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Sample records for high-affinity molecular phenotype

  1. Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

    2005-01-01

    that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... for the fusion constructs was observed. We conclude that the glucagon-like peptide 1 fusion construct mimics the natural interaction of the receptor with (beta)arr2 with respect to binding peptide ligands, G protein-mediated signaling and internalization, and that this distinct molecular phenotype is reminiscent......To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...

  2. Molecular strategies of microbial iron assimilation: from high-affinity complexes to cofactor assembly systems.

    Science.gov (United States)

    Miethke, Marcus

    2013-01-01

    Microorganisms have to cope with restricted iron bioavailability in most environmental habitats as well as during host colonization. The continuous struggle for iron has brought forth a plethora of acquisition and assimilation strategies that share several functional and mechanistic principles. One common theme is the utilization of high-affinity chelators for extracellular iron mobilization, generally known as siderophore-dependent iron acquisition. This basic strategy is related with another central aspect of microbial iron acquisition, which is the release of the mobilized iron from extracellular sources to allow its transfer and incorporation into metabolically active proteins. A variety of mechanisms which are often coupled with high-affinity uptake have evolved to facilitate the removal of iron from siderophore ligands; however, they differ in many key aspects including substrate specificities and release efficiencies. The most sophisticated iron release pathways comprise processes of specific hydrolysis and reduction of ferric siderophores, especially in the case of high-affinity iron complexes with greatly negative redox potentials that often represent crucial factors for virulence development in bacterial and fungal pathogens. During the following steps of iron utilization, the acquired metal is transferred through intracellular trafficking pathways which may include diverse storage compartments in order to be directed to cofactor assembly systems and to final protein targeting. Several of these iron channeling routes have been described recently and provide first insights into the later steps of iron assimilation that characterize an essential part of the cellular iron homeostasis network.

  3. Premature aging phenotype in mice lacking high affinity nicotinic receptors: region specific changes in layer V pyramidal cell morphology

    Directory of Open Access Journals (Sweden)

    Eleni Konsolaki

    2014-02-01

    estimated by multiplying the vertical and the horizontal dimensions of the apical tuft to indicate the extent of its areal coverage and “cell body elongation” was calculated as the horizontal divided by the vertical diameter of the cell body. Cortical thickness was measured as the distance between the pia and ventral border of layer 6 in all sections from which cells were imaged. Our data revealed substantial morphological differences between YFP+ cells of the ACC and V1, in both genotypes, implying different synaptic integration properties and functional role for cells in the two cortical areas. We found an increased susceptibility to aging in cells located in ACC, a region associated with higher cognitive functions. In addition, we found that the lack of the β2 subunit is associated with an appearance of premature aging in layer V pyramidal cells, which is preferentially expressed in ACC. In morphological terms, ACC neurons already look ‘old’ at 4-6 months, whereas V1 cells are minimally affected. Interestingly, the same parameters affected by the mutation are also the ones most prominently affected by aging in normal animals, suggesting possible common underlying mechanisms. In contrast, V1 cells are less affected by aging in WT animals and the impact of the mutation is only apparent in aged individuals. To our knowledge this is the first study that examines the combined effects of aging and genetic predisposition on neuronal subpopulations with distinct areal identities and connectivity patterns but same layer identity and comparable intrinsic properties, thereby allowing an examination of their respective contributions to the aging process. We have shown that high-affinity nicotinic signaling plays a region-specific role both on morphogenesis and/or maintenance of identified layer V pyramidal neurons, as well as on the process of aging per se, by promoting or enhancing the age-related decline in neuronal structure. Hence, the phenotype of YFP+ cells in aged

  4. Premature Aging Phenotype in Mice Lacking High-Affinity Nicotinic Receptors: Region-Specific Changes in Layer V Pyramidal Cell Morphology.

    Science.gov (United States)

    Konsolaki, Eleni; Skaliora, Irini

    2015-08-01

    The mechanisms by which aging leads to alterations in brain structure and cognitive deficits are unclear. Α deficient cholinergic system has been implicated as one of the main factors that could confer a heightened vulnerability to the aging process, and mice lacking high-affinity nicotinic receptors (β2(-/-)) have been proposed as an animal model of accelerated cognitive aging. To date, however, age-related changes in neuronal microanatomy have not been studied in these mice. In the present study, we examine the neuronal structure of yellow fluorescent protein (YFP(+)) layer V neurons in 2 cytoarchitectonically distinct cortical regions in wild-type (WT) and β2(-/-) animals. We find that (1) substantial morphological differences exist between YFP(+) cells of the anterior cingulate cortex (ACC) and primary visual cortex (V1), in both genotypes; (2) in WT animals, ACC cells are more susceptible to aging compared with cells in V1; and (3) β2 deletion is associated with a regionally and temporally specific increase in vulnerability to aging. ACC cells exhibit a prematurely aged phenotype already at 4-6 months, whereas V1 cells are spared in adulthood but strongly affected in old animals. Collectively, our data reveal region-specific synergistic effects of aging and genotype and suggest distinct vulnerabilities in V1 and ACC neurons. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Molecular identification of high-affinity glutamate transporters in sheep and cattle forestomach, intestine, liver, kidney, and pancreas.

    Science.gov (United States)

    Howell, J A; Matthews, A D; Swanson, K C; Harmon, D L; Matthews, J C

    2001-05-01

    Glutamate metabolism is essential to support many facets of metabolism. The objective of this study was to determine the tissue distribution of glutamate transporters known to support the tissue metabolism of glutamate. The expression of proteins capable of high-affinity glutamate transport (system X-(AG)) by epithelia isolated from the rumen, omasum, duodenum, jejunum, ileum, cecum, and colon and homogenates of liver, kidney, and pancreatic tissues from wethers (n = 4; BW = 28.4 +/- 8.4 kg) and steers (n = 3; BW = 426 +/- 32.3 kg) fed forage-based diets was evaluated by immunoblot analysis. Proteins EAAC1 (62, 93 kDa) and GLT-1 (142, 188, >202 kDa) were expressed by every tissue examined. In contrast, GLAST1 (140 kDa) was expressed only by the pancreas, and EAAT4 (67 kDa) was detected only in sheep brain. To corroborate protein expression data, the presence and size of transporter mRNA in ileal, liver, and pancreatic homogenates were evaluated by Northern analysis. GLAST1 mRNA (2.4, 4.3 kb) was detected only in the pancreas, whereas EAAC1 (2.2, 2.8 kb) and GLT-1 (12.1 kb) mRNA transcripts were detected in all three tissues. The expression of EAAT4 and GLT-1 mRNA was confirmed by reverse transcriptase-polymerase chain reaction analyses. Sequencing of the resulting partial-length ovine GLT-1 cDNA revealed 100% identity with the rat homolog. Overall, these data demonstrate that sheep and cattle share the same pattern of system X-(AG) transporter expression, which differed among tissues and transporter isoforms. Accordingly, these data provide the fundamental knowledge to initiate research that determines whether the expression of high-affinity glutamate transporters by ruminants is sensitive to ontogenic and(or) dietary regulation.

  6. Emerging molecular phenotypes of asthma

    Science.gov (United States)

    Ray, Anuradha; Oriss, Timothy B.

    2014-01-01

    Although asthma has long been considered a heterogeneous disease, attempts to define subgroups of asthma have been limited. In recent years, both clinical and statistical approaches have been utilized to better merge clinical characteristics, biology, and genetics. These combined characteristics have been used to define phenotypes of asthma, the observable characteristics of a patient determined by the interaction of genes and environment. Identification of consistent clinical phenotypes has now been reported across studies. Now the addition of various 'omics and identification of specific molecular pathways have moved the concept of clinical phenotypes toward the concept of molecular phenotypes. The importance of these molecular phenotypes is being confirmed through the integration of molecularly targeted biological therapies. Thus the global term asthma is poised to become obsolete, being replaced by terms that more specifically identify the pathology associated with the disease. PMID:25326577

  7. Molecular switch for CLC-K Cl- channel block/activation: optimal pharmacophoric requirements towards high-affinity ligands.

    Science.gov (United States)

    Liantonio, Antonella; Picollo, Alessandra; Carbonara, Giuseppe; Fracchiolla, Giuseppe; Tortorella, Paolo; Loiodice, Fulvio; Laghezza, Antonio; Babini, Elena; Zifarelli, Giovanni; Pusch, Michael; Camerino, Diana Conte

    2008-01-29

    ClC-Ka and ClC-Kb Cl(-) channels are pivotal for renal salt reabsorption and water balance. There is growing interest in identifying ligands that allow pharmacological interventions aimed to modulate their activity. Starting from available ligands, we followed a rational chemical strategy, accompanied by computational modeling and electrophysiological techniques, to identify the molecular requisites for binding to a blocking or to an activating binding site on ClC-Ka. The major molecular determinant that distinguishes activators from blockers is the level of planarity of the aromatic portions of the molecules: only molecules with perfectly coplanar aromatic groups display potentiating activity. Combining several molecular features of various CLC-K ligands, we discovered that phenyl-benzofuran carboxylic acid derivatives yield the most potent ClC-Ka inhibitors so far described (affinity <10 microM). The increase in affinity compared with 3-phenyl-2-p-chlorophenoxy-propionic acid (3-phenyl-CPP) stems primarily from the conformational constraint provided by the phenyl-benzofuran ring. Several other key structural elements for high blocking potency were identified through a detailed structure-activity relationship study. Surprisingly, some benzofuran-based drugs inhibit ClC-Kb with a similar affinity of <10 microM, thus representing the first inhibitors for this CLC-K isoform identified so far. Based on our data, we established a pharmacophore model that will be useful for the development of drugs targeting CLC-K channels.

  8. Characterization of dendritic cell phenotype in allergic conjunctiva: increased expression of Fc(epsilon)RI, the high-affinity receptor for immunoglobulin E.

    Science.gov (United States)

    Manzouri, B; Ohbayashi, M; Leonardi, A; Larkin, D F P; Ono, S J

    2009-11-01

    Dendritic cells (DCs) express the high-affinity receptor for IgE (Fc(epsilon)RI) on their surface, which may enhance their ability to capture and internalize antigens for presentation to T-lymphocytes. The aim of this study was to determine if expression of Fc(epsilon)RI(+) DCs is increased in the conjunctivae of vernal keratoconjunctivitis (VKC) patients compared with those of normal controls. Conjunctival biopsies were obtained from non-atopic and VKC patients. Double immunohistochemical staining was carried out using antibodies against Fc(epsilon)RI and the CD1a antigen, a DC marker. The double-positive cells were counted in five representative fields of view for each conjunctival sample. Fc(epsilon)RI(+) CD1a(+) cells were present in significantly higher numbers in VKC conjunctivae compared with normal controls (mean cell count of 21.3 in VKC vs5.0 in controls, PRI-expressing DCs tended to be confined to the epithelial layer or the superficial substantia propria, but in the VKC samples these Fc(epsilon)RI(+) cells were mainly concentrated in the deeper substantia propria. Fc(epsilon)RI(+) DC numbers are elevated in the conjunctivae of VKC patients, a finding consistent with the results of other studies focusing on atopic conditions. Elevated expression of Fc(epsilon)RI on DCs would facilitate antigen presentation and enhance T-cell priming, thereby contributing to ocular symptoms.

  9. Arabidopsis Sucrose Transporter AtSUC9. High-Affinity Transport Activity, Intragenic Control of Expression, and Early Flowering Mutant Phenotype1[OA

    Science.gov (United States)

    Sivitz, Alicia B.; Reinders, Anke; Johnson, Meghan E.; Krentz, Anthony D.; Grof, Christopher P.L.; Perroux, Jai M.; Ward, John M.

    2007-01-01

    AtSUC9 (At5g06170), a sucrose (Suc) transporter from Arabidopsis (Arabidopsis thaliana) L. Heynh., was expressed in Xenopus (Xenopus laevis) oocytes, and transport activity was analyzed. Compared to all other Suc transporters, AtSUC9 had an ultrahigh affinity for Suc (K0.5 = 0.066 ± 0.025 mm). AtSUC9 showed low substrate specificity, similar to AtSUC2 (At1g22710), and transported a wide range of glucosides, including helicin, salicin, arbutin, maltose, fraxin, esculin, turanose, and α-methyl-d-glucose. The ability of AtSUC9 to transport 10 glucosides was compared directly with that of AtSUC2, HvSUT1 (from barley [Hordeum vulgare]), and ShSUT1 (from sugarcane [Saccharum hybrid]), and results indicate that type I and type II Suc transporters have different substrate specificities. AtSUC9 protein was localized to the plasma membrane by transient expression in onion (Allium cepa) epidermis. Using a whole-gene translational fusion to β-glucuronidase, AtSUC9 expression was found in sink tissues throughout the shoots and in flowers. AtSUC9 expression in Arabidopsis was dependent on intragenic sequence, and this was found to also be true for AtSUC1 (At1g71880) but not AtSUC2. Plants containing mutations in Suc transporter gene AtSUC9 were found to have an early flowering phenotype under short-day conditions. The transport properties of AtSUC9 indicate that it is uniquely suited to provide cellular uptake of Suc at very low extracellular Suc concentrations. The mutant phenotype of atsuc9 alleles indicates that AtSUC9 activity leads to a delay in floral transition. PMID:17098854

  10. NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype

    DEFF Research Database (Denmark)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte

    2002-01-01

    against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed...

  11. Interrogating the Molecular Basis for Substrate Recognition in Serotonin and Dopamine Transporters with High-Affinity Substrate-Based Bivalent Ligands

    DEFF Research Database (Denmark)

    Andersen, Jacob; Ladefoged, Lucy Kate; Kristensen, Trine N. Bjerre

    2016-01-01

    The transporters for the neurotransmitters serotonin and dopamine (SERT and DAT, respectively) are targets for drugs used in the treatment of mental disorders and widely used drugs of abuse. Studies of prokaryotic homologues have advanced our structural understanding of SERT and DAT, but it still...... remains enigmatic whether the human transporters contain one or two high-affinity substrate binding sites. We have designed and employed 24 bivalent ligands possessing a highly systematic combination of substrate moieties (serotonin and/or dopamine) and aliphatic or poly(ethylene glycol) spacers to reveal...... insight into substrate recognition in SERT and DAT. An optimized bivalent ligand comprising two serotonin moieties binds SERT with 3,800-fold increased affinity compared to that of serotonin, suggesting that the human transporters have two distinct substrate binding sites. We show that the bivalent...

  12. High Affinity vs. Native Fibronectin in the Modulation of αvβ3 Integrin Conformational Dynamics: Insights from Computational Analyses and Implications for Molecular Design.

    Directory of Open Access Journals (Sweden)

    Antonella Paladino

    2017-01-01

    Full Text Available Understanding how binding events modulate functional motions of multidomain proteins is a major issue in chemical biology. We address several aspects of this problem by analyzing the differential dynamics of αvβ3 integrin bound to wild type (wtFN10, agonist or high affinity (hFN10, antagonist mutants of fibronectin. We compare the dynamics of complexes from large-scale domain motions to inter-residue coordinated fluctuations to characterize the distinctive traits of conformational evolution and shed light on the determinants of differential αvβ3 activation induced by different FN sequences. We propose an allosteric model for ligand-based integrin modulation: the conserved integrin binding pocket anchors the ligand, while different residues on the two FN10's act as the drivers that reorganize relevant interaction networks, guiding the shift towards inactive (hFN10-bound or active states (wtFN10-bound. We discuss the implications of results for the design of integrin inhibitors.

  13. Molecular and phenotypic characterization of Lactobacillus curvatus ...

    African Journals Online (AJOL)

    Molecular and phenotypic characterization of Lactobacillus curvatus isolated from handmade Brazilian salami. César Milton Baratto, Jane Mary Lafayette Neves Gelinski, Jaqueline Debastiani, Marco Antonio Dalbó ...

  14. Phenotypic characterisation and molecular polymorphism of ...

    African Journals Online (AJOL)

    The study of the phenotypic characterisation and molecular polymorphism of local chicken populations was carried out in Benin on 326 chickens of the Forest ecological area and 316 of the Savannah ecological area, all were 7 months old at least. The collection of blood for the molecular typing was achieved on 121 ...

  15. Phenotypic characterisation and molecular polymorphism of ...

    African Journals Online (AJOL)

    PRECIOUS

    2010-01-18

    Jan 18, 2010 ... The study of the phenotypic characterisation and molecular polymorphism of local chicken populations was carried out in Benin on 326 chickens of the Forest ecological area and 316 of the Savannah ecological area, all were 7 months old at least. The collection of blood for the molecular typing was.

  16. Bivalency and epitope specificity of a high-affinity IgG3 monoclonal antibody to the Streptococcus group A carbohydrate antigen. Molecular modeling of a Fv fragment.

    Science.gov (United States)

    Pitner, J B; Beyer, W F; Venetta, T M; Nycz, C; Mitchell, M J; Harris, S L; Mariño-Albernas, J R; Auzanneau, F I; Forooghian, F; Pinto, B M

    2000-01-29

    The binding of Strep 9, a mouse monoclonal antibody (mAb) of the IgG3 subclass directed against the cell-wall polysaccharide of Group A Streptococcus (GAS), has been characterized. The intact antibody and proteolytic fragments of Strep 9 bind differently to GAS: the intact mAb and F(ab)2' have greater affinity for the carbohydrate epitope than the monomeric Fab or F(ab)'. A mode of binding in which Strep 9 binds bivalently to portions of the polysaccharide on adjacent chains on GAS is proposed. A competitive ELISA protocol using a panel of carbohydrate inhibitors shows that the branched trisaccharide, beta-D-GlcpNAc-(1-->3)-[alpha-L-Rhap-(1-->2)]-alpha-L-Rhap, and an extended surface are key components of the epitope recognized by Strep 9. Microcalorimetry measurements with the mAb and two synthetic haptens, a tetrasaccharide and a hexasaccharide, show enthalpy-entropy compensation as seen in other oligosaccharide-protein interactions. Molecular modeling of the antibody variable region by homology modeling techniques indicates a groove-shaped combining site that can readily accommodate extended surfaces. Visual docking of an oligosaccharide corresponding to the cell-wall polysaccharide into the site provides a putative model for the complex, in which a heptasaccharide unit occupies the site and the GlcpNAc residues of two adjacent branched trisaccharide units occupy binding pockets within the groove-shaped binding site.

  17. Evolution of molecular phenotypes under stabilizing selection

    Science.gov (United States)

    Nourmohammad, Armita; Schiffels, Stephan; Lässig, Michael

    2013-01-01

    Molecular phenotypes are important links between genomic information and organismic functions, fitness, and evolution. Complex phenotypes, which are also called quantitative traits, often depend on multiple genomic loci. Their evolution builds on genome evolution in a complicated way, which involves selection, genetic drift, mutations and recombination. Here we develop a coarse-grained evolutionary statistics for phenotypes, which decouples from details of the underlying genotypes. We derive approximate evolution equations for the distribution of phenotype values within and across populations. This dynamics covers evolutionary processes at high and low recombination rates, that is, it applies to sexual and asexual populations. In a fitness landscape with a single optimal phenotype value, the phenotypic diversity within populations and the divergence between populations reach evolutionary equilibria, which describe stabilizing selection. We compute the equilibrium distributions of both quantities analytically and we show that the ratio of mean divergence and diversity depends on the strength of selection in a universal way: it is largely independent of the phenotype’s genomic encoding and of the recombination rate. This establishes a new method for the inference of selection on molecular phenotypes beyond the genome level. We discuss the implications of our findings for the predictability of evolutionary processes.

  18. Craniosynostoses: Phenotypic/molecular correlations

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, M.M. Jr. [Dalhousie Univ., Nova Scotia (Canada)

    1995-04-10

    From the discovery of the first known human homeobox mutation in MSX2 for craniosynostosis of the Boston type by Jams to the recent report of 2 mutations in FGFR2 in Apert syndrome by Wilkie, it is clear that the molecular aspects of syndromes with craniosynostosis are becoming known at a dizzying pace. Four of the syndromes involve mutations in FGFR2. The first to emerge was Crouzon syndrome followed by Jackson-Weiss syndrome, Pfeiffer syndrome, and finally Apert syndrome. Earlier, Muenke showed that Pfeiffer syndrome was heterogeneous, some families having a mutation in FGFR1. 50 refs., 4 tabs.

  19. Phenotypic and molecular characterization of Salmonella serotypes ...

    African Journals Online (AJOL)

    The presence of Salmonella and human pathogens in unpasteurized milk remains a public health hazard. The study reported the phenotypic and molecular characterization of Salmonella serotypes in cow raw milk, cheese and traditional yoghurt marketed for man's consumption in Nigeria. Isolation of Salmonella was done ...

  20. Phenotypic and Molecular Characterization of Plasmid- Encoded ...

    African Journals Online (AJOL)

    Purpose: To investigate the distribution of plasmid-encoded extended spectrum beta-lacatamases (ESBLs) in Lahore, Pakistan using different phenotypic and molecular methods. Methods: Escherichia coli and Klebsiella spp were obtained over a period of nineteen months (June 2007 to December 2008). Both were tested ...

  1. The Saccharomyces cerevisiae High Affinity Phosphate Transporter Encoded by PHO84 Also Functions in Manganese Homeostasis

    National Research Council Canada - National Science Library

    Laran T. Jensen; Mispa Ajua-Alemanji; Valeria Cizewski Culotta

    2003-01-01

    ... . In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype...

  2. Molecular and phenotypic biomarkers of aging

    OpenAIRE

    Xian Xia; Weiyang Chen; Joseph McDermott; Jing-Dong Jackie Han

    2017-01-01

    Individuals of the same age may not age at the same rate. Quantitative biomarkers of aging are valuable tools to measure physiological age, assess the extent of ‘healthy aging’, and potentially predict health span and life span for an individual. Given the complex nature of the aging process, the biomarkers of aging are multilayered and multifaceted. Here, we review the phenotypic and molecular biomarkers of aging. Identifying and using biomarkers of aging to improve human health, prevent age...

  3. Phenotypic, molecular and technological characterization of ...

    African Journals Online (AJOL)

    The strains were then identified by phenotypic and molecular approaches by amplification and sequencing of 16S rDNA as Lb.casei (C4, C5, V2 and V5), Lb. paracasei (C6) and Lb.plantarum (C7, C8, C10). Virtually all the strains studied, from a technological point of view, produced lactic acid concentrations at or above ...

  4. Molecular and phenotypic biomarkers of aging.

    Science.gov (United States)

    Xia, Xian; Chen, Weiyang; McDermott, Joseph; Han, Jing-Dong Jackie

    2017-01-01

    Individuals of the same age may not age at the same rate. Quantitative biomarkers of aging are valuable tools to measure physiological age, assess the extent of 'healthy aging', and potentially predict health span and life span for an individual. Given the complex nature of the aging process, the biomarkers of aging are multilayered and multifaceted. Here, we review the phenotypic and molecular biomarkers of aging. Identifying and using biomarkers of aging to improve human health, prevent age-associated diseases, and extend healthy life span are now facilitated by the fast-growing capacity of multilevel cross-sectional and longitudinal data acquisition, storage, and analysis, particularly for data related to general human populations. Combined with artificial intelligence and machine learning techniques, reliable panels of biomarkers of aging will have tremendous potential to improve human health in aging societies.

  5. High affinity hemoglobin and Parkinson's disease.

    Science.gov (United States)

    Graham, Jeffrey; Hobson, Douglas; Ponnampalam, Arjuna

    2014-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain. Oxidative damage in this region has been shown to play an important role in the pathogenesis of this disease. Human neurons have been discovered to contain hemoglobin, with an increased concentration seen in the neurons of the SN. High affinity hemoglobin is a clinical entity resulting from mutations that create a functional increase in the binding of hemoglobin to oxygen and an inability to efficiently unload it to tissues. This can result in a number of metabolic compensatory changes, including an elevation in circulating hemoglobin and an increase in the molecule 2,3-diphosphoglycerate (2,3-DPG). Population based studies have revealed that patients with PD have elevated hemoglobin as well as 2,3-DPG levels. Based on these observations, we hypothesize that the oxidative damage seen in PD is related to an underlying high affinity hemoglobin subtype. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Molecular identification of four phenotypes of human Demodex in China.

    Science.gov (United States)

    Hu, Li; Zhao, Ya-E; Cheng, Juan; Ma, Jun-Xian

    2014-07-01

    Traditional classification of Demodex mites by hosts and phenotypic characteristics is defective because of environmental influences. In this study, we proposed molecular identification of four phenotypes of two human Demodex species based on mitochondrial cox1 fragments for the first time. Mites collected from sufferers' facial skin were classified into four phenotypes: phenotype A-C with finger-like terminus, and phenotype D with cone-like terminus. The results of molecular data showed that cox1 sequences were all 429 bp. Divergences, genetic distances and transition/transversion ratios among the three phenotypes with finger-like terminus were 0.0-3.0%, 0.000-0.031, and 6/3-5/0, respectively, in line with intraspecific differences. However, those measures between the phenotype with cone-like terminus and phenotypes with finger-like terminus were 19.6-20.5%, 0.256-0.271, and 0.58 (31/53)-0.66 (35/53), respectively, reaching interspecific level. Phylogenetic trees also showed that the three phenotypes with finger-like terminus clustered as one clade, and the phenotype with cone-like terminus formed another one. Therefore, we conclude that mitochondrial cox1 sequence is a good marker for identification of two human Demodex species. Molecular data indicate no subspecies differentiation. Terminus is an effective character for species identification. Mites with finger-like terminus are Demodex folliculorum, and those with cone-like terminus are Demodex brevis. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Data characterizing the ZMIZ1 molecular phenotype of multiple sclerosis

    National Research Council Canada - National Science Library

    Fewings, N; Gatt, P.N; McKay, F.C; Parnell, G.P; Schibeci, S.D; Edwards, J; Basuki, M.A; Goldinger, A; Fabis-Pedrini, M.J; Kermode, A.G; Manrique, C.P; McCauley, J.L; Nickles, D; Baranzini, S.E; Burke, T; Vucic, S; Stewart, G.J; Booth, D.R

    2017-01-01

    The data presented in this article are related to the research article entitled “The autoimmune risk gene ZMIZ1 is a vitamin D responsived marker of a molecular phenotype of multiple sclerosis” Fewings et al. (2017) [1...

  8. Social parasitism and the molecular basis of phenotypic evolution

    Directory of Open Access Journals (Sweden)

    Alessandro eCini

    2015-02-01

    Full Text Available Contrasting phenotypes arise from similar genomes through a combination of losses, gains, co-option and modifications of inherited genomic material. Understanding the molecular basis of this phenotypic diversity is a fundamental challenge in modern evolutionary biology. Comparisons of the genes and their expression patterns underlying traits in pairs of closely related species offer an unrivalled opportunity to evaluate the extent to which genomic material is reorganised to produce novel traits. Advances in molecular methods now allow us to dissect the molecular machinery underlying phenotypic diversity in almost any organism, from single-celled organisms to the most complex vertebrates. Here we discuss how comparisons of social parasites and their free-living hosts may provide unique insights into the molecular basis of phenotypic evolution. Social parasites evolve from a social ancestor and are specialised to exploit the socially acquired resources of their closely-related, free-living social host. Molecular comparisons of such species pairs can reveal how genomic material is re-organised in the loss of ancestral traits (i.e. of free-living traits in the parasites and the gain of new ones (i.e. specialist traits required for a parasitic lifestyle. We define hypotheses on the molecular basis of phenotypes in the evolution of social parasitism and discuss their wider application in understanding the molecular basis of phenotypic diversity within the theoretical framework of phenotypic plasticity and shifting reaction norms. Currently there are no data available to test these hypotheses, and so we also provide some proof of concept data for our conceptual model using the paper wasp social parasite-host system (Polistes sulcifer - Polistes dominula. This conceptual framework and first empirical data provide a spring-board for directing future genomic analyses on exploiting social parasites as a route to understanding the evolution of phenotypic

  9. Phenotypic and molecular characterization of selected tomato ...

    Indian Academy of Sciences (India)

    2011-08-19

    Aug 19, 2011 ... 1Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario,. Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario,. Suipacha 531, S2002LRK, Rosario, Argentina. 2Facultad de Ciencias Agrarias, Cátedra de Genética, ...

  10. Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

    Directory of Open Access Journals (Sweden)

    Guy Caljon

    Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

  11. Pathway reporter genes define molecular phenotypes of human cells.

    Science.gov (United States)

    Zhang, Jitao David; Küng, Erich; Boess, Franziska; Certa, Ulrich; Ebeling, Martin

    2015-04-24

    The phenotype of a living cell is determined by its pattern of active signaling networks, giving rise to a "molecular phenotype" associated with differential gene expression. Digital amplicon based RNA quantification by sequencing is a useful technology for molecular phenotyping as a novel tool to characterize the state of biological systems. We show here that the activity of signaling networks can be assessed based on a set of established key regulators and expression targets rather than the entire transcriptome. We compiled a panel of 917 human pathway reporter genes, representing 154 human signaling and metabolic networks for integrated knowledge- and data-driven understanding of biological processes. The reporter genes are significantly enriched for regulators and effectors covering a wide range of biological processes, and faithfully capture gene-level and pathway-level changes. We apply the approach to iPSC derived cardiomyocytes and primary human hepatocytes to describe changes in molecular phenotype during development or drug response. The reporter genes deliver an accurate pathway-centric view of the biological system under study, and identify known and novel modulation of signaling networks consistent with literature or experimental data. A panel of 917 pathway reporter genes is sufficient to describe changes in the molecular phenotype defined by 154 signaling cascades in various human cell types. AmpliSeq-RNA based digital transcript imaging enables simultaneous monitoring of the entire pathway reporter gene panel in up to 150 samples. We propose molecular phenotyping as a useful approach to understand diseases and drug action at the network level.

  12. Phenotypic and molecular genetic analysis of Pyruvate Kinase ...

    African Journals Online (AJOL)

    Pyruvate Kinase (PK) deficiency is the most frequent red cell enzymatic defect responsible for hereditary non-spherocytic hemolytic anemia. The disease has been studied in several ethnic groups. However, it is yet an unknown pathology in Tunisia. We report here, the phenotypic and molecular investigation of PK ...

  13. Phenotypic and molecular genetic analysis of Pyruvate Kinase ...

    African Journals Online (AJOL)

    Jaouani Mouna

    2015-09-26

    Sep 26, 2015 ... Abstract Pyruvate Kinase (PK) deficiency is the most frequent red cell enzymatic defect responsi- ble for hereditary non-spherocytic hemolytic anemia. The disease has been studied in several ethnic groups. However, it is yet an unknown pathology in Tunisia. We report here, the phenotypic and molecular ...

  14. Molecular subtypes and imaging phenotypes of breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Nariya [Dept. of Radiology, Seoul National University Hospital, Seoul (Korea, Republic of)

    2016-08-15

    During the last 15 years, traditional breast cancer classifications based on histopathology have been reorganized into the luminal A, luminal B, human epidermal growth factor receptor 2 (HER2), and basal-like subtypes based on gene expression profiling. Each molecular subtype has shown varying risk for progression, response to treatment, and survival outcomes. Research linking the imaging phenotype with the molecular subtype has revealed that non-calcified, relatively circumscribed masses with posterior acoustic enhancement are common in the basal-like subtype, spiculated masses with a poorly circumscribed margin and posterior acoustic shadowing in the luminal subtype, and pleomorphic calcifications in the HER2-enriched subtype. Understanding the clinical implications of the molecular subtypes and imaging phenotypes could help radiologists guide precision medicine, tailoring medical treatment to patients and their tumor characteristics.

  15. Molecular subtypes and imaging phenotypes of breast cancer

    Directory of Open Access Journals (Sweden)

    Nariya Cho

    2016-10-01

    Full Text Available During the last 15 years, traditional breast cancer classifications based on histopathology have been reorganized into the luminal A, luminal B, human epidermal growth factor receptor 2 (HER2, and basal-like subtypes based on gene expression profiling. Each molecular subtype has shown varying risk for progression, response to treatment, and survival outcomes. Research linking the imaging phenotype with the molecular subtype has revealed that non-calcified, relatively circumscribed masses with posterior acoustic enhancement are common in the basal-like subtype, spiculated masses with a poorly circumscribed margin and posterior acoustic shadowing in the luminal subtype, and pleomorphic calcifications in the HER2-enriched subtype. Understanding the clinical implications of the molecular subtypes and imaging phenotypes could help radiologists guide precision medicine, tailoring medical treatment to patients and their tumor characteristics.

  16. Molecular Mechanisms Modulating the Phenotype of Macrophages and Microglia

    Directory of Open Access Journals (Sweden)

    Stephanie A. Amici

    2017-11-01

    Full Text Available Macrophages and microglia play crucial roles during central nervous system development, homeostasis and acute events such as infection or injury. The diverse functions of tissue macrophages and microglia are mirrored by equally diverse phenotypes. A model of inflammatory/M1 versus a resolution phase/M2 macrophages has been widely used. However, the complexity of macrophage function can only be achieved by the existence of varied, plastic and tridimensional macrophage phenotypes. Understanding how tissue macrophages integrate environmental signals via molecular programs to define pathogen/injury inflammatory responses provides an opportunity to better understand the multilayered nature of macrophages, as well as target and modulate cellular programs to control excessive inflammation. This is particularly important in MS and other neuroinflammatory diseases, where chronic inflammatory macrophage and microglial responses may contribute to pathology. Here, we perform a comprehensive review of our current understanding of how molecular pathways modulate tissue macrophage phenotype, covering both classic pathways and the emerging role of microRNAs, receptor-tyrosine kinases and metabolism in macrophage phenotype. In addition, we discuss pathway parallels in microglia, novel markers helpful in the identification of peripheral macrophages versus microglia and markers linked to their phenotype.

  17. Stereoselective synthesis, in vitro, and initial in vivo evaluation of 1-methylpiperidin-4-yl {alpha}-hydroxy-{alpha}-(1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (IPIP): a novel radioiodinated molecular probe with high affinity for the muscarinic receptor

    Energy Technology Data Exchange (ETDEWEB)

    McPherson, Daniel W. E-mail: mcphersod@CSR.NIH.GOV; Breeden, William K.; Beets, Arnold L.; Luo, Huimin; Sood, Victor; Knapp, Furn F

    2001-11-01

    1-Methylpiperidin-4-yl {alpha}-hydroxy-{alpha}-(1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (IPIP) was investigated as a potential radioiodinated molecular probe targeted to the muscarinic receptor complex. The IPIP stereoisomers were synthesized via a chiral intermediate in >95% enantiomeric excess. The R-isomers demonstrated a M{sub 1} to M{sub 2} subtype selectivity of approximately 3 to 1 and the S-isomers demonstrated non-subtype selective binding in vitro. IPIP was radiolabeled with iodide-125 with an average radiochemical yield of 74.4% ({+-}14.8, n 5), specific activities >800 mCi/{mu}mol, and radiochemical purities >97%. In vivo the Z-isomers demonstrated high uniform cerebral uptake suggesting non-subtype selective binding. In contrast, E-R-IPIP, after allowing a low uptake in M{sub 2} rich areas to clear, demonstrated a retention of activity in M{sub 1} and M{sub 4} rich cerebral regions. In addition, the cerebral uptake of E-R-IPIP and Z-S-IPIP were inhibited by 70-90% via pretreatment with R-QNB, an established muscarinic antagonist. An ex vivo metabolism study demonstrated Z-S-IPIP was stable at the receptor site with an absence of radiolabeled metabolites.

  18. Molecular signatures of mammalian hibernation: comparisons with alternative phenotypes.

    Science.gov (United States)

    Xu, Yichi; Shao, Chunxuan; Fedorov, Vadim B; Goropashnaya, Anna V; Barnes, Brian M; Yan, Jun

    2013-08-20

    Mammalian hibernators display phenotypes similar to physiological responses to calorie restriction and fasting, sleep, cold exposure, and ischemia-reperfusion in non-hibernating species. Whether biochemical changes evident during hibernation have parallels in non-hibernating systems on molecular and genetic levels is unclear. We identified the molecular signatures of torpor and arousal episodes during hibernation using a custom-designed microarray for the Arctic ground squirrel (Urocitellus parryii) and compared them with molecular signatures of selected mouse phenotypes. Our results indicate that differential gene expression related to metabolism during hibernation is associated with that during calorie restriction and that the nuclear receptor protein PPARα is potentially crucial for metabolic remodeling in torpor. Sleep-wake cycle-related and temperature response genes follow the same expression changes as during the torpor-arousal cycle. Increased fatty acid metabolism occurs during hibernation but not during ischemia-reperfusion injury in mice and, thus, might contribute to protection against ischemia-reperfusion during hibernation. In this study, we systematically compared hibernation with alternative phenotypes to reveal novel mechanisms that might be used therapeutically in human pathological conditions.

  19. The implications of breast cancer molecular phenotype for radiation oncology

    Directory of Open Access Journals (Sweden)

    Shirin eSioshansi

    2011-06-01

    Full Text Available The identification of distinct molecular subtypes of breast cancer has advanced the understanding and treatment of breast cancer by providing insight into prognosis, patterns of recurrence and effectiveness of therapy. The prognostic significance of molecular phenotype with regard to distant recurrences and overall survival are well established in the literature and has been readily incorporated into systemic therapy management decisions. However, despite the accumulating data suggesting similar prognostic significance for locoregional recurrence, integration of molecular phenotype into local management decision making has lagged. Although there are some conflicting reports, collectively the literature supports a low risk of local recurrence in the hormone receptor positive luminal subtypes compared to hormone receptor negative subtypes (triple negative and HER2-enriched. The development of targeted therapies, such as trastuzumab for the treatment of HER2-enriched subtype, has been shown to mitigate the increased risk of local recurrence. Unfortunately, no such remedy exists to address the increased risk of local recurrence for patients with triple negative tumors, making it a clinical challenge for radiation oncologists. In this review we discuss the correlation between molecular subtype and local recurrence following either breast conservation therapy or mastectomy. We also explore the possible mechanisms for increased local recurrence in triple negative breast cancer and radiotherapeutic implications for this population, such as the safety of breast conservation, consideration of dose escalation and the appropriateness of accelerated partial breast irradiation.

  20. Molecular phenotypes associated with anomalous stamen development in Alternanthera philoxeroides

    Directory of Open Access Journals (Sweden)

    Zhu eZhu

    2015-04-01

    Full Text Available Alternanthera philoxeroides is a perennial amphibious weed native to South America but has now spread to diverse parts of the world. A. philoxeroides reproduces both sexually and asexually in its native range, but propagates solely through vegetative means in its introduced range. Traits associated with sexual reproduction become degraded for sexual dysfunction, with flowers possessing either pistillate stamens or male-sterile anthers. Degradations of sexual characters for loss of sexuality commonly take place in clonal plants. The underlying molecular-genetic processes remain largely unknown. We compared the gene expression profiles of abnormal stamens with that of normal stamens by RNA-Seq analysis, and identified a large number of differentially expressed genes between abnormal and normal stamens. In accordance with flower morphology, the expression of B-class MADS-box genes (ApAP3, ApTM6 and ApPI was markedly reduced in pistillate stamens. However, most of the genes involved in meiosis were expressed normally in stamens with male-sterile anthers. In addition to verifying the expression patterns of genes previously known to be related to stamen and pollen grain development, we also identified previously unknown molecular phenotypes associated with sexual dysfunction in A. philoxeroides, that is helpful for dissecting the molecular mechanisms underpinning various male-sterile phenotypes and the molecular processes underlying the transition from sexuality to asexuality in clonal plants.

  1. The Saccharomyces cerevisiae high affinity phosphate transporter encoded by PHO84 also functions in manganese homeostasis.

    Science.gov (United States)

    Jensen, Laran T; Ajua-Alemanji, Mispa; Culotta, Valeria Cizewski

    2003-10-24

    In the bakers' yeast Saccharomyces cerevisiae, high affinity manganese uptake and intracellular distribution involve two members of the Nramp family of genes, SMF1 and SMF2. In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype. Resistance to zinc, cobalt, and copper ions was also demonstrated for pho84Delta yeast. When challenged with high concentrations of metals, pho84Delta yeast have reduced metal ion accumulation, suggesting that resistance is due to reduced uptake of metal ions. Pho84p accounted for virtually all the manganese accumulated under metal surplus conditions, demonstrating that this transporter is the major source of excess manganese accumulation. The manganese taken in via Pho84p is indeed biologically active and can not only cause toxicity but can also be incorporated into manganese-requiring enzymes. Pho84p is essential for activating manganese enzymes in smf2Delta mutants that rely on low affinity manganese transport systems. A role for Pho84p in manganese accumulation was also identified in a standard laboratory growth medium when high affinity manganese uptake is active. Under these conditions, cells lacking both Pho84p and the high affinity Smf1p transporter accumulated low levels of manganese, although there was no major effect on activity of manganese-requiring enzymes. We conclude that Pho84p plays a role in manganese homeostasis predominantly under manganese surplus conditions and appears to be functioning as a low affinity metal transporter.

  2. The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

    Directory of Open Access Journals (Sweden)

    Alexander Krah

    Full Text Available The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

  3. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    Energy Technology Data Exchange (ETDEWEB)

    Yliniemelae, A.; Gynther, J. (Univ. of Kuopio (Finland)); Konschin, H.; Tylli, H. (Univ. of Helsinki (Finland)); Rouvinen, J. (Univ. of Joensuu (Finland))

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  4. Dephosphorylation of Phytate by Using the Aspergillus niger Phytase with a High Affinity for Phytate

    OpenAIRE

    Nagashima, Tadashi; Tange, Tatsuya; Anazawa, Hideharu

    1999-01-01

    A phytase (EC 3.1.3.8) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 ± 4.6 μM) was statistically analyzed. In regard to the ort...

  5. The Human Phenotype Ontology project: linking molecular biology and disease through phenotype data

    NARCIS (Netherlands)

    Kohler, S.; Doelken, S.C.; Mungall, C.J.; Bauer, S.; Firth, H.V.; Bailleul-Forestier, I.; Black, G.C.M.; Brown, D.L.; Brudno, M.; Campbell, J.; FitzPatrick, D.R.; Eppig, J.T.; Jackson, A.P.; Freson, K.; Girdea, M.; Helbig, I.; Hurst, J.A.; Jahn, J.; Jackson, L.G.; Kelly, A.M.; Ledbetter, D.H.; Mansour, S.; Martin, C.L.; Moss, C.; Mumford, A.; Ouwehand, W.H.; Park, S.M.; Riggs, E.R.; Scott, R.H.; Sisodiya, S.; Vooren, S. van der; Wapner, R.J.; Wilkie, A.O.; Wright, C.F.; Silfhout, A.T. van; Leeuw, N. de; Vries, B. de; Washingthon, N.L.; Smith, C.L.; Westerfield, M.; Schofield, P.; Ruef, B.J.; Gkoutos, G.V.; Haendel, M.; Smedley, D.; Lewis, S.E.; Robinson, P.N.

    2014-01-01

    The Human Phenotype Ontology (HPO) project, available at http://www.human-phenotype-ontology.org, provides a structured, comprehensive and well-defined set of 10,088 classes (terms) describing human phenotypic abnormalities and 13,326 subclass relations between the HPO classes. In addition we have

  6. α4βδ GABA receptors are high-affinity targets for γ-hydroxybutyric acid (GHB)

    DEFF Research Database (Denmark)

    Absalom, N.; Karim, N.; Eghorn, L.F.

    2012-01-01

    γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinit...... and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism....

  7. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  8. Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death

    DEFF Research Database (Denmark)

    Nørholm, Morten Helge Hauberg; Nour-Eldin, Hussam H; Brodersen, Peter

    2006-01-01

    We report the biochemical characterization in Xenopus oocytes of the Arabidopsis thaliana membrane protein, STP13, as a high affinity, hexose-specific H(+)-symporter. Studies with kinase activators suggest that it is negatively regulated by phosphorylation. STP13 promoter GFP reporter lines show ......13 in PCD is supported by microarray data from e.g. plants undergoing senescence and a strong correlation between STP13 transcripts and the PCD phenotype in different accelerated cell death (acd11) mutants....... GFP expression only in the vascular tissue in emerging petals under non-stressed conditions. Quantitative PCR and the pSTP13-GFP plants show induction of STP13 in programmed cell death (PCD) obtained by treatments with the fungal toxin fumonisin B1 and the pathogen Pseudomonas syringae. A role for STP...

  9. Phenotypic and molecular characterization of sweet sorghum accessions for bioenergy production.

    Science.gov (United States)

    da Silva, Michele Jorge; Pastina, Maria Marta; de Souza, Vander Fillipe; Schaffert, Robert Eugene; Carneiro, Pedro Crescêncio Souza; Noda, Roberto Willians; Carneiro, José Eustáquio de Souza; Damasceno, Cynthia Maria Borges; Parrella, Rafael Augusto da Costa

    2017-01-01

    Sweet sorghum [Sorghum bicolor (L.) Moench] is a type of cultivated sorghum characterized by the accumulation of high levels of sugar in the stems and high biomass accumulation, making this crop an important feedstock for bioenergy production. Sweet sorghum breeding programs that focus on bioenergy have two main goals: to improve quantity and quality of sugars in the juicy stem and to increase fresh biomass productivity. Genetic diversity studies are very important for the success of a breeding program, especially in the early stages, where understanding the genetic relationship between accessions is essential to identify superior parents for the development of improved breeding lines. The objectives of this study were: to perform phenotypic and molecular characterization of 100 sweet sorghum accessions from the germplasm bank of the Embrapa Maize and Sorghum breeding program; to examine the relationship between the phenotypic and the molecular diversity matrices; and to infer about the population structure in the sweet sorghum accessions. Morphological and agro-industrial traits related to sugar and biomass production were used for phenotypic characterization, and single nucleotide polymorphisms (SNPs) were used for molecular diversity analysis. Both phenotypic and molecular characterizations revealed the existence of considerable genetic diversity among the 100 sweet sorghum accessions. The correlation between the phenotypic and the molecular diversity matrices was low (0.35), which is in agreement with the inconsistencies observed between the clusters formed by the phenotypic and the molecular diversity analyses. Furthermore, the clusters obtained by the molecular diversity analysis were more consistent with the genealogy and the historic background of the sweet sorghum accessions than the clusters obtained through the phenotypic diversity analysis. The low correlation observed between the molecular and the phenotypic diversity matrices highlights the

  10. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  11. The Human Phenotype Ontology project: linking molecular biology and disease through phenotype data.

    Science.gov (United States)

    Köhler, Sebastian; Doelken, Sandra C; Mungall, Christopher J; Bauer, Sebastian; Firth, Helen V; Bailleul-Forestier, Isabelle; Black, Graeme C M; Brown, Danielle L; Brudno, Michael; Campbell, Jennifer; FitzPatrick, David R; Eppig, Janan T; Jackson, Andrew P; Freson, Kathleen; Girdea, Marta; Helbig, Ingo; Hurst, Jane A; Jähn, Johanna; Jackson, Laird G; Kelly, Anne M; Ledbetter, David H; Mansour, Sahar; Martin, Christa L; Moss, Celia; Mumford, Andrew; Ouwehand, Willem H; Park, Soo-Mi; Riggs, Erin Rooney; Scott, Richard H; Sisodiya, Sanjay; Van Vooren, Steven; Wapner, Ronald J; Wilkie, Andrew O M; Wright, Caroline F; Vulto-van Silfhout, Anneke T; de Leeuw, Nicole; de Vries, Bert B A; Washingthon, Nicole L; Smith, Cynthia L; Westerfield, Monte; Schofield, Paul; Ruef, Barbara J; Gkoutos, Georgios V; Haendel, Melissa; Smedley, Damian; Lewis, Suzanna E; Robinson, Peter N

    2014-01-01

    The Human Phenotype Ontology (HPO) project, available at http://www.human-phenotype-ontology.org, provides a structured, comprehensive and well-defined set of 10,088 classes (terms) describing human phenotypic abnormalities and 13,326 subclass relations between the HPO classes. In addition we have developed logical definitions for 46% of all HPO classes using terms from ontologies for anatomy, cell types, function, embryology, pathology and other domains. This allows interoperability with several resources, especially those containing phenotype information on model organisms such as mouse and zebrafish. Here we describe the updated HPO database, which provides annotations of 7,278 human hereditary syndromes listed in OMIM, Orphanet and DECIPHER to classes of the HPO. Various meta-attributes such as frequency, references and negations are associated with each annotation. Several large-scale projects worldwide utilize the HPO for describing phenotype information in their datasets. We have therefore generated equivalence mappings to other phenotype vocabularies such as LDDB, Orphanet, MedDRA, UMLS and phenoDB, allowing integration of existing datasets and interoperability with multiple biomedical resources. We have created various ways to access the HPO database content using flat files, a MySQL database, and Web-based tools. All data and documentation on the HPO project can be found online.

  12. High-affinity neurotrophin receptors and ligands promote leukemogenesis

    Science.gov (United States)

    Beutel, Gernot; Rhein, Mathias; Meyer, Johann; Koenecke, Christian; Neumann, Thomas; Yang, Min; Krauter, Jürgen; von Neuhoff, Nils; Heuser, Michael; Diedrich, Helmut; Göhring, Gudrun; Wilkens, Ludwig; Schlegelberger, Brigitte; Ganser, Arnold

    2009-01-01

    Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) are high-affinity NT receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients we demonstrate for the first time cell-surface expression of the 3 TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. Although only few point mutations were found in TRK sequences by reverse-transcriptase–polymerase chain reaction (RT-PCR), we observed coexpression of BDNF (ligand for TRKB) in more than 50% of TRKB+ cases (16/30). Activation of TRKA or TRKB by NGF and BDNF, respectively, efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Coexpression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover, activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target. PMID:19059881

  13. OsHAK1, a High-Affinity Potassium Transporter, Positively Regulates Responses to Drought Stress in Rice

    Directory of Open Access Journals (Sweden)

    Guang Chen

    2017-11-01

    Full Text Available Drought is one of the environmental factors that severely restrict plant distribution and crop production. Recently, we reported that the high-affinity potassium transporter OsHAK1 plays important roles in K acquisition and translocation in rice over low and high K concentration ranges, however, knowledge on the regulatory roles of OsHAK1 in osmotic/drought stress is limited. Here, transcript levels of OsHAK1 were found transiently elevated by water deficit in roots and shoots, consistent with the enhanced GUS activity in transgenic plants under stress. Under drought conditions, OsHAK1 knockout mutants (KO presented lower tolerance to the stress and displayed stunted growth at both the vegetative and reproductive stages. Phenotypic analysis of OsHAK1 overexpression seedlings (Ox demonstrated that they present better tolerance to drought stress than wild-type (WT. Compared to WT seedlings, OsHAK1 overexpressors had lower level of lipid peroxidation, higher activities of antioxidant enzymes (POX and CAT and higher proline accumulation. Furthermore, qPCR analysis revealed that OsHAK1 act as a positive regulator of the expression of stress-responsive genes as well as of two well-known rice channel genes (OsTPKb and OsAKT1 involved in K homeostasis and stress responses in transgenic plants under dehydration. Most important, OsHAK1-Ox plants displayed enhanced drought tolerance at the reproductive stage, resulting in 35% more grain yield than WT under drought conditions, and without exhibiting significant differences under normal growth conditions. Consequently, OsHAK1 can be considered to be used in molecular breeding for improvement of drought tolerance in rice.

  14. Dephosphorylation of phytate by using the Aspergillus niger phytase with a high affinity for phytate.

    Science.gov (United States)

    Nagashima, T; Tange, T; Anazawa, H

    1999-10-01

    A phytase (EC 3.1.3.8) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 +/- 4.6 microM) was statistically analyzed. In regard to the orthophosphate released from phytic acid, a significant difference between a low K(m) phytase from A. niger SK-57 and a high K(m) phytase from Aspergillus ficuum was recognized.

  15. How phenotypic plasticity made its way into molecular biology

    Indian Academy of Sciences (India)

    Prakash

    experiments have shown in the last decade that organisms are not only resistant to variations in the environment, but also to internal variations such as those resulting from gene mutations (Morange 2001). On the other hand, phenotypic plasticity can generate a huge change in the properties of an organism in response to ...

  16. How phenotypic plasticity made its way into molecular biology

    Indian Academy of Sciences (India)

    2009-08-03

    Aug 3, 2009 ... Its resurrection has been triggered by a small group of theoreticians, the rise of epigenetic descriptions and the publicized discovery of stem cell plasticity. The notion of phenotypic plasticity remains vague. History shows that too strong a belief in plasticity can be an obstacle to the development of biology.

  17. Acyclic cucurbit[n]uril congeners are high affinity hosts.

    Science.gov (United States)

    Ma, Da; Zavalij, Peter Y; Isaacs, Lyle

    2010-07-16

    We present the design, synthesis via methylene bridged glycoluril tetramer building blocks, and charaterization of acyclic cucurbit[n]uril congeners that function as hosts for a wide variety of ammonium ions in water. The X-ray crystallographic characterization of the free host and its complexes with p-xylylenediamine and spermine establish the flexibility of the methylene bridged backbone of the acyclic cucurbit[n]uril congeners that allow them to adapt to the structural features of the guest. We find that the acyclic cucurbit[n]uril congeners-with their four contiguous methylene bridged glycoluril units and two aromatic o-xylylene walls bearing CO(2)H substituents-bind to ammonium ions in buffered water with values of K(a) ranging from approximately 10(5) M(-1) to greater than 10(9) M(-1). Similar to the cucurbit[n]uril family of hosts, we find that increasing the concentration of metal cations in the buffer reduces the affinity of the acyclic cucurbit[n]uril congener toward guests by competitive binding at the ureidyl C horizontal lineO portals. Although the acyclic cucurbit[n]uril congeners retain the ability to bind to ammonium ions with high affinity, they do so with lower selectivity than cucurbit[n]urils presumably do to the structural flexibility of the hosts. A methylene bridged glycoluril tetramer model compound that lacks the substituted o-xylylene walls is a much lower affinity host, which establishes the importance of these rings on the overall recognition behavior of the acyclic cucurbit[n]uril congeners. Overall, the results in this paper establish that acyclic cucurbit[n]uril receptors that contain four or more contiguous methylene bridged glycoluril units retain many of the excellent recognition properties of the cucurbit[n]uril family.

  18. An overview of potential molecular mechanisms involved in VSMC phenotypic modulation.

    Science.gov (United States)

    Zhang, Ming-Jie; Zhou, Yi; Chen, Lei; Wang, Yan-Qin; Wang, Xu; Pi, Yan; Gao, Chang-Yue; Li, Jing-Cheng; Zhang, Li-Li

    2016-02-01

    The fully differentiated medial vascular smooth muscle cells (VSMCs) of mature vessels keep quiescent and contractile. However, VSMC can exhibit the plasticity in phenotype switching from a differentiated and contractile phenotype to a dedifferentiated state in response to alterations in local environmental cues, which is called phenotypic modulation or switching. Distinguishing from its differentiated state expressing more smooth muscle (SM)-specific/selective proteins, the phenotypic modulation in VSMC is characterized by an increased rate of proliferation, migration, synthesis of extracellular matrix proteins and decreased expression of SM contractile proteins. Although it has been well demonstrated that phenotypic modulation of VSMC contributes to the occurrence and progression of many proliferative vascular diseases, little is known about the details of the molecular mechanisms of VSMC phenotypic modulation. Growing evidence suggests that variety of molecules including microRNAs, cytokines and biochemical factors, membrane receptors, ion channels, cytoskeleton and extracellular matrix play important roles in controlling VSMC phenotype. The focus of the present review is to provide an overview of potential molecular mechanisms involved in VSMC phenotypic modulation in recent years. To clarify VSMC differentiation and phenotypic modulation mechanisms will contribute to producing cell-based therapeutic interventions for aberrant VSMC differentiation-related diseases.

  19. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    Directory of Open Access Journals (Sweden)

    Graham S Baldwin

    Full Text Available The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  20. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    Science.gov (United States)

    Baldwin, Graham S; George, Graham N; Pushie, M Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  1. The neurobehavioral and molecular phenotype of Angelman Syndrome.

    Science.gov (United States)

    Wink, Logan K; Fitzpatrick, Sarah; Shaffer, Rebecca; Melnyk, Sophia; Begtrup, Amber H; Fox, Emma; Schaefer, Tori L; Mathieu-Frasier, Lauren; Ray, Balmiki; Lahiri, Debomoy; Horn, Paul A; Erickson, Craig A

    2015-11-01

    Angelman Syndrome (AS) is a rare neurodevelopmental disorder associated with developmental delay, speech impairment, gait ataxia, and a unique behavioral profile. AS is caused by loss of maternal expression of the paternally imprinted UBE3A gene. In this study we aim to contribute to understanding of the neurobehavioral phenotype of AS with particular focus on the neuropsychiatric presentation of the disorder. We also undertake initial exploration of brain-derived neurotrophic factor (BDNF) plasma levels in AS. Twelve individuals ages 3 years or older with a confirmed genetic diagnosis of AS underwent detailed medical history, phenotypic characterization, and BDNF plasma sampling. The results of this study demonstrate that individuals with AS suffer from significant developmental delay, impaired adaptive behavior, and sleep disruption. Additionally, hyperactivity/impulsivity appears to be the primary behavioral domain noted in these individuals. The majority of individuals in this project met criteria for autism spectrum disorder on the Autism Diagnostic Observation Schedule (ADOS); however, a negative correlation was noted between ADOS score and developmental age. BDNF plasma levels in AS individuals were significantly elevated compared to neurotypical controls. This is the first report of abnormal BDNF levels in AS, and one that necessitates larger future studies. The results provide a clue to understanding abnormal neuronal development in AS and may help guide future AS research. © 2015 Wiley Periodicals, Inc.

  2. Advancing our understanding of infant bronchiolitis through phenotyping and endotyping: clinical and molecular approaches.

    Science.gov (United States)

    Hasegawa, Kohei; Dumas, Orianne; Hartert, Tina V; Camargo, Carlos A

    2016-08-01

    Bronchiolitis is a major public health problem worldwide. However, no effective treatment strategies are available, other than supportive care. Although bronchiolitis has been considered a single disease diagnosed based on clinical characteristics, emerging evidence supports both clinical and pathobiological heterogeneity. The characterization of this heterogeneity supports the concept that bronchiolitis consists of multiple phenotypes or consistent grouping of characteristics. Expert commentary: Using unbiased statistical approaches, multidimentional clinical characteristics will derive bronchiolitis phenotypes. Furthermore, molecular and systems biology approaches will, by linking pathobiology to phenotype, identify endotypes. Large cohort studies of bronchiolitis with comprehensive clinical characterization and system-wide profiling of the '-omics' data (e.g., host genome, transcriptome, epigenome, viral genome, microbiome, metabolome) should enhance our ability to molecularly understand these phenotypes and lead to more targeted and personalized approaches to bronchiolitis treatment.

  3. Drug Repositioning by Kernel-Based Integration of Molecular Structure, Molecular Activity, and Phenotype Data

    Science.gov (United States)

    Wang, Yongcui; Chen, Shilong; Deng, Naiyang; Wang, Yong

    2013-01-01

    Computational inference of novel therapeutic values for existing drugs, i.e., drug repositioning, offers the great prospect for faster and low-risk drug development. Previous researches have indicated that chemical structures, target proteins, and side-effects could provide rich information in drug similarity assessment and further disease similarity. However, each single data source is important in its own way and data integration holds the great promise to reposition drug more accurately. Here, we propose a new method for drug repositioning, PreDR (Predict Drug Repositioning), to integrate molecular structure, molecular activity, and phenotype data. Specifically, we characterize drug by profiling in chemical structure, target protein, and side-effects space, and define a kernel function to correlate drugs with diseases. Then we train a support vector machine (SVM) to computationally predict novel drug-disease interactions. PreDR is validated on a well-established drug-disease network with 1,933 interactions among 593 drugs and 313 diseases. By cross-validation, we find that chemical structure, drug target, and side-effects information are all predictive for drug-disease relationships. More experimentally observed drug-disease interactions can be revealed by integrating these three data sources. Comparison with existing methods demonstrates that PreDR is competitive both in accuracy and coverage. Follow-up database search and pathway analysis indicate that our new predictions are worthy of further experimental validation. Particularly several novel predictions are supported by clinical trials databases and this shows the significant prospects of PreDR in future drug treatment. In conclusion, our new method, PreDR, can serve as a useful tool in drug discovery to efficiently identify novel drug-disease interactions. In addition, our heterogeneous data integration framework can be applied to other problems. PMID:24244318

  4. Phenotypic and molecular characterisation of the causal agent of ...

    African Journals Online (AJOL)

    A study was carried out to confirm the identity of the causal agent of anthracnose, a major fungal disease of mango in Ghana. Forty-five isolates of the pathogen were obtained from diseased mango plant parts, and were identified using morphological, physiological and molecular methods. The results showed that the ...

  5. Computational genes: a tool for molecular diagnosis and therapy of aberrant mutational phenotype

    Directory of Open Access Journals (Sweden)

    Ignatova Zoya

    2007-09-01

    Full Text Available Abstract Background A finite state machine manipulating information-carrying DNA strands can be used to perform autonomous molecular-scale computations at the cellular level. Results We propose a new finite state machine able to detect and correct aberrant molecular phenotype given by mutated genetic transcripts. The aberrant mutations trigger a cascade reaction: specific molecular markers as input are released and induce a spontaneous self-assembly of a wild type protein or peptide, while the mutational disease phenotype is silenced. We experimentally demostrated in in vitro translation system that a viable protein can be autonomously assembled. Conclusion Our work demostrates the basic principles of computational genes and particularly, their potential to detect mutations, and as a response thereafter administer an output that suppresses the aberrant disease phenotype and/or restores the lost physiological function.

  6. Distinct molecular phenotypes in male and female schizophrenia patients.

    Directory of Open Access Journals (Sweden)

    Jordan M Ramsey

    Full Text Available BACKGROUND: In schizophrenia, sex specific dimorphisms related to age of onset, course of illness and response to antipsychotic treatment may be mirrored by sex-related differences in the underlying molecular pathways. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have carried out multiplex immunoassay profiling of sera from 4 independent cohorts of first episode antipsychotic naive schizophrenia patients (n = 133 and controls (n = 133 to identify such sex-specific illness processes in the periphery. The concentrations of 16 molecules associated with hormonal, inflammation and growth factor pathways showed significant sex differences in schizophrenia patients compared with controls. In female patients, the inflammation-related analytes alpha-1-antitrypsin, B lymphocyte chemoattractant BLC and interleukin-15 showed negative associations with positive and negative syndrome scale (PANSS scores. In male patients, the hormones prolactin and testosterone were negatively associated with PANSS ratings. In addition, we investigated molecular changes in a subset of 33 patients before and after 6 weeks of treatment with antipsychotics and found that treatment induced sex-specific changes in the levels of testosterone, serum glutamic oxaloacetic transaminase, follicle stimulating hormone, interleukin-13 and macrophage-derived chemokine. Finally, we evaluated overlapping and distinct biomarkers in the sex-specific molecular signatures in schizophrenia, major depressive disorder and bipolar disorder. CONCLUSIONS/SIGNIFICANCE: We propose that future studies should investigate the common and sex-specific aetiologies of schizophrenia, as the current findings suggest that different therapeutic strategies may be required for male and female patients.

  7. Aberrant Retinoblastoma (RB)-E2F Transcriptional Regulation Defines Molecular Phenotypes of Osteosarcoma.

    Science.gov (United States)

    Scott, Milcah C; Sarver, Aaron L; Tomiyasu, Hirotaka; Cornax, Ingrid; Van Etten, Jamie; Varshney, Jyotika; O'Sullivan, M Gerard; Subramanian, Subbaya; Modiano, Jaime F

    2015-11-20

    We previously identified two distinct molecular subtypes of osteosarcoma through gene expression profiling. These subtypes are associated with distinct tumor behavior and clinical outcomes. Here, we describe mechanisms that give rise to these molecular subtypes. Using bioinformatic analyses, we identified a significant association between deregulation of the retinoblastoma (RB)-E2F pathway and the molecular subtype with worse clinical outcomes. Xenotransplantation models recapitulated the corresponding behavior for each osteosarcoma subtype; thus, we used cell lines to validate the role of the RB-E2F pathway in regulating the prognostic gene signature. Ectopic RB resets the patterns of E2F regulated gene expression in cells derived from tumors with worse clinical outcomes (molecular phenotype 2) to those comparable with those observed in cells derived from tumors with less aggressive outcomes (molecular phenotype 1), providing a functional association between RB-E2F dysfunction and altered gene expression in osteosarcoma. DNA methyltransferase and histone deacetylase inhibitors similarly reset the transcriptional state of the molecular phenotype 2 cells from a state associated with RB deficiency to one seen with RB sufficiency. Our data indicate that deregulation of RB-E2F pathway alters the epigenetic landscape and biological behavior of osteosarcoma. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Effects of lead on the kidney: Roles of high-affinity lead-binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, B.A. (Univ. of Maryland, Baltimore (United States)); DuVal, G. (Univ of Maryland Medical School, Baltimore (United States))

    1991-02-01

    Lead-induced nephropathy produces both tubular and interstitial manifestations of cell injury, but the pathophysiology of these lesions is not completely understood. Delineation of the molecular factors underlying renal handling of lead is one of central importance in understanding the mechanisms of renal cell injury from this agent. Recent studies from this laboratory have identified several distinct high-affinity lead-binding proteins (PbBP) from rat kidney and brain that appear to play critical roles in the intracellular bioavailability of lead to several essential cellular processes in these target tissues at low dose levels. These studies have also shown that the real PbBP is selectively localized in only certain nephrons and only specific segments of the renal proximal tubule. The striking nephron and cell-type specificity of the localization reaction could result from physoiological differences in nephron functional activity or selective molecular uptake mechanisms/metabolism differences that act to define target cell populations in the kidney. In addition, other preliminary studies have shown that short-term, high-dose lead exposure produces increased excretion of this protein into the urine with concomitant decreases in renal concentrations.

  9. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    Energy Technology Data Exchange (ETDEWEB)

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  10. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    OpenAIRE

    Naiwen Cui; Huidan Zhang; Nils Schneider; Ye Tao; Haruichi Asahara; Zhiyi Sun; Yamei Cai; Koehler, Stephan A.; de Greef, Tom F. A.; Alireza Abbaspourrad; Weitz, David A; Shaorong Chong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-...

  11. Quantitative Molecular Phenotyping of Gill Remodeling in a Cichlid Fish Responding to Salinity Stress*

    Science.gov (United States)

    Kültz, Dietmar; Li, Johnathon; Gardell, Alison; Sacchi, Romina

    2013-01-01

    A two-tiered label-free quantitative (LFQ) proteomics workflow was used to elucidate how salinity affects the molecular phenotype, i.e. proteome, of gills from a cichlid fish, the euryhaline tilapia (Oreochromis mossambicus). The workflow consists of initial global profiling of relative tryptic peptide abundances in treated versus control samples followed by targeted identification (by MS/MS) and quantitation (by chromatographic peak area integration) of validated peptides for each protein of interest. Fresh water acclimated tilapia were independently exposed in separate experiments to acute short-term (34 ppt) and gradual long-term (70 ppt, 90 ppt) salinity stress followed by molecular phenotyping of the gill proteome. The severity of salinity stress can be deduced with high technical reproducibility from the initial global label-free quantitative profiling step alone at both peptide and protein levels. However, an accurate regulation ratio can only be determined by targeted label-free quantitative profiling because not all peptides used for protein identification are also valid for quantitation. Of the three salinity challenges, gradual acclimation to 90 ppt has the most pronounced effect on gill molecular phenotype. Known salinity effects on tilapia gills, including an increase in the size and number of mitochondria-rich ionocytes, activities of specific ion transporters, and induction of specific molecular chaperones are reflected in the regulation of abundances of the corresponding proteins. Moreover, specific protein isoforms that are responsive to environmental salinity change are resolved and it is revealed that salinity effects on the mitochondrial proteome are nonuniform. Furthermore, protein NDRG1 has been identified as a novel key component of molecular phenotype restructuring during salinity-induced gill remodeling. In conclusion, besides confirming known effects of salinity on gills of euryhaline fish, molecular phenotyping reveals novel insight into

  12. Quantitative molecular phenotyping of gill remodeling in a cichlid fish responding to salinity stress.

    Science.gov (United States)

    Kültz, Dietmar; Li, Johnathon; Gardell, Alison; Sacchi, Romina

    2013-12-01

    A two-tiered label-free quantitative (LFQ) proteomics workflow was used to elucidate how salinity affects the molecular phenotype, i.e. proteome, of gills from a cichlid fish, the euryhaline tilapia (Oreochromis mossambicus). The workflow consists of initial global profiling of relative tryptic peptide abundances in treated versus control samples followed by targeted identification (by MS/MS) and quantitation (by chromatographic peak area integration) of validated peptides for each protein of interest. Fresh water acclimated tilapia were independently exposed in separate experiments to acute short-term (34 ppt) and gradual long-term (70 ppt, 90 ppt) salinity stress followed by molecular phenotyping of the gill proteome. The severity of salinity stress can be deduced with high technical reproducibility from the initial global label-free quantitative profiling step alone at both peptide and protein levels. However, an accurate regulation ratio can only be determined by targeted label-free quantitative profiling because not all peptides used for protein identification are also valid for quantitation. Of the three salinity challenges, gradual acclimation to 90 ppt has the most pronounced effect on gill molecular phenotype. Known salinity effects on tilapia gills, including an increase in the size and number of mitochondria-rich ionocytes, activities of specific ion transporters, and induction of specific molecular chaperones are reflected in the regulation of abundances of the corresponding proteins. Moreover, specific protein isoforms that are responsive to environmental salinity change are resolved and it is revealed that salinity effects on the mitochondrial proteome are nonuniform. Furthermore, protein NDRG1 has been identified as a novel key component of molecular phenotype restructuring during salinity-induced gill remodeling. In conclusion, besides confirming known effects of salinity on gills of euryhaline fish, molecular phenotyping reveals novel insight into

  13. Aberrant Retinoblastoma (RB)-E2F Transcriptional Regulation Defines Molecular Phenotypes of Osteosarcoma*

    Science.gov (United States)

    Scott, Milcah C.; Sarver, Aaron L.; Tomiyasu, Hirotaka; Cornax, Ingrid; Van Etten, Jamie; Varshney, Jyotika; O'Sullivan, M. Gerard; Subramanian, Subbaya; Modiano, Jaime F.

    2015-01-01

    We previously identified two distinct molecular subtypes of osteosarcoma through gene expression profiling. These subtypes are associated with distinct tumor behavior and clinical outcomes. Here, we describe mechanisms that give rise to these molecular subtypes. Using bioinformatic analyses, we identified a significant association between deregulation of the retinoblastoma (RB)-E2F pathway and the molecular subtype with worse clinical outcomes. Xenotransplantation models recapitulated the corresponding behavior for each osteosarcoma subtype; thus, we used cell lines to validate the role of the RB-E2F pathway in regulating the prognostic gene signature. Ectopic RB resets the patterns of E2F regulated gene expression in cells derived from tumors with worse clinical outcomes (molecular phenotype 2) to those comparable with those observed in cells derived from tumors with less aggressive outcomes (molecular phenotype 1), providing a functional association between RB-E2F dysfunction and altered gene expression in osteosarcoma. DNA methyltransferase and histone deacetylase inhibitors similarly reset the transcriptional state of the molecular phenotype 2 cells from a state associated with RB deficiency to one seen with RB sufficiency. Our data indicate that deregulation of RB-E2F pathway alters the epigenetic landscape and biological behavior of osteosarcoma. PMID:26378234

  14. Molecular and Phenotypic Characterization of Potentially New Shigella dysenteriae Serotype

    Science.gov (United States)

    Coimbra, Roney S.; Lenormand, Pascal; Grimont, Francine; Bouvet, Philippe; Matsushita, Shigeru; Grimont, Patrick A. D.

    2001-01-01

    From September 1997 to November 1998, the French National Center for Salmonella and Shigella received 22 Shigella isolates recovered from 22 different patients suffering from dysentery. None of these isolates reacted with any of the antisera used to identify established Shigella serotypes, but all of them agglutinated in the presence of antisera to a previously described potentially new Shigella dysenteriae serotype (represented by strain 96–204) primarily isolated from stool cultures of imported diarrheal cases in Japan. All French isolates, as well as strain 96–204, showed biochemical reactions typical of S. dysenteriae and gave positive results in a PCR assay for detection of the plasmid ipaH gene coding for invasiveness. No Shiga toxin gene was detected by PCR. These isolates were indistinguishable by molecular analysis of ribosomal DNA (ribotyping) and seemed to be related to S. dysenteriae serotypes 3 and 12. However, further characterization by restriction of the amplified O-antigen gene cluster clearly distinguished this new serotype from all other Shigella or Escherichia coli serotypes. PMID:11158117

  15. An Inquiry-Infused Introductory Biology Laboratory That Integrates Mendel's Pea Phenotypes with Molecular Mechanisms

    Science.gov (United States)

    Kudish, Philip; Schlag, Erin; Kaplinsky, Nicholas J.

    2015-01-01

    We developed a multi-week laboratory in which college-level introductory biology students investigate Mendel's stem length phenotype in peas. Students collect, analyze and interpret convergent evidence from molecular and physiological techniques. In weeks 1 and 2, students treat control and experimental plants with Gibberellic Acid (GA) to…

  16. Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody.

    Science.gov (United States)

    Bedzyk, W D; Weidner, K M; Denzin, L K; Johnson, L S; Hardman, K D; Pantoliano, M W; Asel, E D; Voss, E W

    1990-10-25

    Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.

  17. A high-affinity putrescine-cadaverine transporter from Trypanosoma cruzi

    Science.gov (United States)

    Hasne, Marie-Pierre; Coppens, Isabelle; Soysa, Radika; Ullman, Buddy

    2011-01-01

    Summary Whereas mammalian cells and most other organisms can synthesize polyamines from basic amino acids, the protozoan parasite Trypanosoma cruzi is incapable of polyamine biosynthesis de novo and therefore obligatorily relies upon putrescine acquisition from the host to meet its nutritional requirements. The cell surface proteins that mediate polyamine transport into T. cruzi, as well as most eukaryotes, however, have by-in-large eluded discovery at the molecular level. Here we report the identification and functional characterization of two polyamine transporters, TcPOT1.1 and TcPOT1.2, encoded by alleles from two T. cruzi haplotypes. Overexpression of the TcPOT1.1 and TcPOT1.2 genes in T. cruzi epimastigotes revealed that TcPOT1.1 and TcPOT1.2 were high-affinity transporters that recognized both putrescine and cadaverine but not spermidine or spermine. Furthermore, the activities and subcellular locations of both TcPOT1.1 and TcPOT1.2 in intact parasites were profoundly influenced by extracellular putrescine availability. These results establish TcPOT1.1 and TcPOT1.2 as key components of the T. cruzi polyamine transport pathway, an indispensable nutritional function for the parasite that may be amenable to therapeutic manipulation. PMID:20149109

  18. In silico design of high-affinity ligands for the immobilization of inulinase.

    Science.gov (United States)

    Holyavka, M G; Kondratyev, M S; Samchenko, A A; Kabanov, A V; Komarov, V M; Artyukhov, V G

    2016-04-01

    Using computer modeling, virtual screening of high-affinity ligands for immobilization of inulinase - an enzyme that cleaves inulin and fructose-containing polymers to fructose - has been performed. The inulinase molecule from Aspergillus ficuum (pdb: 3SC7) taken from the database of protein structures was used as a protein model and the target for flexible docking. The set of ligands studied included simple sugars (activators, inhibitors, products of enzymatic catalysis), as well as high-molecular weight compounds (polycation and polyanion exchange resins, glycoproteins, phenylalanine-proline peptide, polylactate, and caffeine). Based on the comparative analysis of the values of the total energy and the localization of ligand binding sites, we made several assumptions concerning the mechanisms of interaction of the suggested matrices for the immobilization of enzyme molecules and the structural features of such complexes. It was also assumed that the candidates for immobilization agents meeting the industrial requirements may be glycoproteins, for which we propose an additional incorporation of cysteine residues into their structure, aimed to create disulfide «anchors» to the surface. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Synthesis of site-heterologous haptens for high-affinity anti-pyraclostrobin antibody generation.

    Science.gov (United States)

    Mercader, Josep V; Agulló, Consuelo; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

    2011-03-07

    The design and synthesis of functional chemical derivatives of small organic molecules is usually a key step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for the generation of high-affinity and selective antibodies. However, due to the usual complexity of the required synthetic procedures, the appropriate comparison of alternative tethering positions has often been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives of pyraclostrobin with the same linkers located at diverse rationally-chosen sites. Activity appraisal of antibodies and bioconjugates was carried out by bidimensional competitive direct and indirect immunoassays, and a superior performance of two of the three synthesized haptens was found. Finally, a detailed analysis of the conformations of the target molecule and the synthesized haptens in aqueous solution was done using computer assisted molecular modeling techniques. This study suggested that the lower titers and affinities of one set of antibodies are most probably due to conformational effects of the spacer arm in the immunizing bioconjugate.

  20. Molecular Ecological Basis of Grasshopper (Oedaleus asiaticus Phenotypic Plasticity under Environmental Selection

    Directory of Open Access Journals (Sweden)

    Xinghu Qin

    2017-10-01

    Full Text Available While ecological adaptation in insects can be reflected by plasticity of phenotype, determining the causes and molecular mechanisms for phenotypic plasticity (PP remains a crucial and still difficult question in ecology, especially where control of insect pests is involved. Oedaleus asiaticus is one of the most dominant pests in the Inner Mongolia steppe and represents an excellent system to study phenotypic plasticity. To better understand ecological factors affecting grasshopper phenotypic plasticity and its molecular control, we conducted a full transcriptional screening of O. asiaticus grasshoppers reared in four different grassland patches in Inner Mongolia. Grasshoppers showed different degrees of PP associated with unique gene expressions and different habitat plant community compositions. Grasshopper performance variables were susceptible to habitat environment conditions and closely associated with plant architectures. Intriguingly, eco-transcriptome analysis revealed five potential candidate genes playing important roles in grasshopper performance, with gene expression closely relating to PP and plant community factors. By linking the grasshopper performances to gene profiles and ecological factors using canonical regression, we first demonstrated the eco-transcriptomic architecture (ETA of grasshopper phenotypic traits (ETAGPTs. ETAGPTs revealed plant food type, plant density, coverage, and height were the main ecological factors influencing PP, while insect cuticle protein (ICP, negative elongation factor A (NELFA, and lactase-phlorizin hydrolase (LCT were the key genes associated with PP. Our study gives a clear picture of gene-environment interaction in the formation and maintenance of PP and enriches our understanding of the transcriptional events underlying molecular control of rapid phenotypic plasticity associated with environmental variability. The findings of this study may also provide new targets for pest control and

  1. Molecular Ecological Basis of Grasshopper (Oedaleus asiaticus) Phenotypic Plasticity under Environmental Selection.

    Science.gov (United States)

    Qin, Xinghu; Hao, Kun; Ma, Jingchuan; Huang, Xunbing; Tu, Xiongbing; Ali, Md Panna; Pittendrigh, Barry R; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Whitman, Douglas W; Zhang, Zehua

    2017-01-01

    While ecological adaptation in insects can be reflected by plasticity of phenotype, determining the causes and molecular mechanisms for phenotypic plasticity (PP) remains a crucial and still difficult question in ecology, especially where control of insect pests is involved. Oedaleus asiaticus is one of the most dominant pests in the Inner Mongolia steppe and represents an excellent system to study phenotypic plasticity. To better understand ecological factors affecting grasshopper phenotypic plasticity and its molecular control, we conducted a full transcriptional screening of O. asiaticus grasshoppers reared in four different grassland patches in Inner Mongolia. Grasshoppers showed different degrees of PP associated with unique gene expressions and different habitat plant community compositions. Grasshopper performance variables were susceptible to habitat environment conditions and closely associated with plant architectures. Intriguingly, eco-transcriptome analysis revealed five potential candidate genes playing important roles in grasshopper performance, with gene expression closely relating to PP and plant community factors. By linking the grasshopper performances to gene profiles and ecological factors using canonical regression, we first demonstrated the eco-transcriptomic architecture (ETA) of grasshopper phenotypic traits (ETAGPTs). ETAGPTs revealed plant food type, plant density, coverage, and height were the main ecological factors influencing PP, while insect cuticle protein (ICP), negative elongation factor A (NELFA), and lactase-phlorizin hydrolase (LCT) were the key genes associated with PP. Our study gives a clear picture of gene-environment interaction in the formation and maintenance of PP and enriches our understanding of the transcriptional events underlying molecular control of rapid phenotypic plasticity associated with environmental variability. The findings of this study may also provide new targets for pest control and highlight the

  2. Identification of differentiation-stage specific molecular markers for the osteoblastic phenotype

    DEFF Research Database (Denmark)

    Twine, Natalie; Chen, Li; Wilkins, Marc

    The phenotype of osteoblastic (OB) cells in culture is currently defined using a limited number of markers of low sensitivity and specificity which belong mostly to extracellular matrix proteins. Also, for clinical use of human skeletal (mesenchymal) stem cells (hMSC) in bone regeneration...... to age-matched control (n=4). Using RNA-seq and cluster analysis, we identified a set of stage-specific molecular markers that define the progression of OB phenotype during ex vivo culture of hMSC, predict in vivo bone formation capacity of hMSC and can be employed to study the mechanisms of impaired...

  3. Substance P downregulates expression of the high affinity IgE receptor (FcepsilonRI) by human mast cells.

    Science.gov (United States)

    McCary, Christine; Tancowny, Brian P; Catalli, Adriana; Grammer, Leslie C; Harris, Kathleen E; Schleimer, Robert P; Kulka, Marianna

    2010-03-30

    The effect of the neuropeptide substance P (SP) on human mast cell (MC) phenotype is poorly understood. In this study, SP effects on human MC expression of the high affinity IgE receptor (FcepsilonRI) were characterized. SP downregulated expression of FcepsilonRI mRNA and protein by approximately 50% and in a concentration dependent manner, the effect was partially mediated by engagement of the neurokinin-1 receptor (NK1R) and resulted in reduced mast cell activation. Sensitization of MC with IgE prior to SP exposure protected MC from SP-mediated FcepsilonRI downregulation. SP release may inhibit MC responses to allergens and these results may have implications in neuroinflammatiion and stress. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

  4. Substance P downregulates expression of the high affinity IgE receptor (FcεRI) by human mast cells

    Science.gov (United States)

    McCary, Christine; Tancowny, Brian P.; Catalli, Adriana; Grammer, Leslie C.; Harris, Kathleen E.; Schleimer, Robert P.; Kulka, Marianna

    2013-01-01

    The effect of the neuropeptide substance P (SP) on human mast cell (MC) phenotype is poorly understood. In this study, SP effects on human MC expression of the high affinity IgE receptor (FcεRI) were characterized. SP downregulated expression of FcεRI mRNA and protein by approximately 50% and in a concentration dependent manner, the effect was partially mediated by engagement of the neurokinin-1 receptor (NK1R) and resulted in reduced mast cell activation. Sensitization of MC with IgE prior to SP exposure protected MC from SP-mediated FcεRI downregulation. SP release may inhibit MC responses to allergens and these results may have implications in neuroinflammatiion and stress. PMID:20117843

  5. Prevalence and molecular characterization of clinical isolates of Escherichia coli expressing an AmpC phenotype

    DEFF Research Database (Denmark)

    Jørgensen, Rikke Lind; Nielsen, Jesper Boye; Friis-Møller, Alice

    2010-01-01

    OBJECTIVES: To establish the prevalence of the AmpC beta-lactamase phenotype in clinical isolates of Escherichia coli and characterize the genetic resistance mechanisms causing the observed phenotype. METHODS: Clinical E. coli (n = 74) with reduced susceptibility to third-generation cephalosporins...... and resistance to cefoxitin were collected from the Department of Clinical Microbiology at Hvidovre Hospital, Denmark, in 2006. The AmpC disc test was used to confirm expression of AmpC, and test-positive strains were selected for further antimicrobial susceptibility testing and molecular characterization....... Sequencing of ampC showed that most isolates were not clonally related. CONCLUSIONS: E. coli expressing an AmpC phenotype occur sporadically and cause significant resistance to cephalosporins. The majority of these are hyperproducing chromosomal ampC although some isolates have acquired pAmpC....

  6. Gene expression profiling of asthma phenotypes demonstrates molecular signatures of atopy and asthma control.

    Science.gov (United States)

    Howrylak, Judie A; Moll, Matthew; Weiss, Scott T; Raby, Benjamin A; Wu, Wei; Xing, Eric P

    2016-05-01

    Recent studies have used cluster analysis to identify phenotypic clusters of asthma with differences in clinical traits, as well as differences in response to therapy with anti-inflammatory medications. However, the correspondence between different phenotypic clusters and differences in the underlying molecular mechanisms of asthma pathogenesis remains unclear. We sought to determine whether clinical differences among children with asthma in different phenotypic clusters corresponded to differences in levels of gene expression. We explored differences in gene expression profiles of CD4(+) lymphocytes isolated from the peripheral blood of 299 young adult participants in the Childhood Asthma Management Program study. We obtained gene expression profiles from study subjects between 9 and 14 years of age after they participated in a randomized, controlled longitudinal study examining the effects of inhaled anti-inflammatory medications over a 48-month study period, and we evaluated the correspondence between our earlier phenotypic cluster analysis and subsequent follow-up clinical and molecular profiles. We found that differences in clinical characteristics observed between subjects assigned to different phenotypic clusters persisted into young adulthood and that these clinical differences were associated with differences in gene expression patterns between subjects in different clusters. We identified a subset of genes associated with atopic status, validated the presence of an atopic signature among these genes in an independent cohort of asthmatic subjects, and identified the presence of common transcription factor binding sites corresponding to glucocorticoid receptor binding. These findings suggest that phenotypic clusters are associated with differences in the underlying pathobiology of asthma. Further experiments are necessary to confirm these findings. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights

  7. A Novel Drug-Mouse Phenotypic Similarity Method Detects Molecular Determinants of Drug Effects.

    Directory of Open Access Journals (Sweden)

    Jeanette Prinz

    2016-09-01

    Full Text Available The molecular mechanisms that translate drug treatment into beneficial and unwanted effects are largely unknown. We present here a novel approach to detect gene-drug and gene-side effect associations based on the phenotypic similarity of drugs and single gene perturbations in mice that account for the polypharmacological property of drugs. We scored the phenotypic similarity of human side effect profiles of 1,667 small molecules and biologicals to profiles of phenotypic traits of 5,384 mouse genes. The benchmarking with known relationships revealed a strong enrichment of physical and indirect drug-target connections, causative drug target-side effect links as well as gene-drug links involved in pharmacogenetic associations among phenotypically similar gene-drug pairs. The validation by in vitro assays and the experimental verification of an unknown connection between oxandrolone and prokineticin receptor 2 reinforces the ability of this method to provide new molecular insights underlying drug treatment. Thus, this approach may aid in the proposal of novel and personalized treatments.

  8. Novel cyclic gamma-hydroxybutyrate (GHB) analogs with high affinity and stereoselectivity of binding to GHB sites in rat brain

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Greenwood, Jeremy R

    2005-01-01

    Gamma-hydroxybutyrate (GHB) is a psychotropic compound endogenous to the brain. Despite its potentially great physiological significance, its exact molecular mechanism of action is unknown. GHB is a weak agonist at GABA(B) receptors, but there is also evidence of specific GHB receptor sites......, the molecular cloning of which remains a challenge. Ligands with high affinity and specificity for the reported GHB binding site are needed for pharmacological dissection of the GHB and GABA(B) effects and for mapping the structural requirements of the GHB receptor-ligand interactions. For this purpose, we have...... analog, HOCPrCA, proved to have 10-fold higher affinity than its enantiomer. Likewise, the R-enantiomers of HOCHCA and HOCPCA selectively inhibited [3H]NCS-382 binding. The best inhibitor of these, (R)-HOCPCA, has an affinity 39 times higher than GHB and is thus among the best GHB ligands reported...

  9. Phenotypic and molecular identification of Fonsecaea pedrosoi strains isolated from chromoblastomycosis patients in Mexico and Venezuela.

    Science.gov (United States)

    Carolina Rojas, O; León-Cachón, Rafael B R; Pérez-Maya, Antonio Alí; Aguirre-Garza, Marcelino; Moreno-Treviño, María G; González, Gloria M

    2015-05-01

    Chromoblastomycosis is a chronic granulomatous disease caused frequently by fungi of the Fonsecaea genus. The objective of this study was the phenotypic and molecular identification of F. pedrosoi strains isolated from chromoblastomycosis patients in Mexico and Venezuela. Ten strains were included in this study. For phenotypic identification, we used macroscopic and microscopic morphologies, carbohydrate assimilation test, urea hydrolysis, cixcloheximide tolerance, proteolitic activity and the thermotolerance test. The antifungal activity of five drugs was evaluated against the isolates. Molecular identification was performed by sequencing the internal transcribed spacer (ITS) ribosomal DNA regions of the isolated strains. The physiological analysis and morphological features were variable and the precise identification was not possible. All isolates were susceptible to itraconazole, terbinafine, voriconazole and posaconazole. Amphotericin B was the least effective drug. The alignment of the 559-nucleotide ITS sequences from our strains compared with sequences of GenBank revealed high homology with F. pedrosoi (EU285266.1). In this study, all patients were from rural areas, six from Mexico and four from Venezuela. Ten isolates were identified by phenotypic and molecular analysis, using ITS sequence and demonstrated that nine isolates from Mexico and Venezuela were 100% homologous and one isolate showed a small genetic distance. © 2015 Blackwell Verlag GmbH.

  10. Molecular and phenotypic characterisation of Phaeomoniella chlamydospora isolates from the demarcated wine region of Dão (Portugal

    Directory of Open Access Journals (Sweden)

    Jorge SOFIA

    2015-09-01

    Full Text Available Sixty-eight isolates of Phaeomoniella chlamydospora obtained from symptomatic esca and Petri disease grapevines, obtained mostly within the Portuguese Dão appellation, were investigated regarding their phenotypic and molecular diversity, in order to determine intraspecific variability and population structure. Phenotypic features such as texture, colour, growing margin zonation, hyphal morphology and diameter growth were evaluated. Molecular characterization was performed through the sequencing of the total ITS region and molecular analyses were used to infer phylogenetic relationships, using the Maximum Likelihood approach. Isolates were divided into two groups, by both phenotypic and molecular analysis, but no clear correspondence was found between the two approaches. Nevertheless, both phenotypic and molecular analysis revealed a strong homogeneity among all isolates, despite their geographical origin, year of isolation and scion/rootstock combination, therefore supporting the clonal reproduction strategy described for this species.

  11. Genetic variability in clones of 'Prata Anã' bananas based on phenotypic and molecular markers

    Directory of Open Access Journals (Sweden)

    Francisco Ermelindo Rodrigues

    2012-01-01

    Full Text Available The objective of this study was to examine the molecular and phenotypic variability of 'Prata Anã' banana clones cultivated in northern Minas Gerais State. For the phenotypic and molecular characterization, the clones were collected from five properties. The morphological characterization of 20 clones was accomplished through a qualitative evaluation of 74 descriptors. For the molecular evaluations, 45 clones were collected, and 14 Random Amplified Polymorphic DNA primers were used. Among the 74 morphological descriptors evaluated in the 20 'Prata Anã' banana tree clones, 53 descriptors presented in the same homogeneous class for all clones. The presence of anthocyanin in the pseudostem (ANT and the shape (BUS and flexion (FLX of the bunch clearly differentiated the 'Prata Anã' banana clones from the commercial cultivar. A total of 176 bands were produced from the molecular analysis using the 14 RAPD primers; there were 116 monomorphic and 60 polymorphic bands, with an average polymorphism percentage of 52.2%. The amplification using the OPP 14 primer resulted in the greatest percentage of polymorphic bands at 88%. The dendrogram indicated that each of the 45 'Prata Anã' clones that were evaluated using RAPD markers clustered with the commercial 'Prata Anã' cultivar.

  12. Relationships of Campanian olive cultivars: comparative analysis of molecular and phenotypic data.

    Science.gov (United States)

    Corrado, Giandomenico; La Mura, Maurizio; Ambrosino, Orsola; Pugliano, Giuseppe; Varricchio, Paola; Rao, Rosa

    2009-08-01

    Estimation of the genetic relatedness of traditional olive cultivars with genetic markers and phenotypic data enables progress in plant breeding, management of genetic resources, and protection of both breeders' rights and certified premium products. We used amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), and quantitative and qualitative morphological traits, including characteristics recommended for variety registration, to study genetic diversity and relationships in the olive at different levels. The 14 varieties analyzed, which are used for the production of Protected Denomination of Origin extra-virgin olive oil, represent the most important cultivars in the Campania region of Italy and typify a regional diversity characteristic of traditional olive cultivation. The genetic distances obtained with the two DNA marker systems were significantly correlated, as were those obtained by quantitative and qualitative traits. A lower but significant correlation was also observed between distances based on molecular markers and quantitative traits, but qualitative traits, even if sampled in high numbers, failed to describe the pattern of molecular similarity. Our data imply that the type and the number of phenotypic traits scored can greatly influence the outcome of the analysis, and care should be taken when qualitative and quantitative data are combined. Furthermore, the data indicate that the two molecular marker systems are useful for investigating genetic relationships, but they may also be used to complement and assist the traditional registration of varieties. We propose that since the information provided by molecular and morphological marker systems in olive is different, they should serve different purposes.

  13. GHB receptor targets in the CNS: Focus on high-affinity binding sites

    DEFF Research Database (Denmark)

    Bay, Tina; Eghorn, Laura Friis; Klein, Anders Bue

    2014-01-01

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects...... effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles...

  14. Evidence for a precursor of the high-affinity metastasis-associated murine laminin receptor

    DEFF Research Database (Denmark)

    Rao, C N; Castronovo, V; Schmitt, M C

    1989-01-01

    The high-affinity cellular receptor for the basement membrane component laminin is differentially expressed during tumor invasion and metastasis. A cDNA clone encoding the murine laminin receptor was isolated and identified on the basis of sequence homology to the human laminin receptor [Wewer et...

  15. High affinity calmodulin target sequence in the signalling molecule PI 3-kinase

    DEFF Research Database (Denmark)

    Fischer, R; Julsgart, J; Berchtold, M W

    1998-01-01

    M-binding peptide derived from the p110gamma isoform interacts with CaM in a calcium-dependent way. Using gel shift analysis and fluorescence spectrophotometry we discovered that the peptide forms a high affinity complex with CaM. Titration experiments using dansylated CaM gave an affinity constant of 5 n...

  16. N-Oxide analogs of WAY-100635 : new high affinity 5-HT (1A) receptor antagonists

    NARCIS (Netherlands)

    Oberwinkler - Marchais, Sandrine; Nowicki, B; Pike, VW; Halldin, C; Sandell, J; Chou, YH; Gulyas, B; Brennum, LT; Farde, L; Wikstrom, H V

    2005-01-01

    WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide] 1 and its O-des-methyl derivative DWAY 2 are well-known high affinity 5-HT1A receptor antagonists. which when labeled with carbon-II (beta(+): t(1/2) 20.4min) in the carbonyl group are effective

  17. [Peroxidative vulnerability of synaptosomal high affinity Ca++-ATPase and pharmacologic effects].

    Science.gov (United States)

    Blaschke, M; Fischer, H D; Schmidt, J

    1988-01-01

    The high affinity Ca++-ATPase participates essentially in the regulation of intrasynaptosomal calcium homeostasis. Related to posthypoxically restricted transmitter release, we examined the influence of newly-generated free radicals (ascorbic acid-ferric salt mixture) or sodium dodecyl sulfate in vitro and of a mild hypobaric hypoxia in vivo on the activity of synaptosomal high affinity Ca++-ATPase. Moreover we tested the effectiveness of piracetam, meclofenoxate hydrochloride, pyritinol and verapamil on the changed enzyme activity subsequent to a hypoxic exposure. The activity of synaptosomal high affinity Ca++-ATPase (1.04 +/- 0.03 mumol Pi/mg.h) is reduced by not more than 40% depending on the concentration of the ascorbic acid-ferric salt mixture used but is nearly totally inhibited by sodium dodecyl sulfate (0.2 mg/ml). Hypobaric hypoxia (18 h, 8.7 kPa) decreases the enzyme activity to 0.79 +/- 0.03 mumol Pi/mg.h. Piracetam, meclofenoxate hydrochloride and pyritinol are protectively effective on the decrease of enzyme activity induced by hypoxia. The results emphasize the importance of intact protein-phospholipid interactions for the enzyme activity and support relations between synaptosomal high affinity Ca++-ATPase and transmitter release.

  18. High-throughput and quantitative genome-wide messenger RNA sequencing for molecular phenotyping.

    Science.gov (United States)

    Collins, John E; Wali, Neha; Sealy, Ian M; Morris, James A; White, Richard J; Leonard, Steven R; Jackson, David K; Jones, Matthew C; Smerdon, Nathalie C; Zamora, Jorge; Dooley, Christopher M; Carruthers, Samantha N; Barrett, Jeffrey C; Stemple, Derek L; Busch-Nentwich, Elisabeth M

    2015-08-05

    We present a genome-wide messenger RNA (mRNA) sequencing technique that converts small amounts of RNA from many samples into molecular phenotypes. It encompasses all steps from sample preparation to sequence analysis and is applicable to baseline profiling or perturbation measurements. Multiplex sequencing of transcript 3' ends identifies differential transcript abundance independent of gene annotation. We show that increasing biological replicate number while maintaining the total amount of sequencing identifies more differentially abundant transcripts. This method can be implemented on polyadenylated RNA from any organism with an annotated reference genome and in any laboratory with access to Illumina sequencing.

  19. Proanthocyanidin oxidation of Arabidopsis seeds is altered in mutant of the high-affinity nitrate transporter NRT2.7

    Science.gov (United States)

    David, Laure C.; Dechorgnat, Julie; Ferrario-Méry, Sylvie

    2014-01-01

    NRT2.7 is a seed-specific high-affinity nitrate transporter controlling nitrate content in Arabidopsis mature seeds. The objective of this work was to analyse further the consequences of the nrt2.7 mutation for the seed metabolism. This work describes a new phenotype for the nrt2.7-2 mutant allele in the Wassilewskija accession, which exhibited a distinctive pale-brown seed coat that is usually associated with a defect in flavonoid oxidation. Indeed, this phenotype resembled those of tt10 mutant seeds defective in the laccase-like enzyme TT10/LAC15, which is involved in the oxidative polymerization of flavonoids such as the proantocyanidins (PAs) (i.e. epicatechin monomers and PA oligomers) and flavonol glycosides. nrt2.7-2 and tt10-2 mutant seeds displayed the same higher accumulation of PAs, but were partially distinct, since flavonol glycoside accumulation was not affected in the nrt2.7-2 seeds. Moreover, measurement of in situ laccase activity excluded a possibility of the nrt2.7-2 mutation affecting the TT10 enzymic activity at the early stage of seed development. Functional complementation of the nrt2.7-2 mutant by overexpression of a full-length NRT2.7 cDNA clearly demonstrated the link between the nrt2.7 mutation and the PA phenotype. However, the PA-related phenotype of nrt2.7-2 seeds was not strictly correlated to the nitrate content of seeds. No correlation was observed when nitrate was lowered in seeds due to limited nitrate nutrition of plants or to lower nitrate storage capacity in leaves of clca mutants deficient in the vacuolar anionic channel CLCa. All together, the results highlight a hitherto-unknown function of NRT2.7 in PA accumulation/oxidation. PMID:24532452

  20. [Modern evolutional developmental biology: mechanical and molecular genetic or phenotypic approaches?].

    Science.gov (United States)

    Vorob'eva, É I

    2010-01-01

    Heightened interest in the evolutionary problems of developmental biology in the 1980s was due to the success of molecular genetics and disappointment in the synthetic theory of evolution, where the chapters of embryology and developmental biology seem to have been left out. Modern evo-devo, which turned out to be antipodean to the methodology of the synthetic theory of evolution, propagandized in the development of evolutionary problems only the mechanical and molecular genetic approach to the evolution of ontogenesis, based on cellular and intercellular interactions. The phonotypical approach to the evaluation of evolutionary occurrences in ontogenesis, which aids in the joining of the genetic and epigenetic levels of research, the theory of natural selection, the nomogenetic conception, and the problem of the wholeness of the organism in onto- and phylogenesis may be against this. The phenotypic approach to ontogenesis is methodologically the most perspective for evolutionary developmental biology.

  1. A massively parallel pipeline to clone DNA variants and examine molecular phenotypes of human disease mutations.

    Directory of Open Access Journals (Sweden)

    Xiaomu Wei

    2014-12-01

    Full Text Available Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H, and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

  2. Phenotypical and Molecular Characterisation of Fusarium circinatum: Correlation with Virulence and Fungicide Sensitivity

    Directory of Open Access Journals (Sweden)

    Martin Mullett

    2017-11-01

    Full Text Available Fusarium circinatum, causing pine pitch canker, is one of the most damaging pathogens of Pinus species. This study investigated the use of phenotypical and molecular characteristics to delineate groups in a worldwide collection of isolates. The groups correlated with virulence and fungicide sensitivity, which were tested in a subset of isolates. Virulence tests of twenty isolates on P. radiata, P. sylvestris and P. pinaster demonstrated differences in host susceptibility, with P. radiata most susceptible and P. sylvestris least susceptible. Sensitivity to the fungicides fludioxonil and pyraclostrobin varied considerably between isolates from highly effective (half-maximal effective concentration (EC50 < 0.1 ppm to ineffective (EC50 > 100 ppm. This study demonstrates the potential use of simply acquired phenotypical (cultural, morphological and molecular metrics to gain a preliminary estimate of virulence and sensitivity to certain fungicides. It also highlights the necessity of including a range of isolates in fungicide tests and host susceptibility assays, particularly of relevance to tree breeding programmes.

  3. Phenotypic and molecular characterization of Brachyspira spp. isolated from laying hens in different housing systems.

    Science.gov (United States)

    Jansson, D S; Fellström, C; Råsbäck, T; Vågsholm, I; Gunnarsson, A; Ingermaa, F; Johansson, K-E

    2008-08-25

    Several species of intestinal spirochaetes, Brachyspira (B.) alvinipulli, B. intermedia and B. pilosicoli, may cause reduced egg production and faecal staining of eggshells in chickens. The aim of this study was to characterize potentially pathogenic and presumably non-pathogenic Brachyspira spp. from commercial laying hens. Selective culture, phenotyping, PCR and 16S rRNA gene sequencing were used and clinical data were collected. Phenotypic profiles were obtained for 489 isolates and 351 isolates obtained after subculture, and 30 isolates were selected for molecular characterization. Seven isolates were positive by a B. intermedia-specific PCR based on the nox gene, and two were positive in a B. hyodysenteriae-specific 23S rRNA gene based PCR. By comparative phylogenetic analysis in combination with PCR and phenotyping, seven isolates were identified as B. intermedia, eight isolates as B. innocens, five as B. murdochii, and three isolates each as B. alvinipulli and "B. pulli". The remaining four isolates could not be assigned to any presently recognized species. Co-infection with several species or genetic variants of Brachyspira spp. were detected in some flocks and samples, suggesting a high level of diversity. Organic flocks with access to outdoor areas were at higher risk (RR=2.3; 95% CI 1.5-3.6) for being colonized than chickens in other housing systems. No significant differences between colonized and non-colonized flocks were found regarding clinical parameters, i.e. mortality, egg production, faecally contaminated eggshells, and wet litter. Our results show that a combination of traditional laboratory diagnostics, molecular tests and phylogeny is needed for identification of Brachyspira sp. from chickens.

  4. Further delineation of the KAT6B molecular and phenotypic spectrum.

    LENUS (Irish Health Repository)

    Gannon, Tamsin

    2015-09-01

    KAT6B sequence variants have been identified previously in both patients with the Say-Barber-Biesecker type of blepharophimosis mental retardation syndromes (SBBS) and in the more severe genitopatellar syndrome (GPS). We report on the findings in a previously unreported group of 57 individuals with suggestive features of SBBS or GPS. Likely causative variants have been identified in 34\\/57 patients and were commonly located in the terminal exons of KAT6B. Of those where parental samples could be tested, all occurred de novo. Thirty out of thirty-four had truncating variants, one had a missense variant and the remaining three had the same synonymous change predicted to affect splicing. Variants in GPS tended to occur more proximally to those in SBBS patients, and genotype\\/phenotype analysis demonstrated significant clinical overlap between SBBS and GPS. The de novo synonymous change seen in three patients with features of SBBS occurred more proximally in exon 16. Statistical analysis of clinical features demonstrated that KAT6B variant-positive patients were more likely to display hypotonia, feeding difficulties, long thumbs\\/great toes and dental, thyroid and patella abnormalities than KAT6B variant-negative patients. The few reported patients with KAT6B haploinsufficiency had a much milder phenotype, though with some features overlapping those of SBBS. We report the findings in a previously unreported patient with a deletion of the KAT6B gene to further delineate the haploinsufficiency phenotype. The molecular mechanisms giving rise to the SBBS and GPS phenotypes are discussed.

  5. Further delineation of the KAT6B molecular and phenotypic spectrum

    Science.gov (United States)

    Gannon, Tamsin; Perveen, Rahat; Schlecht, Hélene; Ramsden, Simon; Anderson, Beverley; Kerr, Bronwyn; Day, Ruth; Banka, Siddharth; Suri, Mohnish; Berland, Siren; Gabbett, Michael; Ma, Alan; Lyonnet, Stan; Cormier-Daire, Valerie; Yilmaz, Rüstem; Borck, Guntram; Wieczorek, Dagmar; Anderlid, Britt-Marie; Smithson, Sarah; Vogt, Julie; Moore-Barton, Heather; Simsek-Kiper, Pelin Ozlem; Maystadt, Isabelle; Destrée, Anne; Bucher, Jessica; Angle, Brad; Mohammed, Shehla; Wakeling, Emma; Price, Sue; Singer, Amihood; Sznajer, Yves; Toutain, Annick; Haye, Damien; Newbury-Ecob, Ruth; Fradin, Melanie; McGaughran, Julie; Tuysuz, Beyhan; Tein, Mark; Bouman, Katelijne; Dabir, Tabib; Van den Ende, Jenneke; Luk, Ho Ming; Pilz, Daniela T; Eason, Jacqueline; Davies, Sally; Reardon, Willie; Garavelli, Livia; Zuffardi, Orsetta; Devriendt, Koen; Armstrong, Ruth; Johnson, Diana; Doco-Fenzy, Martine; Bijlsma, Emilia; Unger, Sheila; Veenstra-Knol, Hermine E; Kohlhase, Jürgen; Lo, Ivan FM; Smith, Janine; Clayton-Smith, Jill

    2015-01-01

    KAT6B sequence variants have been identified previously in both patients with the Say-Barber-Biesecker type of blepharophimosis mental retardation syndromes (SBBS) and in the more severe genitopatellar syndrome (GPS). We report on the findings in a previously unreported group of 57 individuals with suggestive features of SBBS or GPS. Likely causative variants have been identified in 34/57 patients and were commonly located in the terminal exons of KAT6B. Of those where parental samples could be tested, all occurred de novo. Thirty out of thirty-four had truncating variants, one had a missense variant and the remaining three had the same synonymous change predicted to affect splicing. Variants in GPS tended to occur more proximally to those in SBBS patients, and genotype/phenotype analysis demonstrated significant clinical overlap between SBBS and GPS. The de novo synonymous change seen in three patients with features of SBBS occurred more proximally in exon 16. Statistical analysis of clinical features demonstrated that KAT6B variant-positive patients were more likely to display hypotonia, feeding difficulties, long thumbs/great toes and dental, thyroid and patella abnormalities than KAT6B variant-negative patients. The few reported patients with KAT6B haploinsufficiency had a much milder phenotype, though with some features overlapping those of SBBS. We report the findings in a previously unreported patient with a deletion of the KAT6B gene to further delineate the haploinsufficiency phenotype. The molecular mechanisms giving rise to the SBBS and GPS phenotypes are discussed. PMID:25424711

  6. The molecular basis of variable phenotypic severity among common missense mutations causing Rett syndrome.

    Science.gov (United States)

    Brown, Kyla; Selfridge, Jim; Lagger, Sabine; Connelly, John; De Sousa, Dina; Kerr, Alastair; Webb, Shaun; Guy, Jacky; Merusi, Cara; Koerner, Martha V; Bird, Adrian

    2016-02-01

    Rett syndrome is caused by mutations in the X-linked MECP2 gene, which encodes a chromosomal protein that binds to methylated DNA. Mouse models mirror the human disorder and therefore allow investigation of phenotypes at a molecular level. We describe an Mecp2 allelic series representing the three most common missense Rett syndrome (RTT) mutations, including first reports of Mecp2[R133C] and Mecp2[T158M] knock-in mice, in addition to Mecp2[R306C] mutant mice. Together these three alleles comprise ∼25% of all RTT mutations in humans, but they vary significantly in average severity. This spectrum is mimicked in the mouse models; R133C being least severe, T158M most severe and R306C of intermediate severity. Both R133C and T158M mutations cause compound phenotypes at the molecular level, combining compromised DNA binding with reduced stability, the destabilizing effect of T158M being more severe. Our findings contradict the hypothesis that the R133C mutation exclusively abolishes binding to hydroxymethylated DNA, as interactions with DNA containing methyl-CG, methyl-CA and hydroxymethyl-CA are all reduced in vivo. We find that MeCP2[T158M] is significantly less stable than MeCP2[R133C], which may account for the divergent clinical impact of the mutations. Overall, this allelic series recapitulates human RTT severity, reveals compound molecular aetiologies and provides a valuable resource in the search for personalized therapeutic interventions. © The Author 2015. Published by Oxford University Press.

  7. A fluorescent protein scaffold for presenting structurally constrained peptides provides an effective screening system to identify high affinity target-binding peptides.

    Directory of Open Access Journals (Sweden)

    Tetsuya Kadonosono

    Full Text Available Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131-L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides.

  8. A fluorescent protein scaffold for presenting structurally constrained peptides provides an effective screening system to identify high affinity target-binding peptides.

    Science.gov (United States)

    Kadonosono, Tetsuya; Yabe, Etsuri; Furuta, Tadaomi; Yamano, Akihiro; Tsubaki, Takuya; Sekine, Takuya; Kuchimaru, Takahiro; Sakurai, Minoru; Kizaka-Kondoh, Shinae

    2014-01-01

    Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131-L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides.

  9. Molecular identification of four phenotypes of human Demodex mites (Acari: Demodicidae) based on mitochondrial 16S rDNA.

    Science.gov (United States)

    Zhao, Ya-E; Hu, Li; Ma, Jun-Xian

    2013-11-01

    Classification of Demodex mites has long depended on hosts and morphological characteristics. However, the fact that two species coexist in the same host and phenotype is easily influenced by environment causes difficulty and indeterminacy in traditional classification. Genotype, which directly reflects the molecular structure characteristics, is relatively stable. In this study, species identification of four phenotypes of human Demodex mites was conducted. Mites were morphologically classified into four phenotypes: long- and short-bodied Demodex folliculorum with finger-like terminus and Demodex brevis with finger- or cone-like terminus. The mitochondrial 16S ribosomal DNA (rDNA) fragment of individual mite was amplified, cloned, sequenced, and aligned. Sequence divergences, genetic distances, transition/transversion rates, and phylogenetic trees were analyzed. The results demonstrated that the 16S rDNA sequence of three phenotypes with finger-like terminus was 337 bp, and that of phenotype with cone-like terminus was 342 bp. The divergences, genetic distances, and transition/transversion rates among the three phenotypes with finger-like terminus were 0.0-2.7%, 0.000-0.029, and 5.0-7/0 (5/1-7/0), respectively, indicating an intraspecific variation. Yet, those between these three phenotypes and the one with cone-like terminus were 21.6-22.8%, 2.510-2.589, and 0.47-0.59 (22/47-27/46), respectively, suggesting an interspecific variation. The five phylogenetic trees showed that the three phenotypes with finger-like terminus clustered into one branch, while the phenotype with cone-like terminus clustered into another. In conclusion, terminus is a major morphological characteristic for the identification of human Demodex species. The three phenotypes with finger-like terminus belong to D. folliculorum, while the phenotype with cone-like terminus belongs to D. brevis. Molecular identification can verify and replenish morphological identification.

  10. Absence of high-affinity calreticulin autoantibodies in patients with systemic rheumatic diseases and coeliac disease

    DEFF Research Database (Denmark)

    Jørgensen, C S; Hansen, K B; Jacobsen, Søren

    2005-01-01

    Calreticulin has been reported to be an autoantigen in various autoimmune connective tissue diseases and in coeliac disease. Previous studies have used incubation buffers with low salt and low detergent concentrations (low stringency conditions) with serum albumin or other proteins as a blocking...... binding (high stringency conditions). Using the high stringency conditions, we screened sera from 107 patients with systemic lupus erythematosus, sera from patients with other systemic autoimmune diseases and from children with coeliac disease for the presence of high-affinity calreticulin autoantibodies...... by immunoblotting and ELISA. None of the sera contained high-affinity calreticulin antibodies. It is concluded that calreticulin is not a common autoantigen in patients with autoimmune connective tissue diseases or coeliac disease....

  11. MinePath: Mining for Phenotype Differential Sub-paths in Molecular Pathways.

    Directory of Open Access Journals (Sweden)

    Lefteris Koumakis

    2016-11-01

    Full Text Available Pathway analysis methodologies couple traditional gene expression analysis with knowledge encoded in established molecular pathway networks, offering a promising approach towards the biological interpretation of phenotype differentiating genes. Early pathway analysis methodologies, named as gene set analysis (GSA, view pathways just as plain lists of genes without taking into account either the underlying pathway network topology or the involved gene regulatory relations. These approaches, even if they achieve computational efficiency and simplicity, consider pathways that involve the same genes as equivalent in terms of their gene enrichment characteristics. Most recent pathway analysis approaches take into account the underlying gene regulatory relations by examining their consistency with gene expression profiles and computing a score for each profile. Even with this approach, assessing and scoring single-relations limits the ability to reveal key gene regulation mechanisms hidden in longer pathway sub-paths. We introduce MinePath, a pathway analysis methodology that addresses and overcomes the aforementioned problems. MinePath facilitates the decomposition of pathways into their constituent sub-paths. Decomposition leads to the transformation of single-relations to complex regulation sub-paths. Regulation sub-paths are then matched with gene expression sample profiles in order to evaluate their functional status and to assess phenotype differential power. Assessment of differential power supports the identification of the most discriminant profiles. In addition, MinePath assess the significance of the pathways as a whole, ranking them by their p-values. Comparison results with state-of-the-art pathway analysis systems are indicative for the soundness and reliability of the MinePath approach. In contrast with many pathway analysis tools, MinePath is a web-based system (www.minepath.org offering dynamic and rich pathway visualization

  12. Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

    OpenAIRE

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2017-01-01

    Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) te...

  13. Nuclear Choline Acetyltransferase Activates Transcription of a High-affinity Choline Transporter*

    OpenAIRE

    Matsuo, Akinori; Bellier, Jean-Pierre; Nishimura, Masaki; Yasuhara, Osamu; Saito, Naoaki; Kimura, Hiroshi

    2010-01-01

    Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter ...

  14. Selective high-affinity polydentate ligands and methods of making such

    Energy Technology Data Exchange (ETDEWEB)

    Denardo, Sally J.; Denardo, Gerald L.; Balhorn, Rodney L.

    2018-02-06

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  15. Functional Characteristics of the High Affinity IgG Receptor, Fc gamma RI

    NARCIS (Netherlands)

    van der Poel, Cees E.; Spaapen, Robbert M.; van de Winkel, Jan G. J.; Leusen, Jeanette H. W.

    2011-01-01

    IgG FcRs are important mediators of immunity and play a key role during Ab-based immunotherapy. Within the leukocyte IgG receptor family, only Fc gamma RI is capable of IgG binding with high affinity. Fc gamma RI exists as a complex of a ligand binding a-chain and an FcR gamma-chain. The receptors'

  16. Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

    2012-12-18

    A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

  17. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    OpenAIRE

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. CatalaseAbeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro.

  18. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    Directory of Open Access Journals (Sweden)

    Tal Noy-Porat

    2016-03-01

    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  20. Enhanced selection of high affinity DNA-reactive B cells following cyclophosphamide treatment in mice.

    Directory of Open Access Journals (Sweden)

    Daisuke Kawabata

    Full Text Available A major goal for the treatment of patients with systemic lupus erythematosus with cytotoxic therapies is the induction of long-term remission. There is, however, a paucity of information concerning the effects of these therapies on the reconstituting B cell repertoire. Since there is recent evidence suggesting that B cell lymphopenia might attenuate negative selection of autoreactive B cells, we elected to investigate the effects of cyclophosphamide on the selection of the re-emerging B cell repertoire in wild type mice and transgenic mice that express the H chain of an anti-DNA antibody. The reconstituting B cell repertoire in wild type mice contained an increased frequency of DNA-reactive B cells; in heavy chain transgenic mice, the reconstituting repertoire was characterized by an increased frequency of mature, high affinity DNA-reactive B cells and the mice expressed increased levels of serum anti-DNA antibodies. This coincided with a significant increase in serum levels of BAFF. Treatment of transgene-expressing mice with a BAFF blocking agent or with DNase to reduce exposure to autoantigen limited the expansion of high affinity DNA-reactive B cells during B cell reconstitution. These studies suggest that during B cell reconstitution, not only is negative selection of high affinity DNA-reactive B cells impaired by increased BAFF, but also that B cells escaping negative selection are positively selected by autoantigen. There are significant implications for therapy.

  1. Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development.

    Directory of Open Access Journals (Sweden)

    Dorine A Bax

    Full Text Available Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. We have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines.All lines proliferate as adherent monolayers and express glial markers. Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways. Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response.These data demonstrate that glioma cell lines derived from paediatric patients show key molecular differences to those from adults, some of which are well known, whilst others may provide novel targets for evaluation in primary tumours. We thus provide the rationale and demonstrate the practicability of using paediatric glioma cell lines for preclinical and mechanistic studies.

  2. Correlation among phenotypical and molecular techniques in comparing ascomycetous yeast type strains.

    Science.gov (United States)

    De Nicola, Raffaele; Corte, Laura; Lattanzi, Monia; Martini, Alessandro; Fatichenti, Fabrizio; Cardinali, Gianluigi

    2005-01-01

    Different phenotypical or molecular techniques can be used to describe and classify microorganisms for taxonomic, phylogenetic or genetic purposes. In yeast taxonomy the official hierarchic classification, based on morphological and physiological characters, is used together with more convenient molecular techniques such as the DNA sequencing. The question on whether these procedures produce coherent classifications is critical both to interpret taxonomic data consistently and to outline species correctly. In this paper, a set of type strains from the major genera of the budding hemiascomycetes yeast is examined with a series of physiological and molecular techniques, widely employed in taxonomy, in order to compare the among-strains correlations obtained with different methods. Results showed that the level of correlation among different techniques is relatively low, showing that different classifications and species organization could be obtained with diverse approaches. This is particularly interesting, considering that the official description of the yeast species is based on characters different from those becoming increasingly popular in the routine identification.

  3. Design, Synthesis, and in Vitro Pharmacology of New Radiolabeled γ-Hydroxybutyric Acid Analogues Including Photolabile Analogues with Irreversible Binding to the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Sabbatini, Paola; Wellendorph, Petrine; Høg, Signe

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report...

  4. Molecular analysis of spinal muscular atrophy and modification of the phenotype by SMN2.

    Science.gov (United States)

    Mailman, Matthew D; Heinz, John W; Papp, Audrey C; Snyder, Pamela J; Sedra, Mary S; Wirth, Brunhilde; Burghes, Arthur H M; Prior, Thomas W

    2002-01-01

    This study describes SMN1 deletion frequency, carrier studies, and the effect of the modifying SMN2 gene on the spinal muscular atrophy (SMA) phenotype. A novel allele-specific intragenic mutation panel increases the sensitivity of SMN1 testing. From 1995 to 2001, 610 patients were tested for SMN1 deletions and 399 relatives of probands have been tested for carrier status. SMN2 copy number was compared between 52 type I and 90 type III patients, and between type I and type III patients with chimeric SMN genes. A fluorescent allele-specific polymerase chain reaction (PCR) -based strategy detected intragenic mutations in potential compound heterozygotes and was used on 366 patients. Less than half of the patients tested were homozygously deleted for SMN1. A PCR-based panel detected the seven most common intragenic mutations. SMN2 copy number was significantly different between mild and severely affected patients. SMN1 molecular testing is essential for the diagnosis of SMA and allows for accurate carrier testing. Screening for intragenic mutations in SMN1 increases the sensitivity of diagnostic testing. Finally, SMN2 copy number is conclusively shown to ameliorate the phenotype and provide valuable prognostic information.

  5. Comparative Phenotypical and Molecular Analyses of Arabidopsis Grown under Fluorescent and LED Light.

    Science.gov (United States)

    Seiler, Franka; Soll, Jürgen; Bölter, Bettina

    2017-06-13

    Comparative analyses of phenotypic and molecular traits of Arabidopsis thaliana grown under standardised conditions is still a challenge using climatic devices supplied with common light sources. These are in most cases fluorescent lights, which have several disadvantages such as heat production at higher light intensities, an invariable spectral output, and relatively rapid "ageing". This results in non-desired variations of growth conditions and lowers the comparability of data acquired over extended time periods. In this study, we investigated the growth behaviour of Arabidopsis Col0 under different light conditions, applying fluorescent compared to LED lamps, and we conducted physiological as well as gene expression analyses. By changing the spectral composition and/or light intensity of LEDs we can clearly influence the growth behaviour of Arabidopsis and thereby study phenotypic attributes under very specific light conditions that are stable and reproducible, which is not necessarily given for fluorescent lamps. By using LED lights, we can also roughly mimic the sun light emission spectrum, enabling us to study plant growth in a more natural-like light set-up. We observed distinct growth behaviour under the different light regimes which was reflected by physiological properties of the plants. In conclusion, LEDs provide variable emission spectra for studying plant growth under defined, stable light conditions.

  6. Comparative Phenotypical and Molecular Analyses of Arabidopsis Grown under Fluorescent and LED Light

    Directory of Open Access Journals (Sweden)

    Franka Seiler

    2017-06-01

    Full Text Available Comparative analyses of phenotypic and molecular traits of Arabidopsis thaliana grown under standardised conditions is still a challenge using climatic devices supplied with common light sources. These are in most cases fluorescent lights, which have several disadvantages such as heat production at higher light intensities, an invariable spectral output, and relatively rapid “ageing”. This results in non-desired variations of growth conditions and lowers the comparability of data acquired over extended time periods. In this study, we investigated the growth behaviour of Arabidopsis Col0 under different light conditions, applying fluorescent compared to LED lamps, and we conducted physiological as well as gene expression analyses. By changing the spectral composition and/or light intensity of LEDs we can clearly influence the growth behaviour of Arabidopsis and thereby study phenotypic attributes under very specific light conditions that are stable and reproducible, which is not necessarily given for fluorescent lamps. By using LED lights, we can also roughly mimic the sun light emission spectrum, enabling us to study plant growth in a more natural-like light set-up. We observed distinct growth behaviour under the different light regimes which was reflected by physiological properties of the plants. In conclusion, LEDs provide variable emission spectra for studying plant growth under defined, stable light conditions.

  7. Molecular and phenotypic biomarkers of aging [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Xian Xia

    2017-06-01

    Full Text Available Individuals of the same age may not age at the same rate. Quantitative biomarkers of aging are valuable tools to measure physiological age, assess the extent of ‘healthy aging’, and potentially predict health span and life span for an individual. Given the complex nature of the aging process, the biomarkers of aging are multilayered and multifaceted. Here, we review the phenotypic and molecular biomarkers of aging. Identifying and using biomarkers of aging to improve human health, prevent age-associated diseases, and extend healthy life span are now facilitated by the fast-growing capacity of multilevel cross-sectional and longitudinal data acquisition, storage, and analysis, particularly for data related to general human populations. Combined with artificial intelligence and machine learning techniques, reliable panels of biomarkers of aging will have tremendous potential to improve human health in aging societies.

  8. ZrFsy1, a high-affinity fructose/H+ symporter from fructophilic yeast Zygosaccharomyces rouxii.

    Directory of Open Access Journals (Sweden)

    Maria José Leandro

    Full Text Available Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H(+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H(+ symporter, with Km 0.45 ± 0.07 mM and Vmax 0.57 ± 0.02 mmol h(-1 (gdw(-1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2% and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H(+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.

  9. Phenotypic and molecular assessment of seven patients with 6p25 deletion syndrome: Relevance to ocular dysgenesis and hearing impairment

    Directory of Open Access Journals (Sweden)

    Ritch Robert

    2004-06-01

    Full Text Available Abstract Background Thirty-nine patients have been described with deletions involving chromosome 6p25. However, relatively few of these deletions have had molecular characterization. Common phenotypes of 6p25 deletion syndrome patients include hydrocephalus, hearing loss, and ocular, craniofacial, skeletal, cardiac, and renal malformations. Molecular characterization of deletions can identify genes that are responsible for these phenotypes. Methods We report the clinical phenotype of seven patients with terminal deletions of chromosome 6p25 and compare them to previously reported patients. Molecular characterization of the deletions was performed using polymorphic marker analysis to determine the extents of the deletions in these seven 6p25 deletion syndrome patients. Results Our results, and previous data, show that ocular dysgenesis and hearing impairment are the two most highly penetrant phenotypes of the 6p25 deletion syndrome. While deletion of the forkhead box C1 gene (FOXC1 probably underlies the ocular dysgenesis, no gene in this region is known to be involved in hearing impairment. Conclusions Ocular dysgenesis and hearing impairment are the two most common phenotypes of 6p25 deletion syndrome. We conclude that a locus for dominant hearing loss is present at 6p25 and that this locus is restricted to a region distal to D6S1617. Molecular characterization of more 6p25 deletion patients will aid in refinement of this locus and the identification of a gene involved in dominant hearing loss.

  10. VNARs: An Ancient and Unique Repertoire of Molecules That Deliver Small, Soluble, Stable and High Affinity Binders of Proteins

    Directory of Open Access Journals (Sweden)

    Caroline Barelle

    2015-09-01

    Full Text Available At 420 million years, the variable domain of New Antigen Receptors or VNARs are undoubtedly the oldest (and smallest antigen binding single domains identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established and has served to provide a greater understanding of the evolution of humoral immunity; their cellular components and processes as well as the underlying genetic organization and molecular control mechanisms. Intriguingly, unlike the variable domain of the camelid heavy chain antibodies or VHH, VNARs do not conform to all of the characteristic properties of classical antibodies with an ancestral origin that clearly distinguishes them from true immunoglobulin antibodies. However, this uniqueness of their origin only adds to their potential as next generation therapeutic biologics with their structural and functional attributes and commercial freedom all enhancing their profile and current success. In fact their small size, remarkable stability, molecular flexibility and solubility, together with their high affinity and selectivity for target, all reinforce the potential of these domains as drug candidates. The purpose of this review is to provide an overview of the existing basic biology of these unique domains, to highlight the drug-like properties of VNARs and describe current progress in their journey towards the clinic.

  11. Molecular Phenotype of Breast Cancer According to Time Since Last Pregnancy in a Large Cohort of Young Women.

    Science.gov (United States)

    Collins, Laura C; Gelber, Shari; Marotti, Jonathan D; White, Sarah; Ruddy, Kathryn; Brachtel, Elena F; Schapira, Lidia; Come, Steven E; Borges, Virginia F; Schedin, Pepper; Warner, Ellen; Wensley, Taylor; Tamimi, Rulla M; Winer, Eric P; Partridge, Ann H

    2015-07-01

    The increase in breast cancer risk during pregnancy and postpartum is well known; however, the molecular phenotype of breast cancers occurring shortly after pregnancy has not been well studied. Given this, we investigated whether nulliparity and the time interval since pregnancy among parous women affects the breast cancer phenotype in young women. We examined molecular phenotype in relation to time since pregnancy in a prospective cohort of 707 young women (aged ≤40 years) with breast cancer. Parity was ascertained from study questionnaires. Using tumor histologic grade on central review and biomarker expression, cancers were categorized as luminal A- or B-like, HER2 enriched, and triple negative. Overall, 32% were luminal A-like, 41% were luminal B-like, 9% were HER2 enriched, and 18% were triple negative. Although, numerically, patients diagnosed >5 years after pregnancy had more luminal A-like subtypes than women with shorter intervals since pregnancy, there was no evidence of a relationship between these intervals and molecular subtypes once family history of breast cancer and age at diagnosis were considered. Distribution of breast cancer molecular phenotype did not differ significantly among young women by parity or time interval since parturition when important predictors of tumor phenotype such as age and family history were considered. Distribution of breast cancer molecular phenotype did not differ among parous young women by time interval since pregnancy. The implication of these findings for clinical practice suggests that pregnancy-associated breast cancers may be seen up to 5 years beyond parturition. ©AlphaMed Press.

  12. Quantitative molecular phenotyping with topically applied SERS nanoparticles for intraoperative guidance of breast cancer lumpectomy

    Science.gov (United States)

    Wang, Yu; Kang, Soyoung; Khan, Altaz; Ruttner, Gabriel; Leigh, Steven Y.; Murray, Melissa; Abeytunge, Sanjee; Peterson, Gary; Rajadhyaksha, Milind; Dintzis, Suzanne; Javid, Sara; Liu, Jonathan T. C.

    2016-02-01

    There is a need to image excised tissues during tumor-resection procedures in order to identify residual tumors at the margins and to guide their complete removal. The imaging of dysregulated cell-surface receptors is a potential means of identifying the presence of diseases with high sensitivity and specificity. However, due to heterogeneities in the expression of protein biomarkers in tumors, molecular-imaging technologies should ideally be capable of visualizing a multiplexed panel of cancer biomarkers. Here, we demonstrate that the topical application and quantification of a multiplexed cocktail of receptor-targeted surface-enhanced Raman scattering (SERS) nanoparticles (NPs) enables rapid quantitative molecular phenotyping (QMP) of the surface of freshly excised tissues to determine the presence of disease. In order to mitigate the ambiguity due to nonspecific sources of contrast such as off-target binding or uneven delivery, a ratiometric method is employed to quantify the specific vs. nonspecific binding of the multiplexed NPs. Validation experiments with human tumor cell lines, fresh human tumor xenografts in mice, and fresh human breast specimens demonstrate that QMP imaging of excised tissues agrees with flow cytometry and immunohistochemistry, and that this technique may be achieved in less than 15 minutes for potential intraoperative use in guiding breast-conserving surgeries.

  13. Phenotypic and molecular identification of Coccidioides posadasii in a patient evaluated for bilateral lung transplantation.

    Science.gov (United States)

    Fernandez, Analía; Landaburu, Fernanda; Lopez-Daneri, Gabriela; Nagel, Claudia; Di Giorgio, Patricia; Iovannitti, Cristina; Tokumoto, Marta; Mujica, Maria Teresa

    2012-01-01

    Coccidioidomycosis is an endemic fungal infection caused by Coccidioides immitis and Coccidioides posadasii. It can be particularly severe in transplant recipients that have a current or a previous coccidioidal infection. Fatal case of coccidioidomycosis has been described in this group of patients. We report a severe case of pneumonia caused by C. posadassi in a 29 year-old white woman that had been admitted to hospital as part of the evaluation for bilateral lung transplantation. The patient was a native and resident of Catamarca, Argentina. Molecular methodologies contributed to the species identification. Clinical, laboratory records and microbiological tests were carried out to diagnose the infection and to identify C. posadasii. A fungus was isolated from BAL culture. Phenotypic characterization, specific PCR and experimental animal inoculation demonstrated the presence of C. posadasii. The patient responded well to amphotericin B deoxycholate. Lung transplantation was postponed. Specific PCR can be an important alternative for the correct identification of C. immitis or C. posadasii in laboratories with implemented molecular biology tools. This case emphasizes the need for a systematic assessment in organ transplant units of patients inhabiting endemic areas of coccidioidomycosis. Copyright © 2011 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  14. High-affinity antibodies to the 1,4-dihydropyridine Ca2+-channel blockers

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.P.; Sharp, A.; Strom, M.; Kahl, S.D.

    1986-05-01

    Antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers have been produced in rabbits by immunization with dihydropyridine-protein conjugates. Anti-dihydropyridine antibodies were found to specifically bind (/sup 3/H)nitrendipine, (/sup 3/H)-nimodipine, (/sup 3/H)nisoldipine, and (/sup 3/H)PN 200-110 (all 1,4-dihydropyridine Ca2+-channel blockers) with high affinity, while (/sup 3/H)verapamil, (/sup 3/H)diltiazem, and (/sup 3/H)trifluoperazine were not recognized. The average dissociation constant of the (/sup 3/H)nitrendipine-antibody complex was 0.06 (+/- 0.02) X 10(-9) M for an antiserum studied in detail and ranged from 0.01 to 0.24 X 10(-9) M for all antisera. Inhibition of (/sup 3/H)nitrendipine binding was specific for the 1,4-dihydropyridine Ca2+-channel modifiers and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM. Structurally unrelated Ca2+-channel blockers, calmodulin antagonists, inactive metabolites of nitrendipine, and UV-inactivated nisoldipine did not modify (/sup 3/H)nitrendipine binding to the anti-dihydropyridine antibodies. Dihydropyridines without a bulky substituent in the 4-position of the heterocycle were able to displace (/sup 3/H)nitrendipine binding, but the concentrations required for half-maximal inhibition were greater than 800 nM. In summary, anti-dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+-channel blockers.

  15. Regional distribution of high affinity binding of 3H-adenosine in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Traversa, U.; Puppini, P.; de Angelis, L.; Vertua, R.

    1984-06-01

    The high and low affinity adenosine binding sites with Kd values ranging respectively from 0.8 to 1.65 microM and from 3.1 to 13.86 microM were demonstrated in the following rat brain areas: cortex, hippocampus, striatum, cerebellum, diencephalon, and pons-medulla. Adenosine receptors involved in the high affinity binding seem to be mainly Ra-type. The analysis of the regional distribution of 3H-Adenosine showed the highest levels of specific binding in striatum and hippocampus; somewhat smaller values in cortex, cerebellum, and diencephalon, and even lower in pons-medulla.

  16. Similarity of molecular phenotype between known epilepsy gene LGI1 and disease candidate gene LGI2

    Directory of Open Access Journals (Sweden)

    Eisenhaber Frank

    2010-09-01

    Full Text Available Abstract Background The LGI2 (leucine-rich, glioma inactivated 2 gene, a prime candidate for partial epilepsy with pericentral spikes, belongs to a family encoding secreted, beta-propeller domain proteins with EPTP/EAR epilepsy-associated repeats. In another family member, LGI1 (leucine-rich, glioma inactivated 1 mutations are responsible for autosomal dominant lateral temporal epilepsy (ADLTE. Because a few LGI1 disease mutations described in the literature cause secretion failure, we experimentally analyzed the secretion efficiency and subcellular localization of several LGI1 and LGI2 mutant proteins corresponding to observed non-synonymous single nucleotide polymorphisms (nsSNPs affecting the signal peptide, the leucine-rich repeats and the EAR propeller. Results Mapping of disease-causing mutations in the EAR domain region onto a 3D-structure model shows that many of these mutations co-localize at an evolutionary conserved surface region of the propeller. We find that wild-type LGI2 is secreted to the extracellular medium in glycosylated form similarly to LGI1, whereas several mutant proteins tested in this study are secretion-deficient and accumulate in the endoplasmic reticulum. Interestingly, mutations at structurally homologous positions in the EAR domain have the same effect on secretion in LGI1 and LGI2. Conclusions This similarity of experimental mislocalization phenotypes for mutations at homologous positions of LGI2 and the established epilepsy gene LGI1 suggests that both genes share a potentially common molecular pathogenesis mechanism that might be the reason for genotypically distinct but phenotypically related forms of epilepsy.

  17. [FOXP2 and the molecular biology of language: new evidence. I. Phenotypic aspects and animal models].

    Science.gov (United States)

    Benítez-Burraco, A

    FOXP2 is the first gene linked to a hereditary variant of specific language impairment and seems to code for a transcriptional repressor that intervenes in the regulation of development and the functioning of certain thalamic-cortical-striatal circuits. In the last three years significant progress has been made in the analysis of the structural and functional properties of the gene. The most notable advances have been made in the genotypic and phenotypic characterisation of new alterations in its sequencing in human beings; the determination in vivo of the functional properties of the mutated proteins generated from said variants; the cloning and characterisation of new orthologues of the gene; the generation of the first knockout and knockdown organisms for it; and a more precise molecular characterisation of the biological role played by the orthologues corresponding to species that are also capable of learning the articulatory patterns of the vocalisations they use to communicate. The latest clinical evidence and that obtained from analysing animal models generated to date appear to suggest the presence of a 'sensory-motor disorder' as the central deficit behind the different phenotypes associated to the different mutations of the gene in the human species, the functionality of the gene FOXP2 during development of the embryo and during the adult phase, its involvement in the development and functioning of the thalamic-cortical-striatal circuits associated to motor planning, sequential behaviour and procedural learning, and significant old age, in developmental terms, of a part of the neuroanatomical substrate that is involved in processing linguistic stimuli in our species.

  18. Molecular and Phenotypic Characterization of Diarrheagenic Escherichia coli Strains Isolated from Bacteremic Children.

    Science.gov (United States)

    Riveros, Maribel; García, Wilfredo; García, Coralith; Durand, David; Mercado, Erik; Ruiz, Joaquim; Ochoa, Theresa J

    2017-11-01

    Escherichia coli is an important cause of Gram-negative bacteremia. The aim of this study was to characterize at the molecular and phenotypic levels E. coli strains belonging to different diarrheagenic pathotypes [diarrheagenic E. coli (DEC)] isolated from bacteremia in children younger than 5 years of age. Seventy bacteremia E. coli strains were collected in a prospective study in 12 hospitals in Lima, Peru. The presence of virulence genes associated with DEC [enterotoxigenic (lt and st), enteropathogenic (eaeA), shiga toxin-producing (stx1and stx2), enteroinvasive (ipaH), enteroaggregative (aggR), and diffusely adherent (daaD)] was determined by multiplex real-time polymerase chain reaction (PCR). Those positive E. coli strains were further analyzed for 18 additional virulence factors encoding genes and others phenotypic features. Virulence genes associated with DEC were identified in seven bacteremic children (10%), including: one aggR-positive [enteroaggregative E. coli (EAEC)], one eaeA-positive [enteropathogenic E. coli (EPEC)], one st-positive [enterotoxigenic E. coli (ETEC)], one daaD-positive [diffusely adherent E. coli (DAEC)], and three strain positive for aggR and daaD (EAEC/DAEC) at the same time. All strains, except EPEC, had the Ag43 adhesin, and all, except ETEC had the siderophore gene fyuA. The phylogenetic profile of these strains was variable, two (B2), two (D), two (A), and one (B1) strain. These isolates were susceptible to all tested antibacterial agents except to ampicillin and gentamicin. The three EAEC/DAEC strains showed biofilm formation and aggregative adhesion and had the same repetitive extragenic palindromic-PCR patterns. These findings suggest that some DEC strains, especially agg-R and daa-D positive, might cause bacteremia in children.

  19. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-01

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  20. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma.

    Science.gov (United States)

    Zhang, Yi-Fan; Ho, Mitchell

    2016-09-26

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer.

  1. Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter▿

    Science.gov (United States)

    Aktas, Meriyem; Jost, Kathinka A.; Fritz, Christiane; Narberhaus, Franz

    2011-01-01

    Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called “Venus flytrap mechanism” of substrate binding. PMID:21803998

  2. Isolation of Streptomyces sp. PCB7, the first microorganism demonstrating high-affinity uptake of tropospheric H2

    National Research Council Canada - National Science Library

    Constant, Philippe; Poissant, Laurier; Villemur, Richard

    2008-01-01

    .... Studies conducted over the last three decades provide indirect evidences that H(2) soil uptake is mediated by free soil hydrogenases or by unknown microorganisms that have a high affinity for H(2...

  3. Mitogenic effects of urokinase on melanoma cells are independent of high affinity binding to the urokinase receptor.

    Science.gov (United States)

    Koopman, J L; Slomp, J; de Bart, A C; Quax, P H; Verheijen, J H

    1998-12-11

    The structural and functional properties of the urokinase-type plasminogen activator (u-PA) that are involved in the mitogenic effect of this proteolytic enzyme on human melanoma cells M14 and IF6 and the role of the u-PA receptor (u-PAR) in transducing this signal were analyzed. Native u-PA purified from urine induced a mitogenic response in quiescent IF6 and M14 cells that ranged from 25 to 40% of the mitogenic response obtained by fetal calf serum. The half-maximum response in M14 and IF6 cells was reached at u-PA concentrations of approximately 35 and 60 nM, respectively. Blocking the proteolytic activity of u-PA resulted in a 30% decrease of the mitogenic effect, whereas inhibition of plasmin activity did not alter the mitogenic effect. No mitogenic response was elicited by low molecular weight u-PA, lacking the growth factor domain and the kringle domain. The ATF domain of u-PA induced a mitogenic response that was similar to complete u-PA. Defucosylated ATF and recombinant u-PA purified from Escherichia coli lacking all post-translational modifications did not induce a mitogenic response. Blocking the interaction of u-PA with u-PAR, using a specific monoclonal antibody, did not alter the mitogenic effect induced by u-PA. The binding of radiolabeled u-PA to M14 and IF6 cells was characterized by high affinity binding mediated by u-PAR and low affinity binding to an unknown binding site. These results demonstrate that proteolytically inactive u-PA is able to induce a mitogenic response in quiescent melanoma cells in vitro by a mechanism that involves the ATF domain but is independent of high affinity binding to u-PAR. Furthermore, it suggests that u-PA is able to bind with low affinity to a hitherto unidentified membrane associated protein that could be involved in u-PA-induced signal transduction.

  4. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis.

    Science.gov (United States)

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Rajulu, Charukesi; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier; Rubio, Vicente

    2015-09-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. © 2015 American Society of Plant Biologists. All rights reserved.

  5. Structural determinants for high-affinity binding in a Nedd4 WW3* domain-Comm PY motif complex.

    Science.gov (United States)

    Kanelis, Voula; Bruce, M Christine; Skrynnikov, Nikolai R; Rotin, Daniela; Forman-Kay, Julie D

    2006-03-01

    Interactions between the WW domains of Drosophila Nedd4 (dNedd4) and Commissureless (Comm) PY motifs promote axon crossing at the CNS midline and muscle synaptogenesis. Here we report the solution structure of the dNedd4 WW3* domain complexed to the second PY motif (227'TGLPSYDEALH237') of Comm. Unexpectedly, there are interactions between WW3* and ligand residues both N- and C-terminal to the PY motif. Residues Y232'-L236' form a helical turn, following the PPII helical PY motif. Mutagenesis and binding studies confirm the importance of these extensive contacts, not simultaneously observed in other WW domain complexes, and identify a variable loop in WW3* responsible for its high-affinity interaction. These studies expand our general understanding of the molecular determinants involved in WW domain-ligand recognition. In addition, they provide insights into the specific regulation of dNedd4-mediated ubiquitination of Comm and subsequent internalization of Comm or the Comm/Roundabout complex, critical for CNS and muscle development.

  6. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    Science.gov (United States)

    Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang-Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P.; Strømgaard, Kristian

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT. PMID:27089947

  7. Expression of NGF, BDNF and their high-affinity receptors in ovine mammary glands during development and lactation.

    Science.gov (United States)

    Colitti, Monica

    2015-12-01

    The distribution of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their high-affinity tyrosine kinase receptors TrkA and TrkB was investigated by immunohistochemical method in the mammary gland of ewes from prepubertal stage to involution. NGF and BDNF protein expressions were strong during development of glands at prepubertal stage and during pregnancy and decreased during lactation and involution. The expressions localized in both stromal and parenchymal cells of developing gland were mainly arranged in the apical side of secretory cells during lactation. These observations were also confirmed at transcriptional level by RT-PCR analyses. The highest expression of all genes significantly occurred at prepubertal stage. NGF was then down-regulated from pregnancy to involution, and no statistical differences were observed among these stages. The receptor TrkA was also under-expressed from pregnancy to involution, and its expression significantly differed between pregnancy and 30 days of lactation and also between 30 and 60 days of lactation. BDNF was significantly down-regulated at 60 days of lactation in comparison with prepubertal stage and again between pregnancy and 30 days of lactation. The relative abundance of its receptor, TrkB, showed also a significant down-regulation at 60 days of lactation in comparison with pregnancy and involution. Among the myriad of other molecular signals involved in the mammary gland cycle, the local production of neuropeptides and their receptors could be of interest in understanding their potential role in mammary biology.

  8. Drosophila americana as a Model Species for Comparative Studies on the Molecular Basis of Phenotypic Variation

    Science.gov (United States)

    Fonseca, Nuno A.; Morales-Hojas, Ramiro; Reis, Micael; Rocha, Helder; Vieira, Cristina P.; Nolte, Viola; Schlötterer, Christian; Vieira, Jorge

    2013-01-01

    Understanding the molecular basis of within and between species phenotypic variation is one of the main goals of Biology. In Drosophila, most of the work regarding this issue has been performed in D. melanogaster, but other distantly related species must also be studied to verify the generality of the findings obtained for this species. Here, we make the case for D. americana, a species of the virilis group of Drosophila that has been diverging from the model species, D. melanogaster, for approximately 40 Myr. To determine the suitability of this species for such studies, polymorphism and recombination estimates are presented for D. americana based on the largest nucleotide sequence polymorphism data set so far analyzed (more than 100 data sets) for this species. The polymorphism estimates are also compared with those obtained from the comparison of the genome assembly of two D. americana strains (H5 and W11) here reported. As an example of the general utility of these resources, we perform a preliminary study on the molecular basis of lifespan differences in D. americana. First, we show that there are lifespan differences between D. americana populations from different regions of the distribution range. Then, we perform five F2 association experiments using markers for 21 candidate genes previously identified in D. melanogaster. Significant associations are found between polymorphism at two genes (hep and Lim3) and lifespan. For the F2 association study involving the two sequenced strains (H5 and W11), we identify amino acid differences at Lim3 and Hep that could be responsible for the observed changes in lifespan. For both genes, no large gene expression differences were observed between the two strains. PMID:23493635

  9. Phenotypic Characters and Molecular Epidemiology of Campylobacter Jejuni in East China.

    Science.gov (United States)

    Zeng, Dexin; Zhang, Xiaoping; Xue, Feng; Wang, Yanhong; Jiang, Luyan; Jiang, Yuan

    2016-01-01

    In this study, we investigated the distribution, phenotypic and molecular typing characters of Campylobacter jejuni in domestic fowl, and livestock populations in East China, to provide some reference for researches on its molecular epidemiology. A total of 1250 samples were collected from different animal sources, and C. jejuni strains were then isolated and tested for antibiotic sensitivity. Antibiotics-resistance gene and pathogenic genes were detected by polymerase chain reaction. Phylogenic analysis on the C. jejuni strains was performed by multilocus sequence typing (MLST) method. The results showed that 108 out of the 1250 samples (mean 8.64%) were C. jejuni positive. These 108 C. jejuni strains were highly sensitive to antibiotics such as chloramphenicol, amoxicillin, amikacin, cefotaxime, and azithromycin, whereas they were highly resistant to antibiotics such as cefoperazone, cotrimoxazole, cefamandole, sulfamethoxazole, and cefradine. Pathogenicity related gene identification indicated that the mean carrying rate of adhesion related gene cadF and racR, flagellin gene flaA, toxin regulating gene cdtA, cdtB, cdtC, wlaN and virB11, heat shock proteins and transferring proteins related genes dnaJ and ceuE, CiaB and pldA were 92.45%, 38.69%, 73.58%, 71.70%, 52.83%, 96.23%, 12.26%, 1.89%, 0.94%, 65.09%, 39.62% and 9.43%, respectively. A total of 58.82% of these strains contained more than 6 pathogenicity-related genes. MLST typed 58 ST types from the 108 isolated C. jejuni strains, including 24 new types, and ST-21 was the major type, accounting for 39.3% of the total strains. © 2015 Institute of Food Technologists®

  10. Correlation of intercentromeric distance, mosaicism, and sexual phenotype: molecular localization of breakpoints in isodicentric Y chromosomes.

    Science.gov (United States)

    Beaulieu Bergeron, Mélanie; Brochu, Pierre; Lemyre, Emmanuelle; Lemieux, Nicole

    2011-11-01

    Isodicentric chromosomes are among the structural abnormalities of the Y chromosome that are commonly identified in patients. The simultaneous 45,X cell line that is generated in cell division due to instability of the isodicentric Y chromosome [idic(Y)] has long been hypothesized to explain the variable sexual development of these patients, although gonads have been studied in only a subset of cases. We report here on the molecular localization of breakpoints in ten patients with an idic(Y). Breakpoints were mapped by FISH using BACs; gonads and fibroblasts were also analyzed when possible to evaluate the level of mosaicism. First, we demonstrate great tissue variability in the distribution of idic(Y). Second, palindromes and direct repeats were near the breakpoint of several idic(Y), suggesting that these sequences play a role in the formation of idic(Y). Finally, our data suggest that intercentromeric distance has a negative influence on the stability of idic(Y), as a greater proportion of cells with breakage or loss of the idic(Y) were found in idic(Y) with a greater intercentromeric distance. Females had a significantly greater intercentromeric distance on their idic(Y) than did males. In conclusion, our study indicates that the Y chromosome contains sequences that are more prone to formation of isodicentric chromosomes. We also demonstrate that patients with an intercentromeric distance greater than 20 Mb on their idic(Y) are at increased risk of having a female sexual phenotype. Copyright © 2011 Wiley Periodicals, Inc.

  11. Temperature control of molecular circuit switch responsible for virulent phenotype expression in uropathogenic Escherichia coli

    Science.gov (United States)

    Samoilov, Michael

    2010-03-01

    The behavior and fate of biological organisms are to a large extent dictated by their environment, which can be often viewed as a collection of features and constraints governed by physics laws. Since biological systems comprise networks of molecular interactions, one such key physical property is temperature, whose variations directly affect the rates of biochemical reactions involved. For instance, temperature is known to control many gene regulatory circuits responsible for pathogenicity in bacteria. One such example is type 1 fimbriae (T1F) -- the foremost virulence factor in uropathogenic E. coli (UPEC), which accounts for 80-90% of all community-acquired urinary tract infections (UTIs). The expression of T1F is randomly `phase variable', i.e. individual cells switch between virulent/fimbriate and avirulent/afimbriate phenotypes, with rates regulated by temperature. Our computational investigation of this process, which is based on FimB/FimE recombinase-mediated inversion of fimS DNA element, offers new insights into its discrete-stochastic kinetics. In particular, it elucidates the logic of T1F control optimization to the host temperature and contributes further understanding toward the development of novel therapeutic approaches to UPEC-caused UTIs.

  12. MutLα heterodimers modify the molecular phenotype of Friedreich ataxia.

    Directory of Open Access Journals (Sweden)

    Vahid Ezzatizadeh

    Full Text Available Friedreich ataxia (FRDA, the most common autosomal recessive ataxia disorder, is caused by a dynamic GAA repeat expansion mutation within intron 1 of FXN gene, resulting in down-regulation of frataxin expression. Studies of cell and mouse models have revealed a role for the mismatch repair (MMR MutS-heterodimer complexes and the PMS2 component of the MutLα complex in the dynamics of intergenerational and somatic GAA repeat expansions: MSH2, MSH3 and MSH6 promote GAA repeat expansions, while PMS2 inhibits GAA repeat expansions.To determine the potential role of the other component of the MutLα complex, MLH1, in GAA repeat instability in FRDA, we have analyzed intergenerational and somatic GAA repeat expansions from FXN transgenic mice that have been crossed with Mlh1 deficient mice. We find that loss of Mlh1 activity reduces both intergenerational and somatic GAA repeat expansions. However, we also find that loss of either Mlh1 or Pms2 reduces FXN transcription, suggesting different mechanisms of action for Mlh1 and Pms2 on GAA repeat expansion dynamics and regulation of FXN transcription.Both MutLα components, PMS2 and MLH1, have now been shown to modify the molecular phenotype of FRDA. We propose that upregulation of MLH1 or PMS2 could be potential FRDA therapeutic approaches to increase FXN transcription.

  13. Phenotype and molecular diversity evaluation of some wild 2n Solanum species (super series Rotata

    Directory of Open Access Journals (Sweden)

    Dukagjin Zeka

    2015-06-01

    Full Text Available New cultivars are result of the conservation and characterization of potato (Solanum genetic resources in secondary germplasm banks. The objectives of this study were to assess phenotype diversity of 12 clones of 10 wild diploid potato species collection super series Rotata, and to determine their genetic diversity through simple sequence repeat (SSR markers. Totally 63 alleles of 20 cpSSR loci were detected i.e. 3.15 alleles on average per one microsatellite locus. Alleles ranged from two to six per locus. The highest polymorphism was detected in the locus ntcp9 and lowest were recorded having by two alleles in seven of loci. The average value of observed heterozygosity (Ho was 0.61, whereas the mean of polymorphic information contents (PIC was 0.49. Intergenic regions had highest variability (Higr = 0.65 compare with introns (Hin = 0.54 and exons (Hex = 0.45 of the chloroplast genome. Molecular analyses were complemented with tuft morphological measurements according to the descriptor list for the genus Solanum. SSR-based markers highlight a tendency to separate two groups of Rotata wild diploids and show the possibility of duplicities of wild potato genetic resources in the current Czech in vitro collection.

  14. Anti-viral state segregates two molecular phenotypes of pancreatic adenocarcinoma: potential relevance for adenoviral gene therapy

    Directory of Open Access Journals (Sweden)

    Chiorini Jay A

    2010-01-01

    Full Text Available Abstract Background Pancreatic ductal adenocarcinoma (PDAC remains a leading cause of cancer mortality for which novel gene therapy approaches relying on tumor-tropic adenoviruses are being tested. Methods We obtained the global transcriptional profiling of primary PDAC using RNA from eight xenografted primary PDAC, three primary PDAC bulk tissues, three chronic pancreatitis and three normal pancreatic tissues. The Affymetrix GeneChip HG-U133A was used. The results of the expression profiles were validated applying immunohistochemical and western blot analysis on a set of 34 primary PDAC and 10 established PDAC cell lines. Permissivity to viral vectors used for gene therapy, Adenovirus 5 and Adeno-Associated Viruses 5 and 6, was assessed on PDAC cell lines. Results The analysis of the expression profiles allowed the identification of two clearly distinguishable phenotypes according to the expression of interferon-stimulated genes. The two phenotypes could be readily recognized by immunohistochemical detection of the Myxovirus-resistance A protein, whose expression reflects the activation of interferon dependent pathways. The two molecular phenotypes discovered in primary carcinomas were also observed among established pancreatic adenocarcinoma cell lines, suggesting that these phenotypes are an intrinsic characteristic of cancer cells independent of their interaction with the host's microenvironment. The two pancreatic cancer phenotypes are characterized by different permissivity to viral vectors used for gene therapy, as cell lines expressing interferon stimulated genes resisted to Adenovirus 5 mediated lysis in vitro. Similar results were observed when cells were transduced with Adeno-Associated Viruses 5 and 6. Conclusion Our study identified two molecular phenotypes of pancreatic cancer, characterized by a differential expression of interferon-stimulated genes and easily recognized by the expression of the Myxovirus-resistance A protein. We

  15. Molecular and phenotypic distinction of the very recently evolved insular subspecies Mus musculus helgolandicus ZIMMERMANN, 1953.

    Science.gov (United States)

    Babiker, Hiba; Tautz, Diethard

    2015-08-14

    Populations and subspecies of the house mouse Mus musculus were able to invade new regions worldwide in the wake of human expansion. Here we investigate the origin and colonization history of the house mouse inhabiting the small island of Heligoland on the German Bight - Mus musculus helgolandicus. It was first described by Zimmermann in 1953, based on morphological descriptions which were considered to be a mosaic between the subspecies M. m. domesticus and M. m. musculus. Since mice on islands are excellent evolutionary model systems, we have focused here on a molecular characterization and an extended phenotype analysis. The molecular data show that the mice from Heligoland are derived from M. m. domesticus based on mitochondrial D-loop sequences as well as on four nuclear diagnostic markers, including one each from the sex-chromosomes. STRUCTURE analysis based on 21 microsatellite markers assigns Heligoland mice to a distinct population and D-loop network analysis suggests that they are derived from a single colonization event. In spite of mice from the mainland arriving by ships, they are apparently genetically refractory against further immigration. Mutation frequencies in complete mitochondrial genome sequences date the colonization age to approximately 400 years ago. Complete genome sequences from three animals revealed a genomic admixture with M. m. musculus genomic regions with at least 6.5% of the genome affected. Geometric morphometric analysis of mandible shapes including skull samples from two time points during the last century suggest specific adaptations to a more carnivorous diet. The molecular and morphological analyses confirm that M. m. helgolandicus consists of a distinct evolutionary lineage with specific adaptations. It shows a remarkable resilience against genetic mixture with mainland populations of M. m. domesticus despite major disturbances in the past century and a high ship traffic. The genomic admixture with M. m. musculus genetic

  16. Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

    Energy Technology Data Exchange (ETDEWEB)

    Silveira, C.L.; Eldefrawi, A.T.; Eldefrawi, M.E. (Univ. of Maryland, Baltimore (USA))

    1990-05-01

    The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-(3H)cis-methyldioxolane ((3H)CD), which has been used to label a high affinity population of M2 receptors. A single population of sites was detected and (3H)CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with (3H)CD and considered to be of the M2 subtype. Up to 70% of the (3H)CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. (3H)CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of (3H)CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM (3H)CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting (3H)CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration.

  17. Pendred syndrome in two Galician families: insights into clinical phenotypes through cellular, genetic, and molecular studies.

    Science.gov (United States)

    Palos, Fernando; García-Rendueles, María E R; Araujo-Vilar, David; Obregon, Maria Jesús; Calvo, Rosa Maria; Cameselle-Teijeiro, Jose; Bravo, Susana B; Perez-Guerra, Oscar; Loidi, Lourdes; Czarnocka, Barbara; Alvarez, Paula; Refetoff, Samuel; Dominguez-Gerpe, Lourdes; Alvarez, Clara V; Lado-Abeal, Joaquin

    2008-01-01

    We studied two families from Galicia (northwest Spain) with Pendred syndrome (PS) and unusual thyroid phenotypes. In family A, the proposita had a large goiter and hypothyroxinemia but normal TSH and free T3 (FT3). In family B, some affected members showed deafness but not goiter. Our objective was to identify the mutations causing PS and molecular mechanisms underlying the thyroid phenotypes. Interventions included extraction of DNA and of thyroid tissue. Propositi and 10 members of the two families participated in the study. Main outcome measures included SLC26A4 gene analysis, deiodinase activities in thyroid tissue, and c.416-1G-->A effects on SLC26A4 splicing. In addition, a primary PS thyrocyte culture, T-PS2, was obtained from propositus B and compared with another culture of normal human thyrocytes, NT, by Western blotting, confocal microscopy, and iodine uptake kinetics. Proposita A was heterozygous for c.578C-->T and c.279delT, presented with goiter, and had normal TSH and FT3 but low FT4 attributable to high type 1 and type 2 iodothyronine deiodinase activities in the goiter. Propositus B bore c.279delT and a novel mutation c.416-1G-->A; some deaf relatives were homozygous for c.416-1G-->A but did not present goiter. The c.279delT mutation was associated with identical haplotype in the two families. T-PS2 showed truncated pendrin retained intracellularly and high iodine uptake with low efflux leading to iodine retention. c.279delT is a founder mutation in Galicia. Proposita A adapted to poor organification by increasing deiodinase activities in the goiter, avoiding hypothyroidism. Lack of goiter in subjects homozygous for c.416-1G-->A was due to incomplete penetrance allowing synthesis of some wild-type pendrin. Intracellular iodine retention, as seen in T-PS2, could play a role in thyroid alterations in PS.

  18. Glycolytic activity in breast cancer using 18F-FDG PET/CT as prognostic predictor: A molecular phenotype approach.

    Science.gov (United States)

    Garcia Vicente, A M; Soriano Castrejón, A; Amo-Salas, M; Lopez Fidalgo, J F; Muñoz Sanchez, M M; Alvarez Cabellos, R; Espinosa Aunion, R; Muñoz Madero, V

    2016-01-01

    To explore the relationship between basal (18)F-FDG uptake in breast tumors and survival in patients with breast cancer (BC) using a molecular phenotype approach. This prospective and multicentre study included 193 women diagnosed with BC. All patients underwent an (18)F-FDG PET/CT prior to treatment. Maximum standardized uptake value (SUVmax) in tumor (T), lymph nodes (N), and the N/T index was obtained in all the cases. Metabolic stage was established. As regards biological prognostic parameters, tumors were classified into molecular sub-types and risk categories. Overall survival (OS) and disease free survival (DFS) were obtained. An analysis was performed on the relationship between semi-quantitative metabolic parameters with molecular phenotypes and risk categories. The effect of molecular sub-type and risk categories in prognosis was analyzed using Kaplan-Meier and univariate and multivariate tests. Statistical differences were found in both SUVT and SUVN, according to the molecular sub-types and risk classifications, with higher semi-quantitative values in more biologically aggressive tumors. No statistical differences were observed with respect to the N/T index. Kaplan-Meier analysis revealed that risk categories were significantly related to DFS and OS. In the multivariate analysis, metabolic stage and risk phenotype showed a significant association with DFS. High-risk phenotype category showed a worst prognosis with respect to the other categories with higher SUVmax in primary tumor and lymph nodes. Copyright © 2015 Elsevier España, S.L.U. and SEMNIM. All rights reserved.

  19. A linker peptide with high affinity towards silica-containing materials.

    Science.gov (United States)

    Sunna, Anwar; Chi, Fei; Bergquist, Peter L

    2013-06-25

    A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Gidlöf, Ritha; Nielsen, Elsebet Østergaard

    2011-01-01

    Based on a pharmacophore model of the benzodiazepine-binding site of GABA(A) receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K......(i) values of around 0.20nM were determined. They show a structural resemblance with the previously described 2-phenyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones (II) and 2-phenyl-[1,2,4]triazolo[1,5-a]quinoxalin-4(5H)-one (III). The 9-bromo substituted compounds 8a-d were prepared in an 8-step synthesis...

  1. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding

    Science.gov (United States)

    Rosilo, Henna; McKee, Jason R.; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P.; Ikkala, Olli; Kostiainen, Mauri A.

    2014-09-01

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface

  2. Premature aging phenotype in mice lacking high affinity nicotinic receptors: region specific changes in layer V pyramidal cell morphology

    OpenAIRE

    Eleni Konsolaki

    2014-01-01

    The mechanisms by which aging leads to alterations in brain structure and cognitive deficits are unclear. A central yet presently unresolved issue in aging research concerns the distinction between normal/successful aging, consisting of a moderate decline in cognitive performance, and pathological aging, manifested as mild cognitive impairment or full-blown neurodegeneration and dementia. In particular, it has been proposed that the age-related decline in cognitive abilities may be an age-rel...

  3. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  4. Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Katz, D.M.; Kimelberg, H.K.

    1985-07-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; (/sup 3/H)-5-HT). At concentrations in the range of 0.01 to 0.7 microM (/sup 3/H)-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM (/sup 3/H)-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of (/sup 3/H)-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of (/sup 3/H)-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate (/sup 3/H)-5-HT up to 44-fold at an external (/sup 3/H)-5-HT concentration of 10(-7) M. Inhibition of (/sup 3/H)-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of (/sup 3/H)-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on (/sup 3/H)-5-HT uptake.

  5. Molecular characterization and phenotypical study of β-thalassemia in Tucumán, Argentina.

    Science.gov (United States)

    Lazarte, Sandra S; Mónaco, María E; Haro, Ana C; Jiménez, Cecilia L; Ledesma Achem, Myriam E; Issé, Blanca A

    2014-01-01

    The main hereditary hemoglobin (Hb) disorder in Argentina is β-thalassemia (β-thal). Molecular studies performed in the center of the country exhibited a marked prevalence of the codon 39 (C > T) and IVS-I-110 (G > A) mutations. The northwest region of Argentina has a different demographic history characterized by an important Spanish influx. Seventy-one β-thal carriers attending the Instituto de Bioquímica Aplicada, Tucumán, Argentina, were investigated for β-globin gene mutations by real-time polymerase chain reaction (RT-PCR). To examine the genotype-phenotype relationship, mean corpuscular volume (MCV), mean corpuscular Hb (MCH) and Hb A2 were measured. In order to recognize β-thal, Mentzer Index, Shine & Lal and Red Cell Distribution Width Index (RDWI), were calculated. The ethnic background of subjects revealed that 82.0% of the population was of Italian, Spanish and Arab origin. Seven mutations were detected: codon 39 (45.0%), IVS-I-1 (G > A) (22.5%), IVS-I-110 (16.3%), IVS-II-1 (G > A) (4.1%), IVS-I-1 (G > T) (2.0%), IVS-I-6 (T > C) (2.0%) and IVS-II-745 (G > C) (2.0%). In three families (6.1%), β-thal mutations were not determined. These results differed from other Argentinian studies because at present codon 39 and IVS-I-1 are the most prevalent; MCV, MCH and Hb A2 did not correlate with the type of mutation (β(0)/β(+)). Values of MCV (67.0 fL) and Hb A2 (4.85%) were unable to discriminate between them. Significant differences (p Argentina. Differences might represent the influence of Spanish immigration.

  6. Isolation, molecular and phenotypic characterization, and antibiotic susceptibility of Cronobacter spp. from Brazilian retail foods.

    Science.gov (United States)

    Brandão, Marcelo Luiz Lima; Umeda, Natália Scudeller; Jackson, Emily; Forsythe, Stephen James; de Filippis, Ivano

    2017-05-01

    Several Cronobacter species are opportunistic pathogens that cause infections in humans. The aim of this study was to detect Cronobacter spp. from 90 samples of retail foods in Brazil, and characterize the strains by phenotypic tests, molecular assays and antibiotic susceptibility. Three isolation methodologies were evaluated using different selective enrichments and the isolates were identified using Vitek 2.0, PCRs protocols, fusA allele sequencing and multilocus sequence typing (MLST). Thirty-eight samples (42.2%) contained Cronobacter spp., and the highest percentage was found in flours (66.7%, 20/30), followed by spices and herbs (36.7%, 11/30), and cereal mixes for children (23.3%, 7/30). The 45 isolates included four species: C. sakazakii (n = 37), C. malonaticus (n = 3), C. dublinensis (n = 3), and C. muytjensii (n = 2); that presented 20 different fusA alleles. MLST analysis revealed 32 sequence types (STs), 13 of which were newly identified. All strains were sensitive to all antibiotics (n = 10) tested. The combination of CSB/v enrichment with DFI plating was considered the most efficient for Cronobacter spp. isolation. This study revealed the presence of Cronobacter spp. in foods commercialized in Brazil and the isolates showed a high diversity after MLST analysis and included two strains of the C. sakazakii ST4 neonatal meningitic pathovar. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Molecular phenotypes in triple negative breast cancer from African American patients suggest targets for therapy.

    Science.gov (United States)

    Lindner, Robert; Sullivan, Catherine; Offor, Onyinye; Lezon-Geyda, Kimberly; Halligan, Kyle; Fischbach, Neal; Shah, Mansi; Bossuyt, Veerle; Schulz, Vincent; Tuck, David P; Harris, Lyndsay N

    2013-01-01

    Triple negative breast cancer (TNBC) is characterized by high proliferation, poor differentiation and a poor prognosis due to high rates of recurrence. Despite lower overall incidence African American (AA) patients suffer from higher breast cancer mortality in part due to the higher proportion of TNBC cases among AA patients compared to European Americans (EA). It was recently shown that the clinical heterogeneity of TNBC is reflected by distinct transcriptional programs with distinct drug response profiles in preclinical models. In this study, gene expression profiling and immunohistochemistry were used to elucidate potential differences between TNBC tumors of EA and AA patients on a molecular level. In a retrospective cohort of 136 TNBC patients, a major transcriptional signature of proliferation was found to be significantly upregulated in samples of AA ethnicity. Furthermore, transcriptional profiles of AA tumors showed differential activation of insulin-like growth factor 1 (IGF1) and a signature of BRCA1 deficiency in this cohort. Using signatures derived from the meta-analysis of TNBC gene expression carried out by Lehmann et al., tumors from AA patients were more likely of basal-like subtypes whereas transcriptional features of many EA samples corresponded to mesenchymal-like or luminal androgen receptor driven subtypes. These results were validated in The Cancer Genome Atlas mRNA and protein expression data, again showing enrichment of a basal-like phenotype in AA tumors and mesenchymal subtypes in EA tumors. In addition, increased expression of VEGF-activated genes together with elevated microvessel area determined by the AQUA method suggest that AA patients exhibit higher tumor vascularization. This study confirms the existence of distinct transcriptional programs in triple negative breast cancer in two separate cohorts and that these programs differ by racial group. Differences in TNBC subtypes and levels of tumor angiogenesis in AA versus EA patients

  8. Molecular phenotypes in triple negative breast cancer from African American patients suggest targets for therapy.

    Directory of Open Access Journals (Sweden)

    Robert Lindner

    Full Text Available Triple negative breast cancer (TNBC is characterized by high proliferation, poor differentiation and a poor prognosis due to high rates of recurrence. Despite lower overall incidence African American (AA patients suffer from higher breast cancer mortality in part due to the higher proportion of TNBC cases among AA patients compared to European Americans (EA. It was recently shown that the clinical heterogeneity of TNBC is reflected by distinct transcriptional programs with distinct drug response profiles in preclinical models. In this study, gene expression profiling and immunohistochemistry were used to elucidate potential differences between TNBC tumors of EA and AA patients on a molecular level. In a retrospective cohort of 136 TNBC patients, a major transcriptional signature of proliferation was found to be significantly upregulated in samples of AA ethnicity. Furthermore, transcriptional profiles of AA tumors showed differential activation of insulin-like growth factor 1 (IGF1 and a signature of BRCA1 deficiency in this cohort. Using signatures derived from the meta-analysis of TNBC gene expression carried out by Lehmann et al., tumors from AA patients were more likely of basal-like subtypes whereas transcriptional features of many EA samples corresponded to mesenchymal-like or luminal androgen receptor driven subtypes. These results were validated in The Cancer Genome Atlas mRNA and protein expression data, again showing enrichment of a basal-like phenotype in AA tumors and mesenchymal subtypes in EA tumors. In addition, increased expression of VEGF-activated genes together with elevated microvessel area determined by the AQUA method suggest that AA patients exhibit higher tumor vascularization. This study confirms the existence of distinct transcriptional programs in triple negative breast cancer in two separate cohorts and that these programs differ by racial group. Differences in TNBC subtypes and levels of tumor angiogenesis in AA

  9. Contrasting the distribution of phenotypic and molecular variation in the freshwater snail Biomphalaria pfeifferi, the intermediate host of Schistosoma mansoni

    OpenAIRE

    Tian-Bi, Y-NT; Jarne, P; Konan, J-NK; Utzinger, J; N'Goran, E K

    2013-01-01

    Population differentiation was investigated by confronting phenotypic and molecular variation in the highly selfing freshwater snail Biomphalaria pfeifferi, the intermediate host of Schistosoma mansoni. We sampled seven natural populations separated by a few kilometers, and characterized by different habitat regimes (permanent/temporary) and openness (open/closed). A genetic analysis based on five microsatellite markers confirms that B. pfeifferi is a selfer (s≈0.9) and exhi...

  10. In silico investigation of PARP-1 catalytic domains in holo and apo states for the design of high-affinity PARP-1 inhibitors.

    Science.gov (United States)

    Salmas, Ramin Ekhteiari; Unlu, Ayhan; Yurtsever, Mine; Noskov, Sergei Y; Durdagi, Serdar

    2016-01-01

    The rational design of high-affinity inhibitors of poly-ADP-ribose polymerase-1 (PARP-1) is at the heart of modern anti-cancer drug design. While relevance of enzyme to DNA repair processes in cellular environment is firmly established, the structural and functional understanding of the main determinants for high-affinity ligands controlling PARP-1 activity is still lacking. The conserved active site of PARP-1 represents an ideal target for inhibitors and may offer a novel target at the treatment of breast cancer. To fill the gap in the structural knowledge, we report on the combination of molecular dynamics (MD) simulations, principal component analysis (PCA), and conformational analysis that analyzes in great details novel binding mode for a number of inhibitors at the PARP-1. While optimization of the binding affinity for original target is an important goal in the drug design, many of the promising molecules for treatment of the breast cancer are plagued by significant cardiotoxicity. One of the most common side-effects reported for a number of polymerase inhibitors is its off-target interactions with cardiac ion channels and hERG1 channel, in particular. Thus, selected candidate PARP-1 inhibitors were also screened in silico at the central cavities of hERG1 potassium ion channel.

  11. Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri.

    Science.gov (United States)

    Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

    2016-07-22

    Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H(+) or Na(+) electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4',6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Profiling the extended phenotype of plant pathogens: Challenges in Bacterial Molecular Plant Pathology.

    Science.gov (United States)

    Preston, Gail M

    2017-04-01

    One of the most fundamental questions in plant pathology is what determines whether a pathogen grows within a plant? This question is frequently studied in terms of the role of elicitors and pathogenicity factors in the triggering or overcoming of host defences. However, this focus fails to address the basic question of how the environment in host tissues acts to support or restrict pathogen growth. Efforts to understand this aspect of host-pathogen interactions are commonly confounded by several issues, including the complexity of the plant environment, the artificial nature of many experimental infection systems and the fact that the physiological properties of a pathogen growing in association with a plant can be very different from the properties of the pathogen in culture. It is also important to recognize that the phenotype and evolution of pathogen and host are inextricably linked through their interactions, such that the environment experienced by a pathogen within a host, and its phenotype within the host, is a product of both its interaction with its host and its evolutionary history, including its co-evolution with host plants. As the phenotypic properties of a pathogen within a host cannot be defined in isolation from the host, it may be appropriate to think of pathogens as having an 'extended phenotype' that is the product of their genotype, host interactions and population structure within the host environment. This article reflects on the challenge of defining and studying this extended phenotype, in relation to the questions posed below, and considers how knowledge of the phenotype of pathogens in the host environment could be used to improve disease control. What determines whether a pathogen grows within a plant? What aspects of pathogen biology should be considered in describing the extended phenotype of a pathogen within a host? How can we study the extended phenotype in ways that provide insights into the phenotypic properties of pathogens

  13. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    Directory of Open Access Journals (Sweden)

    Thomas Stockner

    Full Text Available The high-resolution crystal structure of the leucine transporter (LeuT is frequently used as a template for homology models of the dopamine transporter (DAT. Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii LeuT and DAT share a rather low overall sequence identity (22% and (iii the extracellular loop 2 (EL2 of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  14. Synthetic 1,2,3-triazole-linked glycoconjugates bind with high affinity to human galectin-3.

    Science.gov (United States)

    Marchiori, Marcelo Fiori; Souto, Dênio Emanuel Pires; Bortot, Leandro Oliveira; Pereira, João Francisco; Kubota, Lauro Tatsuo; Cummings, Richard D; Dias-Baruffi, Marcelo; Carvalho, Ivone; Campo, Vanessa Leiria

    2015-07-01

    This work describes the synthesis of the 1,2,3-triazole amino acid-derived-3-O-galactosides 1-6 and the 1,2,3-triazole di-lactose-derived glycoconjugate 7 as potential galectin-3 inhibitors. The target compounds were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-derived amino acids N3-ThrOBn, N3-PheOBn, N3-N-Boc-TrpOBn, N3-N-Boc-LysOBn, N3-O-tBu-AspOBn and N3-l-TyrOH, and the corresponding alkyne-based sugar 3-O-propynyl-GalOMe, as well as by click chemistry reaction between the azido-lactose and 2-propynyl lactose. Surface plasmon resonance (SPR) assays showed that all synthetic glycoconjugates 1-7 bound to galectin-3 with high affinity, but the highest binders were the amino acids-derived glycoconjugates 2 (KD 7.96μM) and 4 (KD 4.56μM), and the divalent lactoside 7 (KD1 0.15μM/KD2 19μM). Molecular modeling results were in agreement with SPR assays, since more stable interactions with galectin-3 were identified for glycoconjugates 2, 4 and 7. Regarding compounds 2 and 4, they established specific cation-π (Arg144) and ionic (Asp148) interactions, whereas glycoconjugate 7 was capable to bridge two independent galectin-3 CRDs, creating a non-covalent cross-link between two monomers and, thus, reaching a submicromolar affinity towards galectin-3. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.

    Directory of Open Access Journals (Sweden)

    Audrey D McConnell

    Full Text Available A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID. Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.

  16. Melatonin Administration Alters Nicotine Preference Consumption via Signaling Through High-Affinity Melatonin Receptors

    Science.gov (United States)

    Horton, William J.; Gissel, Hannah J.; Saboy, Jennifer E.; Wright, Kenneth P.; Stitzel, Jerry A.

    2015-01-01

    Rationale While it is known that tobacco use varies across the 24-hour day, the time-of-day effects are poorly understood. Findings from several previous studies indicate a potential role for melatonin in these time-of-day effects; however the specific underlying mechanisms have not been well characterized. Understanding of these mechanisms may lead to potential novel smoking cessation treatments. Objective Examine the role of melatonin and melatonin receptors in nicotine free choice consumption Methods A two-bottle oral nicotine choice paradigm was utilized with melatonin supplementation in melatonin deficient mice (C57BL/6J) or without melatonin supplementation in mice proficient at melatonin synthesis (C3H/Ibg) compared to melatonin proficient mice lacking both or one of the high affinity melatonin receptors (MT1 and MT2; double null mutant DM, or MT1 or MT2). Preference for bitter and sweet tastants also was assessed in wild type and MT1 and MT2 DM mice. Finally, home cage locomotor monitoring was performed to determine the effect of melatonin administration on activity patterns. Results Supplemental melatonin in drinking water significantly reduced free-choice nicotine consumption in C57BL/6J mice, which do not produce endogenous melatonin, while not altering activity patterns. Independently, genetic deletion of both MT1 and MT2 receptors in a melatonin proficient mouse strain (C3H) resulted in significantly more nicotine consumption than controls. However single genetic deletion of either the MT1 or MT2 receptor alone did not result in increased nicotine consumption. Deletion of MT1 and MT2 did not impact taste preference. Conclusions This study demonstrates that nicotine consumption can be affected by exogenous or endogenous melatonin and requires at least one of the high-affinity melatonin receptors. The fact that expression of either the MT1 or MT2 melatonin receptor is sufficient to maintain lower nicotine consumption suggests functional overlap and

  17. Melatonin administration alters nicotine preference consumption via signaling through high-affinity melatonin receptors.

    Science.gov (United States)

    Horton, William J; Gissel, Hannah J; Saboy, Jennifer E; Wright, Kenneth P; Stitzel, Jerry A

    2015-07-01

    While it is known that tobacco use varies across the 24-h day, the time-of-day effects are poorly understood. Findings from several previous studies indicate a potential role for melatonin in these time-of-day effects; however, the specific underlying mechanisms have not been well characterized. Understanding of these mechanisms may lead to potential novel smoking cessation treatments. The objective of this study is examine the role of melatonin and melatonin receptors in nicotine free-choice consumption A two-bottle oral nicotine choice paradigm was utilized with melatonin supplementation in melatonin-deficient mice (C57BL/6J) or without melatonin supplementation in mice proficient at melatonin synthesis (C3H/Ibg) compared to melatonin-proficient mice lacking both or one of the high-affinity melatonin receptors (MT1 and MT2; double-null mutant DM, or MT1 or MT2). Preference for bitter and sweet tastants also was assessed in wild-type and MT1 and MT2 DM mice. Finally, home cage locomotor monitoring was performed to determine the effect of melatonin administration on activity patterns. Supplemental melatonin in drinking water significantly reduced free-choice nicotine consumption in C57BL/6J mice, which do not produce endogenous melatonin, while not altering activity patterns. Independently, genetic deletion of both MT1 and MT2 receptors in a melatonin-proficient mouse strain (C3H) resulted in significantly more nicotine consumption than controls. However, single genetic deletion of either the MT1 or MT2 receptor alone did not result in increased nicotine consumption. Deletion of MT1 and MT2 did not impact taste preference. This study demonstrates that nicotine consumption can be affected by exogenous or endogenous melatonin and requires at least one of the high-affinity melatonin receptors. The fact that expression of either the MT1 or MT2 melatonin receptor is sufficient to maintain lower nicotine consumption suggests functional overlap and potential mechanistic

  18. Genetic and Molecular Characterization of Drosophila Brakeless: A Novel Modifier of Merlin Phenotypes

    National Research Council Canada - National Science Library

    LaJeunesse, Dennis

    2004-01-01

    .... Using genetic epistasis, we show that Merlin functions upstream of both scribbler and Cyclin E - demonstrating that Merlin is a dominant second site repressor of loss of function phenotypes for Cyclin E...

  19. Connecting genes, coexpression modules, and molecular signitures to environmental stress phenotypes in plants

    Energy Technology Data Exchange (ETDEWEB)

    Weston, David [ORNL; Gunter, Lee E [ORNL; Rogers, Alistair [ORNL; Wullschleger, Stan D [ORNL

    2008-01-01

    Background One of the eminent opportunities afforded by modern genomic technologies is the potential to provide a mechanistic understanding of the processes by which genetic change translates to phenotypic variation and the resultant appearance of distinct physiological traits. Indeed much progress has been made in this area, particularly in biomedicine where functional genomic information can be used to determine the physiological state (e.g., diagnosis) and predict phenotypic outcome (e.g., patient survival). Ecology currently lacks an analogous approach where genomic information can be used to diagnose the presence of a given physiological state (e.g., stress response) and then predict likely phenotypic outcomes (e.g., stress duration and tolerance, fitness). Results Here, we demonstrate that a compendium of genomic signatures can be used to classify the plant abiotic stress phenotype in Arabidopsis according to the architecture of the transcriptome, and then be linked with gene coexpression network analysis to determine the underlying genes governing the phenotypic response. Using this approach, we confirm the existence of known stress responsive pathways and marker genes, report a common abiotic stress responsive transcriptome and relate phenotypic classification to stress duration. Conclusion Linking genomic signatures to gene coexpression analysis provides a unique method of relating an observed plant phenotype to changes in gene expression that underlie that phenotype. Such information is critical to current and future investigations in plant biology and, in particular, to evolutionary ecology, where a mechanistic understanding of adaptive physiological responses to abiotic stress can provide researchers with a tool of great predictive value in understanding species and population level adaptation to climate change.

  20. Connecting genes, coexpression modules, and molecular signatures to environmental stress phenotypes in plants

    Directory of Open Access Journals (Sweden)

    Rogers Alistair

    2008-02-01

    Full Text Available Abstract Background One of the eminent opportunities afforded by modern genomic technologies is the potential to provide a mechanistic understanding of the processes by which genetic change translates to phenotypic variation and the resultant appearance of distinct physiological traits. Indeed much progress has been made in this area, particularly in biomedicine where functional genomic information can be used to determine the physiological state (e.g., diagnosis and predict phenotypic outcome (e.g., patient survival. Ecology currently lacks an analogous approach where genomic information can be used to diagnose the presence of a given physiological state (e.g., stress response and then predict likely phenotypic outcomes (e.g., stress duration and tolerance, fitness. Results Here, we demonstrate that a compendium of genomic signatures can be used to classify the plant abiotic stress phenotype in Arabidopsis according to the architecture of the transcriptome, and then be linked with gene coexpression network analysis to determine the underlying genes governing the phenotypic response. Using this approach, we confirm the existence of known stress responsive pathways and marker genes, report a common abiotic stress responsive transcriptome and relate phenotypic classification to stress duration. Conclusion Linking genomic signatures to gene coexpression analysis provides a unique method of relating an observed plant phenotype to changes in gene expression that underlie that phenotype. Such information is critical to current and future investigations in plant biology and, in particular, to evolutionary ecology, where a mechanistic understanding of adaptive physiological responses to abiotic stress can provide researchers with a tool of great predictive value in understanding species and population level adaptation to climate change.

  1. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    Science.gov (United States)

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  2. Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis

    DEFF Research Database (Denmark)

    Holst, B; Hastrup, H; Raffetseder, U

    2001-01-01

    either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P...

  3. Early signs of pathological cognitive aging in mice lacking high-affinity nicotinic receptors.

    Directory of Open Access Journals (Sweden)

    Eleni eKonsolaki

    2016-04-01

    Full Text Available In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. Α deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-, which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioural signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviours, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm and extend the hypothesis that β2-/- animals exhibit age-related cognitive impairments, manifested in both spatial learning and recognition memory tasks. In addition, we reveal deficits in spontaneous behaviour and habituation processes earlier in life. To our knowledge, this is the first study to perform an extensive behavioural examination of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioural changes to global dementia due to the combined effect of the neuropathology and aging.

  4. Identification of LAG3 high affinity aptamers by HT-SELEX and Conserved Motif Accumulation (CMA.

    Directory of Open Access Journals (Sweden)

    Mario Martínez Soldevilla

    Full Text Available LAG3 receptor belongs to a family of immune-checkpoints expressed in T lymphocytes and other cells of the immune system. It plays an important role as a rheostat of the immune response. Focus on this receptor as a potential therapeutic target in cancer immunotherapy has been underscored after the success of other immune-checkpoint blockade strategies in clinical trials. LAG3 showcases the interest in the field of autoimmunity as several studies show that LAG3-targeting antibodies can also be used for the treatment of autoimmune diseases. In this work we describe the identification of a high-affinity LAG3 aptamer by High Throughput Sequencing SELEX in combination with a study of potential conserved binding modes according to sequence conservation by using 2D-structure prediction and 3D-RNA modeling using Rosetta. The aptamer with the highest accumulation of these conserved sequence motifs displays the highest affinity to LAG3 recombinant soluble proteins and binds to LAG3-expressing lymphocytes. The aptamer described herein has the potential to be used as a therapeutic agent, as it enhances the threshold of T-cell activation. Nonetheless, in future applications, it could also be engineered for treatment of autoimmune diseases by target depletion of LAG3-effector T lymphocytes.

  5. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination.

    Science.gov (United States)

    Kaur, Kaval; Zheng, Nai-Ying; Smith, Kenneth; Huang, Min; Li, Lie; Pauli, Noel T; Henry Dunand, Carole J; Lee, Jane-Hwei; Morrissey, Michael; Wu, Yixuan; Joachims, Michelle L; Munroe, Melissa E; Lau, Denise; Qu, Xinyan; Krammer, Florian; Wrammert, Jens; Palese, Peter; Ahmed, Rafi; James, Judith A; Wilson, Patrick C

    2015-01-01

    Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.

  6. Glutaraldehyde pretreatment blocks temperature-induced high-affinity (/sup 3/H) tryptamine binding

    Energy Technology Data Exchange (ETDEWEB)

    Serikyaku, S.; Ishitani, R.

    1988-01-01

    The effect of glutaraldehyde (and Azure A) on temperature-sensitive high-affinity (/sup 3/H) tryptamine binding was investigated in rat brain synaptic plasma membranes. In the 0.01-0.1 % concentration range, the glutaraldehyde pretreatment preferentially inhibited only the above-mentioned portion of the binding, whereas the posttreatment of this reagent had no effect. On the other hand, in cases of pretreatment or posttreatment, a concentration of glutaraldehyde as high as 0.1 % was inactive on the basal (/sup 3/H) ligand binding capacity of the membranes. Furthermore, it was revealed that the Scatchard plot of (/sup 3/H) tryptamine binding in membranes pretreated with glutaraldehyde conformed to a straight line, as did a similar plot of temperature-independent binding. And, it was interesting to find that the binding parameters (K/sub D/ and B/sub max/ values) of both samples corresponded closely to each other. On the contrary, in all concentrations, Azure A affected nonspecifically both the temperature-dependent and the independent (/sup 3/H) tryptamine binding to the same degree, regardless of whether or not there was pretreatment or posttreatment. 17 references, 2 figures, 1 table.

  7. Productive common light chain libraries yield diverse panels of high affinity bispecific antibodies

    Science.gov (United States)

    Van Blarcom, Thomas; Melton, Zea; Cheung, Wai Ling; Wagstrom, Chris; McDonough, Dan; Valle Oseguera, Cendy; Ding, Sheng; Rossi, Andrea; Potluri, Shobha; Sundar, Purnima; Sirota, Marina; Yan, Yu; Jones, Jeffrey; Roe-Zurz, Zygy; Srivatsa Srinivasan, Surabhi; Zhai, Wenwu; Pons, Jaume; Rajpal, Arvind; Chaparro-Riggers, Javier

    2018-01-01

    ABSTRACT The commercial success of bispecific antibodies generally has been hindered by the complexities associated with generating appropriate molecules for both research scale and large scale manufacturing purposes. Bispecific IgG (BsIgG) based on two antibodies that use an identical common light chain can be combined with a minimal set of Fc mutations to drive heavy chain heterodimerization in order to address these challenges. However, the facile generation of common light chain antibodies with properties similar to traditional monoclonal antibodies has not been demonstrated and they have only been used sparingly. Here, we describe the design of a synthetic human antibody library based on common light chains to generate antibodies with biochemical and biophysical properties that are indistinguishable to traditional therapeutic monoclonal antibodies. We used this library to generate diverse panels of well-behaved, high affinity antibodies toward a variety of epitopes across multiple antigens, including mouse 4-1BB, a therapeutically important T cell costimulatory receptor. Over 200 BsIgG toward 4-1BB were generated using an automated purification method we developed that enables milligram-scale production of BsIgG. This approach allowed us to identify antibodies with a wide range of agonistic activity that are being used to further investigate the therapeutic potential of antibodies targeting one or more epitopes of 4-1BB. PMID:29227213

  8. Enhanced membrane pore formation through high-affinity targeted antimicrobial peptides.

    Directory of Open Access Journals (Sweden)

    Christopher J Arnusch

    Full Text Available Many cationic antimicrobial peptides (AMPs target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted.

  9. The chromosomal high-affinity binding sites for the Drosophila dosage compensation complex.

    Directory of Open Access Journals (Sweden)

    Tobias Straub

    2008-12-01

    Full Text Available Dosage compensation in male Drosophila relies on the X chromosome-specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC. The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called "high-affinity sites" (HAS. Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features.

  10. Function and Regulation of the Plant COPT Family of High-Affinity Copper Transport Proteins

    Directory of Open Access Journals (Sweden)

    Sergi Puig

    2014-01-01

    Full Text Available Copper (Cu is an essential micronutrient for all eukaryotes because it participates as a redox active cofactor in multiple biological processes, including mitochondrial respiration, photosynthesis, oxidative stress protection, and iron (Fe transport. In eukaryotic cells, Cu transport toward the cytoplasm is mediated by the conserved CTR/COPT family of high-affinity Cu transport proteins. This outlook paper reviews the contribution of our research group to the characterization of the function played by the Arabidopsis thaliana COPT1–6 family of proteins in plant Cu homeostasis. Our studies indicate that the different tissue specificity, Cu-regulated expression, and subcellular localization dictate COPT-specialized contribution to plant Cu transport and distribution. By characterizing lack-of-function Arabidopsis mutant lines, we conclude that COPT1 mediates root Cu acquisition, COPT6 facilitates shoot Cu distribution, and COPT5 mobilizes Cu from storage organelles. Furthermore, our work with copt2 mutant and COPT-overexpressing plants has also uncovered Cu connections with Fe homeostasis and the circadian clock, respectively. Future studies on the interaction between COPT transporters and other components of the Cu homeostasis network will improve our knowledge of plant Cu acquisition, distribution, regulation, and utilization by Cu-proteins.

  11. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination.

    Directory of Open Access Journals (Sweden)

    Kaval Kaur

    Full Text Available Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE. Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.

  12. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  13. Phenotypic and molecular characterization of plants regenerated from non-cryopreserved and cryopreserved wild Solanum lycopersicum mill. Seeds.

    Science.gov (United States)

    Zevallos, B; Cejas, I; Engelmann, F; Carputo, D; Aversano, R; Scarano, M T; Yanes, E; Martinez-Montero, M; Lorenzo, J C

    2014-01-01

    Before cryopreservation is routinely used, its effect on the trueness-to-type of the regenerated plant material needs to be evaluated. In this work, we studied the effect of seed cryopreservation on the phenotypic and molecular characteristics of wild Solanum lycopersicum Mill. plants. Thirty-five morphological traits of plants regenerated from cryopreserved seeds were compared to those measured on plants regenerated from non-cryopreserved seeds. No statistically significant differences were observed between cryopreserved and non-cryopreserved samples, either in the first or in the second generation post-liquid nitrogen exposure. However, at the molecular level, the genetic analyses performed on the second generation plants germinated from control and cryopreserved seeds using 14 nuclear Simple Sequences Repeats (SSR) markers uncovered some changes in microsatellite length between control and cryopreserved samples. These results confirm at the botanical phenotype level the effectiveness of seed cryostorage for conservation and regeneration of true-to-type S. lycopersicum plants. Further experiments are required to clarify potential phenotypic effects of the changes observed in the DNA.

  14. Phenotypic and molecular characterisation of CDK13-related congenital heart defects, dysmorphic facial features and intellectual developmental disorders.

    Science.gov (United States)

    Bostwick, Bret L; McLean, Scott; Posey, Jennifer E; Streff, Haley E; Gripp, Karen W; Blesson, Alyssa; Powell-Hamilton, Nina; Tusi, Jessica; Stevenson, David A; Farrelly, Ellyn; Hudgins, Louanne; Yang, Yaping; Xia, Fan; Wang, Xia; Liu, Pengfei; Walkiewicz, Magdalena; McGuire, Marianne; Grange, Dorothy K; Andrews, Marisa V; Hummel, Marybeth; Madan-Khetarpal, Suneeta; Infante, Elena; Coban-Akdemir, Zeynep; Miszalski-Jamka, Karol; Jefferies, John L; Rosenfeld, Jill A; Emrick, Lisa; Nugent, Kimberly M; Lupski, James R; Belmont, John W; Lee, Brendan; Lalani, Seema R

    2017-08-14

    De novo missense variants in CDK13 have been described as the cause of syndromic congenital heart defects in seven individuals ascertained from a large congenital cardiovascular malformations cohort. We aimed to further define the phenotypic and molecular spectrum of this newly described disorder. To minimise ascertainment bias, we recruited nine additional individuals with CDK13 pathogenic variants from clinical and research exome laboratory sequencing cohorts. Each individual underwent dysmorphology exam and comprehensive medical history review. We demonstrate greater than expected phenotypic heterogeneity, including 33% (3/9) of individuals without structural heart disease on echocardiogram. There was a high penetrance for a unique constellation of facial dysmorphism and global developmental delay, as well as less frequently seen renal and sacral anomalies. Two individuals had novel CDK13 variants (p.Asn842Asp, p.Lys734Glu), while the remaining seven unrelated individuals had a recurrent, previously published p.Asn842Ser variant. Summary of all variants published to date demonstrates apparent restriction of pathogenic variants to the protein kinase domain with clustering in the ATP and magnesium binding sites. Here we provide detailed phenotypic and molecular characterisation of individuals with pathogenic variants in CDK13 and propose management guidelines based upon the estimated prevalence of anomalies identified.

  15. ASK1 (MAP3K5 is transcriptionally upregulated by E2F1 in adipose tissue in obesity, molecularly defining a human dys-metabolic obese phenotype

    Directory of Open Access Journals (Sweden)

    Yulia Haim

    2017-07-01

    Conclusions: AT E2F1 –ASK1 molecularly defines a metabolically-detrimental obese sub-phenotype. Functionally, it may negatively affect AT endocrine function, linking AT stress to whole-body metabolic dysfunction.

  16. A further case of a Prader-Willi syndrome phenotype in a patient with Angelman syndrome molecular defect

    Directory of Open Access Journals (Sweden)

    De Molfetta Greice Andreotti

    2002-01-01

    Full Text Available Angelman syndrome (AS and Prader-Willi syndrome (PWS are distinct human neurogenetic disorders; however, a clinical overlap between AS and PWS has been identified. We report on a further case of a patient showing the PWS phenotype with the AS molecular defect. Despite the PWS phenotype, the DNA methylation analysis of SNRPN revealed an AS pattern. Cytogenetic and FISH analysis showed normal chromosomes 15 and microsatellite analysis showed heterozygous loci inside and outside the 15q11-13 region. The presence of these atypical cases could be more frequent than previously expected and we reinforce that the DNA methylation analysis is important for the correct diagnosis of severe mental deficiency, congenital hypotonia and obesity.

  17. The physiological significance of HKT1, a Na{sup +} - coupled high affinity K{sup +} transporter in `Triticum aestivum`

    Energy Technology Data Exchange (ETDEWEB)

    Box, S.; Schachtman, D.P. [University of Adelaide, SA (Australia). Department of Botany

    1997-12-31

    Full text: Several mechanisms for high affinity K{sup +} uptake by higher plants have been proposed:-an ATP-energised K:+ pump, a K{sup +}/H{sup +} antiport and a H{sup +}coupled carrier. Recently, a Na{sup +}--coupled high affinity K{sup +} transporter, HKT1, was isolated from wheat roots. Whilst Na{sup +}K{sup +} symports have been described in charophyte algae, the cloning of HKT1 from wheat is the first, evidence that this type d transport mechanism may function in higher plants. Is the activity of HKT1 an important mechanism involved in K{sup +} acquisition by wheat? The aim of this study was to assess the physiological significance of Na{sup +}- coupled high affinity K{sup +} uptake in T. aestivum. To determine whether HKT1 plays a significant role in wheat growth, we measured the dry weights and ion content of plants grown in a range of [K{sup +}], with and without Na{sup +}. To directly assess the activity of Na{sup +}- coupled K{sup +} transport, {sup 86}Rb{sup +} and {sup 22}Na{sup +} flux analyses were performed on the elongation zones and whole roots of intact seedlings, expressing a high affinity K{sup +} uptake system. The results of these growth and tracer flux studies will be discussed in relation to the expression of the gene encoding HKT1 in T. aestivum

  18. Novel high-affinity and selective biaromatic 4-substituted ¿-hydroxybutyric acid (GHB) analogues as GHB ligands

    DEFF Research Database (Denmark)

    Høg, Signe; Wellendorph, Petrine; Nielsen, Birgitte

    2008-01-01

    Gamma-hydroxybutyrate (GHB) is a metabolite of gamma-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA(B) receptors and specific high-affinity GHB sites...

  19. Are basophil histamine release and high affinity IgE receptor expression involved in asymptomatic skin sensitization?

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Assing, K; Jensen, Lone Hummelshøj

    2006-01-01

    Immunoglobulin (Ig)E-sensitized persons with positive skin prick test, but no allergy symptoms, are classified as being asymptomatic skin sensitized (AS). The allergic type 1 disease is dependant on IgE binding to the high affinity IgE-receptor (FcepsilonRI) expressed on basophils and mast cells...

  20. A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

    DEFF Research Database (Denmark)

    Zhang, Peng; Jørgensen, Trine Nygaard; Løland, Claus Juul

    2013-01-01

    A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino)prop...

  1. A dualistic conformational response to substrate binding in the human serotonin transporter reveals a high affinity state for serotonin

    DEFF Research Database (Denmark)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida

    2015-01-01

    that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation...

  2. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-Affinity γ-Hydroxybutyrate (GHB) Binding Sites

    DEFF Research Database (Denmark)

    Vogensen, Stine B.; Marek, Ales; Bay, Tina

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screen...

  3. Genotype to phenotype

    National Research Council Canada - National Science Library

    Malcolm, Sue; Goodship, Timothy H. J

    2001-01-01

    ... Disorders Molecular Genetics of Hypertension Human Gene EvolutionAnalysis of Multifactorial Disease Transcription Factors Molecular Genetics of Cancer, Second edition Genotype to Phenotype, second e...

  4. Land-Use Influences the Distribution and Activity of High Affinity CO-Oxidizing Bacteria Associated to type I-coxL Genotype in Soil

    Directory of Open Access Journals (Sweden)

    Liliana eQuiza

    2014-06-01

    Full Text Available Soil carboxydovore bacteria are the biological sink of atmospheric carbon monoxide (CO. The initial oxidation of CO is catalyzed by a CO-dehydrogenase (CODH, and the gene coxL encodes the large subunit of the enzyme. Only a few carboxydovore isolates were shown to oxidize atmospheric CO and little is known about the potential impact of global change on the ecophysiology of this functional group. The main objective of this study was to assess the impact of land-use and soil properties on coxL gene diversity and identify molecular indicators for the soil uptake of atmospheric CO. Soil samples were collected in three neighboring sites encompassing different land-use types, namely deciduous forest, larch plantation and maize field. CO uptake activity was related to total carbon and nitrogen content in soil, with the highest activity observed in deciduous forest. An extensive coxL database was assembled to optimize a PCR detection assay targeting sequences belonging to functional type I-CODH and hypothetical type II-CODH. Fully replicated coxL gene libraries unveiled a unique molecular signature in deciduous forest soil, with enrichment of type I sequences. Genetic profiles of larch and maize monocultures were not statistically different and showed higher level of coxL gene richness than deciduous forest. Soil water content and CO uptake activity explained 38% of the variation of coxL gene profiles in a canonical ordination analysis, leading to the identification of sequences belonging to the δ-Proteobacteria cluster as indicator for high affinity CO uptake activity. Enrichment of type I and δ-Proteobacteria coxL sequences in deciduous forest were confirmed by qPCR in an independent soil survey. CO uptake activity in model carboxydovore bacteria suggested that a significant fraction of detected putative high affinity CO oxidizers were active in soil. Land-use was a driving force separating coxL diversity in deciduous forest from monocultures.

  5. Land-use influences the distribution and activity of high affinity CO-oxidizing bacteria associated to type I-coxL genotype in soil.

    Science.gov (United States)

    Quiza, Liliana; Lalonde, Isabelle; Guertin, Claude; Constant, Philippe

    2014-01-01

    Soil carboxydovore bacteria are the biological sink of atmospheric carbon monoxide (CO). The initial oxidation of CO is catalyzed by a CO-dehydrogenase (CODH), and the gene coxL encodes the large subunit of the enzyme. Only a few carboxydovore isolates were shown to oxidize atmospheric CO and little is known about the potential impact of global change on the ecophysiology of this functional group. The main objective of this study was to assess the impact of land-use and soil properties on coxL gene diversity and identify molecular indicators for the soil uptake of atmospheric CO. Soil samples were collected in three neighboring sites encompassing different land-use types, namely deciduous forest, larch plantation and maize field. CO uptake activity was related to total carbon and nitrogen content in soil, with the highest activity observed in deciduous forest. An extensive coxL database was assembled to optimize a PCR detection assay targeting sequences belonging to functional type I-CODH and hypothetical type II-CODH. Fully replicated coxL gene libraries unveiled a unique molecular signature in deciduous forest soil, with enrichment of type I sequences. Genetic profiles of larch and maize monocultures were not statistically different and showed higher level of coxL gene richness than deciduous forest. Soil water content and CO uptake activity explained 38% of the variation of coxL gene profiles in a canonical ordination analysis, leading to the identification of sequences belonging to the δ-Proteobacteria cluster as indicator for high affinity CO uptake activity. Enrichment of type I and δ-Proteobacteria coxL sequences in deciduous forest were confirmed by qPCR in an independent soil survey. CO uptake activity in model carboxydovore bacteria suggested that a significant fraction of detected putative high affinity CO oxidizers were active in soil. Land-use was a driving force separating coxL diversity in deciduous forest from monocultures.

  6. Analysis of high-affinity binding of protein kinase R to double-stranded RNA.

    Science.gov (United States)

    Husain, Bushra; Mukerji, Ishita; Cole, James L

    2012-11-06

    Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity response to viral infection. PKR is activated upon binding to double-stranded RNA (dsRNA). Our previous analysis of binding of PKR to dsRNAs ranging from 20 to 40 bp supports a dimerization model for activation in which 30 bp represents the minimal length required to bind two PKR monomers and activate PKR via autophosphorylation. These studies were complicated by the formation of protein-RNA aggregates, particularly at low salt concentrations using longer dsRNAs. Here, we have taken advantage of the enhanced sensitivity afforded using fluorescence-detected analytical ultracentrifugation to reduce the RNA concentrations from micromolar to nanomolar. Under these conditions, we are able to characterize high-affinity binding of PKR to longer dsRNAs in 75 mM NaCl. The PKR binding stoichiometries are increased at lower salt concentrations but remain lower than those previously obtained for the dsRNA binding domain. The dependence of the limiting PKR binding stoichiometries on dsRNA length does not conform to standard models for nonspecific binding and suggests that binding to longer sequences occurs via a different binding mode with a larger site size. Although dimerization plays a key role in the PKR activation mechanism, the ability of shorter dsRNAs to bind two PKR monomers is not sufficient to induce autophosphorylation. We propose that activation of PKR by longer RNAs is correlated with an alternative binding mode in which both of the dsRNA binding motifs contact the RNA, inducing PKR to dimerize via a direct interaction of the kinase domains.

  7. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT.

    Directory of Open Access Journals (Sweden)

    Katherine J Fishwick

    Full Text Available Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer's Disease (AD, and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh is closely linked to the activity of the high-affinity choline transporter protein (CHT, but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization.

  8. Integrin alphaVbeta6 is a high-affinity receptor for coxsackievirus A9.

    Science.gov (United States)

    Heikkilä, Outi; Susi, Petri; Stanway, Glyn; Hyypiä, Timo

    2009-01-01

    Coxsackievirus A9 (CAV9), a member of the genus Enterovirus in the family Picornaviridae, possesses an integrin-binding arginine-glycine-aspartic acid (RGD) motif in the C terminus of VP1 capsid protein. CAV9 has been shown to utilize integrins alphaVbeta3 and alphaVbeta6 as primary receptors for cell attachment. While CAV9 RGD-mutants (RGE and RGDdel) are capable of infecting rhabdomyosarcoma (RD) cell line, they grow very poorly in an epithelial lung carcinoma cell line (A549). In this study, the relationships between CAV9 infectivity in A549 and RD cells, receptor expression and integrin binding were analysed. A549 cells were shown to express both integrins alphaVbeta3 and alphaVbeta6, whereas alphaVbeta6 expression was not detected on the RD cells. Native CAV9 but not RGE and RGDdel mutants bound efficiently to immobilized alphaVbeta3 and alphaVbeta6. Adhesion of CAV9 but not RGE/RGDdel to A549 cells was also significantly higher than to RD cells. In contrast, no affinity or adhesion of bacterially produced VP1 proteins to the integrins or to the cells was detected. Function-blocking antibodies against alphaV-integrins blocked CAV9 but not CAV9-RGDdel infectivity, indicating that the viruses use different internalization routes; this may explain the differential infection kinetics of CAV9 and RGDdel. In an affinity assay, soluble alphaVbeta6, but not alphaVbeta3, bound to immobilized CAV9. Similarly, only soluble alphaVbeta6 blocked virus infectivity. These data suggest that CAV9 binding to alphaVbeta6 is a high-affinity interaction, which may indicate its importance in clinical infections; this remains to be determined.

  9. Dual molecular diagnosis contributes to atypical Prader-Willi phenotype in monozygotic twins.

    Science.gov (United States)

    Jehee, Fernanda S; de Oliveira, Valdirene T; Gurgel-Giannetti, Juliana; Pietra, Rafaella X; Rubatino, Fernando V M; Carobin, Natália V; Vianna, Gabrielle S; de Freitas, Mariana L; Fernandes, Karla S; Ribeiro, Beatriz S V; Brüggenwirth, Hennie T; Ali-Amin, Roza; White, Janson J; Akdemir, Zeynep C; Jhangiani, Shalini N; Gibbs, Richard A; Lupski, James R; Varela, Monica C; Koiffmann, Célia; Rosenberg, Carla; Carvalho, Cláudia M B

    2017-09-01

    We describe monozygotic twin girls with genetic variation at two separate loci resulting in a blended phenotype of Prader-Willi syndrome and Pitt-Hopkins syndrome. These girls were diagnosed in early infancy with Prader-Willi syndrome, but developed an atypical phenotype, with apparent intellectual deficiency and lack of obesity. Array-comparative genomic hybridization confirmed a de novo paternal deletion of the 15q11.2q13 region and exome sequencing identified a second mutational event in both girls, which was a novel variant c.145+1G>A affecting a TCF4 canonical splicing site inherited from the mosaic mother. RNA studies showed that the variant abolished the donor splicing site, which was accompanied by activation of an alternative non-canonical splicing-site which then predicts a premature stop codon in the following exon. Clinical re-evaluation of the twins indicated that both variants are likely contributing to the more severe phenotypic presentation. Our data show that atypical clinical presentations may actually be the expression of blended clinical phenotypes arising from independent pathogenic events at two loci. © 2017 Wiley Periodicals, Inc.

  10. Cardiobacterium valvarum infective endocarditis and phenotypic/molecular characterization of 11 Cardiobacterium species strains

    DEFF Research Database (Denmark)

    Chen, Ming; Kemp, Michael; Bruun, Niels E

    2011-01-01

    and prolapse of pulmonary valves in addition to a fluttering excrescence. A mechanical mitral valve and neochordae were inserted successfully. Phenotypically, the two species within the genus Cardiobacterium resemble each other greatly. When using the Vitek 2 Neisseria-Haemophilus identification card...

  11. Phenotypic and molecular characterizations of Yersinia pestis isolates from Kazakhstan and adjacent regions.

    Science.gov (United States)

    Lowell, Jennifer L; Zhansarina, Aigul; Yockey, Brook; Meka-Mechenko, Tatyana; Stybayeva, Gulnaz; Atshabar, Bakyt; Nekrassova, Larissa; Tashmetov, Rinat; Kenghebaeva, Kuralai; Chu, May C; Kosoy, Michael; Antolin, Michael F; Gage, Kenneth L

    2007-01-01

    Recent interest in characterizing infectious agents associated with bioterrorism has resulted in the development of effective pathogen genotyping systems, but this information is rarely combined with phenotypic data. Yersinia pestis, the aetiological agent of plague, has been well defined genotypically on local and worldwide scales using multi-locus variable number tandem repeat analysis (MLVA), with emphasis on evolutionary patterns using old isolate collections from countries where Y. pestis has existed the longest. Worldwide MLVA studies are largely based on isolates that have been in long-term laboratory culture and storage, or on field material from parts of the world where Y. pestis has potentially circulated in nature for thousands of years. Diversity in these isolates suggests that they may no longer represent the wild-type organism phenotypically, including the possibility of altered pathogenicity. This study focused on the phenotypic and genotypic properties of 48 Y. pestis isolates collected from 10 plague foci in and bordering Kazakhstan. Phenotypic characterization was based on diagnostic tests typically performed in reference laboratories working with Y. pestis. MLVA was used to define the genotypic relationships between the central-Asian isolates and a group of North American isolates, and to examine Kazakh Y. pestis diversity according to predefined plague foci and on an intermediate geographical scale. Phenotypic properties revealed that a large portion of this collection lacks one or more plasmids necessary to complete the blocked flea/mammal transmission cycle, has lost Congo red binding capabilities (Pgm-), or both. MLVA analysis classified isolates into previously identified biovars, and in some cases groups of isolates collected within the same plague focus formed a clade. Overall, MLVA did not distinguish unique phylogeographical groups of Y. pestis isolates as defined by plague foci and indicated higher genetic diversity among older biovars.

  12. Monitoring of different vibrio species affecting marine fishes in Lake Qarun and Gulf of Suez: Phenotypic and molecular characterization

    Directory of Open Access Journals (Sweden)

    Mohamed Abdelaziz

    2017-06-01

    Full Text Available Vibriosis is a globally threatening bacterial disease affecting mariculture with high mortalities and severe economic losses. Isolation and Identification of different vibrio species were performed to a total number of one hundred moribund and freshly dead Solea aegyptiaca, Epinephelus marginatus and Mugil cephalus collected from Lake Qarun and Gulf of Suez. Phenotypic picture and molecular identification based on use of 16SrRNA gene sequence confirmed 44 strains as vibrio species. Further molecular identification of retrieved vibrio spp. using species specific primers for collagenase, ToxR and Vvh genes categorized 10 isolates belong to V. alginolyticus, 8 isolates belong to V. parahaemolyticus and 6 isolates belong to V. vulnificus. The total prevalence of vibriosis was 44% where the highest prevalence was recorded in Lake Qarun examined fishes.

  13. The phenotype of the human materno-fetal endothelial barrier: molecular occupancy of paracellular junctions dictate permeability and angiogenic plasticity.

    Science.gov (United States)

    Leach, Lopa

    2002-06-01

    In vitro models predict that molecular occupancy of endothelial junctions may regulate both barrier function and angiogenesis. Whether this is true in human vascular beds undergoing physiological angiogenesis has not been shown. This review presents data which demonstrate there are two distinct junctional phenotypes, 'activated' and 'stable', present in the vascular tree of the human placenta taken from two distinct highly angiogenic gestational periods (first and last trimester). Stability is conferred by the presence of occludin in tight junctions and plakoglobin in adherens junctions. Their localization may be influenced by vascular endothelial growth factor and angiopoietins 1 and 2 that have a similar temporal and site-specific differential expression. The junctional phenotypes are reversible, as shown in studies with endothelial cells isolated from placental microvessels and grown in the presence/absence of cAMP-enhancing agents. Reductions in protein levels and loss of junctional localization of adhesion molecules result in increased permeability to macromolecules, whilst up-regulation and re-targeting of these molecules inhibit cell proliferation and increase transendothelial resistance. These studies suggest junctional adhesion molecules can regulate physiological angiogenesis and vascular re-modelling. Moreover, the activated junctional phenotype of placental microvessels allows them to participate in increased growth and proliferation. This junctional immaturity appears to be at the expense of barrier function resulting in sites of maximal materno-fetal solute exchange.

  14. The molecular genetics and neurobiology of developmental dyslexia as model of a complex phenotype.

    Science.gov (United States)

    Kere, Juha

    2014-09-19

    Among complex disorders, those concerning neuropsychiatric phenotypes involve particular challenges compared to disorders with more easily distinguished clinical signs and measures. One such common and unusually challenging phenotype to disentangle genetically is developmental dyslexia (DD), or reading disability, defined as the inability to learn to read and write for an otherwise normally intelligent child with normal senses and educational opportunity. There is presently ample evidence for the strongly biological etiology for DD, and a dozen susceptibility genes have been suggested. Many of these genes point to common but previously unsuspected biological mechanisms, such as neuronal migration and cilia functions. I discuss here the state-of-the-art in genomic and neurobiological aspects of DD research, starting with short general background to its history. Copyright © 2014 The Author. Published by Elsevier Inc. All rights reserved.

  15. The focal facial dermal dysplasias: phenotypic spectrum and molecular genetic heterogeneity.

    Science.gov (United States)

    Lee, Beom Hee; Aggarwal, Aneel; Slavotinek, Anne; Edelmann, Lisa; Chen, Brenden; Desnick, Robert J

    2017-09-01

    Focal facial dermal dysplasias (FFDDs) are rare genetic/developmental disorders characterised by bilateral 'scar-like' facial lesions. Four subtypes are classified by the bitemporal (FFDD1-3) or preauricular (FFDD4) lesion location. FFDD1-3 are differentiated by additional facial abnormalities and inheritance patterns. Although the genetic defects causing FFDD1 and FFDD2 remain unknown, recent studies identified defects causing FFDD3 and FFDD4. Here, the clinical phenotypes, genetic defects and inheritance of the four FFDD subtypes are described. In addition, the overlapping facial abnormalities in FFDD3 and two other genetic disorders, Ablepharon macrostomia syndrome and Barber-Say syndrome, are noted. Familiarity with the FFDDs by clinicians will further delineate the phenotypes and genetic/developmental defects of these dermal facial disorders. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  16. Excision of Expanded GAA Repeats Alleviates the Molecular Phenotype of Friedreich's Ataxia.

    Science.gov (United States)

    Li, Yanjie; Polak, Urszula; Bhalla, Angela D; Rozwadowska, Natalia; Butler, Jill Sergesketter; Lynch, David R; Dent, Sharon Y R; Napierala, Marek

    2015-06-01

    Friedreich's ataxia (FRDA) is an autosomal recessive neurological disease caused by expansions of guanine-adenine-adenine (GAA) repeats in intron 1 of the frataxin (FXN) gene. The expansion results in significantly decreased frataxin expression. We report that human FRDA cells can be corrected by zinc finger nuclease-mediated excision of the expanded GAA repeats. Editing of a single expanded GAA allele created heterozygous, FRDA carrier-like cells and significantly increased frataxin expression. This correction persisted during reprogramming of zinc finger nuclease-edited fibroblasts to induced pluripotent stem cells and subsequent differentiation into neurons. The expression of FRDA biomarkers was normalized in corrected patient cells and disease-associated phenotypes, such as decreases in aconitase activity and intracellular ATP levels, were reversed in zinc finger nuclease corrected neuronal cells. Genetically and phenotypically corrected patient cells represent not only a preferred disease-relevant model system to study pathogenic mechanisms, but also a critical step towards development of cell replacement therapy.

  17. {sup 99m}Tc(CO){sub 3}-DTMA bombesin conjugates having high affinity for the GRP receptor

    Energy Technology Data Exchange (ETDEWEB)

    Lane, Stephanie R. [Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, MO 65201 (United States); Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Veerendra, Bhadrasetty [Department of Radiology, University of Missouri-Columbia School of Medicine, Columbia, MO 65211 (United States); Rold, Tammy L. [Department of Internal Medicine, University of Missouri-Columbia School of Medicine, Columbia, MO 65211 (United States); Sieckman, Gary L. [Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, MO 65201 (United States); Hoffman, Timothy J. [Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, MO 65201 (United States); Radiopharmaceutical Sciences Institute, University of Missouri-Columbia School of Medicine, Columbia, MO 65211 (United States); Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Jurisson, Silvia S. [Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211 (United States); Smith, Charles J. [Department of Radiology, University of Missouri-Columbia School of Medicine, Columbia, MO 65211 (United States); Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, MO 65201 (United States); University of Missouri Research Reactor Center, University of Missouri-Columbia, Columbia, MO 65211 (United States); Radiopharmaceutical Sciences Institute, University of Missouri-Columbia School of Medicine, Columbia, MO 65211 (United States)], E-mail: smithcj@health.missouri.edu

    2008-04-15

    Introduction: Targeted diagnosis of specific human cancer types continues to be of significant interest in nuclear medicine. {sup 99m}Tc is ideally suited as a diagnostic radiometal for in vivo tumor targeting due to its ideal physical characteristics and diverse labeling chemistries in numerous oxidation states. Methods: In this study, we report a synthetic approach toward design of a new tridentate amine ligand for the organometallic aqua-ion [{sup 99m}Tc(H{sub 2}O){sub 3}(CO){sub 3}]{sup +}. The new chelating ligand framework, 2-(N,N'-Bis(tert-butoxycarbonyl)diethylenetriamine) acetic acid (DTMA), was synthesized from a diethylenetriamine precursor and fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy ({sup 1}H and {sup 13}C). DTMA was conjugated to H{sub 2}N-(X)-BBN(7-14)NH{sub 2}, where X=an amino acid or aliphatic pharmacokinetic modifier and BBN=bombesin peptide, by means of solid phase peptide synthesis. DTMA-(X)-BBN(7-14)NH{sub 2} conjugates were purified by reversed-phase high-performance chromatography and characterized by electrospray-ionization mass spectrometry. Results: The new conjugates were radiolabeled with [{sup 99m}Tc(H{sub 2}O){sub 3}(CO){sub 3}]{sup +} produced via Isolink radiolabeling kits to produce [{sup 99m}Tc(CO){sub 3}-DTMA-(X)-BBN(7-14)NH{sub 2}]. Radiolabeled conjugates were purified by reversed-phase high-performance chromatography. Effective receptor binding behavior was evaluated in vitro and in vivo. Conclusions: [{sup 99m}Tc(CO){sub 3}-DTMA-(X)-BBN(7-14)NH{sub 2}] conjugates displayed very high affinity for the gastrin releasing peptide receptor in vitro and in vivo. Therefore, these conjugates hold some propensity to be investigated as molecular imaging agents that specifically target human cancers uniquely expressing the gastrin releasing peptide receptor subtypes.

  18. Integrating phenotypic data from electronic patient records with molecular level systems biology

    DEFF Research Database (Denmark)

    Brunak, Søren

    2011-01-01

    Electronic patient records remain a rather unexplored, but potentially rich data source for discovering correlations between diseases. We describe a general approach for gathering phenotypic descriptions of patients from medical records in a systematic and non-cohort dependent manner. By extracti...... Classification of Disease ontology and is therefore in principle language independent. As a use case we show how records from a Danish psychiatric hospital lead to the identification of disease correlations, which subsequently are mapped to systems biology frameworks....

  19. Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.

    Science.gov (United States)

    Talbi, S; Hamilton, A E; Vo, K C; Tulac, S; Overgaard, M T; Dosiou, C; Le Shay, N; Nezhat, C N; Kempson, R; Lessey, B A; Nayak, N R; Giudice, L C

    2006-03-01

    Histological evaluation of endometrium has been the gold standard for clinical diagnosis and management of women with endometrial disorders. However, several recent studies have questioned the accuracy and utility of such evaluation, mainly because of significant intra- and interobserver variations in histological interpretation. To examine the possibility that biochemical or molecular signatures of endometrium may prove to be more useful, we have investigated whole-genome molecular phenotyping (54,600 genes and expressed sequence tags) of this tissue sampled across the cycle in 28 normo-ovulatory women, using high-density oligonucleotide microarrays. Unsupervised principal component analysis of all samples revealed that samples self-cluster into four groups consistent with histological phenotypes of proliferative (PE), early-secretory (ESE), mid-secretory (MSE), and late-secretory (LSE) endometrium. Independent hierarchical clustering analysis revealed equivalent results, with two major dendrogram branches corresponding to PE/ESE and MSE/LSE and sub-branching into the four respective phases with heterogeneity among samples within each sub-branch. K-means clustering of genes revealed four major patterns of gene expression (high in PE, high in ESE, high in MSE, and high in LSE), and gene ontology analysis of these clusters demonstrated cycle-phase-specific biological processes and molecular functions. Six samples with ambiguous histology were identically assignable to a cycle phase by both principal component analysis and hierarchical clustering. Additionally, pairwise comparisons of relative gene expression across the cycle revealed genes/families that clearly distinguish the transitions of PE-->ESE, ESE-->MSE, and MSE-->LSE, including receptomes and signaling pathways. Select genes were validated by quantitative RT-PCR. Overall, the results demonstrate that endometrial samples obtained by two different sampling techniques (biopsy and curetting hysterectomy

  20. Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Morocco.

    Science.gov (United States)

    Maâtallah, J; Berraho, E B; Muñoz, S; Sanjuan, J; Lluch, C

    2002-01-01

    To determine the biodiversity of rhizobial strains nodulating Cicer arietinum L. in representative soils from various areas of Morocco. Symbiotic traits, utilization of 49 carbohydrate sources, resistance to antibiotics and heavy metals, tolerance to salinity, to extreme temperatures and pH were studied as phenotypic markers. In addition, restriction fragment length polymorphism (RFLP) of PCR-amplified 16S rDNAs were compared with those of reference strains. Numerical analysis of the phenotypic characteristics showed that the 48 strains studied fell into three distinct groups. RFLP analysis of 16S rRNA genes revealed an additional heterogeneity and four ribotypes were identified. Chickpea rhizobia isolated from Moroccan soils are both phenotypically and genetically diverse. Most of these rhizobia belong to the Mesorhizobium genus. However, some strains originating from a particular soil appeared to have 16S rRNA genes similar to Sinorhizobium as well as very distinct auxanographic characteristics compared with Mesorhizo- bium isolates. A well characterized collection of chickpea-nodulating rhizobia in representative soils of Morocco has been generated, which can be used to develop efficient inoculants for this crop. This is the first report evidencing that chickpeas may be nodulated by bacteria from the Sinorhizobium genus.

  1. Molecular and phenotypic characterization of Agrobacterium species from vineyards allows identification of typical Agrobacterium vitis and atypical biovar 1 strains.

    Science.gov (United States)

    Genov, N; Llop, P; López, M M; Bobev, S G; Álvarez, B

    2015-06-01

    To molecularly and phenotypically characterize a selection of Agrobacterium-like isolates from grapevine canes, crowns, soil and tumours in plants grown under cold conditions. Most of the strains were biovar 3 (Agrobacterium vitis), and the remaining were atypical biovar 1 (Agrobacterium tumefaciens). All of them were tumourigenic on grapevine plants but differences in other hosts were observed. Chromosomal and plasmid-borne traits were analysed by gene amplification with four primer sets. Detection of the pectin enzyme hydrolase gene clearly distinguished A. vitis from the atypical A. tumefaciens. Regarding the virulence sensor gene, limited host range tumour-inducing plasmids were found in the atypical isolates. About opine utilization, most A. vitis and some A. tumefaciens contained octopine/cucumopine plasmids, but the nopaline-type was only detected in one A. tumefaciens. The A. vitis strains were molecularly and phenotypically more homogeneous than those of A. tumefaciens, the latter displaying some typical A. vitis characteristics, suggesting an adaptation to life in grapevine. The findings of this work will help to improve detection procedures of the pathogen, and demonstrate the pathogen diversity in cold vineyards, laying the groundwork for epidemiological studies and development of control strategies of the crown and cane gall disease. © 2015 The Society for Applied Microbiology.

  2. Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Earle, M.; Concas, A.; Yamamura, H.I.

    1986-03-01

    The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 0/C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37/sup 0/C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 3/H)TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists.

  3. Twins in spirit part II: DOTATATE and high-affinity DOTATATE - the clinical experience

    Energy Technology Data Exchange (ETDEWEB)

    Brogsitter, Claudia; Zoephel, Klaus; Hartmann, Holger; Kotzerke, Joerg [Technische Universitaet Dresden, Department of Nuclear Medicine, Dresden (Germany); Schottelius, Margret; Wester, Hans-Juergen [Technische Universitaet Muenchen, Pharmaceutical Radiochemistry and Department of Nuclear Medicine, Muenchen (Germany)

    2014-06-15

    Over recent decades interest in diagnosis and treatment of neuroendocrine tumours (NET) has steadily grown. The basis for diagnosis and therapy of NET with radiolabelled somatostatin (hsst) analogues is the variable overexpression of hsst receptors (hsst1-5 receptors). We hypothesized that radiometal derivatives of DOTA-iodo-Tyr{sup 3}-octreotide analogues might be excellent candidates for somatostatin receptor imaging. We therefore explored the diagnostic potential of {sup 68}Ga-DOTA-iodo-Tyr{sup 3}-octreotate [{sup 68}Ga-DOTA,3-iodo-Tyr{sup 3},Thr{sup 8}]octreotide ({sup 68}Ga-HA-DOTATATE; HA, high-affinity) compared to the established {sup 68}Ga-DOTA-Tyr{sup 3}-octreotate ({sup 68}Ga-DOTATATE) in vivo. The study included 23 patients with known somatostatin receptor-positive metastases from NETs, thyroid cancer or glomus tumours who were investigated with both {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE. A patient-based and a lesion-based comparative analysis was carried out of normal tissue distribution and lesion detectability in a qualitative and a semiquantitative manner. {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE showed comparable uptake in the liver (SUV{sub mean} 8.9 ± 2.2 vs. 9.3 ± 2.5, n.s.), renal cortex (SUV{sub mean} 13.3 ± 3.9 vs. 14.5 ± 3.7, n.s.) and spleen (SUV{sub mean} 24.0 ± 6.7 vs. 22.9 ± 7.3, n.s.). A somewhat higher pituitary uptake was found with {sup 68}Ga-HA-DOTATATE (SUV{sub mean} 6.3 ± 1.8 vs. 5.4 ± 2.1, p < 0.05). On a lesion-by-lesion basis a total of 344 lesions were detected. {sup 68}Ga-HA-DOTATATE demonstrated 328 lesions (95.3 % of total lesions seen), and {sup 68}Ga-DOTATATE demonstrated 332 lesions (96.4 %). The mean SUV{sub max} of all lesions was not significantly different between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE (17.8 ± 11.4 vs. 16.7 ± 10.7, n.s.). Our analysis demonstrated very good concordance between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE PET data. As the availability and use of {sup

  4. High level of molecular and phenotypic biodiversity in Jatropha curcas from Central America compared to Africa, Asia and South America

    Science.gov (United States)

    2014-01-01

    Background The main bottleneck to elevate jatropha (Jatropha curcas L.) from a wild species to a profitable biodiesel crop is the low genetic and phenotypic variation found in different regions of the world, hampering efficient plant breeding for productivity traits. In this study, 182 accessions from Asia (91), Africa (35), South America (9) and Central America (47) were evaluated at genetic and phenotypic level to find genetic variation and important traits for oilseed production. Results Genetic variation was assessed with SSR (Simple Sequence Repeat), TRAP (Target Region Amplification Polymorphism) and AFLP (Amplified fragment length polymorphism) techniques. Phenotypic variation included seed morphological characteristics, seed oil content and fatty acid composition and early growth traits. Jaccard’s similarity and cluster analysis by UPGM (Unweighted Paired Group Method) with arithmetic mean and PCA (Principle Component Analysis) indicated higher variability in Central American accessions compared to Asian, African and South American accessions. Polymorphism Information Content (PIC) values ranged from 0 to 0.65. In the set of Central American accessions. PIC values were higher than in other regions. Accessions from the Central American population contain alleles that were not found in the accessions from other populations. Analysis of Molecular Variance (AMOVA; P jatropha oil significantly differed (P < 0.05) between regions. Conclusions The pool of Central American accessions showed very large genetic variation as assessed by DNA-marker variation compared to accessions from other regions. Central American accessions also showed the highest phenotypic variation and should be considered as the most important source for plant breeding. Some variation in early growth traits was found within a group of accessions from Asia and Africa, while these accessions did not differ in a single DNA-marker, possibly indicating epigenetic variation. PMID:24666927

  5. Naturally occurring tyrosine kinase inserts block high affinity binding of phospholipase C gamma and Shc to TrkC and neurotrophin-3 signaling.

    Science.gov (United States)

    Guiton, M; Gunn-Moore, F J; Glass, D J; Geis, D R; Yancopoulos, G D; Tavaré, J M

    1995-09-01

    Neurotrophin-3 binds to the receptor tyrosine kinase, TrkC. Several naturally occurring splice variants of TrkC exist including those with 14- and 39-amino acid inserts within the tyrosine kinase homology region. When expressed in fibroblasts, full-length TrkC, but not the kinase insert variants, mediated neurotrophin-3-stimulated cell proliferation. We investigated the molecular basis of this signaling defect. The kinase inserts blocked the ability of TrkC to mediate neurotrophin-3 stimulated c-myc and c-fos transcription and activation of the AP-1 transcriptional complex. In cells expressing full-length TrkC, neurotrophin-3 promoted a sustained activation of mitogen-activated protein kinase; TrkC containing kinase inserts only mediated transient activation of mitogen-activated protein kinase. The kinase inserts specifically blocked neurotrophin-3-stimulated autophosphorylation of the phospholipase C gamma binding site on TrkC (tyrosine 789) resulting in a severe reduction in phospholipase C gamma association with TrkC and its tyrosine phosphorylation. Neurotrophin-3-stimulated phosphorylation of the Shc binding site (tyrosine 485) on TrkC, and tyrosine phosphorylation of Shc itself, was unaffected by the kinase inserts; however, the kinase inserts blocked high affinity Shc association with TrkC. It is proposed that the lack of high affinity binding of Shc and/or phospholipase C gamma to the TrkC kinase insert variants may be responsible for the inability of these variants to bring about a full biological response in fibroblasts.

  6. In vivo neutralization of α-cobratoxin with high-affinity llama single-domain antibodies (VHHs and a VHH-Fc antibody.

    Directory of Open Access Journals (Sweden)

    Gabrielle Richard

    Full Text Available Small recombinant antibody fragments (e.g. scFvs and VHHs, which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab'2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α-Cbtx, the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2 was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α-Cbtx. Mouse α-Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20 and the VHH2-Fc antibody effectively neutralized lethality induced by α-Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.

  7. "Velcro" engineering of high affinity CD47 ectodomain as signal regulatory protein α (SIRPα) antagonists that enhance antibody-dependent cellular phagocytosis.

    Science.gov (United States)

    Ho, Chia Chi M; Guo, Nan; Sockolosky, Jonathan T; Ring, Aaron M; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L; Garcia, K Christopher

    2015-05-15

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy. © 2015 by

  8. Molecular phenotypic profiling of a Saccharomyces cerevisiae strain at the single-cell level.

    Science.gov (United States)

    Schmidt, A Mareike; Fagerer, Stephan R; Jefimovs, Konstantins; Buettner, Florian; Marro, Christian; Siringil, Erdem C; Boehlen, Karl L; Pabst, Martin; Ibáñez, Alfredo J

    2014-11-21

    Studying cell-to-cell heterogeneity requires techniques which robustly deliver reproducible results with single-cell sensitivity. Through a new fabrication method for the microarrays for mass spectrometry (MAMS) platform, we now have attained robustness and reproducibility in our single-cell level mass spectrometry measurements that allowed us to combine single-cell MAMS-based measurements from different days and samples. By combining multiple measurements, we were able to identify three co-existing phenotypes in an isogenic population of Saccharomyces cerevisiae characterized by distinctively different levels of glycolytic intermediates.

  9. High Affinity Binding of Chp1 Chromodomain to K9 Methylated Histone H3 is Required to Establish Centromeric Hterochromatin

    Energy Technology Data Exchange (ETDEWEB)

    Schalch, T.; Job, G; Noffsinger, V; Shanker, S; Kuscu, C; Joshua-Tor, L; Partridge, J

    2009-01-01

    In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.

  10. Phenotypic variability of the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA): clinical, molecular and biochemical delineation

    Science.gov (United States)

    2011-01-01

    Background The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA) (OMIM 225400) is a rare inheritable connective tissue disorder characterized by a deficiency of collagen lysyl hydroxylase 1 (LH1; EC 1.14.11.4) due to mutations in PLOD1. Biochemically this results in underhydroxylation of collagen lysyl residues and, hence, an abnormal pattern of lysyl pyridinoline (LP) and hydroxylysyl pyridinoline (HP) crosslinks excreted in the urine. Clinically the disorder is characterized by hypotonia and kyphoscoliosis at birth, joint hypermobility, and skin hyperelasticity and fragility. Severe hypotonia usually leads to delay in gross motor development, whereas cognitive development is reported to be normal. Methods We describe the clinical, biochemical and molecular characterisation, as well as electron microscopy findings of skin, in 15 patients newly diagnosed with this rare type of Ehlers-Danlos syndrome. Results Age at diagnosis ranged from 5 months to 27 years, with only 1/3 of the patients been diagnosed correctly in the first year of life. A similar disease frequency was found in females and males, however a broad disease severity spectrum (intra- and interfamilial), independent of molecular background or biochemical phenotype, was observed. Kyphoscoliosis, one of the main clinical features was not present at birth in 4 patients. Importantly we also noted the occurrence of vascular rupture antenatally and postnatally, as well as developmental delay in 5 patients. Conclusion In view of these findings we propose that EDS VIA is a highly variable clinical entity, presenting with a broad clinical spectrum, which may also be associated with cognitive delay and an increased risk for vascular events. Genotype/phenotype association studies and additional molecular investigations in more extended EDS VIA populations will be necessary to further elucidate the cause of the variability of the disease severity. PMID:21699693

  11. Phenotypic variability of the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA: clinical, molecular and biochemical delineation

    Directory of Open Access Journals (Sweden)

    Kariminejad Ariana

    2011-06-01

    Full Text Available Abstract Background The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VIA (OMIM 225400 is a rare inheritable connective tissue disorder characterized by a deficiency of collagen lysyl hydroxylase 1 (LH1; EC 1.14.11.4 due to mutations in PLOD1. Biochemically this results in underhydroxylation of collagen lysyl residues and, hence, an abnormal pattern of lysyl pyridinoline (LP and hydroxylysyl pyridinoline (HP crosslinks excreted in the urine. Clinically the disorder is characterized by hypotonia and kyphoscoliosis at birth, joint hypermobility, and skin hyperelasticity and fragility. Severe hypotonia usually leads to delay in gross motor development, whereas cognitive development is reported to be normal. Methods We describe the clinical, biochemical and molecular characterisation, as well as electron microscopy findings of skin, in 15 patients newly diagnosed with this rare type of Ehlers-Danlos syndrome. Results Age at diagnosis ranged from 5 months to 27 years, with only 1/3 of the patients been diagnosed correctly in the first year of life. A similar disease frequency was found in females and males, however a broad disease severity spectrum (intra- and interfamilial, independent of molecular background or biochemical phenotype, was observed. Kyphoscoliosis, one of the main clinical features was not present at birth in 4 patients. Importantly we also noted the occurrence of vascular rupture antenatally and postnatally, as well as developmental delay in 5 patients. Conclusion In view of these findings we propose that EDS VIA is a highly variable clinical entity, presenting with a broad clinical spectrum, which may also be associated with cognitive delay and an increased risk for vascular events. Genotype/phenotype association studies and additional molecular investigations in more extended EDS VIA populations will be necessary to further elucidate the cause of the variability of the disease severity.

  12. Functional characterization of the high affinity IgG Receptor : making heads and tails of FcγRI

    NARCIS (Netherlands)

    van der Poel, C.E.

    2011-01-01

    This thesis focuses on human FcγRI, a high affinity receptor for antibodies of the IgG isotype. IgG is the most abundant antibody type in blood and all currently FDA approved therapeutic antibodies are of the IgG isotype. FcγRI, a member of the activating Fcγ receptors, exists as a complex of a

  13. Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells.

    OpenAIRE

    Matthew, E; Laskin, J D; Zimmerman, E A; Weinstein, I B; Hsu, K C; Engelhardt, D L

    1981-01-01

    We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed tha...

  14. Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter

    NARCIS (Netherlands)

    Jorgensen, T.R.; vanKuyk, P.A.; Poulsen, B.R.; Ruijter, G.J.G.; Visser, J.; Iversen, J.J.L.

    2007-01-01

    This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K-s) of a reference strain was about 15 mu M in glucose-limited chemostat culture. Disruption of mstA

  15. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina

    2017-01-01

    γ-Hydroxybutyric acid (GHB) is an endogenous neuroactive substance and proposed neurotransmitter with affinity for both low- and high-affinity binding sites. A radioligand with high and specific affinity toward the high-affinity GHB binding site would be a unique tool toward a more complete...

  16. High-affinity glucose transport in Aspergillus nidulans is mediated by the products of two related but differentially expressed genes.

    Directory of Open Access Journals (Sweden)

    Josep V Forment

    Full Text Available Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation.

  17. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    Science.gov (United States)

    Orito, N.; Umekage, S.; Sato, K.; Kawauchi, S.; Tanaka, H.; Sakai, E.; Tanaka, T.; Kikuchi, Y.

    2012-03-01

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25×10-9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  18. Phenotypic and molecular variation in the green and black poison-dart frog Dendrobates auratus (Anura: Dendrobatidae from Costa Rica

    Directory of Open Access Journals (Sweden)

    Lisa D Patrick

    2009-11-01

    Full Text Available The green and black poison-dart frog Dendrobates auratus exhibits high intraspecific variation in hue color and pattern throughout its range, making it a very popular species in the pet trade. We analyzed the correspondence between color variation and molecular variation of D. auratus from Costa Rica using RAPD analysis. Twenty-six random primers were analyzed for variation in 99 individuals from seven populations. Color pattern was scored from digital images of the dorsal and ventral views. In general, frogs from the Caribbean coast had significantly more light coloration than black color but cannot be grouped by population based only on hue pattern. Only 3 RAPD primers were found to be polymorphic, representing a total of 16 loci. Most of the molecular variation encountered here occurs within populations, thus making unclear the degree of population structure and differentiation. Further examination of COI mtDNA sequences from our samples also supports these results. Partial Mantel correlations suggested that the pattern of molecular variation is not congruent with the variation in color pattern in this species, an outcome that is discussed in terms of phenotypic evolution. Rev. Biol. Trop. 57 (Suppl. 1: 313-321. Epub 2009 November 30.

  19. Molecular genetics of addiction and related heritable phenotypes: genome wide association approaches identify “connectivity constellation” and drug target genes with pleiotropic effects

    OpenAIRE

    Uhl, George R; Drgon, Tomas; Johnson, Catherine; Li, Chuan-Yun; Contoreggi, Carlo; Hess, Judith; Naiman, Daniel; Liu, Qing-Rong

    2008-01-01

    Genome wide association (GWA) can elucidate molecular genetic bases for human individual differences in “complex” phenotypes that include vulnerability to addiction. Here, we review: a) evidence that supports polygenic models with (at least) modest heterogeneity for the genetic architectures of addiction and several related phenotypes; b) technical and ethical aspects of importance for understanding genome wide association data: genotyping in individual samples vs DNA pools, analytic approach...

  20. Phenotypic and molecular differences between rats selectively bred to voluntarily run high vs. low nightly distances.

    Science.gov (United States)

    Roberts, Michael D; Brown, Jacob D; Company, Joseph M; Oberle, Lauren P; Heese, Alexander J; Toedebusch, Ryan G; Wells, Kevin D; Cruthirds, Clayton L; Knouse, John A; Ferreira, J Andries; Childs, Thomas E; Brown, Marybeth; Booth, Frank W

    2013-06-01

    The purpose of the present study was to partially phenotype male and female rats from generations 8-10 (G8-G10) that had been selectively bred to possess low (LVR) vs. high voluntary running (HVR) behavior. Over the first 6 days with wheels, 34-day-old G8 male and female LVRs ran shorter distances (P values, while LVR did not lose or gain fat mass during the 6-day voluntary running period. RNA deep sequencing efforts in the nucleus accumbens showed only eight transcripts to be >1.5-fold differentially expressed between lines in HVR and LVR nonrunners. Interestingly, HVRs presented less Oprd1 mRNA, which ties in to potential differences in dopaminergic signaling between lines. This unique animal model provides further evidence as to how exercise may be mechanistically regulated.

  1. Galactosemia: A strategy to identify new biochemical phenotypes and molecular genotypes

    Energy Technology Data Exchange (ETDEWEB)

    Elsas, L.J.; Langley, S.; Steele, E.; Evinger, J.; Brown, A.; Singh, R.; Fernhoff, P.; Hjelm, L.N.; Dembure, P.P.; Fridovich-Keil, J.L. [Emory Univ. School of Medicine, Atlanta, GA (United States)

    1995-03-01

    We describe a stratagem for identifying new mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. GALT enzyme activity and isoforms were defined in erythrocytes from probands and their first-degree relatives. If the biochemical phenotypes segregated in an autosomal recesssive pattern, we screened for common mutations by using multiplex PCR and restriction endonuclease digestions. If common mutant alleles were not present, the 11 exons of the GALT gene were amplified by PCR, and variations from the normal nucleotide sequences were identified by SSCP. The suspected region(s) was then analyzed by direct DNA sequencing. We identified 86 mutant GALT alleles that reduced erythrocyte GALT activity. Seventy-five of these GALT genomes had abnormal SSCP patterns, of which 41 were sequenced, yielding 12 new and 21 previously reported, rare mutations. Among the novel group of 12 new mutations, an unusual biochemical phenotype was found in a family whose newborn proband has classical galactosemia. He had inherited two mutations in cis (N314D-E204K) from his father, whose GALT activity was near normal, and an additional GALT mutation in the splice-acceptor site of intron C (IVSC) from his mother. The substitution of a positively charged E204K mutation created a unique isoform-banding pattern. An asymptomatic sister`s GALT genes carries three mutations (E203K-N314D/N314D) with eight distinct isoform bands. Surprisingly, her erythrocytes have normal GALT activity. We conclude that the synergism of pedigree, biochemical, SSCP, and direct GALT gene analyses is an efficient protocol for identifying new mutations and speculate that E203K and N314D codon changes produce intra-allelic complementation when in cis. 40 refs., 4 figs., 3 tabs.

  2. Molecular analysis and phenotypic study in 14 Chinese families with Bietti crystalline dystrophy.

    Directory of Open Access Journals (Sweden)

    Houfa Yin

    Full Text Available To investigate the clinical features and cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2 gene mutations in 14 Chinese families with Bietti crystalline dystrophy (BCD.Seventeen patients from 14 unrelated Chinese families with BCD were recruited for complete clinical ophthalmic examination and genetic study. The 11 exons of CYP4V2 were amplified from genomic DNA of all patients and their family members by polymerase chain reaction (PCR and then sequenced. Exons of TIMP3 were also sequenced in BCD patient associated with choroidal neovascularization (CNV. One hundred and seventy unrelated healthy Chinese subjects were screened for mutations in CYP4V2.All 17 patients with BCD had mutations in CYP4V2; one of these mutations was novel (c.219T>A, p.F73L and four other mutations had been reported. The p.F73L mutation was a commonly detected mutation in our study (seven out of 34 alleles, either in the homozygous state or in the heterozygous state. Among the patients, considerable phenotypic variability was detected, both within and between families. Screening of TIMP3 did not find any mutation in the BCD patient associated with CNV.The novel CYP4V2 c.219T>A (p.F73L mutation may be another recurrent mutation in Chinese patients with BCD. Our study expands the mutation spectrum of CYP4V2 and characterizes novel genotype-phenotype associations in Chinese patients with BCD.

  3. Genetic diversity assessment of sesame core collection in China by phenotype and molecular markers and extraction of a mini-core collection

    Science.gov (United States)

    2012-01-01

    Background Sesame (Sesamum indicum L.) is one of the four major oil crops in China. A sesame core collection (CC) was established in China in 2000, but no complete study on its genetic diversity has been carried out at either the phenotypic or molecular level. To provide technical guidance, a theoretical basis for further collection, effective protection, reasonable application, and a complete analysis of sesame genetic resources, a genetic diversity assessment of the sesame CC in China was conducted using phenotypic and molecular data and by extracting a sesame mini-core collection (MC). Results Results from a genetic diversity assessment of sesame CC in China were significantly inconsistent at the phenotypic and molecular levels. A Mantel test revealed the insignificant correlation between phenotype and molecular marker information (r = 0.0043, t = 0.1320, P = 0.5525). The Shannon-Weaver diversity index (I) and Nei genetic diversity index (h) were higher (I = 0.9537, h = 0.5490) when calculated using phenotypic data from the CC than when using molecular data (I = 0.3467, h = 0.2218). A mini-core collection (MC) containing 184 accessions was extracted based on both phenotypic and molecular data, with a low mean difference percentage (MD, 1.64%), low variance difference percentage (VD, 22.58%), large variable rate of coefficient of variance (VR, 114.86%), and large coincidence rate of range (CR, 95.76%). For molecular data, the diversity indices and the polymorphism information content (PIC) for the MC were significantly higher than for the CC. Compared to an alternative random sampling strategy, the advantages of capturing genetic diversity and validation by extracting a MC using an advanced maximization strategy were proven. Conclusions This study provides a comprehensive characterization of the phenotypic and molecular genetic diversities of the sesame CC in China. A MC was extracted using both phenotypic and molecular data. Low MD% and VD%, and large VR% and CR

  4. Genetic diversity assessment of sesame core collection in China by phenotype and molecular markers and extraction of a mini-core collection

    Directory of Open Access Journals (Sweden)

    Zhang Yanxin

    2012-11-01

    Full Text Available Abstract Background Sesame (Sesamum indicum L. is one of the four major oil crops in China. A sesame core collection (CC was established in China in 2000, but no complete study on its genetic diversity has been carried out at either the phenotypic or molecular level. To provide technical guidance, a theoretical basis for further collection, effective protection, reasonable application, and a complete analysis of sesame genetic resources, a genetic diversity assessment of the sesame CC in China was conducted using phenotypic and molecular data and by extracting a sesame mini-core collection (MC. Results Results from a genetic diversity assessment of sesame CC in China were significantly inconsistent at the phenotypic and molecular levels. A Mantel test revealed the insignificant correlation between phenotype and molecular marker information (r = 0.0043, t = 0.1320, P = 0.5525. The Shannon-Weaver diversity index (I and Nei genetic diversity index (h were higher (I = 0.9537, h = 0.5490 when calculated using phenotypic data from the CC than when using molecular data (I = 0.3467, h = 0.2218. A mini-core collection (MC containing 184 accessions was extracted based on both phenotypic and molecular data, with a low mean difference percentage (MD, 1.64%, low variance difference percentage (VD, 22.58%, large variable rate of coefficient of variance (VR, 114.86%, and large coincidence rate of range (CR, 95.76%. For molecular data, the diversity indices and the polymorphism information content (PIC for the MC were significantly higher than for the CC. Compared to an alternative random sampling strategy, the advantages of capturing genetic diversity and validation by extracting a MC using an advanced maximization strategy were proven. Conclusions This study provides a comprehensive characterization of the phenotypic and molecular genetic diversities of the sesame CC in China. A MC was extracted using both phenotypic and molecular data. Low MD% and VD%, and

  5. Molecular analysis of chromosome 21 in a patient with a phenotype of down syndrome and apparently normal karyotype

    Energy Technology Data Exchange (ETDEWEB)

    Ahlbom, B.E.; Wadelius, C.; Zech, L.; Anneren, G. [Uppsala Univ. (Sweden)] [and others

    1996-06-28

    Down syndrome (DS) is caused in most cases by the presence of an extra chromosome 21. It has been shown that the DS phenotype is produced by duplication of only a small part of the long arm of chromosome 21, the 21q22 region, including and distal to locus D21S55. We present molecular investigations on a woman with clinically typical DS but apparently normal chromosomes. Her parents were consanguineous and she had a sister with a DS phenotype, who died at the age of 15 days. Repeated cytogenetic investigations (G-banding and high resolution banding) on the patient and her parents showed apparently normal chromosomes. Autoradiographs of quantitative Southern blots of DNAs from the patient, her parents, trisomy 21 patients, and normal controls were analyzed after hybridization with unique DNA sequences regionally mapped on chromosome 21. Sequences D21S59, D21S1, D21S11, D21S8, D21S17, D21S55, ERG, D21S15, D21S112, and COL6A1 were all found in two copies. Fluorescent in situ hybridization with a chromosome 21-specific genomic library showed no abnormalities and only two copies of chromosome 21 were detected. Nineteen markers from the critical region studied with polymerase chain reaction amplification of di- and tetranucleotide repeats did not indicate any partial trisomy 21. From his study we conclude that the patient does not have any partial submicroscopic trisomy for any segment of chromosome 21. It seems reasonable to assume that she suffers from an autosomal recessive disorder which is phenotypically indistinguishable from DS. 23 refs., 6 figs., 3 tabs.

  6. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

    Science.gov (United States)

    Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

    2015-01-01

    AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants. PMID:25915023

  7. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

    Directory of Open Access Journals (Sweden)

    Aili Bao

    2015-04-01

    Full Text Available AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.

  8. A Novel NOD2-associated Mutation and Variant Blau Syndrome: Phenotype and Molecular Analysis.

    Science.gov (United States)

    Ebrahimiadib, Nazanin; Samra, Khawla Abu; Domina, Aaron M; Stiles, Ethan R; Ewer, Roger; Bocian, Charlie P; Foster, C Stephen

    2016-07-15

    To describe the clinical and molecular implications of a novel mutation in the NOD2/CARD15 gene on a family and its seven affected members. We reviewed the clinical presentations of family members who came to our center for refractory uveitis. Genetic testing and molecular testing was performed. All affected members had adult onset recurrent non-granulomatous panuveitis. The inheritance pattern suggested an autosomal dominant disease and genetic analysis identified a novel mutation in the NOD2 gene that converted amino acid 600 from glutamate to alanine (E600A). Transfection of the E600A NOD2 into human embryonic kidney-293 (HEK293) cells revealed constitutive activation and a reduced ability to respond to the NOD2 ligand, muramyl dipeptide (MDP) as compared with wild-type NOD2. The E600A mutation in the NOD2 gene may confer a higher penetrance of uveitis but a later onset of milder forms of non-ocular involvement.

  9. RANKL/RANK/MMP-1 molecular triad contributes to the metastatic phenotype of breast and prostate cancer cells in vitro.

    Directory of Open Access Journals (Sweden)

    Sandra Casimiro

    Full Text Available The osteolytic nature of bone metastasis results from a tumor-driven increased bone resorption. Bone remodeling is orchestrated by the molecular triad RANK-RANKL-OPG. This process is dysregulated in bone metastases, mostly via induction of RANKL by tumor-derived factors. These factors increase expression of RANKL, which induce osteoclast formation, function, and survival, thereby increasing bone resorption. RANK is unexpectedly expressed by cancer cells, and the activation of RANKL-RANK pathway correlates with an increased invasive phenotype. To investigate the interaction between RANK expression in human breast and prostate cancer cells and their pro-metastatic phenotype we analyzed the activation of RANKL-RANK pathway and its effects on cell migration, invasion, gene expression in vitro, and osteolysis-inducing ability in vivo. RANKL activates kinase signaling pathways, stimulates cell migration, increases cell invasion, and up-regulates MMP-1 expression. In vivo, MMP-1 knockdown resulted in smaller x-ray osteolytic lesions and osteoclastogenesis, and decreased tumor burden. Therefore, RANKL inhibition in bone metastatic disease may decrease the levels of the osteoclastogenesis inducer MMP-1, contributing to a better clinical outcome.

  10. Phenotypic, genetic and molecular characterization of a maize low phytic acid mutant (lpa241)

    DEFF Research Database (Denmark)

    Pilu, R.; Panzeri, D.; Gavazzi, G.

    2003-01-01

    90% reduction of phytic acid and about a tenfold increase in seed-free phosphate content. Although germination rate was decreased by about 30% compared to wild-type, developement of mutant plants was apparentely unaffected. The results of the genetic, biochemical and molecular characterization...... experiments carried out by SSR mapping, MDD-HPLC and RT-PCR are consistent with a mutation affecting the MIPS1S gene, coding for the first enzyme of the phytic acid biosynthetic pathway....

  11. Advances in taxonomy of genus phoma: polyphyletic nature and role of phenotypic traits and molecular systematics.

    Science.gov (United States)

    Rai, Mahendra Kumar; Tiwari, Vaibhav V; Irinyi, László; Kövics, György János

    2014-06-01

    Phoma is a highly polyphyletic genus with its unclear species boundaries. The conventional system of identification is functional but it has its limitations. Besides morphological studies, chemotaxonomy, secondary metabolite and protein profiling have been assessed for the classification and identification of these fungi. Molecular datasets have provided a better outlook towards the phylogenetic and evolutionary trends of Phoma. Molecular markers such as ITS-rDNA, tubulin, actin, translation elongation factor have been widely used by the taxonomists to demarcate species. However, outcomes gained up till now represent preliminary step towards the study of Phoma systematics and a combined approach would be beneficial in the understanding of this polyphyletic group members. Lately, on the base of molecular phylogeny of the type species of the seven Phoma sections a new teleomorph family, Didymellaceae has been established, besides the Phaeosphaeriaceae related to sect. Paraphoma anamorphs, and the Leptosphaeriaceae to sect. Heterospora anamorphs. The estimated ratio is about 70 % of the recognized Phoma-like species can be associated with the Didymellaceae ascomycetous family.

  12. Mepyramine-JNJ7777120-hybrid compounds show high affinity to hH(1)R, but low affinity to hH(4)R.

    Science.gov (United States)

    Wagner, Eva; Wittmann, Hans-Joachim; Elz, Sigurd; Strasser, Andrea

    2011-11-01

    In literature, a synergism between histamine H(1) and H(4) receptor is discussed. Furthermore, it was shown, that the combined application of mepyramine, a H(1) antagonist and JNJ7777120, a H(4) receptor ligand leads to a synergistic effect in the acute murine asthma model. Thus, the aim of this study was to develop new hybrid ligands, containing one H(1) and one H(4) pharmacophor, connected by an appropriate spacer, in order to address both, H(1)R and H(4)R. Within this study, we synthesized nine hybrid compounds, which were pharmacologically characterized at hH(1)R and hH(4)R. The new compounds revealed (high) affinity to hH(1)R, but showed only low affinity to hH(4)R. Additionally, we performed molecular dynamic studies for some selected compounds at hH(1)R, in order to obtain information about the binding mode of these compounds on molecular level. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Phenotypic and molecular identification of Sporothrix isolates of clinical origin in Northeast China.

    Science.gov (United States)

    Yu, Xiaohong; Wan, Zhe; Zhang, Zhenying; Li, Fuqiu; Li, Ruoyu; Liu, Xiaoming

    2013-08-01

    Sporotrichosis is the most common deep mycosis in Northeast China which is an area of high epidemicity due to contact with reeds or cornstalks. In this study, we have characterized a total of 74 clinical isolates from fixed cutaneous, lymphocutaneous and disseminated clinical forms and from Heilongjiang, Jilin, and Liaoning provinces, respectively. All isolates (previously as Sporothrix schenckii) were identified as Sporothrix globosa according to their phenotypic characteristics and calmodulin gene sequences analysis. They were subdivided into two sub-clades (S. globosa I and S. globosa II). Most of our isolates (71/74) presented restricted growth at 37 °C, which differed from a previous report. Up to now, S. globosa is the only pathogenic species in Northeast China, no matter what kind of clinical form and which region it is isolated from. Most of our clinical isolates (68/74) were clustered with three Chinese environmental isolates reported in the literature. The new findings of S. globosa isolates on division and thermotolerance at 37 °C described in this study will help us gain a better understanding of S. globosa.

  14. Phenotypic and molecular assessment of antimicrobial resistance in Lactobacillus paracasei strains of food origin.

    Science.gov (United States)

    Huys, Geert; D'Haene, Klaas; Danielsen, Morten; Mättö, Jaana; Egervärn, Maria; Vandamme, Peter

    2008-02-01

    Antimicrobial resistance data in food-associated lactic acid bacteria (LAB) such as lactobacilli are mostly based on nonstandardized methodologies and/or have been obtained for only a limited number of strains. This susceptibility study included a diverse collection of 115 isolates mainly of food origin originally identified as Lactobacillus paracasei or Lactobacillus casei. Upon reidentification and removal of potential replicate isolates using repetitive DNA element PCR fingerprinting, 65 genotypically unique L. paracasei strains and the L. casei type strain were selected for broth microdilution and Etest assays using the LAB susceptibility test medium. In both methodologies, strains appeared uniformly susceptible to ampicillin and clindamycin but exhibited natural resistance to streptomycin and gentamicin. Three L. paracasei strains from cheese displayed acquired resistance to tetracycline (MIC > or = 32 microg/ml) and/or to erythromycin (MIC >16 microg/ml), which was linked to the presence of a tet(M) or tet(W) gene and/or an erm(B) gene, respectively. Partial sequencing revealed that the tet(M) genes found in two of these strains belonged to two tet(M) sequence homology groups previously found in enterococci. Collectively, phenotypic and genotypic data allowed us to propose tentative epidemiological cutoffs for L. paracasei and L. casei for differentiating susceptible strains from those strains harboring one or more acquired resistance factors.

  15. Tumor Angiogenesis Phenotyping by Nanoparticle-facilitated Magnetic Resonance and Near-infrared Fluorescence Molecular Imaging

    Directory of Open Access Journals (Sweden)

    Peter A Jarzyna

    2012-10-01

    Full Text Available One of the challenges of tailored antiangiogenic therapy is the ability to adequately monitor the angiogenic activity of a malignancy in response to treatment. The αvβ3 integrin, highly overexpressed on newly formed tumor vessels, has been successfully used as a target for Arg-Gly-Asp (RGD-functionalized nanoparticle contrast agents. In the present study, an RGD-functionalized nanocarrier was used to image ongoing angiogenesis in two different xenograft tumor models with varying intensities of angiogenesis (LS174T > EW7. To that end, iron oxide nanocrystals were included in the core of the nanoparticles to provide contrast for T2*-weighted magnetic resonance imaging (MRI, whereas the fluorophore Cy7 was attached to the surface to enable near-infrared fluorescence (NIRF imaging. The mouse tumor models were used to test the potential of the nanoparticle probe in combination with dual modality imaging for in vivo detection of tumor angiogenesis. Pre-contrast and post-contrast images (4 hours were acquired at a 9.4-T MRI system and revealed significant differences in the nanoparticle accumulation patterns between the two tumor models. In the case of the highly vascularized LS174T tumors, the accumulation was more confined to the periphery of the tumors, where angiogenesis is predominantly occurring. NIRF imaging revealed significant differences in accumulation kinetics between the models. In conclusion, this technology can serve as an in vivo biomarker for antiangiogenesis treatment and angiogenesis phenotyping.

  16. Calpastatin ablation aggravates the molecular phenotype in cell and animal models of Huntington disease.

    Science.gov (United States)

    Weber, Jonasz Jeremiasz; Kloock, Simon Johannes; Nagel, Maike; Ortiz-Rios, Midea Malena; Hofmann, Julian; Riess, Olaf; Nguyen, Huu Phuc

    2018-02-02

    Deciphering the molecular pathology of Huntington disease is of particular importance, not only for a better understanding of this neurodegenerative disease, but also to identify potential therapeutic targets. The polyglutamine-expanded disease protein huntingtin was shown to undergo proteolysis, which results in the accumulation of toxic and aggregation-prone fragments. Amongst several classes of proteolytic enzymes responsible for huntingtin processing, the group of calcium-activated calpains has been found to be a significant mediator of the disease protein toxicity. To confirm the impact of calpain-mediated huntingtin cleavage in Huntington disease, we analysed the effect of depleting or overexpressing the endogenous calpain inhibitor calpastatin in HEK293T cells transfected with wild-type or polyglutamine-expanded huntingtin. Moreover, we crossbred huntingtin knock-in mice with calpastatin knockout animals to assess its effect not only on huntingtin cleavage and aggregation but also additional molecular markers. We demonstrated that a reduced or ablated expression of calpastatin triggers calpain overactivation and a consequently increased mutant huntingtin cleavage in cells and in vivo. These alterations were accompanied by an elevated formation of predominantly cytoplasmic huntingtin aggregates. On the other hand, overexpression of calpastatin in cells attenuated huntingtin fragmentation and aggregation. In addition, we observed an enhanced cleavage of DARPP-32, p35 and synapsin-1 in neuronal tissue upon calpain overactivation. Our results corroborate the important role of calpains in the molecular pathogenesis of Huntington disease and endorse targeting these proteolytic enzymes as a therapeutic approach. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Phenotypic and molecular characterization of intrauterine fetal growth restriction in interspecies sheep pregnancy.

    Science.gov (United States)

    Chávez-García, A; Vázquez-Martínez, E R; Murcia, C; Rodríguez, A; Cerbón, M; Mejía, O

    2015-10-01

    Interspecies pregnancies between closely related species are usually performed in livestock to obtain improved and enriched offspring. Indeed, different hybrids have been obtained for research purposes since many years ago, and the maternal-fetal interactions have been studied as a possible strategy for species preservation. The aim of this study was to characterize by physiological and molecular approaches the interspecies pregnancy between bighorn sheep () and domestic sheep (). Hybrids were obtained by artificial insemination; the blood pressure and protein urine levels were measured during the last two-thirds of gestation. After parturition, offspring and placentas were weighed and measured and cotyledons were counted and weighed and their surface area determined. Plasma samples were obtained between wk 8 and 21 of gestation to assess progesterone (P4), vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) levels and cell-free RNA was isolated during the same period to assess hypoxia-inducible factor-1 α (α) gene expression. Hybrid and normal pregnancies were analyzed using physiological and molecular parameters during the last two-thirds of gestation (wk 8-21). The results show that during the measurement period, ewes with a hybrid pregnancy presented normal blood pressure and no alteration in urinary protein content. However, compared with sheep with a normal pregnancy, those with a hybrid pregnancy had a decrease in fetal and placental growth as well as in the cotyledonary surface area. Furthermore, in the hybrid group, there was placental insufficiency, characterized by a decrease in P4 production, as well as indications of endothelial dysfunction, characterized an increase in plasma levels of VEGF and PlGF as well as in plasma gene expression of α. Overall, the results indicate that hybrids of and presented intrauterine growth restriction, essentially due to altered endothelial function and chronic placental insufficiency

  18. Molecular and clinical analysis in a series of patients with Pyknodysostosis reveals some uncommon phenotypic findings.

    Science.gov (United States)

    Valdes-Flores, Margarita; Hidalgo-Bravo, Alberto; Casas-Avila, L; Chima-Galan, Carmen; Hazan-Lasri, Eric J; Pineda-Gomez, Ernesto; Lopez-Estrada, Druso; Zenteno, Juan C

    2014-01-01

    Pyknodysostosis is a rare autosomal recessive skeletal dysplasia characterized by short stature, deformity of the skull, osteosclerosis, hypoplasia of the clavicle, and bone fragility. Radiographs show increased bone density, osteosclerosis, and acroosteolysis of the terminal phalanges. The pycnodysostosis gene is located on chromosome 1q21 and encodes an enzyme called Cathepsin K. Cathepsin K is a cysteine protease lysosomal protein associated with the degradation of bone and cartilage. In the current study, the authors described the clinical, radiological and molecular features of a group of six Mexican patients, including two familial and two sporadic cases, with Pyknodysostosis. One of the patients presented hypoacusia, an unusual finding in this disease.

  19. Molecular studies of phenotype variation in canine RPGR-XLPRA1

    Science.gov (United States)

    Appelbaum, Tatyana; Becker, Doreen; Santana, Evelyn

    2016-01-01

    Purpose Canine X-linked progressive retinal atrophy 1 (XLPRA1) caused by a mutation in retinitis pigmentosa (RP) GTPase regulator (RPGR) exon ORF15 showed significant variability in disease onset in a colony of dogs that all inherited the same mutant X chromosome. Defective protein trafficking has been detected in XLPRA1 before any discernible degeneration of the photoreceptors. We hypothesized that the severity of the photoreceptor degeneration in affected dogs may be associated with defects in genes involved in ciliary trafficking. To this end, we examined six genes as potential disease modifiers. We also examined the expression levels of 24 genes involved in ciliary trafficking (seven), visual pathway (five), neuronal maintenance genes (six), and cellular stress response (six) to evaluate their possible involvement in early stages of the disease. Methods Samples from a pedigree derived from a single XLPRA1-affected male dog outcrossed to unrelated healthy mix-bred or purebred females were used for immunohistochemistry (IHC), western blot, mutational and haplotype analysis, and gene expression (GE). Cell-specific markers were used to examine retinal remodeling in the disease. Single nucleotide polymorphisms (SNPs) spanning the entire RPGR interacting and protein trafficking genes (RAB8A, RPGRIP1L, CEP290, CC2D2A, DFNB31, and RAB11B) were genotyped in the pedigree. Quantitative real-time PCR (qRT-PCR) was used to examine the expression of a total of 24 genes, including the six genes listed. Results Examination of cryosections from XLPRA1-affected animals of similar age (3–4 years) with different disease severity phenotype revealed mislocalization of opsins and upregulation of the Müller cell gliosis marker GFAP. Four to ten haplotypes per gene were identified in RAB8A, RPGRIP1L, CEP290, CC2D2A, DFNB31, and RAB11B for further assessment as potential genetic modifiers of XLPRA1. No correlation was found between the haplotypes and disease severity. During

  20. Molecular studies of phenotype variation in canine RPGR-XLPRA1.

    Science.gov (United States)

    Appelbaum, Tatyana; Becker, Doreen; Santana, Evelyn; Aguirre, Gustavo D

    2016-01-01

    Canine X-linked progressive retinal atrophy 1 (XLPRA1) caused by a mutation in retinitis pigmentosa (RP) GTPase regulator (RPGR) exon ORF15 showed significant variability in disease onset in a colony of dogs that all inherited the same mutant X chromosome. Defective protein trafficking has been detected in XLPRA1 before any discernible degeneration of the photoreceptors. We hypothesized that the severity of the photoreceptor degeneration in affected dogs may be associated with defects in genes involved in ciliary trafficking. To this end, we examined six genes as potential disease modifiers. We also examined the expression levels of 24 genes involved in ciliary trafficking (seven), visual pathway (five), neuronal maintenance genes (six), and cellular stress response (six) to evaluate their possible involvement in early stages of the disease. Samples from a pedigree derived from a single XLPRA1-affected male dog outcrossed to unrelated healthy mix-bred or purebred females were used for immunohistochemistry (IHC), western blot, mutational and haplotype analysis, and gene expression (GE). Cell-specific markers were used to examine retinal remodeling in the disease. Single nucleotide polymorphisms (SNPs) spanning the entire RPGR interacting and protein trafficking genes (RAB8A, RPGRIP1L, CEP290, CC2D2A, DFNB31, and RAB11B) were genotyped in the pedigree. Quantitative real-time PCR (qRT-PCR) was used to examine the expression of a total of 24 genes, including the six genes listed. Examination of cryosections from XLPRA1-affected animals of similar age (3-4 years) with different disease severity phenotype revealed mislocalization of opsins and upregulation of the Müller cell gliosis marker GFAP. Four to ten haplotypes per gene were identified in RAB8A, RPGRIP1L, CEP290, CC2D2A, DFNB31, and RAB11B for further assessment as potential genetic modifiers of XLPRA1. No correlation was found between the haplotypes and disease severity. During mutational analysis, several new

  1. Negative biomarker based male fertility evaluation: Sperm phenotypes associated with molecular-level anomalies

    Directory of Open Access Journals (Sweden)

    Peter Sutovsky

    2015-01-01

    Full Text Available Biomarker-based sperm analysis elevates the treatment of human infertility and ameliorates reproductive performance in livestock. The negative biomarker-based approach focuses on proteins and ligands unique to defective spermatozoa, regardless of their morphological phenotype, lending itself to analysis by flow cytometry (FC. A prime example is the spermatid specific thioredoxin SPTRX3/TXNDC8, retained in the nuclear vacuoles and superfluous cytoplasm of defective human spermatozoa. Infertile couples with high semen SPTRX3 are less likely to conceive by assisted reproductive therapies (ART and more prone to recurrent miscarriage while low SPTRX3 has been associated with multiple ART births. Ubiquitin, a small, proteolysis-promoting covalent posttranslational protein modifier is found on the surface of defective posttesticular spermatozoa and in the damaged protein aggregates, the aggresomes of spermiogenic origin. Semen ubiquitin content correlates negatively with fertility and conventional semen parameters, and with sperm binding of lectins LCA (Lens culinaris agglutinin; reveals altered sperm surface and PNA (Arachis hypogaea/peanut agglutinin; reveals acrosomal malformation or damage. The Postacrosomal Sheath WWI Domain Binding Protein (PAWP, implicated in oocyte activation during fertilization, is ectopic or absent from defective human and animal spermatozoa. Consequently, FC-parameters of PAWP correlate with ART outcomes in infertile couples and with fertility in bulls. Assays based on the above biomarkers have been combined into multiplex FC semen screening protocols, and the surface expression of lectins and ubiquitin has been utilized to develop nanoparticle-based bull semen purification method validated by field artificial insemination trials. These advances go hand-in-hand with the innovation of FC-technology and genomics/proteomics-based biomarker discovery.

  2. Molecular and Phenotypic Characterization of Metschnikowia pulcherrima Strains from Douro Wine Region

    Directory of Open Access Journals (Sweden)

    Catarina Barbosa

    2018-01-01

    Full Text Available Some non-Saccharomyces yeasts, including Metschnikowia pulcherrima, have been proposed as selected starters due to their contribution for the overall aroma and chemical profiles of wines. In this work, we aimed to evaluate the genetic and phenotypic diversity of Metschnikowia pulcherrima strains isolated from different locations of Douro Wine Region, and to explore their potential as co-adjuncts of S. cerevisiae in alcoholic fermentation. For that purpose, a set of 64 M. pulcherrima isolates were used. Polymerase chain reaction (PCR fingerprinting with M13 primers demonstrated to be an efficient tool in intraspecific discrimination of M. pulcherrima strains. No significant associations were found between genotypic profiles and either geographical origin or winery. The isolates were screened for their stress resistance ability (ethanol, SO2, chitosan, copper, H2O2, and Grape Juice Medium, aroma-related activities (resistance to 5, 5′, 5′′-trifluor-d, l-leucine and cerulenin and β-glycosidase, β-lyase and sulfite-reductase activities as well as other relevant technological proprieties (protease activity and biogenic amines production. M. pulcherrima response to the different enological traits evaluated was greatly strain-dependent. The most discriminant features were the ability of the strains to grow in Grape-Juice Medium (GJM and sulfite-reductase, and their β-lyase and protease activities. The enological potential of a selected M. pulcherrima strain in mixed-culture with S. cerevisiae was also assessed in natural grape-juice of a local variety, under two nitrogen regimes. M. pulcherrima proved to be promising for future industrial application as a co-starter, lowering ethanol, acetic acid and, reported here for the first time, lowering hydrogen sulfide levels in the wines.

  3. Molecular identification and phenotypic characterisation of Sporothrix globosa from clinical cases of Eastern Assam, North-east India

    Directory of Open Access Journals (Sweden)

    Reema Nath

    2017-01-01

    Full Text Available Sporotrichosis is known to be endemic in the state of Assam, North-east India, which is situated in the Sub-Himalayan region. This disease is an acute or chronic infection caused by Sporothrix schenckii species complex which currently includes several species of clinical relevance such as Sporothrix brasiliensis, Sporothrix globosa, Sporothrix schenckii sensu stricto, Sporothrix albicans, Sporothrix mexicana, Sporothrix pallida and Sporothrix luriei. S. globosa is the prevalent species in India. Eight culture-positive patients were diagnosed from suspected consecutive cases of two lymphocutaneous and six fixed cutaneous forms over a period of 4 years in a clinical mycology laboratory of a tertiary care centre in Eastern Assam. Phenotypic speciation was inconclusive using the criteria of Marimon et al. because of atypical growth pattern shown by the isolates. Our isolates showed good growth at 37°C ranging from 6 to 27 mm; four of the isolates showed growth of 11–27 mm unlike S. globosa strains reported earlier. Molecular identification was done by sequencing both the internal transcribed spacer (ITS region and the calmodulin (CAL protein encoding gene (partial. All the isolates were identified as S. globosa. Molecular confirmation of species using ITS region and CAL protein encoding gene (partial is necessary for isolates of S. globosa showing atypical biopatterns.

  4. [Frequency of molecular alterations in heterozygous beta-thalassemia in southern Spain and their relation to the hematologic phenotype].

    Science.gov (United States)

    Molina, M A; Romero, M J; Abril, E; Delgado, I; Cano, R M; Garrido, F; de Pablos, J M; Garrido, M L

    1994-08-01

    Heterozygous beta-thalassemia manifests hematologically with microcytosis, reduced red blood cell hemoglobin concentration and high hemoglobin A2 levels. Almost all molecular alterations are due to point mutations. We attempt to determinate the frequency of that mutations in the Oriental Andalusia Area, and its relationship with the hematological phenotype. We have studied 45 heterozygous patients. DNA samples were amplified by PCR, using the printers CD7 and HI1. A 16 Kb fragment corresponding to beta globin gene was obtained and analyzed by Dot Blot assay and hybridized with allelic specific oligonucleotide (ASO) probes to detect the 6 more frequent mutations found in the South of Spain. Codon 39 nonsense mutation (31.1%) was the most frequent finding followed by IVS-1 NT 110 (26.7%). The relationship between hematological parameters and molecular mutations concluded that IVS-I NT 6 mutation developed a minimal anemia. From the practical point of view, this study indicates that we were able to detect more than 90% of heterozygous beta-tal. with 5 out of 6 ASO probes used in this work. Thus, our data also provides a further implication in prenatal diagnosis.

  5. Molecular Characterization of a High-Affinity Xylobiose Transporter of Streptomyces thermoviolaceus OPC-520 and Its Transcriptional Regulation

    Science.gov (United States)

    Tsujibo, Hiroshi; Kosaka, Mitsuo; Ikenishi, Sadao; Sato, Takaji; Miyamoto, Katsushiro; Inamori, Yoshihiko

    2004-01-01

    Streptomyces thermoviolaceus OPC-520 secretes two types of xylanases (StxI and StxII), an acetyl xylan esterase (StxIII), and an α-l-arabinofuranosidase (StxIV) in the presence of xylan. Xylan degradation products (mainly xylobiose) produced by the action of these enzymes entered the cell and were then degraded to xylose by an intracellular β-xylosidase (BxlA). A gene cluster involved in xylanolytic system of the strain was cloned and sequenced upstream of and including a BxlA-encoding gene (bxlA). The gene cluster consisted of four different open reading frames organized in the order bxlE, bxlF, bxlG, and bxlA. Reverse transcriptase PCR analysis revealed that the gene cluster is transcribed as polycistronic mRNA. The deduced gene products, comprising BxlE (a sugar-binding lipoprotein), BxlF (an integral membrane protein), and BxlG (an integral membrane protein), showed similarity to components of the bacterial ATP-binding cassette (ABC) transport system; however, the gene for the ATP binding protein was not linked to the bxl operon. The soluble recombinant BxlE protein was analyzed for its binding activity for xylooligosaccharides. The protein showed high-level affinity for xylobiose (Kd = 8.75 × 10−9 M) and for xylotriose (Kd = 8.42 × 10−8 M). Antibodies raised against the recombinant BxlE recognized the detergent-soluble BxlE isolated from S. thermoviolaceus membranes. The deduced BxlF and BxlG proteins are predicted to be integral membrane proteins. These proteins contained the conserved EAA loop (between the fourth and the fifth membrane-spanning segments) which is characteristic of membrane proteins from binding-protein-dependent ABC transporters. In addition, the bxlR gene located upstream of the bxl operon was cloned and expressed in Escherichia coli. The bxlR gene encoded a 343-residue polypeptide that is highly homologous to members of the GalR/LacI family of bacterial transcriptional regulators. The purified BxlR protein specifically bound to a 4-bp inverted sequence overlapping the −10 region of the bxl operon. The binding of BxlR to the site was inhibited specifically by low concentrations of xylobiose. This site was also present in the region located between stxI and stxIV and in the upstream region of stxII. BxlR specifically bound to the regions containing the inverted sequence. These results suggest that BxlR might act as a repressor of the genes involved not only in the uptake system of xylan degradation products but also in xylan degradation of S. thermoviolaceus OPC-520. PMID:14761997

  6. Rare, high-affinity anti-pathogen antibodies from human repertoires, discovered using microfluidics and molecular genomics.

    Science.gov (United States)

    Adler, Adam S; Mizrahi, Rena A; Spindler, Matthew J; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Jackson; Leong, Renee; Roalfe, Lucy; White, Rebecca; Goldblatt, David; Johnson, David S

    Affinity-matured, functional anti-pathogen antibodies are present at low frequencies in natural human repertoires. These antibodies are often excellent candidates for therapeutic monoclonal antibodies. However, mining natural human antibody repertoires is a challenge. In this study, we demonstrate a new method that uses microfluidics, yeast display, and deep sequencing to identify 247 natively paired anti-pathogen single-chain variable fragments (scFvs), which were initially as rare as 1 in 100,000 in the human repertoires. Influenza A vaccination increased the frequency of influenza A antigen-binding scFv within the peripheral B cell repertoire from <0.1% in non-vaccinated donors to 0.3-0.4% in vaccinated donors, whereas pneumococcus vaccination did not increase the frequency of antigen-binding scFv. However, the pneumococcus scFv binders from the vaccinated library had higher heavy and light chain Replacement/Silent mutation (R/S) ratios, a measure of affinity maturation, than the pneumococcus binders from the corresponding non-vaccinated library. Thus, pneumococcus vaccination may increase the frequency of affinity-matured antibodies in human repertoires. We synthesized 10 anti-influenza A and nine anti-pneumococcus full-length antibodies that were highly abundant among antigen-binding scFv. All 10 anti-influenza A antibodies bound the appropriate antigen at KD<10 nM and neutralized virus in cellular assays. All nine anti-pneumococcus full-length antibodies bound at least one polysaccharide serotype, and 71% of the anti-pneumococcus antibodies that we tested were functional in cell killing assays. Our approach has future application in a variety of fields, including the development of therapeutic antibodies for emerging viral diseases, autoimmune disorders, and cancer.

  7. Molecular phenotyping of human ovarian cancer stem cells unravels the mechanisms for repair and chemoresistance

    DEFF Research Database (Denmark)

    Alvero, Ayesha B; Chen, Rui; Fu, Han-Hsuan

    2009-01-01

    of the tumor and may be the primary source of recurrence. We describe the characterization of human ovarian cancer stem cells (OCSCs). These cells have a distinctive genetic profile that confers them with the capacity to recapitulate the original tumor, proliferate with chemotherapy, and promote recurrence...... to form spheroids in suspension, and the ability to recapitulate in vivo the original tumor. Chemotherapy eliminates the bulk of the tumor but it leaves a core of cancer cells with high capacity for repair and renewal. The molecular properties identified in these cells may explain some of the unique......A major burden in the treatment of ovarian cancer is the high percentage of recurrence and chemoresistance. Cancer stem cells (CSCs) provide a reservoir of cells that can self-renew, can maintain the tumor by generating differentiated cells [non-stem cells (non-CSCs)] which make up the bulk...

  8. Molecular and Phenotypic Descriptions of Cystodermella cinnabarina from Western Himalaya: A New Genus for Pakistan

    Directory of Open Access Journals (Sweden)

    Abdul Razaq

    2013-01-01

    Full Text Available Cystodermella cinnabarina is reported here for the first time from the moist temperate forests of western Himalaya and is the first collection of a Cystodermella from Pakistan. This species is redescribed here using morphological and molecular data. The phylogenetic analysis which is based on internal transcribed spacers (ITS showed that the Pakistani collection clustered distinctly with similar European sequences in the Cystodermella clade. The Italian and north European sequences of this species clustered in two separate subclades and the Pakistani sequences closely matched the Italian sequences. It is evident that the Pakistani population has a very close evolutionary affinity with the Italian individuals rather than those from northern Europe. The species is distributed in Europe, in North America, and now in the western Himalaya of Asia.

  9. ClC-1 mutations in myotonia congenita patients: insights into molecular gating mechanisms and genotype-phenotype correlation.

    Science.gov (United States)

    Imbrici, P; Maggi, L; Mangiatordi, G F; Dinardo, M M; Altamura, C; Brugnoni, R; Alberga, D; Pinter, G Lauria; Ricci, G; Siciliano, G; Micheli, R; Annicchiarico, G; Lattanzi, G; Nicolotti, O; Morandi, L; Bernasconi, P; Desaphy, J-F; Mantegazza, R; Camerino, D Conte

    2015-09-15

    Loss-of-function mutations of the skeletal muscle ClC-1 channel cause myotonia congenita with variable phenotypes. Using patch clamp we show that F484L, located in the conducting pore, probably induces mild dominant myotonia by right-shifting the slow gating of ClC-1 channel, without exerting a dominant-negative effect on the wild-type (WT) subunit. Molecular dynamics simulations suggest that F484L affects the slow gate by increasing the frequency and the stability of H-bond formation between E232 in helix F and Y578 in helix R. Three other myotonic ClC-1 mutations are shown to produce distinct effects on channel function: L198P shifts the slow gate to positive potentials, V640G reduces channel activity, while L628P displays a WT-like behaviour (electrophysiology data only). Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function. Myotonia congenita is an inherited disease caused by loss-of-function mutations of the skeletal muscle ClC-1 chloride channel, characterized by impaired muscle relaxation after contraction and stiffness. In the present study, we provided an in-depth characterization of F484L, a mutation previously identified in dominant myotonia, in order to define the genotype-phenotype correlation, and to elucidate the contribution of this pore residue to the mechanisms of ClC-1 gating. Patch-clamp recordings showed that F484L reduced chloride currents at every tested potential and dramatically right-shifted the voltage dependence of slow gating, thus contributing to the mild clinical phenotype of affected heterozygote carriers. Unlike dominant mutations located at the dimer interface, no dominant-negative effect was observed when F484L mutant subunits were co-expressed with wild type. Molecular dynamics simulations further revealed that F484L affected the slow gate by increasing the frequency and stability of the H-bond formation between the pore residue E232 and the R helix residue Y578. In addition

  10. Insights from the fungus Fusarium oxysporum point to high affinity glucose transporters as targets for enhancing ethanol production from lignocellulose.

    Directory of Open Access Journals (Sweden)

    Shahin S Ali

    Full Text Available Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km((glucose was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.

  11. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen; Nakano, Hiroyasu

    2017-09-21

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  12. Phenotypic Prenatal Diagnosis of Chronic Granulomatous Disease: A Useful Tool in The Absence Of Molecular Diagnosis.

    Science.gov (United States)

    Kulkarni, M; Gupta, M; Madkaikar, M

    2017-12-01

    Chronic granulomatous disease (CGD) is an inherited immunodeficiency disorder affecting the microbicidal function of the phagocytes. It is characterized by susceptibility to recurrent infections leading to significant morbidity and mortality. Antibacterial and antifungal prophylaxis, though, has significantly reduced the rate and severity of the infections; the breakthrough infections still remain a challenge. Currently, allogenic haematopoietic stem cell transplantation is the only curative option which is very expensive and unavailable for many due to lack of suitable donor. Thus, prenatal diagnosis (PND) forms an important component of management in the affected families. PND is challenging in families approaching late in pregnancy with an uncharacterized molecular defect. In such cases, PND can be performed by analysis of NADPH activity of fetal blood (FB) neutrophils at 18-20 weeks of gestation. Cord blood samples at 18 weeks of gestation from healthy control were used to establish normal ranges for NBT and DHR. PND was offered for six pregnancies (NBT: n = 3, DHR: n = 6) with index cases of CGD confirmed by abnormal NBT and DHR analysis. NBT and DHR tests were found to be negative for all the six cases, confirming the same on samples post-delivery. NBT and DHR tests offer a rapid and sensitive PND of CGD in the absence of facilities for molecular diagnosis. It was observed that addition of CD15 along with CD45 led to an accurate DHR analysis. It is recommended to perform the diagnosis with adequate precautions only at centres with considerable experience and expertise in the diagnosis of CGD. © 2017 The Foundation for the Scandinavian Journal of Immunology.

  13. Molecular phenotypes distinguish patients with relatively stable from progressive idiopathic pulmonary fibrosis (IPF.

    Directory of Open Access Journals (Sweden)

    Kathy Boon

    Full Text Available BACKGROUND: Idiopathic pulmonary fibrosis (IPF is a progressive, chronic interstitial lung disease that is unresponsive to current therapy and often leads to death. However, the rate of disease progression differs among patients. We hypothesized that comparing the gene expression profiles between patients with stable disease and those in which the disease progressed rapidly will lead to biomarker discovery and contribute to the understanding of disease pathogenesis. METHODOLOGY AND PRINCIPAL FINDINGS: To begin to address this hypothesis, we applied Serial Analysis of Gene Expression (SAGE to generate lung expression profiles from diagnostic surgical lung biopsies in 6 individuals with relatively stable (or slowly progressive IPF and 6 individuals with progressive IPF (based on changes in DLCO and FVC over 12 months. Our results indicate that this comprehensive lung IPF SAGE transcriptome is distinct from normal lung tissue and other chronic lung diseases. To identify candidate markers of disease progression, we compared the IPF SAGE profiles in stable and progressive disease, and identified a set of 102 transcripts that were at least 5-fold up regulated and a set of 89 transcripts that were at least 5-fold down regulated in the progressive group (P-valuemolecular expression signature of 134 transcripts that sufficiently distinguished relatively stable from progressive IPF. CONCLUSIONS: These findings indicate that molecular signatures from lung parenchyma at the time of diagnosis could prove helpful in predicting the

  14. Gene expression correlations in human cancer cell lines define molecular interaction networks for epithelial phenotype.

    Science.gov (United States)

    Kohn, Kurt W; Zeeberg, Barry M; Reinhold, William C; Pommier, Yves

    2014-01-01

    Using gene expression data to enhance our knowledge of control networks relevant to cancer biology and therapy is a challenging but urgent task. Based on the premise that genes that are expressed together in a variety of cell types are likely to functions together, we derived mutually correlated genes that function together in various processes in epithelial-like tumor cells. Expression-correlated genes were derived from data for the NCI-60 human tumor cell lines, as well as data from the Broad Institute's CCLE cell lines. NCI-60 cell lines that selectively expressed a mutually correlated subset of tight junction genes served as a signature for epithelial-like cancer cells. Those signature cell lines served as a seed to derive other correlated genes, many of which had various other epithelial-related functions. Literature survey yielded molecular interaction and function information about those genes, from which molecular interaction maps were assembled. Many of the genes had epithelial functions unrelated to tight junctions, demonstrating that new function categories were elicited. The most highly correlated genes were implicated in the following epithelial functions: interactions at tight junctions (CLDN7, CLDN4, CLDN3, MARVELD3, MARVELD2, TJP3, CGN, CRB3, LLGL2, EPCAM, LNX1); interactions at adherens junctions (CDH1, ADAP1, CAMSAP3); interactions at desmosomes (PPL, PKP3, JUP); transcription regulation of cell-cell junction complexes (GRHL1 and 2); epithelial RNA splicing regulators (ESRP1 and 2); epithelial vesicle traffic (RAB25, EPN3, GRHL2, EHF, ADAP1, MYO5B); epithelial Ca(+2) signaling (ATP2C2, S100A14, BSPRY); terminal differentiation of epithelial cells (OVOL1 and 2, ST14, PRSS8, SPINT1 and 2); maintenance of apico-basal polarity (RAB25, LLGL2, EPN3). The findings provide a foundation for future studies to elucidate the functions of regulatory networks specific to epithelial-like cancer cells and to probe for anti-cancer drug targets.

  15. High-affinity CCK receptors are coupled to phospholipase A2 pathways to mediate pancreatic amylase secretion.

    Science.gov (United States)

    Tsunoda, Y; Owyang, C

    1995-09-01

    It is well recognized that JMV-180, a cholecystokinin (CCK) analogue, acts as an agonist on the high-affinity CCK receptor in pancreatic acinar cells. It caused Ca2+ oscillations and amylase secretion in a manner independent of the phospholipase C-inositol 1,4,5-trisphosphate (IP3) pathway. We investigated the mechanism by which the high-affinity CCK receptor utilizes IP3-independent Ca2+ signal transduction to mediate amylase secretion. JMV-180 (1-1,000 nM)-stimulated Ca2+ oscillations and amylase secretion were significantly inhibited by the phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (10 microM). Using streptolysin O-permeabilized cells, we showed that a porcine pancreatic anti-PLA2 antibody from rabbit serum (250 ng/ml) inhibited JMV-180-stimulated amylase secretion. In contrast to CCK octapeptide, JMV-180 (1 nM-10 microM) had no effect on intracellular IP3 levels. These concentrations of JMV-180 did, however, increase intracellular levels of arachidonic acid (AA) metabolite by 2.5-fold in a biphasic manner. Application of exogenous AA (10 microM) released 60% of ATP-incorporated 45Ca2+ from permeabilized pancreatic acini within 3 min in a transient manner. We also showed that active phorbol ester (100 nM) inhibited Ca2+ oscillations and amylase secretion stimulated by JMV-180 (10 nM) or CCK-OPE (100 nM). Application of Mn2+ (2 mM) to superfused acini resulted in a rapid quench of fura 2 fluorescence during 10 nM JMV-180 stimulation, suggesting an involvement of extracellular Ca2+ influx. However, the major source of Ca2+ utilized for oscillations during high-affinity CCK receptor activation was intracellular. In conclusion, we have demonstrated that the high-affinity CCK receptors are coupled to PLA2 pathways to produce AA, which mediates cytosolic Ca2+ oscillation and monophasic amylase secretion, in rat pancreatic acinar cells.

  16. Phenotypic and molecular evaluation of cotton hairy roots as a model system for studying nematode resistance.

    Science.gov (United States)

    Wubben, Martin J; Callahan, Franklin E; Triplett, Barbara A; Jenkins, Johnie N

    2009-09-01

    Agrobacterium rhizogenes-induced cotton (Gossypium hirsutum L.) hairy roots were evaluated as a model system for studying molecular cotton-nematode interactions. Hairy root cultures were developed from the root-knot nematode (RKN) (Meloidogyne incognita [Kofoid and White] Chitwood, race 3)-resistant breeding line M315 and from the reniform nematode (RN) (Rotylenchulus reniformis Linford & Oliveira)-resistant accession GB713 (G. barbadense L.) and compared to a nematode-susceptible culture derived from the obsolete cultivar DPL90. M315, GB713, and DPL90 hairy roots differed significantly in their appearance and growth potential; however, these differences were not correlated with transcript levels of the A. rhizogenes T-DNA genes rolB and aux2 which help regulate hairy root initiation and proliferation. DPL90 hairy roots were found to support both RKN and RN reproduction in tissue culture, whereas M315 and GB713 hairy roots were resistant to RKN and RN, respectively. M315 hairy roots showed constitutive up-regulation of the defense gene MIC3 (Meloidogyne Induced Cotton3) compared to M315 whole-plant roots and DPL90 hairy roots. Our data show the potential use of cotton hairy roots in maintaining monoxenic RKN and RN cultures and suggest hairy roots may be useful in evaluating the effect of manipulated host gene expression on nematode resistance in cotton.

  17. Molecular, phenotypic, and sample-associated data to describe pluripotent stem cell lines and derivatives

    Science.gov (United States)

    Daily, Kenneth; Ho Sui, Shannan J.; Schriml, Lynn M.; Dexheimer, Phillip J.; Salomonis, Nathan; Schroll, Robin; Bush, Stacy; Keddache, Mehdi; Mayhew, Christopher; Lotia, Samad; Perumal, Thanneer M.; Dang, Kristen; Pantano, Lorena; Pico, Alexander R.; Grassman, Elke; Nordling, Diana; Hide, Winston; Hatzopoulos, Antonis K.; Malik, Punam; Cancelas, Jose A.; Lutzko, Carolyn; Aronow, Bruce J.; Omberg, Larsson

    2017-01-01

    The use of induced pluripotent stem cells (iPSC) derived from independent patients and sources holds considerable promise to improve the understanding of development and disease. However, optimized use of iPSC depends on our ability to develop methods to efficiently qualify cell lines and protocols, monitor genetic stability, and evaluate self-renewal and differentiation potential. To accomplish these goals, 57 stem cell lines from 10 laboratories were differentiated to 7 different states, resulting in 248 analyzed samples. Cell lines were differentiated and characterized at a central laboratory using standardized cell culture methodologies, protocols, and metadata descriptors. Stem cell and derived differentiated lines were characterized using RNA-seq, miRNA-seq, copy number arrays, DNA methylation arrays, flow cytometry, and molecular histology. All materials, including raw data, metadata, analysis and processing code, and methodological and provenance documentation are publicly available for re-use and interactive exploration at https://www.synapse.org/pcbc. The goal is to provide data that can improve our ability to robustly and reproducibly use human pluripotent stem cells to understand development and disease. PMID:28350385

  18. Glycan-decorated HPMA copolymers as high-affinity lectin ligands

    Czech Academy of Sciences Publication Activity Database

    Bojarová, Pavla; Chytil, Petr; Mikulová, Barbora; Bumba, Ladislav; Konefal, Rafal; Pelantová, Helena; Krejzová, Jana; Slámová, Kristýna; Petrásková, Lucie; Kotrchová, Lenka; Cvačka, Josef; Etrych, Tomáš; Křen, Vladimír

    2017-01-01

    Roč. 8, č. 17 (2017), s. 2647-2658 ISSN 1759-9954 R&D Projects: GA ČR GC15-02578J; GA MZd(CZ) NV16-28594A; GA MŠk(CZ) LD15085; GA MŠk(CZ) LM2015064; GA MŠk(CZ) LO1507 Institutional support: RVO:61388971 ; RVO:61389013 ; RVO:61388963 Keywords : BETA-N-ACETYLHEXOSAMINIDASE * WHEAT-GERM-AGGLUTININ * CLICK CHEMISTRY Subject RIV: CE - Biochemistry; CD - Macromolecular Chemistry (UMCH-V) OBOR OECD: Biochemistry and molecular biology; Polymer science (UMCH-V) Impact factor: 5.375, year: 2016

  19. Detailed phenotypic and molecular analyses of genetically modified mice generated by CRISPR-Cas9-mediated editing.

    Directory of Open Access Journals (Sweden)

    Bijal A Parikh

    Full Text Available The bacterial CRISPR-Cas9 system has been adapted for use as a genome editing tool. While several recent reports have indicated that successful genome editing of mice can be achieved, detailed phenotypic and molecular analyses of the mutant animals are limited. Following pronuclear micro-injection of fertilized eggs with either wild-type Cas9 or the nickase mutant (D10A and single or paired guide RNA (sgRNA for targeting of the tyrosinase (Tyr gene, we assessed genome editing in mice using rapid phenotypic readouts (eye and coat color. Mutant mice with insertions or deletions (indels in Tyr were efficiently generated without detectable off-target cleavage events. Gene correction of a single nucleotide by homologous recombination (HR could only occur when the sgRNA recognition sites in the donor DNA were modified. Gene repair did not occur if the donor DNA was not modified because Cas9 catalytic activity was completely inhibited. Our results indicate that allelic mosaicism can occur following -Cas9-mediated editing in mice and appears to correlate with sgRNA cleavage efficiency at the single-cell stage. We also show that larger than expected deletions may be overlooked based on the screening strategy employed. An unbiased analysis of all the deleted nucleotides in our experiments revealed that the highest frequencies of nucleotide deletions were clustered around the predicted Cas9 cleavage sites, with slightly broader distributions than expected. Finally, additional analysis of founder mice and their offspring indicate that their general health, fertility, and the transmission of genetic changes were not compromised. These results provide the foundation to interpret and predict the diverse outcomes following CRISPR-Cas9-mediated genome editing experiments in mice.

  20. Systems Modeling of Molecular Mechanisms Controlling Cytokine-driven CD4+ T Cell Differentiation and Phenotype Plasticity

    Science.gov (United States)

    Carbo, Adria; Hontecillas, Raquel; Kronsteiner, Barbara; Viladomiu, Monica; Pedragosa, Mireia; Lu, Pinyi; Philipson, Casandra W.; Hoops, Stefan; Marathe, Madhav; Eubank, Stephen; Bisset, Keith; Wendelsdorf, Katherine; Jarrah, Abdul; Mei, Yongguo; Bassaganya-Riera, Josep

    2013-01-01

    Differentiation of CD4+ T cells into effector or regulatory phenotypes is tightly controlled by the cytokine milieu, complex intracellular signaling networks and numerous transcriptional regulators. We combined experimental approaches and computational modeling to investigate the mechanisms controlling differentiation and plasticity of CD4+ T cells in the gut of mice. Our computational model encompasses the major intracellular pathways involved in CD4+ T cell differentiation into T helper 1 (Th1), Th2, Th17 and induced regulatory T cells (iTreg). Our modeling efforts predicted a critical role for peroxisome proliferator-activated receptor gamma (PPARγ) in modulating plasticity between Th17 and iTreg cells. PPARγ regulates differentiation, activation and cytokine production, thereby controlling the induction of effector and regulatory responses, and is a promising therapeutic target for dysregulated immune responses and inflammation. Our modeling efforts predict that following PPARγ activation, Th17 cells undergo phenotype switch and become iTreg cells. This prediction was validated by results of adoptive transfer studies showing an increase of colonic iTreg and a decrease of Th17 cells in the gut mucosa of mice with colitis following pharmacological activation of PPARγ. Deletion of PPARγ in CD4+ T cells impaired mucosal iTreg and enhanced colitogenic Th17 responses in mice with CD4+ T cell-induced colitis. Thus, for the first time we provide novel molecular evidence in vivo demonstrating that PPARγ in addition to regulating CD4+ T cell differentiation also plays a major role controlling Th17 and iTreg plasticity in the gut mucosa. PMID:23592971

  1. The Physiological and Molecular Characterization of a Small Colony Variant of Escherichia coli and Its Phenotypic Rescue.

    Directory of Open Access Journals (Sweden)

    Victor Santos

    Full Text Available Small colony variants (SCVs can be defined as a naturally occurring sub-population of bacteria characterized by their reduced colony size and distinct biochemical properties. SCVs of Staphylococcus aureus have been studied extensively over the past two decades due to their role in recurrent human infections. However, little work has been done on SCVs of Escherichia coli, and this work has focused on the physiology and morphology that define these colonies of E. coli, such as small size and slow growth. E. coli strain JW0623, has a null lipA mutation in the lipoic acid synthase gene (lipA, and is a lipoic acid auxotroph. When the mutant was grown in LB medium to log phase, it showed remarkable resistance to acid (pH 3, hydrogen peroxide, heat and osmotic stress compared to its parent BW25113. Using RT-PCR and real time RT-PCR, the expression of certain genes was compared in the two strains in an attempt to create a molecular profile of Escherichia coli SCVs. These include genes involved in glycolysis, TCA cycle, electron transport, iron acquisition, biofilm formation and cyclopropane fatty acid synthesis. It was also demonstrated that the addition of 5 μg/ml of lipoic acid to LB medium allows for the phenotypic rescue of the mutant; reversing its slow growth, its resistance characteristics, and elevated gene expression. These results indicate that the mutation in lipA leads to an E. coli SCV that resembles an electron transport defective SCV of S. aureus These strains are typically auxotrophs, and are phenotypically rescued by adding the missing metabolite to rich medium. There are global shifts in gene expression which are reversible and depend on whether the auxotrophic molecule is absent or present. Looking at the E. coli SCV from an evolutionary point of view, it becomes evident that its path to survival is to express genes that confer stress resistance.

  2. No Genetic Diversity at Molecular Markers and Strong Phenotypic Plasticity in Populations of Ranunculus nodiflorus, an Endangered Plant Species in France

    Science.gov (United States)

    Noel, Florence; Machon, Nathalie; Porcher, Emmanuelle

    2007-01-01

    Background and Aims Although conservation biology has long focused on population dynamics and genetics, phenotypic plasticity is likely to play a significant role in population viability. Here, an investigation is made into the relative contribution of genetic diversity and phenotypic plasticity to the phenotypic variation in natural populations of Ranunculus nodiflorus, a rare annual plant inhabiting temporary puddles in the Fontainebleau forest (Paris region, France) and exhibiting metapopulation dynamics. Methods The genetic diversity and phenotypic plasticity of quantitative traits (morphological and fitness components) were measured in five populations, using a combination of field measurements, common garden experiments and genotyping at microsatellite loci. Key Results It is shown that populations exhibit almost undetectable genetic diversity at molecular markers, and that the variation in quantitative traits observed among populations is due to a high level of phenotypic plasticity. Despite the lack of genetic diversity, the natural population of R. nodiflorus exhibits large population sizes and does not appear threatened by extinction; this may be attributable to large phenotypic plasticity, enabling the production of numerous seeds under a wide range of environmental conditions. Conclusions Efficient conservation of the populations can only be based on habitat management, to favour the maintenance of microenvironmental variation and the resulting strong phenotypic plasticity. In contrast, classical actions aiming to improve genetic diversity are useless in the present case. PMID:17468109

  3. Molecular genetics of addiction and related heritable phenotypes: genome-wide association approaches identify "connectivity constellation" and drug target genes with pleiotropic effects.

    Science.gov (United States)

    Uhl, George R; Drgon, Tomas; Johnson, Catherine; Li, Chuan-Yun; Contoreggi, Carlo; Hess, Judith; Naiman, Daniel; Liu, Qing-Rong

    2008-10-01

    Genome-wide association (GWA) can elucidate molecular genetic bases for human individual differences in complex phenotypes that include vulnerability to addiction. Here, we review (a) evidence that supports polygenic models with (at least) modest heterogeneity for the genetic architectures of addiction and several related phenotypes; (b) technical and ethical aspects of importance for understanding GWA data, including genotyping in individual samples versus DNA pools, analytic approaches, power estimation, and ethical issues in genotyping individuals with illegal behaviors; (c) the samples and the data that shape our current understanding of the molecular genetics of individual differences in vulnerability to substance dependence and related phenotypes; (d) overlaps between GWA data sets for dependence on different substances; and (e) overlaps between GWA data for addictions versus other heritable, brain-based phenotypes that include bipolar disorder, cognitive ability, frontal lobe brain volume, the ability to successfully quit smoking, neuroticism, and Alzheimer's disease. These convergent results identify potential targets for drugs that might modify addictions and play roles in these other phenotypes. They add to evidence that individual differences in the quality and quantity of brain connections make pleiotropic contributions to individual differences in vulnerability to addictions and to related brain disorders and phenotypes. A "connectivity constellation" of brain phenotypes and disorders appears to receive substantial pathogenic contributions from individual differences in a constellation of genes whose variants provide individual differences in the specification of brain connectivities during development and in adulthood. Heritable brain differences that underlie addiction vulnerability thus lie squarely in the midst of the repertoire of heritable brain differences that underlie vulnerability to other common brain disorders and phenotypes.

  4. Molecular and Phenotypic Characterization of Staphylococcus epidermidis Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection.

    Directory of Open Access Journals (Sweden)

    Luis A Flores-Páez

    Full Text Available Staphylococcus epidermidis is a common commensal of healthy conjunctiva and it can cause endophthalmitis, however its presence in conjunctivitis, keratitis and blepharitis is unknown. Molecular genotyping of S. epidermidis from healthy conjunctiva could provide information about the origin of the strains that infect the eye. In this paper two collections of S. epidermidis were used: one from ocular infection (n = 62, and another from healthy conjunctiva (n = 45. All isolates were genotyped by pulsed field gel electrophoresis (PFGE, multilocus sequence typing (MLST, staphylococcal cassette chromosome mec (SCCmec, detection of the genes icaA, icaD, IS256 and polymorphism type of agr locus. The phenotypic data included biofilm production and antibiotic resistance. The results displayed 61 PFGE types from 107 isolates and they were highly discriminatory. MLST analysis generated a total of 25 STs, of which 11 STs were distributed among the ocular infection isolates and lineage ST2 was the most frequent (48.4%, while 14 STs were present in the healthy conjunctiva isolates and lineage ST5 was the most abundant (24.4%. By means of a principal coordinates analysis (PCoA and a discriminant analysis (DA it was found that ocular infection isolates had as discriminant markers agr III or agr II, SCCmec V or SCCmec I, mecA gene, resistance to tobramycin, positive biofilm, and IS256+. In contrast to the healthy conjunctiva isolates, the discriminating markers were agr I, and resistance to chloramphenicol, ciprofloxacin, gatifloxacin and oxacillin. The discriminant biomarkers of ocular infection were examined in healthy conjunctiva isolates, and it was found that 3 healthy conjunctiva isolates [two with ST2 and another with ST9] (3/45, 6.66% had similar genotypic and phenotypic characteristics to ocular infection isolates, therefore a small population from healthy conjunctiva could cause an ocular infection. These data suggest that the healthy conjunctiva

  5. Molecular and Phenotypic Characterization of Staphylococcus epidermidis Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection

    Science.gov (United States)

    Flores-Páez, Luis A.; Zenteno, Juan C.; Alcántar-Curiel, María D.; Vargas-Mendoza, Carlos F.; Rodríguez-Martínez, Sandra; Cancino-Diaz, Mario E.; Jan-Roblero, Janet; Cancino-Diaz, Juan C.

    2015-01-01

    Staphylococcus epidermidis is a common commensal of healthy conjunctiva and it can cause endophthalmitis, however its presence in conjunctivitis, keratitis and blepharitis is unknown. Molecular genotyping of S. epidermidis from healthy conjunctiva could provide information about the origin of the strains that infect the eye. In this paper two collections of S. epidermidis were used: one from ocular infection (n = 62), and another from healthy conjunctiva (n = 45). All isolates were genotyped by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), detection of the genes icaA, icaD, IS256 and polymorphism type of agr locus. The phenotypic data included biofilm production and antibiotic resistance. The results displayed 61 PFGE types from 107 isolates and they were highly discriminatory. MLST analysis generated a total of 25 STs, of which 11 STs were distributed among the ocular infection isolates and lineage ST2 was the most frequent (48.4%), while 14 STs were present in the healthy conjunctiva isolates and lineage ST5 was the most abundant (24.4%). By means of a principal coordinates analysis (PCoA) and a discriminant analysis (DA) it was found that ocular infection isolates had as discriminant markers agr III or agr II, SCCmec V or SCCmec I, mecA gene, resistance to tobramycin, positive biofilm, and IS256+. In contrast to the healthy conjunctiva isolates, the discriminating markers were agr I, and resistance to chloramphenicol, ciprofloxacin, gatifloxacin and oxacillin. The discriminant biomarkers of ocular infection were examined in healthy conjunctiva isolates, and it was found that 3 healthy conjunctiva isolates [two with ST2 and another with ST9] (3/45, 6.66%) had similar genotypic and phenotypic characteristics to ocular infection isolates, therefore a small population from healthy conjunctiva could cause an ocular infection. These data suggest that the healthy conjunctiva isolates do not

  6. High-affinity single-domain binding proteins with a binary-code interface.

    Science.gov (United States)

    Koide, Akiko; Gilbreth, Ryan N; Esaki, Kaori; Tereshko, Valentina; Koide, Shohei

    2007-04-17

    High degrees of sequence and conformation complexity found in natural protein interaction interfaces are generally considered essential for achieving tight and specific interactions. However, it has been demonstrated that specific antibodies can be built by using an interface with a binary code consisting of only Tyr and Ser. This surprising result might be attributed to yet undefined properties of the antibody scaffold that uniquely enhance its capacity for target binding. In this work we tested the generality of the binary-code interface by engineering binding proteins based on a single-domain scaffold. We show that Tyr/Ser binary-code interfaces consisting of only 15-20 positions within a fibronectin type III domain (FN3; 95 residues) are capable of producing specific binding proteins (termed "monobodies") with a low-nanomolar K(d). A 2.35-A x-ray crystal structure of a monobody in complex with its target, maltose-binding protein, and mutation analysis revealed dominant contributions of Tyr residues to binding as well as striking molecular mimicry of a maltose-binding protein substrate, beta-cyclodextrin, by the Tyr/Ser binary interface. This work suggests that an interaction interface with low chemical diversity but with significant conformational diversity is generally sufficient for tight and specific molecular recognition, providing fundamental insights into factors governing protein-protein interactions.

  7. Structure of IL-22 Bound to Its High-Affinity IL-22R1 Chain

    Energy Technology Data Exchange (ETDEWEB)

    Jones, B.C.; Logsdon, N.J.; Walter, M.R. (UAB)

    2008-09-29

    IL-22 is an IL-10 family cytokine that initiates innate immune responses against bacterial pathogens and contributes to immune disease. IL-22 biological activity is initiated by binding to a cell-surface complex composed of IL-22R1 and IL-10R2 receptor chains and further regulated by interactions with a soluble binding protein, IL-22BP, which shares sequence similarity with an extracellular region of IL-22R1 (sIL-22R1). IL-22R1 also pairs with the IL-20R2 chain to induce IL-20 and IL-24 signaling. To define the molecular basis of these diverse interactions, we have determined the structure of the IL-22/sIL-22R1 complex. The structure, combined with homology modeling and surface plasmon resonance studies, defines the molecular basis for the distinct affinities and specificities of IL-22 and IL-10 receptor chains that regulate cellular targeting and signal transduction to elicit effective immune responses.

  8. High-affinity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions.

    Science.gov (United States)

    Cailliatte, Rémy; Schikora, Adam; Briat, Jean-François; Mari, Stéphane; Curie, Catherine

    2010-03-01

    In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis.

  9. A putative high affinity phosphate transporter, CmPT1, enhances tolerance to Pi deficiency of chrysanthemum.

    Science.gov (United States)

    Liu, Peng; Chen, Sumei; Song, Aiping; Zhao, Shuang; Fang, Weimin; Guan, Zhiyong; Liao, Yuan; Jiang, Jiafu; Chen, Fadi

    2014-01-10

    Inorganic phosphate (Pi) is essential for plant growth, and phosphorus deficiency is a main limiting factor in plant development. Its acquisition is largely mediated by Pht1 transporters, a family of plasma membrane-located proteins. Chrysanthemum is one of the most important ornamental plants, its productivity is usually compromised when grown in phosphate deficient soils, but the study of phosphate transporters in chrysanthemum is limited. We described the isolation from chrysanthemum of a homolog of the Phosphate Transporter 1 (PT1) family. Its predicted product is a protein with 12 transmembrane domains, highly homologous with other high affinity plant Pi transporters. Real-time quantitative PCR analysis revealed that the gene was transcribed strongly in the root, weakly in the stem and below the level of detection in the leaf of chrysanthemum plants growing in either sufficient or deficient Pi conditions. Transcript abundance was greatly enhanced in Pi-starved roots. A complementation assay in yeast showed that CmPT1 partially compensated for the absence of phosphate transporter activity in yeast strain MB192. The estimated Km of CmPT1 was 35.2 μM. Under both Pi sufficient and deficient conditions, transgenic plants constitutively expressing CmPT1 grew taller than the non-transformed wild type, produced a greater volume of roots, accumulated more biomass and took up more phosphate. CmPT1 encodes a typical, root-expressed, high affinity phosphate transporter, plays an important role in coping Pi deficiency of chrysanthemum plants.

  10. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Directory of Open Access Journals (Sweden)

    Eszter Lázár-Molnár

    2017-03-01

    Full Text Available Programmed Cell Death-1 (PD-1 is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1 exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  11. Identification of a high-affinity Ca sup 2+ pump associated with endocytotic vesicles in Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Milne, J.L.; Coukell, M.B. (York Univ., North York, Ontario (Canada))

    1989-11-01

    In the cellular slime mold Dictyostelium discoideum, changes in free cytosolic Ca{sup 2+} are thought to regulate certain processes during cell aggregation and differentiation. To understand the mechanisms controlling free Ca{sup 2+} levels in this organism, the authors previously isolated and characterized an ATP/Mg{sup 2+}-dependent, high-affinity Ca{sup 2+} pump which appeared to be a component of inside-out plasma membrane vesicles. In this report, they demonstrate that a high-affinity Ca{sup 2+} pump, with properties virtually identical to the isolated pump, can be detected in filipin- or digitonin-permeabilized cells of Dictyostelium. Moreover, Ca{sup 2+}-pumping vesicles, which migrate on Percoll/KCl gradients like the vesicles identified earlier, can be isolated from the permeabilized cells. Results of additional experiments suggest that this intracellular Ca{sup 2+} transporter is associated with a high-capacity non-IP{sub 3}-releasable Ca{sup 2+} store which is generated by endocytosis. A possible role for this store in maintaining Ca{sup 2+} homeostasis in Dictyostelium is discussed.

  12. Nickel(II) Inhibits Tet-Mediated 5-Methylcytosine Oxidation by High Affinity Displacement of the Cofactor Iron(II).

    Science.gov (United States)

    Yin, Ruichuan; Mo, Jiezhen; Dai, Jiayin; Wang, Hailin

    2017-06-16

    Ten-eleven translocation (Tet) family proteins are Fe(II)- and 2-oxoglutarate-dependent dioxygenases that regulate the dynamics of DNA methylation by catalyzing the oxidation of DNA 5-methylcytosine (5mC). To exert physiologically important functions, redox-active iron chelated in the catalytic center of Tet proteins directly involves the oxidation of the multiple substrates. To understand the function and interaction network of Tet dioxygenases, it is interesting to obtain high affinity and a specific inhibitor. Surprisingly, here we found that natural Ni(II) ion can bind to the Fe(II)-chelating motif (HXD) with an affinity of 7.5-fold as high as Fe(II). Consistently, we further found that Ni(II) ion can displace the cofactor Fe(II) of Tet dioxygenases and inhibit Tet-mediated 5mC oxidation activity with an estimated IC50 of 1.2 μM. Essentially, Ni(II) can be used as a high affinity and selective inhibitor to explore the function and dynamics of Tet proteins.

  13. Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

    DEFF Research Database (Denmark)

    Axelsen, Trine Veje; Holm, Arne; Birkelund, Svend

    2009-01-01

    The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42......) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope...

  14. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Lázár-Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil; Quinn, Thomas; Sylvestre, Eliezer; Palmieri, Edith; Ramagopal, Udupi A.; Nathenson, Stanley G.; Guha, Chandan; Almo, Steven C.

    2017-03-01

    Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  15. Molecular value predictions: associations with beef quality, carcass, production, behavior, and efficiency phenotypes in Brahman cattle.

    Science.gov (United States)

    Greenwood, P L; Cafe, L M; McIntyre, B L; Geesink, G H; Thompson, J M; Polkinghorne, R; Pethick, D W; Robinson, D L

    2013-12-01

    Data from 2 previously published experiments, New South Wales (NSW; n = 161) and Western Australia (WA; n = 135), were used to test molecular value predictions (MVP), generated from commercially available gene markers, on economically important traits of Bos indicus (Brahman) cattle. Favorable tenderness MVP scores were associated with reduced shear force values of strip loin (LM) steaks aged 7 d from Achilles-hung carcasses (P ≤ 0.06), as well as steaks aged 1 (P ≤ 0.08) or 7 d (P ≤ 0.07) from carcasses hung from the pelvis (tenderstretch). Favorable tenderness MVP scores were also associated with improved consumer tenderness ratings for strip loin steaks aged 7 d and either Achilles hung (P ≤ 0.006) or tenderstretched (P ≤ 0.07). Similar results were observed in NSW for rump (top butt; gluteus medius) steaks, with favorable tenderness MVP scores associated with more tender (P = 0.006) and acceptable (P = 0.008) beef. Favorable marbling MVP scores were associated with improved (P ≤ 0.021) marbling scores and intramuscular fat (IMF) content in the NSW experiment, despite low variation in marbling in the Brahman cattle. For the WA experiment, however, there were no (P ≥ 0.71) relationships between marbling MVP and marbling scores or IMF content. Although residual (net) feed intake (RFI) was not associated (P = 0.63) with the RFI (feed efficiency) MVP, the RFI MVP was adversely associated with LM tenderness and acceptability of 7-d-aged Achilles-hung carcasses in NSW (P ≤ 0.031) and WA (P ≤ 0.037). Some other relationships and trends were noted between the MVP and the other traits, but few reached statistical significance, and none were evident in both experiments. Results from this study provide evidence to support the use of the tenderness MVP. The value of the marbling MVP, which was associated with marbling in only 1 herd, warrants further evaluation; however, there appears to be no evidence to support use of the RFI MVP in Brahman cattle.

  16. Molecular detection of fluoroquinolone-resistance in multi-drug resistant tuberculosis in Cambodia suggests low association with XDR phenotypes

    Directory of Open Access Journals (Sweden)

    Murray Alan

    2011-09-01

    Full Text Available Abstract Background Drug susceptibility testing (DST remains an important concern for implementing treatment of MDR tuberculosis patients. Implementation of molecular tests for drug resistance identification would facilitate DST particularly in developing countries where culturing is difficult to perform. We have characterized multidrug resistant strains in Cambodia using MDTDRsl tests, drug target sequencing and phenotypic tests. Methods A total of 65 non-MDR and 101 MDR TB isolates collected between May 2007 and June 2009 were tested for resistance to fluoroquinolones and aminoglycosides/cyclic peptides using the GenoType® MTBDRsl assay and gene sequencing. Rifampicin resistance (RMP-R was tested using gene sequencing and genotyping was assessed by spoligotyping. Results A total of 95 of the 101 MDR strains were confirmed to be RMP-R by rpoB gene sequencing. Fourteen of the 101 MDR isolates (14% carried a gyrA mutation associated with fluoroquinolone-resistance (FQ-R (detected by the MTBDRsl assay and sequencing compared with only 1 (1.5% of the 65 non-MDR strains. Only 1 (1% of the MDR isolates was found to be XDR TB. The MDR group contained a higher proportion of Beijing or Beijing like strains (58% than the non MDR group (28%. This percentage is higher in MDR FQ-R strains (71%. Conclusions The new GenoType® MTBDRsl assay combined with molecular tests to detect RMP-R and isoniazid resistance (INH-R represents a valuable tool for the detection of XDR TB. In Cambodia there is a low rate of XDR amongst MDR TB including MDR FQ-R TB. This suggests a low association between FQ-R and XDR TB. Strain spoligotyping confirms Beijing strains to be more prone to accumulate antibiotic resistance.

  17. [The study of dynamics of clinical and molecular phenotypes in the generalized form of myasthenia with optimized and combined treatment].

    Science.gov (United States)

    Drozd, O A; Efremov, V V; Romantsov, M G; Sarvilina, I V

    2013-01-01

    The aim of the study was the research of clinical and molecular phenotypes in the generalized form of myasthenia with optimized treantment, which includes the application of basic mode of the treatment and meglumine sodium succinate, and also the combined mode of the treatment, which includes the mode of the treatment and meglumine sodium succinate and ozonotherapy. The most epressed improvement of indicators of the clinical and neurologic status (the eliminaton of motoring breakdown, eye movement disrders, the weaknesses of mimic, respiratory and chewing muscles, bulbar syndrome, the doubling in eye and ptosis, pharyngeal violations and increase in volume of movement of eyes) was observed in the group of patients receiving the combined therapy. The reduction of the epressiveness of the defeat of the neuromotor device and the decrement of the amplitude of the various degree of M-answer in patients with myasthenla is revealed in the application of the combined therapy in the comparison with the application of the basic and optimized therapy. For the first time we have investigaed the dynamics of the intensity of the expression of specfic peptides and proteins in blood serum in patients with the application of basic therapy, reamberin and ozonotherapy, which have allowed to open the new mechanisms of the efficiency ofthe combined therapy of the disease.

  18. Targeted next-generation sequencing makes new molecular diagnoses and expands genotype-phenotype relationship in Ehlers-Danlos syndrome.

    Science.gov (United States)

    Weerakkody, Ruwan A; Vandrovcova, Jana; Kanonidou, Christina; Mueller, Michael; Gampawar, Piyush; Ibrahim, Yousef; Norsworthy, Penny; Biggs, Jennifer; Abdullah, Abdulshakur; Ross, David; Black, Holly A; Ferguson, David; Cheshire, Nicholas J; Kazkaz, Hanadi; Grahame, Rodney; Ghali, Neeti; Vandersteen, Anthony; Pope, F Michael; Aitman, Timothy J

    2016-11-01

    Ehlers-Danlos syndrome (EDS) comprises a group of overlapping hereditary disorders of connective tissue with significant morbidity and mortality, including major vascular complications. We sought to identify the diagnostic utility of a next-generation sequencing (NGS) panel in a mixed EDS cohort. We developed and applied PCR-based NGS assays for targeted, unbiased sequencing of 12 collagen and aortopathy genes to a cohort of 177 unrelated EDS patients. Variants were scored blind to previous genetic testing and then compared with results of previous Sanger sequencing. Twenty-eight pathogenic variants in COL5A1/2, COL3A1, FBN1, and COL1A1 and four likely pathogenic variants in COL1A1, TGFBR1/2, and SMAD3 were identified by the NGS assays. These included all previously detected single-nucleotide and other short pathogenic variants in these genes, and seven newly detected pathogenic or likely pathogenic variants leading to clinically significant diagnostic revisions. Twenty-two variants of uncertain significance were identified, seven of which were in aortopathy genes and required clinical follow-up. Unbiased NGS-based sequencing made new molecular diagnoses outside the expected EDS genotype-phenotype relationship and identified previously undetected clinically actionable variants in aortopathy susceptibility genes. These data may be of value in guiding future clinical pathways for genetic diagnosis in EDS.Genet Med 18 11, 1119-1127.

  19. Molecular analysis of TSC1 and TSC2 genes and phenotypic correlations in Brazilian families with tuberous sclerosis.

    Directory of Open Access Journals (Sweden)

    Clévia Rosset

    Full Text Available Tuberous sclerosis complex (TSC is an autosomal dominant multisystem disorder characterized by the development of multiple hamartomas in many organs and tissues. It occurs due to inactivating mutations in either of the two genes, TSC1 and TSC2, following a second hit in a tumor suppressor gene in most hamartomas. Comprehensive screening for mutations in both the TSC1 and TSC2 loci has been performed in several cohorts of patients and a broad spectrum of pathogenic mutations have been described. In Brazil, there is no data regarding incidence and prevalence of tuberous sclerosis and mutations in TSC1 and TSC2. We analyzed both genes in 53 patients with high suspicion of tuberous sclerosis using multiplex-ligation dependent probe amplification and a customized next generation sequencing panel. Confirmation of all variants was done by the Sanger method. We identified 50 distinct variants in 47 (89% of the patients. Five were large rearrangements and 45 were point mutations. The symptoms presented by our series of patients were not different between male and female individuals, except for the more common occurrence of shagreen patch in women (p = 0.028. In our series, consistent with other studies, TSC2 mutations were associated with a more severe phenotypic spectrum than TSC1 mutations. This is the first study that sought to characterize the molecular spectrum of Brazilian individuals with tuberous sclerosis.

  20. Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1.

    Science.gov (United States)

    Belenky, Peter A; Moga, Tiberiu G; Brenner, Charles

    2008-03-28

    NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.

  1. Scalable high-affinity stabilization of magnetic iron oxide nanostructures by a biocompatible antifouling homopolymer

    KAUST Repository

    Luongo, Giovanni

    2017-10-12

    Iron oxide nanostructures have been widely developed for biomedical applications, due to their magnetic properties and biocompatibility. In clinical application, the stabilization of these nanostructures against aggregation and non-specific interactions is typically achieved using weakly anchored polysaccharides, with better-defined and more strongly anchored synthetic polymers not commercially adopted due to complexity of synthesis and use. Here, we show for the first time stabilization and biocompatibilization of iron oxide nanoparticles by a synthetic homopolymer with strong surface anchoring and a history of clinical use in other applications, poly(2-methacryloyloxyethy phosphorylcholine) (poly(MPC)). For the commercially important case of spherical particles, binding of poly(MPC) to iron oxide surfaces and highly effective individualization of magnetite nanoparticles (20 nm) are demonstrated. Next-generation high-aspect ratio nanowires (both magnetite/maghemite and core-shell iron/iron oxide) are furthermore stabilized by poly(MPC)-coating, with nanowire cytotoxicity at large concentrations significantly reduced. The synthesis approach is exploited to incorporate functionality into the poly(MPC) chain is demonstrated by random copolymerization with an alkyne-containing monomer for click-chemistry. Taking these results together, poly(MPC) homopolymers and random copolymers offer a significant improvement over current iron oxide nanoformulations, combining straightforward synthesis, strong surface-anchoring and well-defined molecular weight.

  2. High affinity human antibody fragments to dengue virus non-structural protein 3.

    Directory of Open Access Journals (Sweden)

    Nicole J Moreland

    Full Text Available BACKGROUND: The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3 are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5' triphosphatase domain which forms the remainder of the 618-aa long protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531 within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. CONCLUSIONS/SIGNIFICANCE: Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

  3. Utilization of molecular, phenotypic and geographical diversity to develop compact composite core collection in the oilseed crop, Safflower (Carthamus tinctorius L. through maximization strategy

    Directory of Open Access Journals (Sweden)

    Shivendra Kumar

    2016-10-01

    Full Text Available Carthamus tinctorius L. is a dryland oilseed crop yielding high quality edible oil. Previous studies have described significant phenotypic variability in the crop and used geographical distribution and phenotypic trait values to develop core collections. However, the molecular diversity component was lacking in the earlier collections thereby limiting their utility in breeding programs. The present study evaluated the phenotypic variability for twelve agronomically important traits during two growing seasons (2011-12 and 2012-13 in a global reference collection of 531 safflower accessions, assessed earlier by our group for genetic diversity and population structure using AFLP markers. Significant phenotypic variation was observed for all the agronomic traits in the representative collection. Cluster analysis of phenotypic data grouped the accessions into five major clusters. Accessions from the Indian Subcontinent and America harboured maximal phenotypic variability with unique characters for a few traits. MANOVA analysis indicated significant interaction between genotypes and environment for both the seasons. Initially, six independent core collections (CC1 – CC6 were developed using molecular marker and phenotypic data for two seasons through POWERCORE and MSTRAT. These collections captured the entire range of trait variability but failed to include complete genetic diversity represented in 19 clusters reported earlier through Bayesian Analysis of Population Structure (BAPS. Therefore, we merged the three POWERCORE core collections (CC1 – CC3 to generate a composite core collection, CartC1 and three MSTRAT core collections (CC4 – CC6to generate another composite core collection, CartC2.The mean difference percentage, variance difference percentage, variable rate of coefficient of variance percentage, coincidence rate of range percentage, Shannon’s diversity index and Nei’s gene diversity for CartC1 were 11.2, 43.7, 132.4, 93.4, 0

  4. Utilization of Molecular, Phenotypic, and Geographical Diversity to Develop Compact Composite Core Collection in the Oilseed Crop, Safflower (Carthamus tinctorius L.) through Maximization Strategy.

    Science.gov (United States)

    Kumar, Shivendra; Ambreen, Heena; Variath, Murali T; Rao, Atmakuri R; Agarwal, Manu; Kumar, Amar; Goel, Shailendra; Jagannath, Arun

    2016-01-01

    Safflower (Carthamus tinctorius L.) is a dryland oilseed crop yielding high quality edible oil. Previous studies have described significant phenotypic variability in the crop and used geographical distribution and phenotypic trait values to develop core collections. However, the molecular diversity component was lacking in the earlier collections thereby limiting their utility in breeding programs. The present study evaluated the phenotypic variability for 12 agronomically important traits during two growing seasons (2011-12 and 2012-13) in a global reference collection of 531 safflower accessions, assessed earlier by our group for genetic diversity and population structure using AFLP markers. Significant phenotypic variation was observed for all the agronomic traits in the representative collection. Cluster analysis of phenotypic data grouped the accessions into five major clusters. Accessions from the Indian Subcontinent and America harbored maximal phenotypic variability with unique characters for a few traits. MANOVA analysis indicated significant interaction between genotypes and environment for both the seasons. Initially, six independent core collections (CC1-CC6) were developed using molecular marker and phenotypic data for two seasons through POWERCORE and MSTRAT. These collections captured the entire range of trait variability but failed to include complete genetic diversity represented in 19 clusters reported earlier through Bayesian analysis of population structure (BAPS). Therefore, we merged the three POWERCORE core collections (CC1-CC3) to generate a composite core collection, CartC1 and three MSTRAT core collections (CC4-CC6) to generate another composite core collection, CartC2. The mean difference percentage, variance difference percentage, variable rate of coefficient of variance percentage, coincidence rate of range percentage, Shannon's diversity index, and Nei's gene diversity for CartC1 were 11.2, 43.7, 132.4, 93.4, 0.47, and 0

  5. Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity: EFFECTS ON EMBRYO SURVIVAL.

    Science.gov (United States)

    Fresco, Victor M; Kern, Christine B; Mohammadi, Moosa; Twal, Waleed O

    2016-09-02

    Fibulin-1 (FBLN1) is a member of a growing family of extracellular matrix glycoproteins that includes eight members and is involved in cellular functions such as adhesion, migration, and differentiation. FBLN1 has also been implicated in embryonic heart and valve development and in the formation of neural crest-derived structures, including aortic arch, thymus, and cranial nerves. Fibroblast growth factor 8 (FGF8) is a member of a large family of growth factors, and its functions include neural crest cell (NCC) maintenance, specifically NCC migration as well as patterning of structures formed from NCC such as outflow tract and cranial nerves. In this report, we sought to investigate whether FBLN1 and FGF8 have cooperative roles in vivo given their influence on the development of the same NCC-derived structures. Surface plasmon resonance binding data showed that FBLN1 binds tightly to FGF8 and prevents its enzymatic degradation by ADAM17. Moreover, overexpression of FBLN1 up-regulates FGF8 gene expression, and down-regulation of FBLN1 by siRNA inhibits FGF8 expression. The generation of a double mutant Fbln1 and Fgf8 mice (Fbln1(-/-) and Fgf8(-/-)) showed that haplo-insufficiency (Fbln1(+/-) and Fgf8(+/-)) resulted in increased embryonic mortality compared with single heterozygote crosses. The mortality of the FGF8/Fbln1 double heterozygote embryos occurred between 14.5 and 16.5 days post-coitus. In conclusion, FBLN1/FGF8 interaction plays a role in survival of vertebrate embryos, and reduced levels of both proteins resulted in added mortality in utero The FBLN1/FGF8 interaction may also be involved in the survival of neural crest cell population during development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Selective Distribution of a High-Affinity Plasminogen-Binding Site among Group A Streptococci Associated with Impetigo

    Science.gov (United States)

    Svensson, Mikael D.; Sjöbring, Ulf; Bessen, Debra E.

    1999-01-01

    Group A streptococci can be classified according to their tendency to cause either impetigo, pharyngitis, or both types of infection. Genotypic markers for tissue site preference lie within emm genes, which encode fibrillar surface proteins that play a key role in virulence. emm gene products (M and M-like proteins) display an extensive array of binding activities for tissue and plasma proteins of the human host. In a previous study, a high-affinity binding site for human plasmin(ogen) was mapped to the emm53 gene product. In this report, a structurally similar plasminogen-binding domain is found to be widely and selectively distributed among group A streptococci harboring the emm gene marker for the skin as the preferred tissue site for infection. The findings are highly suggestive of a central role for bacterial modulation of host plasmin(ogen) during localized infection at the epidermis. PMID:10417156

  7. Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using (3)H-HOCPCA

    DEFF Research Database (Denmark)

    Klein, A B; Bay, T; Villumsen, I S

    2016-01-01

    GHB (γ-hydroxybutyric acid) is a compound endogenous to mammalian brain with high structural resemblance to GABA. GHB possesses nanomolar-micromolar affinity for a unique population of binding sites, but the exact nature of these remains elusive. In this study we utilized the highly selective GHB......, (3)H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that (3)H-HOCPCA labels only the high-affinity specific GHB binding site, found in high...... brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that (3)H-HOCPCA is a highly sensitive radioligand, offering advantages over...

  8. High-level amikacin resistance in Pseudomonas aeruginosa associated with a 3'-phosphotransferase with high affinity for amikacin.

    Science.gov (United States)

    Torres, C; Perlin, M H; Baquero, F; Lerner, D L; Lerner, S A

    2000-08-01

    This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside. Amikacin-resistant P. aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3')-VI. The enzyme from P. aeruginosa PA40 was purified to > 70% homogeneity. The Km of amikacin for this enzyme was 1.4 microM, the Vmax/Km ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3')-IIps. Amikacin resistance in this strain was, therefore, associated with APH(3')-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed.

  9. Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics

    Science.gov (United States)

    Aweda, Tolulope A.; Meares, Claude F.

    2011-01-01

    For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (KA >108 M−1; KD titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate. PMID:21964396

  10. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy.

    Science.gov (United States)

    Lázár-Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil; Quinn, Thomas; Sylvestre, Eliezer; Palmieri, Edith; Ramagopal, Udupi A; Nathenson, Stanley G; Guha, Chandan; Almo, Steven C

    2017-03-01

    Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting.

    Directory of Open Access Journals (Sweden)

    Christopher Ullman

    Full Text Available Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2. Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model.

  12. Histones have high affinity for the glomerular basement membrane. Relevance for immune complex formation in lupus nephritis

    Energy Technology Data Exchange (ETDEWEB)

    Schmiedeke, T.M.; Stoeckl, F.W.W.; Weber, R.; Sugisaki, Y.; Batsford, S.R.; Vogt, A.

    1989-06-01

    An effort has been made to integrate insights on charge-based interactions in immune complex glomerulonephritis with nuclear antigen involvement in lupus nephritis. Attention was focussed on the histones, a group of highly cationic nuclear constituents, which could be expected to bind to fixed anionic sites present in the glomerular basement membrane (GBM). We demonstrated that all histone subfractions, prepared according to Johns, have a high affinity for GBM and the basement membrane of peritubular capillaries. Tissue uptake of /sup 125/I-labeled histones was measured by injecting 200 micrograms of each fraction into the left kidney via the aorta and measuring organ uptake after 15 min. In glomeruli isolated from the left kidneys, the following quantities of histones were found: f1, 13 micrograms; f2a (f2al + f2a2), 17 micrograms; f2b, 17 micrograms; and f3, 32 micrograms. Kinetic studies of glomerular binding showed that f1 disappeared much more rapidly than f2a. The high affinity of histones (pI between 10.5 and 11.0; mol wt 10,000-22,000) for the GBM correlates well with their ability to form aggregates (mol wt greater than 100,000) for comparison lysozyme (pI 11, mol wt 14,000), which does not aggregate spontaneously bound poorly (0.4 micrograms in isolated glomeruli). The quantity of histones and lysozyme found in the isolated glomeruli paralleled their in vitro affinity for a Heparin-Sepharose column (gradient elution studies). This gel matrix contains the sulfated, highly anionic polysaccharide heparin, which is similar to the negatively charged heparan sulfate present in the GBM. Lysozyme eluted with 0.15 M NaCl, f1 with 1 M NaCl, and f2a, f2b, and f3 could not be fully desorbed even with 2 M NaCl; 6 M guanidine-HCl was necessary.

  13. High affinity antigen recognition of the dual specific variants of herceptin is entropy-driven in spite of structural plasticity.

    Directory of Open Access Journals (Sweden)

    Jenny Bostrom

    Full Text Available The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2 antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.

  14. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV

    Directory of Open Access Journals (Sweden)

    Wenjing Qi

    2016-10-01

    Full Text Available Zr(IV can form phosphate and Zr(IV (–PO32−–Zr4+– complex owing to the high affinity between Zr(IV with phosphate. Zr(IV can induce the aggregation of gold nanoparticles (AuNPs, while adenosine triphosphate(ATP can prevent Zr(IV-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRAsensor for ATP have been developed using AuNPs based on the high affinity between Zr(IVwith ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV. After the addition of ATP, ATP reacts with Zr(IV and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV, ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945 with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  15. Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody.

    Science.gov (United States)

    Lord, Dana M; Bird, Julie J; Honey, Denise M; Best, Annie; Park, Anna; Wei, Ronnie R; Qiu, Huawei

    2018-01-15

    Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies.

  16. The role of valence on the high-affinity binding of Griffonia simplicifolia isolectins to type A human erythrocytes.

    Science.gov (United States)

    Knibbs, R N; Takagaki, M; Blake, D A; Goldstein, I J

    1998-12-01

    The Griffonia simplicifolia-I (GS-I) isolectins have been used to probe the effect of lectin valence on their high-affinity binding to human erythrocytes. These tetrameric lectins are composed of A and B subunits and constitute a series of five isolectins (A4, A3B, A2B2, AB3, B4). The A subunit is specific for alpha-D-GalNAc end groups and binds to the blood type A determinant GalNAcalpha1, as well as to terminal alpha-D-Gal groups found on type B cells. The B subunit is specific for alpha-D-Gal end groups, and binds very specifically to type B erythrocytes. This series of isolectins is tetravalent (A4), trivalent (A3B), divalent (A2B2), and monovalent (AB3) for type A erythrocytes; thus, this system provides the opportunity to examine the effect of lectin valency on the association constants of these GS-I isolectins binding to cells. Cell binding experiments carried out using 125I-labeled GS-I isolectins and type A human erythrocytes allowed us to demonstrate that (1) the association constant of the isolectin monovalent for alpha-D-GalNAc (AB3) is virtually identical to its association constant for the haptenic sugar methyl-N-acetyl-alpha-D-galactosaminide, reported previously, and (2) the association constant of the GS-I isolectins for human type A erythrocytes increases with increasing valency of the isolectin. These results indicate that the increased affinity displayed by the GS-I isolectins for human type A erythrocytes is dependent on their multivalency, and not on an extended binding site nor on nonspecific, or noncarbohydrate, interactions of the lectin with the cell surface. These findings should be of general relevance to understanding the high-affinity interactions observed between other multivalent proteins and multivalent ligands (e.g., cell surfaces).

  17. Nerve growth factor, brain-derived neurotrophic factor and their high-affinity receptors are overexpressed in extramammary Paget's disease.

    Science.gov (United States)

    Qian, Yue; Takeuchi, Satoshi; Chen, Shan-Juan; Dugu, Long; Tsuji, Gaku; Xie, Lining; Nakahara, Takeshi; Moroi, Yoichi; Tu, Ya-Ting; Furue, Masutaka

    2010-11-01

    Neurotrophin (NT) systems appear to play important roles in the pathogenesis of several tumors, but their expression in extramammary Paget's disease (EPD) has not been investigated. Thirty-four paraffin-embedded EPD specimens (32 primary EPD and 2 metastatic to lymph nodes) were subject to immunohistochemical staining for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT3, NT4, their high-affinity receptors (TrkA, TrkB and TrkC) and the common low-affinity receptor, p75 NT receptor (p75). All 34 EPD specimens, including 2 metastatic to lymph nodes, showed cytoplasmic overexpression of NGF, BDNF, TrkA and TrkB. The expression (% positive cells) of NGF, BDNF, NT3, NT4, TrkA and TrkB (81.6 ± 14.9, 86.0 ± 10.4, 89.6 ± 14.9, 87.8 ± 17.9, 83 ± 14.4 and 86.2 ± 11.7%) in EPD was significantly higher than in normal skin (21.6 ± 6.5, 27.6 ± 4.5, 19.7 ± 10.1, 8.2 ± 10.0, 25.0 ± 5.3 and 25.4 ± 6.4%), and the expression of these factors in invasive EPD was significantly higher than in noninvasive EPD. Interestingly, Paget cells were negative for p75 and TrkC in all the 34 EPD specimens. These results suggest that overexpression of NGF, BDNF and their high-affinity receptors (TrkA and TrkB) might play a role in the pathogenesis of EPD. Copyright © 2010 John Wiley & Sons A/S.

  18. Selective Adsorption of Tetrahydropalmatine by a Molecularly ...

    African Journals Online (AJOL)

    NICO

    1School of Chemistry and Chemical Engineering, Guangxi University for Nationalities, Nanning 530006, P.R. China. 2Guangxi ... Selective adsorption experiments demonstrated the high affinity and THP selectiv- ... Molecularly imprinted polymer, tetrahydropalmatine, selective adsorption, microcalorimetry, modified rosin. 1.

  19. ROBO1, a tumor suppressor and critical molecular barrier for localized tumor cells to acquire invasive phenotype: Study in African-American and Caucasian prostate cancer models

    OpenAIRE

    Parray, Aijaz; Siddique, Hifzur R; Kuriger, Jacquelyn K.; Mishra, Shrawan K.; Rhim, Johng S.; Nelson, Heather H.; Aburatani, Hiroyuki; Badrinath R Konety; Koochekpour, Shahriar; Saleem, Mohammad

    2014-01-01

    High-risk populations exhibit early transformation of localized prostate cancer (CaP) disease to metastasis which results in the mortality of such patients. The paucity of knowledge about the molecular mechanism involved in acquiring of metastatic behavior by primary tumor cells and non-availability of reliable phenotype-discriminating biomarkers are stumbling blocks in the management of CaP disease. Here, we determine the role and translational relevance of ROBO1 (an organogenesis-associated...

  20. Gene expression reaction norms unravel the molecular and cellular processes underpinning the plastic phenotypes of Alternanthera philoxeroides in contrasting hydrological conditions

    Directory of Open Access Journals (Sweden)

    Lexuan eGao

    2015-11-01

    Full Text Available Alternanthera philoxeroides is an amphibious invasive weed that can colonize both aquatic and terrestrial habitats. Individuals growing in different habitats exhibit extensive phenotypic variation but little genetic differentiation. Little is known about the molecular basis underlying environment-induced phenotypic changes. Variation in transcript abundance in A. philoxeroides was characterized throughout the time-courses of pond and upland treatments using RNA-Sequencing. 7,805 genes demonstrated variable expression in response to different treatments,forming 11 transcriptionally coordinated gene groups. Functional enrichment analysis of plastically expressed genes revealed pathway changes in hormone-mediated signaling, osmotic adjustment, cell wall remodeling and programmed cell death, providing a mechanistic understanding of the biological processes underlying the phenotypic changes in A. philoxeroides. Both transcriptional modulation of environmentally sensitive loci and environmentally dependent control of regulatory loci influenced the plastic responses to the environment. Phenotypic responses and gene expression patterns to contrasting hydrological conditions were compared between A. philoxeroides and its alien congener A. pungens. The terricolous A. pungens displayed limited phenotypic plasticity to different treatments. It was postulated based on gene expression comparison that the interspecific variation in plasticity between A. philoxeroides and A. pungens was not due to environmentally-mediated changes in hormone levels but to variations in the type and relative abundance of different signal transducers and receptors expressed in the target tissue.

  1. The UL8 subunit of the helicase-primase complex of herpes simplex virus promotes DNA annealing and has a high affinity for replication forks.

    Science.gov (United States)

    Bermek, Oya; Weller, Sandra K; Griffith, Jack D

    2017-09-22

    During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination. For instance, replication of the HSV-1 genome produces X- and Y-branched structures, reminiscent of recombination intermediates. HSV-1's replication machinery includes a trimeric helicase-primase composed of helicase (UL5) and primase (UL52) subunits and a third subunit, UL8. UL8 has been reported to stimulate the helicase and primase activities of the complex in the presence of ICP8, an HSV-1 protein that functions as an annealase, a protein that binds complementary single-stranded DNA (ssDNA) and facilitates its annealing to duplex DNA. UL8 also influences the intracellular localization of the UL5/UL52 subunits, but UL8's catalytic activities are not known. In this study we used a combination of biochemical techniques and transmission electron microscopy. First, we report that UL8 alone forms protein filaments in solution. Moreover, we also found that UL8 binds to ssDNAs >50-nucletides long and promotes the annealing of complementary ssDNA to generate highly branched duplex DNA structures. Finally, UL8 has a very high affinity for replication fork structures containing a gap in the lagging strand as short as 15 nucleotides, suggesting that UL8 may aid in directing or loading the trimeric complex onto a replication fork. The properties of UL8 uncovered here suggest that UL8 may be involved in the generation of the X- and Y-branched structures that are the hallmarks of HSV replication. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

    Science.gov (United States)

    Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

    2015-01-01

    Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

  3. Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances

    Energy Technology Data Exchange (ETDEWEB)

    Rannug, A.; Rannug, U.; Rosenkranz, H.S.; Winqvist, L.; Westerholm, R.; Agurell, E.; Grafstroem, A.K.

    1987-11-15

    The purpose of the present study was to determine whether ultraviolet light (UV) irradiation of amino acids produces compounds with affinity for the Ah receptor. Aqueous solutions of L-tryptophan were exposed to radiation from an unfiltered high-pressure mercury lamp. The photoproducts formed were solvent-extracted or concentrated on Sep-Pak C18 cartridges. The concentrated extracts or eluants were treated for their ability to compete with /sup 3/H-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Binding was assayed in liver cytosolic preparations from Sprague-Dawley rats using a technique based on hydroxylapatite separation. Photoproducts with receptor affinity were formed in a time-dependent manner. Histidine and tryptamine also gave products upon UV irradiation that competed with TCDD. Commercial tryptophan, at least aged, contained trace amounts of impurities with receptor affinity. Analysis by TLC and high-pressure liquid chromatography of the photo-products of tryptophan showed a minimum of three different binding compounds. Two of the products were studied in greater detail. One of them, showing UV absorbance and yellow fluorescence, gave a molecular ion (M+) of 284 and the other gave M+ 312 but showed little UV absorption and fluorescence. The concentration, based on mass spectrometry quantifications, of the two compounds that displaced more than 50% of TCDD was found to be extremely low, giving Kd values of 0.44 nM (M+ 312) and 0.07 nM (M+ 284). The existence of high affinity receptors for oxidized amino acids is postulated and their possible role in the proliferative cellular responses to TCDD and tryptophan is discussed briefly.

  4. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome.

    Science.gov (United States)

    Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

    2015-01-01

    Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

  5. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A

    2012-10-01

    Full Text Available Abstract Background For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND, the potent drug for treatment of multiple myeloma (MM, a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. Results In this study, a hapten of LND (N-glutaryl-LND was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA and keyhole limpet hemocyanin (KLH proteins by ethyl-3-(3-dimethylaminopropyl carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. Conclusions The high affinity of the antibody (IC50 = 10 ng/mL will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  6. Preparation of a novel antiserum to aromatase with high affinity and specificity: Its clinicopathological significance on breast cancer tissue.

    Science.gov (United States)

    Kanomata, Naoki; Matsuura, Shiro; Nomura, Tsunehisa; Kurebayashi, Junichi; Mori, Taisuke; Kitawaki, Jo; Moriya, Takuya

    2017-01-01

    Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and

  7. E-cadherin expression phenotypes associated with molecular subtypes in invasive non-lobular breast cancer: evidence from a retrospective study and meta-analysis.

    Science.gov (United States)

    Liu, Jiang-Bo; Feng, Chen-Yi; Deng, Miao; Ge, Dong-Feng; Liu, De-Chun; Mi, Jian-Qiang; Feng, Xiao-Shan

    2017-08-01

    This retrospective study and meta-analysis was designed to explore the relationship between E-cadherin (E-cad) expression and the molecular subtypes of invasive non-lobular breast cancer, especially in early-stage invasive ductal carcinoma (IDC). A total of 156 post-operative cases of early-stage IDCs were retrospectively collected for the immunohistochemistry (IHC) detection of E-cad expression. The association of E-cad expression with molecular subtypes of early-stage IDCs was analyzed. A literature search was conducted in March 2016 to retrieve publications on E-cad expression in association with molecular subtypes of invasive non-lobular breast cancer, and a meta-analysis was performed to estimate the relational statistics. E-cad was expressed in 82.7% (129/156) of early-stage IDCs. E-cad expression was closely associated with the molecular types of early-stage IDCs (P molecular subtypes were an independent factor influencing E-cad expression in early-stage IDCs. A total of 12 observational studies (including our study) were included in the meta-analysis. The meta-analytical results show a significantly greater risk of E-cad expression loss in triple-negative breast cancer (TNBC) than in other molecular subtypes (TNBC vs. luminal A: RR = 3.45, 95% CI = 2.79-4.26; TNBC vs. luminal B: RR = 2.41, 95% CI = 1.49-3.90; TNBC vs. HER2-enriched: RR = 1.95, 95% CI = 1.24-3.07). Early-stage IDCs or invasive non-lobular breast cancers with the TNBC molecular phenotype have a higher risk for the loss of E-cad expression than do tumors with non-TNBC molecular phenotypes, suggesting that E-cad expression phenotypes were closely related to molecular subtypes and further studies are needed to clarify the underlying mechanism.

  8. Molecular definition of deletions of different segments of distal 5p that result in distinct phenotypic features

    Energy Technology Data Exchange (ETDEWEB)

    Church, D.M.; Bengtsson, U.; Wasmuth, J.J. [Univ. of California, Irvine (United States); Niebuhr, E. [Univ. of Copenhagen, CA (United States)

    1995-05-01

    Cri du chat syndrome (CDC) is a segmental aneusomy associated with deletions of chromosome 5p15. In an effort to define regions that produce the phenotypes associated with CDC, we have analyzed deletions from 17 patients. The majority of these patients had atypical CDC features or were asymptomatic. Using these patients, we have mapped several phenotypes associated with deletions of 5p, including speech delay, catlike cry, newborn facial dysmorphism, and adult facial dysmorphism. This phenotypic map should provide a framework with which to begin identification of genes associated with various phenotypic features associated with deletions of distal 5p. We have also analyzed the parental origin of the de novo deletions, to determine if genomic imprinting could be occurring in this region. In addition, we have isolated cosmids that could be useful for both prenatal and postnatal assessments of del5(p) individuals. 25 refs., 4 figs., 3 tabs.

  9. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

    OpenAIRE

    Sabban, Sari; Ye, Hongtu; Helm, Birgit

    2014-01-01

    The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development ...

  10. Molecular Etiology of Hearing Impairment in Inner Mongolia: mutations in SLC26A4 gene and relevant phenotype analysis

    Directory of Open Access Journals (Sweden)

    Wu Bailin

    2008-11-01

    Full Text Available Abstract Background The molecular etiology of hearing impairment in Chinese has not been thoroughly investigated. Study of GJB2 gene revealed that 30.4% of the patients with hearing loss in Inner Mongolia carried GJB2 mutations. The SLC26A4 gene mutations and relevant phenotype are analyzed in this study. Methods One hundred and thirty-five deaf patients were included. The coding exons of SLC26A4 gene were sequence analyzed in 111 patients, not including 22 patients carrying bi-allelic GJB2 mutations or one patient carrying a known GJB2 dominant mutation as well as one patient with mtDNA 1555A>G mutation. All patients with SLC26A4 mutations or variants were subjected to high resolution temporal bone CT scan and those with confirmed enlarged vestibular aqueduct and/or other inner ear malformation were then given further ultrasound scan of thyroid and thyroid hormone assays. Results Twenty-six patients (19.26%, 26/135 were found carrying SLC26A4 mutation. Among them, 17 patients with bi-allelic SLC26A4 mutations were all confirmed to have EVA or other inner ear malformation by CT scan. Nine patients were heterozygous for one SLC26A4 mutation, including 3 confirmed to be EVA or EVA and Mondini dysplasia by CT scan. The most common mutation, IVS7-2A>G, accounted for 58.14% (25/43 of all SLC26A4 mutant alleles. The shape and function of thyroid were confirmed to be normal by thyroid ultrasound scan and thyroid hormone assays in 19 of the 20 patients with EVA or other inner ear malformation except one who had cystoid change in the right side of thyroid. No Pendred syndrome was diagnosed. Conclusion In Inner Mongolia, China, mutations in SLC26A4 gene account for about 12.6% (17/135 of the patients with hearing loss. Together with GJB2 (23/135, SLC26A4 are the two most commonly mutated genes causing deafness in this region. Pendred syndrome is not detected in this deaf population. We established a new strategy that detects SLC26A4 mutations prior to the

  11. A comparative study of myocardial molecular phenotypes of two tfr2β null mice: role in ischemia/reperfusion.

    Science.gov (United States)

    Boero, Martina; Pagliaro, Pasquale; Tullio, Francesca; Pellegrino, Rosa M; Palmieri, Antonietta; Ferbo, Ludovica; Saglio, Giuseppe; De Gobbi, Marco; Penna, Claudia; Roetto, Antonella

    2015-01-01

    Transferrin receptor 2 (Tfr2) is an iron-modulator transcribed in two isoforms, Tfr2α and Tfr2β. The latter is expressed in the heart. We obtained two mouse models with silencing of Tfr2β: one with a normal systemic iron amount (SIA), i.e., Tfr2-KI, and the other, i.e., LCKO-KI, with high SIA due to hepatic Tfr2α silencing. We aimed to assess whether Tfr2β might play a role in myocardial injury and whether Tfr2β silencing might modify proteins of iron metabolism, antioxidant, apoptotic, and survival enzyme activities in the heart undergoing ischemia/reperfusion (I/R). Isolated hearts of wild-type (WT) and Tfr2-null mice were studied before or after an I/R protocol, and proteins/RNA analyzed by Western blot and/or quantitative PCR. Tfr2β increased in WT hearts subject to I/R, and both Tfr2β null mice hearts were protected against I/R injury (about 40% smaller infarct-size compared to WT hearts). RISK kinases (ERK1/2-AKT-PKCε) were found up-regulated after I/R in Tfr2-KI, whereas SAFE enzyme (Stat3) and GSK3β resulted phosphorylated during I/R in LCKO-KI hearts. While HO-1 and HIF-2a were high in both Tfr2β-null mice, Catalase, and proapoptotic factors were upregulated only in LCKO-KI. Finally, Tfr2-KI hearts presented an increased Ferritin-H and a decreased Ferroportin1, whereas LCKO-KI hearts displayed an upregulation of Ferritin-L chain and DMT1/Hamp-RNA. In conclusion, Tfr2β isoform is involved in cardiac iron metabolism and its silencing leads to a protected phenotype (antioxidants, RISK, and/or SAFE upregulation) against I/R challenging. Iron-dependent signals involved in cardioprotection seem to be positively affected by Tfr2β downregulation and subsequent Ferritins upregulation. © 2015 International Union of Biochemistry and Molecular Biology.

  12. ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments.

    Directory of Open Access Journals (Sweden)

    Anuradha Kuchibhatla

    Full Text Available A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0 pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.

  13. Fragile X mental retardation protein recognition of G quadruplex structure per se is sufficient for high affinity binding to RNA.

    Science.gov (United States)

    Bole, Medhavi; Menon, Lakshmi; Mihailescu, Mihaela-Rita

    2008-12-01

    Fragile X syndrome, the most common form of inherited mental retardation is caused by the expansion of a CGG trinucleotide repeat in the fragile X mental retardation 1 (fmr1) gene. The abnormal expansion of the CGG repeat causes hypermethylation and subsequent silencing of the fmr1 gene, resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine rich region (RGG box) to bind to messenger RNAs that form G quadruplex structures. Several studies reported that the G quadruplex RNA recognition alone is not sufficient for FMRP RGG box binding and that an additional stem and/or a G quadruplex-stem junction region may also be important in recognition. In this study we have used biophysical methods such as fluorescence, UV, CD and NMR spectroscopy to demonstrate that the recognition of the RNA G quadruplex structure per se, in the absence of a stem region, is sufficient for the FMRP high affinity and specific binding. These findings indicate that the presence of a stem structure in some of the FMRP G quadruplex forming mRNAs is not a requirement for protein recognition as previously believed, but rather for the proper formation of the correct RNA G quadruplex structure recognized by FMRP.

  14. G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity.

    Science.gov (United States)

    Tatsumi, Kasumi; Sakashita, Gyosuke; Nariai, Yuko; Okazaki, Kosuke; Kato, Hiroaki; Obayashi, Eiji; Yoshida, Hisashi; Sugiyama, Kanako; Park, Sam-Yong; Sekine, Joji; Urano, Takeshi

    2017-03-07

    The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (Kd = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research.

  15. New high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma.

    Science.gov (United States)

    Zhang, Yi-Fan; Phung, Yen; Gao, Wei; Kawa, Seiji; Hassan, Raffit; Pastan, Ira; Ho, Mitchell

    2015-05-21

    Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296-390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers.

  16. A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

    Science.gov (United States)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-01-01

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

  17. A high affinity recombinant antibody to the human EphA3 receptor with enhanced ADCC activity.

    Science.gov (United States)

    Tomasevic, Nenad; Luehrsen, Kenneth; Baer, Mark; Palath, Varghese; Martinez, David; Williams, Jason; Yi, Christina; Sujatha-Bhaskar, Swathi; Lanke, Rohini; Leung, John; Ching, Wendy; Lee, Andreia; Bai, Lu; Yarranton, Geoffrey; Bebbington, Christopher

    2014-12-01

    EphA3 is expressed in solid tumors and leukemias and is an attractive target for the therapy. We have generated a panel of Humaneered® antibodies to the ligand-binding domain using a Fab epitope-focused library that has the same specificity as monoclonal antibody mIIIA4. A high-affinity antibody was selected that competes with the mIIIA4 antibody for binding to EphA3 and has an improved affinity of ∼1 nM. In order to generate an antibody with potent cell-killing activity the variable regions were assembled with human IgG1k constant regions and expressed in a Chinese hamster ovary (CHO) cell line deficient in fucosyl transferase. Non-fucosylated antibodies have been reported to have enhanced binding affinity for the IgG receptor CD16a (FcγRIIIa). The affinity of the antibody for recombinant CD16a was enhanced approximately 10-fold. This resulted in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity against EphA3-expressing leukemic cells, providing a potent antibody for the evaluation as a therapeutic agent.

  18. ZK91587: a novel synthetic antimineralocorticoid displays high affinity for corticosterone (type I) receptors in the rat hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Sutanto, W.; de Kloet, E.R.

    1988-01-01

    In vitro cytosol binding assays have shown the properties of binding of a novel steroid, ZK91587 (15..beta.., 16..beta..b-methylene-mexrenone) in the brain of rats. Scatchard and Woolf analyses of the binding data reveal the binding of (/sup 3/H) ZK91587 to the total hippocampal coritcosteroid receptor sites with high affinity, and low capacity. When 100-fold excess RU28362 was included simultaneously with (/sup 3/H) ZK91587, the labelled steroid binds with the same affinity and capacity. Relative binding affinities (RBA) of various steroids for the Type I or Type II corticosteroid receptor in these animals are: Type I: ZK91587 = corticosterone (B) > cortisol (F); Type II: B > F >>> ZK91587. In the binding kinetic study, ZK91587 has a high association rate of binding in the rat. The steroid dissociates following a one slope pattern, indicating, the present data demonstrate that in the rat hippocampus, ZK91587 binds specifically to the Type I (corticosterone-preferring/mineralocorticoid-like receptor.

  19. Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria.

    Science.gov (United States)

    Willmann, Anita; Weiss, Michael; Nehls, Uwe

    2007-02-01

    A main function of ectomycorrhizas, a symbiosis between certain soil fungi and fine roots of woody plants, is the exchange of plant-derived carbohydrates for fungus-derived nutrients. As it is required in large amounts, nitrogen is of special interest. A gene (AmAMT2) coding for a putative fungal ammonium importer was identified in an EST project of functional Amanita muscaria/poplar ectomycorrhizas. Heterologous expression of the entire AmAMT2 coding region in yeast revealed the corresponding protein to be a high-affinity ammonium importer. In axenically grown Amanita hyphae AmAMT2 expression was strongly repressed by nitrogen, independent of whether the offered nitrogen source was transported by AmAMT2 or not. In functional ectomycorrhizas the AmAMT2 transcript level was further decreased in both hyphal networks (sheath and Hartig net), while extraradical hyphae revealed strong gene expression. Together our data suggest that (1) AmAMT2 expression is regulated by the endogenous nitrogen content of hyphae and (2) fungal hyphae in ectomycorrhizas are well supported with nitrogen even when the extraradical mycelium is nitrogen limited. As a consequence of AmAMT2 repression in mycorrhizas, ammonium can be suggested as a potential nitrogen source delivered by fungal hyphae in symbiosis.

  20. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion (Cornell); (UAB); (Florida)

    2010-08-18

    Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 {angstrom}) crystal structure of the A{sub 3}VP{sub 1} fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 {angstrom}) through the interaction of two noncontiguous regions in the primary sequence. The A{sub 3} repeat of the alanine-rich domain adopts an extended {alpha}-helix that intertwines with the P{sub 1} repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A{sub 3} and P{sub 1} helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

  1. CM156, a high affinity sigma ligand, attenuates the stimulant and neurotoxic effects of methamphetamine in mice.

    Science.gov (United States)

    Kaushal, Nidhi; Seminerio, Michael J; Shaikh, Jamaluddin; Medina, Mark A; Mesangeau, Christophe; Wilson, Lisa L; McCurdy, Christopher R; Matsumoto, Rae R

    2011-01-01

    Methamphetamine (METH) is a highly addictive psychostimulant drug of abuse. Low and high dose administration of METH leads to locomotor stimulation, and dopaminergic and serotonergic neurotoxicity, respectively. The behavioral stimulant and neurotoxic effects of METH can contribute to addiction and other neuropsychiatric disorders, thus necessitating the identification of potential pharmacotherapeutics against these effects produced by METH. METH binds to σ receptors at physiologically relevant concentrations. Also, σ receptors are present on and can modulate dopaminergic and serotonergic neurons. Therefore, σ receptors provide a viable target for the development of pharmacotherapeutics against the adverse effects of METH. In the present study, CM156, a σ receptor ligand with high affinity and selectivity for σ receptors over 80 other non-σ binding sites, was evaluated against METH-induced stimulant, hyperthermic, and neurotoxic effects. Pretreatment of male, Swiss Webster mice with CM156 dose dependently attenuated the locomotor stimulation, hyperthermia, striatal dopamine and serotonin depletions, and striatal dopamine and serotonin transporter reductions produced by METH, without significant effects of CM156 on its own. These results demonstrate the ability of a highly selective σ ligand to mitigate the effects of METH. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Slc5a8, a Na+-coupled high-affinity transporter for short-chain fatty acids, is a conditional tumour suppressor in colon that protects against colitis and colon cancer under low-fibre dietary conditions.

    Science.gov (United States)

    Gurav, Ashish; Sivaprakasam, Sathish; Bhutia, Yangzom D; Boettger, Thomas; Singh, Nagendra; Ganapathy, Vadivel

    2015-07-15

    Mammalian colon harbours trillions of bacteria under physiological conditions; this symbiosis is made possible because of a tolerized response from the mucosal immune system. The mechanisms underlying this tolerogenic phenomenon remain poorly understood. In the present study we show that Slc5a8 (solute carrier gene family 5a, member 8), a Na(+)-coupled high-affinity transporter in colon for the bacterial fermentation product butyrate, plays a critical role in this process. Among various immune cells in colon, dendritic cells (DCs) are unique not only in their accessibility to luminal contents but also in their ability to induce tolerogenic phenotype in T-cells. We found that DCs exposed to butyrate express the immunosuppressive enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2), promote conversion of naive T-cells into immunosuppressive forkhead box P3(+) (FoxP3(+)) Tregs (regulatory T-cells) and suppress conversion of naive T-cells into pro-inflammatory interferon (IFN)-γ-producing cells. Slc5a8-null DCs do not induce IDO1 and Aldh1A2 and do not generate Tregs or suppress IFN-γ-producing T-cells in response to butyrate. We also provide in vivo evidence for an obligatory role for Slc5a8 in suppression of IFN-γ-producing T-cells. Furthermore, Slc5a8 protects against colitis and colon cancer under conditions of low-fibre intake but not when dietary fibre intake is optimal. This agrees with the high-affinity nature of the transporter to mediate butyrate entry into cells. We conclude that Slc5a8 is an obligatory link between dietary fibre and mucosal immune system via the bacterial metabolite butyrate and that this transporter is a conditional tumour suppressor in colon linked to dietary fibre content. © 2015 Authors; published by Portland Press Limited.

  3. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

    DEFF Research Database (Denmark)

    Kandra, L.; Abou Hachem, Maher; Gyemant, G.

    2006-01-01

    as binding barriers. Barley a-amylase I mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile...

  4. High-affinity human leucocyte antigen class I binding variola-derived peptides induce CD4(+) T cell responses more than 30 years post-vaccinia virus vaccination

    DEFF Research Database (Denmark)

    Wang, M.; Tang, Sheila Tuyet; Lund, Ole

    2009-01-01

    Interferon-gamma secreting T lymphocytes against pox virus-derived synthetic 9-mer peptides were tested by enzyme-linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high-affinity human...

  5. A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

    DEFF Research Database (Denmark)

    Bach, Anders*; Clausen, Bettina H; Møller, Magda

    2012-01-01

    Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-d-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors...

  6. Amino propynyl benzoic acid building block in rigid spacers of divalent ligands binding to the Syk SH2 domains with equally high affinity as the natural ligand

    NARCIS (Netherlands)

    Dekker, Frank J; de Mol, Nico J; Fischer, Marcel J E; Liskamp, Rob M J; Dekker, Frank

    2003-01-01

    The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2 domains, which are important in signal transduction. The

  7. Cytokine-induced immune complex binding to the high-affinity IgG receptor, FcγRI, in the presence of monomeric IgG

    NARCIS (Netherlands)

    van der Poel, C.E.; Karssemeijer, R.A.; Boross, P.; van der Linden, J.A.; Blokland - Fromme, M.; van de Winkel, J.G.J.; Leusen, J.H.W.

    2010-01-01

    FcγRI is the sole high-affinity immunoglobulin G (IgG) receptor on leukocytes. Its role in immunity and the clearance of opsonized particles has been challenged, as the receptor function may well be hindered by serum IgG. Here, we document immune complex binding by FcγRI to be readily enhanced by

  8. RNA Aptamer Binds Heparin-Binding Epidermal Growth Factor-Like Growth Factor with High Affinity and Specificity and Neutralizes Its Activity

    Directory of Open Access Journals (Sweden)

    Masaki Yamato

    2017-09-01

    Conclusion: We identified a novel RNA aptamer that bound with high affinity and specificity to rhHB-EGF and potently inhibited the rhHB-EGF-mediated phosphorylation of EGFR. The anti-HB-EGF aptamer may be a promising therapeutic agent for specifically neutralizing HB-EGF signaling.

  9. High-Affinity Sites Form an Interaction Network to Facilitate Spreading of the MSL Complex across the X Chromosome in Drosophila

    NARCIS (Netherlands)

    Ramírez, Fidel; Lingg, Thomas; Toscano, Sarah; Lam, Kin Chung; Georgiev, Plamen; Chung, Ho-Ryun; Lajoie, Bryan R; de Wit, Elzo; Zhan, Ye; de Laat, Wouter; Dekker, Job; Manke, Thomas; Akhtar, Asifa

    2015-01-01

    Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, we show that high-affinity sites (HAS), landing platforms of the male-specific

  10. Synthesis and biological evaluation of disubstituted N6- cyclopentyladenine analogues: The search for a neutral antagonist with high affinity for the adenosine A1 receptor

    NARCIS (Netherlands)

    Ligt, R.A.F. de; Klein, P.A.M. van der; Frijtag Drabbe Künzel, J.K. von; Lorenzen, A.; El Maate, F.A.; Fujikawa, S.; Westhoven, R. van; Hoven, T. van den; Brussee, J.; Ijzerman, A.P.

    2004-01-01

    Novel 3,8- and 8,9-disubstituted N6-cyclopentyladenine derivatives were synthesised in moderate overall yield from 6-chloropurine. The derivatives were made in an attempt to find a new neutral antagonist with high affinity for adenosine A1 receptors. N6-Cyclopentyl-9- methyladenine (N-0840) was used

  11. Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein

    NARCIS (Netherlands)

    van der Wolk, J.P.W.; Klose, M; de Wit, Janny; Blaauwen, T.den; Freudl, R; Driessen, A.J.M.

    1995-01-01

    The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain

  12. Label-free assessment of high-affinity antibody-antigen binding constants. Comparison of bioassay, SPR, and PEIA-ellipsometry

    NARCIS (Netherlands)

    Rispens, T.; te Velthuis, H.; Hemker, P.; Speijer, H.; Hermens, W.; Aarden, L.

    2011-01-01

    Assessment of high-affinity antibody-antigen binding parameters is important in such diverse areas as selection of therapeutic antibodies, detection of unwanted hormones in cattle and sensitive immunoassays in clinical chemistry. Label-free assessment of binding affinities is often carried out by

  13. Soil carbon content and relative abundance of high affinity H2-oxidizing bacteria predict atmospheric H2 soil uptake activity better than soil microbial community composition

    NARCIS (Netherlands)

    Khdhiri, Mondher; Hesse, Laura; Popa, Maria Elena; Quiza, Liliana; Lalonde, Isabelle; Meredith, Laura K.; Röckmann, Thomas; Constant, Philippe

    2015-01-01

    Soil-atmosphere exchange of H2 is controlled by gas diffusion and the microbial production and oxidation activities in soil. Among these parameters, the H2 oxidation activity catalyzed by soil microorganisms harboring high affinity hydrogenase is the most difficult variable to parameterize because

  14. Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

    Science.gov (United States)

    Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

    2001-01-01

    BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

  15. Novel radioiodinated {gamma}-hydroxybutyric acid analogues for radiolabeling and Photolinking of high-affinity {gamma}-hydroxybutyric acid binding sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola

    2010-01-01

    ¿-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a (125)I-labeled GHB analog and characterized its binding in rat brain...

  16. Novel Radioiodinated γ-Hydroxybutyric Acid Analogues for Radiolabeling and Photolinking of High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Høg, Signe; Sabbatini, Paola

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a therapeutic drug, a drug of abuse, and an endogenous substance that binds to low- and high-affinity sites in the mammalian brain. To target the specific GHB binding sites, we have developed a 125I-labeled GHB analog and characterized its binding in rat brain...

  17. Dephosphorylation and quantification of organic phosphorus in poultry litter by purified phytic-acid high affinity Aspergillus phosphohydrolases.

    Science.gov (United States)

    Dao, Thanh H; Hoang, Khanh Q

    2008-08-01

    Extracellular phosphohydrolases mediate the dephosphorylation of phosphoesters and influence bioavailability and loss of agricultural P to the environment to pose risks of impairment of sensitive aquatic ecosystems. Induction and culture of five strains of Aspergillus were conducted to develop a source of high-affinity and robust phosphohydrolases for detecting environmental P and quantifying bioactive P pools in heterogeneous environmental specimens. Enzyme stability and activity against organic P in poultry litter were evaluated in 71 samples collected across poultry producing regions of Arkansas, Maryland, and Oklahoma of the US Differences existed in strains' adaptability to fermentation medium as they showed a wide range of phytate-degrading activity. Phosphohydrolases from Aspergillus ficuum had highest activity when the strain was cultured on a primarily chemical medium, compared to Aspergillus oryzae which preferred a wheat bran-based organic medium. Kinetics parameters of A. ficuum enzymes (K(m)=210 microM; V(max) of 407 nmol s(-1)) indicated phytic acid-degrading potential equivalent to that of commercial preparations. Purified A. ficuum phosphohydrolases effectively quantified litter bioactive P pools, showing that organic P occurred at an average of 54 (+/-14)% of total P, compared to inorganic phosphates, which averaged 41 (+/-12)%. Litter management and land application options must consider the high water-extractable and organic P concentrations and the biological availability of the organic enzyme-labile P pool. Robustness of A. ficuum enzymes and simplicity of the in situ ligand-based enzyme assay may thus increase routine assessment of litter bioactive P composition to sense for on-farm accumulation of such environmentally-sensitive P forms.

  18. High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals.

    Directory of Open Access Journals (Sweden)

    Stefan Ryser

    Full Text Available We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs from peripheral blood mononuclear cells (PBMC of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 105 memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii hemagglutinin (HA of influenza virus and (iii the extracellular domain of anaplastic lymphoma kinase (ALK. One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.

  19. Evaluation of the full evaporation technique for quantitative analysis of high boiling compounds with high affinity for apolar matrices.

    Science.gov (United States)

    van Boxtel, Niels; Wolfs, Kris; van Schepdael, Ann; Adams, Erwin

    2014-06-27

    In order to reduce inaccuracies due to possible matrix effects in conventional static headspace-gas chromatography (sHS-GC), it is standard practice to match the composition of calibration standards towards the composition of the sample to be analysed by adding blank matrix. However, the latter is not always available and in that case the full evaporation technique (FET) could be a solution. With FET a small sample volume is introduced in a HS vial and compounds of interest are completely evaporated. Hence no equilibrium between the condensed phase and vapour phase exists. Without the existence of an equilibrium, matrix effects are less likely to occur. Another issue often encountered with sHS-sampling is that low vapour pressure compounds with a high affinity for the dilution medium show a limited sensitivity. FET has proven to be an appropriate solution to address this problem too. In this work, the applicability of FET for the quantitative analysis of high boiling compounds in different complex apolar matrices is examined. Data show that FET is an excellent tool to overcome matrix effects often encountered with conventional sHS analysis. The tested method shows excellent accuracy with recovery values around 100% as well as repeatability with RSD values around 1% for the quantification of high boiling compounds (bp>200°C) such as camphor, menthol, methyl salicylate and ethyl salicylate in various matrices. LOQ values were found to be around 0.3μg per vial. Following validation of the technique, several topical pharmaceutical formulations like ThermoCream(®), Reflexspray(®), Vicks Vaporub(®) and Radosalil(®) were examined. For the latter, a comparison has been made with a sHS-method described in literature. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Identification and Tumour-Binding Properties of a Peptide with High Affinity to the Disialoganglioside GD2.

    Directory of Open Access Journals (Sweden)

    Jan Müller

    Full Text Available Neuroectodermal tumours are characterized by aberrant processing of disialogangliosides concomitant with high expression of GD2 or GD3 on cell surfaces. Antibodies targeting GD2 are already in clinical use for therapy of neuroblastoma, a solid tumour of early childhood. Here, we set out to identify peptides with high affinity to human disialoganglioside GD2. To this end, we performed a combined in vivo and in vitro screen using a recombinant phage displayed peptide library. We isolated a phage displaying the peptide sequence WHWRLPS that specifically binds to the human disialoganglioside GD2. Binding specificity was confirmed by mutational scanning and by comparative analyses using structurally related disialogangliosides. In vivo, significant enrichment of phage binding to xenografts of human neuroblastoma cells in mice was observed. Tumour-specific phage accumulation could be blocked by intravenous coinjection of the corresponding peptide. Comparative pharmacokinetic analyses revealed higher specific accumulation of 68Ga-labelled GD2-binding peptide compared to 111In-labelled peptide in xenografts of human neuroblastoma. In contrast to 124I-MIBG, which is currently evaluated as a neuroblastoma marker in PET/CT, 68Ga-labelled GD2-specific peptide spared the thyroid but was enriched in the kidneys, which could be partially blocked by infusion of amino acids.In summary, we here report on a novel tumour-homing peptide that specifically binds to the disialoganglioside GD2, accumulates in xenografts of neuroblastoma cells in mice and bears the potential for tumour detection using PET/CT. Thus, this peptide may serve as a new scaffold for diagnosing GD2-positive tumours of neuroectodermal origin.

  1. Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

    Science.gov (United States)

    Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

    2014-10-01

    Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

  2. High level of molecular and phenotypic biodiversity in Jatropha curcas from Central America compared to Africa, Asia and South America

    NARCIS (Netherlands)

    Montes Osorio, L.R.; Torres Salvador, A.F.; Jongschaap, R.E.E.; Azurdia, C.; Berduo, J.; Trindade, L.M.; Visser, R.G.F.; Loo, van E.N.

    2014-01-01

    Background The main bottleneck to elevate jatropha (Jatropha curcas L.) from a wild species to a profitable biodiesel crop is the low genetic and phenotypic variation found in different regions of the world, hampering efficient plant breeding for productivity traits. In this study, 182 accessions

  3. Phenotypic and molecular characterization of resistance to macrolides, lincosamides and type B streptogramin of clinical isolates of Staphylococcus spp. of a university hospital in Recife, Pernambuco, Brazil.

    Science.gov (United States)

    Pereira, Jussyêgles Niedja da Paz; Rabelo, Marcelle Aquino; Lima, Jailton Lobo da Costa; Neto, Armando Monteiro Bezerra; Lopes, Ana Catarina de Souza; Maciel, Maria Amélia Vieira

    2016-01-01

    There is a mechanism of macrolide resistance in Staphylococcus spp. which also affects the lincosamides and type B streptogramins characterizing the so-called MLSB resistance, whose expression can be constitutive (cMLSB) or inducible (iMLSB) and is encoded mainly by ermA and ermC genes. The cMLSB resistance is easily detected by susceptibility testing used in the laboratory routine, but iMLSB resistance is not. Therapy with clindamycin in cases of infection with isolated iMLSB resistance may fail. To characterize the phenotypic (occurrence of cMLSB and iMLSB phenotypes) and molecular (occurrence of ermA and ermC genes) profiles of MLSB resistance of clinical isolates of susceptible and methicillin-resistant Staphylococcus aureus and CNS (coagulase-negative Staphylococcus) from patients of a university hospital, in Pernambuco. The antimicrobial susceptibility of 103 isolates was determined by the disk diffusion technique in Mueller-Hinton agar followed by oxacillin screening. The iMLSB phenotype was detected by D test. Isolates with cMLSB and iMLSB phenotypes were subjected to polymerase chain reaction (PCR) for the detection of ermA and ermC genes. The cMLSB and iMLSB phenotypes were respectively identified in 39 (37.9%) and five (4.9%) isolates. The iMLSB phenotype was found only in four (10.8%) methicillin-susceptible S. aureus and one (4.5%) methicillin-resistant S. aureus. In the 44 isolates subjected to PCR, four (9.1%) only ermA gene was detected, a lower frequency when compared to only ermC 17 (38.6%) gene and to one (2.3%) isolate presenting both genes. In the Staphylococcus spp. analyzed, the ermC gene was found more often than the ermA, although the iMLSB phenotype had been less frequent than the cMLSB. It was important to perform the D test for its detection to guide therapeutic approaches. Copyright © 2016 Elsevier Editora Ltda. All rights reserved.

  4. NovelNR2F1variants likely disrupt DNA binding: molecular modeling in two cases, review of published cases, genotype-phenotype correlation, and phenotypic expansion of the Bosch-Boonstra-Schaaf optic atrophy syndrome.

    Science.gov (United States)

    Kaiwar, Charu; Zimmermann, Michael T; Ferber, Matthew J; Niu, Zhiyv; Urrutia, Raul A; Klee, Eric W; Babovic-Vuksanovic, Dusica

    2017-11-01

    Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS) is a recently described autosomal dominant disorder caused by mutations in the NR2F1 gene. There are presently 28 cases of BBSOAS described in the literature. Its common features include developmental delay, intellectual disability, hypotonia, optic nerve atrophy, attention deficit disorder, autism spectrum disorder, seizures, hearing defects, spasticity, and thinning of the corpus callosum. Here we report two unrelated probands with novel, de novo, missense variants in NR2F1 The first is a 14-yr-old male patient with hypotonia, intellectual disability, optic nerve hypoplasia, delayed bone age, short stature, and altered neurotransmitter levels on cerebrospinal fluid testing. The second is a 5-yr-old female with severe developmental delay, motor and speech delay, and repetitive motion behavior. Whole-exome sequencing identified a novel missense NR2F1 variant in each case, Cys86Phe in the DNA-binding domain in Case 1, and a Leu372Pro in the ligand-binding domain in Case 2. The presence of clinical findings compatible with BBSOAS along with structural analysis at atomic resolution using homology-based molecular modeling and molecular dynamic simulations, support the pathogenicity of these variants for BBSOAS. Short stature, abnormal CNS neurotransmitters, and macrocephaly have not been previously reported for this syndrome and may represent a phenotypic expansion of BBSOAS. A review of published cases along with new evidence from this report support genotype-phenotype correlations for this disorder. © 2017 Kaiwar et al.; Published by Cold Spring Harbor Laboratory Press.

  5. A Case of Bordetella brochiseptica at a Military Medical Facility in Hawai‘i: Phenotypic and Molecular Testing of an Uncommon Human Pathogen

    Science.gov (United States)

    Agee, Willie A; Kajiura, Lauren; Hawley-Molloy, Joshua S; Staege, Catherine M; Barnhill, Jason C

    2015-01-01

    Bordetella bronchiseptica (B. bronchiseptica) is rarely implicated in human disease. Human infections typically occur in the context of immunosuppression and while human infection has been sporadically reported in the literature, the majority of these reports are largely descriptive and do not explore the molecular and phenotypic properties of the isolates in question. Here we report the isolation and characterization of a B. bronchiseptica isolate derived from an HIV positive patient at Tripler Army Medical Center on O‘ahu. This case represents the first published report of human infection of B. bronchiseptica in the state of Hawai‘i and the most detailed description of the biochemical and molecular features of a Hawaiian isolate to date. PMID:26225268

  6. A Case of Bordetella brochiseptica at a Military Medical Facility in Hawai'i: Phenotypic and Molecular Testing of an Uncommon Human Pathogen.

    Science.gov (United States)

    Washington, Michael A; Agee, Willie A; Kajiura, Lauren; Hawley-Molloy, Joshua S; Staege, Catherine M; Barnhill, Jason C

    2015-07-01

    Bordetella bronchiseptica (B. bronchiseptica) is rarely implicated in human disease. Human infections typically occur in the context of immunosuppression and while human infection has been sporadically reported in the literature, the majority of these reports are largely descriptive and do not explore the molecular and phenotypic properties of the isolates in question. Here we report the isolation and characterization of a B. bronchiseptica isolate derived from an HIV positive patient at Tripler Army Medical Center on O'ahu. This case represents the first published report of human infection of B. bronchiseptica in the state of Hawai'i and the most detailed description of the biochemical and molecular features of a Hawaiian isolate to date.

  7. Adsorption and photophysics of fullerene C60 at liquid-zeolite particle interfaces: unusually high affinity for hydrophobic, ultrastabilized zeolite Y.

    Science.gov (United States)

    Ellison, Eric H

    2006-06-15

    O2 at the Y901-toluene interface were 18, 9, and 3 times lower, respectively, relative to rate constants in solution. These differences point out that the approach of molecular quenchers to C60 at the interface is more hindered for larger molecules, an expected result for C60 located in half-supercage bowls. The high affinity of fullerenes for hydrophobic zeolite Y provides a strategy for organizing fullerenes at interfaces and for studies of fullerene photochemistry.

  8. Genetic and Epigenetic Tumor Suppressor Gene Silencing Are Distinct Molecular Phenotypes Driven by Growth Promoting Mutations in Nonsmall Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Carmen J. Marsit

    2008-01-01

    Full Text Available Both genetic and epigenetic alterations characterize human nonsmall cell lung cancer (NSCLC, but the biological processes that create or select these alterations remain incompletely investigated. Our hypothesis posits that a roughly reciprocal relationship between the propensity for promoter hypermethylation and a propensity for genetic deletion leads to distinct molecular phenotypes of lung cancer. To test this hypothesis, we examined promoter hypermethylation of 17 tumor suppressor genes, as a marker of epigenetic alteration propensity, and deletion events at the 3p21 region, as a marker of genetic alteration. To model the complex biology between these somatic alterations, we utilized an item response theory model. We demonstrated that tumors exhibiting LOH at greater than 30% of informative alleles in the 3p21 region have a significantly reduced propensity for hypermethylation. At the same time, tumors with activating KRAS mutations showed a significantly increased propensity for hypermethylation of the loci examined, a result similar to what has been observed in colon cancer. These data suggest that NSCLCs have distinct epigenetic or genetic alteration phenotypes acting upon tumor suppressor genes and that mutation of oncogenic growth promoting genes, such as KRAS, is associated with the epigenetic phenotype.

  9. Molecular phenotyping of multiple mouse strains under metabolic challenge uncovers a role for Elovl2 in glucose-induced insulin secretion.

    Science.gov (United States)

    Cruciani-Guglielmacci, Céline; Bellini, Lara; Denom, Jessica; Oshima, Masaya; Fernandez, Neïké; Normandie-Levi, Priscilla; Berney, Xavier P; Kassis, Nadim; Rouch, Claude; Dairou, Julien; Gorman, Tracy; Smith, David M; Marley, Anna; Liechti, Robin; Kuznetsov, Dmitry; Wigger, Leonore; Burdet, Frédéric; Lefèvre, Anne-Laure; Wehrle, Isabelle; Uphues, Ingo; Hildebrandt, Tobias; Rust, Werner; Bernard, Catherine; Ktorza, Alain; Rutter, Guy A; Scharfmann, Raphael; Xenarios, Ioannis; Le Stunff, Hervé; Thorens, Bernard; Magnan, Christophe; Ibberson, Mark

    2017-04-01

    In type 2 diabetes (T2D), pancreatic β cells become progressively dysfunctional, leading to a decline in insulin secretion over time. In this study, we aimed to identify key genes involved in pancreatic beta cell dysfunction by analyzing multiple mouse strains in parallel under metabolic stress. Male mice from six commonly used non-diabetic mouse strains were fed a high fat or regular chow diet for three months. Pancreatic islets were extracted and phenotypic measurements were recorded at 2 days, 10 days, 30 days, and 90 days to assess diabetes progression. RNA-Seq was performed on islet tissue at each time-point and integrated with the phenotypic data in a network-based analysis. A module of co-expressed genes was selected for further investigation as it showed the strongest correlation to insulin secretion and oral glucose tolerance phenotypes. One of the predicted network hub genes was Elovl2, encoding Elongase of very long chain fatty acids 2. Elovl2 silencing decreased glucose-stimulated insulin secretion in mouse and human β cell lines. Our results suggest a role for Elovl2 in ensuring normal insulin secretory responses to glucose. Moreover, the large comprehensive dataset and integrative network-based approach provides a new resource to dissect the molecular etiology of β cell failure under metabolic stress.

  10. Application of molecular cytogenetic techniques to clarify apparently balanced complex chromosomal rearrangements in two patients with an abnormal phenotype: case report

    Directory of Open Access Journals (Sweden)

    Rongen Michel A

    2009-07-01

    Full Text Available Abstract Background Complex chromosomal rearrangements (CCR are rare cytogenetic findings that are difficult to karyotype by conventional cytogenetic analysis partially because of the relative low resolution of this technique. High resolution genotyping is necessary in order to identify cryptic imbalances, for instance near the multiple breakpoints, to explain the abnormal phenotype in these patients. We applied several molecular techniques to elucidate the complexity of the CCRs of two adult patients with abnormal phenotypes. Results Multicolour fluorescence in situ hybridization (M-FISH showed that in patient 1 the chromosomes 1, 10, 15 and 18 were involved in the rearrangement whereas for patient 2 the chromosomes 5, 9, 11 and 13 were involved. A 250 k Nsp1 SNP-array analysis uncovered a deletion in chromosome region 10p13 for patient 1, harbouring 17 genes, while patient 2 showed no pathogenic gains or losses. Additional FISH analysis with locus specific BAC-probes was performed, leading to the identification of cryptic interstitial structural rearrangements in both patients. Conclusion Application of M-FISH and SNP-array analysis to apparently balanced CCRs is useful to delineate the complex chromosomal rearrangement in detail. However, it does not always identify cryptic imbalances as an explanation for the abnormal phenotype in patients with a CCR.

  11. TRPV4 related skeletal dysplasias: a phenotypic spectrum highlighted byclinical, radiographic, and molecular studies in 21 new families

    Directory of Open Access Journals (Sweden)

    Sillence David

    2011-06-01

    Full Text Available Abstract Background The TRPV4 gene encodes a calcium-permeable ion-channel that is widely expressed, responds to many different stimuli and participates in an extraordinarily wide range of physiologic processes. Autosomal dominant brachyolmia, spondylometaphyseal dysplasia Kozlowski type (SMDK and metatropic dysplasia (MD are currently considered three distinct skeletal dysplasias with some shared clinical features, including short stature, platyspondyly, and progressive scoliosis. Recently, TRPV4 mutations have been found in patients diagnosed with these skeletal phenotypes. Methods and Results We critically analysed the clinical and radiographic data on 26 subjects from 21 families, all of whom had a clinical diagnosis of one of the conditions described above: 15 with MD; 9 with SMDK; and 2 with brachyolmia. We sequenced TRPV4 and identified 9 different mutations in 22 patients, 4 previously described, and 5 novel. There were 4 mutation-negative cases: one with MD and one with SMDK, both displaying atypical clinical and radiographic features for these diagnoses; and two with brachyolmia, who had isolated spine changes and no metaphyseal involvement. Conclusions Our data suggest the TRPV4 skeletal dysplasias represent a continuum of severity with areas of phenotypic overlap, even within the same family. We propose that AD brachyolmia lies at the mildest end of this spectrum and, since all cases described with this diagnosis and TRPV4 mutations display metaphyseal changes, we suggest that it is not a distinct entity but represents the mildest phenotypic expression of SMDK.

  12. A high affinity conformational state on VLA integrin heterodimers induced by an anti-beta 1 chain monoclonal antibody.

    Science.gov (United States)

    Arroyo, A G; García-Pardo, A; Sánchez-Madrid, F

    1993-05-05

    The VLA integrin subfamily includes receptors for extracellular matrix proteins as well as receptors involved in cell-cell adhesive interactions. We have previously described the up-regulation of VLA integrin-mediated cell attachment to different ligands by the anti-beta 1 TS2/16 monoclonal antibody (mAb) (Arroyo, A. G., Sánchez-Mateos, P., Campanero, M. R., Martín-Padura, I., Dejana, E., and Sánchez-Madrid, F. (1992) J. Cell Biol. 117, 659-670). In this report, we have investigated the mechanism involved in this regulatory effect. The anti-beta 1-mediated regulatory effect on cell adhesion did not require "de novo" protein synthesis, since it was not affected by pretreatment with either cycloheximide or actinomycin D. To quantitate the effect of the regulatory anti-beta 1 TS2/16 mAb on the affinity of VLA-5 for its ligand, an RGD-containing fragment of fibronectin (FN80), we performed binding studies of radiolabeled soluble FN80 to U-937 cells. The affinity of VLA-5 for FN80 was enhanced about 4-fold in the presence of TS2/16 mAb (Kd = 0.98 +/- 0.07 microM) compared to the functionally irrelevant anti-beta 1 Alex 1/4 mAb (Kd = 4.23 +/- 0.92 microM), whereas no alteration in the number of binding sites was observed. Indeed, the anti-beta 1 TS2/16 mAb is inducing this high affinity state on VLA heterodimers by a direct change on the conformation of these receptors as demonstrated by affinity chromatography analysis using extracellular matrix proteins covalently bound to Sepharose. The yield of VLA-5 fibronectin receptor bound to FN80-Sepharose columns was strongly increased upon treatment of U-937 cell lysates with mAb TS2/16. Moreover, higher concentrations of EDTA were required for eluting the VLA-5 integrin from this matrix. This up-regulatory effect was also observed with F(ab')2 and Fab fragments of the anti-beta 1 TS2/16 mAb, and was also exerted on the purified VLA-5 receptor. Similarly, the yield of VLA-2 retained on a collagen I-Sepharose column was

  13. Phenotypic and molecular fingerprinting of fast growing rhizobia of field-grown pigeonpea from the eastern edge of the Brazilian Pantanal.

    Science.gov (United States)

    Costa, F M; Schiavo, J A; Brasil, M S; Leite, J; Xavier, G R; Fernandes, P I

    2014-01-21

    The aim of this study was to evaluate the diversity of rhizobial isolates obtained from root nodules of pigeonpea plants grown at the eastern edge of the Brazilian Pantanal. The bacterial isolates were isolated from root nodules from field-growing pigeonpea grown in two rural settlements of the Aquidauana municipality. The bacterial isolates were characterized phenotypically by means of cultural characterization, intrinsic antibiotic resistance (IAR), salt and high incubation temperature tolerance, and amylolytic and cellulolytic activities. The molecular characterization of the bacterial isolates was carried out using amplified ribosomal DNA restriction analysis (ARDRA) and Box-polymerase chain reaction (PCR) techniques. In addition, the symbiotic performance of selected rhizobial isolates was evaluated in a greenhouse experiment using sterile substrate. The phenotypic characterization revealed that the bacterial strains obtained from pigeonpea root nodules presented characteristics that are uncommon among rhizobial isolates, indicating the presence of new species nodulating the pigeonpea plants in the Brazilian Pantanal. The molecular fingerprinting of these bacterial isolates also showed a highly diverse collection, with both techniques revealing less than 25% similarity among bacterial isolates. The evaluation of symbiotic performance also indicated the presence of microorganisms with high potential to increase the growth and nitrogen content at the shoots of pigeonpea plants. The results obtained in this study indicate the presence of a highly diversified rhizobial community nodulating the pigeonpea at the eastern edge of the Brazilian Pantanal.

  14. Germline PRKACA amplification causes variable phenotypes that may depend on the extent of the genomic defect: molecular mechanisms and clinical presentations.

    Science.gov (United States)

    Lodish, Maya B; Yuan, Bo; Levy, Isaac; Braunstein, Glenn D; Lyssikatos, Charalampos; Salpea, Paraskevi; Szarek, Eva; Karageorgiadis, Alexander S; Belyavskaya, Elena; Raygada, Margarita; Faucz, Fabio Rueda; Izzat, Louise; Brain, Caroline; Gardner, James; Quezado, Martha; Carney, J Aidan; Lupski, James R; Stratakis, Constantine A

    2015-06-01

    We have recently reported five patients with bilateral adrenocortical hyperplasia (BAH) and Cushing's syndrome (CS) caused by constitutive activation of the catalytic subunit of protein kinase A (PRKACA). By doing new in-depth analysis of their cytogenetic abnormality, we attempted a better genotype-phenotype correlation of their PRKACA amplification. This study is a case series. Molecular cytogenetic, genomic, clinical, and histopathological analyses were performed in five patients with CS. Reinvestigation of the defects of previously described patients by state-of-the-art molecular cytogenetics showed complex genomic rearrangements in the chromosome 19p13.2p13.12 locus, resulting in copy number gains encompassing the entire PRKACA gene; three patients (one sporadic case and two related cases) were observed with gains consistent with duplications, while two sporadic patients were observed with gains consistent with triplications. Although all five patients presented with ACTH-independent CS, the three sporadic patients had micronodular BAH and underwent bilateral adrenalectomy in early childhood, whereas the two related patients, a mother and a son, presented with macronodular BAH as adults. In at least one patient, PRKACA triplication was associated with a more severe phenotype. Constitutional chromosomal PRKACA gene amplification is a recently identified genetic defect associated with CS, a trait that may be inherited in an autosomal dominant manner or occur de novo. Genomic rearrangements can be complex and can result in different copy number states of dosage-sensitive genes, e.g., duplication and triplication. PRKACA amplification can lead to variable phenotypes clinically and pathologically, both micro- and macro-nodular BAH, the latter of which we speculate may depend on the extent of amplification. © 2015 European Society of Endocrinology.

  15. ROBO1, a tumor suppressor and critical molecular barrier for localized tumor cells to acquire invasive phenotype: study in African-American and Caucasian prostate cancer models.

    Science.gov (United States)

    Parray, Aijaz; Siddique, Hifzur R; Kuriger, Jacquelyn K; Mishra, Shrawan K; Rhim, Johng S; Nelson, Heather H; Aburatani, Hiroyuki; Konety, Badrinath R; Koochekpour, Shahriar; Saleem, Mohammad

    2014-12-01

    High-risk populations exhibit early transformation of localized prostate cancer (CaP) disease to metastasis which results in the mortality of such patients. The paucity of knowledge about the molecular mechanism involved in acquiring of metastatic behavior by primary tumor cells and non-availability of reliable phenotype-discriminating biomarkers are stumbling blocks in the management of CaP disease. Here, we determine the role and translational relevance of ROBO1 (an organogenesis-associated gene) in human CaP. Employing CaP-progression models and prostatic tissues of Caucasian and African-American patients, we show that ROBO1 expression is localized to cell-membrane and significantly lost in primary and metastatic tumors. While Caucasians exhibited similar ROBO1 levels in primary and metastatic phenotype, a significant difference was observed between tumor phenotypes in African-Americans. Epigenetic assays identified promoter methylation of ROBO1 specific to African-American metastatic CaP cells. Using African-American CaP models for further studies, we show that ROBO1 negatively regulates motility and invasiveness of primary CaP cells, and its loss causes these cells to acquire invasive trait. To understand the underlying mechanism, we employed ROBO1-expressing/ROBO1-C2C3-mutant constructs, immunoprecipitation, confocal-microscopy and luciferase-reporter techniques. We show that ROBO1 through its interaction with DOCK1 (at SH3-SH2-domain) controls the Rac-activation. However, loss of ROBO1 results in Rac1-activation which in turn causes E-Cadherin/β-catenin cytoskeleton destabilization and induction of cell migration. We suggest that ROBO1 is a predictive biomarker that has potential to discriminate among CaP types, and could be exploited as a molecular target to inhibit the progression of disease as well as treat metastasis in high-risk populations such as African-Americans. © 2014 UICC.

  16. Typing discrepancy between phenotypic and molecular characterization revealing an emerging biovar 9 variant of smooth phage-resistant B. abortus strain 8416 in China

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    YaoXia eKang

    2015-12-01

    Full Text Available A newly isolated smooth colony morphology phage-resistant (SPR strain 8416 isolated from a 45-year-old cattle farm cleaner with clinical features of brucellosis in China was reported. The most unusual phenotype was its resistance to two Brucella phages Tbilisi and Weybridge, but sensitive to Berkeley 2, a pattern similar to that of B. melitensis biovar 1. VITEK 2 biochemical identification system found that both strain 8416 and B. melitensis strains shared positive ILATk, but negative in other B. abortus strains. However, routine biochemical and phenotypic characteristics of strain 8416 were most similar to that of B. abortus biovar 9 except CO2 requirement. In addition, multiple PCR molecular typing assays including AMOS-PCR, B. abortus special PCR (B-ab PCR and a novel sub-biovar typing PCR, indicated that strain 8416 may belong to either biovar 3b or 9 of B. abortus. Surprisingly, further MLVA typing results showed that strain 8416 was most closely related to B. abortus biovar 3 in the Brucella MLVA database, primarily differing in 4 out of 16 screened loci. Therefore, due to the unusual discrepancy between phenotypic (biochemical reactions and particular phage lysis profile and molecular typing characteristics, strain 8416 couldn’t be exactly classified to any of the existing B. abortus biovars and might be a new variant of B. abortus biovar 9. The present study also indicates that the present phage typing scheme for Brucella spp. is subject to variation and the routine Brucella biovar typing needs further studies.

  17. Meier–Gorlin syndrome genotype–phenotype studies: 35 individuals with pre-replication complex gene mutations and 10 without molecular diagnosis

    Science.gov (United States)

    de Munnik, Sonja A; Bicknell, Louise S; Aftimos, Salim; Al-Aama, Jumana Y; van Bever, Yolande; Bober, Michael B; Clayton-Smith, Jill; Edrees, Alaa Y; Feingold, Murray; Fryer, Alan; van Hagen, Johanna M; Hennekam, Raoul C; Jansweijer, Maaike C E; Johnson, Diana; Kant, Sarina G; Opitz, John M; Ramadevi, A Radha; Reardon, Willie; Ross, Alison; Sarda, Pierre; Schrander-Stumpel, Constance T R M; Schoots, Jeroen; Temple, I Karen; Terhal, Paulien A; Toutain, Annick; Wise, Carol A; Wright, Michael; Skidmore, David L; Samuels, Mark E; Hoefsloot, Lies H; Knoers, Nine V A M; Brunner, Han G; Jackson, Andrew P; Bongers, Ernie M H F

    2012-01-01

    Meier–Gorlin syndrome (MGS) is an autosomal recessive disorder characterized by microtia, patellar aplasia/hypoplasia, and short stature. Recently, mutations in five genes from the pre-replication complex (ORC1, ORC4, ORC6, CDT1, and CDC6), crucial in cell-cycle progression and growth, were identified in individuals with MGS. Here, we report on genotype–phenotype studies in 45 individuals with MGS (27 females, 18 males; age 3 months–47 years). Thirty-five individuals had biallelic mutations in one of the five causative pre-replication genes. No homozygous or compound heterozygous null mutations were detected. In 10 individuals, no definitive molecular diagnosis was made. The triad of microtia, absent/hypoplastic patellae, and short stature was observed in 82% of individuals with MGS. Additional frequent clinical features were mammary hypoplasia (100%) and abnormal genitalia (42% predominantly cryptorchidism and hypoplastic labia minora/majora). One individual with ORC1 mutations only had short stature, emphasizing the highly variable clinical spectrum of MGS. Individuals with ORC1 mutations had significantly shorter stature and smaller head circumferences than individuals from other gene categories. Furthermore, compared with homozygous missense mutations, compound heterozygous mutations appeared to have a more severe effect on phenotype, causing more severe growth retardation in ORC4 and more frequently pulmonary emphysema in CDT1. A lethal phenotype was seen in four individuals with compound heterozygous ORC1 and CDT1 mutations. No other clear genotype–phenotype association was observed. Growth hormone and estrogen treatment may be of some benefit, respectively, to growth retardation and breast hypoplasia, though further studies in this patient group are needed. PMID:22333897

  18. Molecular genetic analysis of the calcium sensing receptor gene in patients clinically suspected to have familial hypocalciuric hypercalcemia: phenotypic variation and mutation spectrum in a Danish population.

    Science.gov (United States)

    Nissen, Peter H; Christensen, Signe E; Heickendorff, Lene; Brixen, Kim; Mosekilde, Leif

    2007-11-01

    The autosomal dominantly inherited condition familial hypocalciuric hypercalcemia (FHH) is characterized by elevated plasma calcium levels, relative or absolute hypocalciuria, and normal to moderately elevated plasma PTH. The condition is difficult to distinguish clinically from primary hyperparathyroidism and is caused by inactivating mutations in the calcium sensing receptor (CASR) gene. We sought to define the mutation spectrum of the CASR gene in a Danish FHH population and to establish genotype-phenotype relationships regarding the different mutations. A total of 213 subjects clinically suspected to have FHH, and 121 subjects enrolled as part of a family-screening program were studied. Genotype-phenotype relationships were established in 66 mutation-positive index patients and family members. We determined CASR gene mutations, and correlating levels of plasma calcium (albumin corrected), ionized calcium (pH 7.4), and PTH were measured. We identified 22 different mutations in 39 FHH families. We evaluated data on circulating calcium and PTH for 11 different mutations, representing a spectrum of clinical phenotypes, ranging from calcium concentrations moderately above the upper reference limit, to calcium levels more than 20% above the upper reference limit. Furthermore, the mean plasma PTH concentration was within the normal range in eight of 11 studied mutations, but mild to moderately elevated in families with the mutations p.C582Y, p.C582F, and p.G553R. The present data add 19 novel mutations to the catalog of inactivating CASR mutations and illustrate a variety of biochemical phenotypes in patients with the molecular genetic diagnosis FHH.

  19. Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons.

    Science.gov (United States)

    Schweitz, H; Heurteaux, C; Bois, P; Moinier, D; Romey, G; Lazdunski, M

    1994-02-01

    Calcicludine (CaC) is a 60-amino acid polypeptide from the venom of Dendroaspis angusticeps. It is structurally homologous to the Kunitz-type protease inhibitor, to dendrotoxins, which block K+ channels, and to the protease inhibitor domain of the amyloid beta protein that accumulates in Alzheimer disease. Voltage-clamp experiments on a variety of excitable cells have shown that CaC specifically blocks most of the high-threshold Ca2+ channels (L-, N-, or P-type) in the 10-100 nM range. Particularly high densities of specific 125I-labeled CaC binding sites were found in the olfactory bulb, in the molecular layer of the dentate gyrus and the stratum oriens of CA3 field in the hippocampal formation, and in the granular layer of the cerebellum. 125I-labeled CaC binds with a high affinity (Kd = 15 pM) to a single class of noninteracting sites in rat olfactory bulb microsomes. The distribution of CaC binding sites in cerebella of three mutant mice (Weaver, Reeler, and Purkinje cell degeneration) clearly shows that the specific high-affinity labeling is associated with granule cells. Electrophysiological experiments on rat cerebellar granule neurons in primary culture have shown that CaC potently blocks the L-type component of the Ca2+ current (K0.5 = 0.2 nM). Then CaC, in the nanomolar range, appears to be a highly potent blocker of an L-subtype of neuronal Ca2+ channels.

  20. High affinity interaction of integrin alpha4beta1 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1) enhances migration of human melanoma cells across activated endothelial cell layers.

    Science.gov (United States)

    Klemke, Martin; Weschenfelder, Tatjana; Konstandin, Mathias H; Samstag, Yvonne

    2007-08-01

    The capacity of tumor cells to form metastatic foci correlates with their ability to interact with and migrate through endothelial cell layers. This process involves multiple adhesive interactions between tumor cells and the endothelium. Only little is known about the molecular nature of these interactions during extravasation of tumor cells. In human melanoma cells, the integrin alphavbeta3 is involved in transendothelial migration and its expression correlates with metastasis. However, many human melanoma cells do not express beta3 integrins. Therefore, it remained unclear how these cells undergo transendothelial migration. In this study we show that human melanoma cells with different metastatic potency, which do not express beta2 or beta3 integrins, express the VCAM-1 receptor alpha4beta1. VCAM-1 is up-regulated on activated endothelial cells and is known to promote transendothelial migration of leukocytes. Interestingly, despite comparable cell surface levels of alpha4beta1, only the highly metastatic melanoma cell lines MV3 and BLM, but not the low metastatic cell lines IF6 and 530, bind VCAM-1 with high affinity without further stimulation, and are therefore able to adhere to and migrate on isolated VCAM-1. Moreover, we demonstrate that function-blocking antibodies against the integrin alpha4beta1, as well as siRNA-mediated knock-down of the alpha4 subunit in these highly metastatic human melanoma cells reduce their transendothelial migration. These data imply that only high affinity interactions between the integrin alpha4beta1 on melanoma cells and VCAM-1 on activated endothelial cells may enhance the metastatic capacity of human beta2/beta3-negative melanoma cells.

  1. Challenges of molecular nutrition research 6: the nutritional phenotype database to store, share and evaluate nutritional systems biology studies

    Science.gov (United States)

    Bouwman, Jildau; Dragsted, Lars O.; Drevon, Christian A.; Elliott, Ruan; de Groot, Philip; Kaput, Jim; Mathers, John C.; Müller, Michael; Pepping, Fre; Saito, Jahn; Scalbert, Augustin; Radonjic, Marijana; Rocca-Serra, Philippe; Travis, Anthony; Wopereis, Suzan; Evelo, Chris T.

    2010-01-01

    The challenge of modern nutrition and health research is to identify food-based strategies promoting life-long optimal health and well-being. This research is complex because it exploits a multitude of bioactive compounds acting on an extensive network of interacting processes. Whereas nutrition research can profit enormously from the revolution in ‘omics’ technologies, it has discipline-specific requirements for analytical and bioinformatic procedures. In addition to measurements of the parameters of interest (measures of health), extensive description of the subjects of study and foods or diets consumed is central for describing the nutritional phenotype. We propose and pursue an infrastructural activity of constructing the “Nutritional Phenotype database” (dbNP). When fully developed, dbNP will be a research and collaboration tool and a publicly available data and knowledge repository. Creation and implementation of the dbNP will maximize benefits to the research community by enabling integration and interrogation of data from multiple studies, from different research groups, different countries and different—omics levels. The dbNP is designed to facilitate storage of biologically relevant, pre-processed—omics data, as well as study descriptive and study participant phenotype data. It is also important to enable the combination of this information at different levels (e.g. to facilitate linkage of data describing participant phenotype, genotype and food intake with information on study design and—omics measurements, and to combine all of this with existing knowledge). The biological information stored in the database (i.e. genetics, transcriptomics, proteomics, biomarkers, metabolomics, functional assays, food intake and food composition) is tailored to nutrition research and embedded in an environment of standard procedures and protocols, annotations, modular data-basing, networking and integrated bioinformatics. The dbNP is an evolving enterprise

  2. Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: Characterization of an active digitonin-solubilized receptor complex

    Energy Technology Data Exchange (ETDEWEB)

    Grigoriadis, D.E.; Zaczek, R.; Pearsall, D.M.; De Souza, E.B. (National Institute on Drug Abuse, Baltimore, MD (USA))

    1989-12-01

    The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of (125I)Tyro-ovine CRF ((125I)oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for (125I) oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, (125I)oCRF labeled the same size receptor complex.

  3. Zinc deficiency up-regulates expression of high-affinity phosphate transporter genes in both phosphate-sufficient and -deficient barley roots.

    Science.gov (United States)

    Huang, C; Barker, S J; Langridge, P; Smith, F W; Graham, R D

    2000-09-01

    Phosphate (P) is taken up by plants through high-affinity P transporter proteins embedded in the plasma membrane of certain cell types in plant roots. Expression of the genes that encode these transporters responds to the P status of the plants, and their transcription is normally tightly controlled. However, this tight control of P uptake is lost under Zn deficiency, leading to very high accumulation of P in plants. We examined the effect of plant Zn status on the expression of the genes encoding the HVPT1 and HVPT2 high-affinity P transporters in barley (Hordeum vulgare L. cv Weeah) roots. The results show that the expression of these genes is intimately linked to the Zn status of the plants. Zn deficiency induced the expression of genes encoding these P transporters in plants grown in either P-sufficient or -deficient conditions. Moreover, the role of Zn in the regulation of these genes is specific in that it cannot be replaced by manganese (a divalent cation similar to Zn). It appears that Zn plays a specific role in the signal transduction pathway responsible for the regulation of genes encoding high-affinity P transporters in plant roots. The significance of Zn involvement in the regulation of genes involved in P uptake is discussed.

  4. Phenotypic and Molecular Evidence Suggest That Decrements in Morning and Evening Energy Are Distinct But Related Symptoms

    Science.gov (United States)

    Aouizerat, Bradley E.; Dhruva, Anand; Paul, Steven M.; Cooper, Bruce A.; Kober, Kord M.; Miaskowski, Christine

    2015-01-01

    Context Little is known about energy levels in oncology patients and their family caregivers (FCs). Objectives This study sought to identify latent classes of participants, based on self-reported energy levels and to evaluate for differences in phenotypic and genotypic characteristics between these classes. Methods Energy subscale scores from the Lee Fatigue Scale were used to determine latent class membership. Morning and evening energy scores were obtained just prior to, during, and for four months following the completion of radiation therapy. Genetic associations were evaluated for fifteen pro- and anti-inflammatory cytokine genes. Results Two latent classes with distinct morning energy trajectories were identified. Participants who were younger, female, not married/partnered, Black, and had more comorbidities, and a lower functional status were more likely to be in the Low Morning Energy class. Two polymorphisms (IL2 rs1479923, NFKB1 rs4648110) were associated with morning energy latent class membership. Two latent classes with distinct evening energy trajectories were identified. Participants who were younger and male and who had more comorbidities, decreased body weight, and a lower functional status were more likely to be in the Moderate Evening Energy class. Five different polymorphisms (IL1R2 rs4141134, IL6 rs4719714, IL17A rs8193036, NFKB2 rs1056890, TNFA rs1800683) were associated with evening energy latent class membership. Conclusion This study provides preliminary evidence that decrements in morning and evening energy are associated with different phenotypic risk factors as well as cytokine gene variations. PMID:26031709

  5. Caracterização fenotípica e molecular de amostras de Burkholderia mallei isoladas na Região Nordeste do Brasil Phenotypic and molecular characterization of Burkholderia mallei isolated in northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Karla P.C. Silva

    2009-05-01

    Full Text Available Objetivou-se com este trabalho realizar o estudo bioquímico e molecular de amostras de Burkholderia mallei isoladas de eqüídeos com diagnóstico clínico e sorológico para o mormo e provenientes da Região Metropolitana do Recife-PE e Zona da Mata dos Estados de Alagoas e Pernambuco. Foram realizadas as técnicas microbiológicas para o isolamento e identificação fenotípica de B. mallei e as técnicas moleculares de ribotipagem-PCR e RAPD-PCR. Das oito amostras estudadas, quatro apresentaram pequenas variações fenotípicas. Nas técnicas moleculares, as amostras formaram quatro grupos de diferentes perfis ribotípicos, demonstrando também quatro perfis genotípicos. Houve associação nos resultados da Ribotipagem-PCR e RAPD-PCR. As variações nos perfis ribotípicos e genotípicos foram associadas às diferentes regiões estudadas. De acordo com os resultados obtidos, conclui-se que as pequenas variações bioquímicas não estão associadas aos diferentes perfis moleculares e que essas diferenças demonstram uma heterogeneidade que está associada à procedência das amostras, indicando que a infecção nos animais ocorre por clones diferentes das amostras analisadas.The objective of this paper was to study the molecular performance and phenotypic characterization of Burkholderia mallei isolated from horses with clinical and serological diagnosis of glanders, originating from the Metropolitan District of Recife and Zona da Mata of Pernambuco and Alagoas. The isolation and biochemical identification of B. mallei was carried out by microbiological and molecular techniques of PCR-fingerprinting and RAPD-PCR. From the eight samples studied, four showed little phenotype variations. In the molecular tests, the samples formed 4 groups of different ribotype profiles and 4 genotype profiles. There was some association of PCR-fingerprinting with RAPD-PCR results. It was concluded that the slight biochemical variations were not associated with

  6. Molecular phenotyping of immune cells from young NOD mice reveals abnormal metabolic pathways in the early induction phase of autoimmune diabetes.

    Science.gov (United States)

    Wu, Jian; Kakoola, Dorothy N; Lenchik, Nataliya I; Desiderio, Dominic M; Marshall, Dana R; Gerling, Ivan C

    2012-01-01

    Islet leukocytic infiltration (insulitis) is first obvious at around 4 weeks of age in the NOD mouse--a model for human type 1 diabetes (T1D). The molecular events that lead to insulitis and initiate autoimmune diabetes are poorly understood. Since TID is caused by numerous genes, we hypothesized that multiple molecular pathways are altered and interact to initiate this disease. We evaluated the molecular phenotype (mRNA and protein expression) and molecular networks of ex vivo unfractionated spleen leukocytes from 2 and 4 week-old NOD mice in comparison to two control strains. Analysis of the global gene expression profiles and hierarchical clustering revealed that the majority (~90%) of the differentially expressed genes in NOD mice were repressed. Furthermore, analysis using a modern suite of multiple bioinformatics approaches identified abnormal molecular pathways that can be divided broadly into 2 categories: metabolic pathways, which were predominant at 2 weeks, and immune response pathways, which were predominant at 4 weeks. Network analysis by Ingenuity pathway analysis identified key genes/molecules that may play a role in regulating these pathways. These included five that were common to both ages (TNF, HNF4A, IL15, Progesterone, and YWHAZ), and others that were unique to 2 weeks (e.g. MYC/MYCN, TGFB1, and IL2) and to 4 weeks (e.g. IFNG, beta-estradiol, p53, NFKB, AKT, PRKCA, IL12, and HLA-C). Based on the literature, genes that may play a role in regulating metabolic pathways at 2 weeks include Myc and HNF4A, and at 4 weeks, beta-estradiol, p53, Akt, HNF4A and AR. Our data suggest that abnormalities in regulation of metabolic pathways in the immune cells of young NOD mice lead to abnormalities in the immune response pathways and as such may play a role in the initiation of autoimmune diabetes. Thus, targeting metabolism may provide novel approaches to preventing and/or treating autoimmune diabetes.

  7. Different phenotypic and molecular mechanisms associated with multidrug resistance in Gram-negative clinical isolates from Egypt

    Directory of Open Access Journals (Sweden)

    Helmy OM

    2017-12-01

    Full Text Available Omneya M Helmy, Mona T Kashef Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt Objectives: We set out to investigate the prevalence, different mechanisms, and clonal relatedness of multidrug resistance (MDR among third-generation cephalosporin-resistant Gram-negative clinical isolates from Egypt.Materials and methods: A total of 118 third-generation cephalosporin-resistant Gram-negative clinical isolates were included in this study. Their antimicrobial susceptibility pattern was determined using Kirby–Bauer disk diffusion method. Efflux pump-mediated resistance was tested by the efflux-pump inhibitor-based microplate assay using chlorpromazine. Detection of different aminoglycoside-, β-lactam-, and quinolone-resistance genes was done using polymerase chain reaction. The genetic diversity of MDR isolates was investigated using random amplification of polymorphic DNA.Results: Most of the tested isolates exhibited MDR phenotypes (84.75%. The occurrence of efflux pump-mediated resistance in the different MDR species tested was 40%–66%. Acinetobacter baumannii isolates showed resistance to most of the tested antibiotics, including imipenem. The blaOXA-23-like gene was detected in 69% of the MDR A. baumannii isolates. The MDR phenotype was detected in 65% of Pseudomonas aeruginosa isolates, of which only 23% exhibited efflux pump-mediated resistance. On the contrary, efflux-mediated resistance to piperacillin and gentamicin was recorded in 47.5% of piperacillin-resistant and 25% of gentamicin-resistant MDR Enterobacteriaceae. Moreover, the plasmid-mediated quinolone-resistance genes (aac(6’-Ib-cr, qnrB, and qnrS were detected in 57.6% and 83.33% of quinolone-resistant MDR Escherichia coli and Klebsiella pneumoniae isolates, respectively. The β-lactamase-resistance gene blaSHV-31 was detected for the first time in one MDR K. pneumoniae isolate from an endotracheal tube specimen in Egypt

  8. Reprint of "Abstraction for data integration: Fusing mammalian molecular, cellular and phenotype big datasets for better knowledge extraction".

    Science.gov (United States)

    Rouillard, Andrew D; Wang, Zichen; Ma'ayan, Avi

    2015-12-01

    With advances in genomics, transcriptomics, metabolomics and proteomics, and more expansive electronic clinical record monitoring, as well as advances in computation, we have entered the Big Data era in biomedical research. Data gathering is growing rapidly while only a small fraction of this data is converted to useful knowledge or reused in future studies. To improve this, an important concept that is often overlooked is data abstraction. To fuse and reuse biomedical datasets from diverse resources, data abstraction is frequently required. Here we summarize some of the major Big Data biomedical research resources for genomics, proteomics and phenotype data, collected from mammalian cells, tissues and organisms. We then suggest simple data abstraction methods for fusing this diverse but related data. Finally, we demonstrate examples of the potential utility of such data integration efforts, while warning about the inherit biases that exist within such data. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression

    Science.gov (United States)

    Centritto, Floriana; Paroni, Gabriela; Bolis, Marco; Garattini, Silvio Ken; Kurosaki, Mami; Barzago, Maria Monica; Zanetti, Adriana; Fisher, James Neil; Scott, Mark Francis; Pattini, Linda; Lupi, Monica; Ubezio, Paolo; Piccotti, Francesca; Zambelli, Alberto; Rizzo, Paola; Gianni', Maurizio; Fratelli, Maddalena; Terao, Mineko; Garattini, Enrico

    2015-01-01

    Forty-two cell lines recapitulating mammary carcinoma heterogeneity were profiled for all-trans retinoic acid (ATRA) sensitivity. Luminal and ER+ (estrogen-receptor-positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity. Indeed, only 2 Basal cell lines (MDA-MB157 and HCC-1599) are highly sensitive to the retinoid. Sensitivity of HCC-1599 cells is confirmed in xenotransplanted mice. Short-term tissue-slice cultures of surgical samples validate the cell-line results and support the concept that a high proportion of Luminal/ER+ carcinomas are ATRA sensitive, while triple-negative (Basal) and HER2-positive tumors tend to be retinoid resistant. Pathway-oriented analysis of the constitutive gene-expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity. RARα3 is the major transcript in ATRA-sensitive cells and tumors. Studies in selected cell lines with agonists/antagonists confirm that RARα is the principal mediator of ATRA responsiveness. RARα over-expression sensitizes retinoid-resistant MDA-MB453 cells to ATRA anti-proliferative action. Conversely, silencing of RARα in retinoid-sensitive SKBR3 cells abrogates ATRA responsiveness. All this is paralleled by similar effects on ATRA-dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti-metastatic effects. We define gene sets of predictive potential which are associated with ATRA sensitivity in breast cancer cell lines and validate them in short-term tissue cultures of Luminal/ER+ and triple-negative tumors. In these last models, we determine the perturbations in the transcriptomic profiles afforded by ATRA. The study provides fundamental information for the development of retinoid-based therapeutic strategies aimed at the stratified treatment of breast cancer subtypes

  10. Performance comparison of phenotypic and molecular methods for detection and differentiation of Candida albicans and Candida dubliniensis.

    Science.gov (United States)

    Ahmad, Suhail; Khan, Ziauddin; Asadzadeh, Mohammad; Theyyathel, Ajmal; Chandy, Rachel

    2012-09-25

    Candida albicans is the most pathogenic Candida species but shares many phenotypic features with Candida dubliniensis and may, therefore, be misidentified in clinical microbiology laboratories. Candidemia cases due to C. dubliniensis are increasingly being reported in recent years. Accurate identification is warranted since mortality rates are highest for C. albicans infections, however, C. dubliniensis has the propensity to develop resistance against azoles more easily. We developed a duplex PCR assay for rapid detection and differentiation of C. albicans from C. dubliniensis for resource-poor settings equipped with basic PCR technology and compared its performance with three phenotypic methods. Duplex PCR was performed on 122 germ tube positive and 12 germ tube negative isolates of Candida species previously identified by assimilation profiles on Vitek 2 ID-YST system. Typical morphologic characteristics on simplified sunflower seed agar (SSA), and reaction with a commercial (Bichro-Dubli) latex agglutination test were also performed. The assay was further applied on 239 clinical yeast and yeast-like fungi and results were confirmed by DNA sequencing of internal transcribed spacer (ITS) region of rDNA. The results of duplex PCR assay for 122 germ tube positive and 12 germ tube negative isolates of Candida species were comparable to their identification by Vitek 2 ID-YST system, colony characteristics on SSA and latex agglutination test. Application of duplex PCR also correctly identified all 148 C. albicans and 50 C. dubliniensis strains among 239 yeast-like fungi. The data show that both, duplex PCR and Bichro-Dubli are reliable tests for rapid (within few hours) identification of clinical yeast isolates as C. dubliniensis or C. albicans. However, duplex PCR may be applied directly on clinical yeast isolates for their identification as C. dubliniensis or C. albicans as it does not require prior testing for germ tube formation or latex Candida agglutination.

  11. Phenotypic and Molecular Evidence Suggests That Decrements in Morning and Evening Energy Are Distinct but Related Symptoms.

    Science.gov (United States)

    Aouizerat, Bradley E; Dhruva, Anand; Paul, Steven M; Cooper, Bruce A; Kober, Kord M; Miaskowski, Christine

    2015-11-01

    Little is known about energy levels in oncology patients and their family caregivers. This study sought to identify latent classes of participants, based on self-reported energy levels and evaluate for differences in phenotypic and genotypic characteristics between these classes. Energy subscale scores from the Lee Fatigue Scale were used to determine latent class membership. Morning and evening energy scores were obtained just before, during, and for four months after the completion of radiation therapy. Genetic associations were evaluated for 15 proinflammatory and anti-inflammatory cytokine genes. Two latent classes with distinct morning energy trajectories were identified. Participants who were younger, female, not married/partnered, black, and had more comorbidities, and a lower functional status were more likely to be in the low morning energy class. Two polymorphisms (IL2 rs1479923 and NFKB1 rs4648110) were associated with morning energy latent class membership. Two latent classes with distinct evening energy trajectories were identified. Participants who were younger and male and who had more comorbidities, decreased body weight, and a lower functional status were more likely to be in the moderate evening energy class. Five different polymorphisms (IL1R2 rs4141134, IL6 rs4719714, IL17A rs8193036, NFKB2 rs1056890, and TNFA rs1800683) were associated with evening energy latent class membership. This study provides preliminary evidence that decrements in morning and evening energy are associated with different phenotypic risk factors and cytokine gene variations. Copyright © 2015 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.

  12. Genetic Variability of the High-affinity IgE Receptor α Subunit (Fc ε RI α is Related to Total Serum IgE levels in Allergic Subjects

    Directory of Open Access Journals (Sweden)

    Marek Sanak

    2007-01-01

    Full Text Available Known susceptibility genes to atopy and asthma have been identified by linkage or associations with clinical phenotypes, including total serum IgE levels. IgE-mediated sensitivity reactions require a high-affinity IgE receptor (FcεRI, which immobilizes the immunoglobulin on the surface of the effector cells, mostly mast cells and basophils. In this mini-review, recent findings are presented on genetic variation of this receptor, as related to atopy. Transcription of FCER1A gene encoding the receptor α subunit can be initiated from two separate promoters, the proximal one and the distal one, which results in a transcript containing two novel untranslated exons (1A, 2A. Our knowledge on the role of this mechanism in allergic diseases is still at an infancy stage. Within regulatory elements of FCER1A some common single nucleotide polymorphisms have functional associations, which were recently reported and replicated in different ethnical groups. Interestingly, these associations do not confer susceptibility to allergic diseases, but rather modulate serum concentrations of IgE. Similarly to the previously investigated β subunit of the receptor, FCER1A is a good candidate for a quantitative trait locus (QTL in allergic diseases, and appears to participate in the systemic regulation of IgE levels.

  13. Efficient mRNA-based Genetic Engineering of Human NK Cells with High-Affinity CD16 and CCR7 Augments Rituximab-induced ADCC against Lymphoma and Targets NK cell Migration Towards the Lymph Node Associated Chemokine CCL19

    Directory of Open Access Journals (Sweden)

    Mattias eCarlsten

    2016-03-01

    Full Text Available For more than a decade, investigators have pursued methods to genetically engineer NK cells for use in clinical therapy against cancer. Despite considerable advances in viral transduction of hematopoietic stem cells and T cells, transduction efficiencies for NK cells have remained disappointingly low. Here we show that NK cells can be efficiently genetically reprogrammed using a cGMP-compliant mRNA electroporation method that induces rapid and reproducible transgene expression in nearly all transfected cells, without negatively influencing their viability, phenotype and cytotoxic function. To study its potential therapeutic application, we used this approach to improve key aspects involved in efficient lymphoma targeting by adoptively infused ex vivo expanded NK cells. Electroporation of NK cells with mRNA coding for the chemokine receptor CCR7 significantly promoted migration towards the lymph node-associated chemokine CCL19. Further, introduction of mRNA coding for the high-affinity antibody-binding receptor CD16 (CD16-158V substantially augmented NK cell cytotoxicity against rituximab-coated lymphoma cells. Based on these data, we conclude this approach can be utilized to genetically modify multiple modalities of NK cells in a highly efficient manner with the potential to improve multiple facets of their in vivo tumor targeting, opening a new arena for the development of more efficacious adoptive NK cell-based cancer immunotherapies.

  14. Evolution and ecology meet molecular genetics: adaptive phenotypic plasticity in two isolated Negev desert populations of Acacia raddiana at either end of a rainfall gradient.

    Science.gov (United States)

    Ward, David; Shrestha, Madan K; Golan-Goldhirsh, Avi

    2012-01-01

    The ecological, evolutionary and genetic bases of population differentiation in a variable environment are often related to the selection pressures that plants experience. We compared differences in several growth- and defence-related traits in two isolated populations of Acacia raddiana trees from sites at either end of an extreme environmental gradient in the Negev desert. We used random amplified polymorphic DNA (RAPD) to determine the molecular differences between populations. We grew plants under two levels of water, three levels of nutrients and three levels of herbivory to test for phenotypic plasticity and adaptive phenotypic plasticity. The RAPD analyses showed that these populations are highly genetically differentiated. Phenotypic plasticity in various morphological traits in A. raddiana was related to patterns of population genetic differentiation between the two study sites. Although we did not test for maternal effects in these long-lived trees, significant genotype × environment (G × E) interactions in some of these traits indicated that such plasticity may be adaptive. The main selection pressure in this desert environment, perhaps unsurprisingly, is water. Increased water availability resulted in greater growth in the southern population, which normally receives far less rain than the northern population. Even under the conditions that we defined as low water and/or nutrients, the performance of the seedlings from the southern population was significantly better, perhaps reflecting selection for these traits. Consistent with previous studies of this genus, there was no evidence of trade-offs between physical and chemical defences and plant growth parameters in this study. Rather, there appeared to be positive correlations between plant size and defence parameters. The great variation in several traits in both populations may result in a diverse potential for responding to selection pressures in different environments.

  15. Evolution and ecology meet molecular genetics: adaptive phenotypic plasticity in two isolated Negev desert populations of Acacia raddiana at either end of a rainfall gradient

    Science.gov (United States)

    Ward, David; Shrestha, Madan K.; Golan-Goldhirsh, Avi

    2012-01-01

    Background and Aims The ecological, evolutionary and genetic bases of population differentiation in a variable environment are often related to the selection pressures that plants experience. We compared differences in several growth- and defence-related traits in two isolated populations of Acacia raddiana trees from sites at either end of an extreme environmental gradient in the Negev desert. Methods We used random amplified polymorphic DNA (RAPD) to determine the molecular differences between populations. We grew plants under two levels of water, three levels of nutrients and three levels of herbivory to test for phenotypic plasticity and adaptive phenotypic plasticity. Key Results The RAPD analyses showed that these populations are highly genetically differentiated. Phenotypic plasticity in various morphological traits in A. raddiana was related to patterns of population genetic differentiation between the two study sites. Although we did not test for maternal effects in these long-lived trees, significant genotype × environment (G × E) interactions in some of these traits indicated that such plasticity may be adaptive. Conclusions The main selection pressure in this desert environment, perhaps unsurprisingly, is water. Increased water availability resulted in greater growth in the southern population, which normally receives far less rain than the northern population. Even under the conditions that we defined as low water and/or nutrients, the performance of the seedlings from the southern population was significantly better, perhaps reflecting selection for these traits. Consistent with previous studies of this genus, there was no evidence of trade-offs between physical and chemical defences and plant growth parameters in this study. Rather, there appeared to be positive correlations between plant size and defence parameters. The great variation in several traits in both populations may result in a diverse potential for responding to selection pressures in

  16. Development of a molecularly imprinted polymer based surface plasmon resonance sensor for theophylline monitoring

    Science.gov (United States)

    Zheng, Rui; Cameron, Brent D.

    2011-03-01

    Molecularly imprinted polymer (MIP) thin films and surface plasmon resonance (SPR) sensing technologies were combined to develop a novel sensing platform for monitoring real-time theophylline concentration, which is a compound of interest in environmental monitoring and a molecular probe for phenotyping certain cytochrome P450 enzymes. The MIPs hydrogel is easy to synthesize and provides shape-selective recognition with high affinity to specific target molecules. Different polymerization formulas were tested and optimized. The influence of the monomer sensitive factors were addressed by SPR. SPR is an evanescent wave optics based sensing technique that is suitable for real-time and label free sensing purposes. Gold nanorods (Au NRs) were uniformly immobilized onto a SPR sensing surface for the construction of a fiber optics based prism-free localized SPR (LSPR) measurement. This technique can be also applied to assess the activities of other small organic molecules by adjusting the polymerization formula, thus, this approach also has many other potential applications.

  17. Sustained productivity in recombinant Chinese Hamster Ovary (CHO cell lines: proteome analysis of the molecular basis for a process-related phenotype

    Directory of Open Access Journals (Sweden)

    Gammell Patrick

    2011-07-01

    Full Text Available Abstract Background The ability of mammalian cell lines to sustain cell specific productivity (Qp over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis and LC-MS/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3 that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

  18. Phenotypic and molecular characteristics of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in slaughterhouse pig-related workers and control workers in Guangdong Province, China.

    Science.gov (United States)

    Wang, X L; Li, L; Li, S M; Huang, J Y; Fan, Y P; Yao, Z J; Ye, X H; Chen, S D

    2017-07-01

    Pig farmers and veterinarians have high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) due to the occupational livestock exposure, while few reported this association on slaughterhouse workers. We conducted this cross-sectional study to explore the phenotypic and molecular characteristics of S. aureus and MRSA in slaughterhouse pig-related workers and control workers in Guangdong Province, China. Participants were interviewed and provided two nasal swabs. Swabs were tested for S. aureus, and isolates were further tested for antimicrobial susceptibility, virulence genes and multi-locus sequence typing. Compared with control workers, pig-related workers have significantly higher prevalence of MRSA carriage (adjusted odd ratio (aOR) 3·70, 95% CI 1·63-8·40). The proportions of MRSA resistant to clindamycin, erythromycin, tetracycline or chloromycetin were significantly higher in pig-related workers than in control workers. The predominant phenotypes of S. aureus were resistant to penicillin, clindamycin, erythromycin and tetracycline. Three MRSA CC9 isolates with livestock-associated characteristics (resistance to tetracycline and absence of immune evasion cluster (IEC) genes) were detected in pig-related workers but not in control workers. For human-associated CCs (CC7, CC59, CC6, and CC188), there was no significant difference in IEC profile or antimicrobial resistance between the groups. These findings reveal that there may be a potential risk for livestock-to-human transmission of LA-MRSA and human-to-human transmission of human-associated MRSA.

  19. Sustained productivity in recombinant Chinese Hamster Ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype

    LENUS (Irish Health Repository)

    Meleady, Paula

    2011-07-24

    Abstract Background The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS\\/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

  20. Cryptic species, phenotypic plasticity, and complex life histories: Assessing deep-sea faunal diversity with molecular markers

    Science.gov (United States)

    Vrijenhoek, Robert C.

    2009-09-01

    Many new species of animals have been discovered during the past 40 years of deep-ocean exploration, particularly in chemosynthetic habitats such as hydrothermal vents and cold-water hydrocarbon seeps. Estimating species diversity in these environments is difficult, however, because insufficient sampling often fails to capture the range of organismic variability in time and space. Molecular systematic studies have revealed a number of taxonomic problems that derive from insufficient sampling and a shallow knowledge base regarding many deep-sea taxa. For example, numerous morphologically cryptic species exist among the vesicomyid clams and lepetodrilid limpets that dominate vents and seeps worldwide, suggesting that species richness may be significantly underestimated in these taxa. In contrast, discrete morphotypes of siboglinid tubeworms that are products of developmental plasticity were assigned synonymous species names. Also, distinct juvenile and adult forms of vent shrimp were assigned synonymous genus and species names. Though molecular studies have resolved many of these problems, they are not a panacea because they also suffer from insufficient sampling of taxa and genes, and from contamination of DNA sequences. Working carefully together, molecular and traditional systematists should eventually generate more accurate species lists that allow unbiased estimates of species richness in deep-sea environments.

  1. Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture

    Directory of Open Access Journals (Sweden)

    Satish Latha

    2012-05-01

    Full Text Available Abstract Background Dupuytren's contracture (DC is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC. Methods To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group. These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments. Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02, hierarchical clustering, concordance mapping and Venn diagram. Results We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that

  2. Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture

    Science.gov (United States)

    2012-01-01

    Background Dupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC. Methods To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram. Results We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen

  3. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    DEFF Research Database (Denmark)

    Rannversson, Hafsteinn; Andersen, Jacob; Hall, Lena Sørensen

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode...

  4. Phenotypic, molecular characterization, antimicrobial susceptibility and draft genome sequence of Corynebacterium argentoratense strains isolated from clinical samples

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    I. Fernández-Natal

    2016-03-01

    Full Text Available During a 12-year period we isolated five Corynebacterium argentoratense strains identified by phenotypic methods, including the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF and 16S rRNA gene sequencing. In addition, antimicrobial susceptibility was determined, and genome sequencing for the detection of antibiotic resistance genes was performed. The organisms were isolated from blood and throat cultures and could be identified by all methods used. All strains were resistant to cotrimoxazole, and resistance to β-lactams was partly present. Two strains were resistant to erythromycin and clindamycin. The draft genome sequences of theses isolates revealed the presence of the erm(X resistance gene that is embedded in the genetic structure of the transposable element Tn5423. Although rarely reported as a human pathogen, C. argentoratense can be involved in bacteraemia and probably in other infections. Our results also show that horizontal transfer of genes responsible for antibiotic resistance is occurring in this species.

  5. Genotype-phenotype correlations and expansion of the molecular spectrum of AP4M1-related hereditary spastic paraplegia

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    Conceição Bettencourt

    2017-11-01

    Full Text Available Abstract Background Autosomal recessive hereditary spastic paraplegia (HSP due to AP4M1 mutations is a very rare neurodevelopmental disorder reported for only a few patients. Methods We investigated a Greek HSP family using whole exome sequencing (WES. Results A novel AP4M1A frameshift insertion, and a very rare missense variant were identified in all three affected siblings in the compound heterozygous state (p.V174fs and p.C319R; the unaffected parents were carriers of only one variant. Patients were affected with a combination of: (a febrile seizures with onset in the first year of life (followed by epileptic non-febrile seizures; (b distinctive facial appearance (e.g., coarse features, bulbous nose and hypomimia; (c developmental delay and intellectual disability; (d early-onset spastic weakness of the lower limbs; and (e cerebellar hypoplasia/atrophy on brain MRI. Conclusions We review genotype-phenotype correlations and discuss clinical overlaps between different AP4-related diseases. The AP4M1 belongs to a complex that mediates vesicle trafficking of glutamate receptors, being likely involved in brain development and neurotransmission.

  6. A mutation in the {beta}-myosin rod associated with hypertrophic cardiomyopathy has an unexpected molecular phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Armel, Thomas Z. [Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 (United States); Leinwand, Leslie A., E-mail: leslie.leinwand@colorado.edu [Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 (United States)

    2010-01-01

    Hypertrophic cardiomyopathy (HCM) is a common, autosomal dominant disorder primarily characterized by left ventricular hypertrophy and is the leading cause of sudden cardiac death in youth. HCM is caused by mutations in several sarcomeric proteins, with mutations in MYH7, encoding {beta}-MyHC, being the most common. While many mutations in the globular head region of the protein have been reported and studied, analysis of HCM-causing mutations in the {beta}-MyHC rod domain has not yet been reported. To address this question, we performed an array of biochemical and biophysical assays to determine how the HCM-causing E1356K mutation affects the structure, stability, and function of the {beta}-MyHC rod. Surprisingly, the E1356K mutation appears to thermodynamically destabilize the protein, rather than alter the charge profile know to be essential for muscle filament assembly. This thermodynamic instability appears to be responsible for the decreased ability of the protein to form filaments and may be responsible for the HCM phenotype seen in patients.

  7. The autoimmune risk gene ZMIZ1 is a vitamin D responsive marker of a molecular phenotype of multiple sclerosis.

    Science.gov (United States)

    Fewings, N L; Gatt, P N; McKay, F C; Parnell, G P; Schibeci, S D; Edwards, J; Basuki, M A; Goldinger, A; Fabis-Pedrini, M J; Kermode, A G; Manrique, C P; McCauley, J L; Nickles, D; Baranzini, S E; Burke, T; Vucic, S; Stewart, G J; Booth, D R

    2017-03-01

    Multiple Sclerosis (MS) is a neurological condition driven in part by immune cells from the peripheral circulation, the targets for current successful therapies. The autoimmune and MS risk gene ZMIZ1 is underexpressed in blood in people with MS. We show that, from three independent sets of transcriptomic data, expression of ZMIZ1 is tightly correlated with that of hundreds of other genes. Further we show expression is partially heritable (heritability 0.26), relatively stable over time, predominantly in plasmacytoid dendritic cells and non-classical monocytes, and that levels of ZMIZ1 protein expression are reduced in MS. ZMIZ1 gene expression is increased in response to calcipotriol (1,25 Vitamin D3) (p < 0.0003) and associated with Epstein Barr Virus (EBV) EBNA-1 antibody titre (p < 0.004). MS therapies fingolimod and dimethyl fumarate altered blood ZMIZ1 gene expression compared to untreated MS. The phenotype indicates susceptibility to MS, and may correspond with clinical response and represent a novel clinical target. Copyright © 2016. Published by Elsevier Ltd.

  8. Sequence specific and high affinity recognition of 5′-ACGCGT-3′ by rationally designed pyrrole-imidazole H-pin polyamides: Thermodynamic and structural studies

    Science.gov (United States)

    Mackay, Hilary; Brown, Toni; Uthe, Peter B.; Westrate, Laura; Sielaff, Alan; Jones, Justin; Lajiness, James P.; Kluza, Jerome; O’Hare, Caroline; Nguyen, Binh; Davis, Zach; Bruce, Chrystal; Wilson, W. David; Hartley, John A.; Lee, Moses

    2013-01-01

    in general agreement with ΔCp values determined from changes in the solvent accessible surface areas using complexes of the H-pins bound to (5′-CCACGCGTGG)2. According to the models, the H-pins fit snugly in the minor groove and the linker comfortably holds both polyamide portions in place, with the oxygen atoms pointing into the solvent. In summary, the H-pin polyamide provides an important molecular design motif for the discovery of future generations of programmable small molecules capable of binding to target DNA sequences with high affinity and selectivity. PMID:18819814

  9. Sequence specific and high affinity recognition of 5'-ACGCGT-3' by rationally designed pyrrole-imidazole H-pin polyamides: thermodynamic and structural studies.

    Science.gov (United States)

    Mackay, Hilary; Brown, Toni; Uthe, Peter B; Westrate, Laura; Sielaff, Alan; Jones, Justin; Lajiness, James P; Kluza, Jerome; O'Hare, Caroline; Nguyen, Binh; Davis, Zach; Bruce, Chrystal; Wilson, W David; Hartley, John A; Lee, Moses

    2008-10-15

    agreement with DeltaC(p) values determined from changes in the solvent accessible surface areas using complexes of the H-pins bound to (5'-CCACGCGTGG)(2). According to the models, the H-pins fit snugly in the minor groove and the linker comfortably holds both polyamide portions in place, with the oxygen atoms pointing into the solvent. In summary, the H-pin polyamide provides an important molecular design motif for the discovery of future generations of programmable small molecules capable of binding to target DNA sequences with high affinity and selectivity.

  10. Molecular and iridescent feather reflectance data reveal recent genetic diversification and phenotypic differentiation in a cloud forest hummingbird.

    Science.gov (United States)

    Ornelas, Juan Francisco; González, Clementina; Hernández-Baños, Blanca E; García-Moreno, Jaime

    2016-02-01

    The present day distribution and spatial genetic diversity of Mesoamerican biota reflects a long history of responses to habitat change. The hummingbird Lampornis amethystinus is distributed in northern Mesoamerica, with geographically disjunct populations. Based on sampling across the species range using mitochondrial DNA (mtDNA) sequences and nuclear microsatellites jointly analysed with phenotypic and climatic data, we (1) test whether the fragmented distribution is correlated with main evolutionary lineages, (2) assess body size and plumage color differentiation of populations in geographic isolation, and (3) evaluate a set of divergence scenarios and demographic patterns of the hummingbird populations. Analysis of genetic variation revealed four main groups: blue-throated populations (Sierra Madre del Sur); two groups of amethyst-throated populations (Trans-Mexican Volcanic Belt and Sierra Madre Oriental); and populations east of the Isthmus of Tehuantepec (IT) with males showing an amethyst throat. The most basal split is estimated to have originated in the Pleistocene, 2.39-0.57 million years ago (MYA), and corresponded to groups of populations separated by the IT. However, the estimated recent divergence time between blue- and amethyst-throated populations does not correspond to the 2-MY needed to be in isolation for substantial plumage divergence, likely because structurally iridescent colors are more malleable than others. Results of species distribution modeling and Approximate Bayesian Computation analysis fit a model of lineage divergence west of the Isthmus after the Last Glacial Maximum (LGM), and that the species' suitable habitat was disjunct during past and current conditions. These results challenge the generality of the contraction/expansion glacial model to cloud forest-interior species and urges management of cloud forest, a highly vulnerable ecosystem to climate change and currently facing destruction, to prevent further loss of genetic

  11. Molecular and phenotypic responses of Japanese medaka (Oryzias latipes) early life stages to environmental concentrations of cadmium in sediment.

    Science.gov (United States)

    Barjhoux, Iris; Gonzalez, Patrice; Baudrimont, Magalie; Cachot, Jérôme

    2016-09-01

    Japanese medaka embryos were exposed to environmental concentrations of cadmium (Cd) to investigate adverse and adaptive responses in fish early life stages. Embryos were exposed during their whole development by static sediment-contact to environmental Cd concentrations (2 and 20 μg/g dry weight). Cd bioaccumulation, developmental defects, biochemical and biomolecular (qRT-PCR) responses were analyzed in embryos and hatchlings. A dose-dependent increase of Cd bioaccumulation and developmental defects was observed at hatching. Cd had clear impacts on heartbeat and cardiac morphogenesis and also induced to spinal deformities. The profile and the level of gene transcription were differentially modulated according to the Cd concentration, the duration of exposure and/or the developmental stage of fish. Pro-apoptotic bax and DNA repair rad51 transcripts were significantly repressed in embryos exposed to the highest Cd concentration. Repression of these genes was correlated to the increase of heart rate in 6-day-old embryos. NADH-dehydrogenase nd5 gene transcription was inhibited in larvae at the lowest concentration suggesting mitochondrial respiratory chain impairment, in association with Cd-induced teratogenicity. Finally, wnt1 gene was overexpressed indicating putative deregulation of Wnt signaling pathway, and suggested to be implied in the occurrence of some spinal and cardiac deformities. Results of this study permitted to propose some promising markers at the transcriptional and phenotypical level, responding to environmental concentrations of Cd. The present work also highlights the usefulness of the modified version of the medaka embryo-larval assay with sediment-contact exposure (MELAc) to investigate the toxicity and the modes of action of sediment-bound pollutants.

  12. Phenotypic and Molecular Characterisation of Extended-Spectrum Beta-Lactamase Producing Escherichia coli Obtained from Animal Fecal Samples in Ado Ekiti, Nigeria

    Directory of Open Access Journals (Sweden)

    Olugbenga Adekunle Olowe

    2015-01-01

    Full Text Available Production of extended-spectrum β-lactamases (ESBLs producing E. coli in animals and different methods of identifications from Ado Ekiti, Ekiti State, Nigeria, were investigated. Three hundred and fifty fecal samples, collected from apparently healthy cattle and pigs, were cultured and identified following standard procedures. ESBL phenotypic detection was carried out using combination disc test, double disc synergism test, and ESBL brilliance agar screening. Molecular detection of TEM, SHV, and CTX-M genes was carried out using standard molecular method. One hundred and fourteen E. coli isolates were recovered from the 350 samples processed, out of which 72 (63.2% isolates were positive for ESBLs with multiple resistance to the antibiotics used. Eighty-one (71% isolates were positive for ESBL by combination disc test, 90 (78.9% were positive for double disc synergism test, and 93 (81.6% were positive for ESBL brilliance agar. TEM and CTX-M genes were detected in 48 (42.1% and 51 (44.7% isolates, respectively. SHV gene was not detected in any of the isolates while TEM and CTX-M were detected in 33 (28.9% isolates. This study showed high resistance of E. coli to antibiotics, particularly to the third generation cephalosporins. Regular monitoring and regulated use of antibiotics in livestock should be encouraged.

  13. Phenotypic and Molecular Characterisation of Extended-Spectrum Beta-Lactamase Producing Escherichia coli Obtained from Animal Fecal Samples in Ado Ekiti, Nigeria.

    Science.gov (United States)

    Olowe, Olugbenga Adekunle; Adewumi, Olufunmilayo; Odewale, Gbolabo; Ojurongbe, Olusola; Adefioye, Olusolabomi Jose

    2015-01-01

    Production of extended-spectrum β-lactamases (ESBLs) producing E. coli in animals and different methods of identifications from Ado Ekiti, Ekiti State, Nigeria, were investigated. Three hundred and fifty fecal samples, collected from apparently healthy cattle and pigs, were cultured and identified following standard procedures. ESBL phenotypic detection was carried out using combination disc test, double disc synergism test, and ESBL brilliance agar screening. Molecular detection of TEM, SHV, and CTX-M genes was carried out using standard molecular method. One hundred and fourteen E. coli isolates were recovered from the 350 samples processed, out of which 72 (63.2%) isolates were positive for ESBLs with multiple resistance to the antibiotics used. Eighty-one (71%) isolates were positive for ESBL by combination disc test, 90 (78.9%) were positive for double disc synergism test, and 93 (81.6%) were positive for ESBL brilliance agar. TEM and CTX-M genes were detected in 48 (42.1%) and 51 (44.7%) isolates, respectively. SHV gene was not detected in any of the isolates while TEM and CTX-M were detected in 33 (28.9%) isolates. This study showed high resistance of E. coli to antibiotics, particularly to the third generation cephalosporins. Regular monitoring and regulated use of antibiotics in livestock should be encouraged.

  14. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

    Directory of Open Access Journals (Sweden)

    Rui Liu

    2016-06-01

    Full Text Available Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples.

  15. Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs

    Science.gov (United States)

    Munday, Jane C.; Eze, Anthonius A.; Baker, Nicola; Glover, Lucy; Clucas, Caroline; Aguinaga Andrés, David; Natto, Manal J.; Teka, Ibrahim A.; McDonald, Jennifer; Lee, Rebecca S.; Graf, Fabrice E.; Ludin, Philipp; Burchmore, Richard J. S.; Turner, C. Michael R.; Tait, Andy; MacLeod, Annette; Mäser, Pascal; Barrett, Michael P.; Horn, David; De Koning, Harry P.

    2014-01-01

    Objectives Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. Conclusions TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter. PMID:24235095

  16. The High-Affinity Phosphate Transporter GmPT5 Regulates Phosphate Transport to Nodules and Nodulation in Soybean1[W][OA

    Science.gov (United States)

    Qin, Lu; Zhao, Jing; Tian, Jiang; Chen, Liyu; Sun, Zhaoan; Guo, Yongxiang; Lu, Xing; Gu, Mian; Xu, Guohua; Liao, Hong

    2012-01-01

    Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N2 fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N2 fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on β-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro 33P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance. PMID:22740613

  17. The C-terminal extension of human telomerase reverse transcriptase is necessary for high affinity binding to telomeric DNA.

    Science.gov (United States)

    Tomlinson, Christopher G; Holien, Jessica K; Mathias, Jordan A T; Parker, Michael W; Bryan, Tracy M

    2016-01-01

    The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from short telomere syndromes, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic analyses and molecular modelling of a disease-associated mutant in the C-terminal extension of the reverse transcriptase subunit of human telomerase. The kinetic analyses revealed that the mutation substantially impacts the affinity of telomerase for telomeric DNA, but the magnitude of this impact varies for primers with different 3' ends. Molecular dynamics simulations corroborate this finding, revealing that the mutation results in greater movement of a nearby loop, impacting the DNA-RNA helix differentially with different DNA primers. Thus, the data indicate that this region is the location of one of the enzyme conformational changes responsible for the long-standing observation that off-rates of telomerase vary with telomeric 3' end sequence. Our data provide a molecular basis for a disease-associated telomerase mutation, and the first direct evidence for a role of the C-terminal extension in DNA binding affinity, a function analogous to the "thumb" domain of retroviral reverse transcriptases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. TU-CD-BRB-02: BEST IN PHYSICS (JOINT IMAGING-THERAPY): Identification of Molecular Phenotypes by Integrating Radiomics and Genomics

    Energy Technology Data Exchange (ETDEWEB)

    Grossmann, P; Velazquez, E Rios; Parmar, C; Aerts, H [Dana-Farber Cancer Institute/Harvard Medical School, Boston, MA (United States); Grove, O; Gillies, R [H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida (United States); El-Hachem, N [Institut de Recherches Cliniques de Montreal, Montreal, Quebec (Canada); Leijenaar, R [Research Institute GROW, Maastricht (Netherlands); Haibe-Kains, B [University Health Network, Toronto, Ontario (Canada); Lambin, P

    2015-06-15

    Purpose: To uncover the mechanistic connections between radiomic features, molecular pathways, and clinical outcomes, to develop radiomic based predictors of pathway activation states in individual patients, and to assess whether combining radiomic with clinical and genomic data improves prognostication. Methods: We analyzed two independent lung cancer cohorts totaling 351 patients, for whom diagnostic computed tomography (CT) scans, gene-expression profiles, and clinical outcomes were available. The tumor phenotype was characterized based on 636 radiomic features describing tumor intensity, texture, shape and size. We performed an integrative analysis by developing and independently validating association modules of coherently expressed radiomic features and molecular pathways. These modules were statistically tested for significant associations to overall survival (OS), TNM stage, and pathologic histology. Results: We identified thirteen radiomic-pathway association modules (p < 0.05), the most prominent of which were associated with the immune system, p53 pathway, and other pathways involved in cell cycle regulation. Eleven modules were significantly associated with clinical outcomes (p < 0.05). Strong predictive power for pathway activation states in individual patients was observed using radiomics; the strongest per module predictions ranged from an intra-tumor heterogeneity feature predicting RNA III polymerase transcription (AUC 0.62, p = 0.03), to a tumor intensity dispersion feature predicting pyruvate metabolism and citric acid TCA cycle (AUC 0.72, p < 10−{sup 6}). Stepwise combinations of radiomic data with clinical outcomes and gene expression profiles resulted in consistent increases of prognostic power to predict OS (concordance index max = 0.73, p < 10−{sup 9}). Conclusion: This study demonstrates that radiomic approaches permit a non-invasive assessment of molecular and clinical characteristics of tumors, and therefore have the unprecedented

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

    Science.gov (United States)

    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.

  20. Species-scanning mutagenesis of the serotonin transporter reveals residues essential in selective, high-affinity recognition of antidepressants

    DEFF Research Database (Denmark)

    Mortensen, O V; Kristensen, A S; Wiborg, O

    2001-01-01

    of the antidepressants citalopram, fluoxetine, paroxetine and imipramine were several-fold higher at hSERT compared with bSERT. No species selectivity was observed for the antidepressants fluvoxamine, and sertraline or for the psychostimulants cocaine, the cocaine analogue beta-carbomethoxy-3beta-(4-iodophenyl......, and phenylalanine-513 and to reveal molecular interactions with individual functional groups of citalopram. We suggest that methionine-180 interacts with the heterocyclic nucleus of citalopram or stabilizes the binding pocket and phenylalanine-513 to be a steric blocker of antidepressant recognition....

  1. Phenotypic and Molecular Antibiotic Resistance Determination of Airborne Coagulase Negative Staphylococcus spp. Strains from Healthcare Facilities in Southern Poland.

    Science.gov (United States)

    Lenart-Boroń, Anna; Wolny-Koładka, Katarzyna; Stec, Joanna; Kasprowic, Andrzej

    2016-10-01

    This study assessed the antimicrobial resistance of airborne Staphylococcus spp. strains isolated from healthcare facilities in southern Poland. A total of 55 isolates, belonging to 10 coagulase-negative staphylococci (CoNS) species, isolated from 10 healthcare facilities (including hospitals and outpatient units) were included in the analysis. The most frequently identified species were Staphylococcus saprophyticus and Staphylococcus warneri, which belong to normal human skin flora, but can also be the cause of common and even severe nosocomial infections. Disk diffusion tests showed that the bacterial strains were most frequently resistant to erythromycin and tetracycline and only 18% of strains were susceptible to all tested antimicrobials. Polymerase chain reaction amplification of specific gene regions was used to determine the presence of the Macrolide-Lincosamide-Streptogramin resistance mechanisms in CoNS. The molecular analysis, conducted using specific primer pairs, identified the msrA1 gene, encoding active efflux pumps in bacterial cells, as the most frequent resistance gene. As many as seven antibiotic resistance genes were found in one isolate, whereas the most common number of resistance genes per isolate was five (n = 17). It may be concluded that drug resistance was widely spread among the tested strains, but the resulting antimicrobial resistance profile indicates that in the case of infection, the use of antibiotics from the basic antibiogram group will be effective in therapy. However, before administering treatment, determination of the specific antimicrobial resistance should be conducted, particularly in the case of hospitalized patients.

  2. High-Affinity Manganese Uptake by the Metal Transporter NRAMP1 Is Essential for Arabidopsis Growth in Low Manganese Conditions[C][W

    Science.gov (United States)

    Cailliatte, Rémy; Schikora, Adam; Briat, Jean-François; Mari, Stéphane; Curie, Catherine

    2010-01-01

    In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis. PMID:20228245

  3. High-affinity cooperative Ca2+binding by MICU1-MICU2 serves as an on-off switch for the uniporter.

    Science.gov (United States)

    Kamer, Kimberli J; Grabarek, Zenon; Mootha, Vamsi K

    2017-08-01

    The mitochondrial calcium uniporter is a Ca 2+ -activated Ca 2+ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca 2+ signals. It is regulated by two paralogous EF-hand proteins-MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca 2+ We reconstitute the MICU1-MICU2 heterodimer and demonstrate that it binds Ca 2+ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria-specific lipid cardiolipin. We determine the minimum Ca 2+ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca 2+ -binding affinity of MICU1-MICU2. We conclude that cooperative, high-affinity interaction of the MICU1-MICU2 complex with Ca 2+ serves as an on-off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca 2+ signals. © 2017 The Authors.

  4. In vivo effector functions of high-affinity mouse IgG receptor FcγRI in disease and therapy models.

    Science.gov (United States)

    Gillis, Caitlin M; Zenatti, Priscila P; Mancardi, David A; Beutier, Héloïse; Fiette, Laurence; Macdonald, Lynn E; Murphy, Andrew J; Celli, Susanna; Bousso, Philippe; Jönsson, Friederike; Bruhns, Pierre

    2017-06-01

    Two activating mouse IgG receptors (FcγRs) have the ability to bind monomeric IgG, the high-affinity mouse FcγRI and FcγRIV. Despite high circulating levels of IgG, reports using FcγRI(-/-) or FcγRIV(-/-) mice or FcγRIV-blocking antibodies implicate these receptors in IgG-induced disease severity or therapeutic Ab efficacy. From these studies, however, one cannot conclude on the effector capabilities of a given receptor, because different activating FcγRs possess redundant properties in vivo, and cooperation between FcγRs may occur, or priming phenomena. To help resolve these uncertainties, we used mice expressing only FcγRI to determine its intrinsic properties in vivo. FcγRI(only) mice were sensitive to IgG-induced autoimmune thrombocytopenia and anti-CD20 and anti-tumour immunotherapy, but resistant to IgG-induced autoimmune arthritis, anaphylaxis and airway inflammation. Our results show that the in vivo roles of FcγRI are more restricted than initially reported using FcγRI(-/-) mice, but confirm effector capabilities for this high-affinity IgG receptor in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor.

    Directory of Open Access Journals (Sweden)

    Bryan L Roth

    Full Text Available In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS-2-(ethylamino-2-(3-methoxyphenylcyclohexanone and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenylcyclohexanamine and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenylcyclohexyl]piperidine and 1-[1-(4-methoxyphenylcyclohexyl]piperidine, were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.

  6. The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor.

    Science.gov (United States)

    Roth, Bryan L; Gibbons, Simon; Arunotayanun, Warunya; Huang, Xi-Ping; Setola, Vincent; Treble, Ric; Iversen, Les

    2013-01-01

    In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone) and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenyl)cyclohexanamine) and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine), were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.

  7. Molecular phenotype of zebrafish ovarian follicle by serial analysis of gene expression and proteomic profiling, and comparison with the transcriptomes of other animals

    Directory of Open Access Journals (Sweden)

    Forgue Jean

    2006-03-01

    Full Text Available Abstract Background The ability of an oocyte to develop into a viable embryo depends on the accumulation of specific maternal information and molecules, such as RNAs and proteins. A serial analysis of gene expression (SAGE was carried out in parallel with proteomic analysis on fully-grown ovarian follicles from zebrafish (Danio rerio. The data obtained were compared with ovary/follicle/egg molecular phenotypes of other animals, published or available in public sequence databases. Results Sequencing of 27,486 SAGE tags identified 11,399 different ones, including 3,329 tags with an occurrence superior to one. Fifty-eight genes were expressed at over 0.15% of the total population and represented 17.34% of the mRNA population identified. The three most expressed transcripts were a rhamnose-binding lectin, beta-actin 2, and a transcribed locus similar to the H2B histone family. Comparison with the large-scale expressed sequence tags sequencing approach revealed highly expressed transcripts that were not previously known to be expressed at high levels in fish ovaries, like the short-sized polarized metallothionein 2 transcript. A higher sensitivity for the detection of transcripts with a characterized maternal genetic contribution was also demonstrated compared to large-scale sequencing of cDNA libraries. Ferritin heavy polypeptide 1, heat shock protein 90-beta, lactate dehydrogenase B4, beta-actin isoforms, tubulin beta 2, ATP synthase subunit 9, together with 40 S ribosomal protein S27a, were common highly-expressed transcripts of vertebrate ovary/unfertilized egg. Comparison of transcriptome and proteome data revealed that transcript levels provide little predictive value with respect to the extent of protein abundance. All the proteins identified by proteomic analysis of fully-grown zebrafish follicles had at least one transcript counterpart, with two exceptions: eosinophil chemotactic cytokine and nothepsin. Conclusion This study provides a

  8. A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

    Directory of Open Access Journals (Sweden)

    Jonathan S Wall

    Full Text Available Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

  9. Morphological variability and molecular identification of Uncinaria spp. (Nematoda: Ancylostomatidae) from grizzly and black bears: new species or phenotypic plasticity?

    Science.gov (United States)

    Catalano, Stefano; Lejeune, Manigandan; van Paridon, Bradley; Pagan, Christopher A; Wasmuth, James D; Tizzani, Paolo; Duignan, Pádraig J; Nadler, Steven A

    2015-04-01

    The hookworms Uncinaria rauschi Olsen, 1968 and Uncinaria yukonensis ( Wolfgang, 1956 ) were formally described from grizzly ( Ursus arctos horribilis) and black bears ( Ursus americanus ) of North America. We analyzed the intestinal tracts of 4 grizzly and 9 black bears from Alberta and British Columbia, Canada and isolated Uncinaria specimens with anatomical traits never previously documented. We applied morphological and molecular techniques to investigate the taxonomy and phylogeny of these Uncinaria parasites. The morphological analysis supported polymorphism at the vulvar region for females of both U. rauschi and U. yukonensis. The hypothesis of morphological plasticity for U. rauschi and U. yukonensis was confirmed by genetic analysis of the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. Two distinct genotypes were identified, differing at 5 fixed sites for ITS-1 (432 base pairs [bp]) and 7 for ITS-2 (274 bp). Morphometric data for U. rauschi revealed host-related size differences: adult U. rauschi were significantly larger in black bears than in grizzly bears. Interpretation of these results, considering the historical biogeography of North American bears, suggests a relatively recent host-switching event of U. rauschi from black bears to grizzly bears which likely occurred after the end of the Wisconsin glaciation. Phylogenetic maximum parsimony (MP) and maximum likelihood (ML) analyses of the concatenated ITS-1 and ITS-2 datasets strongly supported monophyly of U. rauschi and U. yukonensis and their close relationship with Uncinaria stenocephala (Railliet, 1884), the latter a parasite primarily of canids and felids. Relationships among species within this group, although resolved by ML, were unsupported by MP and bootstrap resampling. The clade of U. rauschi, U. yukonensis, and U. stenocephala was recovered as sister to the clade represented by Uncinaria spp. from otariid pinnipeds. These results support the absence of strict

  10. Phenotypic and molecular characterization of antimicrobial resistance and virulence factors in Pseudomonas aeruginosa clinical isolates from Recife, State of Pernambuco, Brazil

    Directory of Open Access Journals (Sweden)

    Paula Regina Luna de Araújo Jácome

    2012-12-01

    Full Text Available INTRODUCTION: The emergence of carbapenem resistance mechanisms in Pseudomonas aeruginosa has been outstanding due to the wide spectrum of antimicrobial degradation of these bacteria, reducing of therapeutic options. METHODS: Sixty-one clinical strains of P. aeruginosa isolated from five public hospitals in Recife, Pernambuco, Brazil, were examined between 2006 and 2010, aiming of evaluating the profiles of virulence, resistance to antimicrobials, presence of metallo-β-lactamase (MBL genes, and clonal relationship among isolates. RESULTS: A high percentage of virulence factors (34.4% mucoid colonies; 70.5% pyocyanin; 93.4% gelatinase positives; and 72.1% hemolysin positive and a high percentage of antimicrobial resistance rates (4.9% pan-resistant and 54.1% multi-drug resistant isolates were observed. Among the 29 isolates resistant to imipenem and/or ceftazidime, 44.8% (13/29 were MBL producers by phenotypic evaluation, and of these, 46.2% (6/13 were positive for the blaSPM-1 gene. The blaIMP and blaVIM genes were not detected. The molecular typing revealed 21 molecular profiles of which seven were detected in distinct hospitals and periods. Among the six positive blaSPM-1 isolates, three presented the same clonal profile and were from the same hospital, whereas the other three presented different clonal profiles. CONCLUSIONS: These results revealed that P. aeruginosa is able to accumulate different resistance and virulence factors, making the treatment of infections difficult. The identification of blaSPM-1 genes and the dissemination of clones in different hospitals, indicate the need for stricter application of infection control measures in hospitals in Recife, Brazil, aiming at reducing costs and damages caused by P. aeruginosa infections.

  11. High-Affinity "Click" RGD Peptidomimetics as Radiolabeled Probes for Imaging αvβ3Integrin.

    Science.gov (United States)

    Piras, Monica; Testa, Andrea; Fleming, Ian N; Dall'Angelo, Sergio; Andriu, Alexandra; Menta, Sergio; Mori, Mattia; Brown, Gavin D; Forster, Duncan; Williams, Kaye J; Zanda, Matteo

    2017-07-20

    Nonpeptidic Arg-Gly-Asp (RGD)-mimic ligands were designed and synthesized by click chemistry between an arginine-azide mimic and an aspartic acid-alkyne mimic. Some of these molecules combine excellent in vitro properties (high α v β 3 affinity, selectivity, drug-like logD, high metabolic stability) with a variety of radiolabeling options (e.g., tritium and fluorine-18, plus compatibility with radio-iodination), not requiring the use of chelators or prosthetic groups. The binding mode of the resulting triazole RGD mimics to α v β 3 or α IIb β 3 receptors was investigated by molecular modeling simulations. Lead compound 12 was successfully radiofluorinated and used for in vivo positron emission tomography/computed tomography (PET/CT) studies in U87 tumor models, which showed only modest tumor uptake and retention, owing to rapid excretion. These results demonstrate that the novel click RGD mimics are excellent radiolabeled probes for in vitro and cell-based studies on α v β 3 integrin, whereas further optimization of their pharmacokinetic and dynamic profiles is necessary for successful use in in vivo imaging. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.

    Science.gov (United States)

    Ferrara, Claudia; Grau, Sandra; Jäger, Christiane; Sondermann, Peter; Brünker, Peter; Waldhauer, Inja; Hennig, Michael; Ruf, Armin; Rufer, Arne Christian; Stihle, Martine; Umaña, Pablo; Benz, Jörg

    2011-08-02

    Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.

  13. Potential relationship between phenotypic and molecular characteristics in revealing livestock-associated Staphylococcus aureus in Chinese humans without occupational livestock contact

    Directory of Open Access Journals (Sweden)

    Yanping Fan

    2016-09-01

    Full Text Available While some studies have defined Staphylococcus aureus based on its clonal complex and resistance pattern, few have explored the relations between the genetic lineages and antibiotic resistance patterns and immune evasion cluster (IEC genes. Our aim was to investigate the potential relationship between phenotypic and molecular characteristics so as to reveal livestock-associated S. aureus in humans. The study participants were interviewed, and they provided two nasal swabs for S. aureus analysis. All S. aureus and methicillin-resistant S. aureus (MRSA were tested for antibiotic susceptibility, multilocus sequence type and IEC genes. Of the 1162 participants, 9.3% carried S. aureus, including MRSA (1.4% and multidrug-resistant S. aureus (MDRSA, 2.8%. The predominant multidrug-resistant pattern among MDRSA isolates was nonsusceptibility to erythromycin, clindamycin and tetracycline. The most common S. aureus genotypes were ST7, ST6, ST188 and ST59, and the predominant MRSA genotype was ST7. Notably, the livestock-associated S. aureus isolates (IEC-negative CC9, IEC-negative tetracycline-resistant CC398, and IEC-negative tetracycline-resistant CC5 were found in people with no occupational livestock contact. These findings reveal a potential relationship between S. aureus CCs and IEC genes and antibiotic resistance patterns in defining livestock-associated S. aureus in humans and support growing concern about the potential livestock-to-human transmission of livestock-associated S. aureus by non-occupational livestock contact.

  14. Engineering of an Extremely Thermostable Alpha/Beta Barrel Scaffold to Serve as a High Affinity Molecular Recognition Element for Use in Sensor Applications

    Science.gov (United States)

    2015-12-23

    could be further developed for use in robust and low cost biosensors . (a) Papers published in peer- reviewed journals (N/A for none) Enter List of...robust and low cost biosensors . 1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND SUBTITLE 13. SUPPLEMENTARY NOTES 12. DISTRIBUTION AVAILIBILITY STATEMENT 6...public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions

  15. Rare, high-affinity mouse anti-PD-1 antibodies that function in checkpoint blockade, discovered using microfluidics and molecular genomics.

    Science.gov (United States)

    Adler, Adam S; Mizrahi, Rena A; Spindler, Matthew J; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Jackson; Leong, Renee; Johnson, David S

    Conventionally, mouse hybridomas or well-plate screening are used to identify therapeutic monoclonal antibody candidates. In this study, we present an alternative to hybridoma-based discovery that combines microfluidics, yeast single-chain variable fragment (scFv) display, and deep sequencing to rapidly interrogate and screen mouse antibody repertoires. We used our approach on six wild-type mice to identify 269 molecules that bind to programmed cell death protein 1 (PD-1), which were present at an average of 1 in 2,000 in the pre-sort scFv libraries. Two rounds of fluorescence-activated cell sorting (FACS) produced populations of PD-1-binding scFv with a mean enrichment of 800-fold, whereas most scFv present in the pre-sort mouse repertoires were de-enriched. Therefore, our work suggests that most of the antibodies present in the repertoires of immunized mice are not strong binders to PD-1. We observed clusters of related antibody sequences in each mouse following FACS, suggesting evolution of clonal lineages. In the pre-sort repertoires, these putative clonal lineages varied in both the complementary-determining region (CDR)3K and CDR3H, while the FACS-selected PD-1-binding subsets varied primarily in the CDR3H. PD-1 binders were generally not highly diverged from germline, showing 98% identity on average with germline V-genes. Some CDR3 sequences were discovered in more than one animal, even across different mouse strains, suggesting convergent evolution. We synthesized 17 of the anti-PD-1 binders as full-length monoclonal antibodies. All 17 full-length antibodies bound recombinant PD-1 with KD < 500 nM (average = 62 nM). Fifteen of the 17 full-length antibodies specifically bound surface-expressed PD-1 in a FACS assay, and nine of the antibodies functioned as checkpoint inhibitors in a cellular assay. We conclude that our method is a viable alternative to hybridomas, with key advantages in comprehensiveness and turnaround time.

  16. The P-glycoprotein (ABCB1) linker domain encodes high-affinity binding sequences to alpha- and beta-tubulins.

    Science.gov (United States)

    Georges, Elias

    2007-06-26

    P-Glycoprotein (or ABCB1) has been shown to cause multidrug resistance in tumor cell lines selected with lipophilic anticancer drugs. ABCB1 encodes a duplicated molecule with two hydrophobic and hydrophilic domains linked by a highly charged region of approximately 90 amino acids, the "linker domain" with as yet unknown function(s). In this report, we demonstrate a role for this domain in binding to other cellular proteins. Using overlapping hexapeptides that encode the entire amino acid sequence of the linker domain of human ABCB1, we show a direct and specific binding between sequences in the linker domain and several intracellular proteins. Three different polypeptide sequences [617EKGIYFKLVTM627 (LDS617-627), 657SRSSLIRKRSTRRSVRGSQA676 (LDS657-676), and 693PVSFWRIMKLNLT705 (LDS693-705)] in the linker domain interacted tightly with several proteins with apparent molecular masses of approximately 80, 57, and 30 kDa. Interestingly, only the 57 kDa protein (or P57) interacted with all three different sequences of the linker domain. Purification and partial N-terminal amino acid sequencing of P57 showed that it encodes the N-terminal amino acids of alpha- and beta-tubulins. The identity of the P57 interacting protein as tubulins was further confirmed by Western blotting using monoclonal antibodies to alpha- and beta-tubulin. Taken together, the results of this study provide the first evidence for ABCB1 protein interaction mediated by sequences in the linker domain. These findings are likely to provide further insight into the functions of ABCB1 in normal and drug resistant tumor cells.

  17. NFM Cross-Reactivity to MOG Does Not Expand a Critical Threshold Level of High-Affinity T Cells Necessary for Onset of Demyelinating Disease.

    Science.gov (United States)

    Blanchfield, Lori; Sabatino, Joseph J; Lawrence, Laurel; Evavold, Brian D

    2017-10-15

    Of interest to the etiology of demyelinating autoimmune disease is the potential to aberrantly activate CD4(+) T cells due to cross-recognition of multiple self-epitopes such as has been suggested for myelin oligodendrocyte glycoprotein epitope 35-55 (MOG35-55) and neurofilament medium protein epitope 15-35 (NFM15-35). NFM15-35 is immunogenic in C57BL/6 mice but fails to induce demyelinating disease by polyclonal T cells despite having the same TCR contact residues as MOG35-55, a known encephalitogenic Ag. Despite reported cross-reactivity with MOG-specific T cells, the polyclonal response to NFM15-35 did not expand threshold numbers of MOG38-49 tetramer-positive T cells. Furthermore, NFM lacked functional synergy with MOG to promote experimental autoimmune encephalomyelitis because NFM-deficient synonymous with knockout mice developed an identical disease course to wild-type mice after challenge with MOG35-55 Single-cell analysis of encephalitogenic T cells using the peptide:MHC monomer-based two-dimensional micropipette adhesion frequency assay confirmed that NFM was not a critical Ag driving demyelinating disease because NFM18-30-specific T cells in the CNS were predominantly reactive to MOG38-49 The absence of NFM contribution to disease allowed mapping of the amino acids required for encephalitogenicity and expansion of high-affinity, MOG-specific T cells that defined the polyclonal response. Alterations of N-terminal residues outside of the NFM15-35 core nonamer promoted expansion of high-affinity, MOG38-49 tetramer-positive T cells and promoted consistent experimental autoimmune encephalomyelitis induction, unlike mice challenged with NFM15-35 Although NFM15-35 is immunogenic and cross-reactive with MOG at the polyclonal level, it fails to expand a threshold level of encephalitogenic, high-affinity MOG-specific T cells. Copyright © 2017 by The American Association of Immunologists, Inc.

  18. Comparison of high affinity binding of {sup 3}H-proadifen and {sup 3}H-(-)-cocaine t rat liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S.B. [Astra Arcus AB, Dept. of Neuropharmacology, Soedertaelje (Sweden)

    1995-06-01

    The characteristics of the binding of {sup 3}H-proadifen to rat liver membranes were studied and compared to those of {sup 3}H-cocaine. It was found that {sup 3}H-proadifen was bound reversibly with high affinity (K{sub D}=1.8{+-}0.5 nM) and large capacity (B{sub max}=2010{+-}340 pmol/g wet tissue) to liver membranes. The corresponding values for the {sup 3}H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of {sup 3}H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 {mu}M CdCl{sub 2} in the incubation buffer it was possible to differentiate between two {sup 3}H-cocaine binding sites with K{sub d} values of 1.6 and 7.7 nM and B{sub max} values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of {sup 3}H-proadifen and {sup 3}H-cocaine inhibited the binding of {sup 3}H-proadifen (IC{sub 50}=10 nM) and proadifen that of {sup 3}H-cocaine (IC{sub 50}=1 nM). There was a high correlation coefficient (r{sub r}=0.972; P<0.01; n=12) in the Spearman rank test between the inhibitory potencies of compounds examined in both systems. Beside some potent alaproclate analogues a couple of compounds had moderately high affinity (IC{sub 50}=100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl{sub 2}, ZnCl{sub 2} and CuCl{sub 2} inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd{sup 2+} on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15{mu}M. The similarity of the characteristics of the binding of these ligands with that of {sup 3}H-alaproclate is discussed. It is suggested that all three compounds bind to the same sites, although additional binding sites seem to exist for proadifen. (au) (9 refs.).

  19. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Moran, Jeffery H. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Arkansas Department of Public Health, Public Health Laboratory, Little Rock, AR 72205 (United States); Prather, Paul L., E-mail: pratherpaull@uams.edu [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States)

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  20. A tyrosine kinase inhibitor-based high-affinity PET radiopharmaceutical targets vascular endothelial growth factor receptor.

    Science.gov (United States)

    Li, Feng; Jiang, Sheng; Zu, Youli; Lee, Daniel Y; Li, Zheng

    2014-09-01

    approved oncologic drug selective for VEGFR demonstrates excellent tumor targeting, particularly for the dimeric form. The multivalent probe yielded a 100-fold improvement in receptor affinity while maintaining pharmacokinetic and biodistribution properties well suited for PET imaging in our preclinical model. These results indicate that a clinically relevant theranostic platform can be rapidly developed from known small molecules that target key cellular receptors. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  1. Identification of High Affinity Polo-like Kinase 1 (Plk1) Polo-box Domain Binding Peptides Using Oxime-based Diversification

    Science.gov (United States)

    Liu, Fa; Park, Jung-Eun; Qian, Wen-Jian; Lim, Dan; Scharow, Andrej; Berg, Thorsten; Yaffe, Michael B.; Lee, Kyung S.; Burke, Terrence R.

    2012-01-01

    In an effort to develop improved binding antagonists of the polo-like kinase 1 (Plk1) polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide, PLHSpT using oxime-based post-solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders-of-magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein-protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues. PMID:22292814

  2. The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity

    DEFF Research Database (Denmark)

    Hansen, B E; Nielsen, Claus Henrik; Madsen, H O

    2011-01-01

    Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype...... as the HLA-DRB1*1501, where the MBP89V is preferred as the p1 anchor. Notably, full-length MBP was able to compete for peptide binding with an affinity similar to that seen for the high-affinity binding peptides, DRa170-83 and IIP53-65. In summary, the HLA-DP2 molecule binds the immunodominant epitope in MS...

  3. Bayesian estimation of genetic parameters for multivariate threshold and continuous phenotypes and molecular genetic data in simulated horse populations using Gibbs sampling

    Directory of Open Access Journals (Sweden)

    Hoeschele Ina

    2007-05-01

    Full Text Available Abstract Background Requirements for successful implementation of multivariate animal threshold models including phenotypic and genotypic information are not known yet. Here simulated horse data were used to investigate the properties of multivariate estimators of genetic parameters for categorical, continuous and molecular genetic data in the context of important radiological health traits using mixed linear-threshold animal models via Gibbs sampling. The simulated pedigree comprised 7 generations and 40000 animals per generation. Additive genetic values, residuals and fixed effects for one continuous trait and liabilities of four binary traits were simulated, resembling situations encountered in the Warmblood horse. Quantitative trait locus (QTL effects and genetic marker information were simulated for one of the liabilities. Different scenarios with respect to recombination rate between genetic markers and QTL and polymorphism information content of genetic markers were studied. For each scenario ten replicates were sampled from the simulated population, and within each replicate six different datasets differing in number and distribution of animals with trait records and availability of genetic marker information were generated. (CoVariance components were estimated using a Bayesian mixed linear-threshold animal model via Gibbs sampling. Residual variances were fixed to zero and a proper prior was used for the genetic covariance matrix. Results Effective sample sizes (ESS and biases of genetic parameters differed significantly between datasets. Bias of heritability estimates was -6% to +6% for the continuous trait, -6% to +10% for the binary traits of moderate heritability, and -21% to +25% for the binary traits of low heritability. Additive genetic correlations were mostly underestimated between the continuous trait and binary traits of low heritability, under- or overestimated between the continuous trait and binary traits of moderate

  4. Metal ion blockage of tritium incorporation into gamma-carboxyglutamic acid of prothrombin. Stoichiometry of gamma-carboxyglutamic acid to Gd3+ for the high affinity sites

    Energy Technology Data Exchange (ETDEWEB)

    Bajaj, S.P.; Saini, R.; Katz, A.; Cai, G.Z.; Maki, S.L.; Brodsky, G.L.

    1988-07-15

    Prothrombin possesses two high affinity and four low affinity gamma-carboxyglutamic acid (Gla)-dependent gadolinium binding sites. Earlier work has shown that tritium can be specifically incorporated at the gamma-carbon of Gla in proteins at pH 5. In the present work we show that inclusion of saturating concentrations of Ca2+ in nondenaturing buffer systems ranging from pH 5.5 to 8.5 prevents the exchange of tritium into all 10 Gla residues of prothrombin. Similarly, saturating concentrations of Gd3+ prevent tritium incorporation into Gla at pH 5.5. Positive cooperativity was observed for the binding of Gd3+ to human prothrombin (at pH 5.5) for the two high affinity sites (Kd congruent to 35 nM). The four low affinity sites bind Gd3+ with a Kd congruent to 5 microM. Incubation of prothrombin ranging in concentrations from 10 to 40 microM with 2 eq of Gd3+ at pH 5.5 prevents 5.7 (average of seven determinations) Gla residues from tritium incorporation. Sedimentation velocity experiments conducted at pH 5.5 indicate that prothrombin in the presence of saturating concentrations of Gd3+ polymerizes, most likely, to a trimer. Further, in the presence of 2 eq of Gd3+, calculated percent weight average concentration of monomer prothrombin is congruent to 100% at 10 microM, approximately equal to 95% at 20 microM, and congruento to 80% at 40 microM protein concentration. Thus, it appears that under conditions in which prothrombin primarily exists as a monomer, occupancy of the initial two metal binding sites by Gd3+ involves six Gla residues.

  5. Single Nucleotide Polymorphisms of the High Affinity IgG Receptor FcγRI Reduce Immune Complex Binding and Downstream Effector Functions.

    Science.gov (United States)

    Brandsma, Arianne M; Ten Broeke, Toine; van Dueren den Hollander, Evelien; Caniels, Thomas G; Kardol-Hoefnagel, Tineke; Kuball, Jürgen; Leusen, Jeanette H W

    2017-10-01

    Binding of IgG Abs to FcγRs on immune cells induces FcγR cross-linking that leads to cellular effector functions, such as phagocytosis, Ab-dependent cellular cytotoxicity, and cytokine release. However, polymorphisms in low affinity FcγRs have been associated with altered avidity toward IgG, thereby substantially impacting clinical outcomes of multimodular therapy when targeting cancer or autoimmune diseases with mAbs as well as the frequency and severity of autoimmune diseases. In this context, we investigated the consequences of three nonsynonymous single nucleotide polymorphisms (SNPs) for the high affinity receptor for IgG, FcγRI. Only SNP V39I, located in the extracellular domain of FcγRI, reduces immune-complex binding of FcγRI whereas monomeric IgG binding is unaffected. This leads to reduced FcγRI effector functions, including Fc receptor γ-chain signaling and intracellular calcium mobilization. SNPs I301M and I338T, located in the transmembrane or intracellular domain, respectively, have no influence on monomeric IgG or immune complex binding, but FcRγ signaling is decreased for both SNPs, especially for I338T. We also found that the frequency of these SNPs in a cohort of healthy Dutch individuals is very low within the population. To our knowledge, this study addresses for the first time the biological consequences of SNPs in the high affinity FcγR, and reveals reduction in several FcγRI functions, which have the potential to alter efficacy of therapeutic Abs. Copyright © 2017 by The American Association of Immunologists, Inc.

  6. Isolation of a high affinity Bet v 1-specific IgG-derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments.

    Science.gov (United States)

    Gadermaier, Elisabeth; Marth, Katharina; Lupinek, Christian; Campana, Raffaela; Hofer, Gerhard; Blatt, Katharina; Smiljkovic, Dubravka; Roder, Uwe; Focke-Tejkl, Margarete; Vrtala, Susanne; Keller, Walter; Valent, Peter; Valenta, Rudolf; Flicker, Sabine

    2018-01-09

    Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild type allergens. However, so far no treatment-induced IgG antibodies have been characterized. To clone, express and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a non-allergic subject. A phage-displayed combinatorial single chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1 and partially suppressed allergen-induced basophil activation. Immunization with unfolded hypoallergenic allergen derivatives induces high affinity antibodies even in non-allergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. “Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis*

    Science.gov (United States)

    Ho, Chia Chi M.; Guo, Nan; Sockolosky, Jonathan T.; Ring, Aaron M.; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L.; Garcia, K. Christopher

    2015-01-01

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don't-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy

  8. Blockade of the high-affinity noradrenaline transporter (NET) by the selective 5-HT reuptake inhibitor escitalopram: an in vivo microdialysis study in mice

    Science.gov (United States)

    Nguyen, Hai T; Guiard, Bruno P; Bacq, Alexandre; David, Denis J; David, Indira; Quesseveur, Gaël; Gautron, Sophie; Sanchez, Connie; Gardier, Alain M

    2013-01-01

    BACKGROUND AND PURPOSE Escitalopram, the S(+)-enantiomer of citalopram is the most selective 5-HT reuptake inhibitor approved. Although all 5-HT selective reuptake inhibitors (SSRIs) increase extracellular levels of 5-HT ([5-HT]ext). some also enhance, to a lesser extent, extracellular levels of noradrenaline ([NA]ext). However, the mechanisms by which SSRIs activate noradrenergic transmission in the brain remain to be determined. EXPERIMENTAL APPROACH This study examined the effects of escitalopram, on both [5-HT]ext and [NA]ext in the frontal cortex (FCx) of freely moving wild-type (WT) and mutant mice lacking the 5-HT transporter (SERT−/−) by using intracerebral microdialysis. We explored the possibilities that escitalopram enhances [NA]ext, either by a direct mechanism involving the inhibition of the low- or high-affinity noradrenaline transporters, or by an indirect mechanism promoted by [5-HT]ext elevation. The forced swim test (FST) was used to investigate whether enhancing cortical [5-HT]ext and/or [NA]ext affected the antidepressant-like activity of escitalopram. KEY RESULTS In WT mice, a single systemic administration of escitalopram produced a significant increase in cortical [5-HT]ext and [NA]ext. As expected, escitalopram failed to increase cortical [5-HT]ext in SERT−/− mice, whereas its neurochemical effects on [NA]ext persisted in these mutants. In WT mice subjected to the FST, escitalopram increased swimming parameters without affecting climbing behaviour. Finally, escitalopram, at relevant concentrations, failed to inhibit cortical noradrenaline and 5-HT uptake mediated by low-affinity monoamine transporters. CONCLUSIONS AND IMPLICATIONS These experiments suggest that escitalopram enhances, although moderately, cortical [NA]extin vivo by a direct mechanism involving the inhibition of the high-affinity noradrenaline transporter (NET). PMID:22233336

  9. Agrobacterium tumefaciens Zur Regulates the High-Affinity Zinc Uptake System TroCBA and the Putative Metal Chaperone YciC, along with ZinT and ZnuABC, for Survival under Zinc-Limiting Conditions.

    Science.gov (United States)

    Chaoprasid, Paweena; Dokpikul, Thanittra; Johnrod, Jaruwan; Sirirakphaisarn, Sirin; Nookabkaew, Sumontha; Sukchawalit, Rojana; Mongkolsuk, Skorn

    2016-06-15

    the high-affinity zinc importer TroCBA. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. Integrated genomic analysis of triple-negative breast cancers reveals novel microRNAs associated with clinical and molecular phenotypes and sheds light on the pathways they control.

    Science.gov (United States)

    de Rinaldis, Emanuele; Gazinska, Patrycja; Mera, Anca; Modrusan, Zora; Fedorowicz, Grazyna M; Burford, Brian; Gillett, Cheryl; Marra, Pierfrancesco; Grigoriadis, Anita; Dornan, David; Holmberg, Lars; Pinder, Sarah; Tutt, Andrew

    2013-09-23

    This study focuses on the analysis of miRNAs expression data in a cohort of 181 well characterised breast cancer samples composed primarily of triple-negative (ER/PR/HER2-negative) tumours with associated genome-wide DNA and mRNA data, extensive patient follow-up and pathological information. We identified 7 miRNAs associated with prognosis in the triple-negative tumours and an additional 7 when the analysis was extended to the set of all ER-negative cases. miRNAs linked to an unfavourable prognosis were associated with a broad spectrum of motility mechanisms involved in the invasion of stromal tissues, such as cell-adhesion, growth factor-mediated signalling pathways, interaction with the extracellular matrix and cytoskeleton remodelling. When we compared different intrinsic molecular subtypes we found 46 miRNAs that were specifically expressed in one or more intrinsic subtypes. Integrated genomic analyses indicated these miRNAs to be influenced by DNA genomic aberrations and to have an overall influence on the expression levels of their predicted targets. Among others, our analyses highlighted the role of miR-17-92 and miR-106b-25, two polycistronic miRNA clusters with known oncogenic functions. We showed that their basal-like subtype specific up-regulation is influenced by increased DNA copy number and contributes to the transcriptional phenotype as well as the activation of oncogenic pathways in basal-like tumours. This study analyses previously unreported miRNA, mRNA and DNA data and integrates these with pathological and clinical information, from a well-annotated cohort of breast cancers enriched for triple-negative subtypes. It provides a conceptual framework, as well as integrative methods and system-level results and contributes to elucidate the role of miRNAs as biomarkers and modulators of oncogenic processes in these types of tumours.

  11. Charged hydrogels for post-loading, release, and molecular imprinting of proteins

    NARCIS (Netherlands)

    Schillemans, J.P.|info:eu-repo/dai/nl/304835137

    2010-01-01

    Molecular imprinting is a technique to create template-shaped cavities in polymer matrices with memory of the template molecules, to be used in molecular recognition. Molecular imprinting of low molecular weight compounds is a well established technique used to create high affinity materials. On the

  12. Pharmacokinetics, pharmacodynamics and safety of QGE031 (ligelizumab), a novel high-affinity anti-IgE antibody, in atopic subjects

    Science.gov (United States)

    Arm, J P; Bottoli, I; Skerjanec, A; Floch, D; Groenewegen, A; Maahs, S; Owen, C E; Jones, I; Lowe, P J

    2014-01-01

    Background Using a monoclonal antibody with greater affinity for IgE than omalizumab, we examined whether more complete suppression of IgE provided greater pharmacodynamic effects, including suppression of skin prick responses to allergen. Objective To explore the pharmacokinetics, pharmacodynamics and safety of QGE031 (ligelizumab), a novel high-affinity humanized monoclonal IgG1κ anti-IgE. Methods Preclinical assessments and two randomized, placebo-controlled, double-blind clinical trials were conducted in atopic subjects. The first trial administered single doses of QGE031 (0.1–10 mg/kg) or placebo intravenously, while the second trial administered two to four doses of QGE031 (0.2– 4 mg/kg) or placebo subcutaneously at 2-week intervals. Both trials included an open-label omalizumab arm. Results Sixty of 73 (82%) and 96 of 110 (87%) subjects completed the intravenous and subcutaneous studies, respectively. Exposure to QGE031 and its half-life depended on the QGE031 dose and serum IgE level. QGE031 had a biexponential pharmacokinetic profile after intravenous administration and a terminal half-life of approximately 20 days. QGE031 demonstrated dose- and time-dependent suppression of free IgE, basophil FcεRI and basophil surface IgE superior in extent (free IgE and surface IgE) and duration to omalizumab. At Day 85, 6 weeks after the last dose, skin prick wheal responses to allergen were suppressed by > 95% and 41% in subjects treated subcutaneously with QGE031 (2 mg/kg) or omalizumab, respectively (P < 0.001). Urticaria was observed in QGE031- and placebo-treated subjects and was accompanied by systemic symptoms in one subject treated with 10 mg/kg intravenous QGE031. There were no serious adverse events. Conclusion and Clinical Relevance These first clinical data for QGE031, a high-affinity IgG1κ anti-IgE, demonstrate that increased suppression of free IgE compared with omalizumab translated to superior pharmacodynamic effects in atopic subjects

  13. Design and Investigation of a [18F]-Labeled Benzamide Derivative as a High Affinity Dual Sigma Receptor Subtype Radioligand for Prostate Tumor Imaging.

    Science.gov (United States)

    Yang, Dongzhi; Comeau, Anthony; Bowen, Wayne D; Mach, Robert H; Ross, Brian D; Hong, Hao; Van Dort, Marcian E

    2017-03-06

    High overexpression of sigma (σ) receptors (σ1 and σ2 subtypes) in a variety of human solid tumors has prompted the development of σ receptor-targeting radioligands, as imaging agents for tumor detection. A majority of these radioligands to date target the σ2 receptor, a potential marker of tumor proliferative status. The identification of approximately equal proportions of both σ receptor subtypes in prostate tumors suggests that a high affinity, dual σ receptor-targeting radioligand could potentially provide enhanced tumor targeting efficacy in prostate cancer. To accomplish this goal, we designed a series of ligands which bind to both σ receptor subtypes with high affinity. Ligand 3a in this series, displaying optimal dual σ receptor subtype affinity (σ1, 6.3 nM; σ2, 10.2 nM) was radiolabeled with fluorine-18 (18F) to give [18F]3a and evaluated as a σ receptor-targeting radioligand in the mouse PC-3 prostate tumor model. Cellular assays with PC-3 cells demonstrated that a major proportion of [18F]3a was localized to cell surface σ receptors, while ∼10% of [18F]3a was internalized within cells after incubation for 3.5 h. Serial PET imaging in mice bearing PC-3 tumors revealed that uptake of [18F]3a was 1.6 ± 0.8, 4.4 ± 0.3, and 3.6 ± 0.6% ID/g (% injection dose per gram) in σ receptor-positive prostate tumors at 15 min, 1.5 h, and 3.5 h postinjection, respectively (n = 3) resulting in clear tumor visualization. Blocking studies conducted with haloperidol (a nonselective inhibitor for both σ receptor subtypes) confirmed that the uptake of [18F]3a was σ receptor-mediated. Histology analysis confirmed similar expression of σ1 and σ2 in PC-3 tumors which was significantly greater than its expression in normal organs/tissues such as liver, kidney, and muscle. Metabolite studies revealed that >50% of radioactivity in PC-3 tumors at 30 min postinjection represented intact [18F]3a. Prominent σ receptor-specific uptake of [18F]3a in prostate tumors

  14. Genome data mining and soil survey for the novel group 5 [NiFe]-hydrogenase to explore the diversity and ecological importance of presumptive high-affinity H(2)-oxidizing bacteria.

    Science.gov (United States)

    Constant, Philippe; Chowdhury, Soumitra Paul; Hesse, Laura; Pratscher, Jennifer; Conrad, Ralf

    2011-09-01

    Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H(2) possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H(2)-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H(2) oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 10(6) to 10(8) hhyL gene copies g (dry weight)(-1). Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H(2)-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H(2) oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H(2) by soil, because high-affinity H(2) oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur.

  15. Molecular initiating events of the intersex phenotype: Low-dose exposure to 17α-ethinylestradiol rapidly regulates molecular networks associated with gonad differentiation in the adult fathead minnow testis

    Energy Technology Data Exchange (ETDEWEB)

    Feswick, April; Loughery, Jennifer R.; Isaacs, Meghan A.; Munkittrick, Kelly R.; Martyniuk, Christopher J., E-mail: cmartyni@yahoo.ca

    2016-12-15

    testis, despite the fact that dmrt1 itself was not different in expression from control males. Transcriptional networks involving forkhead box L2 (foxl2) (transcript involved in ovarian follicle development) were increased in expression in the testis. Noteworthy was that a gene network associated to granulosa cell development was increased over 100%, suggesting that this transcriptome network may be important for monitoring estrogenic exposures. Other cell processes rapidly downregulated by EE2 at the transcript level included glucose homeostasis, response to heavy metal, amino acid catabolism, and the cyclooxygenase pathway. Conversely, lymphocyte chemotaxis, intermediate filament polymerization, glucocorticoid metabolism, carbohydrate utilization, and anterior/posterior axis specification were increased. These data provide new insight into the transcriptional responses that are perturbed prior to gonadal remodeling and intersex following exposure to estrogens. These data demonstrate that low concentrations of EE2 (1) rapidly suppresses male hormone production, (2) down-regulate molecular networks related to male sex differentiation, and (3) induce transcriptional networks related to granulosa cell development in the adult testis. These responses are hypothesized to be key molecular initiating events that occur prior to the development of the intersex phenotype following estrogenic exposures.

  16. GABAA/Benzodiazepine receptor binding in patients with schizophrenia using [11C]Ro15-4513, a radioligand with relatively high affinity for alpha5 subunit.

    Science.gov (United States)

    Asai, Yoshiyuki; Takano, Akihiro; Ito, Hiroshi; Okubo, Yoshiro; Matsuura, Masato; Otsuka, Akihiko; Takahashi, Hidehiko; Ando, Tomomichi; Ito, Shigeo; Arakawa, Ryosuke; Asai, Kunihiko; Suhara, Tetsuya

    2008-02-01

    Dysfunction of the GABA system is considered to play a role in the pathology of schizophrenia. Individual subunits of GABA(A)/Benzodiazepine (BZ) receptor complex have been revealed to have different functional properties. alpha5 subunit was reported to be related to learning and memory. Changes of alpha5 subunit in schizophrenia were reported in postmortem studies, but the results were inconsistent. In this study, we examined GABA(A)/BZ receptor using [(11)C]Ro15-4513, which has relatively high affinity for alpha5 subunit, and its relation to clinical symptoms in patients with schizophrenia. [(11)C]Ro15-4513 bindings of 11 patients with schizophrenia (6 drug-naïve and 5 drug-free) were compared with those of 12 age-matched healthy control subjects using positron emission tomography. Symptoms were assessed using the Positive and Negative Syndrome Scale. [(11)C]Ro15-4513 binding was quantified by binding potential (BP) obtained by the reference tissue model. [(11)C]Ro15-4513 binding in the prefrontal cortex and hippocampus was negatively correlated with negative symptom scores in patients with schizophrenia, although there was no significant difference in BP between patients and controls. GABA(A)/BZ receptor including alpha5 subunit in the prefrontal cortex and hippocampus might be involved in the pathophysiology of negative symptoms of schizophrenia.

  17. Binding site number variation and high-affinity binding consensus of Myb-SANT-like transcription factor Adf-1 in Drosophilidae.

    Science.gov (United States)

    Lang, Michael; Juan, Elvira

    2010-10-01

    There is a growing interest in the evolution of transcription factor binding sites and corresponding functional change of transcriptional regulation. In this context, we have examined the structural changes of the ADF-1 binding sites at the Adh promoters of Drosophila funebris and D. virilis. We detected an expanded footprinted region in D. funebris that contains various adjacent binding sites with different binding affinities. ADF-1 was described to direct sequence-specific DNA binding to sites consisting of the multiple trinucleotide repeat . The ADF-1 recognition sites with high binding affinity differ from this trinucleotide repeat consensus sequence and a new consensus sequence is proposed for the high-affinity ADF-1 binding sites. In vitro transcription experiments with the D. funebris and D. virilis ADF-1 binding regions revealed that stronger ADF-1 binding to the expanded D. funebris ADF-1 binding region only moderately lead to increased transcriptional activity of the Adh gene. The potential of this regional expansion is discussed in the context of different ADF-1 cellular concentrations and maintenance of the ADF-1 stimulus. Altogether, evolutionary change of ADF-1 binding regions involves both, rearrangements of complex binding site cluster and also nucleotide substitutions within sites that lead to different binding affinities.

  18. High-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity in neuronal cultures for mechanistic and drug discovery studies.

    Science.gov (United States)

    Ray, Balmiki; Bailey, Jason A; Simon, Jay R; Lahiri, Debomoy K

    2012-07-01

    Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory-related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzyme A by the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High-affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate-limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well-documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide-based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK-N-SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease.

  19. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.; Ozanich, Richard M.; Miller, Keith D.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cindy J.

    2009-09-01

    A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment was demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.

  20. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1, Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo

    Directory of Open Access Journals (Sweden)

    Tatiana P. Sankova

    2017-11-01

    Full Text Available There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST and the N-terminal domain (ectodomain of human high affinity copper transporter CTR1 (hNdCTR1, which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell’s copper metabolism and its chelating properties are discussed.

  1. The Mitochondrial Metallochaperone SCO1 Is Required to Sustain Expression of the High-Affinity Copper Transporter CTR1 and Preserve Copper Homeostasis

    Directory of Open Access Journals (Sweden)

    Christopher J. Hlynialuk

    2015-02-01

    Full Text Available Human SCO1 fulfills essential roles in cytochrome c oxidase (COX assembly and the regulation of copper (Cu homeostasis, yet it remains unclear why pathogenic mutations in this gene cause such clinically heterogeneous forms of disease. Here, we establish a Sco1 mouse model of human disease and show that ablation of Sco1 expression in the liver is lethal owing to severe COX and Cu deficiencies. We further demonstrate that the Cu deficiency is explained by a functional connection between SCO1 and CTR1, the high-affinity transporter that imports Cu into the cell. CTR1 is rapidly degraded in the absence of SCO1 protein, and we show that its levels are restored in Sco1−/− mouse embryonic fibroblasts upon inhibition of the proteasome. These data suggest that mitochondrial signaling through SCO1 provides a post-translational mechanism to regulate CTR1-dependent Cu import into the cell, and they further underpin the importance of mitochondria in cellular Cu homeostasis.

  2. Induction of high-affinity IgE receptor on lung dendritic cells during viral infection leads to mucous cell metaplasia.

    Science.gov (United States)

    Grayson, Mitchell H; Cheung, Dorothy; Rohlfing, Michelle M; Kitchens, Robert; Spiegel, Daniel E; Tucker, Jennifer; Battaile, John T; Alevy, Yael; Yan, Le; Agapov, Eugene; Kim, Edy Y; Holtzman, Michael J

    2007-10-29

    Respiratory viral infections are associated with an increased risk of asthma, but how acute Th1 antiviral immune responses lead to chronic inflammatory Th2 disease remains undefined. We define a novel pathway that links transient viral infection to chronic lung disease with dendritic cell (DC) expression of the high-affinity IgE receptor (FcepsilonRIalpha). In a mouse model of virus-induced chronic lung disease, in which Sendai virus triggered a switch to persistent mucous cell metaplasia and airway hyperreactivity after clearance of replicating virus, we found that FceRIa(-/-) mice no longer developed mucous cell metaplasia. Viral infection induced IgE-independent, type I IFN receptor-dependent expression of FcepsilonRIalpha on mouse lung DCs. Cross-linking DC FcepsilonRIalpha resulted in the production of the T cell chemoattractant CCL28. FceRIa(-/-) mice had decreased CCL28 and recruitment of IL-13-producing CD4(+) T cells to the lung after viral infection. Transfer of wild-type DCs to FceRIa(-/-) mice restored these events, whereas blockade of CCL28 inhibited mucous cell metaplasia. Therefore, lung DC expression of FcepsilonRIalpha is part of the antiviral response that recruits CD4(+) T cells and drives mucous cell metaplasia, thus linking antiviral responses to allergic/asthmatic Th2 responses.

  3. Botrytis cinerea can import and utilize nucleosides in salvage and catabolism and BcENT functions as high affinity nucleoside transporter.

    Science.gov (United States)

    Daumann, Manuel; Golfier, Philippe; Knüppel, Nathalie; Hahn, Matthias; Möhlmann, Torsten

    2016-08-01

    Nucleotide de novo synthesis is an essential pathway in nearly all organisms. Transport processes as well as salvage and catabolism of nucleotides and pathway intermediates are required to balance nucleotide pools. We have analysed the genome of the fungal plant pathogen Botrytis cinerea for genes involved in nucleotide metabolism and found a complete set of genes necessary for purine and pyrimidine uptake and salvage based on homology of the gene products to corresponding proteins from Aspergillus nidulans. Candidate genes required for a complete purine catabolic sequence were identified in addition. These analyses were complemented by growth tests showing functional transport and salvage activity for pyrimidines. Growth of B. cinerea mycelium in nitrogen free medium could be restored by addition of purines, indicating the presence of a functional purine catabolism, whereas pyrimidines did not support growth. Bcin07g05490 (BcENT) was identified as sole member of the equilibrative nucleoside transporter (ENT) family. The protein synthesized in Saccharomyces cerevisiae revealed high affinity transport of adenosine (KM = 6.81 μM) and uridine (KM=9.04 μM). Furthermore, a BcENT knockout mutant was generated and tested in a range of growth and infection assays. These results provide detailed insight in the use of externally supplied nucleobases and nucleosides by B. cinerea. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  4. LysGH15B, the SH3b domain of staphylococcal phage endolysin LysGH15, retains high affinity