in which such motifs confer degenerate antigen specificity in the context of a highly diverse and largely private set of T cell receptors. PMID:28450864

  14. The mammalian beta-tubulin repertoire: hematopoietic expression of a novel, heterologous beta-tubulin isotype

    PubMed Central

    1986-01-01

    We describe the structure of a novel and unusually heterologous beta- tubulin isotype (M beta 1) isolated from a mouse bone marrow cDNA library, and a second isotype (M beta 3) isolated from a mouse testis cDNA library. Comparison of M beta 1 and M beta 3 with the completed (M beta 4, M beta 5) or extended (M beta 2) sequence of three previously described beta-tubulin isotypes shows that each includes a distinctive carboxy-terminal region, in addition to multiple amino acid substitutions throughout the polypeptide chain. In every case where a mammalian interspecies comparison can be made, both the carboxy- terminal and internal amino acid substitutions that distinguish one isotype from another are absolutely conserved. We conclude that these characteristic differences are important in determining functional distinctions between different kinds of microtubule. The amino acid homologies between M beta 2, M beta 3, M beta 4, and M beta 5 are in the range of 95-97%; however the homology between M beta 1 and all the other isotypes is very much less (78%). The dramatic divergence in M beta 1 is due to multiple changes that occur throughout the polypeptide chain. The overall level of expression of M beta 1 is low, and is restricted to those tissues (bone marrow, spleen, developing liver and lung) that are active in hematopoiesis in the mouse. We predict that the M beta 1 isotype is functionally specialized for assembly into the mammalian marginal band. PMID:3782288

  15. Identification of novel mammalian caspases reveals an important role of gene loss in shaping the human caspase repertoire.

    PubMed

    Eckhart, Leopold; Ballaun, Claudia; Hermann, Marcela; VandeBerg, John L; Sipos, Wolfgang; Uthman, Aumaid; Fischer, Heinz; Tschachler, Erwin

    2008-05-01

    Proteases of the caspase family play central roles in apoptosis and inflammation. Recently, we have described a new gene encoding caspase-15 that has been inactivated independently in different mammalian lineages. To determine the dynamics of gene duplication and loss in the entire caspase gene family, we performed a comprehensive evolutionary analysis of mammalian caspases. By comparative genomics and reverse transcriptase-polymerase chain reaction analyses, we identified 3 novel mammalian caspase genes, which we tentatively named caspases-16 through -18. Caspase-16, which is most similar in sequence to caspase-14, has been conserved in marsupials and placental mammals, including humans. Caspase-17, which is most similar to caspase-3, has been conserved among fish, frog, chicken, lizard, and the platypus but is absent from marsupials and placental mammals. Caspase-18, which is most similar to caspase-8, has been conserved among chicken, platypus, and opossum but is absent from placental mammals. These gene distribution patterns suggest that, in the evolutionary lineage leading to humans, caspase-17 was lost after the split of protherian and therian mammals and caspase-18 was lost after the split of marsupials and placental mammals. In the canine genome, the number of caspases has been reduced by the fusion of the neighboring genes caspases-1 and -4, resulting in a single coding region. Further lineage-specific gene inactivations were found for caspase-10 in murine rodents and caspase-12 in humans, rabbit, and cow. Lineage-specific gene duplications were found for caspases-1, -3, and -12 in opossum and caspase-4 in primates. Other caspases were generally conserved in all mammalian species investigated. Using the positions of introns as stable characters during recent vertebrate evolution, we define 3 phylogenetic clades of caspase genes: caspases-1/-2/-4/-5/-9/-12/-14/-15/-16 (clade I), caspases-3/-6/-7/-17 (clade II), and caspases-8/-10/-18/CFLAR (clade III). We

  16. Aging affects B-cell antigen receptor repertoire diversity in primary and secondary lymphoid tissues.

    PubMed

    Tabibian-Keissar, Hilla; Hazanov, Lena; Schiby, Ginette; Rosenthal, Noemie; Rakovsky, Aviya; Michaeli, Miri; Shahaf, Gitit Lavy; Pickman, Yishai; Rosenblatt, Kinneret; Melamed, Doron; Dunn-Walters, Deborah; Mehr, Ramit; Barshack, Iris

    2016-02-01

    The elderly immune system is characterized by reduced responses to infections and vaccines, and an increase in the incidence of autoimmune diseases and cancer. Age-related deficits in the immune system may be caused by peripheral homeostatic pressures that limit bone marrow B-cell production or migration to the peripheral lymphoid tissues. Studies of peripheral blood B-cell receptor spectratypes have shown that those of the elderly are characterized by reduced diversity, which is correlated with poor health status. In the present study, we performed for the first time high-throughput sequencing of immunoglobulin genes from archived biopsy samples of primary and secondary lymphoid tissues in old (74 ± 7 years old, range 61-89) versus young (24 ± 5 years old, range 18-45) individuals, analyzed repertoire diversities and compared these to results in peripheral blood. We found reduced repertoire diversity in peripheral blood and lymph node repertoires from old people, while in the old spleen samples the diversity was larger than in the young. There were no differences in somatic hypermutation characteristics between age groups. These results support the hypothesis that age-related immune frailty stems from altered B-cell homeostasis leading to narrower memory B-cell repertoires, rather than changes in somatic hypermutation mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. T cell receptor repertoires after adoptive transfer of expanded allogeneic regulatory T cells.

    PubMed

    Theil, A; Wilhelm, C; Kuhn, M; Petzold, A; Tuve, S; Oelschlägel, U; Dahl, A; Bornhäuser, M; Bonifacio, E; Eugster, A

    2017-02-01

    Regulatory T cell (Treg ) therapy has been exploited in autoimmune disease, solid organ transplantation and in efforts to prevent or treat graft-versus-host disease (GVHD). However, our knowledge on the in-vivo persistence of transfused Treg is limited. Whether Treg transfusion leads to notable changes in the overall Treg repertoire or whether longevity of Treg in the periphery is restricted to certain clones is unknown. Here we use T cell receptor alpha chain sequencing (TCR-α-NGS) to monitor changes in the repertoire of Treg upon polyclonal expansion and after subsequent adoptive transfer. We applied TCR-α-NGS to samples from two patients with chronic GVHD who received comparable doses of stem cell donor derived expanded Treg . We found that in-vitro polyclonal expansion led to notable repertoire changes in vitro and that Treg cell therapy altered the peripheral Treg repertoire considerably towards that of the infused cell product, to different degrees, in each patient. Clonal changes in the peripheral blood were transient and correlated well with the clinical parameters. We suggest that T cell clonotype analyses using TCR sequencing should be considered as a means to monitor longevity and fate of adoptively transferred T cells.

  18. CTLA4 blockade broadens the peripheral T cell receptor repertoire

    PubMed Central

    Robert, Lidia; Tsoi, Jennifer; Wang, Xiaoyan; Emerson, Ryan; Homet, Blanca; Chodon, Thinle; Mok, Stephen; Huang, Rong Rong; Cochran, Alistair J.; Comin-Anduix, Begonya; Koya, Richard C.; Graeber, Thomas G.; Robins, Harlan; Ribas, Antoni

    2014-01-01

    Purpose To evaluate the immunomodulatory effects of CTLA-4 blockade with tremelimumab in peripheral blood mononuclear cells (PBMC). Experimental Design We used next generation sequencing to study the complementarity determining region 3 (CDR3) from the rearranged T cell receptor (TCR) variable beta (V-beta) in PBMC of 21 patients, at baseline and 30–60 days after receiving tremelimumab. Results After receiving tremelimumab there was a median of 30% increase in unique productive sequences of TCR V-beta CDR3 in 19 out of 21 patients, and a median decrease of 30% in only 2 out of 21 patients. These changes were significant for richness (p=0.01) and for Shannon index diversity (p=0.04). In comparison, serially collected PBMC from four healthy donors did not show a significant change in TCR V-beta CDR3 diversity over one year. There was a significant difference in the total unique productive TCR V-beta CDR3 sequences between patients experiencing toxicity with tremelimumab compared to patients without toxicity (p=0.05). No relevant differences were noted between clinical responders and non-responders. Conclusions CTLA4 blockade with tremelimumab diversifies the peripheral T cell pool, representing a pharmacodynamic effect of how this class of antibodies modulates the human immune system. PMID:24583799

  19. Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing.

    PubMed

    Freeman, J Douglas; Warren, René L; Webb, John R; Nelson, Brad H; Holt, Robert A

    2009-10-01

    T-cell receptor (TCR) genomic loci undergo somatic V(D)J recombination, plus the addition/subtraction of nontemplated bases at recombination junctions, in order to generate the repertoire of structurally diverse T cells necessary for antigen recognition. TCR beta subunits can be unambiguously identified by their hypervariable CDR3 (Complement Determining Region 3) sequence. This is the site of V(D)J recombination encoding the principal site of antigen contact. The complexity and dynamics of the T-cell repertoire remain unknown because the potential repertoire size has made conventional sequence analysis intractable. Here, we use 5'-RACE, Illumina sequencing, and a novel short read assembly strategy to sample CDR3(beta) diversity in human T lymphocytes from peripheral blood. Assembly of 40.5 million short reads identified 33,664 distinct TCR(beta) clonotypes and provides precise measurements of CDR3(beta) length diversity, usage of nontemplated bases, sequence convergence, and preferences for TRBV (T-cell receptor beta variable gene) and TRBJ (T-cell receptor beta joining gene) gene usage and pairing. CDR3 length between conserved residues of TRBV and TRBJ ranged from 21 to 81 nucleotides (nt). TRBV gene usage ranged from 0.01% for TRBV17 to 24.6% for TRBV20-1. TRBJ gene usage ranged from 1.6% for TRBJ2-6 to 17.2% for TRBJ2-1. We identified 1573 examples of convergence where the same amino acid translation was specified by distinct CDR3(beta) nucleotide sequences. Direct sequence-based immunoprofiling will likely prove to be a useful tool for understanding repertoire dynamics in response to immune challenge, without a priori knowledge of antigen.

  20. HIV-1 Epitope Variability Is Associated with T Cell Receptor Repertoire Instability and Breadth.

    PubMed

    Balamurugan, Arumugam; Claiborne, Deon; Ng, Hwee L; Yang, Otto O

    2017-08-15

    Mutational escape of HIV-1 from HIV-1-specific CD8(+) T lymphocytes (CTLs) is a major barrier for effective immune control. Each epitope typically is targeted by multiple clones with distinct T cell receptors (TCRs). While the clonal repertoire may be important for containing epitope variation, determinants of its composition are poorly understood. We investigate the clonal repertoire of 29 CTL responses against 23 HIV-1 epitopes longitudinally in nine chronically infected untreated subjects with plasma viremia of <3,000 RNA copies/ml over 17 to 179 weeks. The composition of TCRs targeting each epitope varied considerably in stability over time, although clonal stability (Sorensen index) was not significantly time dependent within this interval. However, TCR stability inversely correlated with epitope variability in the Los Alamos HIV-1 Sequence Database, consistent with TCR evolution being driven by epitope variation. Finally, a robust inverse correlation of TCR breadth against each epitope versus epitope variability further suggested that this variability drives TCR repertoire diversification. In the context of studies demonstrating rapidly shifting HIV-1 sequences in vivo, our findings support a variably dynamic process of shifting CTL clonality lagging in tandem with viral evolution and suggest that preventing escape of HIV-1 may require coordinated direction of the CTL clonal repertoire to simultaneously block escape pathways.IMPORTANCE Mutational escape of HIV-1 from HIV-1-specific CD8(+) T lymphocytes (CTLs) is a major barrier to effective immune control. The number of distinct CTL clones targeting each epitope is proposed to be an important factor, but the determinants are poorly understood. Here, we demonstrate that the clonal stability and number of clones for the CTL response against an epitope are inversely associated with the general variability of the epitope. These results show that CTLs constantly lag epitope mutation, suggesting that preventing HIV

  1. Evaluation of the Antigen-Experienced B-Cell Receptor Repertoire in Healthy Children and Adults

    PubMed Central

    IJspeert, Hanna; van Schouwenburg, Pauline A.; van Zessen, David; Pico-Knijnenburg, Ingrid; Driessen, Gertjan J.; Stubbs, Andrew P.; van der Burg, Mirjam

    2016-01-01

    Upon antigen recognition via their B cell receptor (BR), B cells migrate to the germinal center where they undergo somatic hypermutation (SHM) to increase their affinity for the antigen, and class switch recombination (CSR) to change the effector function of the secreted antibodies. These steps are essential to create an antigen-experienced BR repertoire that efficiently protects the body against pathogens. At the same time, the BR repertoire should be selected to protect against responses to self-antigen or harmless antigens. Insights into the processes of SHM, selection, and CSR can be obtained by studying the antigen-experienced BR repertoire. Currently, a large reference data set of healthy children and adults, which ranges from neonates to the elderly, is not available. In this study, we analyzed the antigen-experienced repertoire of 38 healthy donors (HD), ranging from cord blood to 74 years old, by sequencing IGA and IGG transcripts using next generation sequencing. This resulted in a large, freely available reference data set containing 412,890 IGA and IGG transcripts. We used this data set to study mutation levels, SHM patterns, antigenic selection, and CSR from birth to elderly HD. Only small differences were observed in SHM patterns, while the mutation levels increase in early childhood and stabilize at 6 years of age at around 7%. Furthermore, comparison of the antigen-experienced repertoire with sequences from the naive immune repertoire showed that features associated with autoimmunity such as long CDR3 length and IGHV4-34 usage are reduced in the antigen-experienced repertoire. Moreover, IGA2 and IGG2 usage was increased in HD in higher age categories, while IGG1 usage was decreased. In addition, we studied clonal relationship in the different samples. Clonally related sequences were found with different subclasses. Interestingly, we found transcripts with the same CDR1–CDR3 sequence, but different subclasses. Together, these data suggest that

  2. 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

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

    DTIC Science & Technology

    2015-09-02

    distribution is unlimited. Aldehyde Recognition and Discrimination by Mammalian Odorant Receptors via Functional Group-Specific Hydration Chemistry The...Recognition and Discrimination by Mammalian Odorant Receptors via Functional Group-Specific Hydration Chemistry Report Title The mammalian odorant receptors...octanal. Aldehyde Recognition and Discrimination by Mammalian Odorant Receptors via Functional Group-Specific Hydration Chemistry Approved for public

  4. 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.

  5. Genomic variation in the vomeronasal receptor gene repertoires of inbred mice

    PubMed Central

    2012-01-01

    Background Vomeronasal receptors (VRs), expressed in sensory neurons of the vomeronasal organ, are thought to bind pheromones and mediate innate behaviours. The mouse reference genome has over 360 functional VRs arranged in highly homologous clusters, but the vast majority are of unknown function. Differences in these receptors within and between closely related species of mice are likely to underpin a range of behavioural responses. To investigate these differences, we interrogated the VR gene repertoire from 17 inbred strains of mice using massively parallel sequencing. Results Approximately half of the 6222 VR genes that we investigated could be successfully resolved, and those that were unambiguously mapped resulted in an extremely accurate dataset. Collectively VRs have over twice the coding sequence variation of the genome average; but we identify striking non-random distribution of these variants within and between genes, clusters, clades and functional classes of VRs. We show that functional VR gene repertoires differ considerably between different Mus subspecies and species, suggesting these receptors may play a role in mediating behavioural adaptations. Finally, we provide evidence that widely-used, highly inbred laboratory-derived strains have a greatly reduced, but not entirely redundant capacity for differential pheromone-mediated behaviours. Conclusions Together our results suggest that the unusually variable VR repertoires of mice have a significant role in encoding differences in olfactory-mediated responses and behaviours. Our dataset has expanded over nine fold the known number of mouse VR alleles, and will enable mechanistic analyses into the genetics of innate behavioural differences in mice. PMID:22908939

  6. The truncated TrkB receptor influences mammalian sleep

    PubMed Central

    Watson, Adam J.; Henson, Kyle; Dorsey, Susan G.

    2014-01-01

    Brain-derived neurotrophic factor (BDNF) is a neurotrophin hypothesized to play an important role in mammalian sleep expression and regulation. In order to investigate the role of the truncated receptor for BDNF, TrkB.T1, in mammalian sleep, we examined sleep architecture and sleep regulation in adult mice constitutively lacking this receptor. We find that TrkB.T1 knockout mice have increased REM sleep time, reduced REM sleep latency, and reduced sleep continuity. These results demonstrate a novel role for the TrkB.T1 receptor in sleep expression and provide new insights into the relationship between BDNF, psychiatric illness, and sleep. PMID:25502751

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

    USDA-ARS?s Scientific Manuscript database

    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...

  8. A Large Repertoire of Parasite Epitopes Matched by a Large Repertoire of Host Immune Receptors in an Invertebrate Host/Parasite Model

    PubMed Central

    Moné, Yves; Gourbal, Benjamin; Duval, David; Du Pasquier, Louis; Kieffer-Jaquinod, Sylvie; Mitta, Guillaume

    2010-01-01

    For many decades, invertebrate immunity was believed to be non-adaptive, poorly specific, relying exclusively on sometimes multiple but germ-line encoded innate receptors and effectors. But recent studies performed in different invertebrate species have shaken this paradigm by providing evidence for various types of somatic adaptations at the level of putative immune receptors leading to an enlarged repertoire of recognition molecules. Fibrinogen Related Proteins (FREPs) from the mollusc Biomphalaria glabrata are an example of these putative immune receptors. They are known to be involved in reactions against trematode parasites. Following not yet well understood somatic mechanisms, the FREP repertoire varies considerably from one snail to another, showing a trend towards an individualization of the putative immune repertoire almost comparable to that described from vertebrate adaptive immune system. Nevertheless, their antigenic targets remain unknown. In this study, we show that a specific set of these highly variable FREPs from B. glabrata forms complexes with similarly highly polymorphic and individually variable mucin molecules from its specific trematode parasite S. mansoni (Schistosoma mansoni Polymorphic Mucins: SmPoMucs). This is the first evidence of the interaction between diversified immune receptors and antigenic variant in an invertebrate host/pathogen model. The same order of magnitude in the diversity of the parasite epitopes and the one of the FREP suggests co-evolutionary dynamics between host and parasite regarding this set of determinants that could explain population features like the compatibility polymorphism observed in B. glabrata/S. mansoni interaction. In addition, we identified a third partner associated with the FREPs/SmPoMucs in the immune complex: a Thioester containing Protein (TEP) belonging to a molecular category that plays a role in phagocytosis or encapsulation following recognition. The presence of this last partner in this

  9. Localization of mineralocorticoid receptors at mammalian synapses.

    PubMed

    Prager, Eric M; Brielmaier, Jennifer; Bergstrom, Hadley C; McGuire, Jennifer; Johnson, Luke R

    2010-12-15

    In the brain, membrane associated nongenomic steroid receptors can induce fast-acting responses to ion conductance and second messenger systems of neurons. Emerging data suggest that membrane associated glucocorticoid and mineralocorticoid receptors may directly regulate synaptic excitability during times of stress when adrenal hormones are elevated. As the key neuron signaling interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. The lateral amygdala is a key site for synaptic plasticity underlying conditioned fear, which can both trigger and be coincident with the stress response. A large body of electrophysiological data shows rapid regulation of neuronal excitability by steroid hormone receptors. Despite the importance of these receptors, to date, only the glucocorticoid receptor has been anatomically localized to the membrane. We investigated the subcellular sites of mineralocorticoid receptors in the lateral amygdala of the Sprague-Dawley rat. Immunoblot analysis revealed the presence of mineralocorticoid receptors in the amygdala. Using electron microscopy, we found mineralocorticoid receptors expressed at both nuclear including: glutamatergic and GABAergic neurons and extra nuclear sites including: presynaptic terminals, neuronal dendrites, and dendritic spines. Importantly we also observed mineralocorticoid receptors at postsynaptic membrane densities of excitatory synapses. These data provide direct anatomical evidence supporting the concept that, at some synapses, synaptic transmission is regulated by mineralocorticoid receptors. Thus part of the stress signaling response in the brain is a direct modulation of the synapse itself by adrenal steroids.

  10. The anatomy of mammalian sweet taste receptors.

    PubMed

    Chéron, Jean-Baptiste; Golebiowski, Jérôme; Antonczak, Serge; Fiorucci, Sébastien

    2017-02-01

    All sweet-tasting compounds are detected by a single G-protein coupled receptor (GPCR), the heterodimer T1R2-T1R3, for which no experimental structure is available. The sweet taste receptor is a class C GPCR, and the recently published crystallographic structures of metabotropic glutamate receptor (mGluR) 1 and 5 provide a significant step forward for understanding structure-function relationships within this family. In this article, we recapitulate more than 600 single point site-directed mutations and available structural data to obtain a critical alignment of the sweet taste receptor sequences with respect to other class C GPCRs. Using this alignment, a homology 3D-model of the human sweet taste receptor is built and analyzed to dissect out the role of key residues involved in ligand binding and those responsible for receptor activation. Proteins 2017; 85:332-341. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Characterization of the T cell receptor repertoire causing collagen arthritis in mice

    PubMed Central

    1993-01-01

    Collagen type II-induced arthritis (CIA) is generated in susceptible rodent strains by intradermal injections of homologous or heterologous native type II collagen in complete Freund's adjuvant. Symptoms of CIA are analogous to those of the human autoimmune disease, rheumatoid arthritis. CIA is a model system for T cell-mediated autoimmune disease. To study the T cell receptor (TCR) repertoire of bovine type II-specific T cells that may be involved in the pathogenesis of CIA in DBA/1Lac.J (H-2q) mice, 13 clonally distinct T cell hybridomas specific for bovine type II collagen have been established and the alpha and beta chains of their TCRs have been analyzed. These T cell hybridomas recognize epitopes that are shared by type II collagens from distinct species and not by type I collagens, and exhibit a highly restricted TCR-alpha/beta repertoire. The alpha chains of the TCRs employ three V alpha gene subfamilies (V alpha 11, V alpha 8, and V alpha 22) and four J alpha gene segments (J alpha 42, J alpha 24, J alpha 37, and J alpha 32). The V alpha 22 is a newly identified subfamily consisting of approximately four to six members, and exhibits a high degree of polymorphism among four mouse strains of distinct V alpha haplotypes. In addition, the beta chains of the TCRs employ three V beta gene subfamilies (V beta 8, V beta 1, and V beta 6), however the V beta 8.2 gene segment is preferentially utilized (58.3%). In contrast, the J beta gene segment usage is more heterogeneous. On the basis of the highly limited TCR-alpha/beta repertoire of the TCRs of the panel of bovine type II-specific T cell hybrid clones, a significant reduction (60%) of the incidence of arthritis in DBA/1Lac.J mice is accomplished by the use of anti-V beta 8.2 antibody therapy. PMID:8381155

  12. Statistical inference of the generation probability of T-cell receptors from sequence repertoires.

    PubMed

    Murugan, Anand; Mora, Thierry; Walczak, Aleksandra M; Callan, Curtis G

    2012-10-02

    Stochastic rearrangement of germline V-, D-, and J-genes to create variable coding sequence for certain cell surface receptors is at the origin of immune system diversity. This process, known as "VDJ recombination", is implemented via a series of stochastic molecular events involving gene choices and random nucleotide insertions between, and deletions from, genes. We use large sequence repertoires of the variable CDR3 region of human CD4+ T-cell receptor beta chains to infer the statistical properties of these basic biochemical events. Because any given CDR3 sequence can be produced in multiple ways, the probability distribution of hidden recombination events cannot be inferred directly from the observed sequences; we therefore develop a maximum likelihood inference method to achieve this end. To separate the properties of the molecular rearrangement mechanism from the effects of selection, we focus on nonproductive CDR3 sequences in T-cell DNA. We infer the joint distribution of the various generative events that occur when a new T-cell receptor gene is created. We find a rich picture of correlation (and absence thereof), providing insight into the molecular mechanisms involved. The generative event statistics are consistent between individuals, suggesting a universal biochemical process. Our probabilistic model predicts the generation probability of any specific CDR3 sequence by the primitive recombination process, allowing us to quantify the potential diversity of the T-cell repertoire and to understand why some sequences are shared between individuals. We argue that the use of formal statistical inference methods, of the kind presented in this paper, will be essential for quantitative understanding of the generation and evolution of diversity in the adaptive immune system.

  13. Receptor heteromerization expands the repertoire of cannabinoid signaling in rodent neurons.

    PubMed

    Rozenfeld, Raphael; Bushlin, Ittai; Gomes, Ivone; Tzavaras, Nikos; Gupta, Achla; Neves, Susana; Battini, Lorenzo; Gusella, G Luca; Lachmann, Alexander; Ma'ayan, Avi; Blitzer, Robert D; Devi, Lakshmi A

    2012-01-01

    A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB₁R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB₁R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB₁R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB₁R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB₁R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB₁R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB₁R desensitization. Additionally, presence of DOR facilitates signaling via a new CB₁R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB₁R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.

  14. 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

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

    PubMed

    Wang, Kai; Zhao, Huabin

    2015-09-04

    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. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  16. High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire.

    PubMed

    Hou, Xianliang; Lu, Chong; Chen, Sisi; Xie, Qian; Cui, Guangying; Chen, Jianing; Chen, Zhi; Wu, Zhongwen; Ding, Yulong; Ye, Ping; Dai, Yong; Diao, Hongyan

    2016-03-01

    The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals.Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR.Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides.Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination.

  17. High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire

    PubMed Central

    Hou, Xianliang; Lu, Chong; Chen, Sisi; Xie, Qian; Cui, Guangying; Chen, Jianing; Chen, Zhi; Wu, Zhongwen; Ding, Yulong; Ye, Ping; Dai, Yong; Diao, Hongyan

    2016-01-01

    Abstract The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals. Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR. Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides. Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination. PMID:26962778

  18. Glycosaminoglycan receptors facilitate infection of mammalian cells

    USDA-ARS?s Scientific Manuscript database

    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. Localization of Mineralocorticoid Receptors at Mammalian Synapses

    DTIC Science & Technology

    2010-12-15

    receptor balance in health and disease. Endocr Rev 19: 269–301. 58. Patel PD, Lopez JF, Lyons DM, Burke S, Wallace M, et al. (2000) Glucocorticoid and...Corticosterone alters AMPAR mobility and facilitates bidirectional synaptic plasticity. PLoS One 4: e4714. 75. Roozendaal B, Hernandez A, Cabrera SM, Hagewoud R

  20. T cell receptor repertoire in the peripheral blood and intestinal mucosa of coeliac patients.

    PubMed Central

    Lahat, N; Ben-Nun, A; Cohen, L; Kinarty, A; Lerner, A

    1995-01-01

    The alpha beta and gamma delta T cell receptor (TCR) repertoire in the peripheral blood and intestinal mucosa of six coeliac and six age-matched controls was analysed by reverse transcription and polymerase chain reaction (PCR). No TCR alpha and gamma delta restriction was observed in coeliacs and controls. However, V gamma 3 was expressed only in coeliac peripheral and intestinal T cells. V delta 2 was strongly expressed in coeliacs and scarcely transcribed in control cells. The unique expression of these gamma delta TCR in coeliac patients suggests that V gamma 3 and perhaps V delta 2 TCR-bearing lymphocytes may play a role in the pathogenesis of coeliac disease. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7664488

  1. The heterogeneous allelic repertoire of human toll-like receptor (TLR) genes.

    PubMed

    Georgel, Philippe; Macquin, Cécile; Bahram, Seiamak

    2009-11-17

    Toll-Like Receptors (TLR) are critical elements of the innate arm of the vertebrate immune system. They constitute a multigenic family of receptors which collectively bind a diverse array of--exogeneous as well as endogeneous--ligands. An exponential burst of knowledge has defined their biological role in fight against infections and generation/modulation of auto-immune disorders. Hence, they could at least be conceptually recognized--despite being structurally unrelated - as innate counterparts to Major Histocompatibility Complex (MHC) molecules--equally recognizing antigenic ligands (albeit structurally more homogeneous i.e., peptides), again derived from self and/or non-self sources--preeminent this time in adaptive immunity. Our great disparities in face of infections and/or susceptibility to auto-immune diseases have provoked an intense search for genetic explanations, in part satisfied by the extraordinary MHC allelic repertoire. An equally in-depth and systematic analysis of TLR diversity is lacking despite numerous independent reports of a growing number of SNPs within these loci. The work described here aims at providing a preliminary picture of the allelic repertoire--and not purely SNPs--of all 10 human TLR coding sequences (with exception of TLR3) within a single cohort of up to 100 individuals. It appears from our work that TLR are unequally polymorphic: TLR2 (DNA alleles: 7/protein alleles: 3), 4 (4/3), 7 (6/3), 8 (9/2) and 9 (8/3) being comparatively least diverse whereas TLR1 (11/10), 5 (14/12), 6 (10/8) and 10 (15/10) show a substantial number of alleles. In addition to allelic assignment of a large number of SNPs, 10 new polymorphic positions were hereby identified. Hence this work depicts a first overview of the diversity of almost all human TLR genes, a prelude for large-scale population genetics as well as genetic association studies.

  2. 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.

  3. Receptor guanylyl cyclases in mammalian olfactory function

    PubMed Central

    Zufall, Frank; Munger, Steven D.

    2009-01-01

    The contributions of guanylyl cyclases to sensory signaling in the olfactory system have been unclear. Recently, studies of a specialized subpopulation of olfactory sensory neurons (OSNs) located in the main olfactory epithelium have provided important insights into the neuronal function of one receptor guanylyl cyclase, GC-D. Mice expressing reporters such as β-galactosidase and green fluorescent protein in OSNs that normally express GC-D have allowed investigators to identify these neurons in situ, facilitating anatomical and physiological studies of this sparse neuronal population. The specific perturbation of GC-D function in vivo has helped to resolve the role of this guanylyl cyclase in the transduction of olfactory stimuli. Similar approaches could be useful for the study of the orphan receptor GC-G, which is expressed in another distinct subpopulation of sensory neurons located in the Grueneberg ganglion. In this review, we discuss key findings that have reinvigorated the study of guanylyl cyclase function in the olfactory system. PMID:19941039

  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.

  5. Assembly-based inference of B-cell receptor repertoires from short read RNA sequencing data with V’DJer

    PubMed Central

    Mose, Lisle E.; Selitsky, Sara R.; Bixby, Lisa M.; Marron, David L.; Iglesia, Michael D.; Serody, Jonathan S.; Perou, Charles M.; Vincent, Benjamin G.; Parker, Joel S.

    2016-01-01

    Motivation: B-cell receptor (BCR) repertoire profiling is an important tool for understanding the biology of diverse immunologic processes. Current methods for analyzing adaptive immune receptor repertoires depend upon PCR amplification of VDJ rearrangements followed by long read amplicon sequencing spanning the VDJ junctions. While this approach has proven to be effective, it is frequently not feasible due to cost or limited sample material. Additionally, there are many existing datasets where short-read RNA sequencing data are available but PCR amplified BCR data are not. Results: We present here V’DJer, an assembly-based method that reconstructs adaptive immune receptor repertoires from short-read RNA sequencing data. This method captures expressed BCR loci from a standard RNA-seq assay. We applied this method to 473 Melanoma samples from The Cancer Genome Atlas and demonstrate V’DJer’s ability to accurately reconstruct BCR repertoires from short read mRNA-seq data. Availability and Implementation: V’DJer is implemented in C/C ++, freely available for academic use and can be downloaded from Github: https://github.com/mozack/vdjer Contact: benjamin_vincent@med.unc.edu or parkerjs@email.unc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27559159

  6. Structure of a mammalian ryanodine receptor.

    PubMed

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

    2015-01-01

    Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca(2+)) 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-megadalton complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle electron cryomicroscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3,757 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 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 Ca(2+).

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

    PubMed Central

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

    1992-01-01

    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

  9. Bioactivity of the putative apelin proprotein expands the repertoire of apelin receptor ligands.

    PubMed

    Shin, Kyungsoo; Chapman, Nigel A; Sarker, Muzaddid; Kenward, Calem; Huang, Shuya K; Weatherbee-Martin, Nathan; Pandey, Aditya; Dupré, Denis J; Rainey, Jan K

    2017-08-01

    Apelin is a peptide ligand for a class A G-protein coupled receptor called the apelin receptor (AR or APJ) that regulates angiogenesis, the adipoinsular axis, and cardiovascular functions. Apelin has been shown to be bioactive as 13, 17, and 36 amino acid isoforms, C-terminal fragments of the putatively inactive 55-residue proprotein (proapelin or apelin-55). Although intracellular proprotein processing has been proposed, isolation of apelin-55 from colostrum and milk demonstrates potential for secretion prior to processing and the possibility of proapelin-AR interaction. Apelin isoform activity and potency were compared by an In-Cell Western™ assay for ERK phosphorylation using a stably AR-transfected HEK293A cell line. Conformational comparison of apelin isoforms was carried out by circular dichroism and heteronuclear solution-state nuclear magnetic resonance spectroscopy. Apelin-55 is shown to activate the AR, with similar maximum ERK phophorylation response and potency to the shorter isoforms except for apelin-13, which exhibited a greater potency. Correlating to this shared activity, highly similar conformations are exhibited in all apelin isoforms for the shared C-terminal region responsible for receptor binding and activation. AR activation by all apelin isoforms likely hinges upon shared conformation and dynamics in the C-terminus, with apelin-55 providing an alternative bioactive isoform despite the addition of 19N-terminal residues relative to apelin-36. Beyond providing novel insight into the physiology of this system, re-annotation of proapelin to the bioactive apelin-55 isoform adds to the molecular toolkit for dissection of apelin-AR interactions and expands the repertoire of therapeutic targets for the apelinergic system. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Deep Sequencing of the T-cell Receptor Repertoire Demonstrates Polyclonal T-cell Infiltrates in Psoriasis

    PubMed Central

    Harden, Jamie L.; Hamm, David; Gulati, Nicholas; Lowes, Michelle A.; Krueger, James G.

    2015-01-01

    It is well known that infiltration of pathogenic T-cells plays an important role in psoriasis pathogenesis. However, the antigen specificity of these activated T-cells is relatively unknown. Previous studies using T-cell receptor polymerase chain reaction technology (TCR-PCR) have suggested there are expanded T-cell receptor (TCR) clones in psoriatic skin, suggesting a response to an unknown psoriatic antigen. Here we describe the results of high-throughput deep sequencing of the entire αβ- and γδ- TCR repertoire in normal healthy skin and psoriatic lesional and non-lesional skin. From this study, we were able to determine that there is a significant increase in the abundance of unique β- and γ- TCR sequences in psoriatic lesional skin compared to non-lesional and normal skin, and that the entire T-cell repertoire in psoriasis is polyclonal, with similar diversity to normal and non-lesional skin. Comparison of the αβ- and γδ- TCR repertoire in paired non-lesional and lesional samples showed many common clones within a patient, and these close were often equally abundant in non-lesional and lesional skin, again suggesting a diverse T-cell repertoire. Although there were similar (and low) amounts of shared β-chain sequences between different patient samples, there was significantly increased sequence sharing of the γ-chain in psoriatic skin from different individuals compared to those without psoriasis. This suggests that although the T-cell response in psoriasis is highly polyclonal, particular γδ- T-cell subsets may be associated with this disease. Overall, our findings present the feasibility of this technology to determine the entire αβ- and γδ- T-cell repertoire in skin, and that psoriasis contains polyclonal and diverse αβ- and γδ- T-cell populations. PMID:26594339

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

    PubMed

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

    2016-06-03

    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. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  12. 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

  13. Identification of Complete Repertoire of Apis florea Odorant Receptors Reveals Complex Orthologous Relationships with Apis mellifera.

    PubMed

    Karpe, Snehal D; Jain, Rikesh; Brockmann, Axel; Sowdhamini, Ramanathan

    2016-09-26

    We developed a computational pipeline for homology based identification of the complete repertoire of olfactory receptor (OR) genes in the Asian honey bee species, Apis florea Apis florea is phylogenetically the most basal honey bee species and also the most distant sister species to the Western honey bee Apis mellifera, for which all OR genes had been identified before. Using our pipeline, we identified 180 OR genes in A. florea, which is very similar to the number of ORs identified in A. mellifera (177 ORs). Many characteristics of the ORs including gene structure, synteny of tandemly repeated ORs and basic phylogenetic clustering are highly conserved. The composite phylogenetic tree of A. florea and A. mellifera ORs could be divided into 21 clades which are in harmony with the existing Hymenopteran tree. However, we found a few nonorthologous OR relationships between both species as well as independent pseudogenization of ORs suggesting separate evolutionary changes. Particularly, a subgroup of the OR gene clade XI, which had been hypothesized to code cuticular hydrocarbon receptors showed a high number of species-specific ORs RNAseq analysis detected a total number of 145 OR transcripts in male and 162 in female antennae. Most of the OR genes were highly expressed on the female antennae. However, we detected five distinct male-biased OR genes, out of which three genes (AfOr11, AfOr18, AfOr170P) were shown to be male-biased in A. mellifera, too, thus corroborating a behavioral function in sex-pheromone communication.

  14. 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.

  15. Murinization of Internalin Extends Its Receptor Repertoire, Altering Listeria monocytogenes Cell Tropism and Host Responses

    PubMed Central

    Tsai, Yu-Huan; Disson, Olivier; Bierne, Hélène; Lecuit, Marc

    2013-01-01

    Listeria monocytogenes (Lm) is an invasive foodborne pathogen that leads to severe central nervous system and maternal-fetal infections. Lm ability to actively cross the intestinal barrier is one of its key pathogenic properties. Lm crosses the intestinal epithelium upon the interaction of its surface protein internalin (InlA) with its host receptor E-cadherin (Ecad). InlA-Ecad interaction is species-specific, does not occur in wild-type mice, but does in transgenic mice expressing human Ecad and knock-in mice expressing humanized mouse Ecad. To study listeriosis in wild-type mice, InlA has been “murinized” to interact with mouse Ecad. Here, we demonstrate that, unexpectedly, murinized InlA (InlAm) mediates not only Ecad-dependent internalization, but also N-cadherin-dependent internalization. Consequently, InlAm-expressing Lm targets not only goblet cells expressing luminally-accessible Ecad, as does Lm in humanized mice, but also targets villous M cells, which express luminally-accessible N-cadherin. This aberrant Lm portal of entry results in enhanced innate immune responses and intestinal barrier damage, both of which are not observed in wild-type Lm-infected humanized mice. Murinization of InlA therefore not only extends the host range of Lm, but also broadens its receptor repertoire, providing Lm with artifactual pathogenic properties. These results challenge the relevance of using InlAm-expressing Lm to study human listeriosis and in vivo host responses to this human pathogen. PMID:23737746

  16. 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

  17. 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.

  18. Low-dose radiation accelerates aging of the T-cell receptor repertoire in CBA/Ca mice.

    PubMed

    Candéias, Serge M; Mika, Justyna; Finnon, Paul; Verbiest, Tom; Finnon, Rosemary; Brown, Natalie; Bouffler, Simon; Polanska, Joanna; Badie, Christophe

    2017-06-30

    While the biological effects of high-dose-ionizing radiation on human health are well characterized, the consequences of low-dose radiation exposure remain poorly defined, even though they are of major importance for radiological protection. Lymphocytes are very radiosensitive, and radiation-induced health effects may result from immune cell loss and/or immune system impairment. To decipher the mechanisms of effects of low doses, we analyzed the modulation of the T-cell receptor gene repertoire in mice exposed to a single low (0.1 Gy) or high (1 Gy) dose of radiation. High-throughput T-cell receptor gene profiling was used to visualize T-lymphocyte dynamics over time in control and irradiated mice. Radiation exposure induces "aging-like" effects on the T-cell receptor gene repertoire, detectable as early as 1 month post-exposure and for at least 6 months. Surprisingly, these effects are more pronounced in animals exposed to 0.1 Gy than to 1 Gy, where partial correction occurs over time. Importantly, we found that low-dose radiation effects are partially due to the hematopoietic stem cell impairment. Collectively, our findings show that acute low-dose radiation exposure specifically results in long-term alterations of the T-lymphocyte repertoire.

  19. T-cell receptor repertoire variation may be associated with type 2 diabetes mellitus in humans.

    PubMed

    Frankl, Joseph A; Thearle, Marie S; Desmarais, Cindy; Bogardus, Clifton; Krakoff, Jonathan

    2016-03-01

    Recent work in Pima Indians, a population with high rates of obesity and type 2 diabetes mellitus (T2DM), demonstrated that human leukocyte antigen haplotype DRB1*02 carriers have an increased acute insulin response and decreased risk for the development of T2DM, implicating loss of self-tolerance in the pathogenesis of T2DM. Advances in genomic sequencing have made T-cell receptor repertoire analysis a practical mode of investigation. High-throughput sequencing of T-cell receptor complementarity-determining region 3 was carried out in male Pima Indians with normal glucose regulation (n = 11; age = 31 ± 8 years; %fat = 30.2 ± 8.7%) and the protective DRB1*02 haplotype versus those with T2DM without DRB1*02 (n = 7; age = 34 ± 8 years; %fat = 31.2 ± 4.7%). Findings were partially replicated in another cohort by assessing the predictive ability of T-cell receptor variation on risk of T2DM in Pima Indian men (n = 27; age = 28.9 ± 7.1 years; %fat = 28.8 ± 7.1%) and women (n = 20; age = 29 ± 7.0 years; %fat = 37.1 ± 6.8%) with baseline normal glucose regulation but without the protective haplotype who were invited to follow-up examinations as frequently as every 2 years where diabetes status was assessed by a 75-g oral glucose tolerance test. Of these subjects, 13 developed diabetes. T-cell receptor complementarity-determining region 3 length was shorter in those with T2DM, and a one-nucleotide decrease in complementarity-determining region 3 length was associated with a nearly threefold increase in risk for future diabetes. The frequency of one variable gene, TRBV7-8, was higher in those with T2DM. A 1% increase in TRBV7-8 frequency was associated with a greater than threefold increase in diabetes risk. These results indicate that T-cell autoimmunity may be an important component in progression to T2DM in Pima Indians. Copyright © 2015 John Wiley & Sons, Ltd.

  20. 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

  1. Active NMDA glutamate receptors are expressed by mammalian osteoclasts

    PubMed Central

    Espinosa, Leon; Itzstein, Cécile; Cheynel, Hervé; Delmas, Pierre D; Chenu, Chantal

    1999-01-01

    The N-methyl-D-aspartate (NMDA) glutamate receptor, widely distributed in the mammalian nervous system, has recently been identified in bone. In this study, we have investigated whether NMDA receptors expressed by osteoclasts have an electrophysiological activity. Using the patch clamp technique two agonists of the NMDA receptor, L-glutamate (Glu) and NMDA, were shown to activate whole-cell currents recorded in isolated rabbit osteoclasts. The current-voltage (I-V) relationships of the currents induced by Glu (IGlu) and NMDA (INMDA) were studied using Mg2+-free solutions. The agonist-induced currents had a linear I-V relationship with a reversal potential near 0 mV, as expected for a voltage independent and non-selective cationic current. IGlu and INMDA were sensitive to specific blockers of NMDA subtype glutamate receptors, such as magnesium ions, (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten -5,10-imine (MK-801) and 1-(1,2-diphenylethyl) piperidine (DEP). The block of IGlu and INMDA by these specific antagonists was voltage dependent, strong for negative potentials (inward current) and absent for positive potentials (outward current). These results demonstrate that NMDA receptors are functional in rabbit osteoclasts, and that their electrophysiological and pharmacological properties in these cells are similar to those documented for neuronal cells. Active NMDA receptors expressed by osteoclasts may represent a new target for regulating bone resorption. PMID:10373688

  2. Killer immunoglobulin-like receptor repertoire analysis in a Caucasian Spanish cohort with inflammatory bowel disease.

    PubMed

    López-Hernández, Ruth; Campillo, Jose A; Legaz, Isabel; Valdés, Mariano; Salama, Hortensia; Boix, Francisco; Hernández-Martínez, A M; Eguia, Jorge; González-Martínez, G; Moya-Quiles, Maria R; Minguela, Alfredo; García-Alonso, Ana; Carballo, Fernando; Muro, Manuel

    2016-11-01

    Immunological molecules are implicated in inflammatory disorders, including inflammatory bowel disease (IBD; Crohn disease [CD] and ulcerative colitis [UC]). Killer cell immunoglobulin-like receptors (KIRs) are also genetically variable proteins involved in immune function. They are expressed by NK cells and certain T lymphocytes, regulate specificity and function by interaction with HLA Class I molecules, may be either inhibitory or activating and are polymorphic both in terms of alleles and haplotype gene content. Genetic associations between activating KIRs and certain autoimmune and inflammatory diseases have been reported; however, a possible association between KIR and IBD remains unclear. The aim of this study was to determine the relationship between KIR repertoire and IBD pathologies in a Spanish cohort. KIR variability was analyzed using PCR-sequence specific oligonucleotide probes (SSOP). Inhibitory KIR2DL5 was found more frequently in UC and IBD patient groups than in healthy controls (P = 0.028 and P = 0.01, respectively), as was activating KIR2DS1 (P = 0.02, Pc > 0.05, UC vs. Controls; P = 0.001, Pc = 0.01, IBD vs Controls; P = 0.01, Pc > 0.05, Controls vs CR), KIR2DS5 (P = 0.0028, Pc = 0.04, Controls vs UC; P = 0.0001, Pc = 0.0017, Controls vs IBD; P = 0.01, Pc > 0.05, Controls vs CD) and KIR3DS1 (P = 0.012, Pc > 0.05, Controls vs IBD). Our data suggest that imbalance between activating and inhibitory KIR may partially explain the different pathogeneses of these IBDs and that there is a hypothetical role for the telomeric B region (which contains both KIR2DS5 and KIR2DS1) in these diseases.

  3. 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

  4. 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

    2016-04-29

    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.

  5. Odorant-stimulated phosphoinositide signaling in mammalian olfactory receptor neurons

    PubMed Central

    Klasen, K.; Corey, E.A.; Kuck, F.; Wetzel, C.H.; Hatt, H.; Ache, B.W.

    2009-01-01

    Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase Cδ1 (PLCδ1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P2/IP3 and PI(3,4,5)P3, the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2 sec of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction. PMID:19781634

  6. High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

    PubMed Central

    Trimmer, Casey; Snyder, Lindsey L.; Mainland, Joel D.

    2014-01-01

    Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system. PMID:24961834

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

    PubMed Central

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

    2008-01-01

    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 γ-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

  8. 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.

  9. 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.

  10. 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.

  11. NK cell killer Ig-like receptor repertoire acquisition and maturation are strongly modulated by HLA class I molecules.

    PubMed

    Sleiman, Marwan; Brons, Nicolaas H C; Kaoma, Tony; Dogu, Figen; Villa-Forte, Alexandra; Lenoble, Patrick; Hentges, François; Kotsch, Katja; Gadola, Stephan D; Vilches, Carlos; Zimmer, Jacques

    2014-03-15

    The interaction between clonally distributed inhibitory receptors and their activating counterparts on NK cells and HLA class I molecules defines NK cell functions, but the role of HLA class I ligands in the acquisition of their receptors during NK development is still unclear. Although some studies demonstrated that HLA-C affects the expression of killer Ig-like receptors (KIR), other studies showed that NK cells acquire their KIR repertoire in a stochastic manner. Only when infected with human CMV is an expansion of self-specific KIR(+) NKG2C(+) NK cells detected. To gain more insight into this question, we compared the coexpression of different KIR molecules, NKG2A, CD8, and CD57, on NK cells in healthy donors and seven patients with deficient HLA class I expression due to mutations in one of the TAP genes. Our results show a correlation between the presence/absence of HLA class I molecules and the coexpression of their receptors. In an HLA class I low-expression context, an increase in KIR molecules' coexpression is detected on the NKG2A(+) CD8(+) subset. In functional assays, hyporesponsiveness was observed for TAP-deficient NK cells derived from four patients. In contrast, NK cells from patient five were functional, whereas CD107a(+) and IFN-γ(+) CD56(dim) NK cells presented a different pattern of HLA class I receptors compared with healthy donors. Taken together, our results provide strong evidence for the role of HLA class I molecules in NK cell maturation and KIR repertoire acquisition.

  12. Mammalian Nicotinic Acetylcholine Receptors: From Structure to Function

    PubMed Central

    Albuquerque, Edson X.; Pereira, Edna F. R.; Alkondon, Manickavasagom; Rogers, Scott W.

    2009-01-01

    The classical studies of nicotine by Langley at the turn of the 20th century introduced the concept of a “receptive substance,” from which the idea of a “receptor” came to light. Subsequent studies aided by the Torpedo electric organ, a rich source of muscle-type nicotinic receptors (nAChRs), and the discovery of α-bungarotoxin, a snake toxin that binds pseudo-irreversibly to the muscle nAChR, resulted in the muscle nAChR being the best characterized ligand-gated ion channel hitherto. With the advancement of functional and genetic studies in the late 1980s, the existence of nAChRs in the mammalian brain was confirmed and the realization that the numerous nAChR subtypes contribute to the psychoactive properties of nicotine and other drugs of abuse and to the neuropathology of various diseases, including Alzheimer’s, Parkinson’s, and schizophrenia, has since emerged. This review provides a comprehensive overview of these findings and the more recent revelations of the impact that the rich diversity in function and expression of this receptor family has on neuronal and nonneuronal cells throughout the body. Despite these numerous developments, our understanding of the contributions of specific neuronal nAChR subtypes to the many facets of physiology throughout the body remains in its infancy. PMID:19126755

  13. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

  14. 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

  15. Characterization of the diversity of T cell receptor γδ complementary determinant region 3 in human peripheral blood by Immune Repertoire Sequencing.

    PubMed

    Chen, Hui; Zou, Mingjin; Teng, Da; Zhang, Jianmin; He, Wei

    2017-04-01

    γδ T cells function as sentinels in early host response to infections and malignancies. Although γδ T cells are regarded as innate immune cells and recognize antigens in a non-MHC restricted manner, they possess a huge diversity of complementary determinant region 3 (CDR3) of T cell receptor (TCR) generated by the rearrangement of germ-line gene V- (D) -J-C fragments. However, the detailed characteristics of the TCRγδ CDR3 repertoire remain unclear. A comprehensive analysis would answer fundamental questions about the diversity of the TCRγδ CDR3 repertoire and elucidate the mechanism underlying γδ T cell recognition of pathogens and tumor antigens. In this study, we used Immune Repertoire Sequencing (IR-SEQ) to analyze the diversity of TCRγδ CDR3 repertoires from 30 healthy donors. The results show that IR-SEQ had sufficient repeatability to analyze the TCRγδ CDR3 repertoire. The diversity of TCRγδ CDR3 repertoire is quite dispersed and individually different. The TCR δ chain (TRD) repertoire displayed more diversity and less sharing among individuals compared with TCR γ chain (TRG). To our knowledge, this is the first study to use IR-SEQ to characterize the repertoire of TCRγδ CDR3 in human peripheral blood γδ T cells by using IR-SEQ. Our findings provide a basic understanding of the diversity of TCRγδ repertoire in the physiological condition, which provides a clue to the underlying mechanism of γδ T cell recognition of pathogens and tumor antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Distinctive selection mechanisms govern the T cell receptor repertoire of peripheral CD4-CD8- alpha/beta T cells

    PubMed Central

    1992-01-01

    The T cell receptor (TCR) repertoire of CD4+ and CD8+ alpha/beta T cells is heavily influenced by positive and negative selection events that occur during T cell development in the thymus. The coreceptors CD4 and CD8 appear to be essential for this selection to occur. To gain insight into whether T cells that express TCR alpha/beta but lack either coreceptor (CD4- CD8- TCR alpha/beta or alpha/beta double- negative [DN] cells) are also subject to positive and negative selection, and whether selection can occur in the absence of coreceptors, we have performed an extensive immunogenetic analysis of the TCR V beta repertoire of alpha/beta DN cells in lymph nodes of normal mice. Our results show that alpha/beta DN cells appear to be unaffected by clonal deletion of V beta 5 and V beta 11 in I-E- expressing mice, and do not undergo deletion of V beta 6- and V beta 8.1-expressing T cells in Mls-1a-positive mice. They are also unaffected by positive selection of V beta 17a+ T cells in the context of I-Aq. The results suggest that most selection events require the participation of CD4 and CD8, while alpha/beta DN cells are unselected. This argues that most alpha/beta DN cells probably have never expressed CD4 or CD8. However, a unique form of repertoire selection occurs: enrichment of V beta 17a+ alpha/beta DN cells in I-E+ mice. This could be an instance of coreceptor-independent selection. PMID:1512537

  17. Functional characterization of ecdysone receptor gene switches in mammalian cells.

    PubMed

    Panguluri, Siva K; Kumar, Prasanna; Palli, Subba R

    2006-12-01

    Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches. We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand. We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors. Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand, RH-2485. The EcR's heterodimeric partner, ultraspiracle, is required for efficient binding of EcR to the ligand. The essential role of retinoid X receptor (RXR) or its insect homolog, ultraspiracle, in EcR function is shown by RXR knockdown experiments using RNAi. Chromatin immunoprecipitation assays demonstrated that VP16 (activation domain, AD):GAL4(DNA binding domain, DBD):EcR(ligand binding domain, LBD) or GAL4(DBD):EcR(LBD) fusion proteins can bind to GAL4 response elements in the absence of ligand. The VP16(AD) fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion protein of Homo sapiens RXR requires ligand for its heterodimerization with GAL4(DBD):EcR(LBD).

  18. Pseudomonas pili. Studies on antigenic determinants and mammalian cell receptors.

    PubMed

    Paranchych, W; Sastry, P A; Drake, D; Pearlstone, J R; Smillie, L B

    1985-01-01

    P. aeruginosa PAK pili are thin 5.2 nm diameter filaments containing a single 15-kd polypeptide subunit which is 144 amino acid residues in length. Studies on pili binding to a variety of synthetic sugars representing many di- tri- and tetra-saccharide structures found in mammalian glycoproteins and glycolipids failed to reveal any significant binding activity. On the other hand, a wide spectrum of binding activities was observed when a variety of structural proteins and enzymes were used as binding substrates. Of 30 proteins tested, phosphorylase b, pyruvate kinase and aldolase showed highest pilus binding activity. It was concluded that the PAK pilus receptor is probably a polypeptide rather than an oligosaccharide. Using arginine-specific cleavage to produce four large peptides, several proteases to produce subfragments of the large peptides, and antipilus rabbit antiserum, PAK pilin was found to contain four antigenic determinants. Epitopes near the NH2- and COOH-termini were only weakly immunogenic, whereas two epitopes near the center of the pilus protein titrated about 85% of the antipilus antibodies. Cleavage of the pilus protein into smaller peptides resulted in marked decreases in the affinity of antigenic peptides for their specific antibodies, suggesting that the immunodominant epitopes of PAK pilin are conformation-specific.

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

    PubMed

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

    2009-05-25

    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. 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. Our results suggest that ecological niche adaptations such as daily activity patterns may have shaped avian OR gene repertoires.

  20. 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

  1. The dynamics of T-cell receptor repertoire diversity following thymus transplantation for DiGeorge anomaly.

    PubMed

    Ciupe, Stanca M; Devlin, Blythe H; Markert, M Louise; Kepler, Thomas B

    2009-06-01

    T cell populations are regulated both by signals specific to the T-cell receptor (TCR) and by signals and resources, such as cytokines and space, that act independently of TCR specificity. Although it has been demonstrated that disruption of either of these pathways has a profound effect on T-cell development, we do not yet have an understanding of the dynamical interactions of these pathways in their joint shaping of the T cell repertoire. Complete DiGeorge Anomaly is a developmental abnormality that results in the failure of the thymus to develop, absence of T cells, and profound immune deficiency. After receiving thymic tissue grafts, patients suffering from DiGeorge anomaly develop T cells derived from their own precursors but matured in the donor tissue. We followed three DiGeorge patients after thymus transplantation to utilize the remarkable opportunity these subjects provide to elucidate human T-cell developmental regulation. Our goal is the determination of the respective roles of TCR-specific vs. TCR-nonspecific regulatory signals in the growth of these emerging T-cell populations. During the course of the study, we measured peripheral blood T-cell concentrations, TCRbeta V gene-segment usage and CDR3-length spectratypes over two years or more for each of the subjects. We find, through statistical analysis based on a novel stochastic population-dynamic T-cell model, that the carrying capacity corresponding to TCR-specific resources is approximately 1000-fold larger than that of TCR-nonspecific resources, implying that the size of the peripheral T-cell pool at steady state is determined almost entirely by TCR-nonspecific mechanisms. Nevertheless, the diversity of the TCR repertoire depends crucially on TCR-specific regulation. The estimated strength of this TCR-specific regulation is sufficient to ensure rapid establishment of TCR repertoire diversity in the early phase of T cell population growth, and to maintain TCR repertoire diversity in the face of

  2. The Dynamics of T-Cell Receptor Repertoire Diversity Following Thymus Transplantation for DiGeorge Anomaly

    PubMed Central

    Ciupe, Stanca M.; Devlin, Blythe H.; Markert, M. Louise; Kepler, Thomas B.

    2009-01-01

    T cell populations are regulated both by signals specific to the T-cell receptor (TCR) and by signals and resources, such as cytokines and space, that act independently of TCR specificity. Although it has been demonstrated that disruption of either of these pathways has a profound effect on T-cell development, we do not yet have an understanding of the dynamical interactions of these pathways in their joint shaping of the T cell repertoire. Complete DiGeorge Anomaly is a developmental abnormality that results in the failure of the thymus to develop, absence of T cells, and profound immune deficiency. After receiving thymic tissue grafts, patients suffering from DiGeorge anomaly develop T cells derived from their own precursors but matured in the donor tissue. We followed three DiGeorge patients after thymus transplantation to utilize the remarkable opportunity these subjects provide to elucidate human T-cell developmental regulation. Our goal is the determination of the respective roles of TCR-specific vs. TCR-nonspecific regulatory signals in the growth of these emerging T-cell populations. During the course of the study, we measured peripheral blood T-cell concentrations, TCRβ V gene-segment usage and CDR3-length spectratypes over two years or more for each of the subjects. We find, through statistical analysis based on a novel stochastic population-dynamic T-cell model, that the carrying capacity corresponding to TCR-specific resources is approximately 1000-fold larger than that of TCR-nonspecific resources, implying that the size of the peripheral T-cell pool at steady state is determined almost entirely by TCR-nonspecific mechanisms. Nevertheless, the diversity of the TCR repertoire depends crucially on TCR-specific regulation. The estimated strength of this TCR-specific regulation is sufficient to ensure rapid establishment of TCR repertoire diversity in the early phase of T cell population growth, and to maintain TCR repertoire diversity in the face of

  3. Blood T-cell receptor diversity decreases during the course of HIV infection, but the potential for a diverse repertoire persists

    PubMed Central

    Young, Jennifer J.; Schmidt, Diane; Zhang, Qianjun; Hoh, Rebecca; Busch, Michael; Martin, Jeffrey; Deeks, Steven; McCune, Joseph M.

    2012-01-01

    HIV infection results in a decrease in circulating CD4+ T-cell and naive T-cell numbers. If such losses were associated with an erosion of T-cell receptor (TCR) repertoire diversity in the peripheral T-cell pool, this might exacerbate the state of persistent immunodeficiency. Existing methods for the analysis of the TCR repertoire have demonstrated skewed distributions of TCR genes in HIV-infected subjects but cannot directly measure TCR diversity. Here we used AmpliCot, a quantitative assay based on DNA hybridization kinetics, to measure TCR diversity in a cross-sectional comparison of 19 HIV-infected persons to 18 HIV-uninfected controls. HIV-infected persons had a 10-fold decrease in total TCR repertoire diversity in 1.5 mL of blood compared with uninfected controls, with decreased diversity correlating most closely with a lower CD4+ T-cell percentage. Nonetheless, the TCR repertoire diversity of sort-purified T-cell subpopulations in HIV-infected and HIV-uninfected subjects was comparable. These observations suggest that the TCR repertoire diversity changes in whole blood during HIV disease progression are primarily the result of changes in the number and proportion of T-cell subpopulations and that most HIV-infected persons may retain a sufficiently diverse TCR repertoire to permit immune reconstitution with antiretroviral therapy alone, without thymopoiesis. PMID:22371879

  4. Mining human antibody repertoires

    PubMed Central

    2010-01-01

    Human monoclonal antibodies (mAbs) have become drugs of choice for the management of an increasing number of human diseases. Human antibody repertoires provide a rich source for human mAbs. Here we review the characteristics of natural and non-natural human antibody repertoires and their mining with non-combinatorial and combinatorial strategies. In particular, we discuss the selection of human mAbs from naïve, immune, transgenic and synthetic human antibody repertoires using methods based on hybridoma technology, clonal expansion of peripheral B cells, single-cell PCR, phage display, yeast display and mammalian cell display. Our reliance on different strategies is shifting as we gain experience and refine methods to the efficient generation of human mAbs with superior pharmacokinetic and pharmacodynamic properties. PMID:20505349

  5. 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

  6. A comparison of reptilian and avian olfactory receptor gene repertoires: species-specific expansion of group gamma genes in birds.

    PubMed

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

    2009-09-21

    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). 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 (approximately 72%) compared with the chicken (approximately 66%) and the zebra finch (approximately 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 alpha, theta and gamma genes. Interestingly, the vast majority of the avian OR genes are confined to a large expansion of a single branch (the so called gamma-c clade). An analysis of the selective pressure on the paralogous genes of each gamma-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 gamma-c expansions of the two birds do not intermix, i.e., they are lineage-specific. Almost all (group gamma-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. We identified a surprisingly large number of potentially functional avian OR genes

  7. The Missense of Smell: Functional Variability in the Human Odorant Receptor Repertoire

    PubMed Central

    Keller, Andreas; Li, Yun R.; Zhou, Ting; Trimmer, Casey; Snyder, Lindsey L.; Moberly, Andrew H.; Adipietro, Kaylin A.; Liu, Wen Ling L.; Zhuang, Hanyi; Zhan, Senmiao; Lee, Somin S.; Lin, Abigail; Matsunami, Hiroaki

    2014-01-01

    Humans have approximately 400 intact odorant receptors, but each individual has a unique set of genetic variations that lead to variation in olfactory perception. We used a heterologous assay to determine how often genetic polymorphisms in odorant receptors alter receptor function. We identified agonists for 18 odorant receptors and found that 63% of the odorant receptors we examined had polymorphisms that altered in vitro function. On average, two individuals differ functionally at over 30% of their odorant receptor alleles. To show that these in vitro results are relevant to olfactory perception, we verified that variations in OR10G4 genotype explain over 15% of the observed variation in perceived intensity and over 10% of the observed variation in perceived valence for the high affinity in vitro agonist guaiacol, but do not explain phenotypic variation for the lower affinity agonists vanillin and ethyl vanillin. PMID:24316890

  8. A comprehensive profiling of T- and B-lymphocyte receptor repertoires from a Chinese-origin rhesus macaque by high-throughput sequencing.

    PubMed

    Fu, Longfei; Li, Xinyang; Zhang, Wei; Wang, Changxi; Wu, Jinghua; Yang, Huanming; Wang, Jian; Liu, Xiao

    2017-01-01

    Due to the close genetic background, high similarity of physiology, and susceptibility to infectious and metabolic diseases with humans, rhesus macaques have been widely used as an important animal model in biomedical research, especially in the study of vaccine development and human immune-related diseases. In recent years, high-throughput sequencing based immune repertoire sequencing (IR-SEQ) has become a powerful tool to study the dynamic adaptive immune responses. Several previous studies had analyzed the responses of B cells to HIV-1 trimer vaccine or T cell repertoire of rhesus macaques using this technique, however, there are little studies that had performed a comprehensive analysis of immune repertoire of rhesus macaques, including T and B lymphocytes. Here, we did a comprehensive analysis of the T and B cells receptor repertoires of a Chinese rhesus macaque based on the 5'-RACE and IR-SEQ. The detailed analysis includes the distribution of CDR3 length, the composition of amino acids and nucleotides of CDR3, V, J and V-J combination usage, the insertion and deletion length distribution and somatic hypermutation rates of the framework region 3 (FR3). In addition, we found that several positions of FR3 region have high mutation frequencies, which may indicate the existence of new genes/alleles that have not been discovered and/or collected into IMGT reference database. We believe that a comprehensive profiling of immune repertoire of rhesus macaque will facilitate the human immune-related diseases studies.

  9. Comparative genomic analysis identifies an evolutionary shift of vomeronasal receptor gene repertoires in the vertebrate transition from water to land

    PubMed Central

    Shi, Peng; Zhang, Jianzhi

    2007-01-01

    Two evolutionarily unrelated superfamilies of G-protein coupled receptors, V1Rs and V2Rs, bind pheromones and “ordinary” odorants to initiate vomeronasal chemical senses in vertebrates, which play important roles in many aspects of an organism’s daily life such as mating, territoriality, and foraging. To study the macroevolution of vomeronasal sensitivity, we identified all V1R and V2R genes from the genome sequences of 11 vertebrates. Our analysis suggests the presence of multiple V1R and V2R genes in the common ancestor of teleost fish and tetrapods and reveals an exceptionally large among-species variation in the sizes of these gene repertoires. Interestingly, the ratio of the number of intact V1R genes to that of V2R genes increased by ∼50-fold as land vertebrates evolved from aquatic vertebrates. A similar increase was found for the ratio of the number of class II odorant receptor (OR) genes to that of class I genes, but not in other vertebrate gene families. Because V1Rs and class II ORs have been suggested to bind to small airborne chemicals, whereas V2Rs and class I ORs recognize water-soluble molecules, these increases reflect a rare case of adaptation to terrestrial life at the gene family level. Several gene families known to function in concert with V2Rs in the mouse are absent outside rodents, indicating rapid changes of interactions between vomeronasal receptors and their molecular partners. Taken together, our results demonstrate the exceptional evolutionary fluidity of vomeronasal receptors, making them excellent targets for studying the molecular basis of physiological and behavioral diversity and adaptation. PMID:17210926

  10. 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

  11. 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

  12. The scavenger receptor repertoire in six cnidarian species and its putative role in cnidarian-dinoflagellate symbiosis

    PubMed Central

    Neubauer, Emilie F.; Poole, Angela Z.; Davy, Simon K.

    2016-01-01

    Many cnidarians engage in a mutualism with endosymbiotic photosynthetic dinoflagellates that forms the basis of the coral reef ecosystem. Interpartner interaction and regulation includes involvement of the host innate immune system. Basal metazoans, including cnidarians have diverse and complex innate immune repertoires that are just beginning to be described. Scavenger receptors (SR) are a diverse superfamily of innate immunity genes that recognize a broad array of microbial ligands and participate in phagocytosis of invading microbes. The superfamily includes subclades named SR-A through SR-I that are categorized based on the arrangement of sequence domains including the scavenger receptor cysteine rich (SRCR), the C-type lectin (CTLD) and the CD36 domains. Previous functional and gene expression studies on cnidarian-dinoflagellate symbiosis have implicated SR-like proteins in interpartner communication and regulation. In this study, we characterized the SR repertoire from a combination of genomic and transcriptomic resources from six cnidarian species in the Class Anthozoa. We combined these bioinformatic analyses with functional experiments using the SR inhibitor fucoidan to explore a role for SRs in cnidarian symbiosis and immunity. Bioinformatic searches revealed a large diversity of SR-like genes that resembled SR-As, SR-Bs, SR-Es and SR-Is. SRCRs, CTLDs and CD36 domains were identified in multiple sequences in combinations that were highly homologous to vertebrate SRs as well as in proteins with novel domain combinations. Phylogenetic analyses of CD36 domains of the SR-B-like sequences from a diversity of metazoans grouped cnidarian with bilaterian sequences separate from other basal metazoans. All cnidarian sequences grouped together with moderate support in a subclade separately from bilaterian sequences. Functional experiments were carried out on the sea anemone Aiptasia pallida that engages in a symbiosis with Symbiodinium minutum (clade B1

  13. The scavenger receptor repertoire in six cnidarian species and its putative role in cnidarian-dinoflagellate symbiosis.

    PubMed

    Neubauer, Emilie F; Poole, Angela Z; Weis, Virginia M; Davy, Simon K

    2016-01-01

    Many cnidarians engage in a mutualism with endosymbiotic photosynthetic dinoflagellates that forms the basis of the coral reef ecosystem. Interpartner interaction and regulation includes involvement of the host innate immune system. Basal metazoans, including cnidarians have diverse and complex innate immune repertoires that are just beginning to be described. Scavenger receptors (SR) are a diverse superfamily of innate immunity genes that recognize a broad array of microbial ligands and participate in phagocytosis of invading microbes. The superfamily includes subclades named SR-A through SR-I that are categorized based on the arrangement of sequence domains including the scavenger receptor cysteine rich (SRCR), the C-type lectin (CTLD) and the CD36 domains. Previous functional and gene expression studies on cnidarian-dinoflagellate symbiosis have implicated SR-like proteins in interpartner communication and regulation. In this study, we characterized the SR repertoire from a combination of genomic and transcriptomic resources from six cnidarian species in the Class Anthozoa. We combined these bioinformatic analyses with functional experiments using the SR inhibitor fucoidan to explore a role for SRs in cnidarian symbiosis and immunity. Bioinformatic searches revealed a large diversity of SR-like genes that resembled SR-As, SR-Bs, SR-Es and SR-Is. SRCRs, CTLDs and CD36 domains were identified in multiple sequences in combinations that were highly homologous to vertebrate SRs as well as in proteins with novel domain combinations. Phylogenetic analyses of CD36 domains of the SR-B-like sequences from a diversity of metazoans grouped cnidarian with bilaterian sequences separate from other basal metazoans. All cnidarian sequences grouped together with moderate support in a subclade separately from bilaterian sequences. Functional experiments were carried out on the sea anemone Aiptasia pallida that engages in a symbiosis with Symbiodinium minutum (clade B1

  14. IMPre: An Accurate and Efficient Software for Prediction of T- and B-Cell Receptor Germline Genes and Alleles from Rearranged Repertoire Data

    PubMed Central

    Zhang, Wei; Wang, I-Ming; Wang, Changxi; Lin, Liya; Chai, Xianghua; Wu, Jinghua; Bett, Andrew J.; Dhanasekaran, Govindarajan; Casimiro, Danilo R.; Liu, Xiao

    2016-01-01

    Large-scale study of the properties of T-cell receptor (TCR) and B-cell receptor (BCR) repertoires through next-generation sequencing is providing excellent insights into the understanding of adaptive immune responses. Variable(Diversity)Joining [V(D)J] germline genes and alleles must be characterized in detail to facilitate repertoire analyses. However, most species do not have well-characterized TCR/BCR germline genes because of their high homology. Also, more germline alleles are required for humans and other species, which limits the capacity for studying immune repertoires. Herein, we developed “Immune Germline Prediction” (IMPre), a tool for predicting germline V/J genes and alleles using deep-sequencing data derived from TCR/BCR repertoires. We developed a new algorithm, “Seed_Clust,” for clustering, produced a multiway tree for assembly and optimized the sequence according to the characteristics of rearrangement. We trained IMPre on human samples of T-cell receptor beta (TRB) and immunoglobulin heavy chain and then tested it on additional human samples. Accuracy of 97.7, 100, 92.9, and 100% was obtained for TRBV, TRBJ, IGHV, and IGHJ, respectively. Analyses of subsampling performance for these samples showed IMPre to be robust using different data quantities. Subsequently, IMPre was tested on samples from rhesus monkeys and human long sequences: the highly accurate results demonstrated IMPre to be stable with animal and multiple data types. With rapid accumulation of high-throughput sequence data for TCR and BCR repertoires, IMPre can be applied broadly for obtaining novel genes and a large number of novel alleles. IMPre is available at https://github.com/zhangwei2015/IMPre. PMID:27867380

  15. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

    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.

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

    PubMed

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

    2015-11-16

    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.

  18. 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

  19. 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.

  20. 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.

  1. Mammalian glycosylation in immunity.

    PubMed

    Marth, Jamey D; Grewal, Prabhjit K

    2008-11-01

    Glycosylation produces a diverse and abundant repertoire of glycans, which are collectively known as the glycome. Glycans are one of the four fundamental macromolecular components of all cells, and are highly regulated in the immune system. Their diversity reflects their multiple biological functions that encompass ligands for proteinaceous receptors known as lectins. Since the discovery that selectins and their glycan ligands are important for the regulation of leukocyte trafficking, it has been shown that additional features of the vertebrate immune system are also controlled by endogenous cellular glycosylation. This Review focuses on the emerging immunological roles of the mammalian glycome.

  2. Neonicotinoid insecticides differently modulate acetycholine-induced currents on mammalian α7 nicotinic acetylcholine receptor.

    PubMed

    Cartereau, Alison; Martin, Carine; Thany, Steeve H

    2017-08-29

    Neonicotinoid insecticides are described as poor agonists of mammalian nicotinic acetylcholine receptors. In this paper, we provide evidence that they diffenrently act on mammalian nicotinic receptors. Two-electrode voltage-clamp electrophysiology was used to characterized the pharmacology of neonicotinoid insecticides on α7 receptors expressed in Xenopus oocytes. Single and combined application of clothianidin, acetamiprid and thiamethoxam were tested. The neonicotinoid insecticides, clothianidin and acetamiprid were partial agonists of mammalian neuronal α7 nicotinic receptors and thiamethoxam, a neonicotinoid insecticide, which is converted to clothianidin in insect and plant tissues had no effect. Pretreatment of 10 μM clothianidin and acetamiprid with 100 μM acetylcholine, significantly enhanced the subsequent acetylcholine-evoked currents whereas, 10 μM thiamethoxam reduced acetylcholine-induced current amplitudes. Moreover, the combinations of the three neonicotinoids decreased the ACh evoked currents. The present findings suggest that neonicotinoid insecticides differently affect α7 nicotinic acetylcholine receptors and can modulate acetylcholine-induced current. In final, the data indicate a previous unknown modulation of mammalian α7 receptors by combined application of clothianidin, acetamiprid and thiamethoxam. This article is protected by copyright. All rights reserved.

  3. High-throughput T-cell receptor sequencing across chronic liver diseases reveals distinct disease-associated repertoires.

    PubMed

    Liaskou, Evaggelia; Klemsdal Henriksen, Eva Kristine; Holm, Kristian; Kaveh, Fatemeh; Hamm, David; Fear, Janine; Viken, Marte K; Hov, Johannes Roksund; Melum, Espen; Robins, Harlan; Olweus, Johanna; Karlsen, Tom H; Hirschfield, Gideon M

    2016-05-01

    Hepatic T-cell infiltrates and a strong genetic human leukocyte antigen association represent characteristic features of various immune-mediated liver diseases. Conceptually the presence of disease-associated antigens is predicted to be reflected in T-cell receptor (TCR) repertoires. Here, we aimed to determine if disease-associated TCRs could be identified in the nonviral chronic liver diseases primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and alcoholic liver disease (ALD). We performed high-throughput sequencing of the TCRβ chain complementarity-determining region 3 of liver-infiltrating T cells from PSC (n = 20), PBC (n = 10), and ALD (n = 10) patients, alongside genomic human leukocyte antigen typing. The frequency of TCRβ nucleotide sequences was significantly higher in PSC samples (2.53 ± 0.80, mean ± standard error of the mean) compared to PBC samples (1.13 ± 0.17, P < 0.0001) and ALD samples (0.62 ± 0.10, P < 0.0001). An average clonotype overlap of 0.85% was detected among PSC samples, significantly higher compared to the average overlap of 0.77% seen within the PBC (P = 0.024) and ALD groups (0.40%, P < 0.0001). From eight to 42 clonotypes were uniquely detected in each of the three disease groups (≥30% of the respective patient samples). Multiple, unique sequences using different variable family genes encoded the same amino acid clonotypes, providing additional support for antigen-driven selection. In PSC and PBC, disease-associated clonotypes were detected among patients with human leukocyte antigen susceptibility alleles. We demonstrate liver-infiltrating disease-associated clonotypes in all three diseases evaluated, and evidence for antigen-driven clonal expansions. Our findings indicate that differential TCR signatures, as determined by high-throughput sequencing, may represent an imprint of distinctive antigenic repertoires present in the different chronic liver diseases

  4. Comparative analysis of NK cell receptor repertoire in adults and very elderly subjects with cytomegalovirus infection.

    PubMed

    Juárez-Vega, Guillermo; Rangel-Ramírez, Velia; Monsiváis-Urenda, Adriana; Niño-Moreno, Perla; Garcia-Sepúlveda, Christian; Noyola, Daniel E; González-Amaro, Roberto

    2017-03-01

    Human cytomegalovirus (HCMV) infection in children and young adults has been associated with changes in the innate immune system. We herein analyzed the possible effect of very long term HCMV infection on the expression of several NK cell receptors. Ninety HCMV-seropositive individuals were included and classified as young adults (n=30), elderly (n=30) and very elderly subjects (n=30). A peripheral blood sample was obtained and the expression of NK cell receptors (NKG2A, NKG2C, ILT2, CD161, KIR2DL1, KIR3DL1, and KIR3DL2) by NK and other lymphocyte subsets was assessed by flow cytometry. In addition, the frequency of the sixteen KIR genes was analyzed by polymerase chain reaction. We found a significant increase in the number of NKG2C+ NK and T cells in elderly individuals compared to young adults accompanied by an opposite trend in the number of NKG2A+ lymphocytes, and ILT2+ cells were also increased in elderly individuals. A significant increase in the levels of CD3-CD56+NKG2C+CD57+ cells was also detected in the elderly groups. Finally, KIR gene analysis revealed that the KIR genotype 2 was significantly less frequent in the elderly individuals. Our results support that long-term infection by HCMV exerts a significant progressive effect on the innate immune system. Copyright © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  5. Induction of Direct Antimicrobial Activity Through Mammalian Toll-Like Receptors

    NASA Astrophysics Data System (ADS)

    Thoma-Uszynski, Sybille; Stenger, Steffen; Takeuchi, Osamu; Ochoa, Maria Teresa; Engele, Matthias; Sieling, Peter A.; Barnes, Peter F.; Röllinghoff, Martin; Bölcskei, Pal L.; Wagner, Manfred; Akira, Shizuo; Norgard, Michael V.; Belisle, John T.; Godowski, Paul J.; Bloom, Barry R.; Modlin, Robert L.

    2001-02-01

    The mammalian innate immune system retains from Drosophila a family of homologous Toll-like receptors (TLRs) that mediate responses to microbial ligands. Here, we show that TLR2 activation leads to killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages, through distinct mechanisms. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide-dependent killing of intracellular tubercle bacilli, but in human monocytes and alveolar macrophages, this pathway was nitric oxide-independent. Thus, mammalian TLRs respond (as Drosophila Toll receptors do) to microbial ligands and also have the ability to activate antimicrobial effector pathways at the site of infection.

  6. Yeast as a model system for mammalian seven-transmembrane segment receptors

    SciTech Connect

    Jeansonne, N.E.

    1994-05-01

    Investigators have used the budding yeast Saccharomyces cerevisiae as a model system in which to study the {beta}-adrenergic receptor, the T-cell receptor pathway, initiation of mammalian DNA replication, initiation of mammalian transcription, secretion, the CDC2 kinase system, cell cycle control, and aging, as well as the function of oncogenes. This list continues to growth with the discovery of an immunoglobulin heavy-chain binding homologue in yeast, an Rb binding protein homologue, and a possible yeast arrestin. Yeast is relatively easy to maintain, to grow, and to genetically manipulate. A single gene can be overexpressed, selectively mutated or deleted from its chromosomal location. In this way, the in vivo function of a gene can be studied. It has become reasonable to consider yeast as a model system for studying the seven transmembrane segments (7-TMS) receptor family. Currently, subtypes of the {beta}-adrenergic receptor are being studied in yeast. The receptor and its G{sub {alpha}}-G-protein, trigger the mating pheromone receptor pathway. This provides a powerful assay for determining receptor function. Studies expressing the muscarinic cholinergic receptor in yeast are underway. The yeast pheromone receptor belongs to this receptor family, sharing sequences and secondary structure homology. An effective strategy has been to identify a yeast pathway or process which is homologous to a mammalian system. The pathway is delineated in yeast, identifying other genetic components. Then yeast genes are used to screen for human homologues of these components. The putative human homologues are then expressed in yeast and in mammalian cells to determine function. When this type of {open_quotes}mixing and matching{close_quotes} works, yeast genetics can be a powerful tool. 115 refs.

  7. Direct measurement of B-cell receptor repertoire's composition and variation in systemic lupus erythematosus.

    PubMed

    Liu, S; Hou, X L; Sui, W G; Lu, Q J; Hu, Y L; Dai, Y

    2017-01-01

    Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is known to be associated with polyclonal B-cell hyper-reactivity. B-cell receptor (BCR) has a central role in B-cell development, activation, survival and apoptosis, and thus is a critical component of the regulation of both protective and autoreactive B cells. In this study, we applied multiplex PCR and Illumina high-throughput sequencing to study the composition and variation of the BCRs in peripheral blood mononuclear cells from SLE patients and healthy donors (NC). We found that SLE group displayed significantly shorter CDR3 average length (14.86±0.76aa vs 15.70±0.43aa), more arginine percentage of CDR3 amino acids (7.57±0.20% vs 7.32±0.19%) and poorer immunological diversity than the healthy ones. CDR3 sequence YGMDV present in all SLE samples may provide more information in generating more effective B-cell targeted diagnosis/therapies strategies.

  8. Normalization of the peripheral blood T cell receptor V beta repertoire after cultured postnatal human thymic transplantation in DiGeorge syndrome.

    PubMed

    Davis, C M; McLaughlin, T M; Watson, T J; Buckley, R H; Schiff, S E; Hale, L P; Haynes, B F; Markert, M L

    1997-03-01

    Complete DiGeorge syndrome is an immunodeficiency disease characterized by thymic aplasia and the absence of functioning peripheral T cells. A patient with this syndrome was transplanted with cultured postnatal human thymic tissue. Within 5 weeks of transplantation, flow cytometry, T cell receptor V beta sequence analysis, and cell function studies showed the presence of oligoclonal populations of nonfunctional clonally expanded peripheral T cells that were derived from pretransplantation T cells present in the skin. However, at 3 months posttransplantation, a biopsy of the transplanted thymus showed normal intrathymic T cell maturation of host T cells with normal TCR V beta expression on thymocytes. By 9 months postransplantation, peripheral T cell function was restored and the TCR V beta repertoire became polyclonal, coincident with the appearance of normal T cell function. These data suggest that the transplanted thymus was responsible for the establishment of a new T cell repertoire via thymopoiesis in the chimeric thymic graft.

  9. Genomic and expression analyses of Tursiops truncatus T cell receptor gamma (TRG) and alpha/delta (TRA/TRD) loci reveal a similar basic public γδ repertoire in dolphin and human.

    PubMed

    Linguiti, Giovanna; Antonacci, Rachele; Tasco, Gianluca; Grande, Francesco; Casadio, Rita; Massari, Serafina; Castelli, Vito; Consiglio, Arianna; Lefranc, Marie-Paule; Ciccarese, Salvatrice

    2016-08-15

    The bottlenose dolphin (Tursiops truncatus) is a mammal that belongs to the Cetartiodactyla and have lived in marine ecosystems for nearly 60 millions years. Despite its popularity, our knowledge about its adaptive immunity and evolution is very limited. Furthermore, nothing is known about the genomics and evolution of dolphin antigen receptor immunity. Here we report a evolutionary and expression study of Tursiops truncatus T cell receptor gamma (TRG) and alpha/delta (TRA/TRD) genes. We have identified in silico the TRG and TRA/TRD genes and analyzed the relevant mature transcripts in blood and in skin from four subjects. The dolphin TRG locus is the smallest and simplest of all mammalian loci as yet studied. It shows a genomic organization comprising two variable (V1 and V2), three joining (J1, J2 and J3) and a single constant (C), genes. Despite the fragmented nature of the genome assemblies, we deduced the TRA/TRD locus organization, with the recent TRDV1 subgroup genes duplications, as it is expected in artiodactyls. Expression analysis from blood of a subject allowed us to assign unambiguously eight TRAV genes to those annotated in the genomic sequence and to twelve new genes, belonging to five different subgroups. All transcripts were productive and no relevant biases towards TRAV-J rearrangements are observed. Blood and skin from four unrelated subjects expression data provide evidence for an unusual ratio of productive/unproductive transcripts which arise from the TRG V-J gene rearrangement and for a "public" gamma delta TR repertoire. The productive cDNA sequences, shared both in the same and in different individuals, include biases of the TRGV1 and TRGJ2 genes. The high frequency of TRGV1-J2/TRDV1- D1-J4 productive rearrangements in dolphins may represent an interesting oligo-clonal population comparable to that found in human with the TRGV9- JP/TRDV2-D-J T cells and in primates. Although the features of the TRG and TRA/TRD loci organization reflect

  10. Skewed primary Igκ repertoire and V-J joining in C57BL/6 mice: implications for recombination accessibility and receptor editing

    PubMed Central

    Aoki-Ota, Miyo; Torkamani, Ali; Ota, Takayuki; Schork, Nicholas; Nemazee, David

    2012-01-01

    Previous estimates of the diversity of the mouse antibody repertoire have been based on fragmentary data owing to many technical limitations, in particular the many samples necessary to provide adequate coverage. Here, we used 5' coding end amplification of Igκ mRNAs from bone marrow, splenic and lymph node B cells of C57BL/6 mice combined with amplicon pyrosequencing to assess the functional and non-functional Vκ repertoire. To evaluate the potential effects of receptor editing, we also compared V/J associations and usage in bone marrows of mouse mutants under constitutive negative selection or an altered ability to undergo secondary recombination. In order to focus on preimmune B cells, our cell sorting strategy excluded memory B cells and plasma cells. Analysis of approximately 90 Mbp, representing >250,000 individual transcripts from 59 mice, revealed that 101 distinct functionalVκ genes are used, but at frequencies ranging from ~.001% to ~10%.Usage of sevenVκgenes made up over 40% of the repertoire. A small class of transcripts from apparently nonfunctional Vκ genes was found, as were occasional transcripts from several apparently functional genes that carry aberrant recombination signals. Of 404 potential V-J combinations (101 Vκs X 4 Jκs), 398 (98.5%) were found at least once in our sample. For most Vκ transcripts, all Jκs were used, but V-J association biases were common.Usage patterns were remarkably stable in different selective conditions.Overall, the primary κ repertoire is highly skewedby preferred rearrangements, limiting antibody diversity, but potentially facilitating receptor editing. PMID:22287713

  11. 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

  12. A new method for quantitative analysis of the T cell receptor V region repertoires in healthy common marmosets by microplate hybridization assay.

    PubMed

    Kitaura, Kazutaka; Fujii, Yoshiki; Matsutani, Takaji; Shirai, Kenji; Suzuki, Satsuki; Takasaki, Tomohiko; Shimada, Shin; Kametani, Yoshie; Shiina, Takashi; Takabayashi, Shuji; Katoh, Hideki; Ogasawara, Kouetsu; Kurane, Ichiro; Suzuki, Ryuji

    2012-10-31

    The common marmoset, Callithrix jacchus, is one of the smallest primates and is increasingly used for an experimental nonhuman primate model in many research fields. Analysis of T cell receptor (TCR) repertoires is a powerful tool to investigate T cell immunity in terms of antigen specificity and variability of TCR expression. However, monoclonal antibodies specific for many TCR Vα or Vβ chains have not been created. We have recently identified a large number of TCRα chain variable (TRAV) and TCRβ chain variable (TRBV) sequences from a cDNA library of common marmosets. The purpose of this study is to develop a new method for analysis of TCR repertoires in the common marmoset using this sequence information. This method is based on a microplate hybridization technique using 32 TRAV-specific and 32 TRBV-specific oligoprobes following an adaptor-ligation PCR. This enables the easy quantitation of the respective TRAV and TRBV expression levels. No cross-hybridization among specific-oligoprobes and very low variances in repeated measures of the same samples was found, demonstrating high specificity and reproducibility. Furthermore, this method was validated by an antihuman Vβ23 antibody which specifically bound to marmoset Vβ23. Using this method, we analyzed TCR repertoires from various tissue samples (PBMCs, spleen, lymph node and thymus) and isolated T cell subpopulations (CD4+CD8+, CD4+CD8− and CD4−CD8+) from the thymus of 10 common marmosets. Neither tissue-specific nor T cell subpopulation-specific differences was found in TRAV and TRBV repertoires. These results suggest that, unlike mice, TCR repertoires in the common marmoset are not affected by endogenous superantigens and are conserved among individuals, among tissues, and among T cell subpopulations. Thus, TCR repertoire analysis with high specificity and reproducibility is a very useful technique, with the potential to replace flow cytometric analysis using a panel of TRV-specific antibodies, many

  13. Frequent expansions of the bitter taste receptor gene repertoire during evolution of mammals in the Euarchontoglires clade.

    PubMed

    Hayakawa, Takashi; Suzuki-Hashido, Nami; Matsui, Atsushi; Go, Yasuhiro

    2014-08-01

    Genome studies of mammals in the superorder Euarchontoglires (a clade that comprises the orders Primates, Dermoptera, Scandentia, Rodentia, and Lagomorpha) are important for understanding the biological features of humans, particularly studies of medical model animals such as macaques and mice. Furthermore, the dynamic ecoevolutionary signatures of Euarchontoglires genomes may be discovered because many species in this clade are characterized by their successful adaptive radiation to various ecological niches. In this study, we investigated the evolutionary trajectory of bitter taste receptor genes (TAS2Rs) in 28 Euarchontoglires species based on homology searches of 39 whole-genome assemblies. The Euarchontoglires species possessed variable numbers of intact TAS2Rs, which ranged from 16 to 40, and their last common ancestor had at least 26 intact TAS2Rs. The gene tree showed that there have been at least seven lineage-specific events involving massive gene duplications. Gene duplications were particularly evident in the ancestral branches of anthropoids (the anthropoid cluster), which may have promoted the adaptive evolution of anthropoid characteristics, such as a trade-off between olfaction and other senses and the development of herbivorous characteristics. Subsequent whole-gene deletions of anthropoid cluster TAS2Rs in hominoid species suggest ongoing ectopic homologous recombination in the anthropoid cluster. These findings provide insights into the roles of adaptive sensory evolution in various ecological niches and important clues related to the molecular mechanisms that underlie taste diversity in Euarchontoglires mammalian species, including humans.

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

    PubMed

    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

    2015-12-01

    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.

  15. Biased T-cell receptor repertoires in patients with chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome).

    PubMed

    Pierdominici, M; Mazzetta, F; Caprini, E; Marziali, M; Digilio, M C; Marino, B; Aiuti, A; Amati, F; Russo, G; Novelli, G; Pandolfi, F; Luzi, G; Giovannetti, A

    2003-05-01

    Chromosome 22q11.2 deletion (del22q11.2) syndrome (DiGeorge syndrome/velocardiofacial syndrome) is a common syndrome typically consisting of congenital heart disease, hypoparathyroidism, developmental delay and immunodeficiency. Although a broad range of immunologic defects have been described in these patients, limited information is currently available on the diversity of the T-cell receptor (TCR) variable beta (BV) chain repertoire. The TCRBV repertoires of nine patients with del22q11.2 syndrome were determined by flow cytometry, fragment size analysis of the third complementarity determining region (CDR3 spectratyping) and sequencing of V(D)J regions. The rate of thymic output and the phenotype and function of peripheral T cells were also studied. Expanded TCRBV families were detected by flow cytometry in both CD4+ and CD8+ T cells. A decreased diversity of TCR repertoires was also demonstrated by CDR3 spectratyping, showing altered CDR3 profiles in the majority of TCRBV families investigated. The oligoclonal nature of abnormal peaks detected by CDR3 spectratyping was confirmed by the sequence analysis of the V(D)J regions. Thymic output, evaluated by measuring TCR rearrangement excision circles (TRECs), was significantly decreased in comparison with age-matched controls. Finally, a significant up-regulation in the percentage, but not in the absolute count, of activated CD4+ T cells (CD95+, CCR5+, HLA-DR+), IFN-gamma - and IL-2-expressing T cells was detected. These findings suggest that the diversity of CD4 and CD8 TCRBV repertoires is decreased in patients with del22q11.2 syndrome, possibly as a result of either impaired thymic function and/or increased T-cell activation.

  16. Predominant role of T cell receptor (TCR)-alpha chain in forming preimmune TCR repertoire revealed by clonal TCR reconstitution system.

    PubMed

    Yokosuka, Tadashi; Takase, Kan; Suzuki, Misao; Nakagawa, Yohko; Taki, Shinsuke; Takahashi, Hidemi; Fujisawa, Takehiko; Arase, Hisashi; Saito, Takashi

    2002-04-15

    The CDR3 regions of T cell receptor (TCR)-alpha and -beta chains play central roles in the recognition of antigen (Ag)-MHC complex. TCR repertoire is created on the basis of Ag recognition specificity by CDR3s. To analyze the potential spectrum of TCR-alpha and -beta to exhibit Ag specificity and generate TCR repertoire, we established hundreds of TCR transfectants bearing a single TCR-alpha or -beta chain derived from a cytotoxic T cell (CTL) clone, RT-1, specific for HIVgp160 peptide, and randomly picked up TCR-beta or -alpha chains. Surprisingly, one-third of such TCR-beta containing random CDR3 beta from naive T cells of normal mice could reconstitute the antigen-reactive TCR coupling with RT-1 TCR-alpha. A similar dominant function of TCR-alpha in forming Ag-specific TCR, though low-frequency, was obtained for lymphocytic choriomeningitis virus-specific TCR. Subsequently, we generated TCR-alpha and/or -beta transgenic (Tg) mice specific for HIVgp160 peptide, and analyzed the TCR repertoire of Ag-specific CTLs. Similar to the results from TCR reconstitution, TCR-alpha Tg generated CTLs with heterogeneous TCR-beta, whereas TCR-beta Tg-induced CTLs bearing a single TCR-alpha. These findings of Ag recognition with minimum involvement of CDR3 beta expand our understanding regarding the flexibility of the spectrum of TCR and suggest a predominant role of TCR-alpha chain in determining the preimmune repertoire of Ag-specific TCR.

  17. 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

  18. Functional synergy between cholecystokinin receptors CCKAR and CCKBR in mammalian brain development.

    PubMed

    Nishimura, Sayoko; Bilgüvar, Kaya; Ishigame, Keiko; Sestan, Nenad; Günel, Murat; Louvi, Angeliki

    2015-01-01

    Cholecystokinin (CCK), a peptide hormone and one of the most abundant neuropeptides in vertebrate brain, mediates its actions via two G-protein coupled receptors, CCKAR and CCKBR, respectively active in peripheral organs and the central nervous system. Here, we demonstrate that the CCK receptors have a dynamic and largely reciprocal expression in embryonic and postnatal brain. Using compound homozygous mutant mice lacking the activity of both CCK receptors, we uncover their additive, functionally synergistic effects in brain development and demonstrate that CCK receptor loss leads to abnormalities of cortical development, including defects in the formation of the midline and corpus callosum, and cortical interneuron migration. Using comparative transcriptome analysis of embryonic neocortex, we define the molecular mechanisms underlying these defects. Thus we demonstrate a developmental, hitherto unappreciated, role of the two CCK receptors in mammalian neocortical development.

  19. Functional Synergy between Cholecystokinin Receptors CCKAR and CCKBR in Mammalian Brain Development

    PubMed Central

    Nishimura, Sayoko; Bilgüvar, Kaya; Ishigame, Keiko; Sestan, Nenad; Günel, Murat; Louvi, Angeliki

    2015-01-01

    Cholecystokinin (CCK), a peptide hormone and one of the most abundant neuropeptides in vertebrate brain, mediates its actions via two G-protein coupled receptors, CCKAR and CCKBR, respectively active in peripheral organs and the central nervous system. Here, we demonstrate that the CCK receptors have a dynamic and largely reciprocal expression in embryonic and postnatal brain. Using compound homozygous mutant mice lacking the activity of both CCK receptors, we uncover their additive, functionally synergistic effects in brain development and demonstrate that CCK receptor loss leads to abnormalities of cortical development, including defects in the formation of the midline and corpus callosum, and cortical interneuron migration. Using comparative transcriptome analysis of embryonic neocortex, we define the molecular mechanisms underlying these defects. Thus we demonstrate a developmental, hitherto unappreciated, role of the two CCK receptors in mammalian neocortical development. PMID:25875176

  20. Mammalian. beta. /sub 1/- and. beta. /sub 2/-adrenergic receptors: immunological and structural comparison

    SciTech Connect

    Moxham, C.P.; George, S.T.; Graziano, M.P.; Brandwein, H.J.; Malbon, C.C.

    1986-11-05

    ..beta../sub 1/- and ..beta../sub 2/-adrenergic receptors, pharmacologically distinct proteins, have been reported to be structurally dissimilar. In the present study three techniques were employed to compare the nature of mammalian ..beta../sub 1/- and ..beta../sub 2/-adrenergic receptors. Antibodies against each of the receptor subtypes were raised separately. Polyclonal antisera against ..beta../sub 1/-receptors of rat fat cells were raised in mice, and antisera against ..beta../sub 2/-receptors of guinea pig lung were raised in rabbits. Receptors purified from rat fat cells (..beta../sub 1/-), S49 mouse lymphoma cells (..beta../sub 2/-), and rat liver (..beta../sub 2/-) were probed with these antisera. Each anti-receptor antisera demonstrated the ability to immunoprecipitate purified receptors of both ..beta../sub 1/- and ..beta../sub 2/-subtypes. The mobility of ..beta..-receptors subjected to polyacrylamide gel electrophoresis was probed using antireceptor antibodies and nitrocellulose blots of the gels. Fat cell ..beta../sub 1/-adrenergic receptors display M/sub r/ = 67,000 under reducing conditions and M/sub r/ = 54,000 under nonreducing conditions, as previously reported. Both ..beta../sub 1/- and ..beta../sub 2/-receptors displayed this same shift in electrophoretic mobility observed in the presence as compared to the absence of disulfide bridge-reducing agents, as detected both by autoradiography of the radiolabeled receptors and by immunoblotting of native receptors. Finally, isoelectric focusing of purified radioiodinated ..beta../sub 1/- and ..beta../sub 2/-adrenergic receptors revealed identical isoelectric points. These data are the first to provide analyses of immunological, structural, and biochemical features of ..beta../sub 1/- and ..beta../sub 2/-subtypes in tandem and underscore the structural similarities that exist between these pharmacologically distinct receptors.

  1. 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}.

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

    PubMed

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

    2015-09-01

    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.

  3. 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

  4. 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

  5. Phase description of the Huber-Braun neuron model for mammalian cold receptors

    NASA Astrophysics Data System (ADS)

    Freund, J. A.; Finke, C.; Braun, H. A.; Feudel, U.

    2013-10-01

    The spiking activity of mammalian cold receptors is described by the Huber-Braun neuron model. Sweeping temperature as a control parameter across a biologically relevant range this model exhibits a complex bifurcation structure seen in the sequence of interspike intervals. The model's distinctive feature is the interaction between a fast spike generating dynamics and a slow subthreshold oscillation. Viewing the spike generation as a cycle, the dynamics may also be modeled phenomenologically by two phases, one for the spike cycle and the second for the slow subthreshold oscillation. In fact, a phase model of temperature-dependent mammalian cold receptors was already proposed by Roper et al. (2000). Here we follow their approach and investigate to what extent this model is able to reproduce the bifurcation patterns of the Huber-Braun model. Special attention is paid to the tonic firing to bursting transition observed in the low temperature range.

  6. Elabela-Apelin Receptor Signaling Pathway is Functional in Mammalian Systems

    PubMed Central

    Wang, Zhi; Yu, Daozhan; Wang, Mengqiao; Wang, Qilong; Kouznetsova, Jennifer; Yang, Rongze; Qian, Kun; Wu, Wenjun; Shuldiner, Alan; Sztalryd, Carole; Zou, Minghui; Zheng, Wei; Gong, Da-Wei

    2015-01-01

    Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development. PMID:25639753

  7. Elabela-apelin receptor signaling pathway is functional in mammalian systems.

    PubMed

    Wang, Zhi; Yu, Daozhan; Wang, Mengqiao; Wang, Qilong; Kouznetsova, Jennifer; Yang, Rongze; Qian, Kun; Wu, Wenjun; Shuldiner, Alan; Sztalryd, Carole; Zou, Minghui; Zheng, Wei; Gong, Da-Wei

    2015-02-02

    Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development.

  8. Neurotrophins and their receptors in early development of the mammalian nervous system.

    PubMed

    Bartkowska, Katarzyna; Turlejski, Kris; Djavadian, Rouzanna L

    2010-01-01

    Neurotrophins belonging to the class of growth factors and including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) are widely recognized as essential factors in the developing central nervous system (CNS). Neurotrophins are synthesized as precursor forms (proneurotrophins). Mature forms of neurotrophins exert their effect by binding to specific tyrosine kinases receptors (TrkA, TrkB and TrkC) as well as via the p75 receptor, a member of the tumor necrosis factor receptor superfamily while proneurotrophins interact with the receptor p75 or co-receptor complex of p75 and sortilin, that is a Vps10p domain-containing transmembrane protein. Expression of neurotrophins corresponds with the onset of neurogenesis in developing mammalian species. BDNF is low in early embryonic stages of development, while NT-3 highly expresses in the developing CNS. Expression of neurotrophins receptors mainly overlaps at early development. Data concerning early distribution of neurotrophins and their receptors in the nervous system and results in mice with targeted disruptions of neurotrophin or receptor genes show that neurotrophins and their receptors play distinct roles in control and regulation of the most crucial developmental processes such as proliferation, migration, differentiation, survival, apoptosis and synaptic plasticity.

  9. A propofol binding site on mammalian GABAA receptors identified by photolabeling

    PubMed Central

    Yip, Grace M S; Chen, Zi-Wei; Edge, Christopher J; Smith, Edward H; Dickinson, Robert; Hohenester, Erhard; Townsend, R Reid; Fuchs, Karoline; Sieghart, Werner; Evers, Alex S; Franks, Nicholas P

    2014-01-01

    Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA receptors, but where it binds to this receptor is not known and has been a matter of some controversy. We have synthesized a novel propofol analogue photolabeling reagent that has a biological activity very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin which have been identified using X-ray crystallography. Using a combination of the protiated label and a deuterated version, and mammalian receptors labeled in intact membranes, we have identified a novel binding site for propofol in GABAA receptors consisting of both β3 homopentamers and α1β3 heteropentamers. The binding site is located within the β subunit, at the interface between the transmembrane domains and the extracellular domain, and lies close to known determinants of anesthetic sensitivity in transmembrane segments TM1 and TM2. PMID:24056400

  10. Effects of Insecticidal Ketones Present in Mint Plants on GABAA Receptor from Mammalian Neurons.

    PubMed

    Sánchez-Borzone, Mariela Eugenia; Marin, Leticia Delgado; García, Daniel Asmed

    2017-01-01

    The genus Mentha, an important member of the Lamiaceae family, is represented by many species commonly known as mint. The insecticidal activity of Mentha oil and its main components has been tested and established against various insects/pests. Among these, the ketone monoterpenes that are most common in different Mentha species demonstrated insect toxicity, with pulegone being the most active, followed by carvone and menthone. Considering that the GABAA receptor (GABAA-R) is one of the main insecticide targets on neurons, and that pulegone would modulate the insect GABA system, it may be expected that the insecticidal properties of Mentha ketones are mediated by their interaction with this receptor. In order to discern the pharmacological actions of these products when used as insecticides on mammalian organisms, we evaluated the pharmacologic activity of ketones, commonly present in Mentha plants, on native GABAA-R from rats. Determination of ketones effects on allosterically enhanced benzodiazepine binding, using primary cultures of cortical neurons, which express functional receptors and MTT assay to evaluate their cell toxicity. Our results seem to indicate that ketone components of Mentha, with proven repellent or insecticide activity, were able to behave as GABAA-R negative allosteric modulators in murine cells and consequently could exhibit convulsant activity in mammalians. Only pulegone at the highest assayed concentration (2 mM) showed a significant reduction in cell viability after exposure for 24 hr. The present results strongly suggest that the ketone components of Mentha are able to exhibit convulsant activity in mammalian organisms, but functional assays and in vivo experiments would be necessary to corroborate this proposed action. The pharmacological activity of insecticide ketones, commonly present in Mentha plants, was evaluated on native GABAA receptor from mammalian neurons.All studied compounds: pulegone, menthone and dihydrocarvone, were

  11. Effects of Insecticidal Ketones Present in Mint Plants on GABAA Receptor from Mammalian Neurons

    PubMed Central

    Sánchez-Borzone, Mariela Eugenia; Marin, Leticia Delgado; García, Daniel Asmed

    2017-01-01

    Background: The genus Mentha, an important member of the Lamiaceae family, is represented by many species commonly known as mint. The insecticidal activity of Mentha oil and its main components has been tested and established against various insects/pests. Among these, the ketone monoterpenes that are most common in different Mentha species demonstrated insect toxicity, with pulegone being the most active, followed by carvone and menthone. Considering that the GABAA receptor (GABAA-R) is one of the main insecticide targets on neurons, and that pulegone would modulate the insect GABA system, it may be expected that the insecticidal properties of Mentha ketones are mediated by their interaction with this receptor. Objective: In order to discern the pharmacological actions of these products when used as insecticides on mammalian organisms, we evaluated the pharmacologic activity of ketones, commonly present in Mentha plants, on native GABAA-R from rats. Materials and Methods: Determination of ketones effects on allosterically enhanced benzodiazepine binding, using primary cultures of cortical neurons, which express functional receptors and MTT assay to evaluate their cell toxicity. Results: Our results seem to indicate that ketone components of Mentha, with proven repellent or insecticide activity, were able to behave as GABAA-R negative allosteric modulators in murine cells and consequently could exhibit convulsant activity in mammalians. Only pulegone at the highest assayed concentration (2 mM) showed a significant reduction in cell viability after exposure for 24 hr. Conclusion: The present results strongly suggest that the ketone components of Mentha are able to exhibit convulsant activity in mammalian organisms, but functional assays and in vivo experiments would be necessary to corroborate this proposed action. SUMMARY The pharmacological activity of insecticide ketones, commonly present in Mentha plants, was evaluated on native GABAA receptor from mammalian

  12. Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells.

    PubMed

    Chauhan, Anoop Singh; Kumar, Manoj; Chaudhary, Surbhi; Patidar, Anil; Dhiman, Asmita; Sheokand, Navdeep; Malhotra, Himanshu; Raje, Chaaya Iyengar; Raje, Manoj

    2017-03-15

    Prokaryotic pathogens establish infection in mammals by capturing the proteolytic enzyme plasminogen (Plg) onto their surface to digest host extracellular matrix (ECM). One of the bacterial surface Plg receptors is the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In a defensive response, the host mounts an inflammatory response, which involves infiltration of leukocytes to sites of inflammation. This requires macrophage exit from the blood and migration across basement membranes, a phenomenon dependent on proteolytic remodeling of the ECM utilizing Plg. The ability of Plg to facilitate inflammatory cell recruitment critically depends on receptors on the surface of phagocyte cells. Utilizing a combination of biochemical, cellular, knockdown, and in vivo approaches, we demonstrated that upon inflammation, macrophages recruit GAPDH onto their surface to carry out the same task of capturing Plg to digest ECM to aid rapid phagocyte migration and combat the invading pathogens. We propose that GAPDH is an ancient, evolutionarily conserved receptor that plays a key role in the Plg-dependent regulation of macrophage recruitment in the inflammatory response to microbial aggression, thus pitting prokaryotic GAPDH against mammalian GAPDH, with both involved in a conserved role of Plg activation on the surface of their respective cells, to conflicting ends.-Chauhan, A. S., Kumar, M., Chaudhary, S., Patidar, A., Dhiman, A., Sheokand, N., Malhotra, H., Raje, C. I., Raje, M. Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells.

  13. The mammalian clock component PERIOD2 coordinates circadian output by interaction with nuclear receptors.

    PubMed

    Schmutz, Isabelle; Ripperger, Jürgen A; Baeriswyl-Aebischer, Stéphanie; Albrecht, Urs

    2010-02-15

    Mammalian circadian clocks provide a temporal framework to synchronize biological functions. To obtain robust rhythms with a periodicity of about a day, these clocks use molecular oscillators consisting of two interlocked feedback loops. The core loop generates rhythms by transcriptional repression via the Period (PER) and Cryptochrome (CRY) proteins, whereas the stabilizing loop establishes roughly antiphasic rhythms via nuclear receptors. Nuclear receptors also govern many pathways that affect metabolism and physiology. Here we show that the core loop component PER2 can coordinate circadian output with the circadian oscillator. PER2 interacts with nuclear receptors including PPARalpha and REV-ERBalpha and serves as a coregulator of nuclear receptor-mediated transcription. Consequently, PER2 is rhythmically bound at the promoters of nuclear receptor target genes in vivo. In this way, the circadian oscillator can modulate the expression of nuclear receptor target genes like Bmal1, Hnf1alpha, and Glucose-6-phosphatase. The concept that PER2 may propagate clock information to metabolic pathways via nuclear receptors adds an important facet to the clock-dependent regulation of biological networks.

  14. Molecular piracy of mammalian interleukin-8 receptor type B by herpesvirus saimiri.

    PubMed

    Ahuja, S K; Murphy, P M

    1993-10-05

    Viruses are known to acquire and modify the genes of their hosts to attain a survival advantage in the host environment. Herpesvirus saimiri (HVS) is a T-lymphotropic virus that causes fatal lymphoproliferative diseases in several non-human primates. The gene ECRF3 of HVS was most likely acquired from a primate host. ECRF3 encodes a putative seven-transmembrane-domain receptor that is remotely related (approximately 30% amino acid identity) to the known mammalian alpha and beta chemokine receptors, namely interleukin-8 receptor (IL8R) types A and B and the MIP-1 alpha/RANTES receptor, respectively. Chemokines regulate the trafficking, activation, and, in some cases, proliferation of myeloid and lymphoid cell types. We now show that ECRF3 encodes a functional receptor for the alpha chemokines IL-8, GRO/melanoma growth stimulatory activity (MGSA), and NAP-2 but not for beta chemokines, a specificity identical to that of IL8RB. Paradoxically, IL8RA shares 77% amino acid identity with IL8RB but is not a receptor for GRO/MGSA or NAP-2. This is the first functional characterization of a viral seven-transmembrane-domain receptor. It suggests a novel role for alpha chemokines in the pathogenesis of HVS infection by transmembrane signaling via the product of ECRF3.

  15. The T-cell Receptor Repertoire Influences the Tumor Microenvironment and Is Associated with Survival in Aggressive B-cell Lymphoma.

    PubMed

    Keane, Colm; Gould, Clare; Jones, Kimberley; Hamm, David; Talaulikar, Dipti; Ellis, Jonathan; Vari, Frank; Birch, Simone; Han, Erica; Wood, Peter; Le-Cao, Kim-Anh; Green, Michael R; Crooks, Pauline; Jain, Sanjiv; Tobin, Josh; Steptoe, Raymond J; Gandhi, Maher K

    2017-04-01

    Purpose: To investigate the relationship between the intra-tumoral T-cell receptor (TCR) repertoire and the tumor microenvironment (TME) in de novo diffuse large B-cell lymphoma (DLBCL) and the impact of TCR on survival.Experimental Design: We performed high-throughput unbiased TCRβ sequencing on a population-based cohort of 92 patients with DLBCL treated with conventional (i.e., non-checkpoint blockade) frontline "R-CHOP" therapy. Key immune checkpoint genes within the TME were digitally quantified by nanoString. The primary endpoints were 4-year overall survival (OS) and progression-free survival (PFS).Results: The TCR repertoire within DLBCL nodes was abnormally narrow relative to non-diseased nodal tissues (P < 0.0001). In DLBCL, a highly dominant single T-cell clone was associated with inferior 4-year OS rate of 60.0% [95% confidence interval (CI), 31.7%-79.6%], compared with 79.8% in patients with a low dominant clone (95% CI, 66.7%-88.5%; P = 0.005). A highly dominant clone also predicted inferior 4-year PFS rate of 46.6% (95% CI, 22.5%-76.6%) versus 72.6% (95% CI, 58.8%-82.4%, P = 0.008) for a low dominant clone. In keeping, clonal expansions were most pronounced in the EBV(+) DLBCL subtype that is known to express immunogenic viral antigens and is associated with particularly poor outcome. Increased T-cell diversity was associated with significantly elevated PD-1, PD-L1, and PD-L2 immune checkpoint molecules.Conclusions: Put together, these findings suggest that the TCR repertoire is a key determinant of the TME. Highly dominant T-cell clonal expansions within the TME are associated with poor outcome in DLBCL treated with conventional frontline therapy. Clin Cancer Res; 23(7); 1820-8. ©2016 AACR.

  16. 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

  17. T cell receptor variable β gene repertoire in liver and peripheral blood lymphocytes of chronically hepatitis C virus-infected patients with and without mixed cryoglobulinaemia

    PubMed Central

    Russi, S; Lauletta, G; Serviddio, G; Sansonno, S; Conteduca, V; Sansonno, L; De Re, V; Sansonno, D

    2013-01-01

    To characterize the repertoire of T lymphocytes in chronically hepatitis C virus (HCV)-infected patients with and without mixed cryoglobulinaemia (MC). T cell receptor (TCR) variable (V) β clonalities in portal tracts isolated from liver biopsy sections with a laser capture microdissection technique in 30 HCV-positive MC patients were studied by size spectratyping. Complementarity-determining region 3 (CDR3) profiles of liver-infiltrating lymphocytes (LIL) were also compared with those circulating in the blood. The representative results of TCR Vβ by CDR3 were also obtained from liver tissues and peripheral blood lymphocytes (PBL) of 21 chronically HCV-infected patients without MC. LIL were highly restricted, with evidence of TCR Vβ clonotypic expansions in 23 of 30 (77%) and in 15 of 21 (71%) MC and non-MC patients, respectively. The blood compartment contained TCR Vβ expanded clones in 19 (63%) MC and 12 (57%) non-MC patients. The occurrence of LIL clonalities was detected irrespective of the degree of liver damage or circulating viral load, whereas it correlated positively with higher levels of intrahepatic HCV RNA. These results support the notion that TCR Vβ repertoire is clonally expanded in HCV-related MC with features comparable to those found in chronically HCV-infected patients without MC. PMID:23574322

  18. Transcriptional activity and biological effects of mammalian estrogen receptor ligands on three hepatic estrogen receptors in Mozambique tilapia.

    PubMed

    Davis, L K; Katsu, Y; Iguchi, T; Lerner, D T; Hirano, T; Grau, E G

    2010-10-01

    Like other fish species, Mozambique tilapia has three forms of estrogen receptor, ERα, ERβ1, and ERβ2. A primary function of 17β-estradiol (E(2)) in oviparous species is the hepatic induction of the yolk precursor protein, vitellogenin (Vg). To characterize the roles of ERs in Vg production, transactivation assays and an in vivo study were carried out utilizing agonists for mammalian ERα and ERβ, and an antagonist for mammalian ERα, propyl-pyrazole-triol (PPT), diarylpropionitrile (DPN), and methyl-piperidino-pyrazole (MPP), respectively. ERα was more sensitive and responsive to PPT than ERβ1 or ERβ2 in transactivation assays. All ER isoforms indicated equivalent responsiveness to DPN compared with E(2), although sensitivity to DPN was lower. MPP exhibited antagonistic action on transactivation of all ER isoforms and reduced the E(2) effect on Vg and ERα 48h post-injection. DPN increased ERα and Vg expression and plasma Vg post-injection, whereas PPT was without effect; DPN seems to stimulate Vg production through activation of ERα. The ligand binding domain of all tilapia ER forms shares only 60-65% amino acid identity with human ERα and ERβ. This, together with our results, clearly indicates that agonistic or antagonistic characteristics of PPT, DPN and MPP cannot be extrapolated from mammalian to piscine ERs. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. The Ca(2+) influx through the mammalian skeletal muscle dihydropyridine receptor is irrelevant for muscle performance.

    PubMed

    Dayal, Anamika; Schrötter, Kai; Pan, Yuan; Föhr, Karl; Melzer, Werner; Grabner, Manfred

    2017-09-07

    Skeletal muscle excitation-contraction (EC) coupling is initiated by sarcolemmal depolarization, which is translated into a conformational change of the dihydropyridine receptor (DHPR), which in turn activates sarcoplasmic reticulum (SR) Ca(2+) release to trigger muscle contraction. During EC coupling, the mammalian DHPR embraces functional duality, as voltage sensor and L-type Ca(2+) channel. Although its unique role as voltage sensor for conformational EC coupling is firmly established, the conventional function as Ca(2+) channel is still enigmatic. Here we show that Ca(2+) influx via DHPR is not necessary for muscle performance by generating a knock-in mouse where DHPR-mediated Ca(2+) influx is eliminated. Homozygous knock-in mice display SR Ca(2+) release, locomotor activity, motor coordination, muscle strength and susceptibility to fatigue comparable to wild-type controls, without any compensatory regulation of multiple key proteins of the EC coupling machinery and Ca(2+) homeostasis. These findings support the hypothesis that the DHPR-mediated Ca(2+) influx in mammalian skeletal muscle is an evolutionary remnant.In mammalian skeletal muscle, the DHPR functions as a voltage sensor to trigger muscle contraction and as a Ca(2+) channel. Here the authors show that mice where Ca(2+) influx through the DHPR is eliminated display no difference in skeletal muscle function, suggesting that the Ca(2+) influx through this channel is vestigial.

  20. Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm

    PubMed Central

    1995-01-01

    Calcium flux is required for the mammalian sperm acrosome reaction, an exocytotic event triggered by egg binding, which results in a dramatic rise in sperm intracellular calcium. Calcium-dependent membrane fusion results in the release of enzymes that facilitate sperm penetration through the zona pellucida during fertilization. We have characterized inositol 1,4,5-trisphosphate (IP3)-gated calcium channels and upstream components of the phosphoinositide signaling system in mammalian sperm. Peptide antibodies colocalized G alpha q/11 and the beta 1 isoform of phospholipase C (PLC beta 1) to the anterior acrosomal region of mouse sperm. Western blotting using a polyclonal antibody directed against purified brain IP3 receptor (IP3R) identified a specific 260 kD band in 1% Triton X-100 extracts of rat, hamster, mouse and dog sperm. In each species, IP3R immunostaining localized to the acrosome cap. Scatchard analysis of [3H]IP3 binding to rat sperm sonicates revealed a curvilinear plot with high affinity (Kd = 26 nM, Bmax = 30 pmol/mg) and low affinity (Kd = 1.6 microM, Bmax = 550 pmol/mg) binding sites, reflecting among the highest receptor densities in mammalian tissue. Immunoelectron microscopy confirmed the acrosomal localization in rat sperm. The IP3R fractionated with acrosomes by discontinuous sucrose gradient centrifugation and was enriched in the medium of acrosome- reacted sperm. ATP-dependent 45Ca2+ loading of digitonin permeabilized rat sperm was decreased by 45% in the presence of 10 microM IP3. The IP3-mediated release of calcium was blocked by heparin. Thapsigargin, a sequiterpene lactone inhibitor of the microsomal Ca(2+)-ATPase, stimulated the acrosome reaction of mouse sperm to the same extent as the Ca2+ ionophore, A23187. The failure of caffeine and ryanodine to affect calcium accumulation suggested that thapsigargin acted through an IP3-sensitive store. The presence of G alpha q/11, PLC beta 1 and a functional IP3R in the anterior acrosomal region

  1. Motif-based construction of a functional map for mammalian olfactory receptors.

    PubMed

    Liu, Agatha H; Zhang, Xinmin; Stolovitzky, Gustavo A; Califano, Andrea; Firestein, Stuart J

    2003-05-01

    We applied an automatic and unsupervised system to a nearly complete database of mammalian odor receptor genes. The generated motifs and gene classification were subjected to extensive and systematic downstream analysis to obtain biological insights. Two major results from this analysis were: (1) a map of sequence motifs that may correlate with function and (2) the corresponding receptor classes in which members of each class are likely to share specific functions. We have discovered motifs that have been implicated in structural integrity and posttranslational modification, as well as motifs very likely to be directly involved in ligand binding. We further propose a combinatorial molecular hypothesis, based on unique combinations of the observed motifs, that provides a foundation for understanding the generation of a large number of ligand binding sites.

  2. 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

  3. T cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen

    PubMed Central

    1995-01-01

    Two unusual characteristics of the memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, have provided an unique opportunity to investigate self tolerance and T cell receptor (TCR) plasticity in humans. First, the response is exceptionally restricted, dominated by cytotoxic T lymphocytes (CTL) with identical TCR protein sequences (Argaet, V. P., C. W. Schmidt, S. R. Burrows, S. L. Silins, M. G. Kurilla, D. L. Doolan, A. Suhrbier, D. J. Moss, E. Kieff, T. B. Sculley, and I. S. Misko. 1994. J. Exp. Med. 180:2335-2340). Second, CTL expressing this receptor are cross-reactive with the alloantigen HLA B* 4402 on uninfected cells (Burrows, S. R., R. Khanna, J. M. Burrows, and D. J. Moss. 1994. J. Exp. Med. 179:1155-1161). No CTL using this conserved public TCR could be reactivated from the peripheral blood of EBV exposed individuals expressing both HLA B8 and B*4402, demonstrating the clonal inactivation of potentially self- reactive T cells in humans. A significant FLRGRAYGL-specific response was still apparent, however, and TCR sequence analysis of multiple CTL clones revealed an oligoclonal TCR repertoire for this determinant within these individuals, using diverse V and J gene segments and CDR3 regions. In addition, a significant public TCR component was identified in which several distinct alpha/beta rearrangements are shared by CTL clones from a number of unrelated HLA B8+, B*4402+ donors. The striking dominance of public TCR in the response to this EBV epitope suggests a strong genetic bias in TCR gene recombination. Fine specificity analysis using peptide analogues showed that, of six different antigen receptors for FLRGRAYGL/HLA B8, none associate closely with the peptide's full array of potential TCR contact residues. Whereas the HLA B*4402-cross-reactive receptor binds amino acids toward the COOH terminus of the peptide, others preferentially favor an NH2-terminal determinant, presumably evading an area

  4. 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

  5. Coevolution of activating and inhibitory receptors within mammalian carcinoembryonic antigen families

    PubMed Central

    2010-01-01

    Background Most rapidly evolving gene families are involved in immune responses and reproduction, two biological functions which have been assigned to the carcinoembryonic antigen (CEA) gene family. To gain insights into evolutionary forces shaping the CEA gene family we have analysed this gene family in 27 mammalian species including monotreme and marsupial lineages. Results Phylogenetic analysis provided convincing evidence that the primordial CEA gene family in mammals consisted of five genes, including the immune inhibitory receptor-encoding CEACAM1 (CEA-related cell adhesion molecule) ancestor. Our analysis of the substitution rates within the nucleotide sequence which codes for the ligand binding domain of CEACAM1 indicates that the selection for diversification is, perhaps, a consequence of the exploitation of CEACAM1 by a variety of viral and bacterial pathogens as their cellular receptor. Depending on the extent of the amplification of an ancestral CEACAM1, the number of CEACAM1-related genes varies considerably between mammalian species from less than five in lagomorphs to more than 100 in bats. In most analysed species, ITAM (immunoreceptor tyrosine-based activation motifs) or ITAM-like motif-containing proteins exist which contain Ig-V-like, ligand binding domains closely related to that of CEACAM1. Human CEACAM3 is one such protein which can function as a CEACAM1 decoy receptor in granulocytes by mediating the uptake and destruction of specific bacterial pathogens via its ITAM-like motif. The close relationship between CEACAM1 and its ITAM-encoding relatives appears to be maintained by gene conversion and reciprocal recombination. Surprisingly, secreted CEACAMs resembling immunomodulatory CEACAM1-related trophoblast-specific pregnancy-specific glycoproteins (PSGs) found in humans and rodents evolved only in a limited set of mammals. The appearance of PSG-like genes correlates with invasive trophoblast growth in these species. Conclusions These

  6. 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

    2011-02-23

    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

  7. Enterotoxin/guanylin receptor type guanylyl cyclases in non-mammalian vertebrates.

    PubMed

    Nakauchi, Mina; Suzuki, Norio

    2005-05-01

    Cyclic GMP is a ubiquitous intracellular second messenger produced by guanylyl cyclases (GCs). The enterotoxin/guanylin receptor type membrane GC (designated as GC-C in mammals) is activated by exogenous ligands such as heat-stable enterotoxins (STa), small peptides secreted by some pathogenic strains of Escherichia coli which cause severe secretory diarrhea and also activated by endogenous ligands such as guanylin and uroguanylin. The STa/guanylin receptor type membrane GC, as well as other type membrane GCs, is composed of an extracellular domain, a single transmembrane domain, and an intracellular region comprising a kinase-like domain and a catalytic domain. The STa/guanylin receptor type membrane GC is identified in various vertebrates including fishes, amphibians, reptiles, and birds, implying that it serves some important and undefined physiological roles in the intestine of non-mammalian vertebrates, e.g. the regulation of water and salt absorption. In mammals, only a single membrane GC (GC-C) is known to be the STa/guanylin receptor. On the contrary, two membrane GC cDNAs are cloned from the intestine of the European eel Anguilla anguilla (GC-C1 and GC-C2) and the medaka fish Oryzias latipes (OlGC6 and OlGC9). OlGC6 and OlGC9 are structurally distinct and show different ligand responsibility. Accumulated evidences indicate that the transcriptional regulatory mechanism of the human GC-C gene is different from that of the corresponding medaka fish GC gene; the human GC-C gene is regulated by Cdx2 and/or HNF-4, and the medaka fish OlGC6 gene is regulated by OlPC4, which is a medaka fish homologue of the mammalian transcriptional positive co-factor 4 (PC4). Furthermore, the transcriptional regulatory mechanism of the OlGC9 gene is different from those of both the OlGC6 and human GC-C genes, indicating that the study on these two medaka fish GCs will be useful for further understanding of the STa/guanylin receptor type membrane GC in the vertebrates.

  8. Functional coupling of a mammalian somatostatin receptor to the yeast pheromone response pathway.

    PubMed Central

    Price, L A; Kajkowski, E M; Hadcock, J R; Ozenberger, B A; Pausch, M H

    1995-01-01

    A detailed analysis of structural and functional aspects of G-protein-coupled receptors, as well as discovery of novel pharmacophores that exert their effects on members of this class of receptors, will be facilitated by development of a yeast-based bioassay. To that end, yeast strains that functionally express the rat somatostatin receptor subtype 2 (SSTR2) were constructed. High-affinity binding sites for somatostatin ([125I-Tyr-11]S-14) comparable to those in native tissues were detected in yeast membrane extracts at levels equivalent to the alpha-mating pheromone receptor (Ste2p). Somatostatin-dependent growth of strains modified by deletion of genes encoding components of the pheromone response pathway was detected through induction of a pheromone-responsive HIS3 reporter gene, enabling cells to grow on medium lacking histidine. Dose-dependent growth responses to S-14 and related SSTR2 subtype-selective agonists that were proportional to the affinity of the ligands for SSTR2 were observed. The growth response required SSTR2, G alpha proteins, and an intact signal transduction pathway. The sensitivity of the bioassay was affected by intracellular levels of the G alpha protein. A mutation in the SST2 gene, which confers supersensitivity to pheromone, was found to significantly enhance the growth response to S-14. In sst2 delta cells, SSTR2 functionally interacted with both a chimeric yeast/mammalian G alpha protein and the yeast G alpha protein, Gpa1p; to promote growth. These yeast strains should serve as a useful in vivo reconstitution system for examination of molecular interactions of the G-protein-coupled receptors and G proteins. PMID:7565771

  9. 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

  10. Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study.

    PubMed

    Allard, M; Geoffre, S; Legendre, P; Vincent, J D; Simonnet, G

    1989-10-23

    An in vitro binding assay, using 125I-YLFQPQRFamide, a newly synthetized iodinated analog of FLFQPQRFamide, in which Phe1 (F) has been substituted by a Tyr (Y), was developed to demonstrate and characterize putative binding sites of this brain morphine modulating peptide. This radioligand bound in a time-dependent manner to rat spinal cord membrane preparation. This binding was dose-dependent, saturable and reversible. Both kinetic data and saturation measured at equilibrium lead to the existence of a homogenous population of high affinity binding sites with a Kd value of 0.09-0.1 nM and a maximal capacity Bmax of 14.5 +/- 2 fmol/mg protein. Results of competition experiments show that both FLFQPQRFamide and its analog YLFQPQRFamide had a similar capacity to inhibit the 125I-YLFQPQRFamide binding, suggesting that this radioiodinated analog is a good tool to study binding characteristics of FLFQPQRFamide receptors. The related octadecapeptide AGEGLSSPFWSLAAPQRFamide, another mammalian morphine modulating peptide competes for radioligand binding with similar potency. Our results also show that mu, delta and kappa opiate receptor agonists as well as the antagonist naloxone were not able to affect binding either in presence or in absence of 120 mM NaCl. Together, these data demonstrate that FLFQPQRFamide does not function as an endogenous opiate receptor antagonist and that is capacity to reduce opiate-induced analgesia is supported by specific binding sites.

  11. Aldehyde recognition and discrimination by mammalian odorant receptors via functional group-specific hydration chemistry.

    PubMed

    Li, Yadi; Peterlin, Zita; Ho, Jianghai; Yarnitzky, Tali; Liu, Min Ting; Fichman, Merav; Niv, Masha Y; Matsunami, Hiroaki; Firestein, Stuart; Ryan, Kevin

    2014-11-21

    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.

  12. Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors.

    PubMed

    Tomizawa, Motohiro; Casida, John E

    2003-01-01

    Neonicotinoids, the most important new class of synthetic insecticides of the past three decades, are used to control sucking insects both on plants and on companion animals. Imidacloprid (the principal example), nitenpyram, acetamiprid, thiacloprid, thiamethoxam, and others act as agonists at the insect nicotinic acetylcholine receptor (nAChR). The botanical insecticide nicotine acts at the same target without the neonicotinoid level of effectiveness or safety. Fundamental differences between the nAChRs of insects and mammals confer remarkable selectivity for the neonicotinoids. Whereas ionized nicotine binds at an anionic subsite in the mammalian nAChR, the negatively tipped ("magic" nitro or cyano) neonicotinoids interact with a proposed unique subsite consisting of cationic amino acid residue(s) in the insect nAChR. Knowledge reviewed here of the functional architecture and molecular aspects of the insect and mammalian nAChRs and their neonicotinoid-binding site lays the foundation for continued development and use of this new class of safe and effective insecticides.

  13. 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. © 2016 Elsevier Inc. All rights reserved.

  14. The role of tumor necrosis factor receptor superfamily members in mammalian brain development, function and homeostasis

    PubMed Central

    Twohig, Jason P.; Cuff, Simone M.; Yong, Audrey A.; Wang, Eddie C.Y.

    2012-01-01

    Tumor necrosis factor receptor superfamily (TNFRSF) members were initially identified as immunological mediators, and are still commonly perceived as immunological molecules. However, our understanding of the diversity of TNFRSF members’ roles in mammalian physiology has grown significantly since the first discovery of TNFRp55 (TNFRSF1) in 1975. In particular, the last decade has provided evidence for important roles in brain development, function and the emergent field of neuronal homeostasis. Recent evidence suggests that TNFRSF members are expressed in an overlapping regulated pattern during neuronal development, participating in the regulation of neuronal expansion, growth, differentiation and regional pattern development. This review examines evidence for non-immunological roles of TNFRSF members in brain development, function and maintenance under normal physiological conditions. In addition, several aspects of brain function during inflammation will also be described, when illuminating and relevant to the non-immunological role of TNFRSF members. Finally, key questions in the field will be outlined. PMID:21861782

  15. The role of tumor necrosis factor receptor superfamily members in mammalian brain development, function and homeostasis.

    PubMed

    Twohig, Jason P; Cuff, Simone M; Yong, Audrey A; Wang, Eddie C Y

    2011-01-01

    Tumor necrosis factor receptor superfamily (TNFRSF) members were initially identified as immunological mediators, and are still commonly perceived as immunological molecules. However, our understanding of the diversity of TNFRSF members' roles in mammalian physiology has grown significantly since the first discovery of TNFRp55 (TNFRSF1) in 1975. In particular, the last decade has provided evidence for important roles in brain development, function and the emergent field of neuronal homeostasis. Recent evidence suggests that TNFRSF members are expressed in an overlapping regulated pattern during neuronal development, participating in the regulation of neuronal expansion, growth, differentiation and regional pattern development. This review examines evidence for non-immunological roles of TNFRSF members in brain development, function and maintenance under normal physiological conditions. In addition, several aspects of brain function during inflammation will also be described, when illuminating and relevant to the non-immunological role of TNFRSF members. Finally, key questions in the field will be outlined.

  16. Pharmacological profile of zacopride and new quaternarized fluorobenzamide analogues on mammalian α7 nicotinic acetylcholine receptor.

    PubMed

    Bourdin, Céline M; Lebreton, Jacques; Mathé-Allainmat, Monique; Thany, Steeve H

    2015-08-15

    From quaternarization of quinuclidine enantiomers of 2-fluoro benzamide LMA10203 in dichloromethane, the corresponding N-chloromethyl derivatives LMA10227 and LMA10228 were obtained. Here, we compared the agonist action of known zacopride and its 2-fluoro benzamide analogues, LMA10203, LMA10227 and LMA10228 against mammalian homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We found that LMA10203 was a partial agonist of α7 receptor with a pEC50 value of 4.25 ± 0.06 μM whereas LMA10227 and LMA10228 were poorly active on α7 homomeric nicotinic receptor. LMA10227 and LMA10228 were identified as antagonists of acetylcholine-induced currents with IC50 values of 28.4 μM and 39.3 μM whereas LMA10203 and zacopride possessed IC50 values of 8.07 μM and 7.04 μM, respectively. Moreover, despite their IC50 values, LMA10227 was the most potent inhibitor of nicotine-induced current amplitudes (65.7 ± 2.1% inhibition). LMA10203 and LMA10228 had the same inhibitory effects (26.5 ± 7.5% and 33.2 ± 4.1%, respectively), whereas zacopride had no significant inhibitory effect (4.37 ± 4%) on nicotine-induced responses. Our results revealed different pharmacological properties between the four compounds on acetylcholine and nicotine currents. The mode of action of benzamide compounds may need to be reinterpreted with respect to the potential role of α7 receptor.

  17. Next-generation-sequencing-spectratyping reveals public T-cell receptor repertoires in pediatric very severe aplastic anemia and identifies a β chain CDR3 sequence associated with hepatitis-induced pathogenesis

    PubMed Central

    Krell, Pina F. I.; Reuther, Susanne; Fischer, Ute; Keller, Thomas; Weber, Stephan; Gombert, Michael; Schuster, Friedhelm R.; Asang, Corinna; Stepensky, Polina; Strahm, Brigitte; Meisel, Roland; Stoye, Jens; Borkhardt, Arndt

    2013-01-01

    Current diagnostic approaches that characterize T-cell deficiency by analyzing diversity of T-cell receptor sequences effectuate limited informational gain about the actual restrictiveness. For deeper insight into T-cell receptor repertoires we developed next-generation-sequencing-spectratyping, which employs high coverage Roche/454 sequencing of T-cell receptor (β)-chain amplicons. For automated analysis of high-throughput-sequencing data, we developed a freely available software, the TCR profiler. Gene usage, length, encoded amino acid sequence and sequence diversity of the complementarity determining region 3 were determined and comprehensively integrated into a novel complexity score. Repertoires of CD8+ T cells from children with idiopathic or hepatitis-induced very severe aplastic anemia (n=7), children two months after bone marrow transplantation (n=7) and healthy controls (children n=5, adults n=5) were analyzed. Complexity scores clearly distinguished between healthy and diseased, and even between different immune deficiency states. The repertoire of aplastic anemia patients was dominated by public (i.e. present in more than one person) T-cell receptor clonotypes, whereas only 0.2% or 1.9% were public in normal children and adults, respectively. The CDR3 sequence ASSGVGFSGANVLT was highly prevalent in 3 cases of hepatitis-induced anemia (15–32% of all sequences), but was only low expressed in idiopathic aplastic anemia (2–5%, n=4) or healthy controls (<1%). Fifteen high frequent sequences were present exclusively in aplastic anemia patients. Next-generation-sequencing-spectratyping allows in-depth analysis of T-cell receptor repertoires and their restriction in clinical samples. A dominating clonotype was identified in hepatitis-induced anemia that may be associated with disease pathogenesis and several aplastic-anemia-associated, putatively autoreactive clonotypes were sequenced. PMID:23716544

  18. The T Cell Response to the Contact Sensitizer Paraphenylenediamine Is Characterized by a Polyclonal Diverse Repertoire of Antigen-Specific Receptors

    PubMed Central

    Oakes, Theres; Popple, Amy Lee; Williams, Jason; Best, Katharine; Heather, James M.; Ismail, Mazlina; Maxwell, Gavin; Gellatly, Nichola; Dearman, Rebecca J.; Kimber, Ian; Chain, Benny

    2017-01-01

    Paraphenylenediamine (PPD) is a common component of hair dyes and black henna tattoos and can cause skin sensitization and allergic contact dermatitis (ACD). The cutaneous inflammatory reaction associated with ACD is driven by both CD4+ and CD8+ T cells. However, the characteristics of such responses with respect to clonal breadth and magnitude are poorly defined. In this study, we have characterized the in vitro recall response of peripheral blood T cells prepared from PPD-allergic individuals to a PPD–human serum albumin (HSA) conjugate (PPD–HSA). Quantitative high throughput sequencing was used to characterize the changes in the repertoire of T cell receptor (TCR) α and β genes after exposure to antigen in vitro. The PPD conjugate induced expansion of T cells carrying selected TCRs, with around 800 sequences (around 1%) being 8 or more times as abundant after culture than before. The expanded sequences showed strong skewing of V and J usage, consistent with an antigen-driven clonal expansion. The complementarity-determining region 3 sequences of the expanded TCRs could be grouped into several families of related amino acid sequence, but the overall diversity of the expanded sample was not much less than that of a random sample of the same size. The results suggest a model in which PPD–HSA conjugate stimulates a broad diversity of TCRs, with a wide range of stimulation strengths, which manifest as different degrees of in vitro expansion. PMID:28261218

  19. Quantitative T cell repertoire analysis by deep cDNA sequencing of T cell receptor α and β chains using next-generation sequencing (NGS)

    PubMed Central

    Fang, Hua; Yamaguchi, Rui; Liu, Xiao; Daigo, Yataro; Yew, Poh Yin; Tanikawa, Chizu; Matsuda, Koichi; Imoto, Seiya; Miyano, Satoru; Nakamura, Yusuke

    2015-01-01

    Immune responses play a critical role in various disease conditions including cancer and autoimmune diseases. However, to date, there has not been a rapid, sensitive, comprehensive, and quantitative analysis method to examine T-cell or B-cell immune responses. Here, we report a new approach to characterize T cell receptor (TCR) repertoire by sequencing millions of cDNA of TCR α and β chains in combination with a newly-developed algorithm. Using samples from lung cancer patients treated with cancer peptide vaccines as a model, we demonstrate that detailed information of the V-(D)-J combination along with complementary determining region 3 (CDR3) sequences can be determined. We identified extensive abnormal splicing of TCR transcripts in lung cancer samples, indicating the dysfunctional splicing machinery in T lymphocytes by prior chemotherapy. In addition, we found three potentially novel TCR exons that have not been described previously in the reference genome. This newly developed TCR NGS platform can be applied to better understand immune responses in many disease areas including immune disorders, allergies, and organ transplantations. PMID:25964866

  20. 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.

  1. 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

  2. Eye on the B-ALL: B-cell receptor repertoires reveal persistence of numerous B-lymphoblastic leukemia subclones from diagnosis to relapse

    PubMed Central

    Bashford-Rogers, R J M; Nicolaou, K A; Bartram, J; Goulden, N J; Loizou, L; Koumas, L; Chi, J; Hubank, M; Kellam, P; Costeas, P A; Vassiliou, G S

    2016-01-01

    The strongest predictor of relapse in B-cell acute lymphoblastic leukemia (B-ALL) is the level of persistence of tumor cells after initial therapy. The high mutation rate of the B-cell receptor (BCR) locus allows high-resolution tracking of the architecture, evolution and clonal dynamics of B-ALL. Using longitudinal BCR repertoire sequencing, we find that the BCR undergoes an unexpectedly high level of clonal diversification in B-ALL cells through both somatic hypermutation and secondary rearrangements, which can be used for tracking the subclonal composition of the disease and detect minimal residual disease with unprecedented sensitivity. We go on to investigate clonal dynamics of B-ALL using BCR phylogenetic analyses of paired diagnosis-relapse samples and find that large numbers of small leukemic subclones present at diagnosis re-emerge at relapse alongside a dominant clone. Our findings suggest that in all informative relapsed patients, the survival of large numbers of clonogenic cells beyond initial chemotherapy is a surrogate for inherent partial chemoresistance or inadequate therapy, providing an increased opportunity for subsequent emergence of fully resistant clones. These results frame early cytoreduction as an important determinant of long-term outcome. PMID:27211266

  3. Eye on the B-ALL: B-cell receptor repertoires reveal persistence of numerous B-lymphoblastic leukemia subclones from diagnosis to relapse.

    PubMed

    Bashford-Rogers, R J M; Nicolaou, K A; Bartram, J; Goulden, N J; Loizou, L; Koumas, L; Chi, J; Hubank, M; Kellam, P; Costeas, P A; Vassiliou, G S

    2016-12-01

    The strongest predictor of relapse in B-cell acute lymphoblastic leukemia (B-ALL) is the level of persistence of tumor cells after initial therapy. The high mutation rate of the B-cell receptor (BCR) locus allows high-resolution tracking of the architecture, evolution and clonal dynamics of B-ALL. Using longitudinal BCR repertoire sequencing, we find that the BCR undergoes an unexpectedly high level of clonal diversification in B-ALL cells through both somatic hypermutation and secondary rearrangements, which can be used for tracking the subclonal composition of the disease and detect minimal residual disease with unprecedented sensitivity. We go on to investigate clonal dynamics of B-ALL using BCR phylogenetic analyses of paired diagnosis-relapse samples and find that large numbers of small leukemic subclones present at diagnosis re-emerge at relapse alongside a dominant clone. Our findings suggest that in all informative relapsed patients, the survival of large numbers of clonogenic cells beyond initial chemotherapy is a surrogate for inherent partial chemoresistance or inadequate therapy, providing an increased opportunity for subsequent emergence of fully resistant clones. These results frame early cytoreduction as an important determinant of long-term outcome.

  4. The T Cell Response to the Contact Sensitizer Paraphenylenediamine Is Characterized by a Polyclonal Diverse Repertoire of Antigen-Specific Receptors.

    PubMed

    Oakes, Theres; Popple, Amy Lee; Williams, Jason; Best, Katharine; Heather, James M; Ismail, Mazlina; Maxwell, Gavin; Gellatly, Nichola; Dearman, Rebecca J; Kimber, Ian; Chain, Benny

    2017-01-01

    Paraphenylenediamine (PPD) is a common component of hair dyes and black henna tattoos and can cause skin sensitization and allergic contact dermatitis (ACD). The cutaneous inflammatory reaction associated with ACD is driven by both CD4+ and CD8+ T cells. However, the characteristics of such responses with respect to clonal breadth and magnitude are poorly defined. In this study, we have characterized the in vitro recall response of peripheral blood T cells prepared from PPD-allergic individuals to a PPD-human serum albumin (HSA) conjugate (PPD-HSA). Quantitative high throughput sequencing was used to characterize the changes in the repertoire of T cell receptor (TCR) α and β genes after exposure to antigen in vitro. The PPD conjugate induced expansion of T cells carrying selected TCRs, with around 800 sequences (around 1%) being 8 or more times as abundant after culture than before. The expanded sequences showed strong skewing of V and J usage, consistent with an antigen-driven clonal expansion. The complementarity-determining region 3 sequences of the expanded TCRs could be grouped into several families of related amino acid sequence, but the overall diversity of the expanded sample was not much less than that of a random sample of the same size. The results suggest a model in which PPD-HSA conjugate stimulates a broad diversity of TCRs, with a wide range of stimulation strengths, which manifest as different degrees of in vitro expansion.

  5. Analysis of T cell receptor repertoire of muscle-infiltrating T lymphocytes in polymyositis. Restricted V alpha/beta rearrangements may indicate antigen-driven selection.

    PubMed Central

    Mantegazza, R; Andreetta, F; Bernasconi, P; Baggi, F; Oksenberg, J R; Simoncini, O; Mora, M; Cornelio, F; Steinman, L

    1993-01-01

    Polymyositis is an inflammatory myopathy characterized by mononuclear cell infiltration of muscle tissue. Myocytotoxic T lymphocytes have been recognized in the infiltrates, but the muscle antigen, target of the immune attack, has not been identified. Molecular characterization of the variable regions of T cell receptors (TCRs) on the infiltrating lymphocytes can be expected to provide insights into the pathogenic process. The V alpha/beta TCR repertoire was investigated by RNA-PCR in muscle biopsies from 15 polymyositis patients and 16 controls (6 Duchenne muscular dystrophy and 10 with no inflammatory or dystrophic myopathy). A variety of rearranged variable TCR genes was found in polymyositis, V alpha 1, V alpha 5, V beta 1, and V beta 15 being the most common (present in 60-100% of patients). In Duchenne muscular dystrophy patients TCR V alpha or beta rearrangements were found although no restriction was observed; no rearrangements were found in muscles from the other controls. Sequence analysis revealed the presence of the J beta 2.1 region in 90% of the V beta 15 clones studied, no random N additions in the diversity region, and a common motif within the CDR3 region. These results suggest that selection of muscle-infiltrating T lymphocytes is antigen driven in polymyositis. Images PMID:8514895

  6. Assessment of T-cell receptor repertoire and clonal expansion in peripheral T-cell lymphoma using RNA-seq data.

    PubMed

    Gong, Qiang; Wang, Chao; Zhang, Weiwei; Iqbal, Javeed; Hu, Yang; Greiner, Timothy C; Cornish, Adam; Kim, Jo-Heon; Rabadan, Raul; Abate, Francesco; Wang, Xin; Inghirami, Giorgio G; McKeithan, Timothy W; Chan, Wing C

    2017-09-12

    T-cell clonality of peripheral T-cell lymphoma (PTCL) is routinely evaluated with a PCR-based method using genomic DNA. However, there are limitations with this approach. The purpose of this study was to determine the utility of RNA-seq for assessing T-cell clonality and T-cell antigen receptor (TCR) repertoire of the neoplastic T-cells in 108 PTCL samples. TCR transcripts, including complementarity-determining region 3 (CDR3) sequences, were assessed. In normal T cells, the CDR3 sequences were extremely diverse, without any clonotype representing more than 2% of the overall TCR population. Dominant clones could be identified in 65 out of 76 PTCL cases (86%) with adequate TCR transcript expression. In monoclonal cases, the dominant clone varied between 11% and 99% of TCRβ transcripts. No unique Vα or Vβ usage was observed. Small T-cell clones were often observed in T- and NK-cell tumors in a percentage higher than observed in reactive conditions. γ chain expression was very low in tumors expressing TCRαβ, but its expression level was high and clonality was detected in a TCRγδ expressing tumor. NK cell lymphoma (NKCL) did not express significant levels of TCR Vβ or Vγ genes. RNA-seq is a useful tool for detecting and characterizing clonal TCR rearrangements in PTCL.

  7. Invertebrate Iridescent Virus 6, a DNA Virus, Stimulates a Mammalian Innate Immune Response through RIG-I-Like Receptors

    PubMed Central

    Ahlers, Laura R. H.; Bastos, Reginaldo G.; Hiroyasu, Aoi

    2016-01-01

    Insects are not only major vectors of mammalian viruses, but are also host to insect-restricted viruses that can potentially be transmitted to mammals. While mammalian innate immune responses to arboviruses are well studied, less is known about how mammalian cells respond to viruses that are restricted to infect only invertebrates. Here we demonstrate that IIV-6, a DNA virus of the family Iridoviridae, is able to induce a type I interferon-dependent antiviral immune response in mammalian cells. Although IIV-6 is a DNA virus, we demonstrate that the immune response activated during IIV-6 infection is mediated by the RIG-I-like receptor (RLR) pathway, and not the canonical DNA sensing pathway via cGAS/STING. We further show that RNA polymerase III is required for maximal IFN-β secretion, suggesting that viral DNA is transcribed by this enzyme into an RNA species capable of activating the RLR pathway. Finally, we demonstrate that the RLR-driven mammalian innate immune response to IIV-6 is functionally capable of protecting cells from subsequent infection with the arboviruses Vesicular Stomatitis virus and Kunjin virus. These results represent a novel example of an invertebrate DNA virus activating a canonically RNA sensing pathway in the mammalian innate immune response, which reduces viral load of ensuing arboviral infection. PMID:27824940

  8. miRNAome analysis of the mammalian neuronal nicotinic acetylcholine receptor gene family.

    PubMed

    Hogan, Eric M; Casserly, Alison P; Scofield, Michael D; Mou, Zhongming; Zhao-Shea, Rubing; Johnson, Chris W; Tapper, Andrew R; Gardner, Paul D

    2014-12-01

    Nicotine binds to and activates a family of ligand-gated ion channels, neuronal nicotinic acetylcholine receptors (nAChRs). Chronic nicotine exposure alters the expression of various nAChR subtypes, which likely contributes to nicotine dependence; however, the underlying mechanisms regulating these changes remain unclear. A growing body of evidence indicates that microRNAs (miRNAs) may be involved in nAChR regulation. Using bioinformatics, miRNA library screening, site-directed mutagenesis, and gene expression analysis, we have identified a limited number of miRNAs that functionally interact with the 3'-untranslated regions (3' UTRs) of mammalian neuronal nAChR subunit genes. In silico analyses revealed specific, evolutionarily conserved sites within the 3' UTRs through which the miRNAs regulate gene expression. Mutating these sites disrupted miRNA regulation confirming the in silico predictions. In addition, the miRNAs that target nAChR 3' UTRs are expressed in mouse brain and are regulated by chronic nicotine exposure. Furthermore, we show that expression of one of these miRNAs, miR-542-3p, is modulated by nicotine within the mesocorticolimbic reward pathway. Importantly, overexpression of miR-542-3p led to a decrease in the protein levels of its target, the nAChR β2 subunit. Bioinformatic analysis suggests that a number of the miRNAs play a general role in regulating cholinergic signaling. Our results provide evidence for a novel mode of nicotine-mediated regulation of the mammalian nAChR gene family.

  9. 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

  10. [The role of the class A scavenger receptors, SR-A and MARCO, in the immune system. Part 1. The structure of receptors, their ligand binding repertoires and ability to initiate intracellular signaling].

    PubMed

    Józefowski, Szczepan

    2012-02-29

    Recognition of pathogens by innate immune cells is mediated by pattern recognition receptors (PRR), which include scavenger receptors (SR). The class A SR, SR-A/CD204 and MARCO, are characterized by the presence of collagenous and SR cysteine-rich domains in their extracellular portions. Both receptors are expressed mainly on macrophages and dendritic cells. Thanks to their ability to bind to a wide range of polyanionic ligands, the class A SR may participate in numerous functions of these cells, such as endocytosis, and adhesion to extracellular matrix and to other cells. Among SR-A ligands are oxidized lipoproteins and β-amyloid fibrils, which link SR-A to the pathogenesis of arteriosclerosis and Alzheimer's disease. Despite the demonstration of class A SR involvement in so many processes, the lack of selective ligands precluded reaching definite conclusions concerning their signaling abilities. Using specific receptor ligation with antibodies, we showed that SR-A and MARCO trigger intracellular signaling, modulating pro-inflammatory and microbicidal activities of macrophages. Surprisingly, despite similarities in structure and ligand binding repertoires, SR-A and MARCO exert opposite effects on interleukin-12 (IL-12) production in macrophages. SR-A ligation also stimulated H₂O₂ and IL-10 production, but had no effect on the release of several other cytokines. These limited effects of specific SR-A ligation contrast with generalized enhancement of immune responses observed in SR-A-deficient mice. Recent studies have revealed that many of these effects of SR-A deficiency may be caused by compensatory changes in the expression of other receptors and/or disinhibition of signal transduction from receptors belonging to the Toll/IL-1R family, rather than by the loss of the receptor function of SR-A.

  11. T-cell Receptor-optimized Peptide Skewing of the T-cell Repertoire Can Enhance Antigen Targeting*

    PubMed Central

    Ekeruche-Makinde, Julia; Clement, Mathew; Cole, David K.; Edwards, Emily S. J.; Ladell, Kristin; Miles, John J.; Matthews, Katherine K.; Fuller, Anna; Lloyd, Katy A.; Madura, Florian; Dolton, Garry M.; Pentier, Johanne; Lissina, Anna; Gostick, Emma; Baxter, Tiffany K.; Baker, Brian M.; Rizkallah, Pierre J.; Price, David A.; Wooldridge, Linda; Sewell, Andrew K.

    2012-01-01

    Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8+ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A27–35 (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8+ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8+ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201+ individuals that were capable of efficient HLA A*0201+ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties. PMID:22952231

  12. 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

  13. 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…

  14. Intrinsic and Regulated Gonadotropin-Releasing Hormone Receptor Gene Transcription in Mammalian Pituitary Gonadotrophs

    PubMed Central

    Janjic, Marija M.; Stojilkovic, Stanko S.; Bjelobaba, Ivana

    2017-01-01

    The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH), acting via its receptors (GnRHRs) expressed in pituitary gonadotrophs, represents a critical molecule in control of reproductive functions in all vertebrate species. GnRH-activated receptors regulate synthesis of gonadotropins in a frequency-dependent manner. The number of GnRHRs on the plasma membrane determines the responsiveness of gonadotrophs to GnRH and varies in relation to age, sex, and physiological status. This is achieved by a complex control that operates at transcriptional, translational, and posttranslational levels. This review aims to overview the mechanisms of GnRHR gene (Gnrhr) transcription in mammalian gonadotrophs. In general, Gnrhr exhibits basal and regulated transcription activities. Basal Gnrhr transcription appears to be an intrinsic property of native and immortalized gonadotrophs that secures the presence of a sufficient number GnRHRs to preserve their functionality independently of the status of regulated transcription. On the other hand, regulated transcription modulates GnRHR expression during development, reproductive cycle, and aging. GnRH is crucial for regulated Gnrhr transcription in native gonadotrophs but is ineffective in immortalized gonadotrophs. In rat and mouse, both basal and GnRH-induced Gnrhr transcription rely primarily on the protein kinase C signaling pathway, with subsequent activation of mitogen-activated protein kinases. Continuous GnRH application, after a transient stimulation, shuts off regulated but not basal transcription, suggesting that different branches of this signaling pathway control transcription. Pituitary adenylate cyclase-activating polypeptide, but not activins, contributes to the regulated transcription utilizing the protein kinase A signaling pathway, whereas a mechanisms by which steroid hormones modulate Gnrhr transcription has not been well characterized.

  15. Non-mammalian models reveal the role of alternative ligands for thyroid hormone receptors.

    PubMed

    Orozco, Aurea; Lazcano, Iván; Hernández-Puga, Gabriela; Olvera, Aurora

    2017-03-04

    Thyroid hormones, or THs, are well-known regulators of a wide range of biological processes that occur throughout the lifespan of all vertebrates. THs act through genomic mechanisms mediated by thyroid hormone receptors (TRs). The main product of the thyroid gland is thyroxine or T4, which can be further transformed by different biochemical pathways to produce at least 15 active or inactive molecules. T3, a product of T4 outer-ring deiodination, has been recognized as the main bioactive TH. However, growing evidence has shown that other TH derivatives are able to bind to, and/or activate TRs, to induce thyromimetic effects. The compiled data in this review points to at least two of these TR alternative ligands: TRIAC and T2. Taking this into account, non-mammalian models have proven to be advantageous to explore new TH derivatives with potential novel actions, prompting a re-evaluation of the role and mechanism of action of TR alternative ligands that were previously believed to be inactive. The functional implications of these ligands across different vertebrates may require us to reconsider current established notions of thyroid physiology. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. 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.

  17. Pharmacological and biochemical properties of the benzodiazepine-GABA receptor in codfish brain in comparison with mammalian brain

    SciTech Connect

    Deng, L.

    1989-01-01

    The GABA receptor of codfish brain is encoded by an ancestral gene of the mammalian GABA receptor based on phylogenetic studies. The mammalian GABA receptor consists of at least two subunits ({beta} and {alpha}) which could be photoaffinity labeled by the GABA agonist ({sup 3}H)muscimol (57 kDa) and the benzodiazepine (BZ) agonist ({sup 3}H)flunitrazepam (52 kDa), respectively. In contrast, electrophoresis of codfish GABA receptor photoaffinity labeled by the same ligands showed a single radioactive peak on sodium dodecyl surface polyarcylamide gel, giving rise to a relative molecular weight of 56-57 kDa equivalent to the {beta} subunit of 57 kDa in mammals. The homogeneity of purified receptor using benzodiazepine (Ro 7-1986/1) affinity chromatography was further verified by two-dimensional gel electrophoresis based on isoelectric point and molecular weight, in addition to a single band on a silver stained gel and specific activity. The receptor density and affinity constant for ({sup 3}H)muscimol and ({sup 3}H)flunitrazepam are comparable to those in bovine, rate, and human brain.

  18. 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

  19. A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains.

    PubMed

    Kitaura, Kazutaka; Shini, Tadasu; Matsutani, Takaji; Suzuki, Ryuji

    2016-10-11

    High-throughput sequencing of T cell receptor (TCR) genes is a powerful tool for analyses of antigen specificity, clonality and diversity of T lymphocytes. Here, we developed a new TCR repertoire analysis method using 454 DNA sequencing technology in combination with an adaptor-ligation mediated polymerase chain reaction (PCR). This method allows the amplification of all TCR genes without PCR bias. To compare gene usage, diversity and similarity of expressed TCR repertoires among individuals, we conducted next-generation sequencing (NGS) of TRA and TRB genes in peripheral blood mononuclear cells from 20 healthy human individuals. From a total of 267,037 sequence reads from 20 individuals, 149,216 unique sequence reads were identified. Preferential usage of several V and J genes were observed while some recombinations of TRAV with TRAJ appeared to be restricted. The extent of TCR diversity was not significantly different between TRA and TRB, while TRA repertoires were more similar between individuals than TRB repertoires were. The interindividual similarity of TRA depended largely on the frequent presence of shared TCRs among two or more individuals. A publicly available TRA had a near-germline TCR with a shorter CDR3. Notably, shared TRA sequences, especially those shared among a large number of individuals', often contained TCRα related with invariant TCRα derived from invariant natural killer T cells and mucosal-associated invariant T cells. These results suggest that retrieval of shared TCRs by NGS would be useful for the identification of potential new invariant TCRα chains. This NGS method will enable the comprehensive quantitative analysis of TCR repertoires at a clonal level.

  20. Influence of the Route of Infection on Development of T-Cell Receptor β-Chain Repertoires of Reovirus-Specific Cytotoxic T Lymphocytes

    PubMed Central

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

    2004-01-01

    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 β-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 β-chain 6 (Vβ6) expanded in the spleens of mice infected by either route and in CTL lines established from the spleens and draining lymphoid tissues. Adoptively transferred Vβ6+ 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 Vβ6+ cells were enriched for clones utilizing uniform complementarity-determining region 3 (CDR3) lengths. However, sequencing of CDR3β regions from Vβ6+ CD8+ cells indicated that Jβ 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

  1. Impact of cell culture media on the expansion efficiency and T-cell receptor Vbeta (TRBV) repertoire of in vitro expanded T cells using feeder cells.

    PubMed

    Block, Andrea; Rohde, Maria; Erben, Ulrike; Hammer, Markus; Hummel, Michael; Blunert, Katja; Schultheiss, Heinz-Peter; Volk, Hans-Dieter; Noutsias, Michel

    2008-05-01

    The effects of different cell culture media on expansion efficiency and alterations in T-cell receptor V beta (TRBV) expression of in vitro expanded lymphocytes are not well established. Low numbers of CD3+ T cells from peripheral blood lymphocytes of healthy donors were subjected to polyclonal in vitro expansion in the presence of autologous CD3-depleted mononuclear cells as feeder cells (FCs) and their numbers and TRBV expressions were compared in media containing human (HS-RPMI) or fetal bovine serum (FBS-RPMI), Panserin413, or X-Vivo 15TM designed for lymphocyte culture. During three courses of restimulation within 28 days with CD3-antibody (OKT-3), IL-2, and initial CD3+, T-cell: FC ratios of 1:50 lowered to 1:5 and T cells expanded more than 1,000-fold in the media containing complete sera. Loss of cluster formation, associated with expansion failure, was only observed in cultures using synthetic media and resulted in only about 70-fold expansion. Whereas TRVB expression as determined by real-time PCR was substantially altered after 14 days of culture in X-Vivo 15, at day 28 only T cells from long-term culture in HS-RPMI presented the initial TRBV composition. Culture media have substantial impact on in vitro T-cell expansion. In the presence of FCs, medium containing human serum is superior to synthetic media and FBS-RPMI for long-term culture regarding T-cell number and TRBV repertoire. In contrast, the synthetic media Panserin413 and XVivo15 show lower expansion efficiency and reproducibility and, as RPMI1640+10%FBS, can contribute to overstimulation of certain TRBVs at advanced culture time points.

  2. Paracrine regulation of the epithelial Na+ channel in the mammalian collecting duct by purinergic P2Y2 receptor tone.

    PubMed

    Pochynyuk, Oleh; Bugaj, Vladislav; Rieg, Timo; Insel, Paul A; Mironova, Elena; Vallon, Volker; Stockand, James D

    2008-12-26

    Growing evidence implicates a key role for extracellular nucleotides in cellular regulation, including of ion channels and renal function, but the mechanisms for such actions are inadequately defined. We investigated purinergic regulation of the epithelial Na+ channel (ENaC) in mammalian collecting duct. We find that ATP decreases ENaC activity in both mouse and rat collecting duct principal cells. ATP and other nucleotides, including UTP, decrease ENaC activity via apical P2Y2 receptors. ENaC in collecting ducts isolated from mice lacking this receptor have blunted responses to ATP. P2Y2 couples to ENaC via PLC; direct activation of PLC mimics ATP action. Tonic regulation of ENaC in the collecting duct occurs via locally released ATP; scavenging endogenous ATP and inhibiting P2 receptors, in the absence of other stimuli, rapidly increases ENaC activity. Moreover, ENaC has greater resting activity in collecting ducts from P2Y2-/- mice. Loss of collecting duct P2Y2 receptors in the knock-out mouse is the primary defect leading to increased ENaC activity based on the ability of direct PLC stimulation to decrease ENaC activity in collecting ducts from P2Y2-/- mice in a manner similar to ATP in collecting ducts from wild-type mice. These findings demonstrate that locally released ATP acts in an autocrine/paracrine manner to tonically regulate ENaC in mammalian collecting duct. Loss of this intrinsic regulation leads to ENaC hyperactivity and contributes to hypertension that occurs in P2Y2 receptor-/- mice. P2Y2 receptor activation by nucleotides thus provides physiologically important regulation of ENaC and electrolyte handling in mammalian kidney.

  3. Distinct and non-overlapping T cell receptor repertoires expanded by DNA vaccination in wild-type and HER-2 transgenic BALB/c mice.

    PubMed

    Rolla, Simona; Nicoló, Chiara; Malinarich, Silvia; Orsini, Massimiliano; Forni, Guido; Cavallo, Federica; Ria, Francesco

    2006-12-01

    Central tolerance to tumor-associated Ags is an immune-escape mechanism that significantly limits the TCR repertoires available for tumor eradication. The repertoires expanded in wild-type BALB/c and rat-HER-2/neu (rHER-2) transgenic BALB-neuT mice following DNA immunization against rHER-2 were compared by spectratyping the variable (V)beta and the joining (J)beta CDR 3. Following immunization, BALB/c mice raised a strong response. Every mouse used one or more CD8+ T cell rearrangements of the Vbeta9-Jbeta1.2 segments characterized by distinct length of the CDR3 and specific for 63-71 or 1206-1214 rHER-2 peptides. In addition, two CD4+ T cell rearrangements recurred in >50% of mice. Instead, BALB-neuT mice displayed a limited response to rHER-2. Their repertoire is smaller and uses different rearrangements confined to CD4+ T cells. Thus, central tolerance in BALB-neuT mice acts by silencing the BALB/c mice self-reactive repertoire and reducing the size of the CD8+ T cell component. CD8+ and CD4+ T cells from both wild-type and transgenic mice home to tumors. This definition of the T cell repertoires available is critical to the designing of immunological maneuvers able to elicit an effective immune reaction against HER-2-driven carcinogenesis.

  4. T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire

    PubMed Central

    1991-01-01

    We report here the first extensive study of a T cell repertoire for a class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) response. We have found that the T cell receptors (TCRs) carried by 28 H-2Kd-restricted CTL clones specific for a single Plasmodium berghei circumsporozoite nonapeptide are highly diverse in terms of V alpha, J alpha, and J beta segments and aminoacid composition of the junctional regions. However, despite this extensive diversity, a high proportion of the TCRs contain the same V beta segment. These results are in contrast to most previously reported T cell responses towards class II MHC-peptide complexes, where the TCR repertoires appeared to be much more limited. In our study, the finding of a dominant V beta in the midst of otherwise highly diverse TCRs suggests the importance of the V beta segment in shaping the T cell repertoire specific for a given MHC-peptide complex. As an additional finding, we observed that nearly all clones have rearranged both TCR alpha loci. Moreover, as many as one-third of the CTL clones that we analyzed apparently display two productive alpha rearrangements. This argues against a regulated model of sequential recombination at the alpha locus and consequently raises the question of whether allelic exclusion of the TCR alpha chain is achieved at all. PMID:1836010

  5. Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: Possible role in antigen-driven reactions in the absence of germinal centers

    PubMed Central

    Diaz, Marilyn; Greenberg, Andrew S.; Flajnik, Martin F.

    1998-01-01

    The new antigen receptor (NAR) gene in the nurse shark diversifies extensively by somatic hypermutation. It is not known, however, whether NAR somatic hypermutation generates the primary repertoire (like in the sheep) or rather is used in antigen-driven immune responses. To address this issue, the sequences of NAR transmembrane (Tm) and secretory (Sec) forms, presumed to represent the primary and secondary repertoires, respectively, were examined from the peripheral blood lymphocytes of three adult nurse sharks. More than 40% of the Sec clones but fewer than 11% of Tm clones contained five mutations or more. Furthermore, more than 75% of the Tm clones had few or no mutations. Mutations in the Sec clones occurred mostly in the complementarity-determining regions (CDR) with a significant bias toward replacement substitutions in CDR1; in Tm clones there was no significant bias toward replacements and only a low level of targeting to the CDRs. Unlike the Tm clones where the replacement mutational pattern was similar to that seen for synonymous changes, Sec replacements displayed a distinct pattern of mutations. The types of mutations in NAR were similar to those found in mouse Ig genes rather than to the unusual pattern reported for shark and Xenopus Ig. Finally, an oligoclonal family of Sec clones revealed a striking trend toward acquisition of glutamic/aspartic acid, suggesting some degree of selection. These data strongly suggest that hypermutation of NAR does not generate the repertoire, but instead is involved in antigen-driven immune responses. PMID:9826702

  6. Functional expression of mammalian receptors and membrane channels in different cells.

    PubMed

    Eifler, Nora; Duckely, Myriam; Sumanovski, Lazar T; Egan, Terrance M; Oksche, Alexander; Konopka, James B; Lüthi, Anita; Engel, Andreas; Werten, Paul J L

    2007-08-01

    In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other. SFV-transfected mammalian cell lines provide the closest to native environment for the expression of mammalian membrane proteins, and they exhibited the best overall performance. But depending on the protein, baculovirus-infected Sf9 cells performed almost as well as mammalian cells. The lowest expression levels for the proteins tested here were obtained in yeast.

  7. Bispecific T-cells Expressing Polyclonal Repertoire of Endogenous γδ T-cell Receptors and Introduced CD19-specific Chimeric Antigen Receptor

    PubMed Central

    Deniger, Drew C; Switzer, Kirsten; Mi, Tiejuan; Maiti, Sourindra; Hurton, Lenka; Singh, Harjeet; Huls, Helen; Olivares, Simon; Lee, Dean A; Champlin, Richard E; Cooper, Laurence JN

    2013-01-01

    Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR+γδ T cells were expanded on CD19+ artificial antigen-presenting cells (aAPC), which resulted in >109 CAR+γδ T cells from <106 total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR+γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19+ tumor cell lines compared with CARnegγδ T cells. CD19+ leukemia xenografts in mice were reduced with CAR+γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells. PMID:23295945

  8. The N-methyl-D-aspartate neurotransmitter receptor is a mammalian brain target for the dinoflagellate Pfiesteria piscicida toxin.

    PubMed

    El-Nabawi, A; Quesenberry, M; Saito, K; Silbergeld, E; Vasta, G; Eldefrawi, A

    2000-11-15

    Blooms of Pfiesteria piscicida, a dinoflagellate in eastern U.S. coastal rivers, are believed to secrete toxins that kill fish and produce short-term memory loss in humans. Only one or two of Pfiesteria's multiple stages secrete the toxin, and only under certain environmental conditions. Thus, neither the presence of Pfiesteria nor fish kill alone can be indicative of toxin presence. The objective of this study was to identify the mammalian molecular brain target for the toxin that is associated with decrements in memory. Seven rat brain neurotransmitter receptors were selected to study because of their reported roles in cognitive function: receptors for nicotine, muscarine, AMPA/kainate, N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid, and dopamine 1 and 2. The effects of 17 environmental and laboratory samples on radioactive ligand binding to these receptors were studied. Of the seven receptors, binding only to the NMDA receptor was inhibited by only the two Pfiesteria-containing waters (identified by PCR) that also killed fish, and not by any of the other 15 samples tested. It is suggested that inhibition of NMDA-receptor binding is the cause of memory loss in exposed humans. Thus, it could be a useful biomarker for the toxin's presence in rivers for decisions on closures and for identification of the fractions containing the toxin during its purification. Knowledge of the toxin's molecular target, and how it affects its function, also leads to suggestions for therapeutics to use in animal models.

  9. 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

    2008-08-06

    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.

  10. mom identifies a receptor for the Drosophila JAK/STAT signal transduction pathway and encodes a protein distantly related to the mammalian cytokine receptor family

    PubMed Central

    Chen, Hua-Wei; Chen, Xiu; Oh, Su-Wan; Marinissen, Maria J.; Gutkind, J. Silvio; Hou, Steven X.

    2002-01-01

    The JAK/STAT signal transduction pathway controls numerous events in Drosophila melanogaster development. Receptors for the pathway have yet to be identified. Here we have identified a Drosophila gene that shows embryonic mutant phenotypes identical to those in the hopscotch (hop)/JAK kinase and marelle (mrl)/Stat92e mutations. We named this gene master of marelle (mom). Genetic analyses place mom's function between upd (the ligand) and hop. We further show that cultured cells transfected with the mom gene bind UPD and activate the HOP/STAT92E signal transduction pathway. mom encodes a protein distantly related to the mammalian cytokine receptor family. These data show that mom functions as a receptor of the Drosophila JAK/STAT signal transduction pathway. PMID:11825879

  11. 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

  12. Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells

    PubMed Central

    Sapinoro, Ramil; Volcy, Ketna; Shanaka, W.W.; Rodrigo, I.; Schlesinger, Jacob J.; Dewhurst, Stephen

    2008-01-01

    Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcγRI, but not its associated γ chain, and was not supported by other FcγR family members (FcγRIIA, FcγRIIB and FcγRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1) . In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcγRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer. PMID:18191979

  13. Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells.

    PubMed

    Sapinoro, Ramil; Volcy, Ketna; Rodrigo, W W Shanaka I; Schlesinger, Jacob J; Dewhurst, Stephen

    2008-04-10

    Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcgammaRI, but not its associated gamma-chain, and was not supported by other FcgammaR family members (FcgammaRIIA, FcgammaRIIB, and FcgammaRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcgammaRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer.

  14. Insight into pattern of codon biasness and nucleotide base usage in serotonin receptor gene family from different mammalian species.

    PubMed

    Dass, J Febin Prabhu; Sudandiradoss, C

    2012-07-15

    5-HT (5-Hydroxy-tryptamine) or serotonin receptors are found both in central and peripheral nervous system as well as in non-neuronal tissues. In the animal and human nervous system, serotonin produces various functional effects through a variety of membrane bound receptors. In this study, we focus on 5-HT receptor family from different mammals and examined the factors that account for codon and nucleotide usage variation. A total of 110 homologous coding sequences from 11 different mammalian species were analyzed using relative synonymous codon usage (RSCU), correspondence analysis (COA) and hierarchical cluster analysis together with nucleotide base usage frequency of chemically similar amino acid codons. The mean effective number of codon (ENc) value of 37.06 for 5-HT(6) shows very high codon bias within the family and may be due to high selective translational efficiency. The COA and Spearman's rank correlation reveals that the nucleotide compositional mutation bias as the major factors influencing the codon usage in serotonin receptor genes. The hierarchical cluster analysis suggests that gene function is another dominant factor that affects the codon usage bias, while species is a minor factor. Nucleotide base usage was reported using Goldman, Engelman, Stietz (GES) scale reveals the presence of high uracil (>45%) content at functionally important hydrophobic regions. Our in silico approach will certainly help for further investigations on critical inference on evolution, structure, function and gene expression aspects of 5-HT receptors family which are potential antipsychotic drug targets.

  15. Repertoire Is the Curriculum.

    ERIC Educational Resources Information Center

    Reynolds, Robert

    2000-01-01

    Focuses on repertoire selection, addressing issues such as beginning the process, keeping lists and resources for future use, seeing concerts as sharing experiences, and problems to avoid. Discusses the importance of networking and offers suggestions for enabling music educators to grow as teachers. (CMK)

  16. 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.

  17. Histamine H4 receptor antagonists as potent modulators of mammalian vestibular primary neuron excitability

    PubMed Central

    Desmadryl, G; Gaboyard-Niay, S; Brugeaud, A; Travo, C; Broussy, A; Saleur, A; Dyhrfjeld-Johnsen, J; Wersinger, E; Chabbert, C

    2012-01-01

    BACKGROUND AND PURPOSE Betahistine, the main histamine drug prescribed to treat vestibular disorders, is a histamine H3 receptor antagonist. Here, we explored the potential for modulation of the most recently cloned histamine receptor (H4 receptor) to influence vestibular system function, using a selective H4 receptor antagonist JNJ 7777120 and the derivate compound JNJ 10191584. EXPERIMENTAL APPROACH RT-PCR was used to assess the presence of H4 receptors in rat primary vestibular neurons. In vitro electrophysiological recordings and in vivo behavioural approaches using specific antagonists were employed to examine the effect of H4 receptor modulation in the rat vestibular system. KEY RESULTS The transcripts of H4 and H3 receptors were present in rat vestibular ganglia. Application of betahistine inhibited the evoked action potential firing starting at micromolar range, accompanied by subsequent strong neuronal depolarization at higher concentrations. Conversely, reversible inhibitory effects elicited by JNJ 10191584 and JNJ 7777120 began in the nanomolar range, without inducing neuronal depolarization. This effect was reversed by application of the selective H4 receptor agonist 4-methylhistamine. Thioperamide, a H3/H4 receptor antagonist, exerted effects similar to those of H3 and H4 receptor antagonists, namely inhibition of firing at nanomolar range and membrane depolarization above 100 µM. H4 receptor antagonists significantly alleviated the vestibular deficits induced in rats, while neither betahistine nor thioperamide had significant effects. CONCLUSIONS AND IMPLICATIONS H4 receptor antagonists have a pronounced inhibitory effect on vestibular neuron activity. This result highlights the potential role of H4 receptors as pharmacological targets for the treatment of vestibular disorders. PMID:22624822

  18. Classification of inhibitory amino acid receptors in the mammalian nervous system.

    PubMed

    Simmonds, M A

    1986-01-01

    Electrophysiological and pharmacological evidence is summarized for the existence of an inhibitory receptor system operated by glycine and another two separate systems operated by gamma-aminobutyric acid (GABA) through GABA-A and GABA-B receptors, respectively. Claims for subclasses of GABA-A receptor are critically reviewed and found not-proven. A quantitative pharmacological profile of the GABA-A receptor and associated regulatory sites for picrotoxin, barbiturates and benzodiazepines on the dorsal funiculus of the rat cuneate nucleus is described. When compared with this profile and the pharmacological properties of the glycine receptor complex, the effects of taurine cannot be entirely explained by actions on these two receptor systems.

  19. P2Y Receptors in the Mammalian Nervous System: Pharmacology, Ligands and Therapeutic Potential

    PubMed Central

    Weisman, Gary A.; Woods, Lucas T.; Erb, Laurie; Seye, Cheikh I.

    2015-01-01

    P2Y receptors for extracellular nucleotides are coupled to activation of a variety of G proteins and stimulate diverse intracellular signaling pathways that regulate functions of cell types that comprise the central nervous system (CNS). There are 8 different subtypes of P2Y receptor expressed in cells of the CNS that are activated by a select group of nucleotide agonists. Here, the agonist selectivity of these 8 P2Y receptor subtypes is reviewed with an emphasis on synthetic agonists with high potency and resistance to degradation by extracellular nucleotidases that have potential applications as therapeutic agents. In addition, the recent identification of a wide variety of subtype-selective antagonists is discussed, since these compounds are critical for discerning cellular responses mediated by activation of individual P2Y receptor subtypes. The functional expression of P2Y receptor subtypes in cells that comprise the CNS is also reviewed and the role of each subtype in the regulation of physiological and pathophysiological responses is considered. Other topics include the role of P2Y receptors in the regulation of blood-brain barrier integrity and potential interactions between different P2Y receptor subtypes that likely impact tissue responses to extracellular nucleotides in the CNS. Overall, current research suggests that P2Y receptors in the CNS regulate repair mechanisms that are triggered by tissue damage, inflammation and disease and thus P2Y receptors represent promising targets for the treatment of neurodegenerative diseases. PMID:22963441

  20. Erythrina mulungu Alkaloids Are Potent Inhibitors of Neuronal Nicotinic Receptor Currents in Mammalian Cells

    PubMed Central

    Setti-Perdigão, Pedro; Serrano, Maria A. R.; Flausino, Otávio A.; Bolzani, Vanderlan S.; Guimarães, Marília Z. P.; Castro, Newton G.

    2013-01-01

    Crude extracts and three isolated alkaloids from Erythrina mulungu plants have shown anxiolytic effects in different animal models. We investigated whether these alkaloids could affect nicotinic acetylcholine receptors and if they are selective for different central nervous system (CNS) subtypes. Screening experiments were performed using a single concentration of the alkaloid co-applied with acetylcholine in whole cell patch-clamp recordings in three different cell models: (i) PC12 cells natively expressing α3* nicotinic acetylcholine receptors; (ii) cultured hippocampal neurons natively expressing α7* nicotinic acetylcholine receptors; and (iii) HEK 293 cells heterologoulsy expressing α4β2 nicotinic acetylcholine receptors. For all three receptors, the percent inhibition of acetylcholine-activated currents by (+)-11á-hydroxyerysotrine was the lowest, whereas (+)-erythravine and (+)-11á-hydroxyerythravine inhibited the currents to a greater extent. For the latter two substances, we obtained concentration-response curves with a pre-application protocol for the α7* and α4β2 nicotinic acetylcholine receptors. The IC50 obtained with (+)-erythravine and (+)-11á-hydroxyerythravine were 6 µM and 5 µM for the α7* receptors, and 13 nM and 4 nM for the α4β2 receptors, respectively. Our data suggest that these Erythrina alkaloids may exert their behavioral effects through inhibition of CNS nicotinic acetylcholine receptors, particularly the α4β2 subtype. PMID:24349349

  1. Erythrina mulungu alkaloids are potent inhibitors of neuronal nicotinic receptor currents in mammalian cells.

    PubMed

    Setti-Perdigão, Pedro; Serrano, Maria A R; Flausino, Otávio A; Bolzani, Vanderlan S; Guimarães, Marília Z P; Castro, Newton G

    2013-01-01

    Crude extracts and three isolated alkaloids from Erythrina mulungu plants have shown anxiolytic effects in different animal models. We investigated whether these alkaloids could affect nicotinic acetylcholine receptors and if they are selective for different central nervous system (CNS) subtypes. Screening experiments were performed using a single concentration of the alkaloid co-applied with acetylcholine in whole cell patch-clamp recordings in three different cell models: (i) PC12 cells natively expressing α3* nicotinic acetylcholine receptors; (ii) cultured hippocampal neurons natively expressing α7* nicotinic acetylcholine receptors; and (iii) HEK 293 cells heterologoulsy expressing α4β2 nicotinic acetylcholine receptors. For all three receptors, the percent inhibition of acetylcholine-activated currents by (+)-11á-hydroxyerysotrine was the lowest, whereas (+)-erythravine and (+)-11á-hydroxyerythravine inhibited the currents to a greater extent. For the latter two substances, we obtained concentration-response curves with a pre-application protocol for the α7* and α4β2 nicotinic acetylcholine receptors. The IC50 obtained with (+)-erythravine and (+)-11á-hydroxyerythravine were 6 µM and 5 µM for the α7* receptors, and 13 nM and 4 nM for the α4β2 receptors, respectively. Our data suggest that these Erythrina alkaloids may exert their behavioral effects through inhibition of CNS nicotinic acetylcholine receptors, particularly the α4β2 subtype.

  2. Biochemical and immunochemical analysis of avian beta 1 and mammalian beta 2-adrenergic receptors.

    PubMed

    Chapot, M P; Cervantes, P; Kaveri, S; Durieu-Trautmann, O; Delavier-Klutchko, C; Emorine, L; Couraud, P O; Strosberg, A D

    1987-01-01

    We have studied the molecular properties of avian beta 1-adrenergic receptor and human beta 2-adrenergic receptor. The turkey erythrocytes beta 1-receptor has been solubilized in active form by digitonin and has been purified to homogeneity by affinity chromatography followed by electroelution from polyacrylamide gel. The photoactivable ligand, iodocyanopindololdiazirine, labels specifically a major 45 kDa and minor 55 kDa polypeptide in turkey erythrocytes, whereas in A431, it labels two polypeptides of molecular weights 65 kDa and 55 kDa. Both types of receptors are N- and possibly O-glycosylated but the turkey beta 1 receptor has only complex carbohydrates whereas the human beta 2 receptor has in addition oligo mannosidic polysaccharidic moiety. Polyclonal and monoclonal antibodies were raised against the beta 1- and beta 2-adrenergic receptors. Polyclonal antibodies were found to mimic beta-adrenergic agonists by stimulating adenylate cyclase upon binding to the receptors. The monoclonal antibodies precipitated both intact and affinity labeled receptors which they also revealed on immunoblots.

  3. Bakers yeast rises to the challenge: reconstitution of mammalian steroid receptor signalling in S. cerevisiae.

    PubMed

    McEwan, I J

    2001-05-01

    Steroid hormones are an important class of signalling molecule, regulating a diverse range of processes in metazoan eukaryotes. The actions of these hormones are mediated by intracellular receptor proteins that act as ligand-activated transcription factors. The ability to reconstitute steroid receptor signalling in the budding yeast, Saccharomyces cerevisiae, provides a genetically tractable model system in which to investigate steroid receptor structure and function. Through targeted disruption and genetic screening, an increasing number of genes have been identified that are likely to have a role in steroid receptor action.

  4. T cell receptor repertoire differences between African Americans and Caucasians associated with polymorphism of the TCRBV3S1 (V{beta}3.1) gene

    SciTech Connect

    De Inocencio, J.; Glass, D.N.; Hirsch, R.

    1995-05-01

    The generation of TCR diversity occurs primarily through rearrangement of germline DNA. Genetic polymorphism of the TCR chains appears to be a rarer mechanism for generating repertoire differences between races. Flow cytometric analysis of the TCR V{beta} repertoire in a population of healthy African Americans (n = 30) and Caucasians (n = 30) revealed a significant difference in the frequency of cells bearing V{beta}3.1, but not V{beta}2, V{beta}5.1, V{beta}5.2-5.3, V{beta}6.7, V{beta}8.1-8.2, V{beta}12.1, V{beta}13.3, or V{beta}19. African Americans had a significantly lower frequency of V{beta}3.1{sup +} cells, in both the CD4{sup +} (2.55 {+-} 0.36% vs 4.85 {+-} 0.43%, p = 0.0001) and the CD8{sup +} (3.03 {+-} 0.54% vs 5.32 {+-} 0.57%, p = 0.004) population than did Caucasians, and this difference was independent of the age of the individuals. Analysis of genomic DNA revealed that the observed differences in frequency of V{beta}3.1{sup +} cells correlated with a recently described polymorphism of the recombination signal sequence of the TCRBV3S1 gene. Allele 1, associated with a lower frequency of V{beta}3.1{sup +} cells, was more commonly present in African Americans (0.68 vs 0.43), whereas allele 2, associated with a higher frequency of V{beta}3.1{sup +} cells, was more commonly present in Caucasians (0.31 vs 0.56). This study demonstrates the potential for TCR repertoire differences, based on genetic polymorphism, between African Americans and Caucasians. 31 refs., 2 figs., 5 tabs.

  5. Expression of neurotrophins and their receptors in the mammalian ovary is developmentally regulated: changes at the time of folliculogenesis.

    PubMed

    Dissen, G A; Hirshfield, A N; Malamed, S; Ojeda, S R

    1995-10-01

    An emerging body of evidence suggests that neurotrophins not only promote neuronal survival and differentiation, but can also target nonneuronal cells for their actions. Neurotrophins initiate their biological effects by binding to cell membrane tyrosine kinase receptors of the trk protooncogene family. In addition, all neurotrophins recognize with similar affinity a different receptor molecule known as p75 nerve growth factor receptor (p75 NGFR) or low affinity NGFR, which appears to interact with the trk receptors to potentiate their response to neurotrophins. The mature mammalian ovary has been shown to synthesize several neurotrophins, including nerve growth factor (NGF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5). The ovary also expresses some of the neurotrophin receptors, including p75 NGFR, trkB [the receptor for NT-4/5 and brain-derived neurotropic factor (BDNF)], and trkA (the NGF receptor). The present experiments were undertaken to determine whether neurotrophins and their receptors are expressed at the time of definitive ovarian histogenesis, and whether any of them exhibit a developmental pattern of expression related to the completion of folliculogenesis. Immunohistochemical identification of p75 NGFR in rat embryonic ovaries revealed that the receptor is predominantly expressed in mesenchymal cells. By gestational day 18, these cells have formed pockets that enclose presumptive pregranulosa cells and groups of oocytes into ovigerous cords. Immediately after birth, the ovigerous cords are subdivided, resulting in the abrupt formation of primordial follicles between 24-48 h after birth. Consistent with these observations, the p75 NGFR messenger RNA (mRNA) content increased after birth and remained elevated at the time of follicular assembly. The NGF and trkA genes showed a different pattern of expression, as the ovarian content of both NGF and trkA mRNA decreased at the time of folliculogenesis. In contrast to the drop in NGF and trkA m

  6. 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.

  7. Molecular characterization and transcriptional analysis of non-mammalian type Toll like receptor (TLR21) from rock bream (Oplegnathus fasciatus).

    PubMed

    Priyathilaka, Thanthrige Thiunuwan; Elvitigala, Don Anushka Sandaruwan; Whang, Ilson; Lim, Bong-Soo; Jeong, Hyung-Bok; Yeo, Sang-Yeob; Choi, Cheol Young; Lee, Jehee

    2014-12-15

    Toll-like receptors (TLRs) are a large family of pattern recognition receptors, which are involved in triggering host immune responses against various pathogens by detecting their evolutionarily conserved pathogen associated molecular patterns (PAMPs). TLR21 is a non-mammalian type TLR, which recognizes unmethylated CpG DNA, and is considered as a functional homolog of mammalian TLR9. In this study, we attempted to identify and characterize a novel TLR21 counterpart from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at molecular level. The complete coding sequence of RbTLR21 was 2919bp in length, which encodes a polypeptide of 973 amino acids with a predicted molecular mass of 112kDa and a theoretical isoelectric point of 8.6. The structure of the deduced RbTLR21 protein is similar to that of the members of typical TLR family, and includes the ectodomain, which consists of 16 leucine rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. According to the pairwise sequence analysis data, RbTLR21 was homologous to that of the orange-spotted grouper (Epinephelus coioides) with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 is closely related to E. coioides TLR21. The RbTLR21 was ubiquitously expressed in all the tissues tested, but the highest expression was found in spleen. Additionally, upon stimulation with Streptococcus iniae, rock bream iridovirus (RBIV), and Edwardsiella tarda, RbTLR21 mRNA was significantly up-regulated in spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed an ortholog of the TLR21 family and may be important in mounting host immune responses against pathogenic infections. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Characterization of [3H]-CGP54626A binding to heterodimeric GABAB receptors stably expressed in mammalian cells

    PubMed Central

    Green, Andrew; Walls, Steven; Wise, Alan; Green, Richard H; Martin, Amanda K; Marshall, Fiona H

    2000-01-01

    Functional human GABAB(1a,2) and GABAB(1b,2) receptors have been stably expressed in mammalian CHO K1 cells.Detailed characterization of GABAB ligand binding at each of the receptors has been compared using [3H]-CGP54626A. In cell membranes fractions, [3H]-CGP54626A bound to a single site with a KD of 1.51±1.12 nM, Bmax of 2.02±0.17 pmoles mg protein−1 and 0.86±0.20 nM, Bmax of 5.19±0.57 pmoles mg protein−1 for GABAB(1a,2) and GABAB(1b,2) respectively.In competition binding assays the rank order was identical for both GABAB receptors. For known GABAB agonists the rank order was CGP27492>SKF97541=CGP46381>GABA>Baclofen and for GABAB antagonists the rank order was CGP54262A>CGP55845>CGP52432>SCH 50911>CGP51176>CGP36742=CGP35348 ⩾2-OH Saclofen ⩾ABPA.The allosteric effect of calcium cations was also investigated. The effect of removal of CaCl2 from the binding assay conditions was ligand dependent to either cause a decrease in ligand affinity or to have no significant effect. However, these effects were similar for both GABAB receptors.A whole cell, scintillation proximity binding assay was used to determine agonist affinity at exclusively heterodimeric GABAB receptors. In competition assays, the rank order was the same for both GABAB(1a,2) and GABAB(1b,2) and consistent with that seen with cell membrane fractions.These data suggest that, in terms of ligand binding, the currently identified isoforms of the GABAB receptor are pharmacologically indistinguishable. PMID:11139457

  9. 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.

  10. The myelin basic protein-specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential V beta 8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment.

    PubMed

    Kääb, G; Brandl, G; Marx, A; Wekerle, H; Bradl, M

    1996-05-01

    In the Lewis rat, myelin basic protein (MBP)-specific, encephalitogenic T cells preferentially recognize sequence 68-88, and use the V beta 8.2 gene to encode their T cell receptors. To analyze the structural prerequisites for the development of the MBP-specific T cell repertoire, we reconstituted severe-combined immunodeficient (SCID) mice with fetal (embryonic day 15-16) Lewis rat lymphoid tissue, and then isolated MBP-specific T cell lines from the adult chimeras after immunization. Two types of chimera were constructed: SCID mice reconstituted with rat fetal liver cells only, allowing T cell maturation within a chimeric SCID thymus consisting of mouse thymic epithelium and rat interdigitating dendritic cells, and SCID mice reconstituted with rat fetal liver cells and rat fetal thymus grafts, allowing T cell maturation within the chimeric SCID and the intact Lewis rat thymic microenvironment. Without exception, the T cell lines isolated from MBP-immunized SCID chimeras were restricted by MHC class II of the Lewis rat (RT1.B1), and none by I-Ad of the SCID mouse. Most of the T cell lines recognized the immunodominant MBP epitope 68-88. In striking contrast to intact Lewis rats, in SCID mice reconstituted by rat fetal liver only, MBP-specific T cell clones used a seemingly random repertoire of V beta genes without a bias for V beta 8.2. In chimeras containing fetal Lewis liver plus fetal thymus grafted under the kidney capsule, however, dominant utilization of V beta 8.2 was restored. The migration of liver-derived stem cells through rat thymus grafts was documented by combining fetal tissues from wild-type and transgenic Lewis rats. The results confirm that the recognition of the immunodominant epitope 68-88 by MBP-specific encephalitogenic T cells is a genetically determined feature of the Lewis rat T cell repertoire. They further suggest that the formation of the repertoire requires T cell differentiation in a syngeneic thymic microenvironment.

  11. 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

  12. Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors.

    PubMed

    Candelario, Manuel; Cuellar, Erika; Reyes-Ruiz, Jorge Mauricio; Darabedian, Narek; Feimeng, Zhou; Miledi, Ricardo; Russo-Neustadt, Amelia; Limon, Agenor

    2015-08-02

    Withania somnifera (WS) has been traditionally used in Ayurvedic medicine as a remedy for debility, stress, nervous exhaustion, insomnia, loss of memory, and to enhance cognitive function. This study provides an empirical evidence to support the traditional use of WS to aid in mental process engaging GABAergic signaling. We evaluated the effect of aqueous WS root extract (aqWS), and its two main components, withaferin A and withanolide A, on the main inhibitory receptors in the central nervous system: ionotropic GABAA receptors. The pharmacological activity of aqWS, withaferin A and withanolide A, was tested on native rat brain GABAA channels microtransplanted into Xenopus oocytes and GABAρ1 receptors heterologously expressed in oocytes. The GABAergic activity of aqWS compounds was evaluated by the two-electrode voltage-clamp method and the fingerprint of the extract was done by LC-MS. Concentration-dependent inward ion currents were elicited by aqWS in microtransplanted oocytes with an EC50 equivalent to 4.7 mg/mL and a Hill coefficient (nH) of 1.6. The GABAA receptor antagonist bicuculline blocked these currents. Our results show that aqWS activated inotropic GABAA channels but with lower efficacy compared to the endogenous agonist GABA. We also demonstrate for first time that aqWS is a potent agonist of GABAρ1 receptors. GABAρ1 receptors were 27 fold more sensitive to aqWS than GABAA receptors. Furthermore, aqWS activated GABAρ1 receptors eliciting maximum currents that were no significantly different to those produced by GABA (paired t-test; p=0.533). The differential activity on GABAA and GABA ρ1 receptors and the reported lack of significant GABA presence in WS root extract indicates that the GABAergic activity of aqWS is not mediated by GABA. WS main active components, witaferin A and withanolide A, were tested to determine if they were responsible for the activation of the GABA receptors. Neither compound activated GABAA nor GABAρ1 receptors

  13. 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.

  14. Nanoscale organization of beta2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells.

    PubMed

    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(2)-adrenergic receptors (beta(2)AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the beta(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(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(2)AR are observed in the membrane of the HEK293 cells that stably overexpress beta(2)AR-GFP and beta(2)AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use beta(2)AR-Venus fusion proteins as models for beta(2)AR function. These observations are critical for designing future cell models and assays based on beta(2)AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

  15. Lactococcal bacteriophage p2 receptor-binding protein structure suggests a common ancestor gene with bacterial and mammalian viruses.

    PubMed

    Spinelli, Silvia; Desmyter, Aline; Verrips, C Theo; de Haard, Hans J W; Moineau, Sylvain; Cambillau, Christian

    2006-01-01

    Lactococcus lactis is a Gram-positive bacterium used extensively by the dairy industry for the manufacture of fermented milk products. The double-stranded DNA bacteriophage p2 infects specific L. lactis strains using a receptor-binding protein (RBP) located at the tip of its noncontractile tail. We have solved the crystal structure of phage p2 RBP, a homotrimeric protein composed of three domains: the shoulders, a beta-sandwich attached to the phage; the neck, an interlaced beta-prism; and the receptor-recognition head, a seven-stranded beta-barrel. We used the complex of RBP with a neutralizing llama VHH domain to identify the receptor-binding site. Structural similarity between the recognition-head domain of phage p2 and those of adenoviruses and reoviruses, which invade mammalian cells, suggests that these viruses, despite evolutionary distant targets, lack of sequence similarity and the different chemical nature of their genomes (DNA versus RNA), might have a common ancestral gene.

  16. The anthelmintic pyrantel acts as a low efficacious agonist and an open-channel blocker of mammalian acetylcholine receptors.

    PubMed

    Rayes, D; De Rosa, M J; Spitzmaul, G; Bouzat, C

    2001-08-01

    Pyrantel is an anthelmintic which acts as an agonist of nicotinic receptors (AChRs) of nematodes and exerts its therapeutic effects by depolarizing their muscle membranes. Here we explore at the single-channel level the action of pyrantel at mammalian muscle AChR. AChR currents are elicited by pyrantel. However, openings do not appear in clearly identifiable clusters over a range of pyrantel concentrations (1-300 microM). The mean open time decreases as a function of concentration, indicating an additional open-channel block. Single-channel recordings in the presence of high ACh concentrations and pyrantel demonstrate that the anthelmintic acts as a high-affinity open-channel blocker. When analyzed in terms of a sequential blocking scheme, the calculated forward rate constant for the blocking process is 8x10(7) M(-1) x s(-1), the apparent dissociation constant is 8 microM at a membrane potential of -70 mV and the process is voltage dependent. Pyrantel displaces alpha-bungarotoxin binding but the concentration dependence of equilibrium binding is shifted towards higher concentrations with respect to that of ACh binding. Thus, by acting at the binding site pyrantel activates mammalian AChRs with low efficacy, and by sterical blockade of the pore, the activated channels are then rapidly inhibited.

  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. Expression and Function of Serotonin 2A and 2B Receptors in the Mammalian Respiratory Network

    PubMed Central

    Koch, Uwe R.; Bischoff, Anna-Maria; Kron, Miriam; Bock, Nathalie; Manzke, Till

    2011-01-01

    Neurons of the respiratory network in the lower brainstem express a variety of serotonin receptors (5-HTRs) that act primarily through adenylyl cyclase. However, there is one receptor family including 5-HT2A, 5-HT2B, and 5-HT2C receptors that are directed towards protein kinase C (PKC). In contrast to 5-HT2ARs, expression and function of 5-HT2BRs within the respiratory network are still unclear. 5-HT2BR utilizes a Gq-mediated signaling cascade involving calcium and leading to activation of phospholipase C and IP3/DAG pathways. Based on previous studies, this signal pathway appears to mediate excitatory actions on respiration. In the present study, we analyzed receptor expression in pontine and medullary regions of the respiratory network both at the transcriptional and translational level using quantitative RT-PCR and self-made as well as commercially available antibodies, respectively. In addition we measured effects of selective agonists and antagonists for 5-HT2ARs and 5-HT2BRs given intra-arterially on phrenic nerve discharges in juvenile rats using the perfused brainstem preparation. The drugs caused significant changes in discharge activity. Co-administration of both agonists revealed a dominance of the 5-HT2BR. Given the nature of the signaling pathways, we investigated whether intracellular calcium may explain effects observed in the respiratory network. Taken together, the results of this study suggest a significant role of both receptors in respiratory network modulation. PMID:21789169

  19. The complete swine olfactory subgenome: expansion of the olfactory gene repertoire in the pig genome.

    PubMed

    Nguyen, Dinh Truong; Lee, Kyooyeol; Choi, Hojun; Choi, Min-kyeung; Le, Minh Thong; Song, Ning; Kim, Jin-Hoi; Seo, Han Geuk; Oh, Jae-Wook; Lee, Kyungtae; Kim, Tae-Hun; Park, Chankyu

    2012-11-15

    Insects and animals can recognize surrounding environments by detecting thousands of chemical odorants. Olfaction is a complicated process that begins in the olfactory epithelium with the specific binding of volatile odorant molecules to dedicated olfactory receptors (ORs). OR proteins are encoded by the largest gene superfamily in the mammalian genome. We report here the whole genome analysis of the olfactory receptor genes of S. scrofa using conserved OR gene specific motifs and known OR protein sequences from diverse species. We identified 1,301 OR related sequences from the S. scrofa genome assembly, Sscrofa10.2, including 1,113 functional OR genes and 188 pseudogenes. OR genes were located in 46 different regions on 16 pig chromosomes. We classified the ORs into 17 families, three Class I and 14 Class II families, and further grouped them into 349 subfamilies. We also identified inter- and intra-chromosomal duplications of OR genes residing on 11 chromosomes. A significant number of pig OR genes (n = 212) showed less than 60% amino acid sequence similarity to known OR genes of other species. As the genome assembly Sscrofa10.2 covers 99.9% of the pig genome, our analysis represents an almost complete OR gene repertoire from an individual pig genome. We show that S. scrofa has one of the largest OR repertoires, suggesting an expansion of OR genes in the swine genome. A significant number of unique OR genes in the pig genome may suggest the presence of swine specific olfactory stimulation.

  20. Lineweaver-Burk analysis for the blocking effects of mammalian dopamine receptor antagonists on dopamine-induced currents in Achatina giant neurones.

    PubMed

    Emaduddin, M; Takeuchi, H

    1996-10-01

    1. We had demonstrated (Emaduddin et al., 1995) the blocking effects of the three mammalian dopamine receptor antagonists, (+/-)-SKF83566 (mammalian dopamine D1-like receptor antagonist), (+)-UH232 (D2 and D3-like receptor antagonist) and (+/-)-sulpiride (D2-like receptor antagonist) on the dose (pressure duration)-response curves of dopamine in the three giant neurone types, LVMN (left visceral multiple spike neurone), d-RPeAN (dorsal-right pedal anterior neurone) and v-LCDN (ventral-left cerebral distinct neurone), of Achatina fulica Férussac under voltage clamp. In the present study, we analyzed these data by Lineweaver-Burk plot. 2. Dopamine-induced inward currents (Iin) of the two neurone types, LVMN and d-RPeAN, were blocked by (+/-)-SKF83566 and (+)-UH232 in partly noncompetitive and partly uncompetitive manners. (+/-)-Sulpiride had no effect on these currents. 3. In contrast, dopamine-induced outward current (Iout) of v-LCDN was inhibited competitively by (+/-)-sulpiride and noncompetitively by (+)-UH232. (+/-)-SKF83566 had no effect on this current. 4. Therefore, we consider that the pharmacological features of the dopamine receptors of Achatina neurones are not identical in detail to those of the mammalian dopamine receptors.

  1. Epoxygenases and peroxisome proliferator-activated receptors in mammalian vascular biology.

    PubMed

    Wray, Jessica; Bishop-Bailey, David

    2008-01-01

    Epoxygenases, particularly of the CYP2C and CYP2J families, are important lipid-metabolizing enzymes. Epoxygenases are found throughout the cardiovascular system where their lipid products, particularly the epoxyeicosatrienoic acids (EETs), which are arachidonic acid metabolites, have the potential to regulate vascular tone, cellular proliferation, migration, inflammation and cardiac function. The receptors for EETs are, however, poorly understood. The peroxisome proliferator-activated receptors (PPARs) are a family of three (alpha, beta/delta and gamma) nuclear receptors that are activated by lipid metabolites. Activation of PPAR alpha and PPAR gamma, similar to the longer term effects of EETs, causes the inhibition of vascular cell proliferation, migration and inflammation. Interestingly, EETs and their metabolites have recently been found to active both PPAR alpha and PPAR gamma. The epoxygenase-EET-PPAR pathway may therefore represent a novel endogenous protective pathway by which short-lived lipid mediators control vascular cell activation.

  2. 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.

  3. 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

  4. 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…

  5. 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...

  6. 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...

  7. 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.

  8. 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…

  9. The sleep hormone oleamide modulates inhibitory ionotropic receptors in mammalian CNS in vitro

    PubMed Central

    Coyne, Leanne; Lees, George; Nicholson, Russell A; Zheng, Jian; Neufield, Katherine D

    2002-01-01

    We examine the sensitivity of GABAA and glycine receptors (same ionotropic superfamily) to oleamide. We address subunit-dependence/modulatory mechanisms and analogies with depressant drugs. Oleamide modulated human GABAA currents (α1β2γ2L) in oocytes (EC50, 28.94±s.e.mean of 1.4 μM; Maximum 216%±35 of control, n=4). Modulation of human α1 glycine homo-oligomers (significant), was less marked, with a lower EC50 (P<0.05) than GABA receptors (EC50, 22.12±1.4 μM; Maximum 171%±30, n=11). Only the hypnogenic cis geometric isomer enhanced glycine currents (without altering slope or maximal current, it reduced the glycine EC50 from 322 to 239 μM: P<0.001). Modulation was not voltage-dependent or associated with a shift in Er. β1 containing GABAA receptors (insensitive to many depressant drugs) were positively modulated by oleamide. Oleamide efficacy was circa 2× greater at α1β1γ2L than α1β2γ2L (P=0.007). Splice variation in γ subunits did not alter oleamide sensitivity. cis-9,10-Octadecenoamide had no effect on the equilibrium binding of [3H]-muscimol or [3H]-EBOB to mouse brain membranes. It does not directly mimic GABA, or operate as a neurosteroid-, benzodiazepine- or barbiturate-like modulator of GABAA-receptors. The transport of [3H]-GABA into mouse brain synaptoneurosomes was unaffected by high micromolar concentrations of cis-9,10-octadecenoamide. Oleamide does not enhance GABA-ergic currents or prolong IPSCs by inhibiting GABA transport. Oleamide is a non-selective modulator of inhibitory ionotropic receptors. The sleep lipid exerts its effects indirectly, or at a novel recognition site on the GABAA complex. PMID:11959801

  10. The sleep hormone oleamide modulates inhibitory ionotropic receptors in mammalian CNS in vitro.

    PubMed

    Coyne, Leanne; Lees, George; Nicholson, Russell A; Zheng, Jian; Neufield, Katherine D

    2002-04-01

    1. We examine the sensitivity of GABA(A) and glycine receptors (same ionotropic superfamily) to oleamide. We address subunit-dependence/modulatory mechanisms and analogies with depressant drugs. 2. Oleamide modulated human GABA(A) currents (alpha(1)beta(2)gamma(2L)) in oocytes (EC(50), 28.94+/-s.e.mean of 1.4 microM; Maximum 216%+/-35 of control, n=4). Modulation of human alpha1 glycine homo-oligomers (significant), was less marked, with a lower EC(50) (P<0.05) than GABA receptors (EC(50), 22.12+/-1.4 microM; Maximum 171%+/-30, n=11). 3. Only the hypnogenic cis geometric isomer enhanced glycine currents (without altering slope or maximal current, it reduced the glycine EC(50) from 322 to 239 microM: P<0.001). Modulation was not voltage-dependent or associated with a shift in E(r). 4. beta 1 containing GABA(A) receptors (insensitive to many depressant drugs) were positively modulated by oleamide. Oleamide efficacy was circa 2x greater at alpha(1)beta(1)gamma(2L) than alpha(1)beta(2)gamma(2L) (P=0.007). Splice variation in gamma subunits did not alter oleamide sensitivity. 5. cis-9,10-Octadecenoamide had no effect on the equilibrium binding of [(3)H]-muscimol or [(3)H]-EBOB to mouse brain membranes. It does not directly mimic GABA, or operate as a neurosteroid-, benzodiazepine- or barbiturate-like modulator of GABA(A)-receptors. 6. The transport of [(3)H]-GABA into mouse brain synaptoneurosomes was unaffected by high micromolar concentrations of cis-9,10-octadecenoamide. Oleamide does not enhance GABA-ergic currents or prolong IPSCs by inhibiting GABA transport. 7. Oleamide is a non-selective modulator of inhibitory ionotropic receptors. The sleep lipid exerts its effects indirectly, or at a novel recognition site on the GABA(A) complex.

  11. 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

  12. Dynamics of binaural processing in the mammalian sound localization pathway--the role of GABA(B) receptors.

    PubMed

    Grothe, Benedikt; Koch, Ursula

    2011-09-01

    The initial binaural processing in the superior olive represents the fastest computation known in the entire mammalian brain. Although the binaural system has to perform under very different and often highly dynamic acoustic conditions, the integration of binaural information in the superior olivary complex (SOC) has not been considered to be adaptive or dynamic itself. Recent evidence, however, shows that the initial processing of interaural level and interaural time differences relies on well-adjusted interactions of both the excitatory and the inhibitory projections, respectively. Under static conditions, these inputs seem to be tightly balanced, but may also require dynamic adjustment for proper function when the acoustic environment changes. GABA(B) receptors are at least one mechanism rendering the system more dynamic than considered so far. A comprehensive description of how binaural processing in the SOC is dynamically regulated by GABA(B) receptors in adults and in early development is important for understanding how spatial auditory processing changes with acoustic context. Copyright © 2011. Published by Elsevier B.V.

  13. Function and dysfunction of mammalian membrane guanylyl cyclase receptors: lessons from genetic mouse models and implications for human diseases.

    PubMed

    Kuhn, Michaela

    2009-01-01

    Besides soluble guanylyl cyclase (GC), the receptor for NO, there are seven plasma membrane forms of guanylyl cyclase (GC) receptors, enzymes that synthesize the second-messenger cyclic GMP (cGMP). All membrane GCs (GC-A to GC-G) share a basic topology, which consists of an extracellular ligand binding domain, a short transmembrane region, and an intracellular domain that contains the catalytic (GC) region. Although the presence of the extracellular domain suggests that all these enzymes function as receptors, specific ligands have been identified for only four of them (GC-A through GC-D). GC-A mediates the endocrine effects of atrial and B-type natriuretic peptides regulating arterial blood pressure and volume homeostasis and also local antihypertrophic and antifibrotic actions in the heart. GC-B, the specific receptor for C-type natriuretic peptide, has a critical role in endochondral ossification. GC-C mediates the effects of guanylin and uroguanylin on intestinal electrolyte and water transport and epithelial cell growth and differentiation. GC-E and GC-F are colocalized within the same photoreceptor cells of the retina and have an important role in phototransduction. Finally, GC-D and GC-G appear to be pseudogenes in the human. In rodents, GC-D is exclusively expressed in the olfactory neuroepithelium, with chemosensory functions. GC-G is the last member of the membrane GC form to be identified. No other mammalian transmembrane GCs are predicted on the basis of gene sequence repositories. In contrast to the other orphan receptor GCs, GC-G has a broad tissue distribution in rodents, including the lung, intestine, kidney, skeletal muscle, and sperm, raising the possibility that there is another yet to be discovered family of cGMP-generating ligands. This chapter reviews the structure and functions of membrane GCs, with special focus on the insights gained to date from genetically modified mice and the role of alterations of these ligand/receptor systems in human

  14. Ligand Specificity and Evolution of Mammalian Musk Odor Receptors: Effect of Single Receptor Deletion on Odor Detection.

    PubMed

    Sato-Akuhara, Narumi; Horio, Nao; Kato-Namba, Aya; Yoshikawa, Keiichi; Niimura, Yoshihito; Ihara, Sayoko; Shirasu, Mika; Touhara, Kazushige

    2016-04-20

    Musk odors have been used widely for fragrance and medicine for >2000 years because of their fascinating scent and physiological effects. Therefore, fragrance manufacturers have been eager to develop high-quality musk compounds that are safe and easily synthesized. We recently identified muscone-responsive olfactory receptors (ORs) MOR215-1 and OR5AN1 in mice and humans, respectively (Shirasu et al., 2014). In this study, we identified musk ORs that are evolutionarily closely related to MOR215-1 or OR5AN1 in various primates and investigated structure-activity relationships for various musk odorants and related compounds. We found that each species has one or two functional musk ORs that exhibit specific ligand spectra to musk compounds. Some of them, including the human OR5AN1, responded to nitro musks with chemical properties distinct from muscone. The ligand specificity of OR5AN1 reflects the perception of musk odors in humans. Genetic deletion of MOR215-1 in mice resulted in drastic reduction of sensitivity to muscone, suggesting that MOR215-1 plays a critical role in muscone perception. Therefore, the current study reveals a clear link between the identified OR and muscone perception. Moreover, the strategy established for screening ligands for the muscone OR may facilitate the development of novel and commercially useful musk odors. The long-sought musk odor receptor family in mammals was discovered and found to be well conserved and narrowly tuned to musk odors. In mice, deletion of the most sensitive musk receptor resulted in drastic reduction in sensitivity to muscone, demonstrating a strong link between receptor and odor perception. In humans, we found one musk receptor that recognized both macrocyclic and nitro musks that had distinct chemical structures. The structure-activity relationships were in a good agreement with human sensory perception and therefore may be used to develop novel musk aroma in fragrance fields. Finally, identification of a

  15. Early History of Glycine Receptor Biology in Mammalian Spinal Cord Circuits

    PubMed Central

    Callister, Robert John; Graham, Brett Anthony

    2010-01-01

    In this review we provide an overview of key in vivo experiments undertaken in the cat spinal cord in the 1950s and 1960s, and point out their contributions to our present understanding of glycine receptor (GlyR) function. Importantly, some of these discoveries were made well before an inhibitory receptor, or its agonist, was identified. These contributions include the universal acceptance of a chemical mode of synaptic transmission; that GlyRs are chloride channels; are involved in reciprocal and recurrent spinal inhibition; are selectively blocked by strychnine; and can be distinguished from the GABAA receptor by their insensitivity to bicuculline. The early in vivo work on inhibitory mechanisms in spinal neurons also contributed to several enduring principles on synaptic function, such as the time associated with synaptic delay, the extension of Dale's hypothesis (regarding the chemical unity of nerve cells and their terminals) to neurons within the central nervous system, and the importance of inhibition for synaptic integration in motor and sensory circuits. We hope the work presented here will encourage those interested in GlyR biology and inhibitory mechanisms to seek out and read some of the “classic” articles that document the above discoveries. PMID:20577630

  16. Analysis of hairless corepressor mutants to characterize molecular cooperation with the vitamin D receptor in promoting the mammalian hair cycle.

    PubMed

    Hsieh, Jui-Cheng; Slater, Stephanie A; Whitfield, G Kerr; Dawson, Jamie L; Hsieh, Grace; Sheedy, Craig; Haussler, Carol A; Haussler, Mark R

    2010-06-01

    The mammalian hair cycle requires both the vitamin D receptor (VDR) and the hairless (Hr) corepressor, each of which is expressed in the hair follicle. Hr interacts directly with VDR to repress VDR-targeted transcription. Herein, we further map the VDR-interaction domain to regions in the C-terminal half of Hr that contain two LXXLL-like pairs of motifs known to mediate contact of Hr with the RAR-related orphan receptor alpha and with the thyroid hormone receptor, respectively. Site-directed mutagenesis indicates that all four hydrophobic motifs are required for VDR transrepression by Hr. Point mutation of rat Hr at conserved residues corresponding to natural mutants causing alopecia in mice (G985W and a C-terminal deletion DeltaAK) and in humans (P95S, C422Y, E611G, R640Q, C642G, N988S, D1030N, A1040T, V1074M, and V1154D), as well as alteration of residues in the C-terminal Jumonji C domain implicated in histone demethylation activity (C1025G/E1027G and H1143G) revealed that all Hr mutants retained VDR association, and that transrepressor activity was selectively abrogated in C642G, G985W, N988S, D1030N, V1074M, H1143G, and V1154D. Four of these latter Hr mutants (C642G, N988S, D1030N, and V1154D) were found to associate normally with histone deacetylase-3. Finally, we identified three regions of human VDR necessary for association with Hr, namely residues 109-111, 134-201, and 202-303. It is concluded that Hr and VDR interact via multiple protein-protein interfaces, with Hr recruiting histone deacetylases and possibly itself catalyzing histone demethylation to effect chromatin remodeling and repress the transcription of VDR target genes that control the hair cycle. (c) 2010 Wiley-Liss, Inc.

  17. Analysis of Hairless Corepressor Mutants to Characterize Molecular Cooperation with the Vitamin D Receptor in Promoting the Mammalian Hair Cycle

    PubMed Central

    Hsieh, Jui-Cheng; Slater, Stephanie A.; Whitfield, G. Kerr; Dawson, Jamie L.; Hsieh, Grace; Sheedy, Craig; Haussler, Carol A.; Haussler, Mark R.

    2010-01-01

    The mammalian hair cycle requires both the vitamin D receptor (VDR) and the hairless (Hr) corepressor, each of which is expressed in the hair follicle. Hr interacts directly with VDR to repress VDR-targeted transcription. Herein, we further map the VDR-interaction domain to regions in the C-terminal half of Hr that contain two LXXLL-like pairs of motifs known to mediate contact of Hr with the RAR-related orphan receptor alpha and with the thyroid hormone receptor, respectively. Site-directed mutagenesis indicates that all four hydrophobic motifs are required for VDR transrepression by Hr. Point mutation of rat Hr at conserved residues corresponding to natural mutants causing alopecia in mice (G985W and a C-terminal deletion ΔAK) and in humans (P95S, C422Y, E611G, R640Q, C642G, N988S, D1030N, A1040T, V1074M and V1154D), as well as alteration of residues in the C-terminal Jumonji C domain implicated in histone demethylation activity (C1025G/E1027G and H1143G) revealed that all Hr mutants retained VDR association, and that transrepressor activity was selectively abrogated in C642G, G985W, N988S, D1030N, V1074M, H1143G and V1154D. Four of these latter Hr mutants (C642G, N988S, D1030N and V1154D) were found to associate normally with histone deacetylase-3. Finally, we identified three regions of human VDR necessary for association with Hr, namely residues 109–111, 134–201, and 202–303. It is concluded that Hr and VDR interact via multiple protein-protein interfaces, with Hr recruiting histone deacetylases and possibly itself catalyzing histone demethylation to effect chromatin remodeling and repress the transcription of VDR target genes that control the hair cycle. PMID:20512927

  18. Mechanisms of anabolic androgenic steroid inhibition of mammalian ɛ-subunit-containing GABAA receptors

    PubMed Central

    Jones, Brian L; Whiting, Paul J; Henderson, Leslie P

    2006-01-01

    GABAergic transmission regulates the activity of gonadotrophin-releasing hormone (GnRH) neurons in the preoptic area/hypothalamus that control the onset of puberty and the expression of reproductive behaviours. One of the hallmarks of illicit use of anabolic androgenic steroids (AAS) is disruption of behaviours under neuroendocrine control. GnRH neurons are among a limited population of cells that express high levels of the ɛ-subunit of the GABAA receptor. To better understand the actions of AAS on neuroendocrine mechanisms, we have characterized modulation of GABAA receptor-mediated currents in mouse native GnRH neurons and in heterologous cells expressing recombinant α2β3ɛ-receptors. GnRH neurons exhibited robust currents in response to millimolar concentrations of GABA and a picrotoxin (PTX)-sensitive, bicuculline-insensitive current that probably arises from spontaneous openings of GABAA receptors. The AAS 17α-methyltestosterone (17α-MeT) inhibited spontaneous and GABA-evoked currents in GnRH neurons. For recombinant α2β3ɛ-receptors, 17α-MeT inhibited phasic and tonic GABA-elicited responses, accelerated desensitization and slowed paired pulse response recovery. Single channel analysis indicated that GABA-evoked events could be described by three open dwell components and that 17α-MeT enhanced residence in the intermediate dwell state. This AAS also inhibited a PTX-sensitive, spontaneous current (open probability, ∼0.15–0.2) in a concentration-dependent fashion (IC50 ≈ 9 μm). Kinetic modelling indicated that the inhibition induced by 17α-MeT occurs by an allosteric block in which the AAS interacts preferentially with a closed state and promotes accumulation in that state. Finally, studies with a G302S mutant ɛ-subunit suggest that this residue within the transmembrane domain TM2 plays a role in mediating AAS binding and modulation. In sum, our results indicate that inclusion of the ɛ-subunit significantly alters the profile of AAS

  19. 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

  20. Analysis of the T-cell receptor beta-chain variable-region (V beta) repertoire in monozygotic twins discordant for human immunodeficiency virus: evidence for perturbations of specific V beta segments in CD4+ T cells of the virus-positive twins.

    PubMed Central

    Rebai, N; Pantaleo, G; Demarest, J F; Ciurli, C; Soudeyns, H; Adelsberger, J W; Vaccarezza, M; Walker, R E; Sekaly, R P; Fauci, A S

    1994-01-01

    We analyzed the T-cell receptor (TCR) V beta repertoire in human immunodeficiency virus type 1 (HIV-1)-infected individuals at different stages of disease. To circumvent the effect of HLA and other loci on the expressed TCR repertoire, we compared the TCR repertoire in nine pairs of monozygotic twins who were discordant for HIV infection. A semiquantitative polymerase chain reaction (PCR) assay and flow cytometry enabled us to show distinct differences in the V beta repertoire in the HIV-positive twin compared with the HIV-negative twin. By combining PCR and cytofluorometry, these differences were restricted to a specific set of TCR V beta segments, with members of the V beta 13 family perturbed in six out of seven cases and those of the V beta 21 family perturbed in four out of seven cases studied. Most of the other V beta families remained unchanged. Our results provide direct evidence for a skewed TCR repertoire in HIV infection. Images PMID:7906416

  1. 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

    Fève, Marie; Saliou, Jean-Michel; Zeniou, Maria; 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.

  2. Conjugated polymers mediate effective activation of the Mammalian Ion Channel Transient Receptor Potential Vanilloid 1.

    PubMed

    Lodola, F; Martino, N; Tullii, G; Lanzani, G; Antognazza, M R

    2017-08-16

    Selective and rapid regulation of ionic channels is pivotal to the understanding of physiological processes and has a crucial impact in developing novel therapeutic strategies. Transient Receptor Potential (TRP) channels are emerging as essential cellular switches that allow animals to respond to their environment. In particular, the Vanilloid Receptor 1 (TRPV1), besides being involved in the body temperature regulation and in the response to pain, has important roles in several neuronal functions, as cytoskeleton dynamics, injured neurons regeneration, synaptic plasticity. Currently available tools to modulate TRPV1 activity suffer from limited spatial selectivity, do not allow for temporally precise control, and are usually not reversible, thus limiting their application potential. The use of optical excitation would allow for overcoming all these limitations. Here, we propose a novel strategy, based on the use of light-sensitive, conjugated polymers. We demonstrate that illumination of a polymer thin film leads to reliable, robust and temporally precise control of TRPV1 channels. Interestingly, the activation of the channel is due to the combination of two different, locally confined effects, namely the release of thermal energy from the polymer surface and the variation of the local ionic concentration at the cell/polymer interface, both mediated by the polymer photoexcitation.

  3. Acquisition of a Reading Repertoire.

    ERIC Educational Resources Information Center

    Powell, William R.

    Drawing on results from informal reading inventories and studies of oral reading behavior, a theory is presented to describe the process of learning to read. Reading is seen to be broader than a skill and more like a repertoire with many abilities, aptitudes, and special accomplishments performing in concert. This repertoire is acquired in four…

  4. Administrative Leadership: Styles, Competencies, Repertoire.

    ERIC Educational Resources Information Center

    Lay-Dopyera, Margaret; Dopyera, John E.

    1985-01-01

    Although leadership has more frequently been viewed as a style or a set of competencies, the model of leadership as a function of repertoire has the advantage of providing a more adequate basis for selecting and preparing effective leaders for early childhood special education programs. The repertoire model, used with the other models, offers a…

  5. 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

  6. TCR repertoires of intratumoral T-cell subsets.

    PubMed

    Linnemann, Carsten; Mezzadra, Riccardo; Schumacher, Ton N M

    2014-01-01

    The infiltration of human tumors by T cells is a common phenomenon, and over the past decades, it has become increasingly clear that the nature of such intratumoral T-cell populations can predict disease course. Furthermore, intratumoral T cells have been utilized therapeutically in clinical studies of adoptive T-cell therapy. In this review, we describe how novel methods that are either based on T-cell receptor (TCR) sequencing or on cancer exome analysis allow the analysis of the tumor reactivity and antigen-specificity of the intratumoral TCR repertoire with unprecedented detail. Furthermore, we discuss studies that have started to utilize these techniques to probe the link between cancer exomes and the intratumoral TCR pool. Based on the observation that both the cancer epitope repertoire and intratumoral TCR repertoire appear highly individual, we outline strategies, such as 'autologous TCR gene therapy', that exploit the tumor-resident TCR repertoire for the development of personalized immunotherapy.

  7. lynx1, an endogenous toxin-like modulator of nicotinic acetylcholine receptors in the mammalian CNS.

    PubMed

    Miwa, J M; Ibanez-Tallon, I; Crabtree, G W; Sánchez, R; Sali, A; Role, L W; Heintz, N

    1999-05-01

    Elapid snake venom neurotoxins exert their effects through high-affinity interactions with specific neurotransmitter receptors. A novel murine gene, lynx1, is highly expressed in the brain and contains the cysteine-rich motif characteristic of this class of neurotoxins. Primary sequence and gene structure analyses reveal an evolutionary relationship between lynx1 and the Ly-6/neurotoxin gene family. lynx1 is expressed in large projection neurons in the hippocampus, cortex, and cerebellum. In cerebellar neurons, lynx1 protein is localized to a specific subdomain including the soma and proximal dendrites. lynx1 binding to brain sections correlates with the distribution of nAChRs, and application of lynx1 to Xenopus oocytes expressing nAChRs results in an increase in acetylcholine-evoked macroscopic currents. These results identify lynx1 as a novel protein modulator for nAChRs in vitro, which could have important implications in the regulation of cholinergic function in vivo.

  8. 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

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

    PubMed

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

    1993-07-01

    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. 6. The results are important in clarifying the mechanism of anxiety and might explain the anxioselectivity of BZR partial agonists.

  10. 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

  11. Activation of Presynaptic GABAB(1a,2) Receptors Inhibits Synaptic Transmission at Mammalian Inhibitory Cholinergic Olivocochlear–Hair Cell Synapses

    PubMed Central

    Wedemeyer, Carolina; Zorrilla de San Martín, Javier; Ballestero, Jimena; Gómez-Casati, María Eugenia; Torbidoni, Ana Vanesa; Fuchs, Paul A.; Bettler, Bernhard; Elgoyhen, Ana Belén

    2013-01-01

    The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca2+-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca2+-activated small-conductance type 2 (SK2)K+ channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABAB(1a,2) receptors [GABAB(1a,2)Rs] that downregulate the amount of ACh released at the OC–hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABABRs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABAB1–GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABAB1a isoform selectively inhibits release at efferent cholinergic synapses. PMID:24068816

  12. 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.

  13. 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

  14. 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.

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

    PubMed

    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.

  16. VCP, the mammalian homolog of cdc48, is tyrosine phosphorylated in response to T cell antigen receptor activation.

    PubMed Central

    Egerton, M; Ashe, O R; Chen, D; Druker, B J; Burgess, W H; Samelson, L E

    1992-01-01

    Activation of T cells through the T cell antigen receptor (TCR) results in the rapid tyrosine phosphorylation of a number of cellular proteins, one of the earliest being a 100 kDa protein. We have sought to identify this 100 kDa substrate by partially purifying the protein by antiphosphotyrosine (APT) affinity purification, in order to obtain amino acid sequence data and, using this information, to isolate the cDNA clone encoding the molecule. We report here that the amino acid sequence data showed pp100 to be the murine equivalent of porcine valosin containing protein (VCP), a finding confirmed from the cloning and sequencing of the murine pp100 cDNA. Sequence analysis has shown VCP to be a member of a family of ATP binding, homo-oligomeric proteins, and the mammalian homolog of Saccharomyces cerevisiae cdc48p, a protein essential to the completion of mitosis in yeast. We also provide proof that both endogenous and expressed murine VCP are tyrosine phosphorylated in response to T cell activation. Thus we have identified a novel component of the TCR mediated tyrosine kinase activation pathway that may provide a link between TCR ligation and cell cycle control. Images PMID:1382975

  17. [Relaxin-3 and relaxin family peptide receptors--from structure to functions of a newly discovered mammalian brain system].

    PubMed

    Kania, Alan; Lewandowski, Marian H; Błasiak, Anna

    2014-01-01

    Relaxin-3, a member of the relaxin peptide family, was discovered in 2001 as a homologue of relaxin--a well-known reproductive hormone. However, it is the brain which turned out to be a major expression site of this newly discovered peptide. Both its molecular structure and expression pattern were shown to be very conserved among vertebrates. Extensive research carried out since the discovery of relaxin-3 contributed to the significant progress in our knowledge regarding this neuropeptide. The endogenous relaxin-3 receptor (RXFP3) was identified and the anatomy of the yet uncharacterized mammalian brain system was described, with nucleus incertus as the main center of relaxin-3 expression. Not only its diffusive projections throughout the whole brain, which reach various brain structures such as the hippocampus, septum, intergeniculate leaflet or amygdala, but also functional studies of the relaxin-3/RXFP3 signaling system, allowed this brain network to be classified as one of the ascending nonspecific brain systems. Thus far, research depicts the connection of relaxin-3 with phenomena such as feeding behavior, spatial memory, sleep/wake cycle or modulation of pituitary gland hormone secretion. Responsiveness of relaxin-3 neurons to stress factors and the strong orexigenic effect exerted by this peptide suggest its participation in modulation of feeding by stress, in particular of the chronic type. The discovery of relaxin-3 opened a new research field which will contribute to our better understanding of the neurobiological basis of feeding disorders.

  18. Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation

    PubMed Central

    Jadhav, Bhalchandra; McKenna, Michael; Johnson, Nicholas; High, Stephen; Sinning, Irmgard; Pool, Martin R.

    2015-01-01

    Two distinct pathways deliver secretory proteins to the Sec61 protein translocase in the endoplasmic reticulum membrane. The canonical pathway requires the signal recognition particle (SRP) and its cognate receptor (SR), and targets ribosome-associated proteins to the Sec translocase. The SRP-independent pathway requires the Sec translocase-associated ER membrane protein Sec62 and can be uncoupled from translation. Here we show that SR switches translocons to SRP-dependent translocation by displacing Sec62. This activity localizes to the charged linker region between the longin and GTPase domains of SRα. Using truncation variants, crosslinking and translocation assays reveals two elements with distinct functions as follows: one rearranges the translocon, displacing Sec62 from Sec61. A second promotes ribosome binding and is conserved between all eukaryotes. These specific regions in SRα reprogramme the Sec translocon and facilitate recruitment of ribosome-nascent chain complexes. Overall, our study identifies an important function of SR, which mechanistically links two seemingly independent modes of translocation. PMID:26634806

  19. Isotopic labeling of mammalian G protein-coupled receptors (GPCRs) heterologously expressed in Caenorhabditis elegans*

    PubMed Central

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

    2015-01-01

    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 posttranslational 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 15N,13C by providing them with isotopically labeled bacteria. 2H labeling also was achieved by growing C. elegans in 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

  20. Human cytomegalovirus infection downregulates vitamin-D receptor in mammalian cells.

    PubMed

    Rieder, Franz J J; Gröschel, Charlotte; Kastner, Marie-Theres; Kosulin, Karin; Laengle, Johannes; Zadnikar, Rene; Marculescu, Rodrig; Schneider, Martina; Lion, Thomas; Bergmann, Michael; Kallay, Enikö; Steininger, Christoph

    2017-01-01

    Vitamin D (VD) is essential for the human body and involved in a wide variety of critical physiological processes including bone, muscle, and cardiovascular health, as well as innate immunity and antimicrobial responses. Here, we elucidated the significance of the VD system in cytomegalovirus (CMV) infection, which is one of the most common opportunistic infections in immunocompromised or -suppressed patients. We found that expression of vitamin D receptor (VDR) was downregulated in CMV-infected cells within 12h [hrs] post infection [p.i.] to 12% relative to VDR expression in mock-infected fibroblasts and did not recover during the CMV replication cycle of 96h. None of the biologically active metabolites of VD, cholecalciferol, calcidiol, or calcitriol, inhibit CMV replication significantly in human fibroblasts. In a feedback loop, expression of CYP24A1 dropped to 3% by 12h p.i. and expression of CYP27B1 increased gradually during the replication cycle of CMV to 970% probably as a consequence of VDR inhibition. VDR expression was not downregulated during influenza virus or adenovirus replication. The potent synthetic vitamin D analog EB-1089 was not able to inhibit CMV replication or antagonize its effect on VDR expression. Only CMV replication, and none of the other viral pathogens evaluated, inhibited the vitamin D system in vitro. In view of the pleiotropism of VDR, CMV-mediated downregulation may have far-reaching virological, immunological, and clinical implications and thus warrant further evaluations in vitro and in vivo.

  1. Ionotropic glutamate receptors: Which ones, when, and where in the mammalian neocortex.

    PubMed

    Hadzic, Minela; Jack, Alexander; Wahle, Petra

    2017-03-01

    A multitude of 18 iGluR receptor subunits, many of which are diversified by splicing and RNA editing, localize to >20 excitatory and inhibitory neocortical neuron types defined by physiology, morphology, and transcriptome in addition to various types of glial, endothelial, and blood cells. Here we have compiled the published expression of iGluR subunits in the areas and cell types of developing and adult cortex of rat, mouse, carnivore, bovine, monkey, and human as determined with antibody- and mRNA-based techniques. iGluRs are differentially expressed in the cortical areas and in the species, and all have a unique developmental pattern. Differences are quantitative rather than a mere absence/presence of expression. iGluR are too ubiquitously expressed and of limited use as markers for areas or layers. A focus has been the iGluR profile of cortical interneuron types. For instance, GluK1 and GluN3A are enriched in, but not specific for, interneurons; moreover, the interneurons expressing these subunits belong to different types. Adressing the types is still a major hurdle because type-specific markers are lacking, and the frequently used neuropeptide/CaBP signatures are subject to regulation by age and activity and vary as well between species and areas. RNA-seq reveals almost all subunits in the two morphofunctionally characterized interneuron types of adult cortical layer I, suggesting a fairly broad expression at the RNA level. It remains to be determined whether all proteins are synthesized, to which pre- or postsynaptic subdomains in a given neuron type they localize, and whether all are involved in synaptic transmission. J. Comp. Neurol. 525:976-1033, 2017. © 2016 Wiley Periodicals, Inc.

  2. 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

  3. 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

  4. Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells.

    PubMed

    Bergson, C; Mrzljak, L; Lidow, M S; Goldman-Rakic, P S; Levenson, R

    1995-04-11

    To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases.

  5. Evaluation of the repertoire of the TonB-dependent receptors of Haemophilus ducreyi for their role in virulence in humans.

    PubMed

    Leduc, Isabelle; Banks, Keith E; Fortney, Kate R; Patterson, Kristine B; Billings, Steve D; Katz, Barry P; Spinola, Stanley M; Elkins, Christopher

    2008-04-15

    Haemophilus ducreyi contains 3 TonB-dependent receptors: the hemoglobin receptor HgbA, which is required for virulence in humans; the heme receptor TdhA; and an uncharacterized conserved hypothetical protein TdX (HD0646). A double tdX/tdhA mutant (FX527) was constructed on the background of a human-passaged variant of strain 35000 (35000HP). Six volunteers were infected with 35000HP at 3 sites on one arm and with FX527 at 3 sites on the other. The pustule formation rate was 55.6% (95% confidence interval [CI], 35.7%-75.4%) at 18 parent-strain sites and 44.4% (95% CI, 15.0%-73.9%) at 18 mutant-strain sites (P = .51). Similar amounts of 35000HP and FX527 were recovered from pustules in semiquantitative culture. Thus, TdX and TdhA are not necessary for virulence, whereas HgbA is both necessary and sufficient for virulence in humans. The data suggest that hemoglobin is the sole source of heme/iron used by H. ducreyi in vivo and has implications for the potential of HgbA as a vaccine.

  6. 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

  7. High-throughput mapping of the promoters of the mouse olfactory receptor genes reveals a new type of mammalian promoter and provides insight into olfactory receptor gene regulation

    PubMed Central

    Clowney, E. Josephine; Magklara, Angeliki; Colquitt, Bradley M.; Pathak, Nidhi; Lane, Robert P.; Lomvardas, Stavros

    2011-01-01

    The olfactory receptor (OR) genes are the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion in olfactory neurons. Using a high-throughput approach, we mapped the transcription start sites of 1085 of the 1400 murine OR genes and performed computational analysis that revealed potential transcription factor binding sites shared by the majority of these promoters. Our analysis produced a hierarchical model for OR promoter recognition in which unusually high AT content, a unique epigenetic signature, and a stereotypically positioned O/E site distinguish OR promoters from the rest of the murine promoters. Our computations revealed an intriguing correlation between promoter AT content and evolutionary plasticity, as the most AT-rich promoters regulate rapidly evolving gene families. Within the AT-rich promoter category the position of the TATA-box does not correlate with the transcription start site. Instead, a spike in GC composition might define the exact location of the TSS, introducing the concept of “genomic contrast” in transcriptional regulation. Finally, our experiments show that genomic neighborhood rather than promoter sequence correlates with the probability of different OR genes to be expressed in the same olfactory cell. PMID:21705439

  8. Tumor Necrosis Factor Receptor Associated Factor 2 Signaling Provokes Adverse Cardiac Remodeling in the Adult Mammalian Heart

    PubMed Central

    Divakaran, Vijay G.; Evans, Sarah; Topkara, Veli K.; Diwan, Abhinav; Burchfield, Jana; Gao, Feng; Dong, Jianwen; Tzeng, Huei-Ping; Sivasubramanian, Natarajan; Barger, Philip M.; Mann, Douglas L.

    2013-01-01

    Background Tumor necrosis factor (TNF) superfamily ligands that provoke a dilated cardiac phenotype signal through a common scaffolding protein termed TNF receptor associated factor 2 (TRAF2); however, virtually nothing is known with regard to TRAF2 signaling in the adult mammalian heart. Methods and Results We generated multiple founder lines of mice with cardiac restricted overexpression of TRAF2 and characterized the phenotype of mice with higher expression levels of TRAF2 (MHC-TRAF2HC). MHC-TRAF2HC transgenic mice developed a time-dependent increase in cardiac hypertrophy, LV dilation and adverse LV remodeling, and a significant decrease in LV +dP/dt and −dP/dt when compared to littermate (LM) controls (p < 0.05 compared to LM). During the early phases of LV remodeling there was a significant increase in total matrix metalloproteinase (MMP) activity that corresponded with a decrease in total myocardial fibrillar collagen content. As the MHC-TRAF2HC mice aged, there was a significant decrease in total MMP activity accompanied by an increase in total fibrillar collagen content and an increase in myocardial tissue inhibitor of metalloproteinase-1 levels. There was a significant increase in NF-κB activation at 4 – 12 weeks and JNK activation at 4 weeks in the MHCs TRAF2HC mice. Transciptional profiling revealed that > 95% of the hypertrophic/dilated cardiomyopathy-related genes that were significantly upregulated genes in the MHC-TRAF2HC hearts contained κB elements in their promoters. Conclusions These results show for the first time that targeted overexpression of TRAF2 is sufficient to mediate adverse cardiac remodeling in the heart. PMID:23493088

  9. Cell Type Specific Spatial and Functional Coupling Between Mammalian Brain Kv2.1 K+ Channels and Ryanodine Receptors

    PubMed Central

    Mandikian, Danielle; Bocksteins, Elke; Parajuli, Laxmi Kumar; Bishop, Hannah I.; Cerda, Oscar; Shigemoto, Ryuichi; Trimmer, James S.

    2014-01-01

    The Kv2.1 voltage-gated K+ channel is widely expressed throughout mammalian brain where it contributes to dynamic activity-dependent regulation of intrinsic neuronal excitability. Here we show that somatic plasma membrane Kv2.1 clusters are juxtaposed to clusters of intracellular ryanodine receptor (RyR) Ca2+-release channels in mouse brain neurons, most prominently in medium spiny neurons (MSNs) of the striatum. Electron microscopy-immunogold labeling shows that in MSNs, plasma membrane Kv2.1 clusters are adjacent to subsurface cisternae, placing Kv2.1 in close proximity to sites of RyR-mediated Ca2+ release. Immunofluorescence labeling in transgenic mice expressing GFP in specific MSN populations reveals the most prominent juxtaposed Kv2.1-RyR clusters in indirect pathway MSNs. Kv2.1 in both direct and indirect pathway MSNs exhibits markedly lower levels of labeling with phosphospecific antibodies directed against the S453, S563, and S603 phosphorylation site compared to levels observed in neocortical neurons, although labeling for Kv2.1 phosphorylation at S563 was significantly lower in indirect pathway MSNs compared to those in the direct pathway. Finally, acute stimulation of RyRs in heterologous cells causes a rapid hyperpolarizing shift in the voltage-dependence of activation of Kv2.1, typical of Ca2+/calcineurin-dependent Kv2.1 dephosphorylation. Together, these studies reveal that striatal MSNs are distinct in their expression of clustered Kv2.1 at plasma membrane sites juxtaposed to intracellular RyRs, as well as in Kv2.1 phosphorylation state. Differences in Kv2.1 expression and phosphorylation between MSNs in direct and indirect pathways provide a cell- and circuit-specific mechanism for coupling intracellular Ca2+ release to phosphorylation-dependent regulation of Kv2.1 to dynamically impact intrinsic excitability. PMID:24962901

  10. 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

  11. 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

  12. Hacking the genetic code of mammalian cells.

    PubMed

    Schwarzer, Dirk

    2009-07-06

    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.

  13. Cycling Memory CD4+ T Cells in HIV Disease Have a Diverse T Cell Receptor Repertoire and a Phenotype Consistent with Bystander Activation

    PubMed Central

    Jiang, Wei; Younes, Souheil-Antoine; Funderburg, Nicholas T.; Mudd, Joseph C.; Espinosa, Enrique; Davenport, Miles P.; Babineau, Denise C.; Sieg, Scott F.

    2014-01-01

    ABSTRACT The mechanisms of increased memory CD4+ T cell cycling in HIV disease are incompletely understood but have been linked to antigen stimulation, homeostatic signals, or exposure to microbial products and the inflammatory cytokines that they induce. We examined the phenotype and Vβ family distribution in cycling memory CD4+ T cells among 52 healthy and 59 HIV-positive (HIV+) donors. Cycling memory CD4+ T cells were proportionally more frequent in subjects with HIV infection than in controls, more often expressed CD38 and PD-1, and less frequently expressed OX40 and intracellular CD40L. OX40 expression on memory CD4+ T cells was induced in vitro by anti-CD3, interleukin-2 (IL-2), IL-7, or IL-15 but not by Toll-like receptor ligands. In HIV+ donors, memory CD4+ T cell cycling was directly related to plasma lipopolysaccharide (LPS) levels, to plasma HIV RNA levels, and to memory CD8+ T cell cycling and was inversely related to peripheral blood CD4+ T cell counts but not to the levels of IL-2, IL-7, or IL-15, while in HIV-negative donors, memory CD4+ T cell cycling was related to IL-7 levels and negatively related to the plasma levels of LPS. In both controls and HIV+ donors, cycling memory CD4+ T cells had a broad distribution of Vβ families comparable to that of noncycling cells. Increased memory CD4+ T cell cycling in HIV disease is reflective of generalized immune activation and not driven primarily by cognate peptide stimulation or exposure to common gamma-chain cytokines. This cycling may be a consequence of exposure to microbial products, to plasma viremia, or, otherwise, to proinflammatory cytokines. IMPORTANCE This work provides evidence that the increased memory CD4+ T cell cycling in HIV infection is not a result of cognate peptide recognition but, rather, is more likely related to the inflammatory environment of HIV infection. PMID:24522925

  14. Dynamic Perturbations of CD4 and CD8 T Cell Receptor Repertoires in Chronic Hepatitis B Patients upon Oral Antiviral Therapy.

    PubMed

    Xu, Ying; Liu, Yu; Zhao, Miaoxian; Chen, Yunqing; Xie, Cantao; Gong, Mingxing; Deng, Haohui; Li, Xueying; Sun, Jian; Hou, Jinlin; Wu, Hongkai; Wang, Zhanhui

    2017-01-01

    Long-term treatment with nucleos(t)ide analogs (NUCs) can improve the antiviral T cell response in chronic hepatitis B (CHB) patients. Whether and to what extent the T cell response is improved by NUCs in the early stage leading to hepatitis B e antigen (HBeAg) seroconversion remain to be clarified. A total of 22 CHB patients undergoing 2-year telbivudine-based therapy were enrolled, including 10 exhibiting a complete response (CR) and 12 exhibiting a non-complete response (NCR) according to HBeAg seroconversion at week 52. Peripheral CD4(+) and CD8(+) T cells were sorted at baseline, weeks 12, and 24. The T cell receptor β chain (TCRβ) complementarity-determining region 3 was analyzed by unbiased high-throughput sequencing. Compared with NCR group, patients in CR group had a much lower percentage of persistent clonotypes (P < 0.001) but remarkably higher percentages of new and expanded clonotypes (P < 0.05) between any two time points for both CD4 and CD8 subsets. The CD4 T cells exhibited a stronger response than CD8 population in the patients. The number of new and expanded clonotypes was inversely associated with the decline of viral antigen. In conclusion, NUC-based therapy induces a broad and vigorous T cell response with rapid decline of antigenemia during the early stage of treatment. A broad T cell expansion is crucial for HBeAg seroconversion. Our findings suggest that the potent suppression of hepatitis B virus replication by NUC monotherapy complemented with additional immunomodulatory strategies may increase the likelihood of a functional cure for CHB in the future.

  15. 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

  16. 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

    Rosen, Meghan D; Chan, Ivan H; Privalsky, Martin L

    2011-08-01

    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.

  17. 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.

  18. 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

  19. Ultrasensitive detection of amines by a trace amine-associated receptor.

    PubMed

    Zhang, Jingji; Pacifico, Rodrigo; Cawley, Dillon; Feinstein, Paul; Bozza, Thomas

    2013-02-13

    The mammalian main olfactory pathway detects volatile chemicals using two families of G-protein-coupled receptors: a large repertoire of canonical odorant receptors and a much smaller set of trace amine-associated receptors (TAARs). The TAARs are evolutionarily conserved in vertebrates, including humans, suggesting an indispensible role in olfaction. However, little is known about the functional properties of TAARs when expressed in native olfactory sensory neurons. Here we describe experiments using gene targeting, electrophysiology, and optical imaging to study the response properties of TAAR-expressing sensory neurons and their associated glomeruli in mice. We show that olfactory sensory neurons that express a subset of the TAAR repertoire are preferentially responsive to amines. In addition, neurons expressing specific TAARs, TAAR3 or TAAR4, are highly sensitive and are also broadly tuned-responding to structurally diverse amines. Surprisingly, we find that TAAR4 is exquisitely sensitive, with apparent affinities for a preferred ligand, phenylethylamine, rivaling those seen with mammalian pheromone receptors. We provide evidence that this unprecedented sensitivity is mediated via receptor coupling to the canonical odorant transduction cascade. The data suggest that the TAARs are evolutionarily retained in the olfactory receptor repertoire to mediate high-sensitivity detection of a biologically relevant class of odorous stimuli.

  20. Endogenous mammalian RF-amide peptides, including PrRP, kisspeptin and 26RFa, modulate nociception and morphine analgesia via NPFF receptors.

    PubMed

    Elhabazi, Khadija; Humbert, Jean-Paul; Bertin, Isabelle; Schmitt, Martine; Bihel, Frédéric; Bourguignon, Jean-Jacques; Bucher, Bernard; Becker, Jérôme A J; Sorg, Tania; Meziane, Hamid; Petit-Demoulière, Benoit; Ilien, Brigitte; Simonin, Frédéric

    2013-12-01

    Mammalian RF-amide peptides are encoded by five different genes and act through five different G protein-coupled receptors. RF-amide-related peptides-1 and -3, neuropeptides AF and FF, Prolactin releasing peptides, Kisspeptins and RFa peptides are currently considered endogenous peptides for NPFF1, NPFF2, GPR10, GPR54 and GPR103 receptors, respectively. However, several studies suggest that the selectivity of these peptides for their receptors is low and indicate that expression patterns for receptors and their corresponding ligands only partially overlap. In this study, we took advantage of the cloning of the five human RF-amide receptors to systematically examine their affinity for and their activation by all human RF-amide peptides. Binding experiments, performed on membranes from CHO cells expressing GPR10, GPR54 and GPR103 receptors, confirmed their high affinity and remarkable selectivity for their cognate ligands. Conversely, NPFF1 and NPFF2 receptors displayed high affinity for all RF-amide peptides. Moreover, GTPγS and cAMP experiments showed that almost all RF-amide peptides efficiently activate NPFF1 and NPFF2 receptors. As NPFF is known to modulate morphine analgesia, we undertook a systematic analysis in mice of the hyperalgesic and anti morphine-induced analgesic effects of a representative set of endogenous RF-amide peptides. All of them induced hyperalgesia and/or prevented morphine analgesia following intracerebroventricular administration. Importantly, these effects were prevented by administration of RF9, a highly selective NPFF1/NPFF2 antagonist. Altogether, our results show that all endogenous RF-amide peptides display pain-modulating properties and point to NPFF receptors as essential players for these effects.

  1. The brain-uterus connection: brain derived neurotrophic factor (BDNF) and its receptor (Ntrk2) are conserved in the mammalian uterus.

    PubMed

    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.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  4. 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

  5. The carboxy-terminal tail or the intracellular loop 3 is required for β-arrestin-dependent internalization of a mammalian type II GnRH receptor.

    PubMed

    Madziva, Michael T; Mkhize, Nonhlanhla N; Flanagan, Colleen A; Katz, Arieh A

    2015-08-15

    The type II GnRH receptor (GnRH-R2) in contrast to mammalian type I GnRH receptor (GnRH-R1) has a cytosolic carboxy-terminal tail. We investigated the role of β-arrestin 1 in GnRH-R2-mediated signalling and mapped the regions in GnRH-R2 required for recruitment of β-arrestin, employing internalization assays. We show that GnRH-R2 activation of ERK is dependent on β-arrestin and protein kinase C. Appending the tail of GnRH-R2 to GnRH-R1 enabled GRK- and β-arrestin-dependent internalization of the chimaeric receptor. Surprisingly, carboxy-terminally truncated GnRH-R2 retained β-arrestin and GRK-dependent internalization, suggesting that β-arrestin interacts with additional elements of GnRH-R2. Mutating serine and threonine or basic residues of intracellular loop 3 did not abolish β-arrestin 1-dependent internalization but a receptor lacking these basic residues and the carboxy-terminus showed no β-arrestin 1-dependent internalization. Our results suggest that basic residues at the amino-terminal end of intracellular loop 3 or the carboxy-terminal tail are required for β-arrestin dependent internalization.

  6. 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/.

  7. Truncated glucagon-like peptide-1 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Identification of a mammalian analogue of the reptilian peptide exendin-4.

    PubMed

    Raufman, J P; Singh, L; Singh, G; Eng, J

    1992-10-25

    To find mammalian analogues of exendin-4, a peptide from Helodermatidae venoms that interacts with newly discovered exendin receptors on dispersed acini from guinea pig pancreas, we examined the actions of recent additions to the vasoactive intestinal peptide/secretin/glucagon family of regulatory peptides. In every respect tested, the truncated form of glucagon-like peptide-1, GLP-1(7-36)NH2, mimicked the actions of exendin-4. Like exendin-4, GLP-1(7-36)NH2 caused an increase in acinar cAMP without stimulating amylase release. GLP-1(7-36)NH2-induced increases in cAMP were inhibited progressively by increasing concentrations of the specific exendin-receptor antagonist, exendin(9-39)NH2. In dispersed acini from guinea pig and rat pancreas, concentrations of GLP-1(7-36)NH2 that stimulated increases in cAMP caused potentiation of cholecystokinin-induced amylase release. Binding of 125I-[Y39]exendin-4 or 125I-GLP-1(7-36)NH2 to dispersed acini from guinea pig pancreas was inhibited by adding increasing concentrations of unlabeled exendin-4 or GLP-1(7-36)NH2. We conclude that the mammalian peptide GLP-1(7-36)NH2 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Exendin(9-39)NH2, a competitive antagonist of the actions of GLP-1(7-36)NH2 in pancreatic acini, may be a useful tool for examining the physiological actions of this peptide.

  8. Mammalian Alpha Arrestins Link Activated Seven Transmembrane Receptors to Nedd4 Family E3 Ubiquitin Ligases and Interact with Beta Arrestins

    PubMed Central

    Shea, Fortune F.; Rowell, Jennie L.; Li, Yechaowei; Chang, Tien-Hsien; Alvarez, Carlos E.

    2012-01-01

    The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination – which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1–5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the

  9. Cannabinoid receptor agonists modulate oligodendrocyte differentiation by activating PI3K/Akt and the mammalian target of rapamycin (mTOR) pathways

    PubMed Central

    Gomez, O; Sanchez-Rodriguez, A; Le, MQU; Sanchez-Caro, C; Molina-Holgado, F; Molina-Holgado, E

    2011-01-01

    BACKGROUND AND PURPOSE The endogenous cannabinoid system participates in oligodendrocyte progenitor differentiation in vitro. To determine the effect of synthetic cannabinoids on oligodendrocyte differentiation, we exposed differentiating cultures of oligodendrocytes with cannabinoid CB1, CB2 and CB1/CB2 receptor agonists and antagonists. The response of the PI3K/Akt and the mammalian target of rapamycin (mTOR) signalling pathways were studied as effectors of cannabinoid activity. EXPERIMENTAL APPROACH Purified oligodendrocyte progenitor cells (OPC) obtained from primary mixed glial cell cultures were treated for 48 h with CB1, CB2 and CB1/CB2 receptor agonists (ACEA, JWH133 and HU210, respectively) in the presence or absence of the antagonists AM281 (CB1 receptor) and AM630 (CB2 receptor). Moreover, inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt and mTOR pathways (LY294002 and rapamycin, respectively) were used to study the involvement of these pathways on cannabinoid-induced OPC maturation. KEY RESULTS ACEA, JWH133 and HU-210 enhanced OPC differentiation as assessed by the expression of stage specific antigens and myelin basic protein (MBP). Moreover, this effect was blocked by the CB receptor antagonists. ACEA, JWH133 and HU210 induced a time-dependent phosphorylation of Akt and mTOR, whereas the inhibitors of PI3K/Akt (LY294002) or of mTOR (rapamycin) reversed the effects of HU-210 on oligodendrocyte differentiation and kinase activation. CONCLUSIONS AND IMPLICATIONS Activation of cannabinoid CB1 or CB2 receptors with selective agonists accelerated oligodendrocyte differentiation through the mTOR and Akt signalling pathways. LINKED ARTICLES This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7 PMID:21480865

  10. 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; Robins, Harlan S.; O’Donnell, Paul V.; 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

  11. 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.

  12. 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

  13. Ligand specificity and affinity of BT-R1, the Bacillus thuringiensis Cry1A toxin receptor from Manduca sexta, expressed in mammalian and insect cell cultures.

    PubMed Central

    Keeton, T P; Bulla, L A

    1997-01-01

    The Manduca sexta receptor for the Bacillus thuringiensis Cry1Aa, Cry1Ab, and Cry1Ac toxins, BT-R1, has been expressed in heterologous cell culture, and its ligand binding characteristics have been determined. When transfected with the BT-R1 cDNA, insect and mammalian cell cultures produce a binding protein of approximately 195 kDa, in contrast to natural BT-R1 from M. sexia, which has an apparent molecular weight of 210 kDa. Transfection of cultured Spodoptera frugiperda cells with the BT-R1 cDNA imparts Cry1A-specific high-affinity binding activity typical of membranes prepared from larval M. sexta midguts. Competition assays with BT-R1 prepared from larval M. sexta midguts and transiently expressed in cell culture reveal virtually identical affinities for the Cry1Aa, Cry1Ab, and Cry1Ac toxins, clearly demonstrating the absolute specificity of the receptor for toxins of the lepidopteran-specific Cry1A family. BT-R1 therefore remains the only M. sexta Cry1A binding protein to be purified, cloned, and functionally expressed in heterologous cell culture, and for the first time, we are able to correlate the Cry1Aa, Cry1Ab, and Cry1Ac toxin sensitivities of M. sexta to the identity and ligand binding characteristics of a single midgut receptor molecule. PMID:9292994

  14. 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

  15. Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity.

    PubMed

    Du, Lanying; Zhao, Guangyu; Chan, Chris C S; Sun, Shihui; Chen, Min; Liu, Zhonghua; Guo, Hongxiang; He, Yuxian; Zhou, Yusen; Zheng, Bo-Jian; Jiang, Shibo

    2009-10-10

    Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease. The potential recurrence of the disease from animal reservoirs highlights the significance of development of safe and efficient vaccines to prevent a future SARS epidemic. In this study, we expressed the recombinant receptor-binding domain (rRBD) in mammalian (293T) cells, insect (Sf9) cells, and E. coli, respectively, and compared their immunogenicity and protection against SARS-CoV infection in an established mouse model. Our results show that all rRBD proteins expressed in the above systems maintained intact conformation, being able to induce highly potent neutralizing antibody responses and complete protective immunity against SARS-CoV challenge in mice, albeit the rRBD expressed in 293T cells elicited stronger humoral immune responses with significantly higher neutralizing activity (P<0.05) than those expressed in Sf9 and E. coli cells. These results suggest that all three rRBDs are effective in eliciting immune responses and protection against SARS-CoV and any of the above expression systems can be used for production of rRBD-based SARS subunit vaccines. Preference will be given to rRBD expressed in mammalian cells for future evaluation of the vaccine efficacy in a non-human primate model of SARS because of its ability to refold into a native conformation more readily and to induce higher level of neutralizing antibody responses than those expressed in E. coli and insect cells.

  16. VH replacement in primary immunoglobulin repertoire diversification.

    PubMed

    Sun, Amy; Novobrantseva, Tatiana I; Coffre, Maryaline; Hewitt, Susannah L; Jensen, Kari; Skok, Jane A; Rajewsky, Klaus; Koralov, Sergei B

    2015-02-03

    The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VH replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged VHDHJH joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VH element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original VH gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJH allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJH rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.

  17. Modulation of mammalian dendritic GABAA receptor function by the kinetics of Cl− and HCO3− transport

    PubMed Central

    Staley, Kevin J; Proctor, William R

    1999-01-01

    During prolonged activation of dendritic GABAA receptors, the postsynaptic membrane response changes from hyperpolarization to depolarization. One explanation for the change in direction of the response is that opposing HCO3− and Cl− fluxes through the GABAA ionophore diminish the electrochemical gradient driving the hyperpolarizing Cl− flux, so that the depolarizing HCO3− flux dominates. Here we demonstrate that the necessary conditions for this mechanism are present in rat hippocampal CA1 pyramidal cell dendrites. Prolonged GABAA receptor activation in low-HCO3− media decreased the driving force for dendritic but not somatic Cl− currents. Prolonged GABAA receptor activation in low-Cl− media containing physiological HCO3− concentrations did not degrade the driving force for dendritic or somatic HCO3− gradients. Dendritic Cl− transport was measured in three ways: from the rate of recovery of GABAA receptor-mediated currents between paired dendritic GABA applications, from the rate of recovery between paired synaptic GABAA receptor-mediated currents, and from the predicted vs. actual increase in synaptic GABAA receptor-mediated currents at progressively more positive test potentials. These experiments yielded estimates of the maximum transport rate (vmax) for Cl− transport of 5 to 7 mmol l−1 s−1, and indicated that vmax could be exceeded by GABAA receptor-mediated Cl− influx. The affinity of the Cl− transporter was calculated in experiments in which the reversal potential for Cl− (ECl) was measured from the GABAA reversal potential in low-HCO3− media during Cl− loading from the recording electrode solution. The calculated KD was 15 mM. Using a standard model of membrane potential, these conditions are demonstrated to be sufficient to produce the experimentally observed, activity-dependent GABAA depolarizing response in pyramidal cell dendrites. PMID:10457084

  18. 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

  19. Caffeine stimulates locomotor activity in the mammalian spinal cord via adenosine A1 receptor-dopamine D1 receptor interaction and PKA-dependent mechanisms.

    PubMed

    Acevedo, JeanMarie; Santana-Almansa, Alexandra; Matos-Vergara, Nikol; Marrero-Cordero, Luis René; Cabezas-Bou, Ernesto; Díaz-Ríos, Manuel

    2016-02-01

    Caffeine is a potent psychostimulant that can have significant and widely variable effects on the activity of multiple neuronal pathways. The most pronounced caffeine-induced behavioral effect seen in rodents is to increase locomotor activity which has been linked to a dose-dependent inhibition of A1 and A(2A) receptors. The effects of caffeine at the level of the lumbar spinal central pattern generator (CPG) network for hindlimb locomotion are lacking. We assessed the effects of caffeine to the locomotor function of the spinal CPG network via extracellular ventral root recordings using the isolated neonatal mouse spinal cord preparation. Addition of caffeine and of an A1 receptor antagonist significantly decreased the cycle period accelerating the ongoing locomotor rhythm, while decreasing burst duration reversibly in most preparations suggesting the role of A1 receptors as the primary target of caffeine. Caffeine and an A1 receptor antagonist failed to stimulate ongoing locomotor activity in the absence of dopamine or in the presence of a D1 receptor antagonist supporting A1/D1 receptor-dependent mechanism of action. The use of caffeine or an A1 receptor blocker failed to stimulate an ongoing locomotor rhythm in the presence of a blocker of the cAMP-dependent protein kinase (PKA) supporting the need of this intracellular pathway for the modulatory effects of caffeine to occur. These results support a stimulant effect of caffeine on the lumbar spinal network controlling hindlimb locomotion through the inhibition of A1 receptors and subsequent activation of D1 receptors via a PKA-dependent intracellular mechanism.

  20. 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

  1. Trade-offs in antibody repertoires to complex antigens.

    PubMed

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

    2015-09-05

    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.

  2. 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.

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

    PubMed Central

    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.

    2013-01-01

    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

  4. A model for the evolution of the mammalian t-cell receptor α/δ and μ loci based on evidence from the duckbill Platypus.

    PubMed

    Parra, Zuly E; Lillie, Mette; Miller, Robert D

    2012-10-01

    The specific recognition of antigen by T cells is critical to the generation of adaptive immune responses in vertebrates. T cells recognize antigen using a somatically diversified T-cell receptor (TCR). All jawed vertebrates use four TCR chains called α, β, γ, and δ, which are expressed as either a αβ or γδ heterodimer. Nonplacental mammals (monotremes and marsupials) are unusual in that their genomes encode a fifth TCR chain, called TCRµ, whose function is not known but is also somatically diversified like the conventional chains. The origins of TCRµ are also unclear, although it appears distantly related to TCRδ. Recent analysis of avian and amphibian genomes has provided insight into a model for understanding the evolution of the TCRδ genes in tetrapods that was not evident from humans, mice, or other commonly studied placental (eutherian) mammals. An analysis of the genes encoding the TCRδ chains in the duckbill platypus revealed the presence of a highly divergent variable (V) gene, indistinguishable from immunoglobulin heavy (IgH) chain V genes (VH) and related to V genes used in TCRµ. They are expressed as part of TCRδ repertoire (VHδ) and similar to what has been found in frogs and birds. This, however, is the first time a VHδ has been found in a mammal and provides a critical link in reconstructing the evolutionary history of TCRµ. The current structure of TCRδ and TCRµ genes in tetrapods suggests ancient and possibly recurring translocations of gene segments between the IgH and TCRδ genes, as well as translocations of TCRδ genes out of the TCRα/δ locus early in mammals, creating the TCRµ locus.

  5. Developmental expression of the platelet-derived growth factor alpha-receptor gene in mammalian central nervous system.

    PubMed

    Yeh, H J; Silos-Santiago, I; Wang, Y X; George, R J; Snider, W D; Deuel, T F

    1993-03-01

    We recently reported that the platelet-derived growth factor (PDGF) A-chain gene is highly expressed in neurons of embryonic and adult mouse central nervous system and suggested that its secretion by neurons may support development and maintenance of glia. We have now analyzed the levels and sites of expression of the cognate PDGF alpha-receptor gene in brain and spinal cord of embryonic and adult mice by in situ hybridization. The predominant cell populations in both gray and white matter expressing transcripts of the PDGF alpha-receptor gene are glial cells or their precursors. Transcripts consistently were not detected in neurons. Expression of the PDGF alpha-receptor gene was first observed at embryonic day 15, increased through postnatal day 14, and fell to lower levels in adults. Expression of the alpha-receptor gene corresponds in temporal sequence to the developmental period of glial migration and proliferation and to the expression of PDGF A by neurons. The results indicate that glia but not neurons have the potential to respond to PDGF A and suggest that neurons influence glial cell development through paracrine regulation.

  6. Cellular senescence or EGFR signaling induces Interleukin 6 (IL-6) receptor expression controlled by mammalian target of rapamycin (mTOR)

    PubMed Central

    Garbers, Christoph; Kuck, Fabian; Aparicio-Siegmund, Samadhi; Konzak, Kirstin; Kessenbrock, Mareike; Sommerfeld, Annika; Häussinger, Dieter; Lang, Philipp A; Brenner, Dirk; Mak, Tak W.; Rose-John, Stefan; Essmann, Frank; Schulze-Osthoff, Klaus; Piekorz, Roland P; Scheller, Jürgen

    2013-01-01

    Interleukin 6 (IL-6) signaling plays a role in inflammation, cancer, and senescence. Here, we identified soluble IL-6 receptor (sIL-6R) as a member of the senescence-associated secretory phenotype (SASP). Senescence-associated sIL-6R upregulation was mediated by mammalian target of rapamycin (mTOR). sIL-6R was mainly generated by a disintegrin and metalloprotease 10 (ADAM10)-dependent ectodomain shedding to enable IL-6 trans-signaling. In vivo, heterozygous PTEN-knockout mice exhibited higher mTOR activity and increased sIL-6R levels. Moreover, aberrant EGF receptor (EGFR) activation triggered IL-6 synthesis. In analogy to senescence, EGFR-induced activation of mTOR also induced IL-6R expression and sIL-6R generation. Hence, mTOR activation reprograms IL-6 non-responder cells into IL-6 responder cells. Our data suggest that mTOR serves as a central molecular switch to facilitate cellular IL-6 classic and trans-signaling via IL-6R upregulation with direct implications for cellular senescence and tumor development. PMID:24047696

  7. 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.

  8. Mammalian touch catches up

    PubMed Central

    Walsh, Carolyn M.; Bautista, Diana M.; Lumpkin, Ellen A.

    2015-01-01

    An assortment of touch receptors innervate the skin and encode different tactile features of the environment. Compared with invertebrate touch and other sensory systems, our understanding of the molecular and cellular underpinnings of mammalian touch lags behind. Two recent breakthroughs have accelerated progress. First, an arsenal of cell-type-specific molecular markers allowed the functional and anatomical properties of sensory neurons to be matched, thereby unraveling a cellular code for touch. Such markers have also revealed key roles of non-neuronal cell types, such as Merkel cells and keratinocytes, in touch reception. Second, the discovery of Piezo genes as a new family of mechanically activated channels has fueled the discovery of molecular mechanisms that mediate and mechanotransduction in mammalian touch receptors. PMID:26100741

  9. Characterization of NPY receptor subtypes Y2 and Y7 in rainbow trout Oncorhynchus mykiss.

    PubMed

    Larsson, Tomas A; Larson, Earl T; Fredriksson, Robert; Conlon, J Michael; Larhammar, Dan

    2006-06-01

    We report the cloning and pharmacological characterization of two neuropeptide Y (NPY) receptor subtypes, Y2 and Y7, in rainbow trout (Oncorhynchus mykiss). These subtypes are approximately 50% identical to each other and belong to the Y2 subfamily of NPY receptors. The binding properties of the receptors were investigated after expression in human HEK-293 EBNA cells. Both receptors bound the three zebrafish peptides NPY, PYYa, and PYYb, as well as porcine NPY and PYY, with affinities in the nanomolar range that are similar to mammalian Y2. The affinity of the truncated porcine NPY fragments, NPY 13-36 and NPY 18-36 was markedly lower compared to mammalian and chicken Y2. This suggests that mammalian and chicken Y2 are unique among NPY receptors in their ability to bind truncated peptide fragments. The antagonist BIIE0246, developed for mammalian Y2, did not bind either of the two rainbow trout receptors. Our results support the proposed expansion of this gene family by duplications before the gnathostome radiation. They also reveal appreciable differences in the repertoire and characteristics of NPY receptors between fish and tetrapods stressing the importance of lineage-specific gene loss as well as sequence divergence after duplication.

  10. Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines.

    PubMed

    Müller, Marcel A; Raj, V Stalin; Muth, Doreen; Meyer, Benjamin; Kallies, Stephan; Smits, Saskia L; Wollny, Robert; Bestebroer, Theo M; Specht, Sabine; Suliman, Tasnim; Zimmermann, Katrin; Binger, Tabea; Eckerle, Isabella; Tschapka, Marco; Zaki, Ali M; Osterhaus, Albert D M E; Fouchier, Ron A M; Haagmans, Bart L; Drosten, Christian

    2012-12-11

    A new human coronavirus (hCoV-EMC) has emerged very recently in the Middle East. The clinical presentation resembled that of the severe acute respiratory syndrome (SARS) as encountered during the epidemic in 2002/2003. In both cases, acute renal failure was observed in humans. HCoV-EMC is a member of the same virus genus as SARS-CoV but constitutes a sister species. Here we investigated whether it might utilize angiotensin-converting enzyme 2 (ACE2), the SARS-CoV receptor. Knowledge of the receptor is highly critical because the restriction of the SARS receptor to deep compartments of the human respiratory tract limited the spread of SARS. In baby hamster kidney (BHK) cells, lentiviral transduction of human ACE2 (hACE2) conferred permissiveness and replication for SARS-CoV but not for hCoV-EMC. Monkey and human kidney cells (LLC-MK2, Vero, and 769-P) and swine kidney cells were permissive for both viruses, but only SARS-CoV infection could be blocked by anti-hACE2 antibody and could be neutralized by preincubation of virus with soluble ACE2. Our data show that ACE2 is neither necessary nor sufficient for hCoV-EMC replication. Moreover, hCoV-EMC, but not SARS-CoV, replicated in cell lines from Rousettus, Rhinolophus, Pipistrellus, Myotis, and Carollia bats, representing four major chiropteran families from both suborders. As human CoV normally cannot replicate in bat cells from different families, this suggests that hCoV-EMC might use a receptor molecule that is conserved in bats, pigs, and humans, implicating a low barrier against cross-host transmission. IMPORTANCE A new human coronavirus (hCoV) emerged recently in the Middle East. The disease resembled SARS (severe acute respiratory syndrome), causing a fatal epidemic in 2002/2003. Coronaviruses have a reservoir in bats and because this novel virus is related to SARS-CoV, we investigated whether it might replicate in bat cells and use the same receptor (angiotensin-converting enzyme 2 [ACE2]). This knowledge is

  11. Analysis of subcomponents of the gamma-aminobutyric acid/benzodiazepine receptor macromolecular complex in mammalian central nervous system

    SciTech Connect

    McCabe, R.T.

    1987-01-01

    Since the presence of endogenous gamma-aminobutyric acid (GABA) may affect benzodiazepine binding to tissue sections in autoradiographic studies, a protocol designed to check for this influence has been investigated. (/sup 3/H)Flunitrazepam (1 nM) was used to label benzodiazepine receptors for autoradiographic localization. Bicuculline was added to the incubation medium of an additional set of tissue sections to antagonize any potential effect of endogenous GABA. Binding in these sections was compared to that occurring in another set in which excess GABA was added to create further GABA enhancement. Binding was also compared to adjacent sections which were treated similarly but also preincubated in distilled-deionized water to burst the cells by osmotic shock and eliminate endogenous GABA, thereby preventing any effect on benzodiazepine binding. The results indicated that endogenous GABA is indeed present in the slide-mounted tissue sections and is affecting benzodiazepine receptor binding differentially in various regions of the brain depending on the density of GABAergic innervation. Scatchard analysis of saturation data demonstrated that the alteration in BZ binding due to GABA was a result of a change in the affinity rather than number of receptors present.

  12. 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

  13. The herbicide atrazine activates endocrine gene networks via non-steroidal NR5A nuclear receptors in fish and mammalian cells.

    PubMed

    Suzawa, Miyuki; Ingraham, Holly A

    2008-05-07

    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 microg/L), and increased the ratio of female to male fish (22 microg/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, INHalpha, alphaGSU, 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.

  14. Comparative analysis of the feline immunoglobulin repertoire.

    PubMed

    Steiniger, Sebastian C J; Glanville, Jacob; Harris, Douglas W; Wilson, Thomas L; Ippolito, Gregory C; Dunham, Steven A

    2017-01-25

    Next-Generation Sequencing combined with bioinformatics is a powerful tool for analyzing the large number of DNA sequences present in the expressed antibody repertoire and these data sets can be used to advance a number of research areas including antibody discovery and engineering. The accurate measurement of the immune repertoire sequence composition, diversity and abundance is important for understanding the repertoire response in infections, vaccinations and cancer immunology and could also be useful for elucidating novel molecular targets. In this study 4 individual domestic cats (Felis catus) were subjected to antibody repertoire sequencing with total number of sequences generated 1079863 for VH for IgG, 1050824 VH for IgM, 569518 for VK and 450195 for VL. Our analysis suggests that a similar VDJ expression patterns exists across all cats. Similar to the canine repertoire, the feline repertoire is dominated by a single subgroup, namely VH3. The antibody paratope of felines showed similar amino acid variation when compared to human, mouse and canine counterparts. All animals show a similarly skewed VH CDR-H3 profile and, when compared to canine, human and mouse, distinct differences are observed. Our study represents the first attempt to characterize sequence diversity in the expressed feline antibody repertoire and this demonstrates the utility of using NGS to elucidate entire antibody repertoires from individual animals. These data provide significant insight into understanding the feline immune system function.

  15. 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…

  16. Antibody Repertoire Development in Swine.

    PubMed

    Butler, J E; Wertz, Nancy; Sinkora, Marek

    2017-02-08

    We describe the domestication of the species, explore its value to agriculture and bioscience, and compare its immunoglobulin (Ig) genes to those of other vertebrates. For encyclopedic information, we cite earlier reviews and chapters. We provide current gene maps for the heavy and light chain loci and describe their polygeny and polymorphy. B-cell and antibody repertoire development is a major focus, and we present findings that challenge several mouse-centric paradigms. We focus special attention on the role of ileal Peyer's patches, the largest secondary lymphoid tissues in newborn piglets and a feature of all artiodactyls. We believe swine fetal development and early class switch evolved to provide natural secretory IgA antibodies able to prevent translocation of bacteria from the gut while the bacterial PAMPs drive development of adaptive immunity. We discuss the value of using the isolator piglet model to address these issues.

  17. Dysregulation of B Cell Repertoire Formation in Myasthenia Gravis Patients Revealed through Deep Sequencing.

    PubMed

    Vander Heiden, Jason A; Stathopoulos, Panos; Zhou, Julian Q; Chen, Luan; Gilbert, Tamara J; Bolen, Christopher R; Barohn, Richard J; Dimachkie, Mazen M; Ciafaloni, Emma; Broering, Teresa J; Vigneault, Francois; Nowak, Richard J; Kleinstein, Steven H; O'Connor, Kevin C

    2017-02-15

    Myasthenia gravis (MG) is a prototypical B cell-mediated autoimmune disease affecting 20-50 people per 100,000. The majority of patients fall into two clinically distinguishable types based on whether they produce autoantibodies targeting the acetylcholine receptor (AChR-MG) or muscle specific kinase (MuSK-MG). The autoantibodies are pathogenic, but whether their generation is associated with broader defects in the B cell repertoire is unknown. To address this question, we performed deep sequencing of the BCR repertoire of AChR-MG, MuSK-MG, and healthy subjects to generate ∼518,000 unique VH and VL sequences from sorted naive and memory B cell populations. AChR-MG and MuSK-MG subjects displayed distinct gene segment usage biases in both VH and VL sequences within the naive and memory compartments. The memory compartment of AChR-MG was further characterized by reduced positive selection of somatic mutations in the VH CDR and altered VH CDR3 physicochemical properties. The VL repertoire of MuSK-MG was specifically characterized by reduced V-J segment distance in recombined sequences, suggesting diminished VL receptor editing during B cell development. Our results identify large-scale abnormalities in both the naive and memory B cell repertoires. Particular abnormalities were unique to either AChR-MG or MuSK-MG, indicating that the repertoires reflect the distinct properties of the subtypes. These repertoire abnormalities are consistent with previously observed defects in B cell tolerance checkpoints in MG, thereby offering additional insight regarding the impact of tolerance defects on peripheral autoimmune repertoires. These collective findings point toward a deformed B cell repertoire as a fundamental component of MG. Copyright © 2017 by The American Association of Immunologists, Inc.

  18. 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

  19. Comparison of the T cell patterns in leprous and cutaneous sarcoid granulomas. Presence of Valpha24-invariant natural killer T cells in T-cell-reactive leprosy together with a highly biased T cell receptor Valpha repertoire.

    PubMed

    Mempel, M; Flageul, B; Suarez, F; Ronet, C; Dubertret, L; Kourilsky, P; Gachelin, G; Musette, P

    2000-08-01

    The T-cell-reactive (eg, tuberculoid and reversal) forms of leprosy represent a well-defined granulomatous reaction pattern against an invading pathogen. The immune response in cutaneous sarcoidosis is a granulomatous condition that pathologically is very similar to T-cell reactive leprosy. However, it lacks a defined causative agent. In view of the role of NKT cells in murine granulomas induced by mycobacterial cell walls, we have searched for the presence of NKT cells in the cutaneous lesions of both leprosy and sarcoidosis. These cells were present in T-cell-reactive leprosy but were undetectable in cutaneous sarcoidosis. We have also studied the TCR Valpha repertoire in the two diseases. In addition to Valpha24(+) NKT cells, all patients with T-cell-reactive leprosy showed a very restricted T-cell-reactive Valpha repertoire with a strong bias toward the use of the Valpha6 and Valpha14 segments. Valpha6 and Valpha14(+) T cells were polyclonal in terms of CDR3 length and Jalpha usage. In contrast, most sarcoidosis patients showed a diverse usage of Valpha chains associated with clonal or oligoclonal expansions reminiscent of antigen-driven activation of conventional T cells. Thus the origin and perpetuation of the two kinds of granulomatous lesions appear to depend on altogether distinct T-cell recruiting mechanisms.

  20. scAAV-mediated gene transfer of interleukin-1-receptor antagonist to synovium and articular cartilage in large mammalian joints.

    PubMed

    Watson, R S; Broome, T A; Levings, P P; Rice, B L; Kay, J D; Smith, A D; Gouze, E; Gouze, J-N; Dacanay, E A; Hauswirth, W W; Nickerson, D M; Dark, M J; Colahan, P T; Ghivizzani, S C

    2013-06-01

    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 adeno-associated virus (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 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 × 10(11) 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.

  1. Mammalian olfactory receptors: molecular mechanisms of odorant detection, 3D-modeling, and structure-activity relationships.

    PubMed

    Persuy, Marie-Annick; Sanz, Guenhaël; Tromelin, Anne; Thomas-Danguin, Thierry; Gibrat, Jean-François; Pajot-Augy, Edith

    2015-01-01

    This chapter describes the main characteristics of olfactory receptor (OR) genes of vertebrates, including generation of this large multigenic family and pseudogenization. OR genes are compared in relation to evolution and among species. OR gene structure and selection of a given gene for expression in an olfactory sensory neuron (OSN) are tackled. The specificities of OR proteins, their expression, and their function are presented. The expression of OR proteins in locations other than the nasal cavity is regulated by different mechanisms, and ORs display various additional functions. A conventional olfactory signal transduction cascade is observed in OSNs, but individual ORs can also mediate different signaling pathways, through the involvement of other molecular partners and depending on the odorant ligand encountered. ORs are engaged in constitutive dimers. Ligand binding induces conformational changes in the ORs that regulate their level of activity depending on odorant dose. When present, odorant binding proteins induce an allosteric modulation of OR activity. Since no 3D structure of an OR has been yet resolved, modeling has to be performed using the closest G-protein-coupled receptor 3D structures available, to facilitate virtual ligand screening using the models. The study of odorant binding modes and affinities may infer best-bet OR ligands, to be subsequently checked experimentally. The relationship between spatial and steric features of odorants and their activity in terms of perceived odor quality are also fields of research that development of computing tools may enhance.

  2. Chronic Exposure to Anabolic Androgenic Steroids Alters Neuronal Function in the Mammalian Forebrain via Androgen Receptor- and Estrogen Receptor-Mediated Mechanisms

    PubMed Central

    Penatti, Carlos A A; Porter, Donna M; Henderson, Leslie P

    2009-01-01

    Anabolic androgenic steroids (AAS) can promote detrimental effects on social behaviors for which γ-aminobutyric acid type A (GABAA) receptor-mediated circuits in the forebrain play a critical role. While all AAS bind to androgen receptors (AR), they may also be aromatized to estrogens and thus potentially impart effects via estrogen receptors (ER). Chronic exposure of wild type male mice to a combination of chemically distinct AAS increased action potential (AP) frequency, selective GABAA receptor subunit mRNAs, and GABAergic synaptic current decay in the medial preoptic area (mPOA). Experiments performed with pharmacological agents and in AR-deficient Tfm mutant mice suggest that the AAS-dependent enhancement of GABAergic transmission in wild type mice is AR-mediated. In AR-deficient mice, the AAS elicited dramatically different effects, decreasing AP frequency, sIPSC amplitude and frequency and the expression of selective GABAA receptor subunit mRNAs. Surprisingly, in the absence of AR signaling, the data indicate that the AAS do not act as ER agonists, but rather suggest a novel in vivo action in which the AAS inhibit aromatase and impair endogenous ER signaling. These results show that the AAS have the capacity to alter neuronal function in the forebrain via multiple steroid signaling mechanisms and suggest that effects of these steroids in the brain will depend not only on the balance of AR- vs. ER-mediated regulation for different target genes, but also on the ability of these drugs to alter steroid metabolism and thus the endogenous steroid milieu. PMID:19812324

  3. Chronic exposure to anabolic androgenic steroids alters neuronal function in the mammalian forebrain via androgen receptor- and estrogen receptor-mediated mechanisms.

    PubMed

    Penatti, Carlos A A; Porter, Donna M; Henderson, Leslie P

    2009-10-07

    Anabolic androgenic steroids (AAS) can promote detrimental effects on social behaviors for which GABA type A (GABA(A)) receptor-mediated circuits in the forebrain play a critical role. While all AAS bind to androgen receptors (AR), they may also be aromatized to estrogens and thus potentially impart effects via estrogen receptors (ER). Chronic exposure of wild-type male mice to a combination of chemically distinct AAS increased action potential (AP) frequency, selective GABA(A) receptor subunit mRNAs, and GABAergic synaptic current decay in the medial preoptic area (mPOA). Experiments performed with pharmacological agents and in AR-deficient Tfm mutant mice suggest that the AAS-dependent enhancement of GABAergic transmission in wild-type mice is AR-mediated. In AR-deficient mice, the AAS elicited dramatically different effects, decreasing AP frequency, spontaneous IPSC amplitude and frequency and the expression of selective GABA(A) receptor subunit mRNAs. Surprisingly, in the absence of AR signaling, the data indicate that the AAS do not act as ER agonists, but rather suggest a novel in vivo action in which the AAS inhibit aromatase and impair endogenous ER signaling. These results show that the AAS have the capacity to alter neuronal function in the forebrain via multiple steroid signaling mechanisms and suggest that effects of these steroids in the brain will depend not only on the balance of AR- versus ER-mediated regulation for different target genes, but also on the ability of these drugs to alter steroid metabolism and thus the endogenous steroid milieu.

  4. 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

  5. The mammalian 43-kD acetylcholine receptor-associated protein (RAPsyn) is expressed in some nonmuscle cells

    PubMed Central

    1989-01-01

    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 (Frail, D.E., L.S. Musil, a. Bonanno, and J.P. Merlie, 1989. Neuron. 2:1077-1086) 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. PMID:2469679

  6. Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

    PubMed Central

    Vallés, Ana Sofía; Roccamo, Ana M; Barrantes, Francisco J

    2009-01-01

    Aim: Studies of the α7-type neuronal nicotinic acetylcholine receptor (AChR), one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of α7-type AChRs1. The current work aims at obtaining and characterizing a cell line with high functional expression of the human α7 AChR. Methods: Ric-3 cDNA was incorporated into SHE-P1-hα7 cells expressing the α7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [125I]α-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent α-bungarotoxin, anti-α7 antibody, and GFP-α7 were performed on the new clone. Results: The human α7-type AChR was stably expressed in a new cell line, which we coined SHE-P1-hα7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [125I]αBTX saturable binding with an apparent KD of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-P1-hα7-Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-α7 antibodies. In contrast, chaperone-less SHE-P1-hα7 cells expressed only intracellular α7 AChRs and failed to produce detectable single-channel currents. Conclusion: The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal α7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells. PMID:19498422

  7. Disease etiology and diagnosis by TCR repertoire analysis goes viral.

    PubMed

    Attaf, Meriem; Sewell, Andrew K

    2016-11-01

    The importance of T-cell receptor (TCR) repertoire diversity is highlighted in murine models of immunodeficiency and in many human pathologies. However, the true extent of TCR diversity and how this diversity varies in health and disease is poorly understood. In a previous issue of the European Journal of Immunology, Lossius et al. [Eur. J. Immunol. 2014. 44: 3439-3452] dissected the composition of the TCR repertoire in the context of multiple sclerosis (MS) using high-throughput sequencing of TCR-β chains in cerebrospinal fluid samples and blood. The authors demonstrated that the TCR repertoire of the CSF was largely distinct from the blood and enriched in EBV-reactive CD8(+) T cells in MS patients. Studies of this kind have long been hindered by technical limitations and remain scarce in the literature. However, TCR sequencing methodologies are progressing apace and will undoubtedly shed light on the genetic basis of T-cell responses and the ontogeny of T-cell-mediated diseases, such as MS.

  8. Receptor binding profile of neuropeptide gamma and its fragments: comparison with the nonmammalian peptides carassin and ranakinin at three mammalian tachykinin receptors.

    PubMed

    Badgery-Parker, T; Lovas, S; Conlon, J M; Burcher, E

    1993-01-01

    The tachykinin binding site preferences of neuropeptide gamma (NP gamma), its C-terminal fragments AcNP gamma(3-21), AcNP gamma(5-21), AcNP gamma(7-21), and AcNP gamma(9-21), other mammalian tachykinins, and the nonmammalian tachykinins ranakinin and carassin were examined in membrane binding competition studies. [125I]-Bolton-Hunter [Sar9,Met(O2)11]SP (BHSarSP), [125I]-neurokinin A (INKA) and [125I]-Bolton-Hunter scyliorhinin II (BHScyII) were used to investigate NK-1, NK-2, and NK-3 sites, in rat submandibular gland, gastric fundus, and brain, respectively. Elongation of the neurokinin A molecule does not appear to influence binding to rat tachykinin NK-1 and NK-2 binding sites. Ranakinin has affinity for the NK-1 and NK-2 site similar to that of substance P and neurokinin A, respectively, but has low affinity for the NK-3 site. Despite its structural similarities to neuropeptide gamma, carassin has only moderate affinity for rat tachykinin binding sites. Possession of an acidic residue at position 4 appears critical for binding to rat NK-2 sites.

  9. 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

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

    2014-11-01

    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.

  10. 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

  11. Highly sensitive and unbiased approach for elucidating antibody repertoires

    PubMed Central

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

    2016-01-01

    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

  12. 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.

  13. 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

  14. IMonitor: A Robust Pipeline for TCR and BCR Repertoire Analysis

    PubMed Central

    Zhang, Wei; Du, Yuanping; Su, Zheng; Wang, Changxi; Zeng, Xiaojing; Zhang, Ruifang; Hong, Xueyu; Nie, Chao; Wu, Jinghua; Cao, Hongzhi; Xu, Xun; Liu, Xiao

    2015-01-01

    The advance of next generation sequencing (NGS) techniques provides an unprecedented opportunity to probe the enormous diversity of the immune repertoire by deep sequencing T-cell receptors (TCRs) and B-cell receptors (BCRs). However, an efficient and accurate analytical tool is still on demand to process the huge amount of data. We have developed a high-resolution analytical pipeline, Immune Monitor (“IMonitor”) to tackle this task. This method utilizes realignment to identify V(D)J genes and alleles after common local alignment. We compare IMonitor with other published tools by simulated and public rearranged sequences, and it demonstrates its superior performance in most aspects. Together with this, a methodology is developed to correct the PCR and sequencing errors and to minimize the PCR bias among various rearranged sequences with different V and J gene families. IMonitor provides general adaptation for sequences from all receptor chains of different species and outputs useful statistics and visualizations. In the final part of this article, we demonstrate its application on minimal residual disease detection in patients with B-cell acute lymphoblastic leukemia. In summary, this package would be of widespread usage for immune repertoire analysis. PMID:26297338

  15. An electrophysiological analysis of the effects of noradrenaline and alpha-receptor antagonists on neuromuscular transmission in mammalian muscular arteries.

    PubMed

    Holman, M E; Surprenant, A

    1980-01-01

    1 The effects of exogenously applied noradrenaline (NA) and alpha-adrenoceptor antagonists on the mechanical and intracellularly recorded responses to perivascular nerve stimulation were examined in the rabbit ear artery, rabbit saphenous artery and rat tail artery. 2 Excitatory junction potentials (e.j.ps) and action potentials recorded from these smooth muscles were not blocked or depressed by phentolamine, phenoxybenzamine, prazosin, or labetolol in concentrations as high as 10 microgram/ml. Phentolamine (1 to 10 microgram/ml) depressed neurally-evoked contractions of the ear and saphenous, but not the tail artery, and also depressed the contractions produced by direct muscle stimulation in the ear and saphenous arteries. Prazosin and labetolol (0.1 to 10 microgram/ml) had no effect on the neurally evoked contractile response in any of the arteries examined. 3 The amplitude of the steady-state e.j.p. during repetitive stimulation at 0.45 to 2 Hz was increased by phentolamine or phenoxybenzamine but not by prazosin or labetolol. Phentolamine and phenoxybenzamine also increased the amplitude of the e.j.p. evoked by a single stimulus in the majority of the preparations. 4 Concentrations of NA greater than or equal to 1 microgram/ml depolarized the smooth muscle while concentrations greater than or equal to 0.5 microgram/ml depressed the amplitude of the e.j.ps recorded from these arteries. alpha-Antagonists did not suppress either the NA-induced membrane depolarization or depression of e.j.ps. 5 These observations call into question the physiological relevance of both pre- and postsynaptic alpha-receptors in regard to adrenergic neuromuscular transmission in muscular arteries.

  16. Nerve growth factor receptor immunoreactivity is transiently associated with the subplate neurons of the mammalian cerebral cortex

    SciTech Connect

    Allendoerfer, K.L.; Shelton, D.L.; Shooter, E.M.; Shatz, C.J. )

    1990-01-01

    Nerve growth factor and its receptor (NGFR) are known to be present in diverse embryonic and neonatal central nervous system tissues, including the cerebral cortex. However, the identity of the cortical cells expressing NGFR immunoreactivity has not been established. We have used immunolabeling coupled with (3H)thymidine autoradiography to identify such cells in ferret and cat brain. Polyclonal antibodies raised against a synthetic peptide corresponding to a conserved amino acid sequence of the NGFR were used for this purpose. Western (immunologic) blot analyses show that these antibodies specifically recognize NGFR and precursor proteins. In both species, NGFR immunoreactivity is primarily associated with the early generated and transient subplate neuron population of the developing neocortex, as indicated by the following evidence: the immunoreactive cells (i) are located directly beneath the developing cortical plate, (ii) frequently have the inverted pyramid shape characteristic of subplate neurons, and (iii) can be labeled by an injection of (3H)thymidine on embryonic day (E) 28, a time when only subplate neurons are being generated. Intense NGFR immunostaining is seen on the cell bodies of these neurons as early as E30, several days after their last round of cell division, and this immunostaining remains strong for approximately 3 weeks. The NGFR immunoreactivity begins to decline around E52 and has disappeared from the region altogether by E60, at which time subplate neurons begin to die. The cellular localization and timing of expression suggest that the NGFR may play a role in the maintenance of subplate neurons and in the maturation of the cerebral cortex.

  17. Association of CD8(+) T lymphocyte repertoire spreading with the severity of DRESS syndrome.

    PubMed

    Niu, Jun; Jia, Qingzhu; Ni, Qingshan; Yang, Yi; Chen, Gang; Yang, Xichuan; Zhai, Zhifang; Yu, Haili; Guan, Peng; Lin, Regina; Song, Zhiqiang; Li, Qi-Jing; Hao, Fei; Zhong, Hua; Wan, Ying

    2015-04-23

    T-cell receptor (TCR)-mediated cross-recognition is a major mechanism in the pathogenesis of drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome. However, the characteristics of the TCR repertoire and the clinical significance of repertoire reformation throughout the course of DRESS are unknown. Here, we isolated CD4(+) and CD8(+) T-cells from peripheral blood of 8 DRESS patients at 10-day intervals and, sequenced CDR3-regions of the TCRB chain by high-throughput sequencing to analyze the dynamic reformation in the T-cell repertoire hierarchy. Compared with healthy donors, T-cell expanded in peripheral repertoires from DRESS patient. The extent of fluctuation of dominant CD8(+) T-cell clones, but not of CD4(+) counterparts, correlated positively with the clinical severity and helped classify the enrolled subjects into "fluctuant" and "flat" repertoire groups. The anti-herpesvirus response, which was measured using anti-EBV/HHV antibodies, and the proportion of the homologous CD8(+) EBV-specific clonotypes, in the "fluctuant" group was substantial higher than that in the "flat" group. Furthermore, autoimmune sequelae were observed in a cured "fluctuant" patient. Collectively, the clinical relevance of the fluctuant CD8(+) T-cell repertoires supports the notion that herpes virus-mediated continuously de novo priming of newly pathogenic CD8(+) T-cell clones is an alternate mechanism responsible for the pathogenicity of DRESS.

  18. Coevolution of intelligence, behavioral repertoire, and lifespan.

    PubMed

    Ghirlanda, Stefano; Enquist, Magnus; Lind, Johan

    2014-02-01

    Across many taxa, intriguing positive correlations exist between intelligence (measured by proxy as encephalization), behavioral repertoire size, and lifespan. Here we argue, through a simple theoretical model, that such correlations arise from selection pressures for efficient learning of behavior sequences. We define intelligence operationally as the ability to disregard unrewarding behavior sequences, without trying them out, in the search for rewarding sequences. We show that increasing a species' behavioral repertoire increases the number of rewarding behavior sequences that can be performed, but also the time required to learn such sequences. This trade-off results in an optimal repertoire size that decreases rapidly with increasing sequence length. Behavioral repertoire size can be increased by increasing intelligence or lengthening the lifespan, giving rise to the observed correlations between these traits.

  19. Expanding the repertoire of deadenylases.

    PubMed

    Skeparnias, Ilias; Αnastasakis, Dimitrios; Shaukat, Athanasios-Nasir; Grafanaki, Katerina; Stathopoulos, Constantinos

    2017-03-07

    Deadenylases belong to an expanding family of exoribonucleases involved mainly in mRNA stability and turnover, with the exception of PARN which has additional roles in the biogenesis of several important non-coding RNAs, including miRNAs and piRNAs. Recently, PARN in C. elegans and its homolog PNLDC1 in B. mori were reported as the elusive trimmers mediating piRNA biogenesis. In addition, characterization of mammalian PNLDC1 in comparison to PARN, showed that is specifically expressed in embryonic stem and germ cells, as well as during early embryo development. Moreover, its expression is correlated with epigenetic events mediated by the de novo DNMT3b methyltransferase and knockdown in stem cells upregulates important genes that regulate multipotency. The recent data suggest that at least some new deadenylases may have expanded roles in cell metabolism as regulators of gene expression, through mRNA deadenylation, ncRNAs biogenesis and ncRNA-mediated mRNA targeting, linking essential mechanisms that regulate epigenetic control and transition events during differentiation. The possible roles of mammalian PNLDC1 along those dynamic networks are discussed in the light of new extremely important findings.

  20. Insights into immune system development and function from mouse T-cell repertoires

    PubMed Central

    Sethna, Zachary; Elhanati, Yuval; Dudgeon, Chrissy S.; Callan, Curtis G.; Levine, Arnold J.; Mora, Thierry; Walczak, Aleksandra M.

    2017-01-01

    The ability of the adaptive immune system to respond to arbitrary pathogens stems from the broad diversity of immune cell surface receptors. This diversity originates in a stochastic DNA editing process (VDJ recombination) that acts on the surface receptor gene each time a new immune cell is created from a stem cell. By analyzing T-cell receptor (TCR) sequence repertoires taken from the blood and thymus of mice of different ages, we quantify the changes in the VDJ recombination process that occur from embryo to young adult. We find a rapid increase with age in the number of random insertions and a dramatic increase in diversity. Because the blood accumulates thymic output over time, blood repertoires are mixtures of different statistical recombination processes, and we unravel the mixture statistics to obtain a picture of the time evolution of the early immune system. Sequence repertoire analysis also allows us to detect the statistical impact of selection on the output of the VDJ recombination process. The effects we find are nearly identical between thymus and blood, suggesting that our analysis mainly detects selection for proper folding of the TCR receptor protein. We further find that selection is weaker in laboratory mice than in humans and it does not affect the diversity of the repertoire. PMID:28196891

  1. Insights into immune system development and function from mouse T-cell repertoires.

    PubMed

    Sethna, Zachary; Elhanati, Yuval; Dudgeon, Chrissy S; Callan, Curtis G; Levine, Arnold J; Mora, Thierry; Walczak, Aleksandra M

    2017-02-28

    The ability of the adaptive immune system to respond to arbitrary pathogens stems from the broad diversity of immune cell surface receptors. This diversity originates in a stochastic DNA editing process (VDJ recombination) that acts on the surface receptor gene each time a new immune cell is created from a stem cell. By analyzing T-cell receptor (TCR) sequence repertoires taken from the blood and thymus of mice of different ages, we quantify the changes in the VDJ recombination process that occur from embryo to young adult. We find a rapid increase with age in the number of random insertions and a dramatic increase in diversity. Because the blood accumulates thymic output over time, blood repertoires are mixtures of different statistical recombination processes, and we unravel the mixture statistics to obtain a picture of the time evolution of the early immune system. Sequence repertoire analysis also allows us to detect the statistical impact of selection on the output of the VDJ recombination process. The effects we find are nearly identical between thymus and blood, suggesting that our analysis mainly detects selection for proper folding of the TCR receptor protein. We further find that selection is weaker in laboratory mice than in humans and it does not affect the diversity of the repertoire.

  2. Concentration dependence of sodium permeation and sodium ion interactions in the cyclic AMP-gated channels of mammalian olfactory receptor neurons.

    PubMed

    Balasubramanian, S; Lynch, J W; Barry, P H

    1997-09-01

    The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs) on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique. With a saturating concentration (100 microM) of adenosine 3',5'-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied at NaCl concentrations from 25 to 300 mM. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear, reversing close to 0 mV. When the external NaCl concentration was maintained at 150 mM and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the Na+ equilibrium potential indicating that PCl/PNa approximately 0. However, at low external NaCl concentrations (< or = 100 mM) there was some significant chloride permeability. Our results further indicated that Na+ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K(m)s in the range of 100-150 mM and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory CNG channels are significantly different from those of photoreceptor CNG channels.

  3. 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 Central

    SAN, YONG-ZHI; LIU, YU; ZHANG, YU; SHI, PING-PING; ZHU, YU-LAN

    2015-01-01

    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

  4. Single amino acid substitutions in alpha-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor.

    PubMed

    Hogg, R C; Miranda, L P; Craik, D J; Lewis, R J; Alewood, P F; Adams, D J

    1999-12-17

    The alpha-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) antagonists, are emerging as important probes of the role played by different nAChR subtypes in cell function and communication. In this study, the native alpha-conotoxins PnIA and PnIB were found to cause concentration-dependent inhibition of the ACh-induced current in all rat parasympathetic neurons examined, with IC(50) values of 14 and 33 nM, and a maximal reduction in current amplitude of 87% and 71%, respectively. The modified alpha-conotoxin [N11S]PnIA reduced the ACh-induced current with an IC(50) value of 375 nM and a maximally effective concentration caused 91% block. [A10L]PnIA was the most potent inhibitor, reducing the ACh-induced current in approximately 80% of neurons, with an IC(50) value of 1.4 nM and 46% maximal block of the total current. The residual current was not inhibited further by alpha-bungarotoxin, but was further reduced by the alpha-conotoxins PnIA or PnIB, and by mecamylamine. (1)H NMR studies indicate that PnIA, PnIB, and the analogues, [A10L]PnIA and [N11S]PnIA, have identical backbone structures. We propose that positions 10 and 11 of PnIA and PnIB influence potency and determine selectivity among alpha7 and other nAChR subtypes, including alpha3beta2 and alpha3beta4. Four distinct components of the nicotinic ACh-induced current in mammalian parasympathetic neurons have been dissected with these conopeptides.

  5. The mammalian target of rapamycin complex 1 (mTORC1) in breast cancer: the impact of oestrogen receptor and HER2 pathways.

    PubMed

    Jerjees, Dena A; Negm, Ola H; Alabdullah, M Layth; Mirza, Sameer; Alkaabi, Methaq; Hameed, Mohamed R; Abduljabbar, Rezvan; Muftah, Abir; Nolan, Chris C; Green, Andrew R; Tighe, Patrick J; Band, Vimla; Ellis, Ian O; Rakha, Emad A

    2015-02-01

    The mammalian target of rapamycin complex 1 (mTORC1) is a downstream of the PI3K/Akt pathway which affects cancer development. mTORC1 has many downstream signalling effectors that can enhance different cellular responses. This study aims to investigate the expression of mTORC1 in breast cancer (BC) and correlate it with key clinicopathological and molecular features of BC especially to proteins related to oestrogen receptor (ER) and HER2 pathways in different BC classes. Moreover, mTORC1 expression was assessed in 6 BC cell lines including ER+ and ER- cell lines with and without HER2 transfection. Immunohistochemistry was used to assess the expression of phospho (p) mTORC1 in a large (n = 1300) annotated BC series prepared as tissue microarray. Reverse phase protein array (RPPA) was used to assess its expression in the different BC cell lines. The expression of p-mTORC1 was cytoplasmic with moderate/high expression noted in 44 % of BC. p-mTORC1 expression was associated with clinicopathological variables characteristic of good prognosis. Positive correlation with ER, ER-related proteins AKT, PI3K and luminal differentiation markers were observed in the whole series and in the ER+HER2- subgroup. Association with HER2 was mainly observed in the ER-negative class. RPPA indicated that p-mTORC1 expression was mainly related to ER expression and with better outcome in the Akt positive tumours. p-mTORC1 is associated with good prognostic features. Its expression is related to ER and ER related proteins in addition to AKT and PI3K. Its relation with HER2 expression is mainly seen in the absence of ER expression.

  6. FGF signaling repertoire of the indirect developing hemichordate Ptychodera flava.

    PubMed

    Fan, Tzu-Pei; Su, Yi-Hsien

    2015-12-01

    Fibroblast growth factors (FGFs) are a group of ligands that play multiple roles during development by transducing signals through FGF receptors (FGFRs) to downstream factors. At least 22 FGF ligands and 4 receptors have been identified in vertebrates, while six to eight FGF ligands and a single FGFR are present in invertebrate chordates, such as tunicates and amphioxus. The chordate FGFs can be categorized into at least seven subfamilies, and the members of which expanded during the evolution of early vertebrates. In contrast, only one FGF and two FGFRs have been found in sea urchins. Thus, it is unclear whether the FGF subfamilies duplicated in the lineage leading to the chordates, or sea urchins lost several fgf genes. Analyses of the FGF signaling repertoire in hemichordates, which together with echinoderms form the closest group to the chordates, may provide insights into the evolution of FGF signaling in deuterostomes. In this study, we identified five FGFs and three FGFRs from Ptychodera flava, an indirect-developing hemichordate acorn worm. Phylogenetic analyses revealed that hemichordates possess a conserved FGF8/17/18 in addition to several putative hemichordate-specific FGFs. Analyses of sequence similarity and protein domain organizations suggested that the sea urchin and hemichordate FGFRs arose from independent lineage-specific duplications. Furthermore, the acorn worm fgf and fgfr genes were demonstrated to be expressed during P. flava embryogenesis. These results set the foundations for further functional studies of FGF signaling in hemichordates and provided insights into the evolutionary history of the FGF repertoire.

  7. Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein.

    PubMed

    Boulay, G; Zhu, X; Peyton, M; Jiang, M; Hurst, R; Stefani, E; Birnbaumer, L

    1997-11-21

    Hormonal stimulation of Gq-protein coupled receptors triggers Ca2+ mobilization from internal stores. This is followed by a Ca2+ entry through the plasma membrane. Drosophila Trp and Trpl proteins have been implicated in Ca2+ entry and three mammalian homologues of Drosophila Trp/Trpl, hTrp1, hTrp3 and bTrp4 (also bCCE) have been cloned and expressed. Using mouse brain RNA as template, we report here the polymerase chain reaction-based cloning and functional expression of a novel Trp, mTrp6. The cDNA encodes a protein of 930 amino acids, the sequence of which is 36.8, 36.3, 43.1, 38.6, and 74. 1% identical to Drosophila Trp and Trpl, bovine Trp4, and human Trp1 and Trp3, respectively. Transient expression of mTrp6 in COS.M6 cells by transfection of the full-length mTrp6 cDNA increases Ca2+ entry induced by stimulation of co-transfected M5 muscarinic acetylcholine receptor with carbachol (CCh), as seen by dual wavelength fura 2 fluorescence ratio measurements. The mTrp6-mediated increase in Ca2+ entry activity was blocked by SKF-96365 and La3+. Ca2+ entry activity induced by thapsigargin was similar in COS cells transfected with or without the mTrp6 cDNA. The thapsigargin-stimulated Ca2+ entry could not be further stimulated by CCh in control cells but was markedly increased in mTrp6-transfected cells. Records of whole cell transmembrane currents developed in response to voltage ramps from -80 to +40 mV in control HEK cells and HEK cells stably expressing mTrp6 revealed the presence of a muscarinic receptor responsive non-selective cation conductance in Trp6 cells that was absent in control cells. Our data support the hypothesis that mTrp6 encodes an ion channel subunit that mediates Ca2+ entry stimulated by a G-protein coupled receptor, but not Ca2+ entry stimulated by intracellular Ca2+ store depletion.

  8. Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders.

    PubMed

    Hu, Francis Jingxin; Volk, Anna-Luisa; Persson, Helena; Säll, Anna; Borrebaeck, Carl; Uhlen, Mathias; Rockberg, Johan

    2017-08-01

    Surface display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various display systems available, phage display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive bacterial host for display of a library of human antibody genes which, when combined with phage display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive bacterial display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Quantitative Analysis of Repertoire-Scale Immunoglobulin Properties in Vaccine-Induced B-Cell Responses.

    PubMed

    Khavrutskii, Ilja V; Chaudhury, Sidhartha; Stronsky, Sabrina M; Lee, Donald W; Benko, Jacqueline G; Wallqvist, Anders; Bavari, Sina; Cooper, Christopher L

    2017-01-01

    Recent advances in the next-generation sequencing of B-cell receptors (BCRs) enable the characterization of humoral responses at a repertoire-wide scale and provide the capability for identifying unique features of immune repertoires in response to disease, vaccination, or infection. Immunosequencing now readily generates 10(3)-10(5) sequences per sample; however, statistical analysis of these repertoires is challenging because of the high genetic diversity of BCRs and the elaborate clonal relationships among them. To date, most immunosequencing analyses have focused on reporting qualitative trends in immunoglobulin (Ig) properties, such as usage or somatic hypermutation (SHM) percentage of the Ig heavy chain variable (IGHV) gene segment family, and on reducing complex Ig property distributions to simple summary statistics. However, because Ig properties are typically not normally distributed, any approach that fails to assess the distribution as a whole may be inadequate in (1) properly assessing the statistical significance of repertoire differences, (2) identifying how two repertoires differ, and (3) determining appropriate confidence intervals for assessing the size of the differences and their potential biological relevance. To address these issues, we have developed a technique that uses Wilcox' robust statistics toolbox to identify statistically significant vaccine-specific differences between Ig repertoire properties. The advantage of this technique is that it can determine not only whether but also where the distributions differ, even when the Ig repertoire properties are non-normally distributed. We used this technique to characterize murine germinal center (GC) B-cell repertoires in response to a complex Ebola virus-like particle (eVLP) vaccine candidate with known protective efficacy. The eVLP-mediated GC B-cell responses were highly diverse, consisting of thousands of clonotypes. Despite this staggering diversity, we identified statistically

  10. Deciphering the olfactory repertoire of the tiger mosquito Aedes albopictus.

    PubMed

    Lombardo, Fabrizio; Salvemini, Marco; Fiorillo, Carmine; Nolan, Tony; Zwiebel, Laurence J; Ribeiro, José M; Arcà, Bruno

    2017-10-11

    The Asian tiger mosquito Aedes albopictus is a highly invasive species and competent vector of several arboviruses (e.g. dengue, chikungunya, Zika) and parasites (e.g. dirofilaria) of public health importance. Compared to other mosquito species, Ae. albopictus females exhibit a generalist host seeking as well as a very aggressive biting behaviour that are responsible for its high degree of nuisance. Several complex mosquito behaviours such as host seeking, feeding, mating or oviposition rely on olfactory stimuli that target a range of sensory neurons localized mainly on specialized head appendages such as antennae, maxillary palps and the mouthparts. With the aim to describe the Ae. albopictus olfactory repertoire we have used RNA-seq to reveal the transcriptome profiles of female antennae and maxillary palps. Male heads and whole female bodies were employed as reference for differential expression analysis. The relative transcript abundance within each tissue (TPM, transcripts per kilobase per million) and the pairwise differential abundance in the different tissues (fold change values and false discovery rates) were evaluated. Contigs upregulated in the antennae (620) and maxillary palps (268) were identified and relative GO and PFAM enrichment profiles analysed. Chemosensory genes were described: overall, 77 odorant binding proteins (OBP), 82 odorant receptors (OR), 60 ionotropic receptors (IR) and 30 gustatory receptors (GR) were identified by comparative genomics and transcriptomics. In addition, orthologs of genes expressed in the female/male maxillary palps and/or antennae and involved in thermosensation (e.g. pyrexia and arrestin1), mechanosensation (e.g. piezo and painless) and neuromodulation were classified. We provide here the first detailed transcriptome of the main Ae. albopictus sensory appendages, i.e. antennae and maxillary palps. A deeper knowledge of the olfactory repertoire of the tiger mosquito will help to better understand its biology and may

  11. Characterization of human αβTCR repertoire and discovery of D-D fusion in TCRβ chains.

    PubMed

    Liu, Peipei; Liu, Di; Yang, Xi; Gao, Jing; Chen, Yan; Xiao, Xue; Liu, Fei; Zou, Jing; Wu, Jun; Ma, Juncai; Zhao, Fangqing; Zhou, Xuyu; Gao, George F; Zhu, Baoli

    2014-01-01

    The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ~2% of the productive human TCRβ CDR3 sequences.

  12. Highly diverse TCRα chain repertoire of pre-immune CD8+ T cells reveals new insights in gene recombination

    PubMed Central

    Genolet, Raphael; Stevenson, Brian J; Farinelli, Laurent; Østerås, Magne; Luescher, Immanuel F

    2012-01-01

    Although the T-cell receptor αδ (TCRαδ) locus harbours large libraries of variable (TRAV) and junctional (TRAJ) gene segments, according to previous studies the TCRα chain repertoire is of limited diversity due to restrictions imposed by sequential coordinate TRAV-TRAJ recombinations. By sequencing tens of millions of TCRα chain transcripts from naive mouse CD8+ T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similarly to that demonstrated for IgH gene recombination. Generation of the observed highly diverse TCRα chain repertoire necessitates deletion of failed attempts by thymic-positive selection and is essential for the formation of highly diverse TCRαβ repertoires, capable of providing good protective immunity. PMID:22373576

  13. 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.

  14. 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

  15. N-linked glycosylation is required for plasma membrane localization of D5, but not D1, dopamine receptors in transfected mammalian cells.

    PubMed

    Karpa, K D; Lidow, M S; Pickering, M T; Levenson, R; Bergson, C

    1999-11-01

    We have analyzed the role of N-linked glycosylation in functional cell surface expression of the D1 and D5 dopamine receptor subtypes. Treatment of transfected HEK 293 cells with tunicamycin, an inhibitor of N-linked oligosaccharide addition, was found to prevent localization of D5 receptors in the plasma membrane. In contrast, tunicamycin treatment had no effect on the plasma membrane localization of the D1 receptor. Polymerase chain reaction mutagenesis was used to generate a panel of D5 receptors containing mutations in the three predicted sites of N-linked glycosylation. Expression of mutant receptors indicated that glycosylation of residue N7 was the major determinant of D5 receptor plasma membrane localization. Mutation of a comparable site in the D1 receptor at position N5 had no effect on the delivery of the D1 receptor to the cell surface. Tunicamycin treatment during receptor biosynthesis, but not N-glycosidase F digestion of mature receptors, abrogated binding of the D5 receptor antagonist [(3)H]SCH23390, suggesting that while oligosaccharide moieties play a key role in the cell surface expression of D5 receptors, they do not appear to contribute to the receptor's ligand binding properties. Together, our data indicate a differential requirement for N-linked glycosylation in functional cell surface expression of D1 and D5 dopamine receptors.

  16. Comprehensive assessment of peripheral blood TCRβ repertoire in infectious mononucleosis and chronic active EBV infection patients.

    PubMed

    Liu, Shenglin; Zhang, Qian; Huang, Dongli; Zhang, Wenli; Zhong, Fengluan; Feng, Jia; Chen, Xueru; Meng, Qingxiang; Chen, Xiaofan; Zhang, Wei; Zhang, Hongyu

    2017-04-01

    Epstein-Barr virus (EBV) primary infection is usually asymptomatic, but it sometimes progresses to infectious mononucleosis (IM). Occasionally, some people develop chronic active EBV infection (CAEBV) with underlying immunodeficiency, which belongs to a continuous spectrum of EBV-associated lymphoproliferative disorders (EBV(+) LPD) with heterogeneous clinical presentations and high mortality. It has been well established that T cell-mediated immune response plays a critical role in the disease evolution of EBV infection. Recently, high-throughput sequencing of the hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (T cell receptor β (TCRβ)) has emerged as a sensitive approach to assess the T cell repertoire. In this study, we fully characterized the diversity of peripheral blood TCRβ repertoire in IM (n = 6) and CAEBV patients (n = 5) and EBV-seropositive controls (n = 5). Compared with the healthy EBV-seropositive controls, both IM and CAEBV patients demonstrate a significant decrease in peripheral blood TCRβ repertoire diversity, basically, including narrowed repertoire breadth, highly expanded clones, and skewed CDR3 length distribution. However, there is no significant difference between IM and CAEBV patients. Furthermore, we observed some disease-related preferences in TRBV/TRBJ usage and combinations, as well as lots of T cell clones shared by different groups (unique or overlapped) involved in public T cell responses, which provide more detailed insights into the divergent disease evolution.

  17. Evolution and function of the TCR Vgamma9 chain repertoire: It's good to be public.

    PubMed

    Pauza, C David; Cairo, Cristiana

    2015-07-01

    Lymphocytes expressing a T cell receptor (TCR) composed of Vgamma9 and Vdelta2 chains represent a minor fraction of human thymocytes. Extrathymic selection throughout post-natal life causes the proportion of cells with a Vgamma9-JP rearrangement to increase and elevates the capacity for responding to non-peptidic phosphoantigens. Extrathymic selection is so powerful that phosphoantigen-reactive cells comprise about 1 in 40 circulating memory T cells in healthy adults and the subset expands rapidly upon infection or in response to malignancy. Skewing of the gamma delta TCR repertoire is accompanied by selection for public gamma chain sequences such that many unrelated individuals overlap extensive in their circulating repertoire. This type of selection implies the presence of a monomorphic antigen-presenting molecule that is an object of current research but remains incompletely defined. While selection on a monomorphic presenting molecule may seem unusual, similar mechanisms shape the alpha beta T cell repertoire including the extreme examples of NKT or mucosal-associated invariant T cells (MAIT) and the less dramatic amplification of public Vbeta chain rearrangements driven by individual MHC molecules and associated with resistance to viral pathogens. Selecting and amplifying public T cell receptors whether alpha beta or gamma delta, are important steps in developing an anticipatory TCR repertoire. Cell clones expressing public TCR can accelerate the kinetics of response to pathogens and impact host survival.

  18. Gestural communication of the gorilla (Gorilla gorilla): repertoire, intentionality and possible origins.

    PubMed

    Genty, Emilie; Breuer, Thomas; Hobaiter, Catherine; Byrne, Richard W

    2009-05-01

    Social groups of gorillas were observed in three captive facilities and one African field site. Cases of potential gesture use, totalling 9,540, were filtered by strict criteria for intentionality, giving a corpus of 5,250 instances of intentional gesture use. This indicated a repertoire of 102 gesture types. Most repertoire differences between individuals and sites were explicable as a consequence of environmental affordances and sampling effects: overall gesture frequency was a good predictor of universality of occurrence. Only one gesture was idiosyncratic to a single individual, and was given only to humans. Indications of cultural learning were few, though not absent. Six gestures appeared to be traditions within single social groups, but overall concordance in repertoires was almost as high between as within social groups. No support was found for the ontogenetic ritualization hypothesis as the chief means of acquisition of gestures. Many gestures whose form ruled out such an origin, i.e. gestures derived from species-typical displays, were used as intentionally and almost as flexibly as gestures whose form was consistent with learning by ritualization. When using both classes of gesture, gorillas paid specific attention to the attentional state of their audience. Thus, it would be unwarranted to divide ape gestural repertoires into 'innate, species-typical, inflexible reactions' and 'individually learned, intentional, flexible communication'. We conclude that gorilla gestural communication is based on a species-typical repertoire, like those of most other mammalian species but very much larger. Gorilla gestures are not, however, inflexible signals but are employed for intentional communication to specific individuals.

  19. Expanding the genetic code of mammalian cells.

    PubMed

    Italia, James S; Zheng, Yunan; Kelemen, Rachel E; Erickson, Sarah B; Addy, Partha S; Chatterjee, Abhishek

    2017-04-15

    In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. Over the last few years, exciting progress has been made toward expanding the toolbox of genetically encoded UAAs in mammalian cells, improving the efficiency of their incorporation and developing innovative applications. Here, we provide our perspective on these recent developments and highlight the current challenges that must be overcome to realize the full potential of this technology. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  20. 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.

  1. Agonist and antagonists induce homodimerization and mixed ligand heterodimerization of human progesterone receptors in vivo by a mammalian two-hybrid assay.

    PubMed

    Leonhardt, S A; Altmann, M; Edwards, D P

    1998-12-01

    This study utilizes the mammalian two-hybrid system to examine the role of ligand in the dimerization of human progesterone receptor (hPR). The GAL4 DNA-binding domain and the herpes simplex virus VP16 transactivation domain were fused to the amino terminus of full-length hPR (both the A and B isoforms) to produce chimeric proteins. PR dimerization was detected by the ability of cotransfected GAL4/PR and VP16/PR chimeras in COS cells to induce expression of a reporter gene under the control of GAL4-binding sites (pG5CAT). Hormone agonist-dependent interactions were observed between the two like isoforms of PR (A-A and B-B) and between PR-A and PR-B (A-B), indicating that hormone can stimulate the formation of the three possible dimeric forms of PR within cells. In contrast, neither type I (ZK98299) nor type II (RU486, ZK112993) progestin antagonists stimulated interaction between these same hybrid PR proteins. However, activation of the VP16/PR chimera by antagonists on a progesterone response element-controlled reporter gene (DHRE-E1b-CAT) was only a fraction (4-13%) of that stimulated by agonist R5020. One possibility for the failure to detect an induction in the two-hybrid assay is antagonist-induced repression of the activity of the VP16/PR fusion protein rather than a failure of antagonists to stimulate interaction between the hybrid proteins. To test this idea, an UP-1 carboxyl-terminal truncation mutant of PR was used to construct the two-hybrid proteins. PR-UP-1 selectively binds antagonists, but not agonists, and is fully activated in response to antagonists. Both types of progestin antagonists stimulated interactions between GAL4/PR(UP-1) and VP16/PR(UP-1) hybrid proteins, indicating that antagonists are capable of stimulating PR dimerization in cells and do not function by disrupting or preventing dimerization. To determine whether PR bound to an antagonist can dimerize in whole cells with PR bound to agonist, GAL4/PR(UP-1) was paired in the two

  2. Tissue-specific expressed antibody variable gene repertoires.

    PubMed

    Briney, Bryan S; Willis, Jordan R; Finn, Jessica A; McKinney, Brett A; Crowe, James E

    2014-01-01

    Recent developments in genetic technologies allow deep analysis of the sequence diversity of immune repertoires, but little work has been reported on the architecture of immune repertoires in mucosal tissues. Antibodies are the key to prevention of infections at the mucosal surface, but it is currently unclear whether the B cell repertoire at mucosal surfaces reflects the dominant antibodies found in the systemic compartment or whether mucosal tissues harbor unique repertoires. We examined the expressed antibody variable gene repertoires from 10 different human tissues using RNA samples derived from a large number of individuals. The results revealed that mucosal tissues such as stomach, intestine and lung possess unique antibody gene repertoires that differed substantially from those found in lymphoid tissues or peripheral blood. Mutation frequency analysis of mucosal tissue repertoires revealed that they were highly mutated, with little evidence for the presence of naïve B cells, in contrast to blood. Mucosal tissue repertoires possessed longer heavy chain complementarity determining region 3 loops than lymphoid tissue repertoires. We also noted a large increase in frequency of both insertions and deletions in the small intestine antibody repertoire. These data suggest that mucosal immune repertoires are distinct in many ways from the systemic compartment.

  3. High-Throughput Sequencing-Based Immune Repertoire Study during Infectious Disease

    PubMed Central

    Hou, Dongni; Chen, Cuicui; Seely, Eric John; Chen, Shujing; Song, Yuanlin

    2016-01-01

    The selectivity of the adaptive immune response is based on the enormous diversity of T and B cell antigen-specific receptors. The immune repertoire, the collection of T and B cells with functional diversity in the circulatory system at any given time, is dynamic and reflects the essence of immune selectivity. In this article, we review the recent advances in immune repertoire study of infectious diseases, which were achieved by traditional techniques and high-throughput sequencing (HTS) techniques. HTS techniques enable the determination of complementary regions of lymphocyte receptors with unprecedented efficiency and scale. This progress in methodology enhances the understanding of immunologic changes during pathogen challenge and also provides a basis for further development of novel diagnostic markers, immunotherapies, and vaccines. PMID:27630639

  4. Vocal repertoire of the social giant otter.

    PubMed

    Leuchtenberger, Caroline; Sousa-Lima, Renata; Duplaix, Nicole; Magnusson, William E; Mourão, Guilherme

    2014-11-01

    According to the "social intelligence hypothesis," species with complex social interactions have more sophisticated communication systems. Giant otters (Pteronura brasiliensis) live in groups with complex social interactions. It is likely that the vocal communication of giant otters is more sophisticated than previous studies suggest. The objectives of the current study were to describe the airborne vocal repertoire of giant otters in the Pantanal area of Brazil, to analyze call types within different behavioral contexts, and to correlate vocal complexity with level of sociability of mustelids to verify whether or not the result supports the social intelligence hypothesis. The behavior of nine giant otters groups was observed. Vocalizations recorded were acoustically and statistically analyzed to describe the species' repertoire. The repertoire was comprised by 15 sound types emitted in different behavioral contexts. The main behavioral contexts of each sound type were significantly associated with the acoustic variable ordination of different sound types. A strong correlation between vocal complexity and sociability was found for different species, suggesting that the communication systems observed in the family mustelidae support the social intelligence hypothesis.

  5. The vertebrate ancestral repertoire of visual opsins, transducin alpha subunits and oxytocin/vasopressin receptors was established by duplication of their shared genomic region in the two rounds of early vertebrate genome duplications

    PubMed Central

    2013-01-01

    Background Vertebrate color vision is dependent on four major color opsin subtypes: RH2 (green opsin), SWS1 (ultraviolet opsin), SWS2 (blue opsin), and LWS (red opsin). Together with the dim-light receptor rhodopsin (RH1), these form the family of vertebrate visual opsins. Vertebrate genomes contain many multi-membered gene families that can largely be explained by the two rounds of whole genome duplication (WGD) in the vertebrate ancestor (2R) followed by a third round in the teleost ancestor (3R). Related chromosome regions resulting from WGD or block duplications are said to form a paralogon. We describe here a paralogon containing the genes for visual opsins, the G-protein alpha subunit families for transducin (GNAT) and adenylyl cyclase inhibition (GNAI), the oxytocin and vasopressin receptors (OT/VP-R), and the L-type voltage-gated calcium channels (CACNA1-L). Results Sequence-based phylogenies and analyses of conserved synteny show that the above-mentioned gene families, and many neighboring gene families, expanded in the early vertebrate WGDs. This allows us to deduce the following evolutionary scenario: The vertebrate ancestor had a chromosome containing the genes for two visual opsins, one GNAT, one GNAI, two OT/VP-Rs and one CACNA1-L gene. This chromosome was quadrupled in 2R. Subsequent gene losses resulted in a set of five visual opsin genes, three GNAT and GNAI genes, six OT/VP-R genes and four CACNA1-L genes. These regions were duplicated again in 3R resulting in additional teleost genes for some of the families. Major chromosomal rearrangements have taken place in the teleost genomes. By comparison with the corresponding chromosomal regions in the spotted gar, which diverged prior to 3R, we could time these rearrangements to post-3R. Conclusions We present an extensive analysis of the paralogon housing the visual opsin, GNAT and GNAI, OT/VP-R, and CACNA1-L gene families. The combined data imply that the early vertebrate WGD events contributed to the

  6. The vertebrate ancestral repertoire of visual opsins, transducin alpha subunits and oxytocin/vasopressin receptors was established by duplication of their shared genomic region in the two rounds of early vertebrate genome duplications.

    PubMed

    Lagman, David; Ocampo Daza, Daniel; Widmark, Jenny; Abalo, Xesús M; Sundström, Görel; Larhammar, Dan

    2013-11-02

    Vertebrate color vision is dependent on four major color opsin subtypes: RH2 (green opsin), SWS1 (ultraviolet opsin), SWS2 (blue opsin), and LWS (red opsin). Together with the dim-light receptor rhodopsin (RH1), these form the family of vertebrate visual opsins. Vertebrate genomes contain many multi-membered gene families that can largely be explained by the two rounds of whole genome duplication (WGD) in the vertebrate ancestor (2R) followed by a third round in the teleost ancestor (3R). Related chromosome regions resulting from WGD or block duplications are said to form a paralogon. We describe here a paralogon containing the genes for visual opsins, the G-protein alpha subunit families for transducin (GNAT) and adenylyl cyclase inhibition (GNAI), the oxytocin and vasopressin receptors (OT/VP-R), and the L-type voltage-gated calcium channels (CACNA1-L). Sequence-based phylogenies and analyses of conserved synteny show that the above-mentioned gene families, and many neighboring gene families, expanded in the early vertebrate WGDs. This allows us to deduce the following evolutionary scenario: The vertebrate ancestor had a chromosome containing the genes for two visual opsins, one GNAT, one GNAI, two OT/VP-Rs and one CACNA1-L gene. This chromosome was quadrupled in 2R. Subsequent gene losses resulted in a set of five visual opsin genes, three GNAT and GNAI genes, six OT/VP-R genes and four CACNA1-L genes. These regions were duplicated again in 3R resulting in additional teleost genes for some of the families. Major chromosomal rearrangements have taken place in the teleost genomes. By comparison with the corresponding chromosomal regions in the spotted gar, which diverged prior to 3R, we could time these rearrangements to post-3R. We present an extensive analysis of the paralogon housing the visual opsin, GNAT and GNAI, OT/VP-R, and CACNA1-L gene families. The combined data imply that the early vertebrate WGD events contributed to the evolution of vision and the

  7. IgRepertoireConstructor: a novel algorithm for antibody repertoire construction and immunoproteogenomics analysis

    PubMed Central

    Safonova, Yana; Bonissone, Stefano; Kurpilyansky, Eugene; Starostina, Ekaterina; Lapidus, Alla; Stinson, Jeremy; DePalatis, Laura; Sandoval, Wendy; Lill, Jennie; Pevzner, Pavel A.

    2015-01-01

    The analysis of concentrations of circulating antibodies in serum (antibody repertoire) is a fundamental, yet poorly studied, problem in immunoinformatics. The two current approaches to the analysis of antibody repertoires [next generation sequencing (NGS) and mass spectrometry (MS)] present difficult computational challenges since antibodies are not directly encoded in the germline but are extensively diversified by somatic recombination and hypermutations. Therefore, the protein database required for the interpretation of spectra from circulating antibodies is custom for each individual. Although such a database can be constructed via NGS, the reads generated by NGS are error-prone and even a single nucleotide error precludes identification of a peptide by the standard proteomics tools. Here, we present the IgRepertoireConstructor algorithm that performs error-correction of immunosequencing reads and uses mass spectra to validate the constructed antibody repertoires. Availability and implementation: IgRepertoireConstructor is open source and freely available as a C++ and Python program running on all Unix-compatible platforms. The source code is available from http://bioinf.spbau.ru/igtools. Contact: ppevzner@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26072509

  8. Recruitment of activating NK-cell receptors 2B4 and NKG2D to membrane microdomains in mammalian cells is dependent on their transmembrane regions.

    PubMed

    Gütgemann, Stephan A; Sandusky, Mina M; Wingert, Sabine; Claus, Maren; Watzl, Carsten

    2015-04-01

    Membrane microdomains play an important role in the regulation of natural killer (NK) cell activities. These cholesterol-rich membrane domains are enriched at the activating immunological synapse and several activating NK-cell receptors are known to localize to membrane microdomains upon receptor engagement. In contrast, inhibitory receptors do not localize in these specialized membrane domains. In addition, the functional competence of educated NK cells correlates with a confinement of activating receptors in membrane microdomains. However, the molecular basis for this confinement is unknown. Here, we investigate the structural requirements for the recruitment of the human-activating NK-cell receptors NKG2D and 2B4 to detergent-resistant membrane fractions in the murine BA/F3 cell line and in the human NK-cell line NKL. This stimulation-dependent recruitment occurred independently of the intracellular domains of the receptors. However, either interfering with the association between NKG2D and DAP10, or mutating the transmembrane region of 2B4 impacted the recruitment of the receptors to detergent-resistant membrane fractions and modulated the function of 2B4 in NK cells. Our data suggest a potential interaction between the transmembrane region of NK-cell receptors and membrane lipids as a molecular mechanism involved in determining the membrane confinement of activating NK-cell receptors.

  9. The Potential Repertoire of the Innate Immune System in the Bladder: Expression of Pattern Recognition Receptors in the Rat Bladder and a Rat Urothelial Cell Line (MYP3 cells)

    PubMed Central

    Hughes, Francis M.; Turner, David P.; Purves, J. Todd

    2015-01-01

    Purpose The urothelium is a frontline sensor of the lower urinary tract, sampling the bladder lumen and stimulating an immune response to infectious and noxious agents. Pattern recognition receptors (PRRs) recognize such agents and coordinate the innate response, often by forming inflammasomes that activate caspase-1 and the release of Interleukin-1β. We have shown the presence of one PRR (NLRP3) in the urothelia and its central role in the inflammatory response to cyclophosphamide. The purpose of this study was to 1) assess the likely range of the PPR response by assessing the repertior present in the rat bladder and 2) determine the utility of the MYP3 rat urothelia cell line for in vitro studies by assessing it’s PPR repertior and functional responsiveness. Methods Immunohistochemistry was performed for seven PPRs (NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4 and AIM2) on bladder sections and MYP3 cells. For functionality, MYP3 cells were challanged with the quinessential NLRP3 activator ATP and assessed for caspase-1 activation. Results All PPRs examined were expressed in the bladder and localized to the urothelial layer with several also in the detrusor (none in the interstitia). MYP3 cells also expressed all PRRs with a variable intracellular location. ATP stimulated caspase-1 activity in MYP3 cells in a dose-dependent manner that was reduced by knockdown of NLRP3 expression. Conclusion The results suggest that the bladder possesses the capacity to initiate an innate immune response to a wide array of uropathological agents and the MYP3 cells will provide an excellent investigational tool for this field. PMID:26490556

  10. Endogenous antigen presentation by autoantigen-transfected Epstein-Barr virus-lymphoblastoid cells. I. Generation of human thyroid peroxidase-reactive T cells and their T cell receptor repertoire.

    PubMed Central

    Martin, A; Magnusson, R P; Kendler, D L; Concepcion, E; Ben-Nun, A; Davies, T F

    1993-01-01

    To develop a model for endogenous thyroid autoantigen presentation, we transfected EBV-transformed B lymphoblastoid cell lines (EBV-LCL), established from patients with autoimmune thyroid disease and normal controls, with cDNA for the human thyroid autoantigen thyroid peroxidase (hTPO). hTPO-antigen presentation to patient peripheral blood T cells was demonstrated after stimulation in vitro for 7 d with irradiated hTPO-transfected or untransfected autologous EBV-LCL. Anti-hTPO-reactive T cells were subsequently cloned in the presence of irradiated, autologous hTPO-transfected EBV-LCL and IL-2.10 T cell-cloned lines exhibited specific hTPO-induced proliferation (stimulation indices of 2.1-7.9) towards autologous hTPO-transfected EBV-LCL, and were subjected to human T cell receptor (hTCR) V gene analysis, using the PCR for the detection of V alpha and V beta hTcR gene families. The results indicated a preferential use of hTCR V alpha 1 and/or V alpha 3 in 9 of the 10 lines. In contrast, hTCR V beta gene family use was more variable. These data demonstrate a model for the endogenous presentation of human thyroid peroxidase in the absence of other thyroid specific antigens. The high frequency of antigen-specific T cells obtained from PBMC using this technique will facilitate further studies at both the functional and hTCR V gene level. Images PMID:7682574

  11. Identification and characterization of the free fatty acid receptor 2 (FFA2) and a novel functional FFA2-like receptor (FFA2L) for short-chain fatty acids in pigs: evidence for the existence of a duplicated FFA2 gene (FFA2L) in some mammalian species.

    PubMed

    Zhang, J; Cheng, S; Wang, Y; Yu, X; Li, J

    2014-04-01

    Free fatty acid receptor 2 (FFA2, also called GPR43) is reported to play a critical role in mediating the actions of short-chain fatty acids (SCFAs) in humans and mice. However, little is known about the structure, functionality, and tissue expression of FFA2 in other mammalian species, including pigs. In the present study, the full-length cDNAs of FFA2 (pFFA2) and a novel FFA2-like gene (named pFFA2L) were cloned from pig intestines by reverse transcription PCR. Both cloned pFFA2 and pFFA2L are predicted to encode 2 receptors of remarkable structural similarity and share high amino acid sequence identities with FFA2 from other mammalian species. Interestingly, the novel FFA2L could also be identified in 9 other mammalian species, suggesting that FFA2L was likely duplicated from FFA2 in the last common ancestor of these species. With the use of a pGL4-SRE-luciferase reporter assay, we demonstrated that pFFA2 expressed in human embryonic kidney 293 cells could be activated by acetate, propionate, and butyrate equipotently, whereas pFFA2L could be activated only by acetate and propionate, indicating that both pFFA2 and pFFA2L are functional receptors for SCFAs with nonidentical pharmacologic properties. Reverse transcription PCR found that pFFA2 mRNA was widely expressed in nearly all tissues examined, including adipose tissue and gastrointestinal (GI) tract, whereas pFFA2L expression was mainly restricted to the GI tract. Taken together, our findings raise a novel concept that the actions of SCFAs are likely mediated by 2 FFA2s (FFA2 and FFA2L) in target tissues of some mammalian species, such as the GI tract of pigs. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Distinctive properties of identical twins' TCR repertoires revealed by high-throughput sequencing.

    PubMed

    Zvyagin, Ivan V; Pogorelyy, Mikhail V; Ivanova, Marina E; Komech, Ekaterina A; Shugay, Mikhail; Bolotin, Dmitry A; Shelenkov, Andrey A; Kurnosov, Alexey A; Staroverov, Dmitriy B; Chudakov, Dmitriy M; Lebedev, Yuri B; Mamedov, Ilgar Z

    2014-04-22

    Adaptive immunity in humans is provided by hypervariable Ig-like molecules on the surface of B and T cells. The final set of these molecules in each organism is formed under the influence of two forces: individual genetic traits and the environment, which includes the diverse spectra of alien and self-antigens. Here we assess the impact of individual genetic factors on the formation of the adaptive immunity by analyzing the T-cell receptor (TCR) repertoires of three pairs of monozygous twins by next-generation sequencing. Surprisingly, we found that an overlap between the TCR repertoires of monozygous twins is similar to an overlap between the TCR repertoires of nonrelated individuals. However, the number of identical complementary determining region 3 sequences in two individuals is significantly increased for twin pairs in the fraction of highly abundant TCR molecules, which is enriched by the antigen-experienced T cells. We found that the initial recruitment of particular TCR V genes for recombination and subsequent selection in the thymus is strictly determined by individual genetic factors. J genes of TCRs are selected randomly for recombination; however, the subsequent selection in the thymus gives preference to some α but not β J segments. These findings provide a deeper insight into the mechanism of TCR repertoire generation.

  13. Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model

    PubMed Central

    Hunter, Morag Rose; Grimsey, Natasha Lillia; Glass, Michelle

    2016-01-01

    G protein-coupled receptors (GPCRs) are important therapeutic targets and therefore extensively studied. Like most transmembrane proteins, there has been considerable difficulty in developing reliable specific antibodies for them. To overcome this, epitope tags are often used to facilitate antibody recognition in studies on fundamental receptor signalling and trafficking. In our study of cannabinoid CB1/dopamine D2 interactions we sought to generate HEK293 cells expressing FLAG-tagged D2 for use in antibody-based assays of GPCR localisation and trafficking activity, however observed that stable FLAG-hD2 expression was particularly challenging to maintain. In contrast, when expressed in cell lines expressing hCB1 robust and stable FLAG-hD2 expression was observed. We hypothesised that co-expression of CB1 might stabilise surface FLAG-hD2 expression, and therefore investigated this further. Here, we describe the observation that co-expression of either cannabinoid CB1 or CB2 receptors in HEK293 decreases the sulfation of a FLAG epitope appended at the N-terminus of the dopamine D2 receptor. Sulfation alters epitope recognition by some anti-FLAG antibodies, leading to the detection of fewer receptors, even though expression is maintained. This demonstrates that cannabinoid receptor expression modifies posttranslational processing of the FLAG-hD2 receptor, and importantly, has wider implications for the utilisation and interpretation of receptor studies involving epitope tags. PMID:27273047

  14. Exhaustive T-cell repertoire sequencing of human peripheral blood samples reveals signatures of antigen selection and a directly measured repertoire size of at least 1 million clonotypes.

    PubMed

    Warren, René L; Freeman, J Douglas; Zeng, Thomas; Choe, Gina; Munro, Sarah; Moore, Richard; Webb, John R; Holt, Robert A

    2011-05-01

    Massively parallel sequencing is a useful approach for characterizing T-cell receptor diversity. However, immune receptors are extraordinarily difficult sequencing targets because any given receptor variant may be present in very low abundance and may differ legitimately by only a single nucleotide. We show that the sensitivity of sequence-based repertoire profiling is limited by both sequencing depth and sequencing accuracy. At two timepoints, 1 wk apart, we isolated bulk PBMC plus naïve (CD45RA+/CD45RO-) and memory (CD45RA-/CD45RO+) T-cell subsets from a healthy donor. From T-cell receptor beta chain (TCRB) mRNA we constructed and sequenced multiple libraries to obtain a total of 1.7 billion paired sequence reads. The sequencing error rate was determined empirically and used to inform a high stringency data filtering procedure. The error filtered data yielded 1,061,522 distinct TCRB nucleotide sequences from this subject which establishes a new, directly measured, lower limit on individual T-cell repertoire size and provides a useful reference set of sequences for repertoire analysis. TCRB nucleotide sequences obtained from two additional donors were compared to those from the first donor and revealed limited sharing (up to 1.1%) of nucleotide sequences among donors, but substantially higher sharing (up to 14.2%) of inferred amino acid sequences. For each donor, shared amino acid sequences were encoded by a much larger diversity of nucleotide sequences than were unshared amino acid sequences. We also observed a highly statistically significant association between numbers of shared sequences and shared HLA class I alleles.

  15. Presynaptic localization of GluK5 in rod photoreceptors suggests a novel function of high affinity glutamate receptors in the mammalian retina

    PubMed Central

    Frotscher, Michael

    2017-01-01

    Kainate receptors mediate glutamatergic signaling through both pre- and presynaptic receptors. Here, we studied the expression of the high affinity kainate receptor GluK5 in the mouse retina. Double-immunofluoresence labeling and electron microscopic analysis revealed a presynaptic localization of GluK5 in the outer plexiform layer. Unexpectedly, we found GluK5 almost exclusively localized to the presynaptic ribbon of photoreceptor terminals. Moreover, in GluK5-deficient mutant mice the structural integrity of synaptic ribbons was severely altered pointing to a novel function of GluK5 in organizing synaptic ribbons in the presynaptic terminals of rod photoreceptors. PMID:28235022

  16. Interaction of the M4 Segment with Other Transmembrane Segments Is Required for Surface Expression of Mammalian α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors*

    PubMed Central

    Salussolia, Catherine L.; Corrales, Alexandra; Talukder, Iehab; Kazi, Rashek; Akgul, Gulcan; Bowen, Mark; Wollmuth, Lonnie P.

    2011-01-01

    Ionotropic glutamate receptors (GluRs) are ligand-gated ion channels with a modular structure. The ion channel itself shares structural similarity, albeit an inverted membrane topology, with P-loop channels. Like P-loop channels, prokaryotic GluR subunits (e.g. GluR0) have two transmembrane segments. In contrast, eukaryotic GluRs have an additional transmembrane segment (M4), located C-terminal to the ion channel core. However, the structural/functional significance of this additional transmembrane segment is poorly defined. Although topologically similar to GluR0, mammalian AMPA receptor (GluA1) subunits lacking the M4 segment do not display surface expression. This lack of expression is not due to the M4 segment serving as an anchor to the ligand-binding domain because insertion of an artificial polyleucine transmembrane segment does not rescue surface expression. Specific interactions between M4 and the ligand-binding domain are also unlikely because insertion of polyglycines into the linker connecting them has no deleterious effects on function or surface expression. However, tryptophan and cysteine scanning mutagenesis of the M4 segment, as well as recovery of function in the polyleucine background, defined a unique face of the M4 helix that is required for GluR surface expression. In the AMPA receptor structure, this face forms intersubunit contacts with the transmembrane helices of the ion channel core (M1 and M3) from another subunit within the homotetramer. Thus, our experiments show that a highly specific interaction of the M4 segment with an adjacent subunit is required for surface expression of AMPA receptors. This interaction may represent a mechanism for regulating AMPA receptor biogenesis. PMID:21930708

  17. Systematic Comparative Evaluation of Methods for Investigating the TCRβ Repertoire

    PubMed Central

    Zhang, Ruifang; Du, Yuanping; Hong, Xueyu; Cao, Hongzhi; Su, Zheng; Wang, Changxi; Wu, Jinghua; Nie, Chao; Xu, Xun; Kristiansen, Karsten

    2016-01-01

    High-throughput sequencing has recently been applied to profile the high diversity of antibodyome/B cell receptors (BCRs) and T cell receptors (TCRs) among immune cells. To date, Multiplex PCR (MPCR) and 5’RACE are predominately used to enrich rearranged BCRs and TCRs. Both approaches have advantages and disadvantages; however, a systematic evaluation and direct comparison of them would benefit researchers in the selection of the most suitable method. In this study, we used both pooled control plasmids and spiked-in cells to benchmark the MPCR bias. RNA from three healthy donors was subsequently processed with the two methods to perform a comparative evaluation of the TCR β chain sequences. Both approaches demonstrated high reproducibility (R2 = 0.9958 and 0.9878, respectively). No differences in gene usage were identified for most V/J genes (>60%), and an average of 52.03% of the CDR3 amino acid sequences overlapped. MPCR exhibited a certain degree of bias, in which the usage of several genes deviated from 5’RACE, and some V-J pairings were lost. In contrast, there was a smaller rate of effective data from 5’RACE (11.25% less compared with MPCR). Nevertheless, the methodological variability was smaller compared with the biological variability. Through direct comparison, these findings provide novel insights into the two experimental methods, which will prove to be valuable in immune repertoire research and its interpretation. PMID:27019362

  18. Estimating the Diversity, Completeness, and Cross-Reactivity of the T Cell Repertoire

    PubMed Central

    Zarnitsyna, Veronika I.; Evavold, Brian D.; Schoettle, Louis N.; Blattman, Joseph N.; Antia, Rustom

    2013-01-01

    In order to recognize and combat a diverse array of pathogens the immune system has a large repertoire of T cells having unique T cell receptors (TCRs) with only a few clones specific for any given antigen. We discuss how the number of different possible TCRs encoded in the genome (the potential repertoire) and the number of different TCRs present in an individual (the realized repertoire) can be measured. One puzzle is that the potential repertoire greatly exceeds the realized diversity of naïve T cells within any individual. We show that the existing hypotheses fail to explain why the immune system has the potential to generate far more diversity than is used in an individual, and propose an alternative hypothesis of “evolutionary sloppiness.” Another immunological puzzle is why mice and humans have similar repertoires even though humans have over 1000-fold more T cells. We discuss how the idea of the “protecton,” the smallest unit of protection, might explain this discrepancy and estimate the size of “protecton” based on available precursor frequencies data. We then consider T cell cross-reactivity – the ability of a T cell clone to respond to more than one epitope. We extend existing calculations to estimate the extent of expected cross-reactivity between the responses to different pathogens. Our results are consistent with two observations: a low probability of observing cross-reactivity between the immune responses to two randomly chosen pathogens; and the ensemble of memory cells being sufficiently diverse to generate cross-reactive responses to new pathogens. PMID:24421780

  19. Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes.

    PubMed

    Falk-Petersen, Christina B; Søgaard, Rikke; Madsen, Kenneth L; Klein, Anders B; Frølund, Bente; Wellendorph, Petrine

    2017-03-16

    δ-Containing GABAA receptors are located extrasynaptically and mediate tonic inhibition. Their involvement in brain physiology positions them as interesting drug targets. There is thus a continued interest in establishing reliable recombinant expression systems for δ-containing GABAA receptors. Inconveniently, the recombinant expression of especially α4 β1/3 δ receptors has been found to be notoriously difficult, due to mixed receptor populations and/or stoichiometries and differential pharmacology depending on the expression system used. With the aim of developing a facile and robust 96-well format cell-based assay for extrasynaptic α4 β1/3 δ receptors, we have engineered and validated a HEK293 Flp-In(™) cell line stably expressing the human δ-subunit. Upon co-transfection of α4 and β1/3 subunits, at optimized ratios, we have established a well-defined system for expressing α4 β1/3 δ receptors and used the fluorescence-based FLIPR Membrane Potential (FMP) assay to evaluate their pharmacology. Using the known reference compounds GABA and THIP, ternary α4 β1/3 δ and binary α4 β1/3 receptors could be distinguished based on potency and kinetic profiles but not efficacy. As expected, DS2 was able to potentiate only δ-containing receptors, whereas Zn(2+) had an inhibitory effect only at binary receptors. By contrast, the hitherto reported δ-selective compounds, AA29504 and 3-OH-2'MeO6MF were non-selective. The expression system was further validated using patch clamp electrophysiology, in which the superagonism of THIP was confirmed. The established FMP assay setup, based on transient expression of human α4 and β1/3 subunits into a δ-subunit stable HEK293 Flp-In(™) cell line, portrays a simple 96-well format assay as a useful supplement to electrophysiological recordings on δ-containing GABAA receptors. This article is protected by copyright. All rights reserved.

  20. Structural and functional diversity of the lectin repertoire in teleost fish: Relevance to innate and adaptive immunity

    PubMed Central

    Vasta, Gerardo R.; Nita-Lazar, Mihai; Giomarelli, Barbara; Ahmed, Hafiz; Du, Shaojun; Cammarata, Matteo; Parrinello, Nicolò; Bianchet, Mario A.; Amzel, L. Mario

    2012-01-01

    Protein–carbohydrate interactions mediated by lectins have been recognized as key components of innate immunity in vertebrates and invertebrates, not only for recognition of potential pathogens, but also for participating in downstream effector functions, such as their agglutination, immobilization, and complement-mediated opsonization and killing. More recently, lectins have been identified as critical regulators of mammalian adaptive immune responses. Fish are endowed with virtually all components of the mammalian adaptive immunity, and are equipped with a complex lectin repertoire. In this review, we discuss evidence suggesting that: (a) lectin repertoires in teleost fish are highly diversified, and include not only representatives of the lectin families described in mammals, but also members of lectin families described for the first time in fish species; (b) the tissue-specific expression and localization of the diverse lectin repertoires and their molecular partners is consistent with their distinct biological roles in innate and adaptive immunity; (c) although some lectins may bind endogenous ligands, others bind sugars on the surface of potential pathogens; (d) in addition to pathogen recognition and opsonization, some lectins display additional effector roles, such as complement activation and regulation of immune functions; (e) some lectins that recognize exogenous ligands mediate processes unrelated to immunity: they may act as anti-freeze proteins or prevent polyspermia during fertilization. PMID:21896283

  1. Effects of phoneme repertoire on phoneme decision.

    PubMed

    Costa, A; Cutler, A; Sebastián-Gallés, N

    1998-08-01

    In three experiments, listeners detected vowel or consonant targets in lists of CV syllables constructed from five vowels and five consonants. Responses were faster in a predictable context (e.g., listening for a vowel target in a list of syllables all beginning with the same consonant) than in an unpredictable context (e.g., listening for a vowel target in a list of syllables beginning with different consonants). In Experiment 1, the listeners' native language was Dutch, in which vowel and consonant repertoires are similar in size. The difference between predictable and unpredictable contexts was comparable for vowel and consonant targets. In Experiments 2 and 3, the listeners' native language was Spanish, which has four times as many consonants as vowels; here effects of an unpredictable consonant context on vowel detection were significantly greater than effects of an unpredictable vowel context on consonant detection. This finding suggests that listeners' processing of phonemes takes into account the constitution of their language's phonemic repertoire and the implications that this has for contextual variability.

  2. Efficient signal transduction by a chimeric yeast-mammalian G protein alpha subunit Gpa1-Gsalpha covalently fused to the yeast receptor Ste2.

    PubMed Central

    Medici, R; Bianchi, E; Di Segni, G; Tocchini-Valentini, G P

    1997-01-01

    Saccharomyces cerevisiae uses G protein-coupled receptors for signal transduction. We show that a fusion protein between the alpha-factor receptor (Ste2) and the Galpha subunit (Gpa1) transduces the signal efficiently in yeast cells devoid of the endogeneous STE2 and GPA1 genes. To evaluate the function of different domains of Galpha, a chimera between the N-terminal region of yeast Gpa1 and the C-terminal region of rat Gsalpha has been constructed. This chimeric Gpa1-Gsalpha is capable of restoring viability to haploid gpa1Delta cells, but signal transduction is prevented. This is consistent with evidence showing that the C-terminus of the homologous Galpha is required for receptor-G protein recognition. Surprisingly, a fusion protein between Ste2 and Gpa1-Gsalpha is able to transduce the signal efficiently. It appears, therefore, that the C-terminus of Galpha is mainly responsible for bringing the G protein into the close proximity of the receptor's intracellular domains, thus ensuring efficient coupling, rather than having a particular role in transmitting the signal. To confirm this conclusion, we show that two proteins interacting with each other (such as Snf1 and Snf4, or Ras and Raf), each of them fused either to the receptor or to the chimeric Galpha, allow efficient signal transduction. PMID:9405353

  3. The evolution of farnesoid X, vitamin D, and pregnane X receptors: insights from the green-spotted pufferfish (Tetraodon nigriviridis) and other non-mammalian species

    PubMed Central

    2011-01-01

    Background The farnesoid X receptor (FXR), pregnane X receptor (PXR), and vitamin D receptor (VDR) are three closely related nuclear hormone receptors in the NR1H and 1I subfamilies that share the property of being activated by bile salts. Bile salts vary significantly in structure across vertebrate species, suggesting that receptors binding these molecules may show adaptive evolutionary changes in response. We have previously shown that FXRs from the sea lamprey (Petromyzon marinus) and zebrafish (Danio rerio) are activated by planar bile alcohols found in these two species. In this report, we characterize FXR, PXR, and VDR from the green-spotted pufferfish (Tetraodon nigriviridis), an actinopterygian fish that unlike the zebrafish has a bile salt profile similar to humans. We utilize homology modelling, docking, and pharmacophore studies to understand the structural features of the Tetraodon receptors. Results Tetraodon FXR has a ligand selectivity profile very similar to human FXR, with strong activation by the synthetic ligand GW4064 and by the primary bile acid chenodeoxycholic acid. Homology modelling and docking studies suggest a ligand-binding pocket architecture more similar to human and rat FXRs than to lamprey or zebrafish FXRs. Tetraodon PXR was activated by a variety of bile acids and steroids, although not by the larger synthetic ligands that activate human PXR such as rifampicin. Homology modelling predicts a larger ligand-binding cavity than zebrafish PXR. We also demonstrate that VDRs from the pufferfish and Japanese medaka were activated by small secondary bile acids such as lithocholic acid, whereas the African clawed frog VDR was not. Conclusions Our studies provide further evidence of the relationship between both FXR, PXR, and VDR ligand selectivity and cross-species variation in bile salt profiles. Zebrafish and green-spotted pufferfish provide a clear contrast in having markedly different primary bile salt profiles (planar bile alcohols for

  4. Life in groups: the roles of oxytocin in mammalian sociality.

    PubMed

    Anacker, Allison M J; Beery, Annaliese K

    2013-12-11

    In recent decades, scientific understanding of the many roles of oxytocin (OT) in social behavior has advanced tremendously. The focus of this research has been on maternal attachments and reproductive pair-bonds, and much less is known about the substrates of sociality outside of reproductive contexts. It is now apparent that OT influences many aspects of social behavior including recognition, trust, empathy, and other components of the behavioral repertoire of social species. This review provides a comparative perspective on the contributions of OT to life in mammalian social groups. We provide background on the functions of OT in maternal attachments and the early social environment, and give an overview of the role of OT circuitry in support of different mating systems. We then introduce peer relationships in group-living rodents as a means for studying the importance of OT in non-reproductive affiliative behaviors. We review species differences in oxytocin receptor (OTR) distributions in solitary and group-living species of South American tuco-tucos and in African mole-rats, as well as singing mice. We discuss variation in OTR levels with seasonal changes in social behavior in female meadow voles, and the effects of OT manipulations on peer huddling behavior. Finally, we discuss avenues of promise for future investigation, and relate current findings to research in humans and non-human primates. There is growing evidence that OT is involved in social selectivity, including increases in aggression toward social outgroups and decreased huddling with unfamiliar individuals, which may support existing social structures or relationships at the expense of others. OT's effects reach beyond maternal attachment and pair bonds to play a role in affiliative behavior underlying "friendships", organization of broad social structures, and maintenance of established social relationships with individuals or groups.

  5. A TCRα framework-centered codon shapes a biased T cell repertoire through direct MHC and CDR3β interactions.

    PubMed

    Gunnarsen, Kristin Støen; Høydahl, Lene Støkken; Risnes, Louise Fremgaard; Dahal-Koirala, Shiva; Neumann, Ralf Stefan; Bergseng, Elin; Frigstad, Terje; Frick, Rahel; du Pré, M Fleur; Dalhus, Bjørn; Lundin, Knut Ea; Qiao, Shuo-Wang; Sollid, Ludvig M; Sandlie, Inger; Løset, Geir Åge

    2017-09-07

    Selection of biased T cell receptor (TCR) repertoires across individuals is seen in both infectious diseases and autoimmunity, but the underlying molecular basis leading to these shared repertoires remains unclear. Celiac disease (CD) occurs primarily in HLA-DQ2.5+ individuals and is characterized by a CD4+ T cell response against gluten epitopes dominated by DQ2.5-glia-α1a and DQ2.5-glia-α2. The DQ2.5-glia-α2 response recruits a highly biased TCR repertoire composed of TRAV26-1 paired with TRBV7-2 harboring a semipublic CDR3β loop. We aimed to unravel the molecular basis for this signature. By variable gene segment exchange, directed mutagenesis, and cellular T cell activation studies, we found that TRBV7-3 can substitute for TRBV7-2, as both can contain the canonical CDR3β loop. Furthermore, we identified a pivotal germline-encoded MHC recognition motif centered on framework residue Y40 in TRAV26-1 engaging both DQB1*02 and the canonical CDR3β. This allowed prediction of expanded DQ2.5-glia-α2-reactive TCR repertoires, which were confirmed by single-cell sorting and TCR sequencing from CD patient samples. Our data refine our understanding of how HLA-dependent biased TCR repertoires are selected in the periphery due to germline-encoded residues.

  6. Mammalian synthetic biology: emerging medical applications.

    PubMed

    Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M; Krams, Rob

    2015-05-06

    In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON-OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes.

  7. Mammalian synthetic biology: emerging medical applications

    PubMed Central

    Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M.; Krams, Rob

    2015-01-01

    In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON–OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes. PMID:25808341

  8. Mammalian Granulocyte–Macrophage Colony-stimulating Factor Receptor Expressed in Primary Avian Hematopoietic Progenitors: Lineage-specific Regulation of Proliferation and Differentiation

    PubMed Central

    Wessely, Oliver; Deiner, Eva-Maria; Lim, Kim Chew; Mellitzer, Georg; Steinlein, Peter; Beug, Hartmut

    1998-01-01

    The cytokine Granulocyte–Macrophage Colony-Stimulating Factor (GM-CSF) regulates proliferation, differentiation, and apoptosis during myelopoiesis and erythropoiesis. Structure–function relationships of GM-CSF interactions with its receptor (GM-R), the biochemistry of GM-R signal transduction, and GM-CSF action in vivo are relatively well understood. Much less is known, however, about GM-R function in primary hematopoietic cells. In this paper we show that expression of the human GM-R in a heterologous cell system (primary avian erythroid and myeloid cells) confirms respective results in murine or human cell lines, but also provides new insights how the GM-R regulates progenitor proliferation and differentiation. As expected, the hGM-CSF stimulated myeloid progenitor proliferation and differentiation and enhanced erythroid progenitor proliferation during terminal differentiation. In the latter cells, however, the hGM-R only partially substituted for the activities of the erythropoietin receptor (EpoR). It failed to replace the EpoR in its cooperation with c-Kit to induce long-term proliferation of erythroid progenitors. Furthermore, the hGM-R α chain specifically interfered with EpoR signaling, an activity neither seen for the βc subunit of the receptor complex alone, nor for the α chain of the closely related Interleukin-3 receptor. These results point to a novel role of the GM-R α chain in defining cell type–specific functions of the GM-R. PMID:9585421

  9. Adrenomedullin in mammalian embryogenesis.

    PubMed

    Garayoa, Mercedes; Bodegas, Elena; Cuttitta, Frank; Montuenga, Luis M

    2002-04-01

    Here are summarized data supporting that adrenomedullin (AM) is a multifunctional factor involved in the complex regulatory mechanisms of mammalian development. During rodent embryogenesis, AM is first expressed in the heart, followed by a broader but also defined spatio-temporal pattern of expression in vascular, neural, and skeletal-forming tissues as well as in the main embryonic internal organs. AM pattern of expression is suggestive of its involvement in the control of embryonic invasion, proliferation, and differentiation processes, probably through autocrine or paracrine modes of action. AM levels in fetoplacental tissues, uterus, maternal and umbilical plasma are highly increased during normal gestation. These findings in addition to other physiological and gene targeting studies support the importance of AM as a vasorelaxant factor implicated in the regulation of maternal vascular adaptation to pregnancy, as well as of fetal and fetoplacental circulations. AM is also present in amniotic fluid and milk, which is suggestive of additional functions in the maturation and immunological protection of the fetus. Altered expression of AM has been found in some gestational pathologies, although it is not yet clear whether this corresponds to causative or compensatory mechanisms. Future studies in regard to the distribution and expression levels of the molecules known to function as AM receptors, together with data on the action of complement factor H (an AM binding protein), may help to better define the roles of AM during embryonic development. Copyright 2002 Wiley-Liss, Inc.

  10. Evolution of Functional and Sequence Variants of the Mammalian XPR1 Receptor for Mouse Xenotropic Gammaretroviruses and the Human-Derived Retrovirus XMRV ▿ †

    PubMed Central

    Yan, Yuhe; Liu, Qingping; Wollenberg, Kurt; Martin, Carrie; Buckler-White, Alicia; Kozak, Christine A.

    2010-01-01

    Genetic conflicts between retroviruses and their receptors result in the evolution of novel host entry restrictions and novel virus envelopes, and such variants can influence trans-species transmission. We screened rodents and other mammals for sequence variation in the Xpr1 receptor for the mouse xenotropic or polytropic mouse leukemia viruses (X-MLVs or P-MLVs, respectively) of the gammaretrovirus family and for susceptibility to mouse-derived X/P-MLVs and to XMRV (xenotropic murine leukemia virus-related virus), an X-MLV-like virus isolated from humans with prostate cancer and chronic fatigue syndrome. We identified multiple distinct susceptibility phenotypes; these include the four known Xpr1 variants in Mus and a novel fifth Xpr1 gene found in Mus molossinus and Mus musculus. We describe the geographic and species distribution of the Mus Xpr1 variants but failed to find the X-MLV-restrictive laboratory mouse allele in any wild mouse. We used mutagenesis and phylogenetic analysis to evaluate the functional contributions made by constrained, variable, and deleted residues. Rodent Xpr1 is under positive selection, indicating a history of host-pathogen conflicts; several codons under selection have known roles in virus entry. All non-Mus mammals are susceptible to mouse X-MLVs, but some restrict other members of the X/P-MLV family, and the resistance of hamster and gerbil cells to XMRV indicates that XMRV has unique receptor requirements. We show that the hypervariable fourth extracellular XPR1 loop (ECL4) contains three evolutionarily constrained residues that do not contribute to receptor function, we identify two novel residues important for virus entry (I579 and T583), and we describe a unique pattern of ECL4 variation in the three virus-restrictive Xpr1 variants found in MLV-infected house mice; these mice carry different deletions in ECL4, suggesting either that these sites or loop size affects receptor function. PMID:20844050

  11. Leucine Stimulates Insulin Secretion via Down-regulation of Surface Expression of Adrenergic α2A Receptor through the mTOR (Mammalian Target of Rapamycin) Pathway

    PubMed Central

    Yang, Jun; Dolinger, Michael; Ritaccio, Gabrielle; Mazurkiewicz, Joseph; Conti, David; Zhu, Xinjun; Huang, Yunfei

    2012-01-01

    The amino acid leucine is a potent secretagogue, capable of inducing insulin secretion. It also plays an important role in the regulation of mTOR activity, therefore, providing impetus to investigate if a leucine-sensing mechanism in the mTOR pathway is involved in insulin secretion. We found that leucine-induced insulin secretion was inhibited by both the mTOR inhibitor rapamycin as well as the adrenergic α2 receptor agonist clonidine. We also demonstrated that leucine down-regulated the surface expression of adrenergic α2A receptor via activation of the mTOR pathway. The leucine stimulatory effect on insulin secretion was attenuated in diabetic Goto-Kakizaki rats that overexpress adrenergic α2A receptors, confirming the role of leucine in insulin secretion. Thus, our data demonstrate that leucine regulates insulin secretion by modulating adrenergic α2 receptors through the mTOR pathway. The role of the mTOR pathway in metabolic homeostasis led us to a second important finding in this study; retrospective analysis of clinical data showed that co-administration of rapamycin and clonidine was associated with an increased incidence of new-onset diabetes in renal transplantation patients over those receiving rapamycin alone. We believe that inhibition of mTOR by rapamycin along with activation of adrenergic α2 receptors by clonidine represents a double-hit to pancreatic islets that synergistically disturbs glucose homeostasis. This new insight may have important implications for the clinical management of renal transplant patients. PMID:22645144

  12. Evolution of functional and sequence variants of the mammalian XPR1 receptor for mouse xenotropic gammaretroviruses and the human-derived retrovirus XMRV.

    PubMed

    Yan, Yuhe; Liu, Qingping; Wollenberg, Kurt; Martin, Carrie; Buckler-White, Alicia; Kozak, Christine A

    2010-11-01

    Genetic conflicts between retroviruses and their receptors result in the evolution of novel host entry restrictions and novel virus envelopes, and such variants can influence trans-species transmission. We screened rodents and other mammals for sequence variation in the Xpr1 receptor for the mouse xenotropic or polytropic mouse leukemia viruses (X-MLVs or P-MLVs, respectively) of the gammaretrovirus family and for susceptibility to mouse-derived X/P-MLVs and to XMRV (xenotropic murine leukemia virus-related virus), an X-MLV-like virus isolated from humans with prostate cancer and chronic fatigue syndrome. We identified multiple distinct susceptibility phenotypes; these include the four known Xpr1 variants in Mus and a novel fifth Xpr1 gene found in Mus molossinus and Mus musculus. We describe the geographic and species distribution of the Mus Xpr1 variants but failed to find the X-MLV-restrictive laboratory mouse allele in any wild mouse. We used mutagenesis and phylogenetic analysis to evaluate the functional contributions made by constrained, variable, and deleted residues. Rodent Xpr1 is under positive selection, indicating a history of host-pathogen conflicts; several codons under selection have known roles in virus entry. All non-Mus mammals are susceptible to mouse X-MLVs, but some restrict other members of the X/P-MLV family, and the resistance of hamster and gerbil cells to XMRV indicates that XMRV has unique receptor requirements. We show that the hypervariable fourth extracellular XPR1 loop (ECL4) contains three evolutionarily constrained residues that do not contribute to receptor function, we identify two novel residues important for virus entry (I579 and T583), and we describe a unique pattern of ECL4 variation in the three virus-restrictive Xpr1 variants found in MLV-infected house mice; these mice carry different deletions in ECL4, suggesting either that these sites or loop size affects receptor function.

  13. Programming in the Zone: Repertoire Selection for the Large Ensemble

    ERIC Educational Resources Information Center

    Hopkins, Michael

    2013-01-01

    One of the great challenges ensemble directors face is selecting high-quality repertoire that matches the musical and technical levels of their ensembles. Thoughtful repertoire selection can lead to increased student motivation as well as greater enthusiasm for the music program from parents, administrators, teachers, and community members. Common…

  14. An Annotated Guide and Interactive Database for Solo Horn Repertoire

    ERIC Educational Resources Information Center

    Schouten, Sarah

    2012-01-01

    Given the horn's lengthy history, it is not surprising that many scholars have examined the evolution of the instrument from the natural horn to the modern horn and its expansive repertoire. Numerous dissertations, theses, and treatises illuminate specific elements of the horn's solo repertoire; however, no scholar has produced a…

  15. An Annotated Guide and Interactive Database for Solo Horn Repertoire

    ERIC Educational Resources Information Center

    Schouten, Sarah

    2012-01-01

    Given the horn's lengthy history, it is not surprising that many scholars have examined the evolution of the instrument from the natural horn to the modern horn and its expansive repertoire. Numerous dissertations, theses, and treatises illuminate specific elements of the horn's solo repertoire; however, no scholar has produced a…

  16. Programming in the Zone: Repertoire Selection for the Large Ensemble

    ERIC Educational Resources Information Center

    Hopkins, Michael

    2013-01-01

    One of the great challenges ensemble directors face is selecting high-quality repertoire that matches the musical and technical levels of their ensembles. Thoughtful repertoire selection can lead to increased student motivation as well as greater enthusiasm for the music program from parents, administrators, teachers, and community members. Common…

  17. Highly diverse TCRα chain repertoire of pre-immune CD8⁺ T cells reveals new insights in gene recombination.

    PubMed

    Genolet, Raphael; Stevenson, Brian J; Farinelli, Laurent; Osterås, Magne; Luescher, Immanuel F

    2012-04-04

    Although the T-cell receptor αδ (TCRαδ) locus harbours large libraries of variable (TRAV) and junctional (TRAJ) gene segments, according to previous studies the TCRα chain repertoire is of limited diversity due to restrictions imposed by sequential coordinate TRAV-TRAJ recombinations. By sequencing tens of millions of TCRα chain transcripts from naive mouse CD8(+) T cells, we observed a hugely diverse repertoire, comprising nearly all possible TRAV-TRAJ combinations. Our findings are not compatible with sequential coordinate gene recombination, but rather with a model in which contraction and DNA looping in the TCRαδ locus provide equal access to TRAV and TRAJ gene segments, similarly to that demonstrated for IgH gene recombination. Generation of the observed highly diverse TCRα chain repertoire necessitates deletion of failed attempts by thymic-positive selection and is essential for the formation of highly diverse TCRαβ repertoires, capable of providing good protective immunity.

  18. Structural repertoire of immunoglobulin λ light chains.

    PubMed

    Chailyan, Anna; Marcatili, Paolo; Cirillo, Davide; Tramontano, Anna

    2011-05-01

    The immunoglobulin λ isotype is present in nearly all vertebrates and plays an important role in the human immune system. Despite its importance, few systematic studies have been performed to analyze the structural conformation of its variable regions, contrary to what is the case for κ and heavy chains. We show here that an analysis of the structures of λ chains allows the definition of a discrete set of recurring conformations (canonical structures) of their hypervariable loops and, most importantly, the identification of sequence constraints that can be used to predict their structure. We also show that the structural repertoire of λ chains is different and more varied than that of the κ chains, consistently with the current view of the involvement of the two major light-chain families in complementary strategies of the immune system to ensure a fine tuning between diversity and stability in antigen recognition.

  19. Deep sequencing and human antibody repertoire analysis

    PubMed Central

    Boyd, Scott D; Crowe, James E

    2016-01-01

    In the past decade, high-throughput DNA sequencing (HTS) methods and improved approaches for isolating antigen-specific B cells and their antibody genes have been applied in many areas of human immunology. This work has greatly increased our understanding of human antibody repertoires and the specific clones responsible for protective immunity or immune-mediated pathogenesis. Although the principles underlying selection of individual B cell clones in the intact immune system are still under investigation, the combination of more powerful genetic tracking of antibody lineage development and functional testing of the encoded proteins promises to transform therapeutic antibody discovery and optimization. Here, we highlight recent advances in this fast-moving field. PMID:27065089

  20. Degeneracy-driven self-structuring dynamics in selective repertoires.

    PubMed

    Atamas, Sergei P; Bell, Jonathan

    2009-08-01

    Numerous biological interactions, such as interactions between T cell receptors or antibodies with antigens, interactions between enzymes and substrates, or interactions between predators and prey are often not strictly specific. In such less specific, or "sloppy," systems, referred to here as degenerate systems, a given unit of a diverse resource (antigens, enzymatic substrates, prey) is at risk of being recognized and consumed by multiple consumers (lymphocytes, enzymes, predators). In this study, we model generalized degenerate consumer-resource systems of Lotka-Volterra and Verhulst types. In the degenerate systems of Lotka-Volterra, there is a continuum of types of consumer and resource based on variation of a single trait (characteristic, or preference). The consumers experience competition for a continuum of resource types. This non-local interaction system is modeled with partial differential-integral equations and shows spontaneous self-structuring of the consumer population that depends on the degree of interaction degeneracy between resource and consumer, but does not mirror the distribution of resource. We also show that the classical Verhulst (i.e. logistic) single population model can be generalized to a degenerate model, which shows qualitative behavior similar to that in the degenerate Lotka-Volterra model. These results provide better insight into the dynamics of selective systems in biology, suggesting that adaptation of degenerate repertoires is not a simple "mirroring" of the environment by the "fittest" elements of population.

  1. LXRα and LXRβ Nuclear Receptors Evolved in the Common Ancestor of Gnathostomes

    PubMed Central

    Fonseca, Elza; Ruivo, Raquel; Lopes-Marques, Mónica; Zhang, Huixian; Santos, Miguel M.; Venkatesh, Byrappa

    2017-01-01

    Nuclear receptors (NRs) regulate numerous aspects of the endocrine system. They mediate endogenous and exogenous cues, ensuring a homeostatic control of development and metabolism. Gene duplication, loss and mutation have shaped the repertoire and function of NRs in metazoans. Here, we examine the evolution of a pivotal orchestrator of cholesterol metabolism in vertebrates, the liver X receptors (LXRs). Previous studies suggested that LXRα and LXRβ genes emerged in the mammalian ancestor. However, we show through genome analysis and functional assay that bona fide LXRα and LXRβ orthologues are present in reptiles, coelacanth and chondrichthyans but not in cyclostomes. These findings show that LXR duplicated before gnathostome radiation, followed by asymmetric paralogue loss in some lineages. We suggest that a tighter control of cholesterol levels in vertebrates was achieved through the exploitation of a wider range of oxysterols, an ability contingent on ligand-binding pocket remodeling. PMID:28057729

  2. Histone recognition and nuclear receptor co-activator functions of Drosophila Cara Mitad, a homolog of the N-terminal portion of mammalian MLL2 and MLL3

    PubMed Central

    Chauhan, Chhavi; Zraly, Claudia B.; Parilla, Megan; Diaz, Manuel O.; Dingwall, Andrew K.

    2012-01-01

    MLL2 and MLL3 histone lysine methyltransferases are conserved components of COMPASS-like co-activator complexes. In vertebrates, the paralogous MLL2 and MLL3 contain multiple domains required for epigenetic reading and writing of the histone code involved in hormone-stimulated gene programming, including receptor-binding motifs, SET methyltransferase, HMG and PHD domains. The genes encoding MLL2 and MLL3 arose from a common ancestor. Phylogenetic analyses reveal that the ancestral gene underwent a fission event in some Brachycera dipterans, including Drosophila species, creating two independent genes corresponding to the N- and C-terminal portions. In Drosophila, the C-terminal SET domain is encoded by trithorax-related (trr), which is required for hormone-dependent gene activation. We identified the cara mitad (cmi) gene, which encodes the previously undiscovered N-terminal region consisting of PHD and HMG domains and receptor-binding motifs. The cmi gene is essential and its functions are dosage sensitive. CMI associates with TRR, as well as the EcR-USP receptor, and is required for hormone-dependent transcription. Unexpectedly, although the CMI and MLL2 PHDf3 domains could bind histone H3, neither showed preference for trimethylated lysine 4. Genetic tests reveal that cmi is required for proper global trimethylation of H3K4 and that hormone-stimulated transcription requires chromatin binding by CMI, methylation of H3K4 by TRR and demethylation of H3K27 by the demethylase UTX. The evolutionary split of MLL2 into two distinct genes in Drosophila provides important insight into distinct epigenetic functions of conserved readers and writers of the histone code. PMID:22569554

  3. The all-or-none role of innervation in expression of apamin receptor and of apamin-sensitive Ca2+-activated K+ channel in mammalian skeletal muscle.

    PubMed

    Schmid-Antomarchi, H; Renaud, J F; Romey, G; Hugues, M; Schmid, A; Lazdunski, M

    1985-04-01

    The long-lasting after-hyperpolarization(s) (AHP) that follows the action potential in rat myotubes differentiated in culture is due to Ca2+-activated K+ channels. These channels have the property to be specifically blocked by the bee venom toxin apamin at low concentrations. Apamin has been used in this work to analyze, by electrophysiological and biochemical techniques, the role of innervation in expression of these important channels. The main results are as follows: (i) Long-lasting AHP that follows the action potential in rat myotubes in culture disappears when myotubes are cocultured with nerve cells from the spinal cord under the conditions of in vitro innervation. (ii) Extensor digitorum longus muscles from adult rats have action potentials that are not followed by AHP but AHP are systematically recorded after muscle denervation and they are blocked by apamin. (iii) Specific 125I-labeled apamin binding is undetectable in innervated muscle fibers but it becomes detectable 2-4 days after muscle denervation to be maximal 10 days after denervation. (iv) Apamin receptors detected with 125I-labeled apamin are present at fetal stages with biochemical characteristics identical to those found in myotubes in culture. The receptor number decreases as maturation proceeds and 125I-labeled apamin receptors completely disappear after the first week of postnatal life, in parallel with the disappearance of multi-innervation. All these results taken together strongly suggest an all-or-none effect of innervation on the expression of apamin-sensitive Ca2+-activated K+ channels.

  4. Histone recognition and nuclear receptor co-activator functions of Drosophila cara mitad, a homolog of the N-terminal portion of mammalian MLL2 and MLL3.

    PubMed

    Chauhan, Chhavi; Zraly, Claudia B; Parilla, Megan; Diaz, Manuel O; Dingwall, Andrew K

    2012-06-01

    MLL2 and MLL3 histone lysine methyltransferases are conserved components of COMPASS-like co-activator complexes. In vertebrates, the paralogous MLL2 and MLL3 contain multiple domains required for epigenetic reading and writing of the histone code involved in hormone-stimulated gene programming, including receptor-binding motifs, SET methyltransferase, HMG and PHD domains. The genes encoding MLL2 and MLL3 arose from a common ancestor. Phylogenetic analyses reveal that the ancestral gene underwent a fission event in some Brachycera dipterans, including Drosophila species, creating two independent genes corresponding to the N- and C-terminal portions. In Drosophila, the C-terminal SET domain is encoded by trithorax-related (trr), which is required for hormone-dependent gene activation. We identified the cara mitad (cmi) gene, which encodes the previously undiscovered N-terminal region consisting of PHD and HMG domains and receptor-binding motifs. The cmi gene is essential and its functions are dosage sensitive. CMI associates with TRR, as well as the EcR-USP receptor, and is required for hormone-dependent transcription. Unexpectedly, although the CMI and MLL2 PHDf3 domains could bind histone H3, neither showed preference for trimethylated lysine 4. Genetic tests reveal that cmi is required for proper global trimethylation of H3K4 and that hormone-stimulated transcription requires chromatin binding by CMI, methylation of H3K4 by TRR and demethylation of H3K27 by the demethylase UTX. The evolutionary split of MLL2 into two distinct genes in Drosophila provides important insight into distinct epigenetic functions of conserved readers and writers of the histone code.

  5. A phosphorylation cascade in the basal ganglia of the mammalian brain: regulation by the D-1 dopamine receptor. A mathematical model of known biochemical reactions.

    PubMed

    Kebabian, J W

    1997-01-01

    Stimulation of the dopamine D-1 receptor in the corpus striatum initiates a cascade of biochemical events. These events include: activation of adenylate cyclase, stimulation of cAMP-dependent protein kinase, protein phosphorylation and inhibition of phosphoprotein phosphotase-1. This article presents and discusses a mathematical model of these biochemical events (and their dependence upon the concentration of cytosolic calcium). According to this model, the activity of calcineurin (which is regulated by the concentration of cytosolic calcium ions) counterbalances the activity of the "D-1 cascade". The combined activity of the "D-1 cascade" and calcineurin can regulate the activity of calcium- and calmodulin-dependent protein kinase II.

  6. Immune Repertoire Profiling Reveals that Clonally Expanded B and T Cells Infiltrating Diseased Human Kidneys Can Also Be Tracked in Blood

    PubMed Central

    Schaller, Susanne; Suessner, Susanne; Sunzenauer, Judith; Reindl-Schwaighofer, Roman; Weiss, Richard; Winkler, Stephan; Gabriel, Christian; Danzer, Martin; Oberbauer, Rainer

    2015-01-01

    Recent advances in high-throughput sequencing allow for the competitive analysis of the human B and T cell immune repertoire. In this study we compared Immunoglobulin and T cell receptor repertoires of lymphocytes found in kidney and blood samples of 10 patients with various renal diseases based on next-generation sequencing data. We used Biomed-2 primer panels and ImmunExplorer software to sequence, analyze and compare complementarity determining regions and V-(D)-J elements. While generally an individual’s renal receptor repertoire is different from the repertoire present in blood, 94% (30/32) of the lymphocytes with clonal expansion in kidney can also be traced in blood however, not all of these clonotypes are equally abundant. Summarizing the data of all analyzed patients, 68% of highly expanded T cell clonotypes and 30% of the highly expanded B cell clonotypes that have infiltrated the kidney can be found amongst the five most abundant clonotypes in blood. In addition, complementarity determining region 3 sequences of the immunoglobulin heavy chains are on average more diverse than T cell receptor beta chains. Immune repertoire analysis of tissue infiltrating B and T cells adds new approaches to the assessment of adaptive immune response in kidney diseases. Our data suggest that expanded clonotypes in the tissues might be traceable in blood samples in the course of treatment or the natural history of the disease. PMID:26600245

  7. Differences in transcriptomic profile and IgA repertoire between jejunal and ileal Peyer's patches.

    PubMed

    Levast, Benoît; De Monte, Michèle; Melo, Sandrine; Chevaleyre, Claire; Berri, Mustapha; Salmon, Henri; Meurens, François

    2010-02-01

    In many species such as sheep and pig, there are two types of Peyer's patches (PP): several discrete patches in the jejunum and a long and continuous patch in the ileum. Most of the immunoglobulin A in the gut is generated by B-cells in the PP germinal centers. Moreover, swine like ovine ileal PP might be important for antigen independent B-cell repertoire diversification. We examined, by quantitative real-time PCR, the expression of 36 transcripts of antimicrobial peptides, chemokines, interleukines, Toll-like receptors and transcription factors from both PP and we highlighted the differences by a principal component analysis. Ileal PP was characterized by a higher mRNA expression of CCL28, IL5, IL10, TLR2 and TLR4 while jejunal PP showed higher mRNA expression of antimicrobial peptides, CCL25, FOXP3, IL4, T-Bet, TSLP and SOCS2. Then, we analyzed some VDJ rearrangements to assess immunoglobulin repertoire diversity in jejunal and ileal PP from weaned piglets. The IgA and IgM repertoires were more diverse in ileal than in jejunal piglet PP. All these results could be related to the rarefaction of interfollicular T-cell zone and the presence in ileal versus jejunal lumen of a more diversified microflora. These findings shed a light on the functional differences between both PP.

  8. Interaction of three-finger proteins from snake venoms and from mammalian brain with the cys-loop receptors and their models.

    PubMed

    Faure, G; Shelukhina, I V; Porowinska, D; Shulepko, M A; Lyukmanova, E N; Dolgikh, D A; Spirova, E N; Kasheverov, I E; Utkin, Yu N; Corringer, J-P; Tsetlin, V I

    2016-05-01

    With the use of surface plasmon resonance (SPR) it was shown that ws-Lynx1, a water-soluble analog of the three-finger membrane-bound protein Lynx1, that modulates the activity of brain nicotinic acetylcholine receptors (nAChRs), interacts with the acetylcholine-binding protein (AChBP) with high affinity, K D = 62 nM. This result agrees with the earlier demonstrated competition of ws-Lynx1 with radioiodinated α-bungarotoxin for binding to AChBP. For the first time it was shown that ws-Lynx1 binds to GLIC, prokaryotic Cys-loop receptor (K D = 1.3 μM). On the contrary, SPR revealed that α-cobratoxin, a three-finger protein from cobra venom, does not bind to GLIC. Obtained results indicate that SPR is a promising method for analysis of topography of ws-Lynx1 binding sites using its mutants and those of AChBP and GLIC.

  9. Mammalian G-protein-coupled receptor expression in Escherichia coli: I. High-throughput large-scale production as inclusion bodies.

    PubMed

    Michalke, Kerstin; Gravière, Marie-Eve; Huyghe, Céline; Vincentelli, Renaud; Wagner, Renaud; Pattus, Franc; Schroeder, Kathrin; Oschmann, Jan; Rudolph, Rainer; Cambillau, Christian; Desmyter, Aline

    2009-03-15

    G-protein-coupled receptors (GPCRs) represent approximately 3% of human proteome and the most prominent class of pharmacological targets. Despite their important role in many functions, only the X-ray structures of rhodopsin, and more recently of the beta(1)- and beta(2)-adrenergic receptors, have been resolved. Structural studies of GPCRs require that several tedious preliminary steps be fulfilled before setting up the first crystallization experiments: protein expression, detergent solubilization, purification, and stabilization. Here we report on screening expression conditions of approximately 100 GPCRs in Escherichia coli with a view to obtain large amounts of inclusion bodies, a prerequisite to the subsequent refolding step. A set of optimal conditions, including appropriate vectors (Gateway pDEST17oi), strain (C43), and fermentation at high optical density, define the best first instance choice. Beyond this minimal setting, however, the rate of success increases significantly with the number of conditions tested. In contrast with experiments based on a single GPCR expression, our approach provides statistically significant results and indicates that up to 40% of GPCRs can be expressed as inclusion bodies in quantities sufficient for subsequent refolding, solubilization, and purification.

  10. TRAV and TRBV repertoire, clonality and the proliferative history of umbilical cord blood T-cells.

    PubMed

    Li, Yangqiu; Chen, Shaohua; Yang, Lijian; Yin, Qingsong; Geng, Suxia; Wu, Xiuli; Schmidt, Christian A; Przybylski, Grzegorz K

    2007-11-01

    Umbilical cord blood (CB) has been used successfully as a source of hematopoietic stem cells for transplantation. But the distribution and clonality of T-cell receptor alpha variable region (TRAV) and T-cell receptor beta variable region (TRBV) subfamily T-cells, the naïve T-cells level and the diversity of thymic recent output function in CB has not been yet clearly defined. In order to characterize the repertoire of CB T-cells, the CDR3 of 29 TRAV and 24 TRBV subfamily genes were analyzed in T-cells from 12 cord blood samples, using RT-PCR and genescan technique. To determine the proliferative history of CB T-cells, quantitative analysis of deltaRec-psiJalpha signal joint T-cell receptor excision circles (sjTRECs) was performed in mononuclear cells, CD3+, CD4+ and CD8+ T-cells from 20 CB samples by real-time PCR. In addition the analysis of 23 TRBV-TRBD1 sjTRECs in 10 cases of CB CD4+ T-cells and CB CD8+ T-cells was performed by semi-nested PCR. We found a marked restriction of TRBV expression pattern in CBMCs compared to peripheral blood mononuclear cells (PBMC), which expressed all 24 TRBV genes. All PCR products of TRAV and TRBV subfamilies from CB, except for 3 cases, displayed polyclonal rearrangement pattern. The deltaRec-psiJalpha sjTRECs counts were significantly higher in CB, than in PB samples. Also the number of detectable TRBV sjTRECs was higher in CB than in peripheral blood. In conclusion, our results indicate polyclonal but restricted repertoire and a very short proliferative history of CB T-cells. The incomplete repertoire and naivety of CB T-cells might be the reason that CB hematopoietic stem cells transplant recipients are less likely to develop graft vs host disease.

  11. Development of the neonatal B and T cell repertoire in swine: implications for comparative and veterinary immunology.

    PubMed

    Butler, John E; Sinkora, Marek; Wertz, Nancy; Holtmeier, Wolfgang; Lemke, Caitlin D

    2006-01-01

    Birth in all higher vertebrates is at the center of the critical window of development in which newborns transition from dependence on innate immunity to dependence on their own adaptive immunity, with passive maternal immunity bridging this transition. Therefore we have studied immunological development through fetal and early neonatal life. In swine, B cells appear earlier in fetal development than T cells. B cell development begins in the yolk sac at the 20th day of gestation (DG20), progresses to fetal liver at DG30 and after DG45 continues in bone marrow. The first wave of developing T cells is gammadelta cells expressing a monomorphic Vdelta rearrangement. Thereafter, alphabeta T cells predominate and at birth, at least 19 TRBV subgroups are expressed, 17 of which appear highly homologous with those in humans. In contrast to the T cell repertoire and unlike humans and mice, the porcine pre-immune VH (IGHV-D-J) repertoire is highly restricted, depending primarily on CDR3 for diversity. The V-KAPPA (IGKV-J) repertoire and apparently also the V-LAMBDA (IGLV-J) repertoire, are also restricted. Diversification of the pre-immune B cell repertoire of swine and the ability to respond to both T-dependent and T-independent antigen depends on colonization of the gut after birth in which colonizing bacteria stimulate with Toll-like receptor ligands, especially bacterial DNA. This may explain the link between repertoire diversification and the anatomical location of primary lymphoid tissue like the ileal Peyers patches. Improper development of adaptive immunity can be caused by infectious agents like the porcine reproductive and respiratory syndrome virus that causes immune dysregulation resulting in immunological injury and autoimmunity.

  12. Immune Tolerance Maintained by Cooperative Interactions between T Cells and Antigen Presenting Cells Shapes a Diverse TCR Repertoire

    PubMed Central

    Best, Katharine; Chain, Benny; Watkins, Chris

    2015-01-01

    The T cell population in an individual needs to avoid harmful activation by self peptides while maintaining the ability to respond to an unknown set of foreign peptides. This property is acquired by a combination of thymic and extra-thymic mechanisms. We extend current models for the development of self/non-self discrimination to consider the acquisition of self-tolerance as an emergent system level property of the overall T cell receptor repertoire. We propose that tolerance is established at the level of the antigen presenting cell/T cell cluster, which facilitates and integrates cooperative interactions between T cells of different specificities. The threshold for self-reactivity is therefore imposed at a population level, and not at the level of the individual T cell/antigen encounter. Mathematically, the model can be formulated as a linear programing optimization problem that can be implemented as a multiplicative update algorithm, which shows a rapid convergence to a stable state. The model constrains self-reactivity within a predefined threshold, but maintains repertoire diversity and cross reactivity which are key characteristics of human T cell immunity. We show further that the size of individual clones in the model repertoire becomes heterogeneous, and that new clones can establish themselves even when the repertoire has stabilized. Our study combines the salient features of the “danger” model of self/non-self discrimination with the concepts of quorum sensing, and extends repertoire generation models to encompass the establishment of tolerance. Furthermore, the dynamic and continuous repertoire reshaping, which underlies tolerance in this model, suggests opportunities for therapeutic intervention to achieve long-term tolerance following transplantation. PMID:26300880

  13. Kinetic study of N-type calcium current modulation by delta-opioid receptor activation in the mammalian cell line NG108-15.

    PubMed Central

    Toselli, M; Tosetti, P; Taglietti, V

    1999-01-01

    The voltage-dependent inhibition of N-type Ca2+ channel current by the delta-opioid agonist [D-pen2, D-pen5]-enkephalin (DPDPE) was investigated in the mammalian cell line NG108-15 with 10 microM nifedipine to block L-type channels, with whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells DPDPE reversibly decreased omega-conotoxin GVIA-sensitive Ba2+ currents in a concentration-dependent way. Inhibition was maximal with 1 microM DPDPE (66% at 0 mV) and was characterized by a slowing of Ba2+ current activation at low test potentials. Both inhibition and kinetic slowing were attenuated at more positive potentials and could be relieved up to 90% by strong conditioning depolarizations. The kinetics of removal of inhibition (de-inhibition) and of its retrieval (re-inhibition) were also voltage dependent. Both de-inhibition and re-inhibition were single exponentials and, in the voltage range from -20 to +10 mV, had significantly different time constants at a given membrane potential, the time course of re-inhibition being faster than that of de-inhibition. The kinetics of de-inhibition at -20 mV and of reinhibition at -40 mV were also concentration dependent, both processes becoming slower at lower agonist concentrations. The rate of de-inhibition at +80/+120 mV was similar to that of Ca2+ channel activation at the same potentials measured during application of DPDPE (approximately 7 ms), both processes being much slower than channel activation in controls (<1 ms). Moreover, the amplitude but not the time course of tail currents changed as the depolarization to +80/+120 mV was made longer. The state-dependent properties of DPDPE Ca2+ channel inhibition could be simulated by a model that assumes that inhibition by DPDPE results from voltage- and concentration-dependent binding of an inhibitory molecule to the N-type channel. PMID:10233071

  14. Human gut microbiota: repertoire and variations.

    PubMed

    Lagier, Jean-Christophe; Million, Matthieu; Hugon, Perrine; Armougom, Fabrice; Raoult, Didier

    2012-01-01

    The composition of human gut microbiota and their relationship with the host and, consequently, with human health and disease, presents several challenges to microbiologists. Originally dominated by culture-dependent methods for exploring this ecosystem, the advent of molecular tools has revolutionized our ability to investigate these relationships. However, many biases that have led to contradictory results have been identified. Microbial culturomics, a recent concept based on a use of several culture conditions with identification by MALDI-TOF followed by the genome sequencing of the new species cultured had allowed a complementarity with metagenomics. Culturomics allowed to isolate 31 new bacterial species, the largest human virus, the largest bacteria, and the largest Archaea from human. Moreover, some members of this ecosystem, such as Eukaryotes, giant viruses, Archaea, and Planctomycetes, have been neglected by the majority of studies. In addition, numerous factors, such as age, geographic provenance, dietary habits, antibiotics, or probiotics, can influence the composition of the microbiota. Finally, in addition to the countless biases associated with the study techniques, a considerable limitation to the interpretation of studies of human gut microbiota is associated with funding sources and transparency disclosures. In the future, studies independent of food industry funding and using complementary methods from a broad range of both culture-based and molecular tools will increase our knowledge of the repertoire of this complex ecosystem and host-microbiota mutualism.

  15. Immunoglobulins, antibody repertoire and B cell development.