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Sample records for binding anti-human cd3

  1. Regulation and function of the CD3¿ DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro

    DEFF Research Database (Denmark)

    Dietrich, J; Kastrup, J; Nielsen, B L

    1997-01-01

    /CD3gamma chimeras; and in vitro by binding CD3gamma peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3gamma D127xxxLL131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4...... and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3gamma S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization rate...

  2. Structural considerations for functional anti-EGFR × anti-CD3 bispecific diabodies in light of domain order and binding affinity.

    Science.gov (United States)

    Asano, Ryutaro; Nagai, Keisuke; Makabe, Koki; Takahashi, Kento; Kumagai, Takashi; Kawaguchi, Hiroko; Ogata, Hiromi; Arai, Kyoko; Umetsu, Mitsuo; Kumagai, Izumi

    2018-03-02

    We previously reported a functional humanized bispecific diabody (bsDb) that targeted EGFR and CD3 (hEx3-Db) and enhancement of its cytotoxicity by rearranging the domain order in the V domain. Here, we further dissected the effect of domain order in bsDbs on their cross-linking ability and binding kinetics to elucidate general rules regarding the design of functional bsDbs. Using Ex3-Db as a model system, we first classified the four possible domain orders as anti-parallel (where both chimeric single-chain components are variable heavy domain (VH)-variable light domain (VL) or VL-VH order) and parallel types (both chimeric single-chain components are mixed with VH-VL and VL-VH order). Although anti-parallel Ex3-Dbs could cross-link the soluble target antigens, their cross-linking ability between soluble targets had no correlation with their growth inhibitory effects. In contrast, the binding affinity of one of the two constructs with a parallel-arrangement V domain was particularly low, and structural modeling supported this phenomenon. Similar results were observed with E2x3-Dbs, in which the V region of the anti-EGFR antibody clone in hEx3 was replaced with that of another anti-EGFR clone. Only anti-parallel types showed affinity-dependent cancer inhibitory effects in each molecule, and E2x3-LH (both components in VL-VH order) showed the most intense anti-tumor activity in vitro and in vivo . Our results showed that, in addition to rearranging the domain order of bsDbs, increasing their binding affinity may be an ideal strategy for enhancing the cytotoxicity of anti-parallel constructs and that E2x3-LH is particularly attractive as a candidate next-generation anti-cancer drug.

  3. Role of CD3 gamma in T cell receptor assembly

    DEFF Research Database (Denmark)

    Dietrich, J; Neisig, A; Hou, X

    1996-01-01

    . In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta......The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC...... predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression...

  4. Failure to synthesize the CD3-gamma chain. Consequences for T cell antigen receptor assembly, processing, and expression

    DEFF Research Database (Denmark)

    Geisler, C

    1992-01-01

    surface expression of the Ti/CD3 complex. Transfection of the wild-type CD3-gamma gene into JGN reconstituted expression of functional Ti/CD3 complexes, and analysis of T cell lines producing different amounts of CD3-gamma indicated that CD3-gamma and CD3-delta competed for the binding to CD3-epsilon.......The TCR consists of the Ti alpha beta heterodimer and the associated CD3 chains, CD3 gamma delta epsilon zeta 2 or zeta eta. The structural relationships between the subunits of the Ti/CD3 complex are still not fully understood. To explore the roles of the individual CD3 chains for the assembly......, intracellular processing, and expression of the TCR, mutants of the T cell line Jurkat were isolated. One variant, JGN, was found to produce all the Ti/CD3 components with the exception of CD3-gamma. The results indicate that: 1) the tetrameric form (Ti alpha beta-CD3 delta epsilon) of the Ti/CD3 complex...

  5. The 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain and binding analysis to anti-human C2 domain antibodies and phospholipid surfaces.

    Directory of Open Access Journals (Sweden)

    Caileen M Brison

    Full Text Available The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the "non-classical" antibody epitopes. Furthermore, antibody-binding results illustrate that the "classical" 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed.

  6. CD3 immunohistochemical staining in diagnosis of lymphocytic colitis

    DEFF Research Database (Denmark)

    Fiehn, Anne-Marie Kanstrup; Engel, Ulla; Holck, Susanne

    2016-01-01

    and eosin (HE) stainings were available. At the second assessment, a supplementary CD3 immunohistochemical staining was also available. The aim was to evaluate whether a supplementary CD3 would increase the diagnostic agreement among pathologists, and whether a CD3 stain would change the diagnosis based...

  7. Radiolabeled Humanized Anti-CD3 Monoclonal Antibody Visilizumab for Imaging Human T-Lymphocytes

    NARCIS (Netherlands)

    Malviya, Gaurav; D'Alessandria, Calogero; Bonanno, Elena; Vexler, Vladimir; Massari, Roberto; Trotta, Carlo; Scopinaro, Francesco; Dierckx, Rudi; Signore, Alberto

    2009-01-01

    Visilizumab is an IgG(2) humanized monoclonal antibody (mAb) characterized by non-Fc gamma R binding and specific to the CD3 antigen, expressed on more than 95% of circulating resting T-lymphocytes and on activated T-lymphocytes homing in inflamed tissues. We hypothesized that the use of a

  8. Sequencing and expression analysis of CD3γ/δ and CD3ɛ chains in mandarin fish, Siniperca chuatsi

    Science.gov (United States)

    Guo, Zheng; Nie, Pin

    2013-01-01

    The genomic and cDNA sequences of the CD3γ/δ and CD3ɛ homologues in the mandarin fish, Siniperca chuats i, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3γ/δ and CD3ɛ contained conserved residues and motifs, such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs. However, mandarin fish CD3γ/δ and CD3ɛ showed some differences to their mammalian counterparts, specifically the absence of a negatively charged residue in the transmembrane region of CD3γ/δ. Additionally, while an N -glycosylation site was present in CD3ɛ, the site was not observed in CD3γ/δ. The CD3γ/δ and CD3ɛ subunit sequences contain six and five exons, respectively, consistent with homologues from Atlantic salmon, Salmo salar. Phylogenetic analysis also revealed that CD3γ/δ and CD3ɛ in mandarin fish are closely related to their counterparts in Acanthopterygian fish. Real-time PCR showed CD3γ/δ and CD3ɛ were expressed mainly in the thymus and spleen in normal healthy fish and, to a lesser extent, in mucosal-associated lymphoid tissues, such as the intestine and gills. When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin, concanavalin, and polyriboinosinic polyribocytidylic acid, mRNA expression levels of CD3γ/δ and CD3ɛ were significantly elevated within 12 h of treatment. This indicated the presence of T lymphocytes in the head kidney of teleost fish, and also the recognition of mitogens by the lymphocytes. Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3γ/δ and CD3ɛ mRNA, indicating that CD3γ/δ and CD3ɛ lymphocytes are involved in the immune response of this species.

  9. Tetravalent anti-CD20/CD3 bispecific antibody for the treatment of B cell lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chia-Yen; Chen, Gregory J.; Tai, Pei-Han; Yang, Yu-Chen [Institute of Biologics, Development Center for Biotechnology, New Taipei City, Taiwan (China); Hsu, Yu-Shen, E-mail: yshsu@advagene.com.tw [Laboratory of Biopharmaceutical Research, Advagene Biopharma, Taipei, Taiwan (China); Chang, Mingi, E-mail: mingi.chang@advagene.com.tw [Laboratory of Biopharmaceutical Research, Advagene Biopharma, Taipei, Taiwan (China); Hsu, Chuan-Lung, E-mail: fabio@dcb.org.tw [Institute of Biologics, Development Center for Biotechnology, New Taipei City, Taiwan (China)

    2016-05-13

    Bispecific antibodies (bsAbs) are second generation antibodies for therapeutic application in immunotherapy. One of the major strategies of the bsAb platform is the recruitment of immune effector T cells by incorporating an anti-CD3 domain. A bispecific T-cell engager (BiTE), with one end having an affinity for CD3 and the other end with affinity for CD19, has been approved in the US and Europe for the treatment of acute lymphoblastic leukemia. However, due to their small size and lack of Fc region, these single-chain variable fragment (scFv) bsAbs have short half-lives in vivo. Additionally, poor solubility, structural instability, and low production yields have also become major challenges in the bulk production process. To overcome these challenges, we have engineered a tetravalent bsAb with bivalent binding specificity for the CD20 and CD3 antigen in an immunoglobulin G (IgG) format. The fusion of the anti-CD3 scFvs to the CD20 antibody via a linker-hinge domain (LHD) results in improved antibody stabilization and properties. Here we demonstrate this antibody's highly efficient cancer cell elimination in a dose-dependent manner in a CD20-expressing B lymphoblastoid cell line in vitro. Our data suggest the potential clinical application of this bsAb for the treatment of CD20-expressing B cell malignancies. - Highlights: • A bispecific antibody (bsAb) can increase immunotherapeutic efficacy. • A tetravalent bsAb with binding specificity for the CD20 and CD3 antigens is proposed. • A linker-hinge domain (LHD) within the bsAb results in improved antibody properties.

  10. Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma

    NARCIS (Netherlands)

    Luiten, R. M.; Coney, L. R.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

    1996-01-01

    The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250

  11. Failure to synthesize the human T-cell CD3-zeta chain and its consequence for the T-cell receptor-CD3 complex expression

    DEFF Research Database (Denmark)

    Geisler, C; Kuhlmann, J; Plesner, T

    1989-01-01

    components, the human T-cell tumour line Jurkat was chemically mutagenized followed by negative selection with F101.01 (a monoclonal antibody against the TcR-CD3 complex), and cloning. Growing clones were analysed for TcR-CD3 expression by immunofluorescence. One clone, J79, was found to express greatly...... diminished levels of TcR-CD3. This clone produced all the TcR-CD3 components except the CD3-zeta, as demonstrated by metabolic labelling and immunoprecipitation followed by one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These data indicate that the CD3-zeta determines...

  12. Magnetoplasma reflectivity studies on Cd3As2

    NARCIS (Netherlands)

    Schleijpen, H.M.A.; Ortenberg, von M.; Gelten, M.J.; Blom, F.A.P.

    1984-01-01

    Magnetoplasma reflectivity measurements were performed on Cd3As2 in order to obtain more experimental details about the structure of the plasma reflectivity edge. The experimental results can be explained by assuming the existence of a surface layer with a carrier concentration-gradient which is

  13. Weak antilocalization in Cd3As2 thin films.

    Science.gov (United States)

    Zhao, Bo; Cheng, Peihong; Pan, Haiyang; Zhang, Shuai; Wang, Baigeng; Wang, Guanghou; Xiu, Faxian; Song, Fengqi

    2016-03-03

    Recently, it has been theoretically predicted that Cd3As2 is a three dimensional Dirac material, a new topological phase discovered after topological insulators, which exhibits a linear energy dispersion in the bulk with massless Dirac fermions. Here, we report on the low-temperature magnetoresistance measurements on a ~50 nm-thick Cd3As2 film. The weak antilocalization under perpendicular magnetic field is discussed based on the two-dimensional Hikami-Larkin-Nagaoka (HLN) theory. The electron-electron interaction is addressed as the source of the dephasing based on the temperature-dependent scaling behavior. The weak antilocalization can be also observed while the magnetic field is parallel to the electric field due to the strong interaction between the different conductance channels in this quasi-two-dimensional film.

  14. Negative magnetoresistance in Dirac semimetal Cd3As2.

    Science.gov (United States)

    Li, Hui; He, Hongtao; Lu, Hai-Zhou; Zhang, Huachen; Liu, Hongchao; Ma, Rong; Fan, Zhiyong; Shen, Shun-Qing; Wang, Jiannong

    2016-01-08

    A large negative magnetoresistance (NMR) is anticipated in topological semimetals in parallel magnetic fields, demonstrating the chiral anomaly, a long-sought high-energy-physics effect, in solid-state systems. Recent experiments reveal that the Dirac semimetal Cd3As2 has the record-high mobility and positive linear magnetoresistance in perpendicular magnetic fields. However, the NMR has not yet been unveiled. Here we report the observation of NMR in Cd3As2 microribbons in parallel magnetic fields up to 66% at 50 K and visible at room temperatures. The NMR is sensitive to the angle between magnetic and electrical fields, robust against temperature and dependent on the carrier density. The large NMR results from low carrier densities in our Cd3As2 samples, ranging from 3.0 × 10(17) cm(-3) at 300 K to 2.2 × 10(16) cm(-3) below 50 K. We therefore attribute the observed NMR to the chiral anomaly. In perpendicular magnetic fields, a positive linear magnetoresistance up to 1,670% at 14 T and 2 K is also observed.

  15. An anti-CD3/anti-CLL-1 bispecific antibody for the treatment of acute myeloid leukemia.

    Science.gov (United States)

    Leong, Steven R; Sukumaran, Siddharth; Hristopoulos, Maria; Totpal, Klara; Stainton, Shannon; Lu, Elizabeth; Wong, Alfred; Tam, Lucinda; Newman, Robert; Vuillemenot, Brian R; Ellerman, Diego; Gu, Chen; Mathieu, Mary; Dennis, Mark S; Nguyen, Allen; Zheng, Bing; Zhang, Crystal; Lee, Genee; Chu, Yu-Waye; Prell, Rodney A; Lin, Kedan; Laing, Steven T; Polson, Andrew G

    2017-02-02

    Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML. © 2017 by The American Society of Hematology.

  16. CD3-positive B cells: a storage-dependent phenomenon.

    Directory of Open Access Journals (Sweden)

    Angela Nagel

    Full Text Available The majority of clinical studies requires extensive management of human specimen including e.g. overnight shipping of blood samples in order to convey the samples in a central laboratory or to simultaneously analyze large numbers of patients. Storage of blood samples for periods of time before in vitro/ex vivo testing is known to influence the antigen expression on the surface of lymphocytes. In this context, the present results show for the first time that the T cell antigen CD3 can be substantially detected on the surface of human B cells after ex vivo storage and that the degree of this phenomenon critically depends on temperature and duration after blood withdrawal. The appearance of CD3 on the B cell surface seems to be a result of contact-dependent antigen exchange between T and B lymphocytes and is not attributed to endogenous production by B cells. Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations. Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC. Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.

  17. Microstructural Evolution of Secondary Phases in the Cast Duplex Stainless Steels CD3MN and CD3MWCuN

    Science.gov (United States)

    Kim, Yoon-Jun; Ugurlu, Ozan; Jiang, Chao; Gleeson, Brian; Chumbley, L. Scott

    2007-02-01

    The isothermal formation behavior of secondary phases in two types of duplex stainless steels (DSS), CD3MN and CD3MWCuN, was characterized. Samples were heat treated from 1 minute to 30 days at temperatures from 700°C to 900°C. Small carbide (M23C6) and nitride (Cr2N) precipitates, together with the intermetallic phases sigma and chi, were observed using scanning electron microscopy (SEM) and confirmed by transmission electron microscopy (TEM) analyses. Based on SEM analysis, time-temperature-transformation (TTT) curves for the sigma and chi phases were determined by measuring their volume fractions from backscattered electron micrographs of heat-treated and quenched sample cross sections. Resulting TTT curves showed that the maximum formation temperature for chi is lower than that for sigma, while the time to reach 1 vol pct formation is much less for sigma than it is for chi. The thermodynamic driving forces associated with the sigma and chi formation were assessed using Thermo-Calc.

  18. Cloning of a novel cell type from human fetal liver expressing cytoplasmic CD3 delta and epsilon but not membrane CD3

    NARCIS (Netherlands)

    Hori, T.; de Waal Malefyt, R.; Duncan, B. W.; Harrison, M. R.; Roncarolo, M. G.; Spits, H.

    1991-01-01

    Seventeen-week human fetal liver cells cultured with a feeder cell mixture of irradiated PBL, irradiated JY cells (an EBV-transformed B cell line) and PHA contained a subpopulation of CD3- cells in addition to a major population of T cells with the mature phenotype. After 12 days in culture, CD3-

  19. Assembly of the T-cell antigen receptor. Participation of the CD3 omega chain

    DEFF Research Database (Denmark)

    Neisig, A; Vangsted, A; Zeuthen, J

    1993-01-01

    The human TCR is composed of the Ti alpha beta heterodimer in association with the CD3 chains CD3 gamma delta epsilon zeta 2. Another chain, referred to as CD3 omega, has recently been described in T cells. CD3 omega is an intracellular protein transiently associated with the CD3 complex during...... the assembly of the TCR in the endoplasmic reticulum (ER) and it is not expressed on the cell surface. The function of CD3 omega is unknown but it has been suggested that it plays an important role in the assembly of the TCR. We have studied the possible function of CD3 omega in the human leukemic T-cell line...... Jurkat and different variants of this cell line. Cells were metabolically labeled, subjected to lysis, immunoprecipitated, and analyzed by SDS-PAGE. The results indicate that: 1) CD3 omega associates primarily with the CD3 delta epsilon complex; 2) CD3 omega is not associated with single Ti alpha or Ti...

  20. Down regulation of the TCR complex CD3 ζ-chain on CD3+ T cells: a potential mechanism for helminth mediated immune modulation

    Directory of Open Access Journals (Sweden)

    Laura Jane Appleby

    2015-02-01

    Full Text Available The CD3ζ forms part of the T cell receptor (TCR where it plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways leading to T cell effector functions. Down regulation of CD3ζ leads to impairment of immune responses including reduced cell proliferation and cytokine production. In experimental models helminth parasites have been shown to modulate immune responses directed against them and unrelated antigens, so called bystander antigens, but there is a lack of studies validating these observations in humans. This study focused on investigated the relationship between expression levels of the TCR CD3ζ chain with lymphocyte cell proliferation during human infection with the helminth parasite, Schistosoma haematobium which causes uro-genital schistosomiasis. Using flow cytometry, peripheral blood mononuclear cells (PBMCs from individuals naturally exposed to S. haematobium in rural Zimbabwe were phenotyped, and expression levels of CD3ζ on T cells were related to intensity of infection. In this population, parasite infection intensity was inversely related to CD3ζ expression levels (p<0.05, consistent with down-regulation of CD3ζ expression during helminth infection. Furthermore, PBMC proliferation was positively related to expression levels of CD3ζ (p<0.05 after allowing for confounding variables (host age, sex, infection level. CD3ζ expression levels had a differing relationship between immune correlates of susceptibility and immunity, measured by antibody responses, indicating a complex relationship between immune activation status and immunity. The relationships between the CD3ζ chain of the TCR and schistosome infection, PBMC proliferation and schistosome-specific antibody responses have not previously been reported, and these results may indicate a mechanism for the impaired T cell proliferative responses observed during human schistosome infection.

  1. DNA fragmentation and cell death mediated by T cell antigen receptor/CD3 complex on a leukemia T cell line.

    Science.gov (United States)

    Takahashi, S; Maecker, H T; Levy, R

    1989-10-01

    An anti-T cell receptor (TcR) monoclonal antibody (mAb), LC4, directed against a human leukemic T cell line, SUP-T13, caused DNA fragmentation ("apoptosis") and cell death upon binding to this cell line. Cross-linking of receptor molecules was necessary for this effect since F(ab')2, but not Fab', fragments of LC4 could induce cell death. Five anti-CD3 mAb tested also caused apoptosis, but only when they were presented on a solid phase. Interestingly, soluble anti-CD3 mAb induced calcium flux and had an additive effect on the calcium flux and interleukin 2 receptor expression induced by LC4, but these anti-CD3 mAb reversed the growth inhibition and apoptosis caused by LC4. The calcium ionophore A23187, but not the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), also induced apoptosis, suggesting that protein kinase C activation alone does not cause apoptosis, although PMA is growth inhibitory. These results suggest that two distinct biological phenomena can accompany stimulation of the TcR/CD3 complex. In both cases, calcium flux and interleukin 2 receptor expression is induced, but only in one case is apoptosis and cell death seen. The signal initiating apoptosis can be selectively prevented by binding CD3 portion of the receptor in this cell line. This difference in signals mediated by the TcR/CD3 complex may be important in explaining the process of thymic selection, as well as in choosing anti-TcR mAb for therapeutic use.

  2. Production and characterization of anti-human IgG F(ab')2 antibody fragment.

    Science.gov (United States)

    Valedkarimi, Zahra; Nasiri, Hadi; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Esparvarinha, Mojghan; Majidi, Jafar

    2018-04-10

    In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab')2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab')2. Non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment resulted in one band in 100 kDa corresponds to F(ab')2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab')2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab')2 fragment worked as efficiently as the commercial one.

  3. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-02-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

  4. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-01-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG 1 ), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 μg/100 μCi of 99m Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of 99m Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14±2.50 %ID/g, 5.06±2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy

  5. Modulation of Cytokine Production by Drugs with Antiepileptic or Mood Stabilizer Properties in Anti-CD3- and Anti-CD40-Stimulated Blood In Vitro

    Directory of Open Access Journals (Sweden)

    Hubertus Himmerich

    2014-01-01

    Full Text Available Increased cytokine production possibly due to oxidative stress has repeatedly been shown to play a pivotal role in the pathophysiology of epilepsy and bipolar disorder. Recent in vitro and animal studies of valproic acid (VPA report antioxidative and anti-inflammatory properties, and suppression of interleukin (IL-6 and tumor necrosis factor (TNF-α. We tested the effect of drugs with antiepileptic or mood stabilizer properties, namely, primidone (PRM, carbamazepine (CBZ, levetiracetam (LEV, lamotrigine (LTG, VPA, oxcarbazepine (OXC, topiramate (TPM, phenobarbital (PB, and lithium on the production of the following cytokines in vitro: interleukin (IL-1β, IL-2, IL-4, IL-6, IL-17, IL-22, and TNF-α. We performed a whole blood assay with stimulated blood of 14 healthy female subjects. Anti-human CD3 monoclonal antibody OKT3, combined with 5C3 antibody against CD40, was used as stimulant. We found a significant reduction of IL-1 and IL-2 levels with all tested drugs other than lithium in the CD3/5C3-stimulated blood; VPA led to a decrease in IL-1β, IL-2, IL-4, IL-6, IL-17, and TNF-α production, which substantiates and adds knowledge to current hypotheses on VPA’s anti-inflammatory properties.

  6. Dissociation mechanisms and dynamics of doubly charged CD3CN observed by PEPIPICO spectroscopy

    International Nuclear Information System (INIS)

    Harada, C.; Tada, S.; Yamamoto, K.; Senba, Y.; Yoshida, H.; Hiraya, A.; Wada, S.; Tanaka, K.; Tabayashi, K.

    2006-01-01

    Dissociation of free acetonitrile-d 3 molecule, CD 3 CN induced by core level excitation was studied near the nitrogen K-edge by time-of-flight fragment mass spectroscopy. A variety of atomic and molecular fragment cations such as D + , CD n + , C 2 D n + , and CD n CN + were detected using the effusive CD 3 CN beam. Photoelectron-photoion-photoion coincidence technique was applied to analyse the dissociation mechanisms and dynamics of doubly charged CD 3 CN 2+ following the N(1s-π * ) excitation. The charge separation mechanisms of core-excited CD 3 CN were discussed in connection with Auger final state distributions

  7. Dissection of a circulating CD3+ CD20+ T cell subpopulation in patients with psoriasis.

    Science.gov (United States)

    Niu, J; Zhai, Z; Hao, F; Zhang, Y; Song, Z; Zhong, H

    2018-05-01

    CD3 + CD20 + T cells are a population of CD3 + T cells that express CD20 and identified in healthy donors and autoimmune diseases. However, the nature and role of these cells in patients with psoriasis remain unclear. In this study, we aimed to investigate the level, phenotype, functional and clinical relevance of CD3 + CD20 + T cells in the peripheral blood of patients with psoriasis. We found that a small subset of CD3 + T cells expressed CD20 molecule in the peripheral blood of patients with psoriasis, and their levels were similar to those in healthy donors. Circulating CD3 + CD20 + T cells in patients with psoriasis were enriched in CD4 + cells and displayed an activated effector phenotype, as these cells contained fewer CD45RA + -naive and CCR7 + cells with increased activity than those of CD3 + T cells lacking CD20. In addition, compared with healthy donors, circulating CD3 + CD20 + T cells in patients with psoriasis produced more cytokines, interleukin (IL)-17A, tumour necrosis factor (TNF)-α and IL-21, but not IL-4 and IFN-γ. Furthermore, a significantly positive correlation was found between the levels of IL-17A, TNF-α and IL-21-production CD3 + CD20 + T cells with Psoriasis Area and Severity Index scores. Our findings suggest that CD3 + CD20 + T cells may play a role in the pathogenesis of psoriasis. © 2018 British Society for Immunology.

  8. Saudi anti-human cancer plants database (SACPD): A collection of plants with anti-human cancer activities.

    Science.gov (United States)

    Al-Zahrani, Ateeq Ahmed

    2018-01-30

    Several anticancer drugs have been developed from natural products such as plants. Successful experiments in inhibiting the growth of human cancer cell lines using Saudi plants were published over the last three decades. Up to date, there is no Saudi anticancer plants database as a comprehensive source for the interesting data generated from these experiments. Therefore, there was a need for creating a database to collect, organize, search and retrieve such data. As a result, the current paper describes the generation of the Saudi anti-human cancer plants database (SACPD). The database contains most of the reported information about the naturally growing Saudi anticancer plants. SACPD comprises the scientific and local names of 91 plant species that grow naturally in Saudi Arabia. These species belong to 38 different taxonomic families. In Addition, 18 species that represent16 family of medicinal plants and are intensively sold in the local markets in Saudi Arabia were added to the database. The website provides interesting details, including plant part containing the anticancer bioactive compounds, plants locations and cancer/cell type against which they exhibit their anticancer activity. Our survey revealed that breast, liver and leukemia were the most studied cancer cell lines in Saudi Arabia with percentages of 27%, 19% and 15%, respectively. The current SACPD represents a nucleus around which more development efforts can expand to accommodate all future submissions about new Saudi plant species with anticancer activities. SACPD will provide an excellent starting point for researchers and pharmaceutical companies who are interested in developing new anticancer drugs. SACPD is available online at https://teeqrani1.wixsite.com/sapd.

  9. Identification of CD3+ T lymphocytes in the green turtle Chelonia mydas

    Science.gov (United States)

    Munoz, F.A.; Estrada-Parra, S.; Romero-Rojas, A.; Work, Thierry M.; Gonzalez-Ballesteros, E.; Estrada-Garcia, I.

    2009-01-01

    To understand the role of the immune system with respect to disease in reptiles, there is the need to develop tools to assess the host's immune response. An important tool is the development of molecular markers to identify immune cells, and these are limited for reptiles. We developed a technique for the cryopreservation of peripheral blood mononuclear cells and showed that a commercially available anti-CD3 epsilon chain antibody detects a subpopulation of CD3 positive peripheral blood lymphocytes in the marine turtle Chelonia mydas. In the thymus and in skin inoculated with phytohemagglutinin, the same antibody showed the classical staining pattern observed in mammals and birds. For Western blot, the anti-CD3 antibodies identified a 17.6 kDa band in membrane proteins of peripheral blood mononuclear cell compatible in weight to previously described CD3 molecules. This is the first demostration of CD3+ cells in reptiles using specific antibodies.

  10. Induction of CD3 delta epsilon omega by phorbol 12-myristate 13-acetate

    DEFF Research Database (Denmark)

    Vangsted, A; Neisig, A; Wallin, H

    1993-01-01

    The effect of phorbol 12-myristate 13-acetate (PMA) on the synthesis, assembly and processing of the components of the T cell receptor (TcR) was studied with special focus on the CD3 omega chain. Treatment of the human leukemic T cell line Jurkat with PMA increased the synthesis of the Ti alpha, CD......3 gamma and CG3 zeta chains two- to threefold and the synthesis of Ti beta and CD3 delta epsilon omega complexes five- to sevenfold as assessed by metabolic labeling, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by scanning densitometry. The amount...... of TcR complexes expressed on the cell surface was decreased after 16 h of PMA treatment. Based on these results we propose a role of CD3 omega in retention of TcR complexes. From PMA-treated CEM cells more than 50-fold the amount of CD3 delta epsilon omega complexes was immunoprecipitated as compared...

  11. Experimental studies of the dissociative recombination processes for the dimethyl ether ions CD3OCD2+ and (CD3)2OD+

    Science.gov (United States)

    Hamberg, M.; Österdahl, F.; Thomas, R. D.; Zhaunerchyk, V.; Vigren, E.; Kaminska, M.; Af Ugglas, M.; Källberg, A.; Simonsson, A.; Paál, A.; Larsson, M.; Geppert, W. D.

    2010-05-01

    Aims: Determination of branching fractions, cross sections and thermal rate coefficients for the dissociative recombination of CD3OCD2+ (0-0.3 eV) and (CD3)2OD+ (0-0.2 eV) at the low relative kinetic energies encountered in the interstellar medium. Methods: The measurements were carried out using merged electron and ion beams at the CRYRING storage ring, Stockholm, Sweden. Results: For (CD3)2OD+ we have experimentally determined the branching fraction for ejection of a single hydrogen atom in the DR process to be maximally 7% whereas 49% of the reactions involve the break up of the COC chain into two heavy fragments and 44% ruptures both C-O bonds. The DR of CD3OCD2+ is dominated by fragmentation of the COC chain into two heavy fragments. The measured thermal rate constants and cross sections are k(T) = 1.7±0.5 × 10-6(T/300)-0.77±0.01 cm3 s-1, σ = 1.2±0.4 × 10-15(Ecm[eV])-1.27±0.01 cm2 and k(T) = 1.7±0.6 × 10-6(T/300)-0.70 ± 0.02 cm3 s-1, σ = 1.7±0.6 × 10-15(Ecm[eV])-1.20±0.02 cm2 for CD3OCD2+ and (CD3)2OD+, respectively.

  12. Synthesis of 1-[13CD3]-9-cis-retinoic acid

    International Nuclear Information System (INIS)

    Bennani, Y.L.

    1995-01-01

    1-[ 13 CD 3 ]-9-cis-Retinoic acid was prepared in 8 steps from 2,6-dimethylcyclohexanone. Alkylation of 2,6-dimethylcyclohexanone under LiHMDS/MnBr 2 / 13 CD 3 I gave the corresponding labeled 2-[ 13 CD 3 ]-2,2,6-trimethylcyclohexanone 4 in good yield. Further functionalization of 4 to 6-[ 13 CD 3 ]-β-cyclocitral 6 proceeded through a Shapiro reaction. Aldehyde 6 was condensed with ethyl 3,3-dimethylacrylate to afford the corresponding bicyclic pyranone 7. Reduction of 7 to lactol 8, followed by acid-catalyzed ring opening gave the 9-cis-aldehyde 9. Wittig-Horner olefination and saponification afforded the title compound in good overall yield and in excellent isotopic purity. (Author)

  13. Expression of cytoplasmic CD3 epsilon proteins in activated human adult natural killer (NK) cells and CD3 gamma, delta, epsilon complexes in fetal NK cells. Implications for the relationship of NK and T lymphocytes

    NARCIS (Netherlands)

    Lanier, L. L.; Chang, C.; Spits, H.; Phillips, J. H.

    1992-01-01

    NK cells have been defined as CD3-, CD16+, and/or CD56+ lymphocytes that mediate MHC-unrestricted cytotoxicity against certain tumors and virus-infected cells. Although CD3 epsilon transcripts have been detected in some NK clones, it has generally been thought that NK cells do not express CD3

  14. Rapid and preferential distribution of blood-borne αCD3εAb to the liver is followed by local stimulation of T cells and natural killer T cells

    Science.gov (United States)

    Wingender, Gerhard; Schumak, Beatrix; Schurich, Anna; Gessner, J Engelbert; Endl, Elmar; Limmer, Andreas; Knolle, Percy A

    2006-01-01

    Dissemination of soluble molecules or antigens via the blood stream is considered to lead to a uniform distribution in the various organs of the body, but organ-specific microarchitecture and vascularization may influence this. Following intravenous injection of αCD3ε antibody (αCD3εAb) we observed clear differences in antibody binding to Fcγ receptor (FcγR)+ antigen-presenting cells (APCs) or T lymphocytes in different organs. Significant binding of blood-borne αCD3εAb was only detected in the spleen and liver and not in the thymus or lymph node. In the spleen, only 10% of dendritic cells/macrophages and 40% of T-cell receptor (TCR)-β+ cells were positive for αCD3εAb, and, dependent on FcγR-mediated cross-linking of αCD3εAb, a similar percentage of splenic TCR-β+ cells were stimulated and became CD69+. Stimulation of TCR-β+ cells in the liver was at least as efficient as in the spleen, but almost all T cells and all scavenger liver sinusoidal endothelial cells bound αCD3εAb. In contrast to CD69 up-regulation, only CD4+ natural killer T (NKT) cells and CD11ahigh CD8+ T cells were activated by αCD3εAb and expressed interferon (IFN)-γ. Again, IFN-γ release from NKT/T cells was at least as efficient in the liver as in the spleen. Taken together, our results support the notion that the combination of extensive hepatic vascularization and very high scavenger activity allows the liver to fulfill its metabolic tasks and to promote stimulation of the large but widely distributed hepatic population of NKT/T cells. PMID:16423047

  15. Pretargeting vs. direct targeting of human betalox5 islet cells subcutaneously implanted in mice using an anti-human islet cell antibody

    International Nuclear Information System (INIS)

    Liu Guozheng; Dou Shuping; Akalin, Ali; Rusckowski, Mary; Streeter, Philip R.; Shultz, Leonard D.; Greiner, Dale L.

    2012-01-01

    Introduction: We previously demonstrated MORF/cMORF pretargeting of human islets and betalox 5 cells (a human beta cell line) transplanted subcutaneously in mice with the anti-human islet antibody, HPi1. We now compare pretargeting with direct targeting in the beta cell transplant model to evaluate the degree to which target/non-target (T/NT) ratios may be improved by pretargeting. Methods: Specific binding of an anti-human islet antibody HPi1 to the beta cells transplanted subcutaneously in mice was examined against a negative control antibody. We then compared pretargeting by MORF-HPi1 plus 111 In-labeled cMORF to direct targeting by 111 In-labeled HPi1. Results: HPi1 binding to betalox5 human cells in the transplant was shown by immunofluorescence. Normal organ 111 In backgrounds by pretargeting were always lower, although target accumulations were similar. More importantly, the transplant to pancreas and liver ratios was, respectively, 26 and 10 by pretargeting as compared to 9 and 0.6 by direct targeting. Conclusions: Pretargeting greatly improves the T/NT ratios, and based on the estimated endocrine to exocrine ratio within a pancreas, pretargeting may be approaching the sensitivity required for successful imaging of human islets within this organ.

  16. PbS/Cd3P2 quantum heterojunction colloidal quantum dot solar cells

    International Nuclear Information System (INIS)

    Cao, Hefeng; Xu, Songman; Liu, Huan; Liu, Zeke; Zhu, Xiangxiang; Peng, Jun; Ma, Wanli; Hu, Long; Luo, Miao; Tang, Jiang

    2015-01-01

    Here, we demonstrated the quantum heterojunction colloidal quantum dot (CQD) solar cells employing the PbS CQDs/Cd 3 P 2 CQDs architecture in which both the p-type PbS and n-type Cd 3 P 2 CQD layers are quantum-tunable and solution-processed light absorbers. We synthesized well-crystallized and nearly monodispersed tetragonal Cd 3 P 2 CQDs and then engineered their energy band alignment with the p-type PbS by tuning the dot size and hence the bandgap to achieve efficient light absorbing and charge separation. We further optimized the device through the Ag-doping strategy of PbS CQDs that may leverage an expanded depletion region in the n-layer, which greatly enhances the photocurrent. The resulting devices showed an efficiency of 1.5%. (paper)

  17. Electronic structure and magnetism of titanium substituted Cd3P2: An ab-initio study

    Science.gov (United States)

    Jaiganesh, G.; Jaya, S. Mathi

    2018-05-01

    Using the ab-initio computations that are based on the density functional theory, we have investigated the magnetism and electronic properties of one and two Ti atom substituted Cd3P2 compound. The magnetic stability of the substituted compounds was obtained by analyzing the minimum total energies in nonmagnetic, ferromagnetic and antiferromagnetic phases. Our results indicated the formation of magnetic order in one and two Ti atom substituted Cd3P2 as well as metallic characteristics in these systems. A significant value of the magnetic moment of Ti atom is observed from our calculations. We further find that the neighboring Cd and P atoms too acquire a small magnetic moment.

  18. CD3+CD8+CD161high Tc17 cells are depleted in HIV-infection

    DEFF Research Database (Denmark)

    Gaardbo, Julie Christine; Hartling, Hans Jakob; Thorsteinsson, Kristina

    2012-01-01

    CD8+ Tc17 cells with pro-inflammatory properties have only recently been acknowledged, and Tc17 cells in HIV-infection are undescribed. CD3+CD8+CD161 Tc17 cells and the production of Interleukin-17 were examined in untreated and treated HIV-infected patients, HIV-HCV co-infected patients...... and healthy controls. Depletion of CD3+CD8+CD161 Tc17 cells and diminished production of Interleukin-17 in HIV-infected patients was found. The level of Tc17 cells was associated with the level of the CD4+ count in treated patients....

  19. The structure of the CD3 ζζ transmembrane dimer in POPC and raft-like lipid bilayer: a molecular dynamics study.

    Science.gov (United States)

    Petruk, Ariel Alcides; Varriale, Sonia; Coscia, Maria Rosaria; Mazzarella, Lelio; Merlino, Antonello; Oreste, Umberto

    2013-11-01

    Plasma membrane lipids significantly affect assembly and activity of many signaling networks. The present work is aimed at analyzing, by molecular dynamics simulations, the structure and dynamics of the CD3 ζζ dimer in palmitoyl-oleoyl-phosphatidylcholine bilayer (POPC) and in POPC/cholesterol/sphingomyelin bilayer, which resembles the raft membrane microdomain supposed to be the site of the signal transducing machinery. Both POPC and raft-like environment produce significant alterations in structure and flexibility of the CD3 ζζ with respect to nuclear magnetic resonance (NMR) model: the dimer is more compact, its secondary structure is slightly less ordered, the arrangement of the Asp6 pair, which is important for binding to the Arg residue in the alpha chain of the T cell receptor (TCR), is stabilized by water molecules. Different interactions of charged residues with lipids at the lipid-cytoplasm boundary occur when the two environments are compared. Furthermore, in contrast to what is observed in POPC, in the raft-like environment correlated motions between transmembrane and cytoplasmic regions are observed. Altogether the data suggest that when the TCR complex resides in the raft domains, the CD3 ζζ dimer assumes a specific conformation probably necessary to the correct signal transduction. © 2013.

  20. The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes

    Directory of Open Access Journals (Sweden)

    Proud Christopher G

    2002-05-01

    Full Text Available Abstract Background Activation of human resting T lymphocytes results in an immediate increase in protein synthesis. The increase in protein synthesis after 16–24 h has been linked to the increased protein levels of translation initiation factors. However, the regulation of protein synthesis during the early onset of T cell activation has not been studied in great detail. We studied the regulation of protein synthesis after 1 h of activation using αCD3 antibody to stimulate the T cell receptor and αCD28 antibody to provide the co-stimulus. Results Activation of the T cells with both antibodies led to a sustained increase in the rate of protein synthesis. The activities and/or phosphorylation states of several translation factors were studied during the first hour of stimulation with αCD3 and αCD28 to explore the mechanism underlying the activation of protein synthesis. The initial increase in protein synthesis was accompanied by activation of the guanine nucleotide exchange factor, eukaryotic initiation factor (eIF 2B, and of p70 S6 kinase and by dephosphorylation of eukaryotic elongation factor (eEF 2. Similar signal transduction pathways, as assessed using signal transduction inhibitors, are involved in the regulation of protein synthesis, eIF2B activity and p70 S6 kinase activity. A new finding was that the p38 MAPK α/β pathway was involved in the regulation of overall protein synthesis in primary T cells. Unexpectedly, no changes were detected in the phosphorylation state of the cap-binding protein eIF4E and the eIF4E-binding protein 4E-BP1, or the formation of the cap-binding complex eIF4F. Conclusions Both eIF2B and p70 S6 kinase play important roles in the regulation of protein synthesis during the early onset of T cell activation.

  1. TCR/CD3 ligation of a TCR-transgenic T lymphoma blocks its proliferation in vitro but does not affect its growth in vivo

    DEFF Research Database (Denmark)

    Reimann, J; Rudolphi, A; Tcherepnev, G

    1994-01-01

    A backcrossed mouse line was established homozygous for the autosomal recessive mutation scid (severe combined immunodeficiency) and carrying T cells which express transgenic (tg) T cell receptor (TCR) alpha and beta chains that mediate H-2 class I (Db)-restricted recognition of a male (H...... of TL1 cells resembled that of small-to-medium lymphoblasts. The cells had the following phenotype: CD3 + TCR alpha T+TCR beta T+CD4-CD8- CD44-CD45RB+LECAM-1 + IL-2R- and low H-2 expression. Exposure of TL1 cells to TCR-binding monoclonal antibodies or lectins blocked in their in vitro proliferation...

  2. Distinguishing diffuse alopecia areata (AA) from pattern hair loss (PHL) using CD3(+) T cells.

    Science.gov (United States)

    Kolivras, Athanassios; Thompson, Curtis

    2016-05-01

    Distinguishing between diffuse subacute alopecia areata (AA), in which the peribulbar infiltrate is absent, and pattern hair loss is challenging, particularly in cases that lack marked follicular miniaturization and a marked catagen/telogen shift. We sought to distinguish diffuse AA from pattern hair loss using CD3(+) T lymphocytes. A total of 28 cases of subacute AA and 31 cases of pattern hair loss were selected and a 4-mm punch biopsy was performed. All the specimens were processed using the "HoVert" (horizontal and vertical) technique. In all cases, hematoxylin-eosin and immunohistochemical stains for CD3, CD4, CD8, and CD20 were performed. The presence of CD3(+) lymphocytes within empty follicular fibrous tracts (stela), even without a concomitant peribulbar infiltrate, is a reliable histopathological clue in supporting a diagnosis of AA (sensitivity 0.964, specificity 1, P ≤ .001). Limited tissue for analysis remained in the clinical sample tissue blocks. The presence of CD3(+) T-cells within empty follicular fibrous tracts (stela) supports a diagnosis of AA. Copyright © 2015 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  3. The Function of CD3+CD56+ NKT-Like Cells in HIV-Infected Individuals

    Directory of Open Access Journals (Sweden)

    Yongjun Jiang

    2014-01-01

    Full Text Available CD3+CD56+ NKT-like cells are one of the critical effectors in the immune response to viral infection and tumors, but the functional features of NKT-like cells in HIV infection have been rarely reported. In this study, we observed and described the state of NKT-like cell functions in primary HIV-infected individuals (PHIs, chronic HIV-infected individuals (CHIs, long-term nonprogressors (LTNPs, and HIV-negative controls (NCs. The results showed that the percentage of IFN-γ+CD3+CD56+ NKT-like cells was notably higher in LTNPs compared with CHIs, and the proportion of CD3+CD56+ NKT-like cells with dual function (IFN-γ+CD107a+ NKT-like cells in LTNPs was also much higher than in CHIs. Additionally, the percentages of IFN-γ+CD107a+ NKT-like cells negatively correlated with viral load. Taken together, our data demonstrated that good functions of CD3+CD56+ NKT-like cells in LTNPs likely occurred as a protective mechanism that slows down HIV disease progression.

  4. The function of CD3+CD56+ NKT-like cells in HIV-infected individuals.

    Science.gov (United States)

    Jiang, Yongjun; Cui, Xiaojian; Cui, Chen; Zhang, Jian; Zhou, Fangyuan; Zhang, Zining; Fu, Yajing; Xu, Junjie; Chu, Zhenxing; Liu, Jing; Han, Xiaoxu; Liao, Christina; Wang, Yanan; Cao, Yaming; Shang, Hong

    2014-01-01

    CD3(+)CD56(+) NKT-like cells are one of the critical effectors in the immune response to viral infection and tumors, but the functional features of NKT-like cells in HIV infection have been rarely reported. In this study, we observed and described the state of NKT-like cell functions in primary HIV-infected individuals (PHIs), chronic HIV-infected individuals (CHIs), long-term nonprogressors (LTNPs), and HIV-negative controls (NCs). The results showed that the percentage of IFN-γ(+)CD3(+)CD56(+) NKT-like cells was notably higher in LTNPs compared with CHIs, and the proportion of CD3(+)CD56(+) NKT-like cells with dual function (IFN-γ(+)CD107a(+) NKT-like cells) in LTNPs was also much higher than in CHIs. Additionally, the percentages of IFN-γ(+)CD107a(+) NKT-like cells negatively correlated with viral load. Taken together, our data demonstrated that good functions of CD3(+)CD56(+) NKT-like cells in LTNPs likely occurred as a protective mechanism that slows down HIV disease progression.

  5. Molecular beam epitaxy of Cd3As2 on a III-V substrate

    Directory of Open Access Journals (Sweden)

    Timo Schumann

    2016-12-01

    Full Text Available Epitaxial, strain-engineered Dirac semimetal heterostructures promise tuning of the unique properties of these materials. In this study, we investigate the growth of thin films of the recently discovered Dirac semimetal Cd3As2 by molecular beam epitaxy. We show that epitaxial Cd3As2 layers can be grown at low temperatures (110 °C–220 °C, in situ, on (111 GaSb buffer layers deposited on (111 GaAs substrates. The orientation relationship is described by ( 112 Cd 3 As 2 || (111 GaSb and [ 1 1 ¯ 0 ] Cd 3 As 2 || [ 1 ¯ 01 ] GaSb . The films are shown to grow in the low-temperature, vacancy ordered, tetragonal Dirac semimetal phase. They exhibit high room temperature mobilities of up to 19300 cm2/Vs, despite a three-dimensional surface morphology indicative of island growth and the presence of twin variants. The results indicate that epitaxial growth on more closely lattice matched buffer layers, such as InGaSb or InAlSb, which allow for imposing different degrees of epitaxial coherency strains, should be possible.

  6. Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

    International Nuclear Information System (INIS)

    Qian Weizhu; Wang Ling; Li Bohua; Wang Hao; Hou Sheng; Hong Xueyu; Zhang Dapeng; Guo Yajun

    2008-01-01

    Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application

  7. Development of CD3 cell quantitation algorithms for renal allograft biopsy rejection assessment utilizing open source image analysis software.

    Science.gov (United States)

    Moon, Andres; Smith, Geoffrey H; Kong, Jun; Rogers, Thomas E; Ellis, Carla L; Farris, Alton B Brad

    2018-02-01

    Renal allograft rejection diagnosis depends on assessment of parameters such as interstitial inflammation; however, studies have shown interobserver variability regarding interstitial inflammation assessment. Since automated image analysis quantitation can be reproducible, we devised customized analysis methods for CD3+ T-cell staining density as a measure of rejection severity and compared them with established commercial methods along with visual assessment. Renal biopsy CD3 immunohistochemistry slides (n = 45), including renal allografts with various degrees of acute cellular rejection (ACR) were scanned for whole slide images (WSIs). Inflammation was quantitated in the WSIs using pathologist visual assessment, commercial algorithms (Aperio nuclear algorithm for CD3+ cells/mm 2 and Aperio positive pixel count algorithm), and customized open source algorithms developed in ImageJ with thresholding/positive pixel counting (custom CD3+%) and identification of pixels fulfilling "maxima" criteria for CD3 expression (custom CD3+ cells/mm 2 ). Based on visual inspections of "markup" images, CD3 quantitation algorithms produced adequate accuracy. Additionally, CD3 quantitation algorithms correlated between each other and also with visual assessment in a statistically significant manner (r = 0.44 to 0.94, p = 0.003 to algorithms presents salient correlations with established methods of CD3 quantitation. These analysis techniques are promising and highly customizable, providing a form of on-slide "flow cytometry" that can facilitate additional diagnostic accuracy in tissue-based assessments.

  8. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    International Nuclear Information System (INIS)

    Berger, Christian; Madshus, Inger Helene; Stang, Espen

    2012-01-01

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: ► Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. ► Antibody combination causes internalization of EGFR by macropinocytosis. ► Antibody-induced internalization of EGFR is independent of EGFR kinase activity. ► Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  9. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene [Institute of Pathology, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Stang, Espen, E-mail: espsta@rr-research.no [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway)

    2012-12-10

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  10. Altered expression of the TCR signaling related genes CD3 and FcεRIγ in patients with aplastic anemia

    Directory of Open Access Journals (Sweden)

    Li Bo

    2012-03-01

    Full Text Available Abstract Background Aplastic anemia (AA is characterized by pancytopenia and bone marrow hypoplasia, which results from immune-mediated hematopoiesis suppression. Understanding the pathophysiology of the immune system, particularly T cells immunity, has led to improved AA treatment over the past decades. However, primary and secondary failure after immunosuppressive therapy is frequent. Thus, knowledge of the immune mechanisms leading to AA is crucial to fundamentally understand the disease. Findings To elucidate the T cell receptor (TCR signal transduction features in AA, the expression levels of CD3γ, δ, ε and ζ chain and FcεRIγ genes, which are involved in TCR signal transduction, and the negative correlation of the expression levels between the CD3ζ and FcεRIγ genes in T cells from peripheral blood mononuclear cells (PBMCs were analyzed. Real-time RT-PCR using the SYBR Green method was used to detect the expression level of these genes in PBMCs from 18 patients with AA and 14 healthy individuals. The β2microglobulin gene (β2M was used as an endogenous reference. The expression levels of the CD3γ, CD3δ, CD3ε and CD3ζ genes in patients with AA were significantly increased compared to a healthy control group, whereas the FcεRIγ gene expression level was significantly decreased in patients with AA in comparison with the healthy control group. Moreover, the negative correlation of the expression levels between the CD3ζ and FcεRIγ genes was lost. Conclusions To our knowledge, this is the first report of the CD3γ, CD3δ, CD3ε, CD3ζ and FcεRIγ gene expression in patients with AA. The abnormally expressed TCR signaling related genes may relate to T cells dysfunction in AA.

  11. Quantum oscillation and nontrivial transport in the Dirac semimetal Cd_3As_2 nanodevice

    International Nuclear Information System (INIS)

    Pan, Haiyang; Wei, Zhongxia; Zhao, Bo; Song, Fengqi; Wang, Baigeng; Zhang, Kang; Gao, Ming; Wang, Xuefeng; Zhang, Rong; Wang, Jue; Han, Min; Pi, Li

    2016-01-01

    Here, we report on the Shubnikov-de Haas oscillation in high-quality Cd_3As_2 nanowires grown by a chemical vapor deposition approach. The dominant transport of topological Dirac fermions is evident by the nontrivial Berry phase in the Landau Fan diagram. The quantum oscillations rise at a small field of 2 T and preserves up to 100 K, revealing a sizeable Landau level gap and a device mobility of 2138 cm"2" V"−"1" s"−"1. The angle-variable oscillations indicate the isotropy of the bulk Dirac transport. The large estimated mean free path makes the Cd_3As_2 nanowire a promising platform for the one-dimensional transport of Dirac semimetals.

  12. Neutron scattering from (CD3ND3)2MnCl4

    International Nuclear Information System (INIS)

    Lehner, N.

    1978-12-01

    For the perovskite-type crystal (CD 3 ND 3 ) 2 MnCl 4 neutron scattering experiments were performed concerning the magnetic behaviour, critical phenomena and lattice dynamics. The crystal is built up from layers, resulting in a quasi two-dimensional antiferromagnetic order, whereas the lattice dynamics shows a more three-dimensional behaviour; this can be explained by long-range Coulomb forces. Only the correlation lengths, which were determined by critical scattering, show a pronounced anisotropy. (orig.) [de

  13. T CD3+CD8+ Lymphocytes Are More Susceptible for Apoptosis in the First Trimester of Normal Human Pregnancy

    Directory of Open Access Journals (Sweden)

    Dorota Darmochwal-Kolarz

    2014-01-01

    Full Text Available Aims. Normal human pregnancy is a complex process of many immunoregulatory mechanisms which protect fetus from the activation of the maternal immune system. The aim of the study was to investigate the apoptosis of lymphocytes in peripheral blood of normal pregnant patients and healthy nonpregnant women. Methods. Sixty pregnant women and 17 nonpregnant women were included in the study. Lymphocytes were isolated and labeled with anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies. Apoptosis was detected by CMXRos staining and analyzed using the flow cytometric method. Results. We found significantly higher apoptosis of total lymphocytes in peripheral blood of pregnant patients when compared to healthy nonpregnant women. The percentage of apoptotic T CD3+CD8+ cells in the first trimester was significantly higher when compared to the third trimester of normal pregnancy. The ratio of T CD3+CD4+ : T CD3+CD8+ apoptotic lymphocytes was significantly lower in the first trimester when compared to other trimesters of pregnancy and to both of the phases of the menstrual cycle. Conclusions. The higher apoptosis of T CD3+CD8+ lymphocytes and the lower ratio of T CD3+CD4+ : T CD3+CD8+ apoptotic cells in the first trimester of normal pregnancy may suggest a higher susceptibility of T CD3+CD8+ cells for apoptosis as a protective mechanism at the early stage of pregnancy.

  14. Unexpected low thermal conductivity and large power factor in Dirac semimetal Cd3As2

    Science.gov (United States)

    Cheng, Zhang; Tong, Zhou; Sihang, Liang; Junzhi, Cao; Xiang, Yuan; Yanwen, Liu; Yao, Shen; Qisi, Wang; Jun, Zhao; Zhongqin, Yang; Faxian, Xiu

    2016-01-01

    Thermoelectrics has long been considered as a promising way of power generation for the next decades. So far, extensive efforts have been devoted to the search of ideal thermoelectric materials, which require both high electrical conductivity and low thermal conductivity. Recently, the emerging Dirac semimetal Cd3As2, a three-dimensional analogue of graphene, has been reported to host ultra-high mobility and good electrical conductivity as metals. Here, we report the observation of unexpected low thermal conductivity in Cd3As2, one order of magnitude lower than the conventional metals or semimetals with a similar electrical conductivity, despite the semimetal band structure and high electron mobility. The power factor also reaches a large value of 1.58 mW·m-1·K-2 at room temperature and remains non-saturated up to 400 K. Corroborating with the first-principles calculations, we find that the thermoelectric performance can be well-modulated by the carrier concentration in a wide range. This work demonstrates the Dirac semimetal Cd3As2 as a potential candidate of thermoelectric materials. Project supported by the National Young 1000 Talent Plan China, the Pujiang Talent Plan in Shanghai, China, the National Natural Science Foundation of China (Grant Nos. 61322407 and 11474058), the Fund for Fostering Talents in Basic Science of the National Natural Science Foundation of China (Grant No. J1103204), and the National Basic Research Program of China (Grant No. 2011CB921803).

  15. CD3 Positive Gastric Plasmablastic Lymphoma in A HIV Negative Patient: A Case Report

    Directory of Open Access Journals (Sweden)

    Betül Bolat Küçükzeybek,

    2017-03-01

    Full Text Available Plasmablastic lymphoma is a rare and aggressive lymphoma characterized by the diffuse proliferation of large neoplastic cells resembling immunoblasts with an immunophenotype of plasma cells. A 47-year-old male was referred to our hospital with gastrointestinal bleeding, and a mass 10 cm in diameter, was detected. An endoscopic biopsy was performed subsequently. Histopathological examination of the biopsy material revealed ulcer, alterations associated with ulcer, and further presented a diffuse infiltration of atypical cells with abundant cytoplasm and pleomorphic nuclei, some with crush artifacts in lamina propria. Immunohistochemically, the tumor cells were negative for cytokeratin, CD2, CD20, and PAX5; but they were positive for CD3, MUM1, CD38 and CD138. Ki67 proliferation index was as high as 95%. The case was signed out as CD3-positive plasmablastic lymphoma with clinical, histopathological and immunohisto-chemical findings. The plasmablastic lymphoma case with an aberrant CD3 expression has been presented here, which is rarely observed in stomach.

  16. Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: Differing impact on CD8 T cell phenotype and responsiveness to restimulation

    Directory of Open Access Journals (Sweden)

    Kurlander Roger J

    2010-10-01

    Full Text Available Abstract Background The ability to expand virus- or tumor-specific T cells without damaging their functional capabilities is critical for success adoptive transfer immunotherapy of patients with opportunistic infection or tumor. Careful comparisons can help identify expansion methods better suited for particular clinical settings and identify recurrent deficiencies requiring new innovation. Methods We compared the efficacy of magnetic beads coated with anti-CD3 and anti-CD28 (anti-CD3/CD28 beads, and soluble anti-CD3 plus mixed mononuclear cells (designated a rapid expansion protocol or REP in expanding normal human T cells. Results Both anti-CD3/CD28 beads and soluble anti-CD3 promoted extensive expansion. Beads stimulated greater CD4 cell growth (geometric mean of 56- versus 27-fold (p Conclusions Anti-CD3/CD28 beads are highly effective for expanding CD4 cells, but soluble anti-CD3 has significant potential advantages for expanding CD8 T cells, particularly where preservation of phenotypically "young" CD8 cells would be desirable, or where the T cells of interest have been antigen-stimulated in vitro or in vivo in the recent past.

  17. First clinical evaluation of radioimmunoimaging using anti-human lung cancer monoclonal antibodies

    International Nuclear Information System (INIS)

    Zhou Qian

    1991-01-01

    Anti-human large cell lung cancer monoclonal antibodies (McAb) 2E3 and 6D1 were produced in the laboratory. Immunohistochemical studies and radiobinding assay showed these antibodies possessed high specificity against lung cancer cells. 28 patients with lung masses were investigated with 131 I-labeled McAb 6D1 and/or 2E3 scintigraphy. 19 of them were histologically proven and 13 were diagnosed primary lung carcinoma. Radioimmunoimaging visualized 10/13 of the primary lung cancers with a detection rate of 77%. Only 1 case of the non-cancer patients and a false localization, giving a true negative rate of 83%. Pathologically the squamous cell lung carcinoma had the highest localization and the small cell lung carcinoma next, but the detection rate was 100% for both. The adenocarcinoma of lung was less sensitive to these McAbs, with a detection rate of only 33% (1 of 3 cases). We conclude that radioimmunoimaging with anti-human large cell lung cancer McAbs is more specific and effective in detecting primary lung cancers and differentiating lung masses than with antibodies against other tumor associated antigens

  18. Mesenchymal stromal cells as vehicles of tetravalent bispecific Tandab (CD3/CD19 for the treatment of B cell lymphoma combined with IDO pathway inhibitor d-1-methyl-tryptophan

    Directory of Open Access Journals (Sweden)

    Xiaolong Zhang

    2017-02-01

    Full Text Available Abstract Background Although blinatumomab, a bispecific T cell engaging antibody, exhibits high clinical response rates in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia (B-ALL and B cell non-Hodgkin’s lymphoma (B-NHL, it still has some limitations because of its short half-life. Mesenchymal stromal cells (MSCs represent an attractive approach for delivery of therapeutic agents to cancer sites owing to their tropism towards tumors, but their immunosuppression capabilities, especially induced by indoleamine 2,3-dioxygenase (IDO, should also be taken into consideration. Methods Human umbilical cord-derived MSCs (UC-MSCs were genetically modified to secrete Tandab (CD3/CD19, a tetravalent bispecific tandem diabody with two binding sites for CD3 and two for CD19. The tropism of MSCs towards Raji cells in vitro was determined by migration assays, and the homing property of MSCs in vivo was analyzed with firefly luciferase-labeled MSCs (MSC-Luc by bioluminescent imaging (BLI. The cytotoxicity of T cells induced by MSC-secreting Tandab (CD3/CD19 was detected in vitro and in vivo in combination with d-1-methyl-tryptophan (D-1MT, an IDO pathway inhibitor. Results The purified Tandab (CD3/CD19 was functional with high-binding capability both for CD3-positive cells and CD19-positive cells and was able to induce specific lysis of CD19-positive cell lines (Raji, Daudi, and BJAB in the presence of T cells. Additionally, results from co-culture killing experiments demonstrated that Tandab (CD3/CD19 secreted from MSCs was also effective. Then, we confirmed that D-1MT could enhance the cytotoxicity of T cells triggered by MSC-Tandab through reversing T cell anergy with down-regulation of CD98 and Jumonji and restoring the proliferation capacity of T cells. Furthermore, MSC-Luc could selectively migrate to tumor site in a BALB/c nude mouse model with Raji cells. And mice injected with MSC-Tandab in combination with D-1MT

  19. Altered phenotypic and functional characteristics of CD3+CD56+ NKT-like cells in human gastric cancer.

    Science.gov (United States)

    Peng, Liu-Sheng; Mao, Fang-Yuan; Zhao, Yong-Liang; Wang, Ting-Ting; Chen, Na; Zhang, Jin-Yu; Cheng, Ping; Li, Wen-Hua; Lv, Yi-Pin; Teng, Yong-Sheng; Guo, Gang; Luo, Ping; Chen, Weisan; Zou, Quan-Ming; Zhuang, Yuan

    2016-08-23

    CD3+CD56+ natural killer T (NKT)-like cells are a group of CD3+ T cells sharing characteristics of NK and T cells and constitute a major component of host anti-tumor immune response in human cancer. However, the nature, function and clinical relevance of CD3+CD56+ NKT-like cells in human gastric cancer (GC) remain unclear. In this study, we showed that the frequencies of CD3+CD56+NKT-like cells in GC tumors were significantly decreased and low levels of tumor-infiltrating CD3+CD56+ NKT-like cells were positively correlated with poor survival and disease progression. Most CD3+CD56+NKT-like cells in GC tumors were CD45RA-CD27+/- central/effector-memory cells with decreased activity and lower expression levels of CD69, NKG2D and DNAM-1 than those in non-tumor tissues. We further observed that tumor-infiltrating CD3+CD56+ NKT-like cells had impaired effector function as shown by decreased IFN-γ, TNF-α, granzyme B and Ki-67 expression. Moreover, in vitro studies showed that soluble factors released from GC tumors could induce the functional impairment of CD3+CD56+ NKT-like cells. Collectively, our data indicate that decreased tumor-infiltrating CD3+CD56+ NKT-like cells with impaired effector function are associated with tumor progression and poor survival of GC patients, which may contribute to immune escape of GC.

  20. Raman polarizabilities of the ν2, ν5 bands of CD3Cl

    International Nuclear Information System (INIS)

    Escribano, R.; Hernandez, M.G.; Mejias, M.; Brodersen, S.

    1985-01-01

    The Raman spectrum of the Coriolis interacting ν 2 , ν 5 bands of CD 3 Cl was recorded with a resolution of ca 0.6 cm -1 . Using the vibrational-rotational parameters of Yamada and Hirota, a computer simulation of the Raman contour was performed, yielding relative values of Raman polarizability derivatives for these bands. By comparison with the absolute intensity measurement of Orza et al., absolute values of the Raman polarizabilities were obtained. The sign of the Raman intensity perturbation was found to be negative. (author)

  1. The feasibility research of galactosyl-anti-mouse CD3 monoclonal antibody being used as carrier of immunotherapy after surgical operation of liver cancer

    International Nuclear Information System (INIS)

    Li Yunchun; Guan Changtian; Yang Xiaochuan; He Sheng; Jiang Ping; Yuan Lin

    2000-01-01

    Objective: To probe into the feasibility of galactosyl-anti-mouse CD 3 monoclonal antibody (Gal-Ant-CD 3 McAb) being used as carrier of immunotherapy after surgical operation of liver cancer. Methods: Gal-Ant-CD 3 McAb was prepared by the covalent coupling of anti-mouse CD 3 monoclonal antibody (Ant-CD 3 McAb) with a bifunctional reagent, 2-imino-2-methoxyethyl-1-thio-galactose. After Gal-Ant-CD 3 McAb and Ant-CD 3 McAb were labelled with 131 I or 125 I, the data of biodistribution in mice, and of imaging in rabbit were obtained. After tumour infiltrating lymphocytes (TIL) and Gal-Ant-CD 3 McAb were coupled into Gal-Ant-CD 3 McAb-TIL, its liver taxis and cytotoxic activity against autologous cancer cells were measured in vitro. Results: Gal-Ant-CD 3 McAb had remarkable livertaxis and its uptake in per gram liver was (59.0 +- 2.1)% that was more than two-fold higher than that of Ant-CD 3 McAb. Gal-Ant-CD 3 McAb-TIL had an obvious liver taxis and cytotoxic activity against autologous cancer cells in vitro. Conclusion: Gal-Ant-CD 3 McAb can be used as the carrier of immunotherapy after surgical operation of liver cancer

  2. The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling

    DEFF Research Database (Denmark)

    Dietrich, J; Bäckström, T; Lauritsen, J P

    1998-01-01

    The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the ......The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR...... the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed....

  3. The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling

    DEFF Research Database (Denmark)

    Dietrich, J; Backstrom, T; Lauritsen, JP

    1998-01-01

    The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR...... the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed....... and the mechanisms involved in the sorting events following PKC-induced internalization are not known. In this study, we demonstrated that following PKC-induced internalization, the TCR is recycled back to the cell surface in a functional state. TCR recycling was dependent on dephosphorylation of CD3gamma, probably...

  4. [IL-2 stimulated responses of CD3(+) CD56(+) NKT cells in pulmonary tuberculosis patients].

    Science.gov (United States)

    Yao, Chun-Yan; Wang, Zhao-Hua; Jiang, Li-Na; Peng, Mei-Yu; Wang, Jing; Li, Bai-Qing

    2010-09-01

    To observe the activation and proliferation characteristics of IL-2 stimulated CD3(+);CD56(+); NKT cells in pulmonary tuberculosis (PTB) patients. Peripheral blood mononuclear cells (PBMCs) from PTB patients and normal subjects were stimulated with IL-2 and cultured for different time points. The CD69 expression on and amount of the CD3(+);CD56(+); NKT cells were detected by multi fluorescence staining and flow cytometry at different time of stimulation and culture. There was no significant difference in percentage of NKT cells between PTB patients and normal healthy controls before culture. When IL-2 was used to stimulate for 0 h, 8 h, 16 h, 40 h and 64 h, the expression of CD69 on NKT cells in normal controls and PTB patients increased significantly, but the CD69 expression level of NKT cells in PTB patients was significantly higher than that in normal persons(PNKT cells increased from (3.44+/-1.20)x10(4); to (323.23+/-75.98) x10(4); (PNKT cells increased from (5.57+/-5.16)x10(4); to (1475.05+/-868.98)x10(4); (PNKT cells in PTB patients present with high activation but low proliferation after stimulated by IL-2.

  5. Increased NY-ESO-1 Expression and Reduced Infiltrating CD3+ T Cells in Cutaneous Melanoma

    Directory of Open Access Journals (Sweden)

    Mara Giavina-Bianchi

    2015-01-01

    Full Text Available NY-ESO-1 is a cancer-testis antigen aberrantly expressed in melanomas, which may serve as a robust and specific target in immunotherapy. NY-ESO-1 antigen expression, tumor features, and the immune profile of tumor infiltrating lymphocytes were assessed in primary cutaneous melanoma. NY-ESO-1 protein was detected in 20% of invasive melanomas (16/79, rarely in in situ melanoma (1/10 and not in benign nevi (0/20. Marked intratumoral heterogeneity of NY-ESO-1 protein expression was observed. NY-ESO-1 expression was associated with increased primary tumor thickness (P=0.007 and inversely correlated with superficial spreading melanoma (P<0.02. NY-ESO-1 expression was also associated with reduced numbers and density of CD3+ tumor infiltrating lymphocytes (P=0.017. When NY-ESO-1 protein was expressed, CD3+ T cells were less diffusely infiltrating the tumor and were more often arranged in small clusters (P=0.010 or as isolated cells (P=0.002 than in large clusters of more than five lymphocytes. No correlation of NY-ESO-1 expression with gender, age, tumor site, ulceration, lymph node sentinel status, or survival was observed. NY-ESO-1 expression in melanoma was associated with tumor progression, including increased tumor thickness, and with reduced tumor infiltrating lymphocytes.

  6. Lineage determination of CD7+ CD5- CD2- and CD7+ CD5+ CD2- lymphoblasts: studies on phenotype, genotype, and gene expression of myeloperoxidase, CD3 epsilon, and CD3 delta.

    Science.gov (United States)

    Yoneda, N; Tatsumi, E; Teshigawara, K; Nagata, S; Nagano, T; Kishimoto, Y; Kimura, T; Yasunaga, K; Yamaguchi, N

    1994-04-01

    The gene expression of myeloperoxidase (MPO), CD3 epsilon, and CD3 delta molecules, the gene rearrangement of T-cell receptor (TCR) delta, gamma, and beta and immunoglobulin heavy (IgH) chain, and the expression of cell-surface antigens were investigated in seven cases of CD7+ CD5- CD2- and four cases of CD7+ CD5+ CD2- acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LBL) blasts, which were negative for cytochemical myeloperoxidase (cyMPO). More mature T-lineage blasts were also investigated in a comparative manner. In conclusion, the CD7+ CD5- CD2- blasts included four categories: undifferentiated blasts without lineage commitment, T-lineage blasts, T-/myeloid lineage blasts, and cyMPO-negative myeloblasts. The CD7+ CD5+ CD2- blasts included two categories; T-lineage and T-/myeloid lineage blasts. The 11 cases were of the germ-line gene (G) for TCR beta and IgH. Four cases were G for TCR delta and TCR gamma. The others were of the monoclonally rearranged gene (R) for TCR delta and G for TCR gamma or R for both TCR delta and TCR gamma. The expression or in vitro induction of CD13 and/or CD33 antigens correlated with the immaturity of these neoplastic T cells, since it was observed in all 11 CD7+ CD5- CD2- and CD7+ CD5+ CD2-, and some CD7+ CD5+ CD2+ (CD3- CD4- CD8-) cases, but not in CD3 +/- CD4+ CD8+ or CD3+ CD4+ CD8- cases. CD3 epsilon mRNA, but not CD3 delta mRNA, was detected in two CD7+ CD5- CD2- cases, while mRNA of neither of the two CD3 molecules was detected in the other tested CD7+ CD5- CD2- cases. In contrast, mRNA of both CD3 epsilon and CD3 delta were detected in all CD7+ CD5+ CD2- cases, indicating that CD7+ CD5- CD2- blasts at least belong to T-lineage. The blasts of two CD7+ CD5- CD2- cases with entire germ-line genes and without mRNA of the three molecules (MPO, CD3 epsilon, and CD3 delta) were regarded as being at an undifferentiated stage prior to their commitment to either T- or myeloid-lineage. The co-expression of the genes of MPO

  7. Radiolabelled anti-human fibrin antibody: a new thrombus-detecting agent

    International Nuclear Information System (INIS)

    Bosnjakovic, V.; Jankovic, B.D.; Horvat, J.; Cvoric, J.

    1977-01-01

    Rabbit anti-human fibrin globulin (A.F.G.) was labelled with iodine ( 131 I) and used as a thrombus-detecting agent. 131 I-A.F.G. labelled thrombi were displayed by means of a gamma scintillation camera. Normal subjects and patients with thrombo-phlebitis of legs, acute fibrin depositions other than thrombi, and chronic varicosities were examined. The 131 I-A.F.G. technique detected both formed thrombi and those that were forming and could discriminate between acute thrombosis and chronic varicosities. Thrombo-phlebitis and extravascular fibrin depositions were best demonstrated between 24 and 72 hours after 131 I-A.F.G. injection. Radiolabelled A.F.G. in normal veins and chronic varicosities was best displayed within 6 hours of injection. (author)

  8. Pre-TCRα supports CD3-dependent reactivation and expansion of TCRα-deficient primary human T-cells

    Directory of Open Access Journals (Sweden)

    Román Galetto

    2014-01-01

    Full Text Available Chimeric antigen receptor technology offers a highly effective means for increasing the anti-tumor effects of autologous adoptive T-cell immunotherapy, and could be made widely available if adapted to the use of allogeneic T-cells. Although gene-editing technology can be used to remove the alloreactive potential of third party T-cells through destruction of either the α or β T-cell receptor (TCR subunit genes, this approach results in the associated loss of surface expression of the CD3 complex. This is nonetheless problematic as it results in the lack of an important trophic signal normally mediated by the CD3 complex at the cell surface, potentially compromising T-cell survival in vivo, and eliminating the potential to expand TCR-knockout cells using stimulatory anti-CD3 antibodies. Here, we show that pre-TCRα, a TCRα surrogate that pairs with TCRβ chains to signal proper TCRβ folding during T-cell development, can be expressed in TCRα knockout mature T-cells to support CD3 expression at the cell surface. Cells expressing pre-TCR/CD3 complexes can be activated and expanded using standard CD3/CD28 T-cell activation protocols. Thus, heterologous expression of pre-TCRα represents a promising technology for use in the manufacturing of TCR-deficient T-cells for adoptive immunotherapy applications.

  9. Giant anisotropic magnetoresistance and planar Hall effect in the Dirac semimetal Cd3As2

    Science.gov (United States)

    Li, Hui; Wang, Huan-Wen; He, Hongtao; Wang, Jiannong; Shen, Shun-Qing

    2018-05-01

    Anisotropic magnetoresistance is the change tendency of resistance of a material on the mutual orientation of the electric current and the external magnetic field. Here, we report experimental observations in the Dirac semimetal Cd3As2 of giant anisotropic magnetoresistance and its transverse version, called the planar Hall effect. The relative anisotropic magnetoresistance is negative and up to -68% at 2 K and 10 T. The high anisotropy and the minus sign in this isotropic and nonmagnetic material are attributed to a field-dependent current along the magnetic field, which may be induced by the Berry curvature of the band structure. This observation not only reveals unusual physical phenomena in Weyl and Dirac semimetals, but also finds additional transport signatures of Weyl and Dirac fermions other than negative magnetoresistance.

  10. Dynamics for CH3F and CD3F isolated in rare gas solids

    International Nuclear Information System (INIS)

    Jones, L.H.; Swanson, B.I.

    1982-01-01

    High resolution infrared spectra have been obtained for CH 3 F and CD 3 F in argon and krypton matrices from 3 to 39 K. Low-temperature spectra show structure indicative of two stable monomer sites. As temperature is raised, the structure collapses to give symmetric peaks for each mode. The line shapes and their broadening with temperature show large dependence on the modes, isotope, and host matrix. On the basis of the absence of rovibrational structure and the very different line shapes for the various modes, we conclude that free or hindered rotation does not contribute to the absorption lines of methyl fluoride in argon or krypton matrices below 40 K. The mode dependent line broadening with increasing temperatures has been attributed to vibrational dephasing involving low energy site local phonon modes. Implications with regard to vibrational energy relaxation, which is currently thought to be dominated by a vibration to rotation energy transfer, are discussed

  11. Application of four anti-human interferon-alpha monoclonal antibodies for immunoassay and comparative analysis of natural interferon-alpha mixtures

    International Nuclear Information System (INIS)

    Andersson, G.; Lundgren, E.; Ekre, H.P.

    1991-01-01

    Four different mouse monoclonal antibodies to human interferon-alpha (IFN-alpha) were evaluated for application in quantitative and comparative analysis of natural IFN-alpha mixtures. Binding to IFN-alpha subtypes in solution revealed individual reactivity patterns. These patterns changed if the IFN-alpha molecules were immobilized either passively to a surface or bound by another antibody. Also, substitution of a single amino acid in IFN-alpha 2 affected the binding, apparently by altering the conformation. Isoelectric focusing of three natural IFN-alpha preparations from different sources, followed by immunoblotting, resulted in individual patterns with each of the four mAbs and also demonstrated variation in the composition of the IFN-alpha preparations. None of the mAbs was subtype specific, but by combining the different mAbs, and also applying polyclonal anti-human IFN-alpha antibodies, it was possible to design sensitive sandwich ELISAs with broad or more limited IFN-alpha subtype specificity

  12. Raman study of vibrational dephasing in liquid CH3CN and CD3CN

    International Nuclear Information System (INIS)

    Schroeder, J.; Schiemann, V.H.; Sharko, P.T.; Jonas, J.

    1977-01-01

    The Raman line shapes of the ν 1 (a 1 ) C--H and C--D fundamentals in liquid acetonitrile and acetonitrile-d 3 have been measured as a function of pressure up to 4 kbar within the temperature interval 30--120 degreeC. Densities have also been determined. From the isotropic component of the vibrational Raman band shape the vibrational relaxation times have been obtained as a function of temperature and pressure (density). The experimental results can be summarized as follows: (i) as T increases at constant density rho, the vibrational relaxation rate (tau/sub vib/) -1 increases; (ii) at constant T as density is raised tau/sub vib/ -1 increases; (iii) at constant pressure the T increase produces higher tau/sub vib/ -1 , however, the change is more pronounced for the CD 3 CN liquid. Isotopic dilution studies of the CH 3 CN/CD 3 CN mixtures shows no significant effect on (tau/sub vib/ -1 ). The experimental data are interpreted in terms of the Kubo stochastic line shape theory and the dephasing model of Fischer and Laubereau. The results based on Kubo formalism indicate that dephasing is the dominant relaxation mechanism and that the modulation is fast. The isolated binary collision model proposed by Fischer and Laubereau for vibrational dephasing reproduces the essential features of the density and temperature dependence of the (tau/sub vib/) -1 and suggests that pure dephasing is the dominant broadening mechanism for the isotropic line shapes studied. In the calculation the elastic collision times were approximated by the Enskog relaxation times

  13. Different signaling pathways induced by alpha-CD3 monoclonal antibody versus alloantigen on the basis of differential ornithine sensitivity.

    Science.gov (United States)

    Mehrotra nee Tandon, P; Lind, D S; Bear, H D; Susskind, B M

    1992-08-01

    Previously we reported that 10 mM ornithine (Orn) selectively inhibits the development of CD8+ CTL in MLC. Herein we show that induction by alpha-CD3 mAb of CD8+ killer cells which manifest antibody-redirected cytotoxicity (ARC) of FcR+ targets is not Orn sensitive. Orn resistance was independent of activation kinetics or alpha-CD3 mAb concentration. alpha-CD3 mAb added to the cytotoxicity assay did not reveal a cytolytic potential in CTL from an Orn-treated MLC when the target cells bore both the inducing alloantigen and FcR. Addition of alpha-CD3 mAb to MLC failed to overcome Orn inhibition of CTL and yet induced ARC activity in the same culture. Expression of mRNA for pore-forming proteins (PFP) and granzyme B was inhibited by Orn in CTL but not in ARC killer cells. Our results demonstrate differences in the T cell activation process stimulated by alloantigen or alpha-CD3 mAb.

  14. T cell receptor zeta allows stable expression of receptors containing the CD3gamma leucine-based receptor-sorting motif

    DEFF Research Database (Denmark)

    Dietrich, J; Geisler, C

    1998-01-01

    The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently of phosph......The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently...... of phosphorylation and leads to rapid internalization and sorting of these chimeras to lysosomal degradation. Because the TCRzeta chain rescues incomplete TCR complexes from lysosomal degradation and allows stable surface expression of fully assembled TCR, we addressed the question whether TCRzeta has the potential...... to mask the CD3gamma leucine-based motif. By studying CD4/CD3gamma and CD16/CD3gamma chimeras, we found that CD16/CD3gamma chimeras associated with TCRzeta. The CD16/CD3gamma-TCRzeta complexes were stably expressed at the cell surface and had a low spontaneous internalization rate, indicating...

  15. [Preparation and preliminary application of rabbit anti-human PON2 antibodies(paraoxonase-2)].

    Science.gov (United States)

    Chen, Miao; Yang, Jin-Ju; Li, Shu-Zhen; Liu, Xiao-Lan; Liu, Ying; Zhang, Lin-Jie; Gao, Jian-En; Sun, Qi-Hong

    2008-07-01

    To preparation and characterize the rabbit polyclonal antibodies against human PON2 (paraoxonase-2). A fragment of human PON2 gene which was of low homology with rabbits but of higher hydrophilicity and immunogenicity was selected for recombinant expression in prokaryotic expression system. The rabbits were immunized with the purified GST fusion protein 3 times. The specificity and sensitivity of the anti-human PON2 polyclonal antibodies were detected by Western blot and indirect immunofluorescence. The GST-PON2 fusion protein was highly expressed in Ecoli with a molecular weight of 46 kDa. Western blot analysis proved the rabbit polyclonal antibodies could specifically recognize 39 kDa native PON2 protein expressed in several cells and tissues, such as HeLa cells, U937 cells, and human liver tissue. Indirect immunofluorescence analysis confirmed that PON2 protein was located in the cytoplasm of SY5Y cells. The rabbit polyclonal antibodies against human PON2 can specifically recognize natural protein expressed in human cells and tissues, Which can be used for further study and clinical detection of human PON2.

  16. Communication: Reactivity borrowing in the mode selective chemistry of H + CHD3 → H2 + CD3

    Science.gov (United States)

    Ellerbrock, Roman; Manthe, Uwe

    2017-12-01

    Quantum state-resolved reaction probabilities for the H + CHD3 → H2 + CD3 reaction are calculated by accurate full-dimensional quantum dynamics calculations using the multi-layer multi-configurational time-dependent Hartree approach and the quantum transition state concept. Reaction probabilities of various ro-vibrational states of the CHD3 reactant are investigated for vanishing total angular momentum. While the reactivity of the different vibrational states of CHD3 mostly follows intuitive patterns, an unusually large reaction probability is found for CHD3 molecules triply excited in the CD3 umbrella-bending vibration. This surprising reactivity can be explained by a Fermi resonance-type mixing of the single CH-stretch excited and the triple CD3 umbrella-bend excited vibrational states of CHD3. These findings show that resonant energy transfer can significantly affect the mode-selective chemistry of CHD3 and result in counter-intuitive reactivity patterns.

  17. Single-cell analysis reveals a link between CD3- and CD59-mediated signaling pathways in Jurkat T cells

    International Nuclear Information System (INIS)

    Lipp, A. M.

    2012-01-01

    Elevation of intracellular free calcium concentration ([Ca2+]i) is a key signal during T cell activation and is commonly used as a read-out parameter for stimulation of T cell signaling. Upon T cell stimulation a variety of calcium signals is produced by individual cells of the T cell population and the type of calcium signal strongly influences cell fate decisions. The heterogeneous nature of T cells is masked in ensemble measurements, which highlights the need for single-cell measurements. In this study we used single-cell calcium measurements in Jurkat cells to investigate signaling pathways, which are triggered by different proteins, namely CD3 and CD59. By application of an automated cluster algorithm the presented assay provides unbiased analysis of a large data set of individual calcium time traces generated by the whole cell population. By using this method we could demonstrate that the Jurkat population generates heterogeneous calcium signals in a stimulus-dependent manner. Furthermore, our data revealed the existence of a link between CD3- and CD59-mediated signaling pathways. Single-cell calcium measurements in Jurkat cells expressing different levels of the T cell receptor (TCR) complex indicated that CD59-mediated calcium signaling is critically dependent on TCR surface expression levels. In addition, triggering CD59-mediated calcium signaling resulted in down-regulation of TCR surface expression levels, which is known to happen upon direct TCR triggering too. Moreover, by using siRNA-mediated protein knock-downs and protein knock-out Jurkat mutants we could show that CD3- and CD59-mediated calcium signaling require identical key proteins. We therefore explored by which mechanism CD59-mediated signaling couples into TCR-mediated signaling. Fluorescence recovery after photobleaching (FRAP) experiments and live-cell protein-protein interaction assays provided no evidence of a direct physical interaction between CD3- and CD59-mediated signaling pathways

  18. Genetic Association of CD247 (CD3ζ) with SLE in a Large-Scale Multiethnic Study

    OpenAIRE

    Martins, Madalena; Williams, Adrienne H.; Comeau, Mary; Marion, Miranda; Ziegler, Julie T.; Freedman, Barry I.; Merrill, Joan T.; Glenn, Stuart B.; Kelly, Jennifer A.; Sivils, Kathy M.; James, Judith A.; Guthridge, Joel M.; Alarcón-Riquelme, Marta E.; Bae, Sang-Cheol; Kim, Jae-Hoon

    2015-01-01

    A classic T-cell phenotype in systemic lupus erythematosus (SLE) is the downregulation and replacement of the CD3ζ chain that alters T-cell receptor signaling. However, genetic associations with SLE in the human CD247 locus that encodes CD3ζ are not well established and require replication in independent cohorts. Our aim was therefore to examine, localize and validate CD247-SLE association in a large multiethnic population. We typed 44 contiguous CD247 single-nucleotide polymorphisms (SNPs) i...

  19. An FDA oncology analysis of CD3 bispecific constructs and first-in-human dose selection.

    Science.gov (United States)

    Saber, Haleh; Del Valle, Pedro; Ricks, Tiffany K; Leighton, John K

    2017-11-01

    We retrospectively examined the nonclinical studies conducted with 17 CD3 bispecific constructs in support of first-in-human (FIH) trials in oncology. We also collected information on the design of dose-finding clinical trials. Sponsors have used different MABEL approaches for FIH dose selection. To better assess acceptable approaches, FIH doses were computed from nonclinical studies and compared to the maximum tolerated doses (MTDs) in patients, to the highest human doses (HHDs) when an MTD was not identified, or to the recommended human dose (RHD) for blinatumomab. We concluded that approaches based on receptor occupancy, highest non-severely toxic dose, or no-observed adverse effect level are not acceptable for selecting the FIH dose as they resulted in doses close to or above the MTDs, HHDs, or the RHD. A FIH dose corresponding to 10%-30% pharmacologic activity (PA) was an acceptable approach. A FIH dose corresponding to 50% PA was acceptable for all except one construct, potentially due to its biological or structural properties. The most common toxicities in animals and patients were those related to cytokine release. Doses were better tolerated when intra-animal or intra-patient dose escalation was used. Exposing naïve patients to an MTD achieved with intra-patient dose escalation design may be unsafe. Published by Elsevier Inc.

  20. Kinetics of the CH3 + HCl/DCl → CH4/CH3D + Cl and CD3 + HCl/DCl → CD3H/CD4 + Cl reactions: An experimental H atom tunneling investigation

    International Nuclear Information System (INIS)

    Eskola, Arkke J.; Seetula, Jorma A.; Timonen, Raimo S.

    2006-01-01

    The kinetics of the radical reactions of CH 3 with HCl or DCl and CD 3 with HCl or DCl have been investigated in a temperature controlled tubular reactor coupled to a photoionization mass spectrometer. The CH 3 (or CD 3 ) radical, R, was produced homogeneously in the reactor by a pulsed 193 nm exciplex laser photolysis of CH 3 COCH 3 (or CD 3 COCD 3 ). The decay of CH 3 /CD 3 was monitored as a function of HCl/DCl concentration under pseudo-first-order conditions to determine the rate constants as a function of temperature, typically from 188 to 500 K. The rate constants of the CH 3 and CD 3 reactions with HCl had strong non-Arrhenius behavior at low temperatures. The rate constants were fitted to a modified Arrhenius expression k = QA exp (-E a /RT) (error limits stated are 1σ + Students t values, units in cm 3 molecule -1 s -1 ): k(CH 3 + HCl) = [1.004 + 85.64 exp (-0.02438 x T/K)] x (3.3 ± 1.3) x 10 -13 exp [-(4.8 ± 0.6) kJ mol -1 /RT] and k(CD 3 + HCl) = [1.002 + 73.31 exp (-0.02505 x T/K)] x (2.7 ± 1.2) x 10 -13 exp [-(3.5 ± 0.5) kJ mol -1 /RT]. The radical reactions with DCl were studied separately over a wide ranges of temperatures and in these temperature ranges the rate constants determined were fitted to a conventional Arrhenius expression k = A exp (-E a /RT) (error limits stated are 1σ + Students t values, units in cm 3 molecule -1 s -1 ): k(CH 3 + DCl) = (2.4 ± 1.6) x 10 -13 exp [-(7.8 ± 1.4) kJ mol -1 /RT] and k(CD 3 + DCl) = (1.2 ± 0.4) x 10 -13 exp [-(5.2 ± 0.2) kJ mol -1 /RT] cm 3 molecule -1 s -1 . Curvature in the Arrhenius plots of the H-atom abstraction reactions at low temperatures was analyzed by considering H-atom tunneling through the reaction barrier and primary kinetic isotope effect. Contribution of tunneling in it was concluded to be negligible. In addition, secondary isotope effect was measured

  1. A novel recombinantly produced banana lectin isoform is a valuable tool for glycoproteomics and a potent modulator of the proliferation response in CD3(+), CD4(+), and CD8(+) populations of human PBMCs

    DEFF Research Database (Denmark)

    Gavrovic-Jankulovic, M; Poulsen, Knud; Brckalo, T

    2008-01-01

    Lectins as carbohydrate-binding proteins have been employed in various biological assays for the detection and characterization of glycan structures on glycoproteins, including clinical biomarkers in disease states. A mannose-specific banana lectin (BanLec) is unique in its specificity for internal......, the immunomodulatory potential of rBanLec and nBanLec were comparable as assessed by an inhibition assay and a human T cell proliferation assay where they induced a strong proliferation response in CD3(+), CD4(+), and CD8(+) populations of human PBMCs. This recombinant BanLec is a useful reagent for glycoproteomics...

  2. Clinical significance of serum anti-human papillomavirus 16 and 18 antibodies in cervical neoplasia.

    Science.gov (United States)

    Chay, Doo Byung; Cho, Hanbyoul; Kim, Bo Wook; Kang, Eun Suk; Song, Eunseop; Kim, Jae-Hoon

    2013-02-01

    To estimate the clinical significance of serum anti-human papillomavirus (HPV) antibodies and high-risk cervical HPV DNA in cervical neoplasia. The study population comprised patients who were histopathologically diagnosed with cervical intraepithelial neoplasia (CIN) 1 (n=64), CIN 2 and 3 (n=241), cervical cancer (n=170), and normal control participants (n=975). Cervical HPV DNA tests were performed through nucleic acid hybridization assay tests, and serum anti-HPV 16 and 18 antibodies were measured by competitive immunoassay. The associations of HPV DNA and anti-HPV antibodies were evaluated with demographic characteristics and compared according to the levels of disease severity. Anti-HPV antibodies were also investigated with clinicopathologic parameters, including survival data. Among various demographic characteristics, factors involving sexual behavior had a higher tendency of HPV DNA positivity and HPV seropositivity. Human papillomavirus DNA mean titer and positivity were both increased in patients with cervical neoplasia compared with those with normal control participants, but there was no statistical difference among types of cervical neoplasia. Serum anti-HPV 16 antibodies were also able to differentiate cervical neoplasia from a normal control participant and furthermore distinguished CIN 1 from CIN 2 and 3 (odd ratio 2.87 [1.43-5.78], P=.002). In cervical cancer, HPV 16 seropositivity was associated with prolonged disease-free survival according to the univariable analysis (hazard ratio=0.12 [0.01-0.94], P=.044). Serum anti-HPV 16 antibodies can distinguish cervical neoplasia from a normal control and has the advantage of identifying high-grade CIN. Moreover, in cervical cancer, HPV 16 seropositivity may be associated with a more favorable prognosis. II.

  3. Immunohistochemical Analysis of Inflammatory Rheumatoid Synovial Tissues Using Anti-Human Podoplanin Monoclonal Antibody Panel.

    Science.gov (United States)

    Suzuki, Tomoto; Takakubo, Yuya; Oki, Hiroharu; Liu, Xing; Honma, Ryusuke; Naganuma, Yasushi; Goodman, Stuart B; Kaneko, Mika K; Kato, Yukinari; Takagi, Michiaki

    2018-02-01

    Podoplanin (PDPN) is a transmembrane sialoglycoprotein, which is expressed in several normal tissues and malignant tumors. Although PDPN expression in rheumatoid arthritis (RA) has been reported, the role of PDPN in RA and other arthritic conditions has not been fully elucidated. In this study, we examined PDPN expression in inflammatory synovial tissues using an anti-human PDPN (hPDPN) monoclonal antibody (mAb) panel to select the most useful one for evaluation of synovitis. Synovial tissue samples were obtained from 11 RA patients and 9 osteoarthritis (OA) patients undergoing joint surgery. PDPN-positive cells were immunostained by a panel of PDPN mAbs (NZ-1, LpMab-3, LpMab-7, LpMab-10, LpMab-12, LpMab-13, and LpMab-17), followed by cell grading of inflammation and cell counting of PDPN-positivity by a quantitative analyzer. Immunohistochemistry showed that PDPN was markedly expressed in both macrophage-like type A and fibroblast-like type B lining cells of the hyperplastic synovial lining cell layer, and macrophages and fibroblasts in the stroma of RA. Among anti-PDPN mAbs, LpMab-12 showed the highest score. In inflammatory OA synovium, PDPN expression was also detectable. Although LpMab-12 also showed the highest score in OA, the difference was not statistically significant. The inflammatory synovitis score of RA was significantly higher than that of OA. PDPN was expressed in inflammatory lining cells and sublining stroma of RA and OA synovium. In the seven anti-hPDPN antibodies examined, LpMab-12 was the most stainable antibody for PDPN in RA synovitis. Thus, LpMab-12 for PDPN has a possible and promising specific biomarker for evaluating synovitis in RA and inflammatory OA.

  4. Anti-human SIRPα antibody is a new tool for cancer immunotherapy.

    Science.gov (United States)

    Murata, Yoji; Tanaka, Daisuke; Hazama, Daisuke; Yanagita, Tadahiko; Saito, Yasuyuki; Kotani, Takenori; Oldenborg, Per-Arne; Matozaki, Takashi

    2018-02-23

    Interaction of signal regulatory protein α (SIRPα) expressed on the surface of macrophages with its ligand CD47 expressed on target cells negatively regulates phagocytosis of the latter cells by the former. We recently showed that blocking Abs to mouse SIRPα enhanced both the Ab-dependent cellular phagocytosis (ADCP) activity of mouse macrophages for Burkitt's lymphoma Raji cells opsonized with an Ab to CD20 (rituximab) in vitro as well as the inhibitory effect of rituximab on the growth of tumors formed by Raji cells in nonobese diabetic (NOD)/SCID mice. However, the effects of blocking Abs to human SIRPα in preclinical cancer models have remained unclear given that such Abs have failed to interact with endogenous SIRPα expressed on macrophages of immunodeficient mice. With the use of Rag2 -/- γ c -/- mice harboring a transgene for human SIRPα under the control of human regulatory elements (hSIRPα-DKO mice), we here show that a blocking Ab to human SIRPα significantly enhanced the ADCP activity of macrophages derived from these mice for human cancer cells. The anti-human SIRPα Ab also markedly enhanced the inhibitory effect of rituximab on the growth of tumors formed by Raji cells in hSIRPα-DKO mice. Our results thus suggest that the combination of Abs to human SIRPα with therapeutic Abs specific for tumor antigens warrants further investigation for potential application to cancer immunotherapy. In addition, humanized mice, such as hSIRPα-DKO mice, should prove useful for validation of the antitumor effects of checkpoint inhibitors before testing in clinical trials. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  5. Approaches to lung cancer treatment using the CD3E x GP-2-directed bispecific monoclonal antibody BIS-1

    NARCIS (Netherlands)

    Kroesen, BJ; Nieken, J; Sleijfer, DT; Molema, G; deVries, EGE; Groen, HJM; Helfrich, W; The, TH; Mulder, NH; deLeij, L

    1997-01-01

    The bispecific monoclonal antibody (bsAb) BIS-1 combines a monoclonal-antibody(mAb)-defined specificity for the CD3 complex, as present on all T lymphocytes, with a mAb-defined specificity for the pancarcinoma/epithelium associated glycoprotein EGP-2. In vitro studies indicate that BIS-1 can direct

  6. Decreased Numbers of CD57+CD3- Cells Identify Potential Innate Immune Differences in Patients with Autism Spectrum Disorder.

    Science.gov (United States)

    Siniscalco, Dario; Mijatovic, Tatjana; Bosmans, Eugene; Cirillo, Alessandra; Kruzliak, Peter; Lombardi, Vincent C; De Meirleir, Kenny; Antonucci, Nicola

    2016-01-01

    Autism spectrum disorders (ASD) are complex, and severe heterogeneous neurodevelopmental pathologies with accepted but complex immune system abnormalities. Additional knowledge regarding potential immune dysfunctions may provide a greater understanding of this malady. The aim of this study was to evaluate the CD57(+)CD3(-) mature lymphocyte subpopulation of natural killer cells as a marker of immune dysfunction in ASD. Three-color flow cytometry-based analysis of fresh peripheral blood samples from children with autism was utilized to measure CD57(+)CD3(-) lymphocytes. A reduction of CD57(+)CD3(-) lymphocyte count was recorded in a significant number of patients with autism. We demonstrated that the number of peripheral CD57(+)CD3(-) cells in children with autism often falls below the clinically accepted normal range. This implies that a defect in the counter-regulatory functions necessary for balancing pro-inflammatory cytokines exists, thus opening the way to chronic inflammatory conditions associated with ASD. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  7. ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy

    DEFF Research Database (Denmark)

    Harwood, Seandean Lykke; Alvarez-Cienfuegos, Ana; Alanes, Natalia Nuñez del Prado

    2018-01-01

    The redirection of T cell activity using bispecific antibodies is one of the most promising cancer immunotherapy approaches currently in development, but it is limited by cytokine storm-related toxicities, as well as the pharmacokinetics and tumor-penetrating capabilities of current bispecific an...

  8. Anthocyanin contents in the seed coat of black soya bean and their anti-human tyrosinase activity and antioxidative activity.

    Science.gov (United States)

    Jhan, J-K; Chung, Y-C; Chen, G-H; Chang, C-H; Lu, Y-C; Hsu, C-K

    2016-06-01

    The seed coat of black soya bean (SCBS) contains high amount of anthocyanins and shows antioxidant and anti-mushroom tyrosinase activities. The objectives of this study were to analyse the anthocyanins in SCBS with different solvents and to find the relationship between anthocyanin profile with anti-human and anti-mushroom tyrosinase activities. SCBS was extracted with hot water, 50 and 80% ethanol, 50 and 80% acetone and 50 and 80% acidified acetone. Total phenol and total flavonoid contents in the extracts were determined. Anthocyanins in the extracts were analysed using HPLC and LC/MS/MS. A genetically engineered human tyrosinase was used to evaluate the anti-tyrosinase potential of the extracts from SCBS. 80% acetone extract from SCBS obtained the highest total phenol, total flavonoid and cyanidin-3-O-glucoside (C3G) contents among all the extracts, whereas the hot water extract showed the lowest antioxidant contents. Three anthocyanin compounds were found in all the extracts from SCBS, and the analysis of HPLC and LC/MS/MS indicated that they were C3G, delphinidin-3-O-glucoside (D3G) and peonidin-3-O-glucoside (P3G). The ratios of C3G (2.84 mg g(-1) ), D3G (0.34 mg g(-1) ) and P3G (0.35 mg g(-1) ) in 80% acidified acetone extract were 76.6, 9.1 and 9.3%, respectively. All the extracts from SCBS possessed anti-human tyrosinase activity. Moreover, a good correlation was found between the anti-human tyrosinase activities and C3G contents in the extracts. Antioxidants in SCBS also possess anti-human and anti-mushroom tyrosinase activities. © 2015 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  9. Signaling signatures and functional properties of anti-human CD28 superagonistic antibodies.

    Directory of Open Access Journals (Sweden)

    Zoe Waibler

    Full Text Available Superagonistic CD28 antibodies (CD28SAs activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4+ T cells but not in cynomolgus T lymphocytes. The sustained Ca++-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-gamma and TNF-alpha. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-gamma and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells.

  10. The Adaptor Protein SAP Directly Associates with CD3ζ Chain and Regulates T Cell Receptor Signaling

    Science.gov (United States)

    Proust, Richard; Bertoglio, Jacques; Gesbert, Franck

    2012-01-01

    Mutations altering the gene encoding the SLAM associated protein (SAP) are responsible for the X-linked lymphoproliferative disease or XLP1. Its absence is correlated with a defective NKT cells development, a decrease in B cell functions and a reduced T cells and NK cells cytotoxic activities, thus leading to an immunodeficiency syndrome. SAP is a small 128 amino-acid long protein that is almost exclusively composed of an SH2 domain. It has been shown to interact with the CD150/SLAM family of receptors, and in a non-canonical manner with SH3 containing proteins such as Fyn, βPIX, PKCθ and Nck1. It would thus play the role of a minimal adaptor protein. It has been shown that SAP plays an important function in the activation of T cells through its interaction with the SLAM family of receptors. Therefore SAP defective T cells display a reduced activation of signaling events downstream of the TCR-CD3 complex triggering. In the present work, we evidence that SAP is a direct interactor of the CD3ζ chain. This direct interaction occurs through the first ITAM of CD3ζ, proximal to the membrane. Additionally, we show that, in the context of the TCR-CD3 signaling, an Sh-RNA mediated silencing of SAP is responsible for a decrease of several canonical T cell signaling pathways including Erk, Akt and PLCγ1 and to a reduced induction of IL-2 and IL-4 mRNA. Altogether, we show that SAP plays a central function in the T cell activation processes through a direct association with the CD3 complex. PMID:22912825

  11. T−B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3ζ subunit of the T-cell antigen receptor complex

    OpenAIRE

    Roberts, Joseph L.; Lauritsen, Jens Peter H.; Cooney, Myriah; Parrott, Roberta E.; Sajaroff, Elisa O.; Win, Chan M.; Keller, Michael D.; Carpenter, Jeffery H.; Carabana, Juan; Krangel, Michael S.; Sarzotti, Marcella; Zhong, Xiao-Ping; Wiest, David L.; Buckley, Rebecca H.

    2007-01-01

    CD3ζ is a subunit of the T-cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. We report here a patient with T−B+NK+ severe combined immunodeficiency (SCID) who was homozygous for a single C insertion following nucleotide 411 in exon 7 of the CD3ζ gene. The few T cells present contained no detectable CD3ζ protein, expressed low levels of cell surface CD3ε, and were nonfunctional. CD4+CD8−CD...

  12. Close linkage of the mouse and human CD3 γ- and δ-chain genes suggests that their transcription is controlled by common regulatory elements

    International Nuclear Information System (INIS)

    Saito, H.; Koyama, T.; Georgopoulos, K.; Clevers, H.; Haser, W.G.; LeBien, T.; Tonegawa, S.; Terhorst, C.

    1987-01-01

    Antigen receptors on the T-cell surface are noncovalently associated with at least four invariant polypeptide chains, CD3-γ, -δ, -epsilon, and -zeta. The mouse CD3-γ gene, consisting of seven exons, was found to be highly homologous to the CD3-γ described earlier. Both the high level of sequence homology and the exon/intron organization indicate that the CD3-γ and -δ genes arose by gene duplication. Surprisingly, murine and human genomic DNA clones could be isolated that contained elements of both the CD3-γ and CD3-δ genes. In fact, the putative transcription start site of the mouse CD3-γ gene is less than 1.4 kilobases from the transcription initiation site of the mouse CD3-δ gene. Common elements that regulate the divergent transcription of the two genes are therefore proposed to be located in the intervening 1.4-kilobase DNA segment. This might contribute to the coordinate expression of the CD3-γ and -δ genes during intrathymic maturation of T lymphocytes

  13. Radiolabeling of anti-human prostatic specific membrane antigen antibody with 99Tcm and its biodistribution in nude mice bearing human prostate cancer

    International Nuclear Information System (INIS)

    Tu Shaohua; Shen Jiangfan; Tao Rong; Ji Xiaowen; Wang Yancheng

    2012-01-01

    Objective: To study the binding affinity of 99 Tc m labeled anti-human prostatic specific membrane antigen (PSMA) monoclonal antibody (McAb) J591 to prostate cancer cells and the biodistribution of 99 Tc m -J591 in nude mice bearing human prostate cancer. Methods: The McAb J591 was labeled with vTcm by improved Schwarz method and the labeled McAb was purified by Sephadex G-50. The binding affinity of J591 with prostate cancer cells was measured by Flow Cytometry. The nude mice bearing PSMA-positive C4-2 prostate carcinoma xenografts were served as experiment groups, mice with PSMA-negative pc3 tumors served as controls. The biodistribution of 99 Tc m -J591 were carried out in both model nude mice. Results: The radiolabeling efficiency of 99 Tc m -J591 was 78.9±6.2%, and radiochemical purity was more than 90% after purification. The 99 Tc m -J591 showed a good combination with PSMA-positive C4-2 cells and no combination with PSMA-negative PC3 cells in vitro. The biodistribution results showed that 99 Tcm-J591 was accumulated in tumor tissue during the 2-24 hours after injection in experiment groups, and no significant uptake in control group. The uptake of 99 Tcm-J591 in tumor tissue reached a maximum 15.91±5.16 % ID/g in experimental group at 12h post-injection. There was a significant difference compared with controls (P 0.05). Conclusion: The monoclonal antibody J591 exhibits an excellent immuno-reactivity and tumor targeting property, and it may be used in diagnosis and target therapy of prostate cancer. (authors)

  14. Thickness dependence of the quantum Hall effect in films of the three-dimensional Dirac semimetal Cd3As2

    Directory of Open Access Journals (Sweden)

    Manik Goyal

    2018-02-01

    Full Text Available Low-temperature magnetotransport studies are reported for (112Cd3As2 films grown on (111CdTe by molecular beam epitaxy as a function of the Cd3As2 film thickness. All films show Shubnikov-de Haas oscillations. An even-integer quantum Hall effect is observed for films thinner than 70 nm. For the thinnest films, the bulk is gapped and transport at low temperatures occurs only via the gapless, two-dimensional states. The lowest Landau level is reached at ∼10 T, and the longitudinal resistance nearly vanishes at the plateaus in the Hall resistance. The results are discussed in the context of the current theoretical understanding of topological surface states in three-dimensional Dirac semimetals.

  15. Anisotropic Fermi Surface and Quantum Limit Transport in High Mobility Three-Dimensional Dirac Semimetal Cd_{3}As_{2}

    Directory of Open Access Journals (Sweden)

    Yanfei Zhao

    2015-09-01

    Full Text Available Three-dimensional topological Dirac semimetals have a linear dispersion in 3D momentum space and are viewed as the 3D analogues of graphene. Here, we report angle-dependent magnetotransport on the newly revealed Cd_{3}As_{2} single crystals and clearly show how the Fermi surface evolves with crystallographic orientations. Remarkably, when the magnetic field lies in the [112] or [441[over ¯

  16. Caracterización de la cadena CD3 épsilon en el primate del nuevo mundo Aotus nancymaae

    Directory of Open Access Journals (Sweden)

    Jean Paul Vernot

    2008-06-01

    Conclusiones. La estructura primaria determinada aquí sugiere que la proteína de Aotus tiene una funcionalidad similar y que los pasos iniciales de activación de la célula T se suceden como en los humanos; por otra parte, la gran variabilidad encontrada en el ectodominio, permite explicar por qué algunos anticuerpos monoclonales dirigidos contra el complejo CD3 de humano no reconocen estas estructuras en el Aotus.

  17. Immunomodulatory effects of royal jelly on aorta CD3, CD68 and eNOS expression in hypercholesterolaemic rats

    Directory of Open Access Journals (Sweden)

    Amal Attia El-Morsy Ibrahim

    2014-08-01

    RJ administration (300 mg/kg/day with CH, produced a counteractive effect, represented in recompense of biochemical alteration, CD4 and CD8 expression. RJ intake also amended the histological picture. The immunohistochemical picture revealed a decrease in CD3, CD86 and eNOS in the aortic tissue. These findings attributed to the significant immunomodulatory effect of RJ remedy suppress deleterious effects of hypercholesterolaemia.

  18. A highly conserved phenylalanine in the alpha, beta-T cell receptor (TCR) constant region determines the integrity of TCR/CD3 complexes

    DEFF Research Database (Denmark)

    Caspar-Bauguil, S; Arnaud, J; Huchenq, A

    1994-01-01

    In the present study, we have investigated the importance of a phenylalanine (phe195) in the Tcr-C alpha region on Tcr-alpha,beta/CD3 membrane expression. An exchange of phe195 with a tyrosine residue does not affect Tcr/CD3 membrane expression; however, exchange with aspartic acid, histidine or ...

  19. Decline in peripheral blood NKG2D+CD3+CD56+ NKT cells in metastatic colorectal cancer patients.

    Science.gov (United States)

    Gharagozloo, M; Rezaei, A; Kalantari, H; Bahador, A; Hassannejad, N; Maracy, M; Nouri, N; Sedghi, M; Ghazanfari, H; Bayat, B

    2018-01-01

    Colorectal cancer (CRC) is one of the main causes of cancer deaths in the world. This cancer can be divided into non-metastatic and metastatic CRC stages. CD3+CD56+ NKT cell subsets are a minor T cell subset in peripheral blood and conduct the killing of tumor cells in direct manner. Little is obvious about levels and surface markers of these cells such as NKG2D in different cancers, especially in CRC. We included 15 non-metastatic (low-grade), 11 non-metastatic (high-grade), 10 metastatic colorectal cancer patients and 18 healthy controls. The percentages of CD3+CD56+ NKT cells and NKG2D+CD56+ NKT cells from samples were analyzed by flow cytometry in peripheral blood mononuclear cells (PBMCs) of samples. We found that there was a significantly lower number of NKG2D+CD3+CD56+ cells in peripheral blood of patients with metastatic colorectal cancer compared with normal controls (77.53 ± 5.79 % vs 90.74 ± 9.84 %; pNKT cells was significantly lower in patients with metastatic colorectal cancer compared to healthy controls strengthens the hypothesis that NKT cells can play a substantial role in the protection against human colorectal cancer, and this opens up avenues for novel studies about elucidating the other aspects of tumor surveillance in CRC progression and immunotherapy (Tab. 2, Fig. 2, Ref. 46).

  20. Synergistic reversal of type 1 diabetes in NOD mice with anti-CD3 and interleukin-1 blockade

    DEFF Research Database (Denmark)

    Ablamunits, Vitaly; Henegariu, Octavian; Hansen, Jakob Bondo

    2012-01-01

    (ab')(2) fragments of anti-CD3 mAb with or without IL-1 receptor antagonist (IL-1RA), or anti-IL-1ß mAb. We studied the reversal of diabetes and effects of treatment on the immune system. Mice that received a combination of anti-CD3 mAb with IL-1RA showed a more rapid rate of remission of diabetes than......Inflammatory cytokines are involved in autoimmune diabetes: among the most prominent is interleukin (IL)-1ß. We postulated that blockade of IL-1ß would modulate the effects of anti-CD3 monoclonal antibody (mAb) in treating diabetes in NOD mice. To test this, we treated hyperglycemic NOD mice with F...... arginase expression in macrophages and dendritic cells, and had delayed adoptive transfer of diabetes. After 1 month, there were increased concentrations of IgG1 isotype antibodies and reduced intrapancreatic expression of IFN-¿, IL-6, and IL-17 despite normal splenocyte cytokine secretion. These studies...

  1. Two-Carrier Transport Induced Hall Anomaly and Large Tunable Magnetoresistance in Dirac Semimetal Cd3As2 Nanoplates.

    Science.gov (United States)

    Li, Cai-Zhen; Li, Jin-Guang; Wang, Li-Xian; Zhang, Liang; Zhang, Jing-Min; Yu, Dapeng; Liao, Zhi-Min

    2016-06-28

    Cd3As2 is a model material of Dirac semimetal with a linear dispersion relation along all three directions in the momentum space. The unique band structure of Cd3As2 is made with both Dirac and topological properties. It can be driven into a Weyl semimetal by symmetry breaking or a topological insulator by enhancing the spin-orbit coupling. Here we report the temperature and gate voltage-dependent magnetotransport properties of Cd3As2 nanoplates with Fermi level near the Dirac point. The Hall anomaly demonstrates the two-carrier transport accompanied by a transition from n-type to p-type conduction with decreasing temperature. The carrier-type transition is explained by considering the temperature-dependent spin-orbit coupling. The magnetoresistance exhibits a large nonsaturating value up to 2000% at high temperatures, which is ascribed to the electron-hole compensation in the system. Our results are valuable for understanding the experimental observations related to the two-carrier transport in Dirac/Weyl semimetals, such as Na3Bi, ZrTe5, TaAs, NbAs, and HfTe5.

  2. NSOM/QD-Based Visualization of GM1 Serving as Platforms for TCR/CD3 Mediated T-Cell Activation

    Directory of Open Access Journals (Sweden)

    Liyun Zhong

    2013-01-01

    Full Text Available Direct molecular imaging of nanoscale relationship between T-cell receptor complexes (TCR/CD3 and gangliosidosis GM1 before and after T-cell activation has not been reported. In this study, we made use of our expertise of near-field scanning optical microscopy(NSOM/immune-labeling quantum dots- (QD-based dual-color imaging system to visualize nanoscale profiles for distribution and organization of TCR/CD3, GM1, as well as their nanospatial relationship and their correlation with PKCθ signaling cascade during T-cell activation. Interestingly, after anti-CD3/anti-CD28 Ab co-stimulation, both TCR/CD3 and GM1 were clustered to form nanodomains; moreover, all of TCR/CD3 nanodomains were colocalized with GM1 nanodomains, indicating that the formation of GM1 nanodomains was greatly correlated with TCR/CD3 mediated signaling. Specially, while T-cells were pretreated with PKCθ signaling inhibitor rottlerin to suppress IL-2 cytokine production, no visible TCR/CD3 nanodomains appeared while a lot of GM1 nanodomains were still observed. However, while T-cells are pretreated with PKCαβ signaling inhibitor GÖ6976 to suppress calcium-dependent manner, all of TCR/CD3 nanodomains were still colocalized with GM1 nanodomains. These findings possibly support the notion that the formation of GM1 nanodomains indeed serves as platforms for the recruitment of TCR/CD3 nanodomains, and TCR/CD3 nanodomains are required for PKCθ signaling cascades and T-cell activation

  3. NKp46+CD3+ cells - a novel non-conventional T-cell subset in cattle exhibiting both NK cell and T-cell features

    Science.gov (United States)

    Connelley, Timothy K.; Longhi, Cassandra; Burrells, Alison; Degnan, Kathryn; Hope, Jayne; Allan, Alasdair; Hammond, John A.; Storset, Anne K.; Morrison, W. Ivan

    2014-01-01

    The NKp46 receptor demonstrates a high degree of lineage-specificity, being expressed almost exclusively in natural killer cells. Previous studies have demonstrated NKp46 expression by T-cells, but NKp46+CD3+ cells are rare and almost universally associated with NKp46 acquisition by T-cells following stimulation. In this study we demonstrate the existence of a population of NKp46+CD3+ cells resident in normal bovine PBMC which include cells of both the αβ TCR+ and γδ TCR+ lineages and is present at a frequency of 0.1-1.7%. NKp46+CD3+ cells express transcripts for a broad repertoire of both natural killer (NKR) and T-cell receptors (TCR) and also the CD3ζ, DAP10 and FcεR1γ but not DAP12 adaptor proteins. In vitro functional analysis of NKp46+CD3+ cells confirm that NKp46, CD16 and CD3 signalling pathways are all functionally competent and capable of mediating-re-direct cytolysis. However, only CD3 cross-ligation elicits IFN-γ release. NKp46+CD3+ cells exhibit cytotoxic activity against autologous Theileria parva infected cells in vitro and during in vivo challenge with this parasite an expansion of NKp46+CD3+ cells was observed in some animals, indicating the cells have the potential to act as an anti-pathogen effector population. The results presented herein identifies and describes a novel non-conventional NKp46+CD3+ T-cell subset that is phenotypically and functionally distinct from conventional NK and T-cells. The ability to exploit both NKR and TCR suggests these cells may fill a functional niche at the interface of innate and adaptive immune responses. PMID:24639352

  4. Regional and systemic distribution of anti-tumor x anti-CD3 heteroaggregate antibodies and cultured human peripheral blood lymphocytes in a human colon cancer xenograft

    International Nuclear Information System (INIS)

    Nelson, H.; Ramsey, P.S.; Kerr, L.A.; McKean, D.J.; Donohue, J.H.

    1990-01-01

    Anti-tumor antibody (317G5) covalently coupled to an anti-CD3 antibody (OKT3) produces a heteroaggregate (HA) antibody that can target PBL to lyse tumor cells expressing the appropriate tumor Ag. The i.v. and i.p. distribution of radiolabeled HA antibody 317G5 x OKT3 and of radiolabeled cultured human PBL were studied in athymic nude mice bearing solid intraperitoneal tumor established from the human colon tumor line, LS174T. Mice were injected with 125I-labeled HA antibody, 125I-labeled anti-tumor mAb, or 111In-labeled PBL, and at designated timepoints tissues were harvested and measured for radioactivity. 125I-317G5 x OKT3 localized specifically to tumor sites. Tumor radioactivity levels (percent injected dose/gram) were lower with 125I-317G5 x OKT3 HA antibody than with 125I-317G5 anti-tumor mAb, but were similar to levels reported for other anti-tumor mAb. The major difference in radioactivity levels observed between i.v. and i.p. administration of 125I-317G5 x OKT3 was an increase in hepatic radioactivity after i.v. HA antibody administration. HA antibodies produced from F(ab')2 fragments, which exhibit decreased m. w. and decreased Fc receptor-mediated binding, demonstrated improved tumor:tissue ratios as compared to intact antibody HA. 125I-317G5 F(ab')2 x OKT3 F(ab')2 antibody levels were equivalent to intact HA antibody levels in tumor, but were lower than intact HA antibody levels in the blood, bowel, and liver. Tumor:bowel ratios (20:1 at 48 h) were highest when 317G5 F(ab')2 x OKT3 F(ab')2 was injected i.p. Autoradiography confirmed that anti-tumor x anti-CD3 HA antibodies localized specifically to intraperitoneal tumor; that i.p. administered HA antibodies penetrated tumor directly; and that i.v. administered HA antibodies distributed along tumor vasculature

  5. Targeted radiotherapy potentiates the cytotoxicity of a novel anti-human DR5 monoclonal antibody and the adenovirus encoding soluble TRAIL in prostate cancer

    International Nuclear Information System (INIS)

    Arafat, W.; Arafat, W.; Zhou, T.; Naoum, G.E.; Buchsbaum, D.J.

    2015-01-01

    TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces a death signal following binding to death receptors (DR4, DR5). We have developed a novel anti-human DR-5 monoclonal antibody (TRA-8) and adenoviral encoding TRAIL (Ad/TRAIL). Herein, we are testing the combined effect of radiotherapy and TRA-8 or Ad TRAIL in prostate cancer cells. Human prostate cancer cell lines LnCap, PC-3 and DU145 were used in this study. Cells were treated either with TRA-8 alone or Ad/TRAIL, radiation alone, or a combination of each at different doses and intervals. Cell survival using the MTS assay and colony forming assay were used to determine radiosensitization. Immunohistochemistry was used to detect bax and bcl-2. Real-time PCR was performed on mRNA of treated prostate cancer cell lines. Finally, a murine model of subcutaneous prostate cancer was used to evaluate the in vivo effect. Cell survival assays detected by MTS assay showed that prostate cell lines treated with a combination of radiation and TRA-8 showed significantly lower survival than cells treated with either radiation or TRA-8 alone. Colony forming assay and cell proliferation assays showed increased killing after combination treatment with TRA-8 or Ad/TRAIL and radiation, than either single agent alone. Mechanistic studies showed that the killing effect was due to induction of apoptosis mostly by increased expression of bax in TRA-8 or Ad/TRAIL treated cells. Additionally, RT-PCR showed an increased copy number of bax in most cells treated with TRA-8 and radiation. It is concluded that radiation and TRA-8 or Ad/ TRAIL produced a synergistic effect in refractory prostrate cancer.

  6. The CD3 gamma leucine-based receptor-sorting motif is required for efficient ligand-mediated TCR down-regulation

    DEFF Research Database (Denmark)

    von Essen, Marina; Menné, Charlotte; Nielsen, Bodil L

    2002-01-01

    . The other pathway is dependent on protein kinase C (PKC)-mediated activation of the CD3 gamma di-leucine-based receptor-sorting motif. Previous studies have failed to demonstrate a connection between ligand- and PKC-induced TCR down-regulation. Thus, although an apparent paradox, the dogma has been...... that ligand- and PKC-induced TCR down-regulations are not interrelated. By analyses of a newly developed CD3 gamma-negative T cell variant, freshly isolated and PHA-activated PBMC, and a mouse T cell line, we challenged this dogma and demonstrate in this work that PKC activation and the CD3 gamma di...

  7. What is the effect of variations optimization of the transition state on α-deuterium secondary kinetic isotope effects? A prototype: CD3H + H right-reversible CD3 + H2

    International Nuclear Information System (INIS)

    Lu, Dahong; Maurice, D.; Truhlar, D.G.

    1990-01-01

    Variational Transition state theory calculations with semiclassical transmission coefficients have been carried out for a prototype case of α-deuterium secondary kinetic isotope effects (KIEs) in a reaction involving the transformation of an sp 3 carbon to sp 2 , in particular for the reactions of CH 4 and CD 3 H with H and D. The authors also study the KIE for the reverse direction and for the reactions of CH 4 and CD 3 H with D. They find that the variational transition states lead to significantly different nontunneling KIEs than the conventional ones, e.g., 1.22 vs. 1.07, and the inclusion of multidimensional tunneling effects increases the discrepancy even more. The origins of these variations and tunneling effects are examined in detail in terms of structures, vibrational frequencies, and the curvature of the reaction path. The conclusions have wide implications for the validity of conventional treatments of kinetic isotope effects. They predict some particularly large secondary KIEs at low temperature, and these predictions can be tested by future experiments

  8. Differential effect on TCR:CD3 stimulation of a 90-kD glycoprotein (gp90/Mac-2BP), a member of the scavenger receptor cysteine-rich domain protein family

    DEFF Research Database (Denmark)

    Silvestri, B; Calderazzo, F; Coppola, V

    1998-01-01

    We studied the effects of a 90-kD glycoprotein (gp90/Mac-2BP) belonging to the scavenger receptor family, present in normal serum and at increased levels in inflammatory disease and cancer patients, on some T cell function parameters. Whereas the lymphocyte proliferative response to non-specific ......We studied the effects of a 90-kD glycoprotein (gp90/Mac-2BP) belonging to the scavenger receptor family, present in normal serum and at increased levels in inflammatory disease and cancer patients, on some T cell function parameters. Whereas the lymphocyte proliferative response to non......-specific mitogens such as phytohaemagglutinin (PHA) and concanavalin A (Con A), but not pokeweed mitogen (PWM), was strongly reduced, probably due to the lectin-binding properties of gp90/Mac-2BP, the response to T cell receptor (TCR) agonists such as superantigens and allogeneic cells was potentiated. When...... lymphocytes were stimulated with different anti-TCR:CD3 MoAbs, both in soluble and solid-phase form, gp90/Mac-2BP was able to down-regulate the proliferative response to anti-CD3 MoAb, whereas the response to anti-TCR alphabeta MoAb was enhanced. A similar differential effect was observed when a MoAb against...

  9. Clinical Trials Using Anti-CD19/CD28/CD3zeta CAR Gammaretroviral Vector-transduced Autologous T Lymphocytes KTE-C19

    Science.gov (United States)

    NCI supports clinical trials that test new and more effective ways to treat cancer. Find clinical trials studying anti-cd19/cd28/cd3zeta car gammaretroviral vector-transduced autologous t lymphocytes kte-c19.

  10. Fine-tuning of T-cell development by the CD3γ di-leucine-based TCR-sorting motif

    DEFF Research Database (Denmark)

    Lauritsen, Jens Peter Holst; Boding, Lasse; Buus, Terkild B

    2015-01-01

    The CD3γ di-leucine-based (diL) receptor-sorting motif plays a central role in TCR down-regulation and in clonal expansion of virus-specific T cells. However, the role of the CD3γ diL motif in T-cell development is not known. In this study, we show that protein kinase C-induced TCR down-regulatio......The CD3γ di-leucine-based (diL) receptor-sorting motif plays a central role in TCR down-regulation and in clonal expansion of virus-specific T cells. However, the role of the CD3γ diL motif in T-cell development is not known. In this study, we show that protein kinase C-induced TCR down...

  11. Rapid Recovery of CD3+CD8+ T Cells on Day 90 Predicts Superior Survival after Unmanipulated Haploidentical Blood and Marrow Transplantation.

    Directory of Open Access Journals (Sweden)

    Deng-Mei Tian

    Full Text Available Rapid immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT is significantly associated with lower infection, relapse and possibly secondary malignancy rates. The aim of this study was to investigate the role of peripheral lymphocyte subsets, especially CD3+CD8+ cytotoxic T cell recovery, in predicting transplant outcomes, including the overall survival (OS and non-relapse mortality (NRM rates after unmanipulated haploidentical blood and marrow transplantation (HBMT.Peripheral blood samples were obtained from 214 HBMT recipients with hematological malignancies. The peripheral lymphocyte subsets (CD3+ T cells, CD3+CD4+ helper T cells, CD3+CD8+ cytotoxic T cells, and CD19+ B cells were analyzed by flow cytometry at days 30, 60, 90, 180, 270 and 360 after HBMT.The CD3+CD8+ cytotoxic T cell recovery at day 90 (CD3+CD8+-90 was correlated with bacterial infection (P = 0.001, NRM (P = 0.001, leukemia-free survival (LFS, P = 0.005, and OS (P = 0.001 at a cutoff value of 375 cells/μL CD3+CD8+ T cells. The incidence of bacterial infection in patients with the CD3+CD8+-90 at ≥375 cells/μL was significantly lower than that of cases with the CD3+CD8+-90 at <375 cells/μL after HBMT (14.6% versus 41.6%, P<0.001. Multivariate analysis showed the rapid recovery of CD3+CD8+ T cells at day 90 after HBMT was strongly associated with a lower incidence of NRM (HR = 0.30; 95% CI: 0.15-0.60; P = 0.000 and superior LFS (HR = 0.51; 95% CI: 0.32-0.82; P = 0.005 and OS (HR = 0.38; 95% CI: 0.23-0.63; P = 0.000.The results suggest that the rapid recovery of CD3+CD8+ cytotoxic T cells at day 90 following HBMT could predict superior transplant outcomes.

  12. Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

    Directory of Open Access Journals (Sweden)

    Begoña Barriuso

    2016-06-01

    Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

  13. Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

    Science.gov (United States)

    Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

    2016-06-10

    Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

  14. Surface expression of anti-CD3scfv stimulates locoregional immunotherapy against hepatocellular carcinoma depending on the E1A-engineered human umbilical cord mesenchymal stem cells.

    Science.gov (United States)

    Zhang, Qing; Yuan, Xiang-Fei; Lu, Yang; Li, Zhen-Zhen; Bao, Shi-Qi; Zhang, Xiao-Long; Yang, Yuan-Yuan; Fan, Dong-Mei; Zhang, Yi-Zhi; Wu, Chen-Xuan; Guo, Hong-Xing; Zhang, Yan-Jun; Ye, Zhou; Xiong, Dong-Sheng

    2017-10-01

    Tumor antigens is at the core of cancer immunotherapy, however, the ideal antigen selection is difficult especially in poorly immunogenic tumors. In this study, we designed a strategy to modify hepatocellular carcinoma (HCC) cells by surface expressing anti-CD3scfv within the tumor site strictly, which depended on the E1A-engineered human umbilical cord mesenchymal stem cells (HUMSC.E1A) delivery system. Subsequently, membrane-bound anti-CD3scfv actived the lymphocytes which lysed HCC cells bypassing the expression of antigens or MHC restriction. First, we constructed the anti-CD3scfv gene driven by human α-fetoprotein (AFP) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. Our results showed that anti-CD3scfv could specifically express on the surface of HCC cells and activate the lymphocytes to kill target cells effectively in vitro. HUMSC infected by AdCD3scfv followed by LentiR.E1A could support the adenoviral replication and packaging in vitro 36 h after LentiR.E1A infection. Using a subcutaneous HepG2 xenograft model, we confirmed that AdCD3scfv and LentiR.E1A co-transfected HUMSC could migrate selectively to the tumor site and produce considerable adenoviruses. The new generated AdCD3scfv infected and modified tumor cells successfully. Mice injected with the MSC.E1A.AdCD3scfv and lymphocytes significantly inhibited the tumor growth compared with control groups. Furthermore, 5-fluorouracil (5-FU) could sensitize adenovirus infection at low MOI resulting in improved lymphocytes cytotoxicity in vitro and in vivo. In summary, this study provides a promising strategy for solid tumor immunotherapy. © 2017 UICC.

  15. Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey) by Anti-Human Monoclonal Antibody

    Science.gov (United States)

    de Souza, Hugo Amorim dos Santos; Costa-Correa, Edmar Henrique; Bianco-Junior, Cesare; Andrade, Márcia Cristina Ribeiro; Lima-Junior, Josué da Costa; Pratt-Riccio, Lilian Rose; Daniel-Ribeiro, Cláudio Tadeu; Totino, Paulo Renato Rivas

    2017-01-01

    Non-human primates (NHP) are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors—such as the signal regulatory protein α (SIRPα)—in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC). As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model. PMID:29312325

  16. Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey by Anti-Human Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Hugo Amorim dos Santos de Souza

    2017-12-01

    Full Text Available Non-human primates (NHP are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors—such as the signal regulatory protein α (SIRPα—in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC. As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model.

  17. Infrared absorption of CH3OSO and CD3OSO radicals produced upon photolysis of CH3OS(O)Cl and CD3OS(O)Cl in p-H2 matrices

    International Nuclear Information System (INIS)

    Lee, Yu-Fang; Kong, Lin-Jun; Lee, Yuan-Pern

    2012-01-01

    Irradiation at 239 ± 20 nm of a p-H 2 matrix containing methoxysulfinyl chloride, CH 3 OS(O)Cl, at 3.2 K with filtered light from a medium-pressure mercury lamp produced infrared (IR) absorption lines at 3028.4 (attributable to ν 1 , CH 2 antisymmetric stretching), 2999.5 (ν 2 , CH 3 antisymmetric stretching), 2950.4 (ν 3 , CH 3 symmetric stretching), 1465.2 (ν 4 , CH 2 scissoring), 1452.0 (ν 5 , CH 3 deformation), 1417.8 (ν 6 , CH 3 umbrella), 1165.2 (ν 7 , CH 3 wagging), 1152.1 (ν 8 , S=O stretching mixed with CH 3 rocking), 1147.8 (ν 9 , S=O stretching mixed with CH 3 wagging), 989.7 (ν 10 , C-O stretching), and 714.5 cm -1 (ν 11 , S-O stretching) modes of syn-CH 3 OSO. When CD 3 OS(O)Cl in a p-H 2 matrix was used, lines at 2275.9 (ν 1 ), 2251.9 (ν 2 ), 2083.3 (ν 3 ), 1070.3 (ν 4 ), 1056.0 (ν 5 ), 1085.5 (ν 6 ), 1159.7 (ν 7 ), 920.1 (ν 8 ), 889.0 (ν 9 ), 976.9 (ν 10 ), and 688.9 (ν 11 ) cm -1 appeared and are assigned to syn-CD 3 OSO; the mode numbers correspond to those used for syn-CH 3 OSO. The assignments are based on the photolytic behavior and a comparison of observed vibrational wavenumbers, infrared intensities, and deuterium isotopic shifts with those predicted with the B3P86/aug-cc-pVTZ method. Our results extend the previously reported four transient IR absorption bands of gaseous syn-CH 3 OSO near 2991, 2956, 1152, and 994 cm -1 to 11 lines, including those associated with C-O, O-S, and S=O stretching modes. Vibrational wavenumbers of syn-CD 3 OSO are new. These results demonstrate the advantage of a diminished cage effect of solid p-H 2 such that the Cl atom, produced via UV photodissociation of CH 3 OS(O)Cl in situ, might escape from the original cage to yield isolated CH 3 OSO radicals.

  18. Infrared absorption of CH3OSO and CD3OSO radicals produced upon photolysis of CH3OS(O)Cl and CD3OS(O)Cl in p-H2 matrices.

    Science.gov (United States)

    Lee, Yu-Fang; Kong, Lin-Jun; Lee, Yuan-Pern

    2012-03-28

    Irradiation at 239 ± 20 nm of a p-H(2) matrix containing methoxysulfinyl chloride, CH(3)OS(O)Cl, at 3.2 K with filtered light from a medium-pressure mercury lamp produced infrared (IR) absorption lines at 3028.4 (attributable to ν(1), CH(2) antisymmetric stretching), 2999.5 (ν(2), CH(3) antisymmetric stretching), 2950.4 (ν(3), CH(3) symmetric stretching), 1465.2 (ν(4), CH(2) scissoring), 1452.0 (ν(5), CH(3) deformation), 1417.8 (ν(6), CH(3) umbrella), 1165.2 (ν(7), CH(3) wagging), 1152.1 (ν(8), S=O stretching mixed with CH(3) rocking), 1147.8 (ν(9), S=O stretching mixed with CH(3) wagging), 989.7 (ν(10), C-O stretching), and 714.5 cm(-1) (ν(11), S-O stretching) modes of syn-CH(3)OSO. When CD(3)OS(O)Cl in a p-H(2) matrix was used, lines at 2275.9 (ν(1)), 2251.9 (ν(2)), 2083.3 (ν(3)), 1070.3 (ν(4)), 1056.0 (ν(5)), 1085.5 (ν(6)), 1159.7 (ν(7)), 920.1 (ν(8)), 889.0 (ν(9)), 976.9 (ν(10)), and 688.9 (ν(11)) cm(-1) appeared and are assigned to syn-CD(3)OSO; the mode numbers correspond to those used for syn-CH(3)OSO. The assignments are based on the photolytic behavior and a comparison of observed vibrational wavenumbers, infrared intensities, and deuterium isotopic shifts with those predicted with the B3P86∕aug-cc-pVTZ method. Our results extend the previously reported four transient IR absorption bands of gaseous syn-CH(3)OSO near 2991, 2956, 1152, and 994 cm(-1) to 11 lines, including those associated with C-O, O-S, and S=O stretching modes. Vibrational wavenumbers of syn-CD(3)OSO are new. These results demonstrate the advantage of a diminished cage effect of solid p-H(2) such that the Cl atom, produced via UV photodissociation of CH(3)OS(O)Cl in situ, might escape from the original cage to yield isolated CH(3)OSO radicals.

  19. Towards neuroimmunotherapy for cancer: the neurotransmitters glutamate, dopamine and GnRH-II augment substantially the ability of T cells of few head and neck cancer patients to perform spontaneous migration, chemotactic migration and migration towards the autologous tumor, and also elevate markedly the expression of CD3zeta and CD3epsilon TCR-associated chains.

    Science.gov (United States)

    Saussez, Sven; Laumbacher, Barbara; Chantrain, Gilbert; Rodriguez, Alexandra; Gu, Songhai; Wank, Rudolf; Levite, Mia

    2014-08-01

    In previous studies we found that several Neurotransmitters and Neuropeptides among them: Glutamate, Dopamine, Gonadotropin-releasing-hormone (GnRH) I and II, Somatostatin, CGRP and Neuropeptide Y, can each by itself, at low physiological concentration (~10 nM) bind its receptors in human T cells and trigger several key T cell functions. These findings showed that the nervous system, via Neurotransmitters and Neuropeptides, can 'talk' directly to the immune system, and stimulate what we coined 'Nerve-Driven Immunity': immune responses dictated by the nervous system. In various human cancers, the immune system of the patients, and their T cells in particular, are not functioning well enough against the cancer due to several reasons, among them the suppressive effects on the immune system induced by: (1) the cancer itself, (2) the chemotherapy and radiotherapy, (3) the ongoing/chronic stress, anxiety, depression and pain felt by the cancer patients. In Head and Neck Cancer (HNC), 5-year survival rate remains below 50%, primarily because of local recurrences or second primary tumors. Two-thirds of HNC patients are diagnosed at advanced clinical stage and have significantly poorer prognosis. Most HNC patients have multiple severe immunological defects especially in their T cells. A major defect in T cells of patients with HNC or other types of cancer is low CD3zeta expression that correlates with poor prognosis, decreased proliferation, apoptotic profile, abnormal cytokine secretion and poor abilities of destructing cancer cells. T cells of cancer patients are often also unable to migrate properly towards the tumor. In this study we asked if Glutamate, Dopamine or GnRH-II can augment the spontaneous migration, chemotactic migration and towards autologous HNC migration, and also increase CD3zeta and CD3epsilon expression, of peripheral T cells purified from the blood of five HNC patients. These HNC patients had either primary tumor or recurrence, and have been already

  20. Preparation and functional studies of hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody

    Directory of Open Access Journals (Sweden)

    Yang J

    2014-05-01

    Full Text Available Jingjing Yang,1,3,* Xiaoping Huang,1,3,* Fanghong Luo,1 Xiaofeng Cheng,3 Lianna Cheng,3 Bin Liu,4 Lihong Chen,2 Ruyi Hu,1,3 Chunyan Shi,1,3 Guohong Zhuang,1,3 Ping Yin2 1Anti-Cancer Research Center, Medical College, Xiamen University, Fujian, People's Republic of China, 2The Department of Pathology, Zhongshan Hospital, Xiamen University, Xiamen, People's Republic of China, 3Organ transplantation institution, Xiamen University, Xiamen, People's Republic of China, 4Jilin Vocational College of Industry and Technology, Jilin, People's Republic of China  *These authors contributed equally to this work Objective: To prepare hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody, and study their characteristics, functions, and mechanisms of action. Materials and methods: The anti-human death receptor 5 single-chain antibody was constructed and expressed. Protein-loaded hydroxyethyl chitosan nanoparticles were prepared, and their size, morphology, particle-size distribution and surface zeta potential were measured by scanning electron microscopy and laser particle-size analysis. Mouse H22 hepatocellular carcinoma cells were cultured, and growth inhibition was examined using the CellTiter-Blue cell-viability assay. Flow cytometry and Hoechst 33342 were employed to measure cell apoptosis. Kunming mice with H22 tumor models were treated with protein-loaded hydroxyethyl chitosan nanoparticles, and their body weight and tumor size were measured, while hematoxylin and eosin staining was used to detect antitumor effects in vivo and side effects from tumors. Results: The protein-loaded hydroxyethyl chitosan nanoparticles had good stability; the zeta potential was -24.2±0.205, and the dispersion index was 0.203. The inhibition of the protein-loaded hydroxyethyl chitosan nanoparticles on H22 growth was both time- and dose-dependent. Increased expressions of active caspase 8, active caspase 3, and BAX were detected

  1. Distribution of intrahepatic T, NK and CD3(+)CD56(+)NKT cells alters after liver transplantation: Shift from innate to adaptive immunity?

    Science.gov (United States)

    Werner, Jens M; Lang, Corinna; Scherer, Marcus N; Farkas, Stefan A; Geissler, Edward K; Schlitt, Hans J; Hornung, Matthias

    2011-07-01

    The liver is an immunological organ containing a large number of T, NK and NKT cells, but little is known about intrahepatic immunity after LTx. Here, we investigated whether the distribution of T, NK and CD3(+)CD56(+)NKT cells is altered in transplanted livers under different circumstances. Core biopsies of transplanted livers were stained with antibodies against CD3 and CD56. Several cell populations including T (CD3(+)CD56(-)), NK (CD3(-)CD56(+)) and NKT cells (CD3(+)CD56(+)) were studied by fluorescence microscopy. Cell numbers were analyzed in relation to the time interval after LTx, immunosuppressive therapy and stage of acute graft rejection (measured with the rejection activity index: RAI) compared to tumor free liver tissue from patients after liver resection due to metastatic disease as control. Recruitment of CD3(+)CD56(+)NKT cells revealed a significant decrease during high RAI scores in comparison to low and middle RAI scores (RAI 7-9: 0.03±0.01/HPF vs. RAI 4-6: 0.1±0.005/HPF). CD3(+)CD56(+)NKT cells were also lower during immunosuppressive therapy with tacrolimus (0.03±0.01/HPF) than with cyclosporine (0.1±0.003/HPF), cyclosporine/MMF (0.1±0.003/HPF) or sirolimus (0.1±0.01/HPF) treatment. Intrahepatic T cell numbers increased significantly 50days after LTx compared to control liver tissue (4.5±0.2/HPF vs. 1.9±0.1/HPF). In contrast, NK cells (0.3±0.004/HPF) were significantly fewer in all biopsies after LTx compared to the control (0.7±0.04/HPF). These data indicate significant alterations in the hepatic recruitment of T, NK and CD3(+)CD56(+)NKT cells after LTx. The increase in T cells and the decrease in NK and CD3(+)CD56(+)NKT cells suggest a shift from innate to adaptive hepatic immunity in the liver graft. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Cd3(MoO4)(TeO3)2: A Polar 3D Compound Containing d10-d0 SCALP-Effect Cations.

    Science.gov (United States)

    Feng, Yuquan; Fan, Huitao; Zhong, Zhiguo; Wang, Hongwei; Qiu, Dongfang

    2016-11-21

    The new polar 3D cadmium molybdotellurite Cd 3 (MoO 4 )(TeO 3 ) 2 was obtained by means of a high-temperature solid-state method. Cd 3 (MoO 4 )(TeO 3 ) 2 is a monoclinic crystal system, and it exhibits the polar space group P2 1 (No. 4). The structure of Cd 3 (MoO 4 )(TeO 3 ) 2 can be viewed as a complicated 3D architecture that is composed of distorted CdO n (n = 6, 7) polyhedra, TeO 3 trigonal pyramids, and MoO 4 polyhedra. The compound features the first 3D NCS cadmium molybdotellurite with 1D 4- and 6-MR channels and a polar structure originating from the TeO 3 groups, MoO 4 groups, and displacements of d 10 Cd 2+ cations. The results were further confirmed by calculations of the net polarization. The UV-vis spectrum and thermal properties indicate that Cd 3 (MoO 4 )(TeO 3 ) 2 exhibits a broad transparent region and excellent thermal stability. SHG tests of Cd 3 (MoO 4 )(TeO 3 ) 2 revealed that its response is approximately the same as that of KH 2 PO 4 at the same grain size between 105 and 150 μm and that it is phase-matchable.

  3. The anti-human trafficking collaboration model and serving victims: Providers' perspectives on the impact and experience.

    Science.gov (United States)

    Kim, Hea-Won; Park, Taekyung; Quiring, Stephanie; Barrett, Diana

    2018-01-01

    A coalition model is often used to serve victims of human trafficking but little is known about whether the model is adequately meeting the needs of the victims. The purpose of this study was to examine anti-human trafficking collaboration model in terms of its impact and the collaborative experience, including challenges and lessons learned from the service providers' perspective. Mixed methods study was conducted to evaluate the impact of a citywide anti-trafficking coalition model from the providers' perspectives. Web-based survey was administered with service providers (n = 32) and focus groups were conducted with Core Group members (n = 10). Providers reported the coalition model has made important impacts in the community by increasing coordination among the key agencies, law enforcement, and service providers and improving quality of service provision. Providers identified the improved and expanded partnerships among coalition members as the key contributing factor to the success of the coalition model. Several key strategies were suggested to improve the coalition model: improved referral tracking, key partner and protocol development, and information sharing.

  4. Kinetic isotope effects in the gas phase reactions of OH and Cl with CH3Cl, CD3Cl, and 13CH3Cl

    Directory of Open Access Journals (Sweden)

    A. A. Gola

    2005-01-01

    Full Text Available The kinetic isotope effects in the reactions of CH3Cl, 13CH3Cl and CD3Cl with OH radicals and Cl atoms were studied in relative rate experiments at 298±2 K and 1013±10 mbar. The reactions were carried out in a smog chamber using long path FTIR detection and the spectroscopic data analyzed employing a non-linear least squares spectral fitting method using measured high-resolution infrared spectra as well as absorption cross sections from the HITRAN database. The reaction rates of 13CH3Cl and CD3Cl with OH and Cl were determined relative to CH3Cl as: kOH+CH3ClkOH+CH3Cl/kOH+13CH3Cl}kOH+13CH3Cl=1.059±0.008, kOH+CH3ClkOH+CH3Cl/kOH+CD3ClkOH+CD3Cl=3.9±0.4, kCl+CH3ClkCl+CH3Cl/kCl+13CH3ClkCl+13CH3Cl =1.070±0.010 and kCl+CH3ClkCl+CH3Cl/kCl+CD3ClkCl+CD3Cl=4.91±0.07. The uncertainties given are 2σ from the statistical analyses and do not include possible systematic errors. The unexpectedly large 13C kinetic isotope effect in the OH reaction of CH3Cl has important implications for the global emission inventory of CH3Cl.

  5. Impact of CD68/(CD3+CD20 ratio at the invasive front of primary tumors on distant metastasis development in breast cancer.

    Directory of Open Access Journals (Sweden)

    Noemí Eiró

    Full Text Available Tumors are infiltrated by macrophages, T and B-lymphocytes, which may favor tumor development by promoting angiogenesis, growth and invasion. The aim of this study was to investigate the clinical relevance of the relative amount of macrophages (CD68⁺, T-cells (CD3⁺ and B-cells (CD20⁺ at the invasive front of breast carcinomas, and the expression of matrix metalloproteases (MMPs and their inhibitors (TIMPs either at the invasive front or at the tumor center. We performed an immunohistochemical study counting CD3, CD20 and CD68 positive cells at the invasive front, in 102 breast carcinomas. Also, tissue sections were stained with MMP-2, -9, -11, -14 and TIMP-2 antibodies, and immunoreactivity location, percentage of reactive area and intensity were determined at the invasive front and at the tumor center. The results showed that an increased CD68 count and CD68/(CD3+CD20 ratio were directly associated with both MMP-11 and TIMP-2 expression by mononuclear inflammatory cells at the tumor center (p = 0.041 and p = 0.025 for CD68 count and p = 0.001 and p = 0.045 for ratio, respectively for MMP-11 and TIMP-2. In addition, a high CD68/(CD3+CD20 ratio (>0.05 was directly associated with a higher probability of shortened relapse-free survival. Multivariate analysis revealed that CD68/(CD3+CD20 ratio was an independent factor associated with distant relapse-free survival (RR: 2.54, CI: (1.23-5.24, p<0.01. Therefore, CD68/(CD3+CD20 ratio at the invasive front could be used as an important prognostic marker.

  6. Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

    Science.gov (United States)

    Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

    2017-02-01

    Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

  7. Masking of the CD3 gamma di-leucine-based motif by zeta is required for efficient T-cell receptor expression

    DEFF Research Database (Denmark)

    Lauritsen, Jens Peter H; Bonefeld, Charlotte Menné; von Essen, Marina

    2004-01-01

    containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced...... TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine...

  8. The CD3-zeta chimeric antigen receptor overcomes TCR Hypo-responsiveness of human terminal late-stage T cells.

    Directory of Open Access Journals (Sweden)

    Gunter Rappl

    Full Text Available Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+ CD57(+ CD7(- phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter.

  9. Accuracy of the centrifugal sudden approximation in the H + CHD3 → H2 + CD3 reaction

    International Nuclear Information System (INIS)

    Zhang, Zhaojun; Chen, Jun; Liu, Shu; Zhang, Dong H.

    2014-01-01

    The initial state selected time-dependent wave packet method has been extended to calculate the coupled-channel reaction probabilities with total angular momentum J tot > 0 for the title reaction with seven degrees of freedom included. Fully converged integral cross sections were obtained for the ground and a number of vibrational excited initial states on a new potential energy surface recently constructed by this group using neural network fitting. As found from a previous study with the centrifugal sudden (CS) approximation, all these initial vibrational excitations investigated in this study enhance the reactivity considerably at a given collision energy, in particular the CH stretch excited state. The energy initially deposited in CH stretch motion is more effective than translational energy on promoting the reaction in the entire energy region, while for CH bending or CD 3 umbrella excitations only at the high collision energy the vibrational energy becomes more effective. Our calculations also revealed that the accuracy of the CS approximation considerably deteriorates with the increase of J tot , in particular on the threshold energy. The CS approximation underestimates the integral cross sections for all these initial states, albeit not very severely. In general, it works better at high collision energies and for vibrationally excited initial states, with the increase of integral cross section

  10. A Preclinical Population Pharmacokinetic Model for Anti-CD20/CD3 T-Cell-Dependent Bispecific Antibodies.

    Science.gov (United States)

    Ferl, Gregory Z; Reyes, Arthur; Sun, Liping L; Cheu, Melissa; Oldendorp, Amy; Ramanujan, Saroja; Stefanich, Eric G

    2018-05-01

    CD20 is a cell-surface receptor expressed by healthy and neoplastic B cells and is a well-established target for biologics used to treat B-cell malignancies. Pharmacokinetic (PK) and pharmacodynamic (PD) data for the anti-CD20/CD3 T-cell-dependent bispecific antibody BTCT4465A were collected in transgenic mouse and nonhuman primate (NHP) studies. Pronounced nonlinearity in drug elimination was observed in the murine studies, and time-varying, nonlinear PK was observed in NHPs, where three empirical drug elimination terms were identified using a mixed-effects modeling approach: i) a constant nonsaturable linear clearance term (7 mL/day/kg); ii) a rapidly decaying time-varying, linear clearance term (t ½  = 1.6 h); and iii) a slowly decaying time-varying, nonlinear clearance term (t ½  = 4.8 days). The two time-varying drug elimination terms approximately track with time scales of B-cell depletion and T-cell migration/expansion within the central blood compartment. The mixed-effects NHP model was scaled to human and prospective clinical simulations were generated. © 2018 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

  11. Biodistribution of 99mTc-labeled anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody h-R3 in a xenograft model of human lung adenocarcinoma

    International Nuclear Information System (INIS)

    Morales-Morales, Alejo; Duconge, Jorge; Caballero-Torres, Idania; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Iznaga-Escobar, Normando

    1999-01-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody (MAb) h-R3 is an (IgG 1 ), which binds to an extracellular domain of EGF-R. It was used to evaluate the biodistribution on nude mice xenografted with H-125 human lung adenocarcinoma cell line. Results were compared with its murine version of the MAb ior-egf/r3. Twenty-one athymic female 4NMRI nu/nu mice were injected intraperitoneally with 10 μg/100 μCi of 99m Tc-labeled MAbs. Immunoreactivity of 99m Tc-labeled MAbs were measured by enzyme-linked immunosorbent assay (ELISA) on H-125 cell line and the immunoreactive fractions was determined by the Lindmo method. Among all organs, significant accumulation was found in serum (27.05 ± 2.08 %ID/g) and tumor (3.903 ± 0.89 %ID/g) at 4 h after injection. These values decreased to 5.03 ± 0.50 %ID/g and 2.19 ± 0.56 %ID/g for serum and tumor, respectively. The immunoreactive fraction was found to be 0.70, with a correlation coefficient r=0.9984. With the good biodistribution and tumor uptake of the 99m Tc-labeled humanized antibody h-R3, a phase I diagnostic clinical trial of tumor with epithelial origin should be pursued

  12. Biodistribution of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody h-R3 in a xenograft model of human lung adenocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Morales-Morales, Alejo; Duconge, Jorge; Caballero-Torres, Idania; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Iznaga-Escobar, Normando E-mail: normando@ict.cim.sld.cu

    1999-04-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody (MAb) h-R3 is an (IgG{sub 1}), which binds to an extracellular domain of EGF-R. It was used to evaluate the biodistribution on nude mice xenografted with H-125 human lung adenocarcinoma cell line. Results were compared with its murine version of the MAb ior-egf/r3. Twenty-one athymic female 4NMRI nu/nu mice were injected intraperitoneally with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled MAbs. Immunoreactivity of {sup 99m}Tc-labeled MAbs were measured by enzyme-linked immunosorbent assay (ELISA) on H-125 cell line and the immunoreactive fractions was determined by the Lindmo method. Among all organs, significant accumulation was found in serum (27.05 {+-} 2.08 %ID/g) and tumor (3.903 {+-} 0.89 %ID/g) at 4 h after injection. These values decreased to 5.03 {+-} 0.50 %ID/g and 2.19 {+-} 0.56 %ID/g for serum and tumor, respectively. The immunoreactive fraction was found to be 0.70, with a correlation coefficient r=0.9984. With the good biodistribution and tumor uptake of the {sup 99m}Tc-labeled humanized antibody h-R3, a phase I diagnostic clinical trial of tumor with epithelial origin should be pursued.

  13. Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII.

    OpenAIRE

    Broze, G J; Hickman, S; Miletich, J P

    1985-01-01

    Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on We...

  14. Ontogeny of human natural killer (NK) cells: fetal NK cells mediate cytolytic function and express cytoplasmic CD3 epsilon,delta proteins

    NARCIS (Netherlands)

    Phillips, J. H.; Hori, T.; Nagler, A.; Bhat, N.; Spits, H.; Lanier, L. L.

    1992-01-01

    Natural killer (NK) cells have been defined as CD3 epsilon-, CD16+ and/or CD56+ lymphocytes that mediate major histocompatibility complex (MHC)-unrestricted cytotoxicity against certain tumors and virus-infected cells. Unlike T lymphocytes, NK cells do not rearrange or productively express T cell

  15. Target Antigen Density Governs the Efficacy of Anti-CD20-CD28-CD3 zeta Chimeric Antigen Receptor-Modified Effector CD8(+) T Cells

    NARCIS (Netherlands)

    Watanabe, Keisuke; Terakura, Seitaro; Martens, Anton C.; van Meerten, Tom; Uchiyama, Susumu; Imai, Misa; Sakemura, Reona; Goto, Tatsunori; Hanajiri, Ryo; Imahashi, Nobuhiko; Shimada, Kazuyuki; Tomita, Akihiro; Kiyoi, Hitoshi; Nishida, Tetsuya; Naoe, Tomoki; Murata, Makoto

    2015-01-01

    The effectiveness of chimeric Ag receptor (CAR)-transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second-and later-generation CARs simultaneously transmit costimulatory signals with CD3 zeta signals upon ligation, but may lead to severe adverse effects

  16. Early CD3+/CD15+ peripheral blood leukocyte chimerism patterns correlate with long-term engraftment in non-malignant hematopoietic SCT.

    Science.gov (United States)

    Ketterl, T G; Flesher, M; Shanley, R; Miller, W

    2014-04-01

    Following hematopoietic SCT (HSCT) for non-malignant disorders (NMDs) variable donor chimerism among lympho-hematopoietic lines may be observed. We retrospectively evaluated early post-HSCT, lineage-sorted (CD3+ and CD15+) peripheral blood leukocyte chimerism data to characterize patterns and assess for association with long-term CD15+ engraftment. 'Early' was defined as the first value obtained between days +14 and +42, 'late' as the last recorded value after day +90. 'High' donor chimerism was defined as 80% on either fraction at all time-points. Patients were classified into four subgroups with respect to early CD3+/CD15+ chimerism patterns (high/low) then analyzed for long-term CD15+ chimerism status. A total of 135 transplants were evaluable, with all three time-points available in 97. Underlying disease, graft source, patient age and conditioning intensity varied. 'Split' early chimerism (discordant high/low CD3+/CD15+ status) was common. Multivariable analysis revealed strong association between conditioning regimen and primary disease on early CD3+/CD15+ chimerism patterns and a dominant predictive effect of early CD15+ chimerism on long-term CD15+ donor engraftment (observed at median day +365). These data may guide real-time clinician decisions (restraint vs intervention, when available) when faced with unfavorable or unusual early lympho-hematopoietic chimerism patterns following HSCT for NMD.

  17. TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3 gamma di-leucine-based motif-dependent mechanism

    DEFF Research Database (Denmark)

    Bonefeld, Charlotte Menné; Rasmussen, B. A.; Lauritsen, J P

    2003-01-01

    of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based...

  18. Higher CD3(+) and CD34(+) cell doses in the graft increase the incidence of acute GVHD in children receiving BMT for thalassemia.

    Science.gov (United States)

    Gaziev, J; Isgrò, A; Marziali, M; Daniele, N; Gallucci, C; Sodani, P; Simone, M D; Adorno, G; Paciaroni, K; Andreani, M; Lanti, A; Del Proposto, G; Testi, M; De Angelis, G; Roveda, A; Alfieri, C; Saltarelli, F; Lucarelli, G

    2012-01-01

    We evaluated the incidence of GVHD, risk factors and the impact of graft composition on acute GVHD (aGVHD) in 92 children who underwent BMT for thalassemia following busulfan/cyclophosphamide (BUCY)-based conditioning regimens and GVHD prophylaxis with CSA/short-MTX and methylprednisolone. The incidence of grade 2-4 and 3-4 aGVHD was 35% (95% confidence interval (CI) 25-44) and 9% (95% CI 4-16), respectively. We found that CD3(+) and CD34(+) cell doses above the median were associated with high incidence of grade 2-4 aGVHD (49 vs 20%, P=0.005 and 46 vs 23%, P=0.021, respectively). In multivariate analysis, high CD3(+) (hazard ratio (HR) 4.6; 95% CI 1.4-14.7; P=0.010) and CD34(+) (HR 4.3; 95% CI 1.4-12.7; P=0.011) cell doses were associated with grade 2-4 aGVHD. We further examined the effect of CD3(+) and CD34(+) cell doses on aGVHD using quartile cutoff points and found a minimum threshold for CD3(+) (38 × 10(6)/kg) and CD34(+) (4 × 10(6)/kg) cells above which the incidence of grade 2-4 aGVHD is significantly increased. This study shows for the first time a positive correlation between the number of CD3(+) and CD34(+) cells and aGVHD in children receiving sibling BMT, and indicates that using tailored and more intensive post transplant immunosuppression may permit to better control aGVHD.

  19. The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: a study of 500 cases.

    Science.gov (United States)

    Janossy, G; Coustan-Smith, E; Campana, D

    1989-03-01

    Current views about the origin of acute lymphoid leukemia (ALL) emphasize the importance of maturation arrest at a precursor cell level. Recently, the CD22 antigen has been identified in the cytoplasm of normal bone marrow-borne immature B lineage cells, while the CD3 antigen (epsilon chain) has been detected within normal immature thymic blasts. In the first part our study performed on 100 cases of known acute leukemias, the expression of such cytoplasmic molecules, referred to as cCD22 and cCD3, was analyzed together with their appearance in the leukemic cells' membrane (mCD22 and mCD3). The presence of cCD22 in B-lineage ALL and that of cCD3 in T-ALL has indeed fully confirmed the diagnosis reached by other markers, and mCD22 and mCD3 were expressed on only a few cases of B- and T-lineage ALL, also revealing a degree of developmental asynchrony within leukemic blasts. In the subsequent analysis both cCD22 and cCD3 have been included in a standard panel of diagnostic reagents applied on 500 consecutive cases of acute leukemia. Here the aim was to analyze both the diagnostic precision of individual markers and the heterogeneity of various leukemic types in terms of the expression of membrane and intracellular antigens and their cytochemical features (Sudan Black B and esterases). It has been found that cCD22 and cCD3 are exquisitely specific for B-precursor ALL (TdT+, CD19+) and T-ALL (TdT+, CD7+), respectively, while both markers are absent in acute myeloblastic leukemia (AML) and acute myelomonocytic and monocytic leukemia (AMML/AMoL). These observations contrast the findings which demonstrate that 31% of cases among nonlymphoid acute leukemia (including AML and AMML) express CD7 and/or TdT. The study of myeloid antigens detected by CD13, CD33, and CD14 is also informative and complementary, both in diagnosing and subdividing the AML and AMML/AMoL groups. The peculiar main observation of this study is that only with the combined use of these markers in a

  20. CD3+/CD16+CD56+ cell numbers in peripheral blood are correlated with higher tumor burden in patients with diffuse large B-cell lymphoma

    Directory of Open Access Journals (Sweden)

    Anna Twardosz

    2011-04-01

    Full Text Available Diffuse large B-cell lymphoma is the commonest histological type of malignant lymphoma, andremains incurable in many cases. Developing more efficient immunotherapy strategies will require betterunderstanding of the disorders of immune responses in cancer patients. NKT (natural killer-like T cells wereoriginally described as a unique population of T cells with the co-expression of NK cell markers. Apart fromtheir role in protecting against microbial pathogens and controlling autoimmune diseases, NKT cells havebeen recently revealed as one of the key players in the immune responses against tumors. The objective of thisstudy was to evaluate the frequency of CD3+/CD16+CD56+ cells in the peripheral blood of 28 diffuse largeB-cell lymphoma (DLBCL patients in correlation with clinical and laboratory parameters. Median percentagesof CD3+/CD16+CD56+ were significantly lower in patients with DLBCL compared to healthy donors(7.37% vs. 9.01%, p = 0.01; 4.60% vs. 5.81%, p = 0.03, although there were no differences in absolute counts.The frequency and the absolute numbers of CD3+/CD16+CD56+ cells were lower in advanced clinical stagesthan in earlier ones. The median percentage of CD3+/CD16+CD56+ cells in patients in Ann Arbor stages 1–2 was5.55% vs. 3.15% in stages 3–4 (p = 0.02, with median absolute counts respectively 0.26 G/L vs. 0.41 G/L (p == 0.02. The percentage and absolute numbers of CD3+/CD16+CD56+ cells were significantly higher in DL-BCL patients without B-symptoms compared to the patients with B-symptoms, (5.51% vs. 2.46%, p = 0.04;0.21 G/L vs. 0.44 G/L, p = 0.04. The percentage of CD3+/CD16+CD56+ cells correlated adversely with serumlactate dehydrogenase (R= –445; p < 0.05 which might influence NKT count. These figures suggest a relationshipbetween higher tumor burden and more aggressive disease and decreased NKT numbers. But it remains tobe explained whether low NKT cell counts in the peripheral blood of patients with DLBCL are the result

  1. Cellular reactions of CD3+ CD4+ CD45RO+ T-lymphocytes on dexamethason in in normal patients and in patients with with rheumatoid arthritis in vitro

    Directory of Open Access Journals (Sweden)

    L. S. Litvinova

    2017-01-01

    Full Text Available The aim of the study was to analyze the influence of glucocorticoid (GC dexamethasone (Dex on changes in CD4+ T-cells expressing the surface molecule of activation (CD25, CD71, HLA-DR and CD95 and their ability to produce proinflammatory mediators in cultures of TCR-stimulated CD3+CD45RO+ T-lymphocytes obtained from healthy donors and patients with rheumatoid arthritis in vitro.Materials and methods. The study included 50 patients and 20 healthy donors. T-cell cultures (CD3+ CD45RO+ were obtained from mononuclear leukocytes of immunomagnetic separation (MACS® technology. As an activator of T-lymphocytes, antibiotic particles with biotinylated antibodies against CD2+, CD3+, CD28+, which simulate the process of costimulation of T cells by antigen-presenting cells, were used. The following concentrations of dexamethasone (2, 8, 16, 32, 64 mg were used in the experiment. The change in the immunophenotype of T-lymphocytes was analyzed by flow cytofluoometry. The secretion of CD3+CD45RO+ T-cells of proinflammatory cytokines IL-2, IFNγ, TNFα, IL-17 and IL-21 was evaluated by enzyme-linked immunosorbent assay.Results. The general suppressor effect of Dex on CD3+CD45RO+ T-cell cultures mediated by a decrease in the number of CD4 + T cells expressing activation molecules (CD25 and proliferation (CD71, as well as inhibition of the production of inflammatory mediators: IFNγ, IL-2 and TNFα. It is shown that against the background of TCR activation Dex increases the number of CD4+CD95+HLA-DR+ cells in CD3+CD45RO+ cultures obtained from RA patients and does not change their content in the control. The correlations between the number of proinflammatory factors (IL-17, IL-21 and TNFα in CD4+CD45RO+CD95+HLA-DR+ T cells in supernatants of cell cultures in RA patients indicate the presence of a pro-inflammatory potential of this population of T cells. We assume that the resistance of CD4+CD45RO+CD95+HLA-DR+ T cells in RA patients to the suppressor effect of

  2. Ontogenic development of kidney, thymus and spleen and phenotypic expression of CD3 and CD4 receptors on the lymphocytes of cobia (Rachycentroncanadum

    Directory of Open Access Journals (Sweden)

    MARTA C. KLOSTERHOFF

    2015-12-01

    Full Text Available ABSTRACT In the present study was evaluated the ontogenic of immunocompetent organs of cobia up to 53 days after hatching (dah through histology and immunohistochemistry techniques. The kidney was the first lymphohematopoietic organ to appear, at 1 dah, followed by the spleen at 5 dah and the thymus at 7 dah. The first CD3 receptors on the lymphocytes were observed in 27% of the thymic tissue at 7 dah and in 99% at 53 dah. The phenotypic expression of CD3 receptors was registered in 10% of the kidney at 8 dah and in 32% at 53 dah. CD4 receptors were observed in 5% and 63% of the thymic area at 7 and 53 dah, respectively. In the kidney, T4 lymphocytes were first observed at 13 dah in 9% of the organ and in 28% at 53 dah, defining the functional development of the specific system associated with immunological memory capacity.

  3. Ontogenic development of kidney, thymus and spleen and phenotypic expression of CD3 and CD4 receptors on the lymphocytes of cobia (Rachycentroncanadum).

    Science.gov (United States)

    Klosterhoff, Marta C; Pereira Júnior, Joaber; Rodrigues, Ricardo V; Gusmão, Emeline P; Sampaio, Luís A; Tesser, Marcelo B; Romano, Luis A

    2015-01-01

    In the present study was evaluated the ontogenic of immunocompetent organs of cobia up to 53 days after hatching (dah) through histology and immunohistochemistry techniques. The kidney was the first lymphohematopoietic organ to appear, at 1 dah, followed by the spleen at 5 dah and the thymus at 7 dah. The first CD3 receptors on the lymphocytes were observed in 27% of the thymic tissue at 7 dah and in 99% at 53 dah. The phenotypic expression of CD3 receptors was registered in 10% of the kidney at 8 dah and in 32% at 53 dah. CD4 receptors were observed in 5% and 63% of the thymic area at 7 and 53 dah, respectively. In the kidney, T4 lymphocytes were first observed at 13 dah in 9% of the organ and in 28% at 53 dah, defining the functional development of the specific system associated with immunological memory capacity.

  4. Comprehensive Approach for Identifying the T Cell Subset Origin of CD3 and CD28 Antibody-Activated Chimeric Antigen Receptor-Modified T Cells.

    Science.gov (United States)

    Schmueck-Henneresse, Michael; Omer, Bilal; Shum, Thomas; Tashiro, Haruko; Mamonkin, Maksim; Lapteva, Natalia; Sharma, Sandhya; Rollins, Lisa; Dotti, Gianpietro; Reinke, Petra; Volk, Hans-Dieter; Rooney, Cliona M

    2017-07-01

    The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45RO + CCR7 - effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28-activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell-derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro-expanded T cells. Copyright © 2017 by The American Association of Immunologists, Inc.

  5. Anti-CD3 Antibody Ameliorates Transfusion-Associated Graft-Versus-Host Disease in a Chemotherapy-Based Mouse Model With Busulfan and Fludarabine

    Directory of Open Access Journals (Sweden)

    Xiaofan Li

    2017-05-01

    Full Text Available ABSTRACT To establish a transfusion-associated graft-versus-host disease (TA-GVHD mouse model with busulfan and fludarabine for effective treatment evaluation. BALB/c (H-2d mice were injected with busulfan (15 mg/kg and fludarabine (30 mg/kg twice a day for 4 days. The mice were transfused with 106 T cell-depleted bone marrow (TCD-BM and cells in different groups 3 days after chemotherapy: syngeneic BALB/c, MHC minor mismatch DBA/2 (H-2d, or MHC major mismatch C57BL/6(H2-b. Recipient BALB/c mice were injected with either blood only or blood+splenocyte. TA-GVHD was monitored in terms of body weight loss, clinical scores, and survival. Dexamethasone (50 mg/kg, cyclophosphamide (50 mg/kg, cyclosporine A (30 mg/kg, and anti-CD3 (1 mg/kg were injected to each group to examine the treatments. Blood transfusion alone is insufficient to induce TA-GVHD in a chemotherapy-based mouse model. A MHC-mismatched TA-GVHD model can be induced by splenocyte and blood transfusion. This MHC-mismatched TA-GVHD model was resistant to dexamethasone treatment. Treatment based on anti-CD3 monoclonal antibody slightly ameliorated TA-GVHD. Treatment effectiveness was associated with T-cell depletion following activation by anti-CD3. Busulfan and fludarabine chemotherapy regimen can be used to establish a TA-GVHD mouse model. Anti-CD3 monoclonal antibody is a potential alternative to treat TA-GVHD.

  6. CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes

    Science.gov (United States)

    Yao, Shuyu; Huang, Dan; Chen, Crystal Y.; Halliday, Lisa; Wang, Richard C.; Chen, Zheng W.

    2014-01-01

    The possibility that CD4+ T cells can act as “innate-like” cells to contain very-early M. tuberculosis (Mtb) dissemination and function as master helpers to sustain multiple effector functions of CD8+ T cells and CD3-negative lymphocytes during development of adaptive immunity against primary tuberculosis(TB) has not been demonstrated. We showed that pulmonary Mtb infection of CD4-depleted macaques surprisingly led to very-early extrathoracic Mtb dissemination, whereas CD4 deficiency clearly resulted in rapid TB progression. CD4 depletion during Mtb infection revealed the ability of CD8+ T cells to compensate and rapidly differentiate to Th17-like/Th1-like, and cytotoxic-like effectors, but these effector functions were subsequently unsustainable due to CD4 deficiency. While CD3-negative non-T lymphocytes in presence of CD4+ T cells developed predominant Th22-like and NK-like (perforin production) responses to Mtb infection, CD4 depletion abrogated these Th22-/NK-like effector functions and favored IL-17 production by CD3-negative lymphocytes. CD4-depleted macaques exhibited no or few pulmonary T effector cells constitutively producing IFN-γ, TNFα, IL-17, IL-22, and perforin at the endpoint of more severe TB, but presented pulmonary IL-4+ T effectors. TB granulomas in CD4-depleted macaques contained fewer IL-22+ and perforin+ cells despite presence of IL-17+ and IL-4+ cells. These results implicate previously-unknown “innate-like” ability of CD4+ T cells to contain extrathoracic Mtb dissemination at very early stage. Data also suggest that CD4+ T cells are required to sustain multiple effector functions of CD8+ T cells and CD3-negative lymphocytes and to prevent rapid TB progression during Mtb infection of nonhuman primates. PMID:24489088

  7. Polymorphisms in the genes ERCC2, XRCC3 and CD3EAP influence treatment outcome in multiple myeloma patients undergoing autologous bone marrow transplantation

    DEFF Research Database (Denmark)

    Vangsted, Annette; Gimsing, Peter; Klausen, Tobias W

    2007-01-01

    ) of polymorphism in the DNA repair genes ERCC1, ERCC2 and XRCC3, and in the apoptotic genes PPP1R13L and CD3EAP in 348 patients with multiple myeloma undergoing autologous bone marrow transplantation. Carriers of the variant C-allele of ERCC2 K751Q, the variant T-allele of XRCC3 T241M and the variant A...... the outcome for patients treated with autologous stem cell transplantation. Udgivelsesdato: 2007-Mar-1...

  8. In Vivo Murine-Matured Human CD3+ Cells as a Preclinical Model for T Cell-Based Immunotherapies

    Directory of Open Access Journals (Sweden)

    Kevin G. Haworth

    2017-09-01

    Full Text Available Adoptive cellular immunotherapy is a promising and powerful method for the treatment of a broad range of malignant and infectious diseases. Although the concept of cellular immunotherapy was originally proposed in the 1990s, it has not seen successful clinical application until recent years. Despite significant progress in creating engineered receptors against both malignant and viral epitopes, no efficient preclinical animal models exist for rapidly testing and directly comparing these engineered receptors. The use of matured human T cells in mice usually leads to graft-versus-host disease (GvHD, which severely limits the effectiveness of such studies. Alternatively, adult apheresis CD34+ cells engraft in neonatal non-obese diabetic (NOD-severe combined immunodeficiency (SCID-common γ chain–/– (NSG mice and lead to the development of CD3+ T cells in peripheral circulation. We demonstrate that these in vivo murine-matured autologous CD3+ T cells from humans (MATCH can be collected from the mice, engineered with lentiviral vectors, reinfused into the mice, and detected in multiple lymphoid compartments at stable levels over 50 days after injection. Unlike autologous CD3+ cells collected from human donors, these MATCH mice did not exhibit GvHD after T cell administration. This novel mouse model offers the opportunity to screen different immunotherapy-based treatments in a preclinical setting.

  9. Torsional energy levels of CH3OH+/CH3OD+/CD3OD+ studied by zero-kinetic energy photoelectron spectroscopy and theoretical calculations

    International Nuclear Information System (INIS)

    Dai, Zuyang; Gao, Shuming; Wang, Jia; Mo, Yuxiang

    2014-01-01

    The torsional energy levels of CH 3 OH + , CH 3 OD + , and CD 3 OD + have been determined for the first time using one-photon zero kinetic energy photoelectron spectroscopy. The adiabatic ionization energies for CH 3 OH, CH 3 OD, and CD 3 OD are determined as 10.8396, 10.8455, and 10.8732 eV with uncertainties of 0.0005 eV, respectively. Theoretical calculations have also been performed to obtain the torsional energy levels for the three isotopologues using a one-dimensional model with approximate zero-point energy corrections of the torsional potential energy curves. The calculated values are in good agreement with the experimental data. The barrier height of the torsional potential energy without zero-point energy correction was calculated as 157 cm −1 , which is about half of that of the neutral (340 cm −1 ). The calculations showed that the cation has eclipsed conformation at the energy minimum and staggered one at the saddle point, which is the opposite of what is observed in the neutral molecule. The fundamental C–O stretch vibrational energy level for CD 3 OD + has also been determined. The energy levels for the combinational excitation of the torsional vibration and the fundamental C–O stretch vibration indicate a strong torsion-vibration coupling

  10. The G-protein coupled receptor, GPR84 regulates IL-4 production by T lymphocytes in response to CD3 crosslinking.

    Science.gov (United States)

    Venkataraman, Chandrasekar; Kuo, Frederick

    2005-11-15

    The orphan G-protein coupled receptor, GPR84 is highly expressed in the bone marrow, and in splenic T cells and B cells. In this study, GPR84-deficient mice were generated to understand the biological function of this orphan receptor. The proliferation of T and B cells in response to various mitogens was normal in GPR84-deficient mice. Interestingly, primary stimulation of T cells with anti-CD3 resulted in increased IL-4 but not IL-2 or IFN-gamma production in GPR84(-/-) mice compared to wild-type mice. Augmented IL-4 production in GPR84-deficient T cells was not related to increased frequency of IL-4-secreting cells in response to anti-CD3 stimulation. In fact, stimulation with anti-CD3 and anti-CD28 resulted in increased levels of IL-4 but not IFN-gamma steady-state mRNA in GPR84(-/-) T cells. In addition, Th2 effector cells generated in vitro from GPR84(-/-) mice produced higher levels of IL-4, IL-5 and IL-13 compared to wild-type mice. However, there was no detectable difference in the extent of IL-4 and IL-5 production between the two groups of mice in response to antigen stimulation of spleen cells, isolated from mice previously immunized with OVA in alum. These studies reveal a novel role for GPR84 in regulating early IL-4 gene expression in activated T cells.

  11. Linker for activation of T cells is displaced from lipid rafts and decreases in lupus T cells after activation via the TCR/CD3 pathway.

    Science.gov (United States)

    Abdoel, Nursamaa; Brun, Susana; Bracho, Carmen; Rodríguez, Martín A; Blasini, Ana M

    2012-03-01

    Systemic lupus erythematosus (SLE) is characterized by abnormal signal transduction mechanisms in T lymphocytes. Linker for activation of T cells (LAT) couples TCR/CD3 activation with downstream signaling pathways. We reported diminished ERK 1/2 kinase activity in TCR/CD3 stimulated lupus T cells. In this study we evaluated the expression, phosphorylation, lipid raft and immunological synapse (IS) localization and colocalization of LAT with key signalosome molecules. We observed a diminished expression and an abnormal localization of LAT in lipid rafts and at the IS in activated lupus T cells. LAT phosphorylation, capture by GST-Grb2 fusion protein, and coupling to Grb2 and PLCγ1, was similar in healthy control and lupus T cells. Our results suggest that an abnormal localization of LAT within lipid rafts and its accelerated degradation after TCR/CD3 activation may compromise the assembly of the LAT signalosome and downstream signaling pathways required for full MAPK activation in lupus T cells. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. TOM1L Is Involved in a Novel Signaling Pathway Important for the IL-2 Production in Jurkat T Cells Stimulated by CD3/CD28 CoLigation

    Directory of Open Access Journals (Sweden)

    Ahmed Elmarghani

    2009-01-01

    Full Text Available TOM1L (target of Myb-1 Like was identified as a binding partner for the full length and catalytically-active Lck in a yeast 2-hybrid screening assay. Here we show that in Jurkat T cells stimulated by CD3/CD28 coligation where the expression of TOM1L is reduced by lenti virus mediated-siRNA results in a dramatically lower IL-2 production. The production of IL-2 in siRNA treated cells stimulated with PMA/ionomycin was not affected indicating an involvement of TOM1L in a pathway proximal of TCR and CD28. The coexpression of Fyn with TOM1L increased the level of the phosphorylated form of Fyn indicating that TOM1L has the ability to activate Fyn. The ability of TOM1L to activate Fyn was further shown in a kinase assay using angiotensin II as a substrate. By confocal microscopy, we show that the expression of TOM1L in non-treated HeLa and SK-N-SH cells colocalizes with the mitochondrial membrane but not with lysosomal compartments or the trans-Golgi network. Furthermore, we show that the over-expression of TOM1L in Jurkat cells causes an increase of the STAT3 expression . Based on our results, we here propose that TOM1L is involved in a novel signaling pathway that is important for the IL-2 production in T cells.

  13. Study on immobilizations of ovine anti-human IgG and MCAb against EHF on radiation-modified silicone films

    Energy Technology Data Exchange (ETDEWEB)

    Jinhui, Guo; Hongfei, Ha; Yuhua, Zhang [Beijing Univ., BJ (China). Dept. of Technical Physics

    1990-08-01

    Films of silicone (silastic) were grafted by monomer acrylamide vis {gamma}-radiation technology and then the ovine anti-human IgG, Epidemic hemorrhagic fever (EHF)-MCAb were immobilized on the silastic-AAM films with different grafting yields passthrough associate reactions. Measruements of relationships between grafting yields. Contents of immobilized antibodies and immunoactivities for immobilized silastic-AAM films were performed by using {sup 125}I method ELISA method was used to measure the immunoactivities for the immobilized monoantibody. The results showed that the antibodies used can be immobilized on radiation-grafted silicone films and this immobilization method has its potential significance in clinical practice.

  14. Study on immobilizations of ovine anti-human IgG and MCAb against EHF on radiation-modified silicone films

    International Nuclear Information System (INIS)

    Guo Jinhui; Ha Hongfei; Zhang Yuhua

    1990-01-01

    Films of silicone (silastic) were grafted by monomer acrylamide vis γ-radiation technology and then the ovine anti-human IgG, Epidemic hemorrhagic fever (EHF)-MCAb were immobilized on the silastic-AAM films with different grafting yields passthrough associate reactions. Measruements of relationships between grafting yields. Contents of immobilized antibodies and immunoactivities for immobilized silastic-AAM films were performed by using 125 I method ELISA method was used to measure the immunoactivities for the immobilized monoantibody. The results showed that the antibodies used can be immobilized on radiation-grafted silicone films and this immobilization method has its potential significance in clinical practice

  15. Allelic Variation in CXCL16 Determines CD3+ T Lymphocyte Susceptibility to Equine Arteritis Virus Infection and Establishment of Long-Term Carrier State in the Stallion

    Science.gov (United States)

    Cook, R. Frank; Eberth, John; Chelvarajan, R. Lakshman; Artiushin, Sergey; Timoney, Peter J.

    2016-01-01

    Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of horses and other equid species. Following natural infection, 10–70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s) of CD3+ T lymphocytes that are susceptible to in vitro EAV infection and that this phenotypic trait is associated with long-term carrier status following exposure to the virus. In contrast, stallions not possessing the CD3+ T lymphocyte susceptible phenotype are at less risk of becoming long-term virus carriers. A genome wide association study (GWAS) using the Illumina Equine SNP50 chip revealed that the ability of EAV to infect CD3+ T lymphocytes and establish long-term carrier status in stallions correlated with a region within equine chromosome 11. Here we identified the gene and mutations responsible for these phenotypes. Specifically, the work implicated three allelic variants of the equine orthologue of CXCL16 (EqCXCL16) that differ by four non-synonymous nucleotide substitutions (XM_00154756; c.715 A → T, c.801 G → C, c.804 T → A/G, c.810 G → A) within exon 1. This resulted in four amino acid changes with EqCXCL16S (XP_001504806.1) having Phe, His, Ile and Lys as compared to EqCXL16R having Tyr, Asp, Phe, and Glu at 40, 49, 50, and 52, respectively. Two alleles (EqCXCL16Sa, EqCXCL16Sb) encoded identical protein products that correlated strongly with long-term EAV persistence in stallions (P<0.000001) and are required for in vitro CD3+ T lymphocyte susceptibility to EAV infection. The third (EqCXCL16R) was associated with in vitro CD3+ T lymphocyte resistance to EAV infection and a significantly lower probability for establishment of the long-term carrier state (viral persistence) in the male reproductive tract. EqCXCL16Sa and Eq

  16. Allelic Variation in CXCL16 Determines CD3+ T Lymphocyte Susceptibility to Equine Arteritis Virus Infection and Establishment of Long-Term Carrier State in the Stallion.

    Directory of Open Access Journals (Sweden)

    Sanjay Sarkar

    2016-12-01

    Full Text Available Equine arteritis virus (EAV is the causative agent of equine viral arteritis (EVA, a respiratory, systemic, and reproductive disease of horses and other equid species. Following natural infection, 10-70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s of CD3+ T lymphocytes that are susceptible to in vitro EAV infection and that this phenotypic trait is associated with long-term carrier status following exposure to the virus. In contrast, stallions not possessing the CD3+ T lymphocyte susceptible phenotype are at less risk of becoming long-term virus carriers. A genome wide association study (GWAS using the Illumina Equine SNP50 chip revealed that the ability of EAV to infect CD3+ T lymphocytes and establish long-term carrier status in stallions correlated with a region within equine chromosome 11. Here we identified the gene and mutations responsible for these phenotypes. Specifically, the work implicated three allelic variants of the equine orthologue of CXCL16 (EqCXCL16 that differ by four non-synonymous nucleotide substitutions (XM_00154756; c.715 A → T, c.801 G → C, c.804 T → A/G, c.810 G → A within exon 1. This resulted in four amino acid changes with EqCXCL16S (XP_001504806.1 having Phe, His, Ile and Lys as compared to EqCXL16R having Tyr, Asp, Phe, and Glu at 40, 49, 50, and 52, respectively. Two alleles (EqCXCL16Sa, EqCXCL16Sb encoded identical protein products that correlated strongly with long-term EAV persistence in stallions (P<0.000001 and are required for in vitro CD3+ T lymphocyte susceptibility to EAV infection. The third (EqCXCL16R was associated with in vitro CD3+ T lymphocyte resistance to EAV infection and a significantly lower probability for establishment of the long-term carrier state (viral persistence in the male reproductive tract. EqCXCL16Sa and

  17. Non-equilibrium reaction and relaxation dynamics in a strongly interacting explicit solvent: F + CD3CN treated with a parallel multi-state EVB model.

    Science.gov (United States)

    Glowacki, David R; Orr-Ewing, Andrew J; Harvey, Jeremy N

    2015-07-28

    We describe a parallelized linear-scaling computational framework developed to implement arbitrarily large multi-state empirical valence bond (MS-EVB) calculations within CHARMM and TINKER. Forces are obtained using the Hellmann-Feynman relationship, giving continuous gradients, and good energy conservation. Utilizing multi-dimensional Gaussian coupling elements fit to explicitly correlated coupled cluster theory, we built a 64-state MS-EVB model designed to study the F + CD3CN → DF + CD2CN reaction in CD3CN solvent (recently reported in Dunning et al. [Science 347(6221), 530 (2015)]). This approach allows us to build a reactive potential energy surface whose balanced accuracy and efficiency considerably surpass what we could achieve otherwise. We ran molecular dynamics simulations to examine a range of observables which follow in the wake of the reactive event: energy deposition in the nascent reaction products, vibrational relaxation rates of excited DF in CD3CN solvent, equilibrium power spectra of DF in CD3CN, and time dependent spectral shifts associated with relaxation of the nascent DF. Many of our results are in good agreement with time-resolved experimental observations, providing evidence for the accuracy of our MS-EVB framework in treating both the solute and solute/solvent interactions. The simulations provide additional insight into the dynamics at sub-picosecond time scales that are difficult to resolve experimentally. In particular, the simulations show that (immediately following deuterium abstraction) the nascent DF finds itself in a non-equilibrium regime in two different respects: (1) it is highly vibrationally excited, with ∼23 kcal mol(-1) localized in the stretch and (2) its post-reaction solvation environment, in which it is not yet hydrogen-bonded to CD3CN solvent molecules, is intermediate between the non-interacting gas-phase limit and the solution-phase equilibrium limit. Vibrational relaxation of the nascent DF results in a spectral

  18. Synthesis, Transport, and Thermoelectric Studies of Topological Dirac Semimetal Cd3As2 for Room Temperature Waste Heat Recovery and Energy Conversion

    Science.gov (United States)

    Hosseini, Tahereh A.

    Rising rates of the energy consumption and growing concerns over the climate change worldwide have made energy efficiency an urgent problem to address. Nowadays, almost two-thirds of the energy produced by burning fossil fuels to generate electrical power is lost in the form of the heat. On this front, increasing electrical power generation through a waste heat recovery remains one of the highly promising venues of the energy research. Thermo-electric generators (TEGs) directly convert thermal energy into electrical and are the prime candidates for application in low-grade thermal energy/ waste heat recovery. The key commercial TE materials, e.g. PbTe and Bi2Te 3, have room temperature ZT of less than 1, whereas ZT exceeding 3 is required for a TEG to be economically viable. With the thermoelectric efficiency typically within a few percent range and a low efficiency-to-cost ratio of TEGs, there has been a resurgence in the search for new class of thermo-electric materials for developing high efficiency thermo-to-electric energy conversion systems, with phonon-glass electron-crystal materials holding the most promise. Herein, we focus on synthesis, characterization and investigation of electrical, thermo-electrical and thermal characteristics of crystalline Cd 3As2, a high performance 3D topological Dirac semimetal with Dirac fermions dispersing linearly in k3-space and possessing one of the largest electron mobilities known for crystalline materials, i.e. 104-105cm2V-1 s-1. Suppression of carrier backscattering, ultra-high charge carrier mobility, and inherently low thermal conductivity make this semimetal a key candidate for demonstrating high, device-favorable S and in turn ZT. In this work, a low-temperature vapor-based crystallization pathway was developed and optimized to produce free standing 2D cm-size crystals in Cd 3As2. Compared to the bulk crystals produced in previous studies, e.g. Piper-Polich, Bridgman, or flux method, Cd3As 2 samples were synthesized

  19. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity.

    Science.gov (United States)

    Dorr, Patrick; Westby, Mike; Dobbs, Susan; Griffin, Paul; Irvine, Becky; Macartney, Malcolm; Mori, Julie; Rickett, Graham; Smith-Burchnell, Caroline; Napier, Carolyn; Webster, Rob; Armour, Duncan; Price, David; Stammen, Blanda; Wood, Anthony; Perros, Manos

    2005-11-01

    Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin (geometric mean 90% inhibitory concentration of 2.0 nM). Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. There was little difference in the sensitivity of the 200 viruses to maraviroc, as illustrated by the biological cutoff in this assay (= geometric mean plus two standard deviations [SD] of 1.7-fold). The mechanism of action of maraviroc was established using cell-based assays, where it blocked binding of viral envelope, gp120, to CCR5 to prevent the membrane fusion events necessary for viral entry. Maraviroc did not affect CCR5 cell surface levels or associated intracellular signaling, confirming it as a functional antagonist of CCR5. Maraviroc has no detectable in vitro cytotoxicity and is highly selective for CCR5, as confirmed against a wide range of receptors and enzymes, including the hERG ion channel (50% inhibitory concentration, >10 microM), indicating potential for an excellent clinical safety profile. Studies in preclinical in vitro and in vivo models predicted maraviroc to have human pharmacokinetics consistent with once- or twice-daily dosing following oral administration. Clinical trials are ongoing to further investigate the potential of using maraviroc for the treatment of HIV-1 infection and AIDS.

  20. Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

    International Nuclear Information System (INIS)

    Nagai, Kentaro; Nakahata, Shingo; Shimosaki, Shunsuke; Tamura, Tomohiro; Kondo, Yuudai; Baba, Takashi; Taki, Tomohiko; Taniwaki, Masafumi; Kurosawa, Gene; Sudo, Yukio; Okada, Seiji; Sakoda, Sumio; Morishita, Kazuhiro

    2014-01-01

    Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Up to 20% of oral dysplasia cases have been suggested to undergo malignant transformation to OSCC; however, there are no methods to predict OSCC development. In this study, to identify the genes associated with oral dysplasia progression, we performed genomic copy number analyses of genomic DNA samples isolated from primary oral dysplasia and OSCC via the microdissection method and found elevated expression of transferrin receptor C (TfR1/TFRC) with genomic amplification in oral dysplasia and OSCC. The expression rate of TFRC in OSCC was significantly higher than that in dysplasia, suggesting that OSCC disease progression might be related to TFRC expression. Additionally, we investigated the in vitro and in vivo impacts of a newly established anti-human TFRC monoclonal antibody, which was isolated from a human cDNA library using the phage-display method, on cell proliferation and survival. The anti-TFRC antibody blocked the interaction between transferrin and TFRC and consequently inhibited iron uptake, leading to the iron deprivation-mediated suppression of cell growth and induction of apoptosis. Moreover, we demonstrated that the anti-TFRC antibody efficiently inhibited tumor growth in a murine xenograft OSCC model. Therefore, we suggest our developed complete human anti-human TFRC antibody as a useful, novel treatment for oral dysplasia and OSCC

  1. The role of intrahepatic CD3 +/CD4 −/CD8 − double negative T (DN T) cells in enhanced acetaminophen toxicity

    International Nuclear Information System (INIS)

    Getachew, Yonas; Cusimano, Frank A.; James, Laura P.; Thiele, Dwain L.

    2014-01-01

    The role of the immune system, specifically NK, NKT and CD3 cells, in acetaminophen (APAP) induced liver injury remains inconsistently defined. In the present study, wild type (C57BL/6J) mice and granzyme B deficient (GrB −/−) mice were treated with acetaminophen to assess the role of the immune system in acute liver injury. Doses of acetaminophen that induced sub lethal liver injury in wild type mice unexpectedly produced fatal hepatotoxicity in granzyme B deficient (GrB −/−) mice. Analysis revealed that GrB −/− mice had an increased population of intrahepatic CD3 (+), CD4 (−), and CD8 (−) lymphocytes expressing the CD69 activation marker and Fas ligand. Depletion of these cells in the GrB −/− and wild type mice made them less susceptible to APAP injury, while depletion of NK1.1 (+) cells or both CD4 (+) and CD8 (+) T cells failed to provide the same hepatoprotection. Transfer of the GrB −/− IHLs further exacerbated liver injury and increased mortality in wild type mice but not in LRP/LPR mice, lacking fas expression. Conclusions: Acetaminophen toxicity is enhanced by the presence of activated, FasL expressing intrahepatic CD3 (+), CD4 (−), CD8 (−), NK1.1 (−) T cells. Depletion of these cells from GrB −/− mice and wild type mice greatly reduces mortality and improves the course of liver injury recovery. - Highlights: • Intrahepatic lymphocytes (IHLs) from GrB −/− mice harbor activated DNT cells. • IHLs from GrB −/− mice exhibit enhanced Fas ligand expression. • Acetaminophen toxicity is enhanced by activated, FasL expressing DNT cells

  2. T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy.

    Science.gov (United States)

    Roszik, János; Sebestyén, Zsolt; Govers, Coen; Guri, Yakir; Szöor, Arpád; Pályi-Krekk, Zsuzsanna; Vereb, György; Nagy, Peter; Szöllosi, János; Debets, Reno

    2011-05-01

    T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and β chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. The role of intrahepatic CD3 +/CD4 −/CD8 − double negative T (DN T) cells in enhanced acetaminophen toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Getachew, Yonas, E-mail: yonas.getachew@utsouthwestern.edu [Division of Digestive and Liver Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9151 (United States); Cusimano, Frank A. [Division of Digestive and Liver Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9151 (United States); James, Laura P. [Department of Pediatrics, University of Arkansas, Little Rock, AR (United States); Thiele, Dwain L. [Division of Digestive and Liver Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9151 (United States)

    2014-10-15

    The role of the immune system, specifically NK, NKT and CD3 cells, in acetaminophen (APAP) induced liver injury remains inconsistently defined. In the present study, wild type (C57BL/6J) mice and granzyme B deficient (GrB −/−) mice were treated with acetaminophen to assess the role of the immune system in acute liver injury. Doses of acetaminophen that induced sub lethal liver injury in wild type mice unexpectedly produced fatal hepatotoxicity in granzyme B deficient (GrB −/−) mice. Analysis revealed that GrB −/− mice had an increased population of intrahepatic CD3 (+), CD4 (−), and CD8 (−) lymphocytes expressing the CD69 activation marker and Fas ligand. Depletion of these cells in the GrB −/− and wild type mice made them less susceptible to APAP injury, while depletion of NK1.1 (+) cells or both CD4 (+) and CD8 (+) T cells failed to provide the same hepatoprotection. Transfer of the GrB −/− IHLs further exacerbated liver injury and increased mortality in wild type mice but not in LRP/LPR mice, lacking fas expression. Conclusions: Acetaminophen toxicity is enhanced by the presence of activated, FasL expressing intrahepatic CD3 (+), CD4 (−), CD8 (−), NK1.1 (−) T cells. Depletion of these cells from GrB −/− mice and wild type mice greatly reduces mortality and improves the course of liver injury recovery. - Highlights: • Intrahepatic lymphocytes (IHLs) from GrB −/− mice harbor activated DNT cells. • IHLs from GrB −/− mice exhibit enhanced Fas ligand expression. • Acetaminophen toxicity is enhanced by activated, FasL expressing DNT cells.

  4. Inefficient Nef-mediated downmodulation of CD3 and MHC-I correlates with loss of CD4+T cells in natural SIV infection.

    Directory of Open Access Journals (Sweden)

    Michael Schindler

    2008-07-01

    Full Text Available Recent data suggest that Nef-mediated downmodulation of TCR-CD3 may protect SIVsmm-infected sooty mangabeys (SMs against the loss of CD4+ T cells. However, the mechanisms underlying this protective effect remain unclear. To further assess the role of Nef in nonpathogenic SIV infection, we cloned nef alleles from 11 SIVsmm-infected SMs with high (>500 and 15 animals with low (<500 CD4+ T-cells/microl in bulk into proviral HIV-1 IRES/eGFP constructs and analyzed their effects on the phenotype, activation, and apoptosis of primary T cells. We found that not only efficient Nef-mediated downmodulation of TCR-CD3 but also of MHC-I correlated with preserved CD4+ T cell counts, as well as with high numbers of Ki67+CD4+ and CD8+CD28+ T cells and reduced CD95 expression by CD4+ T cells. Moreover, effective MHC-I downregulation correlated with low proportions of effector and high percentages of naïve and memory CD8+ T cells. We found that T cells infected with viruses expressing Nef alleles from the CD4low SM group expressed significantly higher levels of the CD69, interleukin (IL-2 and programmed death (PD-1 receptors than those expressing Nefs from the CD4high group. SIVsmm Nef alleles that were less active in downmodulating TCR-CD3 were also less potent in suppressing the activation of virally infected T cells and subsequent cell death. However, only nef alleles from a single animal with very low CD4+ T cell counts rendered T cells hyper-responsive to activation, similar to those of HIV-1. Our data suggest that Nef may protect the natural hosts of SIV against the loss of CD4+ T cells by at least two mechanisms: (i downmodulation of TCR-CD3 to prevent activation-induced cell death and to suppress the induction of PD-1 that may impair T cell function and survival, and (ii downmodulation of MHC-I to reduce CTL lysis of virally infected CD4+ T cells and/or bystander CD8+ T cell activation.

  5. CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor

    DEFF Research Database (Denmark)

    Dietrich, J; Hou, X; Wegener, A M

    1994-01-01

    -regulation of the TCR. Furthermore, analysis of a series of CD3 gamma truncation mutants indicated that in addition to S126 phosphorylation a motif C-terminal of S126 was required for TCR down-regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane-proximal di-leucine motif (L131......, indicating that the TCR was down-regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di-leucine- or tyrosine-based motifs is proposed....

  6. Double negative (CD3+ 4- 8- TCR alphabeta splenic cells from young NOD mice provide long-lasting protection against type 1 diabetes.

    Directory of Open Access Journals (Sweden)

    Beverly Duncan

    2010-07-01

    Full Text Available Double negative CD3(+4(-8(- TCR alphabeta splenic cells (DNCD3 can suppress the immune responses to allo and xenografts, infectious agents, tumors, and some autoimmune disorders. However, little is known about their role in autoimmune diabetes, a disease characterized by the reduction of insulin production subsequent to destruction of pancreatic beta-cells by a polyclonal population of self-reactive T-cells. Herein, we analyzed the function and phenotype of DNCD3 splenic cells in young NOD mice predisposed to several autoimmune disorders among which, the human-like autoimmune diabetes.DNCD3 splenic cells from young NOD mice (1 provided long-lasting protection against diabetes transfer in NOD/Scid immunodeficient mice, (2 proliferated and differentiated in the spleen and pancreas of NOD/Scid mice and pre-diabetic NOD mice into IL-10-secreting T(R-1 like cells in a Th2-like environment, and (3 their anti-diabetogenic phenotype is CD3(+(CD4(-CD8(-CD28(+CD69(+CD25(low Foxp3(- iCTLA-4(-TCR alphabeta(+ with a predominant Vbeta13 gene usage.These findings delineate a new T regulatory component in autoimmune diabetes apart from that of NKT and CD4(+CD25(high Foxp3(+T-regulatory cells. DNCD3 splenic cells could be potentially manipulated towards the development of autologous cell therapies in autoimmune diabetes.

  7. CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1.

    Science.gov (United States)

    Osada, Takuya; Patel, Sandip P; Hammond, Scott A; Osada, Koya; Morse, Michael A; Lyerly, H Kim

    2015-06-01

    Bispecific T cell-engaging (BiTE) antibodies recruit polyclonal cytotoxic T cells (CTL) to tumors. One such antibody is carcinoembryonic antigen (CEA) BiTE that mediates T cell/tumor interaction by simultaneously binding CD3 expressed by T cells and CEA expressed by tumor cells. A widely operative mechanism for mitigating cytotoxic T cell-mediated killing is the interaction of tumor-expressed PD-L1 with T cell-expressed PD-1, which may be partly reversed by PD-1/PD-L1 blockade. We hypothesized that PD-1/PD-L1 blockade during BiTE-mediated T cell killing would enhance CTL function. Here, we determined the effects of PD-1 and PD-L1 blockade during initial T cell-mediated killing of CEA-expressing human tumor cell lines in vitro, as well as subsequent T cell-mediated killing by T lymphocytes that had participated in tumor cell killing. We observed a rapid upregulation of PD-1 expression and diminished cytolytic function of T cells after they had engaged in CEA BiTE-mediated killing of tumors. T cell cytolytic activity in vitro could be maximized by administration of anti-PD-1 or anti-PD-L1 antibodies alone or in combination if applied prior to a round of T cell killing, but T cell inhibition could not be fully reversed by this blockade once the T cells had killed tumor. In conclusion, our findings demonstrate that dual blockade of PD-1 and PD-L1 maximizes T cell killing of tumor directed by CEA BiTE in vitro, is more effective if applied early, and provides a rationale for clinical use.

  8. Targeted delivery of polyamidoamine-paclitaxel conjugate functionalized with anti-human epidermal growth factor receptor 2 trastuzumab

    Directory of Open Access Journals (Sweden)

    Ma P

    2015-03-01

    Full Text Available Pengkai Ma,1 Xuemei Zhang,1 Ling Ni,2 Jinming Li,2 Fengpu Zhang,1 Zheng Wang,1 Shengnan Lian,1 Kaoxiang Sun1 1School of Pharmacy, Yantai University, Yantai, Shandong Province, People’s Republic of China; 2State Key Laboratory of Long-acting and Targeting Drug Delivery System, Yantai, Shandong Province, People’s Republic of China Background: Antibody-dendrimer conjugates have the potential to improve the targeting and release of chemotherapeutic drugs at the tumor site while reducing adverse side effects caused by drug accumulation in healthy tissues. In this study, trastuzumab (TMAB, which binds to human epidermal growth factor receptor 2 (HER2, was used as a targeting agent in a TMAB-polyamidoamine (PAMAM conjugate carrying paclitaxel (PTX specifically to cells overexpressing HER2. Methods: TMAB was covalently linked to a PAMAM dendrimer via bifunctional polyethylene glycol (PEG. PTX was conjugated to PAMAM using succinic anhydride as a cross-linker, yielding TMAB-PEG-PAMAM-PTX. Dynamic light scattering and transmission electron microscopy were used to characterize the conjugates. The cellular uptake and in vivo biodistribution were studied by fluorescence microscopy, flow cytometry, and Carestream In Vivo FX, respectively. Results: Nuclear magnetic resonance spectroscopy demonstrated that PEG, PTX, fluorescein isothiocyanate, and cyanine7 were conjugated to PAMAM. Ultraviolet-visible spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that TMAB was conjugated to PEG-PAMAM. Dynamic light scattering and transmission electron microscopy measurements revealed that the different conjugates ranged in size between 10 and 35 nm and had a spherical shape. In vitro cellular uptake demonstrated that the TMAB-conjugated PAMAM was taken up by HER2-overexpressing BT474 cells more efficiently than MCF-7 cells that expressed lower levels of HER2. Co-localization experiments indicated that TMAB-conjugated PAMAM was

  9. Chimeric bispecific OC/TR monoclonal antibody mediates lysis of tumor cells expressing the folate-binding protein (MOv18) and displays decreased immunogenicity in patients

    NARCIS (Netherlands)

    Luiten, R. M.; Warnaar, S. O.; Sanborn, D.; Lamers, C. H.; Bolhuis, R. L.; Litvinov, S. V.; Zurawski, V. R.; Coney, L. R.

    1997-01-01

    The bispecific OC/TR monoclonal antibody (mAb) cross-links the CD3 molecule on T cells with the human folate-binding protein (FBP), which is highly expressed on nonmucinous ovarian carcinomas. Clinical trials of patients with ovarian carcinoma with the OC/TR mAb have shown some complete and partial

  10. Thermal conductivity and electrical resistivity of cadmium arsenide (Cd3As2) in the temperature range 4.2-40K1

    International Nuclear Information System (INIS)

    Bartkowski, K.; Ratalowicz, J.; Zdanowicz, W.

    1986-01-01

    Results on electrical resistivity and thermal conductivity measured in the temperature range 4.2-40 K are presented for single-crystal and polycrystalline samples of Cd 3 As 2 . Hall effect has been studied at temperatures of 4.2, 77, and 300K. The calculated value of the conduction electron concentration was in the range 1.87-1.95 10 24 m -3 . Electrical resistivity of all investigated samples was independent of temperature up to about 10K and increased slowsly at higher temperatures. The thermal conductivity shows a maximum in the region in which the lattice component of thermal conductivity dominates. The strong anistropy of the lattice component determines the anisotropy of the total thermal conductivity. The electronic component of thermal conductivity does not exhibit any anisotropy and shows a maximum at a temperature of about 300 K

  11. An increase in CD3+CD4+CD25+ regulatory T cells after administration of umbilical cord-derived mesenchymal stem cells during sepsis.

    Directory of Open Access Journals (Sweden)

    Yu-Hua Chao

    Full Text Available Sepsis remains an important cause of death worldwide, and vigorous immune responses during sepsis could be beneficial for bacterial clearance but at the price of collateral damage to self tissues. Mesenchymal stem cells (MSCs have been found to modulate the immune system and attenuate sepsis. In the present study, MSCs derived from bone marrow and umbilical cord were used and compared. With a cecal ligation and puncture (CLP model, the mechanisms of MSC-mediated immunoregulation during sepsis were studied by determining the changes of circulating inflammation-associated cytokine profiles and peripheral blood mononuclear cells 18 hours after CLP-induced sepsis. In vitro, bone marrow-derived MSCs (BMMSCs and umbilical cord-derived MSCs (UCMSCs showed a similar morphology and surface marker expression. UCMSCs had stronger potential for osteogenesis but lower for adipogenesis than BMMSCs. Compared with rats receiving PBS only after CLP, the percentage of circulating CD3+CD4+CD25+ regulatory T (Treg cells and the ratio of Treg cells/T cells were elevated significantly in rats receiving MSCs. Further experiment regarding Treg cell function demonstrated that the immunosuppressive capacity of Treg cells from rats with CLP-induced sepsis was decreased, but could be restored by administration of MSCs. Compared with rats receiving PBS only after CLP, serum levels of interleukin-6 and tumor necrosis factor-α were significantly lower in rats receiving MSCs after CLP. There were no differences between BMMSCs and UCMSCs. In summary, this work provides the first in vivo evidence that administering BMMSCs or UCMSCs to rats with CLP-induced sepsis could increase circulating CD3+CD4+CD25+ Treg cells and Treg cells/T cells ratio, enhance Treg cell suppressive function, and decrease serum levels of interleukin-6 and tumor necrosis factor-α, suggesting the immunomodulatory association of Treg cells and MSCs during sepsis.

  12. IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells.

    Science.gov (United States)

    da Silva, Thiago Aparecido; Mariano, Vania Sammartino; Sardinha-Silva, Aline; de Souza, Maria Aparecida; Mineo, Tiago Wilson Patriarca; Roque-Barreira, Maria Cristina

    2016-01-01

    ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production

  13. IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells.

    Directory of Open Access Journals (Sweden)

    Thiago Aparecido da Silva

    Full Text Available ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs, as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, Artin

  14. Distinct Biomarker Profiles in Ex Vivo T Cell Depletion Graft Manipulation Strategies: CD34+ Selection versus CD3+/19+ Depletion in Matched Sibling Allogeneic Peripheral Blood Stem Cell Transplantation.

    Science.gov (United States)

    Cantilena, Caroline R; Ito, Sawa; Tian, Xin; Jain, Prachi; Chinian, Fariba; Anandi, Prathima; Keyvanfar, Keyvan; Draper, Debbie; Koklanaris, Eleftheria; Hauffe, Sara; Superata, Jeanine; Stroncek, David; Muranski, Pawel; Barrett, A John; Battiwalla, Minoo

    2018-03-01

    Various approaches have been developed for ex vivo T cell depletion in allogeneic stem cell transplantation to prevent graft-versus-host disease (GVHD). Direct comparisons of T cell depletion strategies have not been well studied, however. We evaluated cellular and plasma biomarkers in 2 different graft manipulation strategies, CD3 + CD19 + cell depletion (CD3/19D) versus CD34 + selection (CD34S), and their associations with clinical outcomes. Identical conditions, including the myeloablative preparative regimen, HLA-identical sibling donor, GVHD prophylaxis, and graft source, were used in the 2 cohorts. Major clinical outcomes were similar in the 2 groups in terms of overall survival, nonrelapse mortality, and cumulative incidence of relapse; however, the cumulative incidence of acute GVHD trended to be higher in the CD3/19D cohort compared with the CD34S cohort. A distinct biomarker profile was noted in the CD3/19D cohort: higher levels of ST2, impaired Helios - FoxP3 + Treg reconstitution, and rapid reconstitution of naïve, Th2, and Th17 CD4 cells in the early post-transplantation period. In vitro graft replication studies confirmed that CD3/19D disproportionately depleted Tregs and other CD4 subset repertoires in the graft. This study confirms the utility of biomarker monitoring, which can be directly correlated with biological consequences and possible future therapeutic indications. Published by Elsevier Inc.

  15. Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the developement aplastic crises

    Directory of Open Access Journals (Sweden)

    SANT'ANNA Anadayr L.M.

    2002-01-01

    Full Text Available The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE, Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140 have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05 was seen between IgG antibody prevalence in male (27.8% and female (35.5% patients. Anti-B19 IgG antibodies were more frequent in older (37.6% than younger (28.2% than 20 years old patients, although this difference had no statistical significance (p > 0.05. Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

  16. CD3 TRIGA users conference

    International Nuclear Information System (INIS)

    2008-01-01

    The Sixteenth European TRIGA Users Conference was held in Pitesti, Romania, on 25-28 September, 2000, under the sponsorship of the Institute for Nuclear Research at Pitesti. The papers which follow in this document are presented in the same order as listed in the Conference Program. All papers which were received for publication (44) have been included. Those papers which were presented but not received for publication are presented in abstract form (4 papers). It was very interesting for the Conference attendees from the West to learn about the large scope of excellent work conducted in Romania, especially at the Institute of Nuclear Research in Pitesti. Similarly, it was fortunate that a large attendance of Romanian researchers (53) from many institutes, universities, and government agencies could attend the Conference and interact with their counterparts from outside Romania. The European TRIGA9 Owners' Group was fortunate to be hosted by the owners and users of the world's largest TRIGA reactor - the 14-MW Romanian research and test reactor. The Opening Session talk was given by Radu Berceanu, Minister of Industries and Commerce. It was followed by the following presentations: R and D - Support for Nuclear Power Development by Ioan Rotaru (General Manager of SNNE); Overview of TRIGA Reactor and other Programs at GENERAL ATOMICS by Junaid Razvi (General Manager TRIGA Reactor at GA); Development strategies connected to National Power and Energy Program by Mircea Ionescu (Director Nuclear Energy Department of M.I.C.); Contribution of INR R and D Programs to Sustain Peaceful and Safe utilization of Nuclear Energy in Romania by Constantin Gheorghiu (Scientific Deputy Director at SCN). A Technical visit to TRIGA Reactor at INR Pitesti took place. Opening Session was followed by five sessions dedicated to the following subjects: Session 1 (8 papers) - TRIGA reactors operation, repair and maintenance; Session 2 (10 papers) - Future developments and future goals of TRIGA utilization; Session 3 (12 papers) - Computer codes for nuclear and thermohydraulic analysis; Session 4 (9 papers) - Decommissioning and spent fuel transportation and storage; Session 5 (10 papers) - Core management, in-core experiments and nuclear safety. Eighty seven participants from Austria, Brazil. Indonesia, Italy, Germany, Finland, Slovenia, Turkey, USA and Romania. The conference ended with a final discussion session

  17. A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy.

    Science.gov (United States)

    Alvarez-Fernández, C; Escribà-Garcia, L; Vidal, S; Sierra, J; Briones, J

    2016-07-19

    Immunotherapy based on the adoptive transfer of gene modified T cells is an emerging approach for the induction of tumor-specific immune responses. Memory stem T cells, due to their enhanced antitumor and self-renewal capacity, have become potential candidate for adoptive T cell therapy of cancer. Methods to generate memory stem T cells ex vivo rely on CD3/CD28 costimulation and the use of cytokines such as IL-7 and IL-15 during the entire culture period. However, a strong costimulation may induce differentiation of memory stem T cells to effector memory T cells. Here we show that manipulation of the length of the costimulation and addition of IL-21 enhance the ex vivo expansion of memory stem T cells. Purified naïve T cells from healthy donors were cultured in the presence of anti-CD3/CD28 coated beads, IL-7, IL-15 and/or IL-21 (25 ng/ml). T cells phenotype from the different memory and effector subpopulations were analyzed by multiparametric flow cytometry. A short anti-CD3/CD28 costimulation of naïve T cells, combined with IL-7 and IL-15 significantly increased the frequencies of CD4(+) and CD8(+) memory stem T cells ex vivo, compared to a prolonged costimulation (34.6 ± 4.4 % vs 15.6 ± 4.24 % in CD4(+); p = 0.008, and 20.5 ± 4.00 % vs 7.7 ± 2.53 % in CD8(+); p = 0.02). Moreover, the addition of IL-21 to this condition further enhanced the enrichment and expansion of CD4(+) and CD8(+) memory stem T cells with an increase in the absolute numbers (0.7 × 10(6) ± 0.1 vs 0.26 × 10(6) ± 0.1 cells for CD4(+); p = 0.002 and 1.1 × 10(6) ± 0.1 vs 0.27 × 10(6) ± 0.1 cells for CD8(+); p = 0.0002; short + IL-21 vs long). These new in vitro conditions increase the frequencies and expansion of memory stem T cells and may have relevant clinical implications for the generation of this memory T cell subset for adoptive cell therapy of patients with cancer.

  18. M-ficolin, an innate immune defence molecule, binds patterns of acetyl groups and activates complement

    DEFF Research Database (Denmark)

    Frederiksen, Pernille Dorthea; Thiel, Steffen; Larsen, Claus Bindslev

    2005-01-01

    Ficolins play a role in the innate immune defence as pathogen-associated molecular pattern recognition molecules. Three ficolins are found in humans: H-ficolin, L-ficolin and M-ficolin. L-ficolin and H-ficolin circulate in blood in complexes with mannan-binding lectin-associated serine proteases...... (MASPs) and are capable of activating the complement system. L-ficolin shows affinity for acetylated compounds and binds to various capsulated strains of bacteria. H-ficolin has been shown to bind Aerococcus viridans. Less is known about M-ficolin, but it is thought to be present only on monocytes. We...... system. We developed a monoclonal rat anti-human-M/L-ficolin antibody and verified by flow cytometric analysis the presence of ficolin on the surface of peripheral blood monocytes....

  19. Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110

    Science.gov (United States)

    Deisting, Wibke; Raum, Tobias; Kufer, Peter; Baeuerle, Patrick A.; Münz, Markus

    2015-01-01

    Background Bispecific T cell engager (BiTE®) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE® antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-β), interleukin-10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells. Methods The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE®-induced proliferation of previously resting human peripheral T cells. Findings An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-βand PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE®-induced lysis, and even various combinations could not. Conclusions Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE® antibody construct. PMID:26510188

  20. Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110.

    Directory of Open Access Journals (Sweden)

    Wibke Deisting

    Full Text Available Bispecific T cell engager (BiTE® are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE® antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-β, interleukin-10 (IL-10 and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells.The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE®-induced proliferation of previously resting human peripheral T cells.An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-β and PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE®-induced lysis, and even various combinations could not.Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE® antibody construct.

  1. CD3+/CD8+ T-cell density and tumoral PD-L1 predict survival irrespective of rituximab treatment in Chinese diffuse large B-cell lymphoma patients.

    Science.gov (United States)

    Shi, Yunfei; Deng, Lijuan; Song, Yuqin; Lin, Dongmei; Lai, Yumei; Zhou, LiXin; Yang, Lei; Li, Xianghong

    2018-05-10

    To investigate the prognostic value of tumor-infiltrating T-cell density and programmed cell death ligand-1 (PD-L1) expression in diffuse large B cell lymphoma (DLBCL). One-hundred-twenty-five Chinese DLBCL patients were enrolled in our study and provided samples; 76 of all cases were treated with rituximab (R). Tumor tissues were immunostained and analyzed for CD3+ and CD8+ tumor-infiltrating T-cell density, tumoral PD-L1, and microenvironmental PD-L1 (mPD-L1). The density of CD3 was rated as high in 33.6% cases, while 64.0% of DLBCLs were classified as high CD8 density. Of all cases, 16.8% were PD-L1+. Of the remaining PD-L1-DLBCLs, 29.8% positively expressed mPD-L1. Both CD3 high density and CD8 high density were associated with mPD-L1 positivity (P = 0.001 and P = 0.0001). In multivariate analysis, independently, high CD3 density predicted better OS (P = 0.023), while CD8 high density and PD-L1 positivity were both associated with prolonged PFS (P = 0.013 and P = 0.036, respectively). Even in the subgroup treated with R, univariate analyses indicated that high CD3 density and PD-L1 positivity were associated with better OS (P = 0.041) and PFS (P = 0.033), respectively. The infiltrating densities of CD3+ T-cells, CD8+ T-cells, and PD-L1 expression are predictive of survival in DLBCLs, irrespective of R usage.

  2. Selective T-cell depletion targeting CD45RA reduces viremia and enhances early T-cell recovery compared with CD3-targeted T-cell depletion.

    Science.gov (United States)

    Triplett, Brandon M; Muller, Brad; Kang, Guolian; Li, Ying; Cross, Shane J; Moen, Joseph; Cunningham, Lea; Janssen, William; Mamcarz, Ewelina; Shook, David R; Srinivasan, Ashok; Choi, John; Hayden, Randall T; Leung, Wing

    2018-02-01

    T-cell depletion (TCD) effectively reduces severe graft-versus-host disease in recipients of HLA-mismatched allografts. However, TCD is associated with delayed immune recovery and increased infections. We hypothesized that specific depletion of CD45RA+ naive T cells, rather than broad depletion of CD3+ T cells, can preserve memory-immunity in the allografts and confer protection against important viral infections in the early post-transplant period. Sixty-seven patients who received TCD haploidentical donor transplantation for hematologic malignancy on 3 consecutive trials were analyzed. Patients receiving CD45RA-depleted donor grafts had 2000-fold more donor T cells infused, significantly higher T-cell counts at Day +30 post transplant (550/μL vs 10/μL; P depleted grafts were more likely to experience adenovirus viremia (27% vs 4%, P = .02). CD45RA-depletion provided a large number of donor memory T cells to the recipients and was associated with enhanced early T-cell recovery and protection against viremia. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Neutron scattering studies on phase transitions in (CD3ND3)2CuCl4 and MnCl2.4H2O

    International Nuclear Information System (INIS)

    Steijger, J.J.M.

    1982-10-01

    In this thesis the results of neutron scattering experiments and measurements of the susceptibility on some compounds which display magnetic and/or structural phase transitions, are described. Following an introductory chapter, chapter 2 shows that neutron scattering can be used as a tool for unravelling problems in crystallographic and magnetic structure. The qualitative different scattering patterns for scatters are described. In chapters 3 and 4 an investigation on the layered ferromagnets (CH 3 NH 3 ) 2 CuCl 4 and (CD 3 ND 3 ) 2 CuCl 4 is described. In these materials the copper ions, which carry the magnetic moment, are more closely spaced in the ab-planes, and consequently the magnetic interactions in these planes are stronger than those in the direction perpendicular to these planes by about a factor of 10 5 . Chapter 5 presents a discussion and a calculation of demagnetizing and dipole fields. The second part of this thesis is concerned with the transition from the antiferromagnetic to the paramagnetic phase in MnCl 2 .4H 2 O in the presence of a magnetic field applied perpendicular to the preferred direction of the magnetic moments. The theory is reviewed in chapter 6 and in chapter 7 the correction procedure for inhomogeneous internal fields is applied to the measurements on MnCl 2 .4H 2 O. (Auth./C.F.)

  4. Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

    Science.gov (United States)

    Munisvaradass, Rusheni; Kumar, Suresh; Govindasamy, Chandramohan; Alnumair, Khalid S.; Mok, Pooi Ling

    2017-01-01

    Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non–transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours. PMID:28885562

  5. Highly efficient elimination of colorectal tumor-initiating cells by an EpCAM/CD3-bispecific antibody engaging human T cells.

    Directory of Open Access Journals (Sweden)

    Ines Herrmann

    2010-10-01

    Full Text Available With their resistance to genotoxic and anti-proliferative drugs and potential to grow tumors and metastases from very few cells, cancer stem or tumor-initiating cells (TICs are a severe limitation for the treatment of cancer by conventional therapies. Here, we explored whether human T cells that are redirected via an EpCAM/CD3-bispecific antibody called MT110 can lyse colorectal TICs and prevent tumor growth from TICs. MT110 recognizes EpCAM, a cell adhesion molecule expressed on TICs from diverse human carcinoma, which was recently shown to promote tumor growth through engagement of elements of the wnt pathway. MT110 was highly potent in mediating complete redirected lysis of KRAS-, PI3 kinase- and BRAF-mutated colorectal TICs, as demonstrated in a soft agar assay. In immunodeficient mice, MT110 prevented growth of tumors from a 5,000-fold excess of a minimally tumorigenic TIC dose. T cells engaged by MT110 may provide a potent therapeutic means to eradicate TICs and bulk tumor cells derived thereof.

  6. "Anomalous" excitation in hydrogen-bonded molecular crystals - a Raman scattering study of specifically deuterated acetanilide (C 6D 5-CONH-CD 3)

    Science.gov (United States)

    Sauvajol, J. L.; De Nunzio, G.; Almairac, R.; Moret, J.; Barthés, M.; Bataillon, Place E.

    1991-01-01

    The focus of experimental and theoretical works about crystalline Acetanilide has been the "anomalous" temperature-dependent ir absorption and Raman peaks at about 1650 cm -1 and the multiband structure in the N-H stretch region. A lively discussion about the assignment of these "anomalous" bands has arisen and is still in progress. The present Raman experiments should be placed in this context as an attempt to identify the molecular degrees of freedom which originate the "anomalous" bands. In this aim Raman experiments have been performed on specifically deuterated Acetanilide [C 6D 5-CONH-CD 3] single crystal in the low-frequency (phonon) and C=O stretching regions. On cooling a distinct band at about 1495 cm -1 increases in intensity. We assign this peak to the equivalent of the 1650 cm -1 band in Acetanilide. The temperature dependence of this Raman line was studied. The results are discussed in the light of the models proposed to explain the anomalous behaviour of the 1650 cm -1 Raman line in Acetanilide.

  7. PKCθ/β and CYLD are antagonistic partners in the NFκB and NFAT transactivation pathways in primary mouse CD3+ T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Nikolaus Thuille

    Full Text Available In T cells PKCθ mediates the activation of critical signals downstream of TCR/CD28 stimulation. We investigated the molecular mechanisms by which PKCθ regulates NFκB transactivation by examining PKCθ/β single and double knockout mice and observed a redundant involvement of PKCθ and PKCβ in this signaling pathway. Mechanistically, we define a PKCθ-CYLD protein complex and an interaction between the positive PKCθ/β and the negative CYLD signaling pathways that both converge at the level of TAK1/IKK/I-κBα/NFκB and NFAT transactivation. In Jurkat leukemic T cells, CYLD is endoproteolytically processed in the initial minutes of stimulation by the paracaspase MALT1 in a PKC-dependent fashion, which is required for robust IL-2 transcription. However, in primary T cells, CYLD processing occurs with different kinetics and an altered dependence on PKC. The formation of a direct PKCθ/CYLD complex appears to regulate the short-term spatial distribution of CYLD, subsequently affecting NFκB and NFAT repressional activity of CYLD prior to its MALT1-dependent inactivation. Taken together, our study establishes CYLD as a new and critical PKCθ interactor in T cells and reveals that antagonistic PKCθ/β-CYLD crosstalk is crucial for the adjustment of immune thresholds in primary mouse CD3(+ T cells.

  8. Anti-Human Trafficking Interventions

    Science.gov (United States)

    Davy, Deanna

    2016-01-01

    Since the early 2000s, a significant number of programs and policies have been developed and implemented to prevent and combat human trafficking. At the international, regional and national levels, government, and international, and nongovernment organizations have established plans of action, conducted training, developed policy tools, and…

  9. Interleukin-9 receptor α chain mRNA formation in CD8+ T cells producing anti-human immunodeficiency virus type 1 substance(s)

    International Nuclear Information System (INIS)

    Hossain, M.M.; Tsuchie, H.; Detorio, M.A.; Shirono, H.; Hara, C.; Nishimoto, A.; Saji, A.; Koga, J.; Takata, N.; Maniar, J.K.; Saple, D.G.; Taniguchi, K.; Kageyama, S.; Ichimura, H.; Kurimura, T.

    1998-01-01

    A search for gene(s) associated with anti-human immunodeficiency virus type 1 (HIV-l) activity of CD8 + T cells was attempted using molecular cloning and the relation between the anti-HIV activity of CD8 + T cells and the interleukin-9 receptor a chain (IL-9R-α) mRNA expression from the cDNA clones obtained was examined. The anti-HIV-l activity of CD8 + T cell culture supernatants was assessed by measuring the level of HIV-l replication in a CD4 + T cell line transfected with an infectious HIV-l DNA clone. IL-9R-a mRNA was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR). Of 5 cases showing high level of anti-HIV-l activity (more than 80% suppression of HIV-l replication), the mRNA was detected in 4 cases. Of 10 cases showing low level of anti-HIV-l activity (less than 80% suppression of HIV-l replication), the mRNA was detected in one case. Soluble recombinant human IL-9 receptor (rhIL-9sR) did not suppress HIV-l replication at a concentration of 1 μg/ml. These data suggest that the IL-9R-a mRNA formation in CD8 + T cells may correlate with and play some role in the anti-HIV-l activity of CD8+ T cells from HIV-l-infected individuals. Key words: CD8+ T cells; anti-HIV-l activity; cytokines; interleukin-9 receptor (authors)

  10. Anti-human neutrophil antigen-1a, -1b, and -2 antibodies in neonates and children with immune neutropenias analyzed by extracted granulocyte antigen immunofluorescence assay.

    Science.gov (United States)

    Onodera, Rie; Kurita, Emi; Taniguchi, Kikuyo; Karakawa, Shuhei; Okada, Satoshi; Kihara, Hirotaka; Fujii, Teruhisa; Kobayashi, Masao

    2017-11-01

    Anti-human neutrophil antigen (HNA) antibodies have been implicated in the development of neonatal alloimmune neutropenia (NAN) and autoimmune neutropenia (AIN). There are many conventional assay methods that detect anti-HNA antibodies. However, a method to measure multiple samples and detect several anti-HNA antibodies simultaneously is needed. We developed a new method, the extracted granulocyte antigen immunofluorescence assay (EGIFA), to analyze anti-HNA-1a, -1b, and -2 antibodies in sera. The results obtained by EGIFA were evaluated in comparison with those from several standard assay methods. Anti-HNA antibodies in serum samples from nine familial cases with suspected NAN (n = 19) and children with suspected AIN (n = 88) were also measured by EGIFA. The evaluation of nine serum samples with anti-HNA antibodies suggested that EGIFA demonstrated equivalent specificity and superior sensitivity to monoclonal antibody-specific immobilization of granulocyte antigens and had comparable sensitivity to the granulocyte indirect immunofluorescence test. EGIFA successfully detected anti-HNA-1a or -1b antibodies in seven of nine familial cases with suspected NAN. EGIFA detected anti-HNA antibodies in 40.9% of children with suspected AIN. Among them, isolated anti-HNA-1a or -1b antibody was detected in 4.5 or 12.5% of children, respectively, and anti-HNA-2 antibody was identified in 3.4% of children. The 30.8% (16 of 52) of children negative for anti-HNA antibody by EGIFA were positive for anti-HLA antibody. EGIFA facilitated the measurement of anti-HNA-1a, -1b, and/or -2 antibodies in sera. The prompt measurement of anti-HNA antibodies will improve the diagnosis and clinical management of patients with suspected NAN or AIN. © 2017 AABB.

  11. Pharmacokinetics, biodistribution and dosimetry of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats

    Energy Technology Data Exchange (ETDEWEB)

    Escobar, Normando Iznaga; Morales, Alejo Morales; Duconge, Jorge; Torres, Idania Caballero; Fernandez, Eduardo; Gomez, Jose A

    1998-01-01

    The pharmacokinetics, biodistribution and dosimetry of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t{sub (1(2{alpha}}{sub ))}) of 0.250 h and a mean elimination (t{sub (1(2{beta}}{sub ))}) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with {sup 99m}Tc-labeled humanized MAb R3 conjugate in patients should be supported.

  12. Pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats

    International Nuclear Information System (INIS)

    Escobar, Normando Iznaga; Morales, Alejo Morales; Duconge, Jorge; Torres, Idania Caballero; Fernandez, Eduardo; Gomez, Jose A.

    1998-01-01

    The pharmacokinetics, biodistribution and dosimetry of 99m Tc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t (1(2α)) ) of 0.250 h and a mean elimination (t (1(2β)) ) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with 99m Tc-labeled humanized MAb R3 conjugate in patients should be supported

  13. Maraviroc (UK-427,857), a Potent, Orally Bioavailable, and Selective Small-Molecule Inhibitor of Chemokine Receptor CCR5 with Broad-Spectrum Anti-Human Immunodeficiency Virus Type 1 Activity

    OpenAIRE

    Dorr, Patrick; Westby, Mike; Dobbs, Susan; Griffin, Paul; Irvine, Becky; Macartney, Malcolm; Mori, Julie; Rickett, Graham; Smith-Burchnell, Caroline; Napier, Carolyn; Webster, Rob; Armour, Duncan; Price, David; Stammen, Blanda; Wood, Anthony

    2005-01-01

    Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades a...

  14. Human T cell leukemia virus type I prevents cell surface expression of the T cell receptor through down-regulation of the CD3-gamma, -delta, -epsilon, and -zeta genes

    NARCIS (Netherlands)

    de Waal Malefyt, R.; Yssel, H.; Spits, H.; de Vries, J. E.; Sancho, J.; Terhorst, C.; Alarcon, B.

    1990-01-01

    Infection and transformation by human T cell leukemia virus type I (HTLV-I) up-regulates expression of several inducible genes including those coding for cytokines involved in the proliferation of normal and leukemic T cells. We demonstrate that HTLV-I can also shut off expression of the CD3-gamma,

  15. Ex vivo expansion of CD3depleted cord blood-MNCs in the presence of bone marrow stromal cells; an appropriate strategy to provide functional NK cells applicable for cellular therapy

    Directory of Open Access Journals (Sweden)

    Ehteramolsadat Hosseini

    2017-03-01

    Full Text Available Considering umbilical cord blood (UCB as a rich source of hematopoietic stem cells, we introduced a cost-effective approach to expand CD3depleted UCB-MNCs into functional NK cells. CD3depleted UCB-MNCs were expanded in the presence or absence of a feeder [bone marrow stem cells (BMSCs or osteoblasts], with or without cytokines and their differentiation into NK cells was determined by flow cytometry. NK cell function was quantified by LAMP-1/CD107a expression, TNF-α/IFN-γ release, and LDH release/PI staining in targets. Higher expansion of NK cells was observed after two weeks in the presence of BMSCs and cytokines (104 ± 15 compared to osteoblasts and cytokines (84 ± 29, p < 0.05. On day 14, CD3depleted UCB-MNCs in the presence of BMSCs and cytokines showed lower expression of CD3, CD19, CD14, CD15 and CD69 as well as higher expression of CD2 and CD7, which were suggestive of cell differentiation into mature NK cell lineage. Strong cytotoxicity of expanded cells was also identified with higher LDH release and PI% in targets. Significant upregulation of LAMP-1 with decreased release of IFN-γ and TNF-α from effectors were observed. We demonstrate an effective expansion of UCB-NK cells that maintained their functional capabilities applicable for cellular therapies.

  16. The importance of a sub-region on chromosome 19q13.3 for prognosis of multiple myeloma patients after high-dose treatment and stem cell support: a linkage disequilibrium mapping in RAI and CD3EAP

    DEFF Research Database (Denmark)

    Vangsted, Annette Juul; Klausen, Tobias Wirenfeldt; Gimsing, Peter

    2011-01-01

    The gene RAI was originally described as an inhibitor of RelA/p65 subunit of nuclear factor ¿B (NF-¿B). Here, we analyse the association between genetic variation in the genes RAI and CD3EAP and outcome of 348 myeloma patients treated with high-dose treatment (HDT), 146 patients treated with inte......The gene RAI was originally described as an inhibitor of RelA/p65 subunit of nuclear factor ¿B (NF-¿B). Here, we analyse the association between genetic variation in the genes RAI and CD3EAP and outcome of 348 myeloma patients treated with high-dose treatment (HDT), 146 patients treated...... with interferon-a (INF-a) as maintenance treatment, 177 patients treated with thalidomide, and 74 patients treated with bortezomib at relapse and address if the effects of polymorphisms in CD3EAP and RAI are modified by a functional polymorphism in NF¿B1. By linkage disequilibrium mapping, we found that variant...... alleles of several polymorphisms in a sub-region of 19q13.3 spanning the regions RAI-intron1-1 to RAI intron1-3 and the region exon1 to exon3-6 in CD3EAP were associated with prolonged time-to-treatment failure (TTF; p¿=¿0.003) and overall survival (OS; p¿=¿0.02). Haplotype analyses revealed that none...

  17. The importance of a sub-region on chromosome 19q13.3 for prognosis of multiple myeloma patients after high-dose treatment and stem cell support: a linkage disequilibrium mapping in RAI and CD3EAP

    DEFF Research Database (Denmark)

    Vangsted, Annette J.; Klausen, Tobias Wirenfeldt; Gimsing, Peter

    2011-01-01

    The gene RAI was originally described as an inhibitor of RelA/p65 subunit of nuclear factor κB (NF–κB). Here, we analyse the association between genetic variation in the genes RAI and CD3EAP and outcome of 348 myeloma patients treated with high-dose treatment (HDT), 146 patients treated with inte......The gene RAI was originally described as an inhibitor of RelA/p65 subunit of nuclear factor κB (NF–κB). Here, we analyse the association between genetic variation in the genes RAI and CD3EAP and outcome of 348 myeloma patients treated with high-dose treatment (HDT), 146 patients treated...... with interferon-α (INF-α) as maintenance treatment, 177 patients treated with thalidomide, and 74 patients treated with bortezomib at relapse and address if the effects of polymorphisms in CD3EAP and RAI are modified by a functional polymorphism in NFКB1. By linkage disequilibrium mapping, we found that variant...... alleles of several polymorphisms in a sub-region of 19q13.3 spanning the regions RAI-intron1-1 to RAI intron1-3 and the region exon1 to exon3–6 in CD3EAP were associated with prolonged time-to-treatment failure (TTF; p = 0.003) and overall survival (OS; p = 0.02). Haplotype analyses revealed that none...

  18. Role of polymorphic Fc receptor Fc gammaRIIa in cytokine release and adverse effects of murine IgG1 anti-CD3/T cell receptor antibody (WT31).

    Science.gov (United States)

    Tax, W J; Tamboer, W P; Jacobs, C W; Frenken, L A; Koene, R A

    1997-01-15

    Anti-CD3 monoclonal antibody (mAb) OKT3 is immunosuppressive, but causes severe adverse effects during the first administration ("first-dose reaction"). These adverse effects are presumably caused by cytokine release that results from T-cell activation. In vitro, T-cell activation by anti-CD3 mAb requires interaction with monocyte Fc receptors. The Fc receptor for murine IgG1, Fc gammaRIIa, is polymorphic. In some individuals, murine IgG1 anti-CD3 mAb causes T-cell proliferation and cytokine release in vitro (high responders [HR]), whereas in individuals with the low-responder (LR) phenotype it does not. We have now investigated the role of this Fc gammaRIIa polymorphism in the release of cytokines in vivo and the occurrence of adverse effects after the administration of WT31, a murine IgG1 anti-CD3/T cell receptor mAb. WT31 caused an increase of plasma tumor necrosis factor-alpha in all four HR patients and none of the five LR patients. In all HR patients except one, plasma gamma-interferon and interleukin 6 also increased, and a first-dose response was observed, whereas no cytokine release or adverse effects occurred in any of the LR patients. WT31 caused lymphopenia in all HR and none of the LR patients. FACS analysis demonstrated that in HR patients, after the initial disappearance of CD3+ cells from peripheral blood, modulation of CD3 occurred, whereas in LR patients a high degree of coating of the lymphocytes was observed. Surprisingly, WT31 also induced a marked granulocytopenia, as well as a decrease of thrombocytes, in three of the four HR patients (and in none of the LR patients). These data provide direct clinical evidence that Fc receptor interaction determines the release of cytokines and the occurrence of adverse effects after administration of anti-CD3/T cell receptor mAb. Furthermore, these data suggest that tumor necrosis factor-alpha by itself is not sufficient to induce the first-dose reaction.

  19. Change in CD3 positive T-cell expression in psoriatic arthritis synovium correlates with change in DAS28 and magnetic resonance imaging synovitis scores following initiation of biologic therapy - a single centre, open-label study

    LENUS (Irish Health Repository)

    Pontifex, Eliza K

    2011-01-27

    Abstract Introduction With the development of increasing numbers of potential therapeutic agents in inflammatory disease comes the need for effective biomarkers to help screen for drug efficacy and optimal dosing regimens early in the clinical trial process. This need has been recognized by the Outcome Measures in Rheumatology Clinical Trials (OMERACT) group, which has established guidelines for biomarker validation. To seek a candidate synovial biomarker of treatment response in psoriatic arthritis (PsA), we determined whether changes in immunohistochemical markers of synovial inflammation correlate with changes in disease activity scores assessing 28 joints (ΔDAS28) or magnetic resonance imaging synovitis scores (ΔMRI) in patients with PsA treated with a biologic agent. Methods Twenty-five consecutive patients with PsA underwent arthroscopic synovial biopsies and MRI scans of an inflamed knee joint at baseline and 12 weeks after starting treatment with either anakinra (first 10 patients) or etanercept (subsequent 15 patients) in two sequential studies of identical design. DAS28 scores were measured at both time points. Immunohistochemical staining for CD3, CD68 and Factor VIII (FVIII) was performed on synovial samples and scored by digital image analysis (DIA). MRI scans performed at baseline and at 12 weeks were scored for synovitis semi-quantitatively. The ΔDAS28 of the European League Against Rheumatism good response definition (>1.2) was chosen to divide patients into responder and non-responder groups. Differences between groups (Mann Whitney U test) and correlations between ΔDAS28 with change in immunohistochemical and MRI synovitis scores (Spearman\\'s rho test) were calculated. Results Paired synovial samples and MRI scans were available for 21 patients (8 anakinra, 13 etanercept) and 23 patients (8 anakinra, 15 etanercept) respectively. Change in CD3CD3) and CD68 expression in the synovial sublining layer (ΔCD68sl) was significantly greater in

  20. Arylamine N-acetyltransferase 1 in situ N-acetylation on CD3+ peripheral blood mononuclear cells correlate with NATb mRNA and NAT1 haplotype.

    Science.gov (United States)

    Salazar-González, Raúl A; Turiján-Espinoza, Eneida; Hein, David W; Niño-Moreno, Perla C; Romano-Moreno, Silvia; Milán-Segovia, Rosa C; Portales-Pérez, Diana P

    2018-02-01

    Human arylamine N-acetyltransferase 1 (NAT1) is responsible for the activation and elimination of xenobiotic compounds and carcinogens. Genetic polymorphisms in NAT1 modify both drug efficacy and toxicity. Previous studies have suggested a role for NAT1 in the development of several diseases. The aim of the present study was to evaluate NAT1 protein expression and in situ N-acetylation capacity in peripheral blood mononuclear cells (PBMC), as well as their possible associations with the expression of NAT1 transcript and NAT1 genotype. We report NAT1 protein, mRNA levels, and N-acetylation in situ activity for PBMC obtained from healthy donors. NAT1-specific protein expression was higher in CD3+ cells than other major immune cell subtypes (CD19 or CD56 cells). N-acetylation of pABA varied markedly among the PBMC of participants, but correlated very significantly with levels of NAT1 transcripts. NAT1*4 subjects showed significantly (p = 0.017) higher apparent pABA V max of 71.3 ± 3.7 versus the NAT1*14B subjects apparent V max of 58.5 ± 2.5 nmoles Ac-pABA/24 h/million cells. Levels of pABA N-acetylation activity at each concentration of substrate evaluated also significantly correlated with NAT1 mRNA levels for all samples (p N-acetylation in PBMC is higher in T cell than in other immune cell subtypes and that individual variation in N-acetylation capacity is dependent upon NAT1 mRNA and NAT1 haplotype.

  1. TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Brian Tomkowicz

    Full Text Available TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3 is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion. Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK, the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i NF-kB/NFAT activation, ii CD69 expression, and iii suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

  2. Binding of tissue plasminogen activator to human umbilical vein endothelial cells

    International Nuclear Information System (INIS)

    Beebe, D.P.

    1987-01-01

    The binding of purified, recombinant tissue plasminogen activator (tPA) to human umbilical vein endothelial cells (HUVEC) was studied in vitro using immunofluorescence as well as radiolabeled tPA. Immunofluorescence was performed on HUVEC grown on round glass coverslips using rabbit anti-human tPA and fluorescein-conjugated anti-rabbit immunoglobulin. Positive fluorescence was observed only after incubation of HUVEC with tPA. HUVEC were grown to confluence in 24-well tissue culture plates, washed, and incubated with a constant amount of 125 I-tPA and various concentrations of unlabeled tPA. The binding of tPA to HUVEC was found to be specific, saturable, and reversible. Scatchard analysis yielded as equilibrium constant (K/sub eq/) of 4.2 x 10 6 M -1 and 1.2 x 10 7 binding sites per cell. Binding was inhibited by positively charged amino acids and by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone but not by carbohydrates including mannose, galactose, N-acetyl glucosamine and N-acetyl galactosamine. Neat human plasma abrogates but does not totally inhibit binding of tPA to HUVEC. Binding was neither enhanced nor inhibited by fibronectin. Although the affinity of binding of tPA to HUVEC is low, the endothelial cell may be involved in regulating plasma levels of tPA in vivo which may have therapeutic significance

  3. Anti-human tissue factor antibody ameliorated intestinal ischemia reperfusion-induced acute lung injury in human tissue factor knock-in mice.

    Directory of Open Access Journals (Sweden)

    Xiaolin He

    Full Text Available BACKGROUND: Interaction between the coagulation and inflammation systems plays an important role in the development of acute respiratory distress syndrome (ARDS. Anti-coagulation is an attractive option for ARDS treatment, and this has promoted development of new antibodies. However, preclinical trials for these antibodies are often limited by the high cost and availability of non-human primates. In the present study, we developed a novel alternative method to test the role of a humanized anti-tissue factor mAb in acute lung injury with transgenic mice. METHODOLOGY/PRINCIPAL FINDINGS: Human tissue factor knock-in (hTF-KI transgenic mice and a novel humanized anti-human tissue factor mAb (anti-hTF mAb, CNTO859 were developed. The hTF-KI mice showed a normal and functional expression of hTF. The anti-hTF mAb specifically blocked the pro-coagulation activity of brain extracts from the hTF-KI mice and human, but not from wild type mice. An extrapulmonary ARDS model was used by intestinal ischemia-reperfusion. Significant lung tissue damage in hTF-KI mice was observed after 2 h reperfusion. Administration of CNTO859 (5 mg/kg, i.v. attenuated the severity of lung tissue injury, decreased the total cell counts and protein concentration in bronchoalveolar lavage fluid, and reduced Evans blue leakage. In addition, the treatment significantly reduced alveolar fibrin deposition, and decreased tissue factor and plasminogen activator inhibitor-1 activity in the serum. This treatment also down-regulated cytokine expression and reduced cell death in the lung. CONCLUSIONS: This novel anti-hTF antibody showed beneficial effects on intestinal ischemia-reperfusion induced acute lung injury, which merits further investigation for clinical usage. In addition, the use of knock-in transgenic mice to test the efficacy of antibodies against human-specific proteins is a novel strategy for preclinical studies.

  4. Serological analysis of human anti-human antibody responses in colon cancer patients treated with repeated doses of humanized monoclonal antibody A33.

    Science.gov (United States)

    Ritter, G; Cohen, L S; Williams, C; Richards, E C; Old, L J; Welt, S

    2001-09-15

    Mouse monoclonal antibody A33 (mAb A33) recognizes a M(r) 43,000 cell surface glycoprotein (designated A33) expressed in human colonic epithelium and colon cancer but absent from most other normal tissues. In patients, mAb A33 localizes with high specificity to colon cancer and is retained for up to 6 weeks in the cancer but cleared rapidly from normal colon (5-6 days). As a carrier of (125)I or (131)I, mAb A33 has shown antitumor activity. Induction of strong human anti-mouse antibody (immunoglobulin; HAMA) responses in patients, however, limits the use of the murine mAb A33 to very few injections. A humanized version of this antibody (huAb A33) has been prepared for Phase I and II clinical studies in patients with colon cancer. In those studies, immunogenicity of huAb A33 has been monitored using a novel, highly sensitive BIACORE method, which allows measurement of human anti-human antibodies (HAHAs) without the use of secondary reagents. We found that 63% (26 of 41) of the patients treated with repeated doses of huAb A33 developed HAHAs against a conformational antigenic determinant located in the V(L) and V(H) regions of huAb A33. Detailed serological analysis showed two distinct types of HAHAs. HAHA of type I (49% of patients) was characterized by an early onset with peak HAHA levels after 2 weeks of treatment, which declined with ongoing huAb A33 treatment. HAHA of type II (17% of patients) was characterized by a typically later onset of HAHA than in type I and by progressively increasing HAHA levels with each subsequent huAb A33 administration. Colon cancer patients with type I HAHAs did not develop infusion-related adverse events. In contrast, HAHA of type II was indicative of infusion-related adverse events. By using this new method, we were able to distinguish these two types of HAHAs in patients while on antibody treatment, allowing patients to be removed from study prior to the onset of severe infusion-related adverse events.

  5. Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA-transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy.

    Science.gov (United States)

    Hung, Chien-Fu; Xu, Xuequn; Li, Linhong; Ma, Ying; Jin, Qiu; Viley, Angelia; Allen, Cornell; Natarajan, Pachai; Shivakumar, Rama; Peshwa, Madhusudan V; Emens, Leisha A

    2018-04-02

    CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to "on-target off-tumor" toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of "on-target off-tumor" toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 10 9 peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival

  6. Labelling of anti-human bladder tumor chimeric antibody with 99Tcm and radioimmunoimaging of bladder carcinoma xenograft in nude mice

    International Nuclear Information System (INIS)

    Zhang Chunli; Wang Rongfu; Fu Zhanli; Bai Yin; Ding Yi; Yu Lizhang

    2003-01-01

    Objective: To study the in vitro immunoreactivity and in vivo tissue distribution, tumor targeting property of anti-human bladder tumor human-murine chimeric antibody (ch-BDI) labeled with 99 Tc m and to investigate its possibility for being used in guiding diagnosis and guiding therapy of bladder cancer. Methods: The ch-BDI was labeled with 99 Tc m by improved Schwarz method and the labeled antibody was purified by Sephadex G-50. Labeling yield and radiochemical purity were measured by paper chromatography. The immunoreactive fraction and association constant (K a ) were measured by Lindmo method and Scatchard analysis, respectively. 11.1 MBq (30 μg) 99 Tc m -ch-BDI was intravenously injected into nude mice bearing human bladder cancer xenografts in the right thigh and radioimmunoimaging (RII) was performed 2, 6, 20 and 24 h postinjection. The images were processed by region of interest (ROI) method to acquire the counts of whole body and the tumor and the counts ratios of tumor to contralateral normal tissue or to tissues of other non-tumor bearing organs. The mice were killed after 24 h postinjection imaging and tissue distribution was measured. %ID/g and target to nontarget (T/NT) ratios were calculated. Results: The labeling yield and radiochemical purity of 99 Tc m -ch-BDI were (66.5±7.3)% and >90%, respectively. The immunoreactive fraction was 76% and K a was 3.56 x 10 9 L/mol. RII showed that the tumor was clearly visualized 6 h postinjection and becoming clearer along with time prolonging. The radioactivity of whole body decreased rapidly with time, whereas the radioactivity of the tumor decreased slowly. The T/NT ratios was increased with time. Biodistribution results showed that tumor uptake was 17.4%ID/g 24 h postinjection. T/NT ratios were very high except for the kidney. T/NT ratios for brain, muscle, intestinal wall, bone and heart wall were 136.0, 55.1, 39.3, 29.7 and 27.9, respectively. Conclusion: 99 Tc m -ch-BDI exhibits excellent

  7. An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

    Science.gov (United States)

    Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

    2016-01-01

    Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells. PMID:27028870

  8. Total iron binding capacity

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003489.htm Total iron binding capacity To use the sharing features on this page, please enable JavaScript. Total iron binding capacity (TIBC) is a blood test to ...

  9. IL-15 improves the cytotoxicity of cytokine-induced killer cells against leukemia cells by upregulating CD3+CD56+ cells and downregulating regulatory T cells as well as IL-35.

    Science.gov (United States)

    Tao, Qianshan; Chen, Tianping; Tao, Lili; Wang, Huiping; Pan, Ying; Xiong, Shudao; Zhai, Zhimin

    2013-01-01

    Cytokine-induced killer (CIK) cells are usually generated from peripheral blood mononuclear cells with the stimulation of IL-2 in vitro. Unlike the conventional IL-2-stimulated CIK cells (IL-2-CIK cells), we investigated the characteristics and potential mechanism of IL-15-stimulated CIK cells (IL-15-CIK cells) in this study. Compared with IL-2-CIK cells, the percentage of CD3CD56 cells was significantly increased in IL-15-CIK cells, but the expression of regulatory T (Treg) cells and IL-35 was significantly decreased in IL-15-CIK cells. Meanwhile, the in vitro cytotoxicity against human myeloid leukemia cells K562 of IL-15-CIK cells was significantly augmented compared with IL-2-CIK cells. These data suggest that IL-15 may improve the cytotoxicity of CIK cells against leukemia cells by upregulating CD3CD56 cells and downregulating Treg cells and IL-35.

  10. H-D exchange in metal carbene complexes: Structure of cluster (μ-H)(μ-OCD3)Os3(CO)9{:C(CD3)NC2H8O}

    Science.gov (United States)

    Savkov, Boris; Maksakov, Vladimir; Kuratieva, Natalia

    2015-10-01

    X-ray and spectroscopic data for the new complex (μ-H)(μ-OCH3)Os3(CO)9{:C(CD3)NC2H8O} (2) obtained in the reaction of the (μ-H)(μ-Cl)Os3(CO)9{:C(CH3)NC2H8O} (1) with NaOCD3 in CD3OD solution are reported. It is shown that cluster 1 has the property of CH-acidity inherent of Fisher type carbenes. This had demonstrated using hydrogen deuterium exchange reaction in the presence of a strong base. Bridging chlorine to metoxide ligand substitution takes place during the reaction. The molecular structure of 2 is compared with known analogues.

  11. A High RORγT/CD3 Ratio is a Strong Prognostic Factor for Postoperative Survival in Advanced Colorectal Cancer: Analysis of Helper T Cell Lymphocytes (Th1, Th2, Th17 and Regulatory T Cells).

    Science.gov (United States)

    Yoshida, Naohiro; Kinugasa, Tetsushi; Miyoshi, Hiroaki; Sato, Kensaku; Yuge, Kotaro; Ohchi, Takafumi; Fujino, Shinya; Shiraiwa, Sachiko; Katagiri, Mitsuhiro; Akagi, Yoshito; Ohshima, Koichi

    2016-03-01

    Tumor-infiltrating lymphocytes (TILs), part of the host immune response, have been widely reported as influential factors in the tumor microenvironment for the clinical outcome of colorectal cancer (CRC). However, the network of helper T cells is very complex, and which T-cell subtypes affect the progression of CRC and postoperative prognosis remains unclear. This study investigated the expression of several subtypes of TILs including T helper type 1 (Th1), Th2, Th17, and regulatory T (Treg) cells to determine their correlation with clinicopathologic features and postoperative prognosis. The study investigated the expression of TILs using immunohistochemistry of tissue microarray samples for 199 CRC patients. The number of each T-cell subtype infiltrating tumors was counted using ImageJ software. The relationship between TIL marker expression, clinicopathologic features, and prognosis was analyzed. A high RORγT/CD3 ratio (Th17 ratio) was significantly correlated with lymph node metastasis (p = 0.002), and a high of Foxp3/CD3 ratio (Treg ratio) was correlated with tumor location in the colon (p = 0.04), as shown by the Chi square test. In multivariate analysis, a high RORγT/CD3 ratio was the only independent prognostic factor for overall survival (p = 0.04; hazard ratio [HR], 1.84; 95% confidence interval [CI] 1.02-3.45). This study confirmed a high RORγT/CD3 ratio as a strong prognostic marker for postoperative survival. The immunohistochemistry results suggest that Th17 may affect lymph node metastasis in CRC. If new immunotherapies reducing Th17 expression are established, they may improve the efficiency of cancer treatment and prolong the survival of patients with CRC.

  12. Genome-Wide Association Study among Four Horse Breeds Identifies a Common Haplotype Associated with In Vitro CD3+ T Cell Susceptibility/Resistance to Equine Arteritis Virus Infection ▿

    Science.gov (United States)

    Go, Yun Young; Bailey, Ernest; Cook, Deborah G.; Coleman, Stephen J.; MacLeod, James N.; Chen, Kuey-Chu; Timoney, Peter J.; Balasuriya, Udeni B. R.

    2011-01-01

    Previously, we have shown that horses could be divided into susceptible and resistant groups based on an in vitro assay using dual-color flow cytometric analysis of CD3+ T cells infected with equine arteritis virus (EAV). Here, we demonstrate that the differences in in vitro susceptibility of equine CD3+ T lymphocytes to EAV infection have a genetic basis. To investigate the possible hereditary basis for this trait, we conducted a genome-wide association study (GWAS) to compare susceptible and resistant phenotypes. Testing of 267 DNA samples from four horse breeds that had a susceptible or a resistant CD3+ T lymphocyte phenotype using both Illumina Equine SNP50 BeadChip and Sequenom's MassARRAY system identified a common, genetically dominant haplotype associated with the susceptible phenotype in a region of equine chromosome 11 (ECA11), positions 49572804 to 49643932. The presence of a common haplotype indicates that the trait occurred in a common ancestor of all four breeds, suggesting that it may be segregated among other modern horse breeds. Biological pathway analysis revealed several cellular genes within this region of ECA11 encoding proteins associated with virus attachment and entry, cytoskeletal organization, and NF-κB pathways that may be associated with the trait responsible for the in vitro susceptibility/resistance of CD3+ T lymphocytes to EAV infection. The data presented in this study demonstrated a strong association of genetic markers with the trait, representing de facto proof that the trait is under genetic control. To our knowledge, this is the first GWAS of an equine infectious disease and the first GWAS of equine viral arteritis. PMID:21994447

  13. MHC class I ligation of human T cells activates the ZAP70 and p56lck tyrosine kinases, leads to an alternative phenotype of the TCR/CD3 zeta-chain, and induces apoptosis

    DEFF Research Database (Denmark)

    Skov, S; Bregenholt, S; Claesson, Mogens Helweg

    1997-01-01

    Cross-linking of MHC class I (MHC-I) molecules on human T cells induces signal-transduction events, including activation of tyrosine kinases, tyrosine phosphorylation of phospholipase C-gamma 1, and elevation of the intracellular free calcium concentration. In this study, we demonstrate...... that the ZAP70 tyrosine kinase is tyrosine phosphorylated in Jurkat T cells and in purified peripheral T cells after MHC-I ligation. The tyrosine-phosphorylated ZAP70 kinase exhibits a particular phenotype with low affinities for proteins at 21, 40, 60, and 120 kDa, proteins normally co-precipitated with ZAP70...... after TCR/CD3 stimulation. The phosphorylation of ZAP70 after MHC-I ligation was dependent on TCR/CD3 surface expression. One of the natural substrates for ZAP70 is the zeta-chain dimer of the TCR/CD3 complex. MHC-I cross-linking induces a phosphorylated zeta-protein that migrates as a dimer at 42 k...

  14. Feature Binding in Zebrafish

    Directory of Open Access Journals (Sweden)

    P Neri

    2012-07-01

    Full Text Available Binding operations are primarily ascribed to cortex or similarly complex avian structures. My experiments show that the zebrafish, a lower vertebrate lacking cortex, supports visual feature binding of form and motion for the purpose of social behavior. These results challenge the notion that feature binding may require highly evolved neural structures and demonstrate that the nervous system of lower vertebrates can afford unexpectedly complex computations.

  15. Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

    Science.gov (United States)

    Iwamoto, Ryo; Takagi, Mika; Akatsuka, Jun-Ichi; Ono, Ken-Ichiro; Kishi, Yoshiro; Mekada, Eisuke

    2016-04-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

  16. A Chemical Activation Study of the Unimolecular Reactions of CD3CD2CHCl2 and CHCl2CHCl2 with Analysis of the 1,1-HCl Elimination Pathway.

    Science.gov (United States)

    Larkin, Allie C; Nestler, Matthew J; Smith, Caleb A; Heard, George L; Setser, Donald W; Holmes, Bert E

    2016-10-03

    Chemically activated C2D5CHCl2 molecules were generated with 88 kcal mol-1 of vibrational energy by the recombination of C2D5 and CHCl2 radicals in a room temperature bath gas. The competing 2,1-DCl and 1,1-HCl unimolecular reactions were identified by the observation of the CD3CD=CHCl and CD3CD=CDCl products. The initial CD3CD2C-Cl carbene product from 1,1-HCl elimination rearranges to CD3CD=CDCl under the conditions of the experiments. The experimental rate constants were 2.7 x107 and 0.47 x107 s-1 for 2,1-DCl and 1,1-HCl elimination reactions, respectively, which corresponds to branching fractions of 0.84 and 0.16. The experimental rate constants were compared to calculated statistical rate constants to assign threshold energies of 54 and ≈ 66 kcal mol-1 for the 1,2-DCl and 1,1-HCl reactions, respectively. The statistical rate constants were obtained from models developed from electronic-structure calculations for the molecule and its transition states. The rate constant (5.3 x 107 s-1) for the unimolecular decomposition of CHCl2CHCl2 molecules formed with 82 kcal mol-1 of vibrational energy by the recombination of CHCl2 radicals also is reported. Based upon the magnitude of the calculated rate constant, 1,1-HCl elimination must contribute less than 15% to the reaction; 1,2-HCl elimination is the major reaction and the threshold energy is 59 kcal mol-1. Calculations also were done to analyze previously published rate constants for chemically activated CD2Cl-CHCl2 molecules with 86 kcal mol-1 of energy in order to obtain a better overall description of the nature of the 1,1-HCl pathway for 1,1-dichloroalkanes. The interplay of the threshold energies for the 2,1-HCl and 1,1-HCl reactions and the available energy determines the product branching fractions for individual molecules. The unusual nature of the transition state for 1,1-HCl elimination is discussed.

  17. Melanin-binding radiopharmaceuticals

    International Nuclear Information System (INIS)

    Packer, S.; Fairchild, R.G.; Watts, K.P.; Greenberg, D.; Hannon, S.J.

    1980-01-01

    The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed

  18. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  19. Molecular binding thermodynamics of spherical guests by β-cyclodextrins bearing aromatic substituents

    Energy Technology Data Exchange (ETDEWEB)

    Li, Nan; Chen, Yong; Zhang, Ying-Ming; Wang, Li-Hua; Mao, Wen-Zhao; Liu, Yu, E-mail: yuliu@nankai.edu.cn

    2014-01-20

    Graphical abstract: - Highlights: • Different conformation of β-CD derivatives. • Enthalpy gain. • High binding ability. - Abstract: The molecular binding behaviors of two β-cyclodextrin (β-CD) derivatives bearing 1,2,3-triazole moieties, i.e. mono-6-deoxy-6-{4-(8-oxymethylquinolino)[1,2,3]triazolyl}-β-CD (1) and mono-6-deoxy-6-{4-(8-oxymethylnaphthol)[1,2,3]triazolyl}-β-CD (3), and their analogs without 1,2,3-triazole moieties, i.e. mono-6-deoxy-6-(8-oxymethylquinolino)-β-CD (2) and mono-6-deoxy-6-(8-oxymethylnaphthol)-β-CD (4) toward spherical guests (±)-borneol and (±)-camphor were investigated to elucidate how substituent moiety of host affects the binding abilities by 2D NMR as well as microcalorimetric titrations in aqueous phosphate buffer solution (pH 7.20) at 298.15 K. The binding modes of host–guest interactions obtained from 2D NMR displayed that host CDs without triazole moieties gave better induce-fit efficiency between hosts and guests, leading to stronger binding abilities. Thermodynamically, the inclusion complexation was driven by enthalpy with the stoichiometry of 1:1. Another factor contributed to the enhanced binding abilities was the enthalpy gain with the smaller entropy loss.

  20. Sunitinib indirectly enhanced anti-tumor cytotoxicity of cytokine-induced killer cells and CD3⁺CD56⁺ subset through the co-culturing dendritic cells.

    Directory of Open Access Journals (Sweden)

    Adisak Wongkajornsilp

    Full Text Available Cytokine-induced killer (CIK cells have reached clinical trials for leukemia and solid tumors. Their anti-tumor cytotoxicity had earlier been shown to be intensified after the co-culture with dendritic cells (DCs. We observed markedly enhanced anti-tumor cytotoxicity activity of CIK cells after the co-culture with sunitinib-pretreated DCs over that of untreated DCs. This cytotoxicity was reliant upon DC modulation by sunitinib because the direct exposure of CIK cells to sunitinib had no significant effect. Sunitinib promoted Th1-inducing and pro-inflammatory phenotypes (IL-12, IFN-γ and IL-6 in DCs at the expense of Th2 inducing phenotype (IL-13 and regulatory phenotype (PD-L1, IDO. Sunitinib-treated DCs subsequently induced the upregulation of Th1 phenotypic markers (IFN-γ and T-bet and the downregulation of the Th2 signature (GATA-3 and the Th17 marker (RORC on the CD3⁺CD56⁺ subset of CIK cells. It concluded that sunitinib-pretreated DCs drove the CD3⁺CD56⁺ subset toward Th1 phenotype with increased anti-tumor cytotoxicity.

  1. New quaternary alkali metal cadmium selenites, A2Cd(SeO3)2 (A = K, Rb, and Cs) and Li2Cd3(SeO3)4

    Science.gov (United States)

    Lü, Minfeng; Jo, Hongil; Oh, Seung-Jin; Ok, Kang Min

    2017-12-01

    A series of new alkali metal cadmium selenites, A2Cd(SeO3)2 (A = K, Rb, and Cs) and Li2Cd3(SeO3)4 have been synthesized in phase pure forms through hydrothermal and solid-state reactions. Structural analyses using single crystal X-ray diffraction indicate that while A2Cd(SeO3)2 and Li2Cd3(SeO3)4 reveal layered structures consisting of CdO6 and SeO3 polyhedra, their symmetry, bonding modes, and the orientation of lone pairs on Se4+ cations are different. A closer examination suggests that the observed structural variations found in the reported materials are attributed to the structure-directing effect of alkali metal cations with different sizes. Scanning electron microscopy/energy dispersive analysis by X-ray, thermogravimetric analysis, Infrared and UV-vis diffuse reflectance spectroscopy, transformation reactions under hydrothermal conditions, and local dipole moment calculations for the reported materials are also reported.

  2. Cloning and expression of canine CD25 for validation of an anti-human CD25 antibody to compare T regulatory lymphocytes in healthy dogs and dogs with osteosarcoma.

    Science.gov (United States)

    Rissetto, K C; Rindt, H; Selting, K A; Villamil, J A; Henry, C J; Reinero, C R

    2010-05-15

    T regulatory cells (Tregs) are a unique subset of T helper cells that serve to modify/inhibit effector cells of the immune system and thus are essential to prevent autoimmunity. Overzealous Treg activity may contribute to impaired immune responses to cancer. Tregs can be phenotypically identified by proteins expressed on the cell surface (CD4 and CD25) and inside the cell (forkhead box3 (FoxP3)), although in dogs, no anti-canine CD25 antibody exists. We hypothesized that a mouse anti-human CD25 antibody definitively recognizes the canine protein and can be used to identify Tregs in dogs. We describe cloning and transfection of the canine CD25 gene into human HeLa cells with subsequent expression of the canine protein on the cell surface detected using an anti-human CD25 antibody in a flow cytometric assay. Validation of this antibody was used to identify CD4+CD25+FoxP3+ Tregs in 39 healthy dogs and 16 dogs with osteosarcoma (OSA). Results were expressed in five different ways and showed significantly fewer %CD4+CD25+ T lymphocytes expressing FoxP3 in blood of older dogs (>/=7 years) compared with the other two age groups (<2 and 2-6 years) (p<0.001) and fewer %CD4+CD25+FoxP3+ Tregs in the tumor draining lymph nodes of OSA patients compared to the unrelated lymph node (p=0.049). However, there was no significant difference in % Tregs in the peripheral blood or lymph nodes between the control dogs and those with OSA. While the CD25 antibody can be successfully used in a flow cytometric assay to identify Tregs, this study does not support clinical utility of phenotypic recognition of Tregs in dogs with OSA. Copyright 2010 Elsevier B.V. All rights reserved.

  3. SHBG (Sex Hormone Binding Globulin)

    Science.gov (United States)

    ... Links Patient Resources For Health Professionals Subscribe Search Sex Hormone Binding Globulin (SHBG) Send Us Your Feedback ... As Testosterone-estrogen Binding Globulin TeBG Formal Name Sex Hormone Binding Globulin This article was last reviewed ...

  4. Noncooperative cadmium(II) binding to human metallothionein 1a

    International Nuclear Information System (INIS)

    Sutherland, Duncan E.K.; Stillman, Martin J.

    2008-01-01

    The two-domain (βα) mammalian metallothionein binds seven divalent metals, however, the binding mechanism is not well characterized and recent reports require the presence of the partially metallated protein. In this paper, step-wise metallation of the metal-free, two-domain βα-rhMT and the isolated β-rhMT using Cd(II) is shown to proceed in a noncooperative manner by analysis of electrospray ionization mass spectrometric data. Under limiting amounts of Cd(II), all intermediate metallation states up to the fully metallated Cd 3 -β-rhMT and Cd 7 -βα-rhMT were observed. Addition of excess Cd(II), resulted in formation of the supermetallated (metallation in excess of normal levels) Cd 4 -β- and Cd 8 -βα-metallothionein species. These data establish that noncooperative cadmium metallation is a property of each isolated domain and the complete two-domain protein. Our data now also establish that supermetallation is a property that may provide information about the mechanism of metal transfer to other proteins

  5. Distribution of cyclophilin B-binding sites in the subsets of human peripheral blood lymphocytes.

    Science.gov (United States)

    Denys, A; Allain, F; Foxwell, B; Spik, G

    1997-08-01

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway and released in biological fluids. We have recently demonstrated that both free CyPB and CyPB-CsA complex specifically bind to peripheral blood T lymphocytes and are internalized. These results suggest that CyPB might promote the targeting of the drug into sensitive cells. Peripheral blood lymphocytes are subdivided in several populations according to their biological functions and sensitivity to CsA. We have investigated the binding of CyPB to these different subsets using a CyPB derivatized by fluorescein through its single cysteine which retains its binding properties. We have confirmed that only T cells were involved in the interaction with CyPB. The ligand binding was found to be heterogeneously distributed on the different T-cell subsets and surface-bound CyPB was mainly associated with the CD4-positive cells. No significant difference was noted between the CD45RA and CD45RO subsets, demonstrating that CyPB-binding sites were equally distributed between native and memory T cells. CD3 stimulation of T lymphocytes led to a decrease in the CyPB-binding capacity, that may be explained by a down-regulation of the CyPB-receptor expression upon T-cell activation. Finally, we demonstrated that CyPB-receptor-positive cells, isolated on CyPB sulphydryl-coupled affinity matrices, are more sensitive to CyPB-complexed CsA than mixed peripheral blood lymphocytes, suggesting that CyPB potentiates CsA activity through the binding of the complex. Taken together, our results demonstrate that CyPB-binding sites are mainly associated with resting cells of the helper T lymphocyte, and that CyPB might modulate the distribution of CsA through the drug targeting to sensitive cells.

  6. Genetic Polymorphisms in XRCC1, CD3EAP, PPP1R13L, XPB, XPC, and XPF and the Risk of Chronic Benzene Poisoning in a Chinese Occupational Population.

    Directory of Open Access Journals (Sweden)

    Ping Xue

    Full Text Available Individual variations in the capacity of DNA repair machinery to relieve benzene-induced DNA damage may be the key to developing chronic benzene poisoning (CBP, an increasingly prevalent occupational disease in China. ERCC1 (Excision repair cross complementation group 1 is located on chromosome 19q13.2-3 and participates in the crucial steps of Nucleotide Excision Repair (NER; moreover, we determined that one of its polymorphisms, ERCC1 rs11615, is a biomarker for CBP susceptibility in our previous report. Our aim is to further explore the deeper association between some genetic variations related to ERCC1 polymorphisms and CBP risk.Nine single nucleotide polymorphisms (SNPs of XRCC1 (X-ray repair cross-complementing 1, CD3EAP (CD3e molecule, epsilon associated protein, PPP1R13L (protein phosphatase 1, regulatory subunit 13 like, XPB (Xeroderma pigmentosum group B, XPC (Xeroderma pigmentosum group C and XPF (Xeroderma pigmentosum group F were genotyped by the Snapshot and TaqMan-MGB® probe techniques, in a study involving 102 CBP patients and 204 controls. The potential interactions between these SNPs and lifestyle factors, such as smoking and drinking, were assessed using a stratified analysis.An XRCC1 allele, rs25487, was related to a higher risk of CBP (P<0.001 even after stratifying for potential confounders. Carriers of the TT genotype of XRCC1 rs1799782 who were alcohol drinkers (OR = 8.000; 95% CI: 1.316-48.645; P = 0.022, male (OR = 9.333; 95% CI: 1.593-54.672; P = 0.019, and had an exposure of ≤12 years (OR = 2.612; 95% CI: 1.048-6.510; P = 0.035 had an increased risk of CBP. However, the T allele in PPP1R13L rs1005165 (P<0.05 and the GA allele in CD3EAP rs967591 (OR = 0.162; 95% CI: 0039~0.666; P = 0.037 decreased the risk of CBP in men. The haplotype analysis of XRCC1 indicated that XRCC1 rs25487A, rs25489G and rs1799782T (OR = 15.469; 95% CI: 5.536-43.225; P<0.001 were associated with a high risk of CBP.The findings showed that

  7. CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

    DEFF Research Database (Denmark)

    1995-01-01

    Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

  8. In vitro propagation and dynamics of T cells from skin biopsies by methods using interleukins-2 and -4 or anti-CD3/CD28 antibody-coated microbeads

    DEFF Research Database (Denmark)

    Hashizume, Hideo; Hansen, Anker; Poulsen, Lars K

    2010-01-01

    In order to explore the mechanisms of inflammatory skin disorders, we established two methods of expanding skin-derived lymphocytes, one using high levels of interleukin (IL)-2 and IL-4 (method A) and the other using low levels of cytokines and anti-CD3/CD28 microbeads (method B). Both methods...... provide advantages for functional studies. With either of these two, we could obtain more than 10(7) cells/ from a 3 mm skin biopsy in 21 days from 23 out of 26 biopsies of various skin diseases. The relevance of these cells was confirmed by shifted T-cell receptor beta chain variable region (TCR......-Vbeta) repertoire and antigen-dependent proliferation in antigen-driven skin disorders. The propagation of skin-resident lymphocytes, seen especially in method A, seems to be mediated by a functional defect of regulatory T cells residing in skin sequentially expanding under the conditions of our methods....

  9. Sequential memory: Binding dynamics

    Science.gov (United States)

    Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

    2015-10-01

    Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories—episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

  10. Cellulose binding domain proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  11. Binding and Bulgarian

    NARCIS (Netherlands)

    Schürcks-Grozeva, Lilia Lubomirova

    2003-01-01

    In haar proefschrift analyseert Lilia Schürcks de anaforische verschijnselen in de Bulgaarse taal. Het gaat dan om wederkerende aspecten, uitgedrukt bij woorden als ‘zich’ en ‘elkaar’. De situatie in het Bulgaars blijkt moeilijk in te passen in de klassieke Binding Theory van Noam Chomsky. Bron: RUG

  12. Malignant mesothelioma effusions are infiltrated by CD3+ T cells highly expressing PD-L1 and the PD-L1+ tumor cells within these effusions are susceptible to ADCC by the anti-PD-L1 antibody avelumab

    Science.gov (United States)

    Khanna, Swati; Thomas, Anish; Abate-Daga, Daniel; Zhang, Jingli; Morrow, Betsy; Steinberg, Seth M.; Orlandi, Augusto; Ferroni, Patrizia; Schlom, Jeffrey; Guadagni, Fiorella; Hassan, Raffit

    2016-01-01

    INTRODUCTION The functional aspects of programmed death 1 (PD-1) and PD ligand 1 (PD-L1) immune checkpoints in malignant mesothelioma have not been studied. METHODS Tumor samples from 65 patients with mesothelioma were evaluated for PD-L1 expression by immunohistochemistry and its prognostic significance. Malignant effusions from patients with pleural and peritoneal mesothelioma were evaluated for PD-1+ and PD-L1+ infiltrating lymphocytes and their role in inducing tumor cell PD-L1 expression. Antibody dependent cellular cytotoxicity (ADCC) of avelumab, a fully humanized IgG1 anti PD-L1 antibody towards primary mesothelioma cell lines was evaluated in presence of autologous and allogeneic NK cells. RESULTS Of 65 pleural and peritoneal mesothelioma tumors examined, 41 (63%) were PD-L1 positive, which was associated with slightly inferior overall survival compared to patients with PD-L1 negative tumors (median 23.0 vs. 33.3 months; p=0.35). The frequency of PD-L1 expression was similar in pleural and peritoneal mesothelioma patients with 62% and 64% of samples positive, respectively. Of nine mesothelioma effusion samples evaluated, the fraction of cells expressing PD-L1 ranged from 12 to 83%. Of 7 patients with paired malignant effusion and peripheral blood mononuclear cells (PBMC) samples, PD-L1 expression was significantly higher on CD3+ T cells present in malignant effusions as compared with PBMC (p=0.016). In addition, CD14+PD-1+ cells were elevated in malignant effusions compared with PBMC (p=0.031). The lymphocytes present in malignant effusions recognized autologous tumor cells and induced IFN-γ-mediated PD-L1 expression on the tumor cell surface. Of the three primary mesothelioma cell lines tested, two were susceptible to avelumab mediated ADCC in presence of autologous NK cells. CONCLUSION The majority of pleural as well as peritoneal mesothelioma express PD-L1. Malignant effusions in this disease are characterized by presence of tumor cells and CD3+ T

  13. Temperaturedependent After-Effects of the Cobalt-57-Iron-57 Electron-Capture Reaction in Cd3[57Co(CN)6]2,12H2O

    DEFF Research Database (Denmark)

    Fenger, Jes; Olsen, Jørgen

    1974-01-01

    The Mössbauer emission spectrum of Cd3[57Co(CN)6]2,12H2O was measured at temperatures between 78 and 323 K. Above 200 K the spectra can be resolved into two doublets; one is ascribed to 57Fe(CN)63– and one to 57Fe(CN)52–; it is suggested that the pentacyanide is formed by a Coulomb explosion...... following an Auger process associated with the 57Co(EC)57Fe-decay. Below 200 K a third species is detected; it may be Fe2+ formed by cyanide linkage isomerism. Measurements of the total intensity of the spectrum and the relative intensities of the individual doublets as a function of temperature permit...... in principle a determination of the f-factors for the species formed. It is estimated that the 57Fe(CN)52– has a formal Debye temperature of 170 K; at room temperature it is detected with a probability of only 50% of that of the 57Fe(CN)63–....

  14. Carboplatin binding to histidine

    Energy Technology Data Exchange (ETDEWEB)

    Tanley, Simon W. M. [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom); Diederichs, Kay [University of Konstanz, D-78457 Konstanz (Germany); Kroon-Batenburg, Loes M. J. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Levy, Colin [University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom); Schreurs, Antoine M. M. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Helliwell, John R., E-mail: john.helliwell@manchester.ac.uk [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom)

    2014-08-29

    An X-ray crystal structure showing the binding of purely carboplatin to histidine in a model protein has finally been obtained. This required extensive crystallization trials and various novel crystal structure analyses. Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described.

  15. Optical Binding of Nanowires

    Czech Academy of Sciences Publication Activity Database

    Simpson, Stephen Hugh; Zemánek, Pavel; Marago, O.M.; Jones, P.H.; Hanna, S.

    2017-01-01

    Roč. 17, č. 6 (2017), s. 3485-3492 ISSN 1530-6984 R&D Projects: GA ČR GB14-36681G Grant - others:AV ČR(CZ) CNR-16-12 Program:Bilaterální spolupráce Institutional support: RVO:68081731 Keywords : optical binding nanowires * Brownian motion * self-organization * non-equilibrium thermodynamics * non-equilibrium steady state * spin-orbit coupling * emergent phenomena Subject RIV: BH - Optics, Masers, Lasers OBOR OECD: Optics (including laser optics and quantum optics) Impact factor: 12.712, year: 2016

  16. IGF binding proteins.

    Science.gov (United States)

    Bach, Leon A

    2017-12-18

    Insulin-like growth factor binding proteins (IGFBPs) 1-6 bind IGFs but not insulin with high affinity. They were initially identified as serum carriers and passive inhibitors of IGF actions. However, subsequent studies showed that, although IGFBPs inhibit IGF actions in many circumstances, they may also potentiate these actions. IGFBPs are widely expressed in most tissues, and they are flexible endocrine and autocrine/paracrine regulators of IGF activity, which is essential for this important physiological system. More recently, individual IGFBPs have been shown to have IGF-independent actions. Mechanisms underlying these actions include (i) interaction with non-IGF proteins in compartments including the extracellular space and matrix, the cell surface and intracellularly; (ii) interaction with and modulation of other growth factor pathways including EGF, TGF- and VEGF; and (iii) direct or indirect transcriptional effects following nuclear entry of IGFBPs. Through these IGF-dependent and IGF-independent actions, IGFBPs modulate essential cellular processes including proliferation, survival, migration, senescence, autophagy and angiogenesis. They have been implicated in a range of disorders including malignant, metabolic, neurological and immune diseases. A more complete understanding of their cellular roles may lead to the development of novel IGFBP-based therapeutic opportunities.

  17. Simultaneous assessment of cytotoxic T lymphocyte responses against multiple viral infections by combined usage of optimal epitope matrices, anti- CD3 mAb T-cell expansion and "RecycleSpot"

    Directory of Open Access Journals (Sweden)

    Wong Johnson T

    2005-05-01

    Full Text Available Abstract The assessment of cellular anti-viral immunity is often hampered by the limited availability of adequate samples, especially when attempting simultaneous, high-resolution determination of T cell responses against multiple viral infections. Thus, the development of assay systems, which optimize cell usage, while still allowing for the detailed determination of breadth and magnitude of virus-specific cytotoxic T lymphocyte (CTL responses, is urgently needed. This study provides an up-to-date listing of currently known, well-defined viral CTL epitopes for HIV, EBV, CMV, HCV and HBV and describes an approach that overcomes some of the above limitations through the use of peptide matrices of optimally defined viral CTL epitopes in combination with anti-CD3 in vitro T cell expansion and re-use of cells from negative ELISpot wells. The data show that, when compared to direct ex vivo cell preparations, antigen-unspecific in vitro T cell expansion maintains the breadth of detectable T cell responses and demonstrates that harvesting cells from negative ELISpot wells for re-use in subsequent ELISpot assays (RecycleSpot, further maximized the use of available cells. Furthermore when combining T cell expansion and RecycleSpot with the use of rationally designed peptide matrices, antiviral immunity against more than 400 different CTL epitopes from five different viruses can be reproducibly assessed from samples of less than 10 milliliters of blood without compromising information on the breadth and magnitude of these responses. Together, these data support an approach that facilitates the assessment of cellular immunity against multiple viral co-infections in settings where sample availability is severely limited.

  18. The Ratio of Regulatory (FOXP3+) to Total (CD3+) T Cells Determined by Epigenetic Cell Counting and Cardiovascular Disease Risk: A Prospective Case-cohort Study in Non-diabetics.

    Science.gov (United States)

    Barth, Sebastian Dietmar; Kaaks, Rudolf; Johnson, Theron; Katzke, Verena; Gellhaus, Katharina; Schulze, Janika Josephin; Olek, Sven; Kühn, Tilman

    2016-09-01

    Experimental and clinical evidence indicate that inflammatory processes in atherogenesis and the development of cardiovascular complications are promoted by a loss of regulatory T cell (Treg)-mediated immunological tolerance to plaque antigens. Yet, the association between alterations of systemic Treg frequency and cardiovascular disease incidence remains uncertain. A nested case-cohort study was conducted within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg, comprising a random subcohort (n=778) and primary cases of myocardial infarction (MI, n=276) and ischemic stroke (n=151). Pre-diagnostic FOXP3+ Treg and total CD3+ T-lymphocyte (tTL) frequencies in blood were measured by epigenetic-based, quantitative real-time PCR-assisted cell counting. Multivariate, Prentice-weighted Cox regression analyses revealed that lower Treg/tTL ratios were not associated with the risk of either MI (lowest vs. highest sex-specific quartile; hazard ratio: 0.72, 95% confidence interval: 0.46 to 1.13; P trend =0.51) or stroke (HR: 0.90, 95% CI: 0.51 to 1.60; P trend =0.78). There were no correlations of Treg/tTL ratios with C-reactive protein, HbA1c, and various lipid parameters. Among middle-aged adults from the general population, imbalances in the relative frequency of Tregs within the total T cell compartment do not confer an increased risk of MI or stroke. Copyright © 2016 Forschungsgesellschaft für Arbeitsphysiologie und Arbeitschutz e.V. Published by Elsevier B.V. All rights reserved.

  19. Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

    Science.gov (United States)

    Adams, Ralph; Griffin, Laura; Compson, Joanne E; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair Dg; Humphreys, David P; Heywood, Sam

    2016-10-01

    We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.

  20. Pictorial binding: endeavor to classify

    Directory of Open Access Journals (Sweden)

    Zinchenko S.

    2015-01-01

    Full Text Available The article is devoted to the classification of bindings of the 1-19th centuries with a unique and untypical book binding decoration technique (encaustic, tempera and oil paintings. Analysis of design features, materials and techniques of art decoration made it possible to identify them as a separate type - pictorial bindings and divide them into four groups. The first group consists of Coptic bindings, decorated with icon-painting images in encaustic technique. The second group is made up of leather Western bindings of the 13-14th centuries, which have the decoration and technique of ornamentation close to iconography. The third group involves parchment bindings, ornamentation technique of which is closer to the miniature. The last group comprises bindings of East Slavic origin of the 15-19th centuries, decorated with icon-painting pictures made in the technique of tempera or oil painting. The proposed classification requires further basic research as several specific kinds of bindings have not yet been investigated

  1. Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism*

    Science.gov (United States)

    Gunawardane, Ruwanthi N.; Fordstrom, Preston; Piper, Derek E.; Masterman, Stephanie; Siu, Sophia; Liu, Dongming; Brown, Mike; Lu, Mei; Tang, Jie; Zhang, Richard; Cheng, Janet; Gates, Andrew; Meininger, David; Chan, Joyce; Carlson, Tim; Walker, Nigel; Schwarz, Margrit; Delaney, John; Zhou, Mingyue

    2016-01-01

    Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease. PMID:26644477

  2. CD4+, CD8+, CD3+ cell counts and CD4+/CD8+ ratio among patients with mycobacterial diseases (leprosy, tuberculosis), HIV infections, and normal healthy adults: a comparative analysis of studies in different regions of India.

    Science.gov (United States)

    Hussain, Tahziba; Kulshreshtha, K K; Yadav, V S; Katoch, Kiran

    2015-01-01

    In this study, we estimated the CD4+, CD8+, CD3+ cell counts and the CD4/CD8 ratio among normal healthy controls (adults and children), leprosy patients (without any complications and during reactional states), TB patients (with and without HIV), and HIV-positive patients (early infection and full-blown AIDS) and correlated the changes with disease progression. In our study, it was observed that among adults, CD4+ cell counts ranged from 518-1098, CD8+ from 312-952, whereas CD4/CD8 ratio from 0.75-2.30. Among children, both CD4+ and CD8+ cells were more and the CD4/CD8 ratio varied from 0.91-3.17. With regard to leprosy patients, we observed that CD4+ and CD8+ cell counts were lower among PB (pauci-bacillary) and MB (multi-bacillary) patients. CD4/CD8 ratio was 0.99 ± 0.28 among PB patients while the ratio was lower, 0.78 ± 0.20, among MB patients. CD4+ cell counts were raised during RR (reversal reactions) and ENL (erythema nodosum leprosum) among the PB and MB patients whereas the CD8+ cell counts were lower among PB and MB patients. CD4/CD8 ratio doubled during reactional episodes of RR and ENL. Among the HIV-negative tuberculosis (TB) patients, both the CD4+ and CD8+ cell counts were found to be less and the CD4/CD8 ratio varied between 0.53-1.75. Among the HIV-positive TB patients and HIV-positive patients, both the CD4+ and CD8+ cells were very less and ratio drops significantly. In the initial stages of infection, as CD4+ counts drop, an increase in the CD8+ cell counts was observed and the ratio declines. In full-blown cases, CD4+ cell counts were very low, 3-4 to 54 cells, CD8+ cells from 12-211 and the ratio drops too low. This study is the first of its kind in this region of the country and assumes importance since no other study has reported the values of CD4+ and CD8+ T-lymphocyte counts among patients with mycobacterial diseases (leprosy and TB), HIV infections along with normal healthy individuals of the region, and correlation with clinical

  3. Megalin binds and mediates cellular internalization of folate binding protein

    DEFF Research Database (Denmark)

    Birn, Henrik; Zhai, Xiaoyue; Holm, Jan

    2005-01-01

    Folate is an essential vitamin involved in a number of biological processes. High affinity folate binding proteins (FBPs) exist both as glycosylphosphatidylinositol-linked, membrane associated folate binding proteins and as soluble FBPs in plasma and some secretory fluids such as milk, saliva...... to express high levels of megalin, is inhibitable by excess unlabeled FBP and by receptor associated protein, a known inhibitor of binding to megalin. Immortalized rat yolk sac cells, representing an established model for studying megalin-mediated uptake, reveal (125)I-labeled FBP uptake which is inhibited...

  4. Evaluation of the binding interaction between bovine serum albumin and dimethyl fumarate, an anti-inflammatory drug by multispectroscopic methods

    Science.gov (United States)

    Jattinagoudar, Laxmi; Meti, Manjunath; Nandibewoor, Sharanappa; Chimatadar, Shivamurti

    2016-03-01

    The information of the quenching reaction of bovine serum albumin with dimethyl fumarate is obtained by multi-spectroscopic methods. The number of binding sites, n and binding constants, KA were determined at different temperatures. The effect of increasing temperature on Stern-Volmer quenching constants (KD) indicates that a dynamic quenching mechanism is involved in the interaction. The analysis of thermodynamic quantities namely, ∆H° and ∆S° suggested hydrophobic forces playing a major role in the interaction between dimethyl fumarate and bovine serum albumin. The binding site of dimethyl fumarate on bovine serum albumin was determined by displacement studies, using the site probes viz., warfarin, ibuprofen and digitoxin. The determination of magnitude of the distance of approach for molecular interactions between dimethyl fumarate and bovine serum albumin is calculated according to the theory of Förster energy transfer. The CD, 3D fluorescence spectra, synchronous fluorescence measurements and FT-IR spectral results were indicative of the change in secondary structure of the protein. The influence of some of the metal ions on the binding interaction was also studied.

  5. [3]tetrahydrotrazodone binding. Association with serotonin binding sites

    International Nuclear Information System (INIS)

    Kendall, D.A.; Taylor, D.P.; Enna, S.J.

    1983-01-01

    High (17 nM) and low (603 nM) affinity binding sites for [ 3 ]tetrahydrotrazodone ([ 3 ] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [ 3 ]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [ 3 ] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [ 3 ]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

  6. Protein binding of psychotropic agents

    International Nuclear Information System (INIS)

    Hassan, H.A.

    1990-01-01

    Based upon fluorescence measurements, protein binding of some psychotropic agents (chlorpromazine, promethazine, and trifluoperazine) to human IgG and HSA was studied in aqueous cacodylate buffer, PH7. The interaction parameters determined from emission quenching of the proteins. The interaction parameters determined include the equilibrium constant (K), calculated from equations derived by Borazan and coworkers, the number of binding sites (n) available to the monomer molecules on a single protein molecule. The results revealed a high level of affinity, as reflected by high values of K, and the existence of specific binding sites, since a limited number of n values are obtained. 39 tabs.; 37 figs.; 83 refs

  7. Superresolution microscopy with transient binding.

    Science.gov (United States)

    Molle, Julia; Raab, Mario; Holzmeister, Susanne; Schmitt-Monreal, Daniel; Grohmann, Dina; He, Zhike; Tinnefeld, Philip

    2016-06-01

    For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Analysis of changes in the percentage of B (CD19) and T (CD3) lymphocytes, nk cells, subsets CD4, CD8 in differentiated thyroid cancer patients treated with iodine-131

    International Nuclear Information System (INIS)

    Luo Quanyong; Yu Yongli; Chen Libo; Lu Hankui; Zhu Ruisen

    2004-01-01

    Objective: To evaluate the changes in the percentage of B (CD19) and T (CD3) lymphocytes, NK cells, subsets CD4, CD8 in patients with differentiated thyroid carcinoma (DTC) who received iodine-131 for therapeutic purposes. Methods: In this study, 102 DTC patients were divided into three groups. Group A, 8 cases received 1850 MBq of iodine-131 for the remnant thyroid ablation. Group B, 43 cases received 3700 MBq of iodine-131 for the treatment of cervical lymph node metastasis. Group C, 51 cases received 7400 MBq of iodine-131 for remote metastasis. All patients were in a hypothyroid state at the time of administration of iodine-131 and resumed L-thyroxine (2μg/Kg/day) 5 days after iodine-131 administration. The percentage of B and T lymphocytes, NK cells, subsets CD4, CD8 in peripheral blood were serially analyzed at baseline and at days 7, 30 and 90 after iodine-131 administration using a Coulter EPICS XL cytometer. Ten healthy individuals were used as a control group for lymphocyte subset values. Results: Comparing the basal lymphocyte subset levels in groups A, B and C with the control group, only NK cells showed significantly higher levels in patients than in controls (P=0.043). In group A, only the percentage of NK cells (P=0.031) and B cells (P =0.024) were reduced at day 7. In group B, a decrease in the percentage of NK cells at days 7(P=0.005), 30 (P=0.021) was observed, while a significant decrease in the percentage of B cells was only observed at day 7(P=0.006). Among T cells, only CD4+ was obviously affected, resulting in a reduction in the CD4+/CD8+ ratio at day 30 (P=0.034). In group C, patients showed a decrease in the percentage of NK cells at days 7 (P=0.023), 30 (P=0.006). A decrease in the percentage of both B and T lymphocytes was observed at days 7(P=0.020, 0.018 respectively), 30(P=0.041, 0.025 respectively). Among T cells, a decrease in the percentage of CD4+ and an increase in the percentage of CD8+ were observed, resulting in a marked

  9. A novel recombinantly produced banana lectin isoform is a valuable tool for glycoproteomics and a potent modulator of the proliferation response in CD3(+), CD4(+), and CD8(+) populations of human PBMCs

    DEFF Research Database (Denmark)

    Gavrovic-Jankulovic, M; Poulsen, Knud; Brckalo, T

    2008-01-01

    Lectins as carbohydrate-binding proteins have been employed in various biological assays for the detection and characterization of glycan structures on glycoproteins, including clinical biomarkers in disease states. A mannose-specific banana lectin (BanLec) is unique in its specificity for internal......, the gene of banana lectin was cloned, sequenced and a recombinant protein was produced in Escherichia coli. The obtained cDNA revealed a novel banana lectin isoform, with an open reading frame of 426 nucleotides, encoding a cytoplasmatic protein of 141 amino acids. The molecular mass of rBanLec determined...

  10. To bind or not to bind? Different temporal binding effects from voluntary pressing and releasing actions.

    Science.gov (United States)

    Zhao, Ke; Chen, Yu-Hsin; Yan, Wen-Jing; Fu, Xiaolan

    2013-01-01

    Binding effect refers to the perceptual attraction between an action and an outcome leading to a subjective compression of time. Most studies investigating binding effects exclusively employ the "pressing" action without exploring other types of actions. The present study addresses this issue by introducing another action, releasing action or the voluntary lifting of the finger/wrist, to investigate the differences between voluntary pressing and releasing actions. Results reveal that releasing actions led to robust yet short-lived temporal binding effects, whereas pressing condition had steady temporal binding effects up to super-seconds. The two actions also differ in sensitivity to changes in temporal contiguity and contingency, which could be attributed to the difference in awareness of action. Extending upon current models of "willed action," our results provide insights from a temporal point of view and support the concept of a dual system consisting of predictive motor control and top-down mechanisms.

  11. Binding energies of cluster ions

    International Nuclear Information System (INIS)

    Parajuli, R.; Matt, S.; Scheier, P.; Echt, O.; Stamatovic, A.; Maerk, T.D.

    2002-01-01

    The binding energy of charged clusters may be measured by analyzing the kinetic energy released in the metastable decay of mass selected parent ions. Using finite heat bath theory to determine the binding energies of argon, neon, krypton, oxygen and nitrogen from their respective average kinetic energy released were carried out. A high-resolution double focussing two-sector mass spectrometer of reversed Nier-Johnson type geometry was used. MIKE ( mass-analysed ion kinetic energy) were measured to investigate decay reactions of mass-selected ions. For the inert gases neon (Ne n + ), argon (Ar n + ) and krypton (Kr n + ), it is found that the binding energies initially decrease with increasing size n and then level off at a value above the enthalpy of vaporization of the condensed phase. Oxygen cluster ions shown a characteristic dependence on cluster size (U-shape) indicating a change in the metastable fragmentation mechanism when going from the dimer to the decamer ion. (nevyjel)

  12. Metal binding by food components

    DEFF Research Database (Denmark)

    Tang, Ning

    for zinc binding by the investigated amino acids, peptides and proteins. The thiol group or imidazole group containing amino acids, peptides and proteins which exhibited strong zinc binding ability were further selected for interacting with zinc salts in relation to zinc absorption. The interactions...... between the above selected food components and zinc citrate or zinc phytate will lead to the enhanced solubility of zinc citrate or zinc phytate. The main driving force for this observed solubility enhancement is the complex formation between zinc and investigated food components as revealed by isothermal...... titration calorimetry and quantum mechanical calculations. This is due to the zinc binding affinity of the relatively softer ligands (investigated food components) will become much stronger than citrate or phytate when they present together in aqueous solution. This mechanism indicates these food components...

  13. Skyrmions with low binding energies

    Energy Technology Data Exchange (ETDEWEB)

    Gillard, Mike, E-mail: m.n.gillard@leeds.ac.uk; Harland, Derek, E-mail: d.g.harland@leeds.ac.uk; Speight, Martin, E-mail: speight@maths.leeds.ac.uk

    2015-06-15

    Nuclear binding energies are investigated in two variants of the Skyrme model: the first replaces the usual Skyrme term with a term that is sixth order in derivatives, and the second includes a potential that is quartic in the pion fields. Solitons in the first model are shown to deviate significantly from ansätze previously assumed in the literature. The binding energies obtained in both models are lower than those obtained from the standard Skyrme model, and those obtained in the second model are close to the experimental values.

  14. Skyrmions with low binding energies

    International Nuclear Information System (INIS)

    Gillard, Mike; Harland, Derek; Speight, Martin

    2015-01-01

    Nuclear binding energies are investigated in two variants of the Skyrme model: the first replaces the usual Skyrme term with a term that is sixth order in derivatives, and the second includes a potential that is quartic in the pion fields. Solitons in the first model are shown to deviate significantly from ansätze previously assumed in the literature. The binding energies obtained in both models are lower than those obtained from the standard Skyrme model, and those obtained in the second model are close to the experimental values

  15. Skyrmions with low binding energies

    Directory of Open Access Journals (Sweden)

    Mike Gillard

    2015-06-01

    Full Text Available Nuclear binding energies are investigated in two variants of the Skyrme model: the first replaces the usual Skyrme term with a term that is sixth order in derivatives, and the second includes a potential that is quartic in the pion fields. Solitons in the first model are shown to deviate significantly from ansätze previously assumed in the literature. The binding energies obtained in both models are lower than those obtained from the standard Skyrme model, and those obtained in the second model are close to the experimental values.

  16. Cellulose binding domain fusion proteins

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  17. When is protein binding important?

    Science.gov (United States)

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. Copyright © 2013 Wiley Periodicals, Inc.

  18. p56Lck and p59Fyn Regulate CD28 Binding to Phosphatidylinositol 3-Kinase, Growth Factor Receptor-Bound Protein GRB-2, and T Cell-Specific Protein-Tyrosine Kinase ITK: Implications for T-Cell Costimulation

    Science.gov (United States)

    Raab, Monika; Cai, Yun-Cai; Bunnell, Stephen C.; Heyeck, Stephanie D.; Berg, Leslie J.; Rudd, Christopher E.

    1995-09-01

    T-cell activation requires cooperative signals generated by the T-cell antigen receptor ξ-chain complex (TCRξ-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, ξ-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

  19. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen

    2012-01-01

    Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

  20. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...

  1. Ligand binding by PDZ domains

    DEFF Research Database (Denmark)

    Chi, Celestine N.; Bach, Anders; Strømgaard, Kristian

    2012-01-01

    , for example, are particularly rich in these domains. The general function of PDZ domains is to bring proteins together within the appropriate cellular compartment, thereby facilitating scaffolding, signaling, and trafficking events. The many functions of PDZ domains under normal physiological as well...... as pathological conditions have been reviewed recently. In this review, we focus on the molecular details of how PDZ domains bind their protein ligands and their potential as drug targets in this context....

  2. Calcium binding by dietary fibre

    International Nuclear Information System (INIS)

    James, W.P.T.; Branch, W.J.; Southgate, D.A.T.

    1978-01-01

    Dietary fibre from plants low in phytate bound calcium in proportion to its uronic-acid content. This binding by the non-cellulosic fraction of fibre reduces the availability of calcium for small-intestinal absorption, but the colonic microbial digestion of uronic acids liberates the calcium. Thus the ability to maintain calcium balance on high-fibre diets may depend on the adaptive capacity on the colon for calcium. (author)

  3. Material Binding Peptides for Nanotechnology

    Directory of Open Access Journals (Sweden)

    Urartu Ozgur Safak Seker

    2011-02-01

    Full Text Available Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.

  4. Anion binding in biological systems

    Energy Technology Data Exchange (ETDEWEB)

    Feiters, Martin C [Department of Organic Chemistry, Institute for Molecules and Materials, Faculty of Science, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen (Netherlands); Meyer-Klaucke, Wolfram [EMBL Hamburg Outstation at DESY, Notkestrasse 85, D-22607 Hamburg (Germany); Kostenko, Alexander V; Soldatov, Alexander V [Faculty of Physics, Southern Federal University, Sorge 5, Rostov-na-Donu, 344090 (Russian Federation); Leblanc, Catherine; Michel, Gurvan; Potin, Philippe [Centre National de la Recherche Scientifique and Universite Pierre et Marie Curie Paris-VI, Station Biologique de Roscoff, Place Georges Teissier, BP 74, F-29682 Roscoff cedex, Bretagne (France); Kuepper, Frithjof C [Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA37 1QA, Scotland (United Kingdom); Hollenstein, Kaspar; Locher, Kaspar P [Institute of Molecular Biology and Biophysics, ETH Zuerich, Schafmattstrasse 20, Zuerich, 8093 (Switzerland); Bevers, Loes E; Hagedoorn, Peter-Leon; Hagen, Wilfred R, E-mail: m.feiters@science.ru.n [Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft (Netherlands)

    2009-11-15

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L{sub 3} (2p{sub 3/2}) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  5. Anion binding in biological systems

    International Nuclear Information System (INIS)

    Feiters, Martin C; Meyer-Klaucke, Wolfram; Kostenko, Alexander V; Soldatov, Alexander V; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Kuepper, Frithjof C; Hollenstein, Kaspar; Locher, Kaspar P; Bevers, Loes E; Hagedoorn, Peter-Leon; Hagen, Wilfred R

    2009-01-01

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L 3 (2p 3/2 ) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  6. Anion binding in biological systems

    Science.gov (United States)

    Feiters, Martin C.; Meyer-Klaucke, Wolfram; Kostenko, Alexander V.; Soldatov, Alexander V.; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Küpper, Frithjof C.; Hollenstein, Kaspar; Locher, Kaspar P.; Bevers, Loes E.; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2009-11-01

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L3 (2p3/2) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  7. Solute-vacancy binding in aluminum

    International Nuclear Information System (INIS)

    Wolverton, C.

    2007-01-01

    Previous efforts to understand solute-vacancy binding in aluminum alloys have been hampered by a scarcity of reliable, quantitative experimental measurements. Here, we report a large database of solute-vacancy binding energies determined from first-principles density functional calculations. The calculated binding energies agree well with accurate measurements where available, and provide an accurate predictor of solute-vacancy binding in other systems. We find: (i) some common solutes in commercial Al alloys (e.g., Cu and Mg) possess either very weak (Cu), or even repulsive (Mg), binding energies. Hence, we assert that some previously reported large binding energies for these solutes are erroneous. (ii) Large binding energies are found for Sn, Cd and In, confirming the proposed mechanism for the reduced natural aging in Al-Cu alloys containing microalloying additions of these solutes. (iii) In addition, we predict that similar reduction in natural aging should occur with additions of Si, Ge and Au. (iv) Even larger binding energies are found for other solutes (e.g., Pb, Bi, Sr, Ba), but these solutes possess essentially no solubility in Al. (v) We have explored the physical effects controlling solute-vacancy binding in Al. We find that there is a strong correlation between binding energy and solute size, with larger solute atoms possessing a stronger binding with vacancies. (vi) Most transition-metal 3d solutes do not bind strongly with vacancies, and some are even energetically strongly repelled from vacancies, particularly for the early 3d solutes, Ti and V

  8. Polymeric competitive protein binding adsorbents for radioassay

    International Nuclear Information System (INIS)

    Adams, R.J.

    1976-01-01

    Serum protein comprising specific binding proteins such as antibodies, B 12 intrinsic factor, thyroxin binding globulin and the like may be copolymerized with globulin constituents of serum by the action of ethylchloroformate to form readily packed insoluble precipitates which, following purification as by washing, are eminently suited for employment as competitive binding protein absorbents in radioassay procedures. 10 claims, no drawings

  9. Insulin binding to individual rat skeletal muscles

    International Nuclear Information System (INIS)

    Koerker, D.J.; Sweet, I.R.; Baskin, D.G.

    1990-01-01

    Studies of insulin binding to skeletal muscle, performed using sarcolemmal membrane preparations or whole muscle incubations of mixed muscle or typical red (soleus, psoas) or white [extensor digitorum longus (EDL), gastrocnemius] muscle, have suggested that red muscle binds more insulin than white muscle. We have evaluated this hypothesis using cryostat sections of unfixed tissue to measure insulin binding in a broad range of skeletal muscles; many were of similar fiber-type profiles. Insulin binding per square millimeter of skeletal muscle slice was measured by autoradiography and computer-assisted densitometry. We found a 4.5-fold range in specific insulin tracer binding, with heart and predominantly slow-twitch oxidative muscles (SO) at the high end and the predominantly fast-twitch glycolytic (FG) muscles at the low end of the range. This pattern reflects insulin sensitivity. Evaluation of displacement curves for insulin binding yielded linear Scatchard plots. The dissociation constants varied over a ninefold range (0.26-2.06 nM). Binding capacity varied from 12.2 to 82.7 fmol/mm2. Neither binding parameter was correlated with fiber type or insulin sensitivity; e.g., among three muscles of similar fiber-type profile, the EDL had high numbers of low-affinity binding sites, whereas the quadriceps had low numbers of high-affinity sites. In summary, considerable heterogeneity in insulin binding was found among hindlimb muscles of the rat, which can be attributed to heterogeneity in binding affinities and the numbers of binding sites. It can be concluded that a given fiber type is not uniquely associated with a set of insulin binding parameters that result in high or low binding

  10. Ebola Virus Binding to Tim-1 on T Lymphocytes Induces a Cytokine Storm

    Directory of Open Access Journals (Sweden)

    Patrick Younan

    2017-09-01

    Full Text Available Ebola virus (EBOV disease (EVD results from an exacerbated immunological response that is highlighted by a burst in the production of inflammatory mediators known as a “cytokine storm.” Previous reports have suggested that nonspecific activation of T lymphocytes may play a central role in this phenomenon. T-cell immunoglobulin and mucin domain-containing protein 1 (Tim-1 has recently been shown to interact with virion-associated phosphatidylserine to promote infection. Here, we demonstrate the central role of Tim-1 in EBOV pathogenesis, as Tim-1−/− mice exhibited increased survival rates and reduced disease severity; surprisingly, only a limited decrease in viremia was detected. Tim-1−/− mice exhibited a modified inflammatory response as evidenced by changes in serum cytokines and activation of T helper subsets. A series of in vitro assays based on the Tim-1 expression profile on T cells demonstrated that despite the apparent absence of detectable viral replication in T lymphocytes, EBOV directly binds to isolated T lymphocytes in a phosphatidylserine–Tim-1-dependent manner. Exposure to EBOV resulted in the rapid development of a CD4Hi CD3Low population, non-antigen-specific activation, and cytokine production. Transcriptome and Western blot analysis of EBOV-stimulated CD4+ T cells confirmed the induction of the Tim-1 signaling pathway. Furthermore, comparative analysis of transcriptome data and cytokine/chemokine analysis of supernatants highlight the similarities associated with EBOV-stimulated T cells and the onset of a cytokine storm. Flow cytometry revealed virtually exclusive binding and activation of central memory CD4+ T cells. These findings provide evidence for the role of Tim-1 in the induction of a cytokine storm phenomenon and the pathogenesis of EVD.

  11. Adaptive evolution of transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Berg Johannes

    2004-10-01

    Full Text Available Abstract Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. Results We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background mutation rate, the selection coefficient, and the effective population size. Conclusions The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.

  12. Synthetic LPS-Binding Polymer Nanoparticles

    Science.gov (United States)

    Jiang, Tian

    Lipopolysaccharide (LPS), one of the principal components of most gram-negative bacteria's outer membrane, is a type of contaminant that can be frequently found in recombinant DNA products. Because of its strong and even lethal biological effects, selective LPS removal from bioproducts solution is of particular importance in the pharmaceutical and health care industries. In this thesis, for the first time, a proof-of-concept study on preparing LPS-binding hydrogel-like NPs through facile one-step free-radical polymerization was presented. With the incorporation of various hydrophobic (TBAm), cationic (APM, GUA) monomers and cross-linkers (BIS, PEG), a small library of NPs was constructed. Their FITC-LPS binding behaviors were investigated and compared with those of commercially available LPS-binding products. Moreover, the LPS binding selectivity of the NPs was also explored by studying the NPs-BSA interactions. The results showed that all NPs obtained generally presented higher FITC-LPS binding capacity in lower ionic strength buffer than higher ionic strength. However, unlike commercial poly-lysine cellulose and polymyxin B agarose beads' nearly linear increase of FITC-LPS binding with particle concentration, NPs exhibited serious aggregation and the binding quickly saturated or even decreased at high particle concentration. Among various types of NPs, higher FITC-LPS binding capacity was observed for those containing more hydrophobic monomers (TBAm). However, surprisingly, more cationic NPs with higher content of APM exhibited decreased FITC-LPS binding in high ionic strength conditions. Additionally, when new cationic monomer and cross-linker, GUA and PEG, were applied to replace APM and BIS, the obtained NPs showed improved FITC-LPS binding capacity at low NP concentration. But compared with APM- and BIS-containing NPs, the FITC-LPS binding capacity of GUA- and PEG-containing NPs saturated earlier. To investigate the NPs' binding to proteins, we tested the NPs

  13. Sex hormone binding globulin phenotypes

    DEFF Research Database (Denmark)

    Cornelisse, M M; Bennett, Patrick; Christiansen, M

    1994-01-01

    Human sex hormone binding globulin (SHBG) is encoded by a normal and a variant allele. The resulting SHBG phenotypes (the homozygous normal SHBG, the heterozygous SHBG and the homozygous variant SHBG phenotype) can be distinguished by their electrophoretic patterns. We developed a novel detection....... This method of detection was used to determine the distribution of SHBG phenotypes in healthy controls of both sexes and in five different pathological conditions characterized by changes in the SHBG level or endocrine disturbances (malignant and benign ovarian neoplasms, hirsutism, liver cirrhosis...... on the experimental values. Differences in SHBG phenotypes do not appear to have any clinical significance and no sex difference was found in the SHBG phenotype distribution....

  14. DNA Binding Hydroxyl Radical Probes.

    Science.gov (United States)

    Tang, Vicky J; Konigsfeld, Katie M; Aguilera, Joe A; Milligan, Jamie R

    2012-01-01

    The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA.

  15. Asymmetric cation-binding catalysis

    DEFF Research Database (Denmark)

    Oliveira, Maria Teresa; Lee, Jiwoong

    2017-01-01

    The employment of metal salts is quite limited in asymmetric catalysis, although it would provide an additional arsenal of safe and inexpensive reagents to create molecular functions with high optical purity. Cation chelation by polyethers increases the salts' solubility in conventional organic...... solvents, thus increasing their applicability in synthesis. The expansion of this concept to chiral polyethers led to the emergence of asymmetric cation-binding catalysis, where chiral counter anions are generated from metal salts, particularly using BINOL-based polyethers. Alkali metal salts, namely KF...... highly enantioselective silylation reactions in polyether-generated chiral environments, and leading to a record-high turnover in asymmetric organocatalysis. This can lead to further applications by the asymmetric use of other inorganic salts in various organic transformations....

  16. The helical structure of DNA facilitates binding

    International Nuclear Information System (INIS)

    Berg, Otto G; Mahmutovic, Anel; Marklund, Emil; Elf, Johan

    2016-01-01

    The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction–diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general. (paper)

  17. Fundamental considerations in ski binding analysis.

    Science.gov (United States)

    Mote, C D; Hull, M L

    1976-01-01

    1. The static adjustment of a ski binding by hand or by available machines is only an adjustment and is neither a static nor a dynamic evaluation of the binding design. Bindings of different design with identical static adjustments will perform differently in environments in which the forces are static or dynamic. 2. The concept of binding release force is a useful measure of binding adjustment, but it is inappropriate as a criterion for binding evaluation. First, it does not direct attention toward the injury causing mechanism, strain, or displacement in the leg. Second, it is only part of the evaluation in dynamic problems. 3. The binding release decision in present bindings is displacement controlled. The relative displacement of the boot and ski is the system variable. For any specified relative displacement the binding force can be any of an infinite number of possibilities determined by the loading path. 4. The response of the leg-ski system to external impulses applied to the ski is independent of the boot-ski relative motion as long as the boot recenters quickly in the binding. Response is dependent upon the external impulse plus system inertia, damping and stiffness. 5. When tested under half sinusoidal forces applied to a test ski, all bindings will demonstrate static and impulse loading regions. In the static region the force drives the binding to a relative release displacement. In the impulse region the initial velocity of the ski drives the binding to a release displacement. 6. The transition between the static and impulse loading regions is determined by the binding's capacity to store and dissipate energy along the principal loading path. Increased energy capacity necessitates larger external impulses to produce release. 7. In all bindings examined to date, the transmitted leg displacement or strain at release under static loading exceeds leg strain under dynamic or impact loading. Because static loading is responsible for many injuries, a skier

  18. Lead-Binding Proteins: A Review

    Directory of Open Access Journals (Sweden)

    Harvey C. Gonick

    2011-01-01

    Full Text Available Lead-binding proteins are a series of low molecular weight proteins, analogous to metallothionein, which segregate lead in a nontoxic form in several organs (kidney, brain, lung, liver, erythrocyte. Whether the lead-binding proteins in every organ are identical or different remains to be determined. In the erythrocyte, delta-aminolevulinic acid dehydratase (ALAD isoforms have commanded the greatest attention as proteins and enzymes that are both inhibitable and inducible by lead. ALAD-2, although it binds lead to a greater degree than ALAD-1, appears to bind lead in a less toxic form. What may be of greater significance is that a low molecular weight lead-binding protein, approximately 10 kDa, appears in the erythrocyte once blood lead exceeds 39 μg/dL and eventually surpasses the lead-binding capacity of ALAD. In brain and kidney of environmentally exposed humans and animals, a cytoplasmic lead-binding protein has been identified as thymosin β4, a 5 kDa protein. In kidney, but not brain, another lead-binding protein has been identified as acyl-CoA binding protein, a 9 kDa protein. Each of these proteins, when coincubated with liver ALAD and titrated with lead, diminishes the inhibition of ALAD by lead, verifying their ability to segregate lead in a nontoxic form.

  19. Drug binding properties of neonatal albumin

    DEFF Research Database (Denmark)

    Brodersen, R; Honoré, B

    1989-01-01

    Neonatal and adult albumin was isolated by gel chromatography on Sephacryl S-300, from adult and umbilical cord serum, respectively. Binding of monoacetyl-diamino-diphenyl sulfone, warfarin, sulfamethizole, and diazepam was studied by means of equilibrium dialysis and the binding data were analyzed...... by the method of several acceptable fitted curves. It was found that the binding affinity to neonatal albumin is less than to adult albumin for monoacetyl-diamino-diphenyl sulfone and warfarin. Sulfamethizole binding to the neonatal protein is similarly reduced when more than one molecule of the drug is bound...

  20. Bitopic Ligands and Metastable Binding Sites

    DEFF Research Database (Denmark)

    Fronik, Philipp; Gaiser, Birgit I; Sejer Pedersen, Daniel

    2017-01-01

    of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding...... that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites....

  1. Retinoid-binding proteins: similar protein architectures bind similar ligands via completely different ways.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Zhang

    Full Text Available BACKGROUND: Retinoids are a class of compounds that are chemically related to vitamin A, which is an essential nutrient that plays a key role in vision, cell growth and differentiation. In vivo, retinoids must bind with specific proteins to perform their necessary functions. Plasma retinol-binding protein (RBP and epididymal retinoic acid binding protein (ERABP carry retinoids in bodily fluids, while cellular retinol-binding proteins (CRBPs and cellular retinoic acid-binding proteins (CRABPs carry retinoids within cells. Interestingly, although all of these transport proteins possess similar structures, the modes of binding for the different retinoid ligands with their carrier proteins are different. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we analyzed the various retinoid transport mechanisms using structure and sequence comparisons, binding site analyses and molecular dynamics simulations. Our results show that in the same family of proteins and subcellular location, the orientation of a retinoid molecule within a binding protein is same, whereas when different families of proteins are considered, the orientation of the bound retinoid is completely different. In addition, none of the amino acid residues involved in ligand binding is conserved between the transport proteins. However, for each specific binding protein, the amino acids involved in the ligand binding are conserved. The results of this study allow us to propose a possible transport model for retinoids. CONCLUSIONS/SIGNIFICANCE: Our results reveal the differences in the binding modes between the different retinoid-binding proteins.

  2. New DNA-binding radioprotectors

    Science.gov (United States)

    Martin, Roger

    The normal tissue damage associated with cancer radiotherapy has motivated the development at Peter Mac of a new class of DNA-binding radioprotecting drugs that could be applied top-ically to normal tissues at risk. Methylproamine (MP), the lead compound, reduces radiation induced cell kill at low concentrations. For example, experiments comparing the clonogenic survival of transformed human keratinocytes treated with 30 micromolar MP before and dur-ing various doses of ionising radiation, with the radiation dose response for untreated cells, indicate a dose reduction factor (DRF) of 2. Similar survival curve experiments using various concentrations of MP, with parallel measurements of uptake of MP into cell nuclei, have en-abled the relationship between drug uptake and extent of radioprotection to be established. Radioprotection has also been demonstrated after systemic administration to mice, for three different endpoints, namely lung, jejunum and bone marrow (survival at 30 days post-TBI). The results of pulse radiolysis studies indicated that the drugs act by reduction of transient radiation-induced oxidative species on DNA. This hypothesis was substantiated by the results of experiments in which MP radioprotection of radiation-induced DNA double-strand breaks, assessed as -H2AX foci, in the human keratinocyte cell line. For both endpoints, the extent of radioprotection increased with MP concentration up to a maximal value. These results are consistent with the hypothesis that radioprotection by MP is mediated by attenuation of the extent of initial DNA damage. However, although MP is a potent radioprotector, it becomes cytotoxic at higher concentrations. This limitation has been addressed in an extensive program of lead optimisation and some promising analogues have emerged from which the next lead will be selected. Given the clinical potential of topical radioprotection, the new analogues are being assessed in terms of delivery to mouse oral mucosa. This is

  3. Binding of (/sup 3/H)imipramine to human platelet membranes with compensation for saturable binding to filters and its implication for binding studies with brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, O.M.; Wood, K.M.; Williams, D.C.

    1984-08-01

    Apparent specific binding of (/sup 3/H)imipramine to human platelet membranes at high concentrations of imipramine showed deviation from that expected of a single binding site, a result consistent with a low-affinity binding site. The deviation was due to displaceable, saturable binding to the glass fibre filters used in the assays. Imipramine, chloripramine, desipramine, and fluoxetine inhibited binding to filters whereas 5-hydroxytryptamine and ethanol were ineffective. Experimental conditions were developed that eliminated filter binding, allowing assay of high- and low-affinity binding to membranes. Failure to correct for filter binding may lead to overestimation of binding parameters, Bmax and KD for high-affinity binding to membranes, and may also be misinterpreted as indicating a low-affinity binding component in both platelet and brain membranes. Low-affinity binding (KD less than 2 microM) of imipramine to human platelet membranes was demonstrated and its significance discussed.

  4. Cyclophilin B mediates cyclosporin A incorporation in human blood T-lymphocytes through the specific binding of complexed drug to the cell surface.

    Science.gov (United States)

    Allain, F; Denys, A; Spik, G

    1996-07-15

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein located within intracellular vesicles and released in biological fluids. We recently reported the specific binding of this protein to T-cell surface receptor which is internalized even in the presence of CsA. These results suggest that CyPB might target the drug to lymphocytes and consequently modify its activity. To verify this hypothesis, we have first investigated the binding capacity and internalization of the CsA-CyPB complex in human peripheral blood T-lymphocytes and secondly compared the inhibitory effect of both free and CyPB-complexed CsA on the CD3-induced activation and proliferation of T-cells. Here, we present evidence that both the CsA-CyPB complex and free CyPB bind to the T-lymphocyte surface, with similar values of Kd and number of sites. At 37 degrees C, the complex is internalized but, in contrast to the protein, the drug is accumulated within the cell. Moreover, CyPB receptors are internalized together with the ligand and rapidly recycled to the cell surface. Finally, we demonstrate that CyPB-complexed CsA remains as efficient as uncomplexed CsA and that CyPB enhances the immunosuppressive activity of the drug. Taken together, our results support the hypothesis that surface CyPB receptors may be related to the selective and variable action of CsA, through specific binding and targeting of the CyPB-CsA complex to peripheral blood T-lymphocytes.

  5. Multiple binding modes of ibuprofen in human serum albumin identified by absolute binding free energy calculations

    KAUST Repository

    Evoli, Stefania

    2016-11-10

    Human serum albumin possesses multiple binding sites and transports a wide range of ligands that include the anti-inflammatory drug ibuprofen. A complete map of the binding sites of ibuprofen in albumin is difficult to obtain in traditional experiments, because of the structural adaptability of this protein in accommodating small ligands. In this work, we provide a set of predictions covering the geometry, affinity of binding and protonation state for the pharmaceutically most active form (S-isomer) of ibuprofen to albumin, by using absolute binding free energy calculations in combination with classical molecular dynamics (MD) simulations and molecular docking. The most favorable binding modes correctly reproduce several experimentally identified binding locations, which include the two Sudlow\\'s drug sites (DS2 and DS1) and the fatty acid binding sites 6 and 2 (FA6 and FA2). Previously unknown details of the binding conformations were revealed for some of them, and formerly undetected binding modes were found in other protein sites. The calculated binding affinities exhibit trends which seem to agree with the available experimental data, and drastically degrade when the ligand is modeled in a protonated (neutral) state, indicating that ibuprofen associates with albumin preferentially in its charged form. These findings provide a detailed description of the binding of ibuprofen, help to explain a wide range of results reported in the literature in the last decades, and demonstrate the possibility of using simulation methods to predict ligand binding to albumin.

  6. Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Sigurskjold, B W; Kragelund, B B

    1996-01-01

    Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-...

  7. Identification of Glossina morsitans morsitans odorant binding ...

    African Journals Online (AJOL)

    Tsetse flies are vectors of trypanosome parasites, causative agents of Trypanosomiasis in humans and animals. Odorant Binding Proteins (OBPs) are critical in insect olfaction as they bind volatile odours from the environment and transport them to receptors within olfactory receptor neurons for processing providing critical ...

  8. Triazatriangulene as binding group for molecular electronics

    DEFF Research Database (Denmark)

    Wei, Zhongming; Wang, Xintai; Borges, Anders

    2014-01-01

    The triazatriangulene (TATA) ring system was investigated as a binding group for tunnel junctions of molecular wires on gold surfaces. Self-assembled monolayers (SAMs) of TATA platforms with three different lengths of phenylene wires were fabricated, and their electrical conductance was recorded ...... with its high stability and directionality make this binding group very attractive for molecular electronic measurements and devices. (Figure Presented)....

  9. Localization-enhanced biexciton binding in semiconductors

    DEFF Research Database (Denmark)

    Langbein, Wolfgang Werner; Hvam, Jørn Märcher

    1999-01-01

    The influence of excitonic localization on the binding energy of biexcitons is investigated for quasi-three-dimensional and quasi-two-dimensional AlxGa1-xAs structures. An increase of the biexciton binding energy is observed for localization energies comparable to or larger than the free biexcito...

  10. Binding of corroded ions to human saliva.

    Science.gov (United States)

    Mueller, H J

    1985-05-01

    Employing equilibrium dialysis, the binding abilities of Cu, Al, Co and Cr ions from corroded Cu-Al and Co-Cr dental casting alloys towards human saliva and two of its gel chromatographic fractions were determined. Results indicate that both Cu and Co bind to human saliva i.e. 0.045 and 0.027 mg/mg protein, respectively. Besides possessing the largest binding ability, Cu also possessed the largest binding capacity. The saturation of Cu binding was not reached up to the limit of 0.35 mg protein/ml employed in the tests, while Co reached full saturation at about 0.2 mg protein/ml. Chromium showed absolutely no binding to human saliva while Al ions did not pass through the dialysis membranes. Compared to the binding with solutions that were synthetically made up to contain added salivary-type proteins, it is shown that the binding to human saliva is about 1 order of magnitude larger, at least for Cu ions.

  11. Intentional binding of visual effects.

    Science.gov (United States)

    Ruess, Miriam; Thomaschke, Roland; Kiesel, Andrea

    2018-04-01

    When an action produces an effect, the effect is perceived earlier in time compared to a stimulus without preceding action. This temporal bias is called intentional binding (IB) and serves as an implicit measure of sense of agency. Typically, IB is investigated by presenting a rotating clock hand while participants execute an action and perceive a resulting tone. Participants are asked to estimate the time point of tone onset by referring to the clock hand position. This time point estimate is compared to a time point estimate of a tone in a condition in which the tone occurs without preceding action. Studies employing this classic clock paradigm employed auditory action effects. We modified this paradigm to investigate potential IB of visual action effects, and, additionally, to investigate how IB differs for visual action effects (Experiment 1) in comparison to auditory action effects (Experiment 2). Our results show that, like the IB of an auditory effect, the time point of a visual action effect is shifted toward the causing action, and that the size of the IB depends on the delay duration of the effect. Comparable to auditory action effects, earlier action effects showed stronger IB compared to later action effects. Yet overall IB of the visual effects was weaker than IB of the auditory effects. As IB is seen as an indicator of sense of agency, this may have important implications for the design of human-machine interfaces.

  12. Characterization of chlorophyll binding to LIL3.

    Science.gov (United States)

    Mork-Jansson, Astrid Elisabeth; Eichacker, Lutz Andreas

    2018-01-01

    The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to in vitro expressed LIL3 requires pre-incubation of reaction partners at room temperature. We show chlorophyll a but not chlorophyll b binding to LIL3 at a molar ratio of 1:1. Neither dynamic light scattering nor native PAGE, enabled a discrimination between binding of chlorophyll a and/or b to LIL3.

  13. (TH) diazepam binding to human granulocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bond, P.A.; Cundall, R.L.; Rolfe, B.

    1985-07-08

    (TH)-diazepam binds to sites on human granulocyte membranes, with little or no binding to platelets or lymphocytes. These (TH)-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki=6.23nM) but only weakly by clonazepam (Ki=14 M). Binding of (TH) diazepam at 0 is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109 +/- 17f moles per mg of protein and K/sub D/ of 3.07 +/- 0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 +/- 0.014. Binding is time dependent and reversible and it varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein. 11 references, 3 figures, 1 table.

  14. Factor VIIa binding and internalization in hepatocytes

    DEFF Research Database (Denmark)

    Hjortoe, G; Sorensen, B B; Petersen, L C

    2005-01-01

    The liver is believed to be the primary clearance organ for coagulation proteases, including factor VIIa (FVIIa). However, at present, clearance mechanisms for FVIIa in liver are unknown. To obtain information on the FVIIa clearance mechanism, we investigated the binding and internalization...... no effect. HEPG2 cells internalized FVIIa with a rate of 10 fmol 10(-5) cells h(-1). In contrast to HEPG2 cells, FVIIa binding to primary rat hepatocytes was completely independent of TF, and excess unlabeled FVIIa partly reduced the binding of 125I-FVIIa to rat hepatocytes. Further, compared with HEPG2...... cells, three- to fourfold more FVIIa bound to rat primary hepatocytes, and the bound FVIIa was internalized at a faster rate. Similar FVIIa binding and internalization profiles were observed in primary human hepatocytes. Plasma inhibitors had no effect on FVIIa binding and internalization in hepatocytes...

  15. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  16. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-01-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  17. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    International Nuclear Information System (INIS)

    Serck-Hanssen, G.; Soevik, O.

    1987-01-01

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of 125 I-insulin was carried out at 15 0 C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table

  18. Malignant Mesothelioma Effusions Are Infiltrated by CD3+ T Cells Highly Expressing PD-L1 and the PD-L1+ Tumor Cells within These Effusions Are Susceptible to ADCC by the Anti-PD-L1 Antibody Avelumab.

    Science.gov (United States)

    Khanna, Swati; Thomas, Anish; Abate-Daga, Daniel; Zhang, Jingli; Morrow, Betsy; Steinberg, Seth M; Orlandi, Augusto; Ferroni, Patrizia; Schlom, Jeffrey; Guadagni, Fiorella; Hassan, Raffit

    2016-11-01

    The functional aspects of programmed death 1 (PD-1) and PD ligand 1 (PD-L1) immune checkpoints in malignant mesothelioma have not been studied. Tumor samples from 65 patients with mesothelioma were evaluated for PD-L1 expression by immunohistochemistry, and its prognostic significance was examined. Malignant effusions from patients with pleural and peritoneal mesothelioma were evaluated for PD-1-positive and PD-L1-positive infiltrating lymphocytes and their role in inducing PD-L1 expression in tumor cells. Antibody-dependent cellular cytotoxicity (ADCC) of avelumab, a fully humanized immunoglobulin G1 anti PD-L1 antibody against primary mesothelioma cell lines, was evaluated in presence of autologous and allogeneic natural killer cells. Of 65 pleural and peritoneal mesothelioma tumors examined, 41 (63%) were PD-L1-positive, which was associated with slightly inferior overall survival compared to patients with PD-L1-negative tumors (median 23.0 versus 33.3 months, p = 0.35). The frequency of PD-L1 expression was similar in patients with pleural and peritoneal mesothelioma, with 62% and 64% of samples testing positive, respectively. In nine mesothelioma effusion samples evaluated, the fraction of cells expressing PD-L1 ranged from 12% to 83%. In seven patients with paired malignant effusion and peripheral blood mononuclear cell (PBMC) samples, PD-L1 expression was significantly higher on CD3-positive T cells present in malignant effusions as compared with PBMCs (p = 0.016). In addition, the numbers of CD14-positive PD-1-positive cells were increased in malignant effusions compared with PBMCs (p = 0.031). The lymphocytes present in malignant effusions recognized autologous tumor cells and induced interferon-γ-mediated PD-L1 expression on the tumor cell surface. Of the three primary mesothelioma cell lines tested, two were susceptible to avelumab-mediated ADCC in the presence of autologous natural killer cells. Most pleural as well as peritoneal mesotheliomas

  19. Human liver aldehyde dehydrogenase: coenzyme binding

    International Nuclear Information System (INIS)

    Kosley, L.L.; Pietruszko, R.

    1987-01-01

    The binding of [U- 14 C] NAD to mitochondrial (E2) and cytoplasmin(E1) aldehyde dehydrogenase was measured by gel filtration and sedimentation techniques. The binding data for NAD and (E1) yielded linear Scatchard plots giving a dissociation constant of 25 (+/- 8) uM and the stoichiometry of 2 mol of NAD bound per mol of E1. The binding data for NAD and (E2) gave nonlinear Scatchard plots. The binding of NADH to E2 was measured via fluorescence enhancement; this could not be done with E1 because there was no signal. The dissociation constant for E2 by this technique was 0.7 (+/- 0.4) uM and stoichiometry of 1.0 was obtained. The binding of [U- 14 C] NADH to (E1) and (E2) was also measured by the sedimentation technique. The binding data for (E1) and NADH gave linear Scatchard plots giving a dissociation constant of 13 (+/- 6) uM and the stoichiometry of 2.0. The binding data for NADH to (E2) gave nonlinear Scatchard plots. With (E1), the dissociation constants for both NAD and NADH are similar to those determined kinetically, but the stoichiometry is only half of that found by stopped flow technique. With (E2) the dissociation constant by fluorometric procedure was 2 orders of magnitude less than that from catalytic reaction

  20. Increased thyrotropin binding in hyperfunctioning thyroid nodules.

    Science.gov (United States)

    Müller-Gärtner, H W; Schneider, C; Bay, V; Tadt, A; Rehpenning, W; de Heer, K; Jessel, M

    1987-08-01

    The object of this study was to investigate TSH receptors in hyperfunctioning thyroid nodules (HFN). In HFN, obtained from seven patients, 125-I-TSH binding as determined by equilibrium binding analysis on particulate membrane preparations, was found to be significantly increased as compared with normal thyroid tissues (five patients; P less than 0.001). Scatchard analysis of TSH-binding revealed two kinds of binding sites for both normal thyroid tissue and HFN, and displayed significantly increased association constants of high- and low-affinity binding sites in HFN (Ka = 11.75 +/- 6.8 10(9) M-1, P less than 0.001 and Ka = 2.1 +/- 1.0 10(7) M-1, P less than 0.025; x +/- SEM) as compared with normal thyroid tissue (Ka = 0.25 +/- 0.06 10(9) M-1, Ka = 0.14 +/- 0.03 10(7) M-1; x +/- SEM). The capacity of the high-affinity binding sites in HFN was found to be decreased (1.8 +/- 1.1 pmol/mg protein, x +/- SEM) in comparison with normal thyroid tissue (4.26 +/- 1.27 pmol/mg protein; x +/- SEM). TSH-receptor autoradiography applied to cryostatic tissue sections confirmed increased TSH binding of the follicular epithelium in HFN. These data suggest that an increased affinity of TSH-receptor sites in HFN in iodine deficient areas may be an important event in thyroid autonomy.

  1. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    Science.gov (United States)

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  2. Hardware device binding and mutual authentication

    Science.gov (United States)

    Hamlet, Jason R; Pierson, Lyndon G

    2014-03-04

    Detection and deterrence of device tampering and subversion by substitution may be achieved by including a cryptographic unit within a computing device for binding multiple hardware devices and mutually authenticating the devices. The cryptographic unit includes a physically unclonable function ("PUF") circuit disposed in or on the hardware device, which generates a binding PUF value. The cryptographic unit uses the binding PUF value during an enrollment phase and subsequent authentication phases. During a subsequent authentication phase, the cryptographic unit uses the binding PUF values of the multiple hardware devices to generate a challenge to send to the other device, and to verify a challenge received from the other device to mutually authenticate the hardware devices.

  3. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit......Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length...... than 5 amino acids showed binding and a clear correlation with hydrophobicity was demonstrated for oligomers of different hydrophobic amino acids. Insertion of hydrophilic amino acids in a hydrophobic sequence diminished or abolished binding. In conclusion our results show that calreticulin has...

  4. Fatty Acid Binding Proteins in Prostate Cancer

    National Research Council Canada - National Science Library

    Jett, Marti

    2000-01-01

    We have shown that there is a distinct pattern of fatty acid binding protein (FAEP) expression in prostate cancer vs normal cells and that finding has be confirmed in patient samples of biopsy specimens...

  5. Bilirubin Binding Capacity in the Preterm Neonate.

    Science.gov (United States)

    Amin, Sanjiv B

    2016-06-01

    Total serum/plasma bilirubin (TB), the biochemical measure currently used to evaluate and manage hyperbilirubinemia, is not a useful predictor of bilirubin-induced neurotoxicity in premature infants. Altered bilirubin-albumin binding in premature infants limits the usefulness of TB in premature infants. In this article, bilirubin-albumin binding, a modifying factor for bilirubin-induced neurotoxicity, in premature infants is reviewed. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Exciton Binding Energy of Monolayer WS2

    Science.gov (United States)

    Zhu, Bairen; Chen, Xi; Cui, Xiaodong

    2015-03-01

    The optical properties of monolayer transition metal dichalcogenides (TMDC) feature prominent excitonic natures. Here we report an experimental approach to measuring the exciton binding energy of monolayer WS2 with linear differential transmission spectroscopy and two-photon photoluminescence excitation spectroscopy (TP-PLE). TP-PLE measurements show the exciton binding energy of 0.71 +/- 0.01 eV around K valley in the Brillouin zone.

  7. P-shell hyperon binding energies

    International Nuclear Information System (INIS)

    Koetsier, D.; Amos, K.

    1991-01-01

    A shell model for lambda hypernuclei has been used to determine the binding energy of the hyperon in nuclei throughout the p shell. Conventional (Cohen and Kurath) potential energies for nucleon-nucleon interactions were used with hyperon-nucleon interactions taken from Nijmegen one boson exchange potentials. The hyperon binding energies calculated from these potentials compare well with measured values. 7 refs., 2 figs

  8. Extracellular and intracellular steroid binding proteins

    International Nuclear Information System (INIS)

    Wagner, R.K.

    1978-01-01

    Steroid hormone binding proteins can be measured, after the removal of endogenous steroids, as specific complexes with radio-labelled hormones. In this study all the requirements for a quantitative determination of steroid hormone binding proteins are defined. For different methods, agargel electrophoresis, density gradient centrifugation, equilibrium dialysis and polyacrylamide electrophoresis have been evaluated. Agar electrophoresis at low temperature was found to be the simplest and most useful procedure. With this method the dissociation rates of high affinity complexes can be assessed and absolute binding protein concentrations can be determined. The dissociation rates of the oestradiol-oestrogen receptor complex and the R-5020-progestin receptor complex are low (1-2% per h run time.) In contrast, that of complexes between androgen receptor and dihydrotestosterone (17β-hydroxy-5α-androstan-3-one (DHT), progestin receptor and progesterone, corticosteroid binding globulin (CBG) and cortisol or progesterone and sex hormone binding globulin (SHBG) and DHT were hign (16-27% per h run time). Target tissue extracts (cytosols) contain, besides soluble tissue proteins, large amounts of plasma proteins. The extent of this plasma contamination can be determined by measuring the albumin concentration in cytosols by immunodiffusion. In cytosols of 4 different human target tissues the albumin content varied from 20-30% corresponding to an even higher whole plasma concentration. Steroid binding plasma proteins, such as CBG and SHBG are constituents of this containment. (author)

  9. The readiness potential reflects intentional binding

    Directory of Open Access Journals (Sweden)

    Han-Gue eJo

    2014-06-01

    Full Text Available When a voluntary action is causally linked with a sensory outcome, the action and its consequent effect are perceived as being closer together in time. This effect is called intentional binding. Although many experiments were conducted on this phenomenon, the underlying neural mechanisms are not well understood. While intentional binding is specific to voluntary action, we presumed that preconscious brain activity (the readiness potential, RP, which occurs before an action is made, might play an important role in this binding effect. In this study, the brain dynamics were recorded with electroencephalography (EEG and analyzed in single-trials in order to estimate whether intentional binding is correlated with the early neural processes. Moreover, we were interested in different behavioral performance between meditators and non-meditators since meditators are expected to be able to keep attention more consistently on a task. Thus, we performed the intentional binding paradigm with twenty mindfulness meditators and compared them to matched controls. Although, we did not observe a group effect on either behavioral data or EEG recordings, we found that self-initiated movements following ongoing negative deflections of slow cortical potentials (SCPs result in a stronger binding effect compared to positive potentials, especially regarding the perceived time of the consequent effect. Our results provide the first direct evidence that the early neural activity within the range of SCPs affects perceived time of a sensory outcome that is caused by intentional action.

  10. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

    Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

  11. LIBRA: LIgand Binding site Recognition Application.

    Science.gov (United States)

    Hung, Le Viet; Caprari, Silvia; Bizai, Massimiliano; Toti, Daniele; Polticelli, Fabio

    2015-12-15

    In recent years, structural genomics and ab initio molecular modeling activities are leading to the availability of a large number of structural models of proteins whose biochemical function is not known. The aim of this study was the development of a novel software tool that, given a protein's structural model, predicts the presence and identity of active sites and/or ligand binding sites. The algorithm implemented by ligand binding site recognition application (LIBRA) is based on a graph theory approach to find the largest subset of similar residues between an input protein and a collection of known functional sites. The algorithm makes use of two predefined databases for active sites and ligand binding sites, respectively, derived from the Catalytic Site Atlas and the Protein Data Bank. Tests indicate that LIBRA is able to identify the correct binding/active site in 90% of the cases analyzed, 90% of which feature the identified site as ranking first. As far as ligand binding site recognition is concerned, LIBRA outperforms other structure-based ligand binding sites detection tools with which it has been compared. The application, developed in Java SE 7 with a Swing GUI embedding a JMol applet, can be run on any OS equipped with a suitable Java Virtual Machine (JVM), and is available at the following URL: http://www.computationalbiology.it/software/LIBRAv1.zip. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Zinc Binding by Lactic Acid Bacteria

    Directory of Open Access Journals (Sweden)

    Jasna Mrvčić

    2009-01-01

    Full Text Available Zinc is an essential trace element in all organisms. A common method for the prevention of zinc deficiency is pharmacological supplementation, especially in a highly available form of a metalloprotein complex. The potential of different microbes to bind essential and toxic heavy metals has recently been recognized. In this work, biosorption of zinc by lactic acid bacteria (LAB has been investigated. Specific LAB were assessed for their ability to bind zinc from a water solution. Significant amount of zinc ions was bound, and this binding was found to be LAB species-specific. Differences among the species in binding performance at a concentration range between 10–90 mg/L were evaluated with Langmuir model for biosorption. Binding of zinc was a fast process, strongly influenced by ionic strength, pH, biomass concentration, and temperature. The most effective metal-binding LAB species was Leuconostoc mesenteroides (27.10 mg of Zn2+ per gram of dry mass bound at pH=5 and 32 °C, during 24 h. FT-IR spectroscopy analysis and electron microscopy demonstrated that passive adsorption and active uptake of the zinc ions were involved.

  13. Radiation damage to DNA-binding proteins

    International Nuclear Information System (INIS)

    Culard, G.; Eon, S.; DeVuyst, G.; Charlier, M.; Spotheim-Maurizot, M.

    2003-01-01

    The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

  14. Mannose-Binding Lectin Binds to Amyloid Protein and Modulates Inflammation

    Directory of Open Access Journals (Sweden)

    Mykol Larvie

    2012-01-01

    Full Text Available Mannose-binding lectin (MBL, a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid β peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aβ are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

  15. High levels of serum mannose-binding lectin are associated with the severity of clinical signs of leptospirosis

    Directory of Open Access Journals (Sweden)

    K.A. Miranda

    2009-04-01

    Full Text Available The clinical heterogeneity observed in leptospirosis may be associated with host factors or bacteria virulence. Human serum mannose-binding lectin (MBL recognizes many pathogens, and low levels of this lectin are associated with susceptibility to infection. MBL is also implicated in the modulation of the inflammatory process. We determined the levels of serum MBL during leptospirosis infection. A double-antibody sandwich ELISA was used to detect the immunoreactive serum MBL. The ELISA plates were coated with monoclonal antibody to MBL and bound MBL or recombinant human MBL were detected by rabbit anti-human MBL serum. HRPO-conjugated goat anti-rabbit antibody was used for detection of the reaction. Two groups of patients seen at referral hospitals in Recife, PE, Brazil, were divided according to the year of infection, 2001 (N = 61 or 2002 (N = 57 and compared in terms of disease severity and levels of serum MBL. A group of healthy volunteers (N = 97 matched by age, gender, and ethnic background was used as control. Patients infected in 2001 had more severe outcomes than those infected in 2002, including jaundice, hemorrhage, respiratory alteration, and renal complication (P = 0.0009; chi-square test. The frequency of patients producing serum MBL >1000 ng/mL was higher in the 2001 group than in the 2002 and control groups (P < 0.01, suggesting an association of MBL level with disease severity. The involvement of MBL and genetic variation of the MBL2 gene should be further evaluated to establish the role of this lectin in the pathogenesis of leptospirosis.

  16. Recent improvements to Binding MOAD: a resource for protein–ligand binding affinities and structures

    Science.gov (United States)

    Ahmed, Aqeel; Smith, Richard D.; Clark, Jordan J.; Dunbar, James B.; Carlson, Heather A.

    2015-01-01

    For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein–ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23 269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. PMID:25378330

  17. Recent improvements to Binding MOAD: a resource for protein-ligand binding affinities and structures.

    Science.gov (United States)

    Ahmed, Aqeel; Smith, Richard D; Clark, Jordan J; Dunbar, James B; Carlson, Heather A

    2015-01-01

    For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein-ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23,269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Crystal structure of the botulinum neurotoxin type G binding domain: insight into cell surface binding.

    Science.gov (United States)

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R; Stevens, Raymond C

    2010-04-16

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-A X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent. Copyright (c) 2010. Published by Elsevier Ltd.

  19. Structure-function relationships of Na+, K+, ATP, or Mg2+ binding and energy transduction in Na,K-ATPase

    DEFF Research Database (Denmark)

    Jorgensen, Peter L.; Pedersen, Per Amstrup

    2000-01-01

    Na,K-ATPase; Mutagenesis; Na+ binding; K+ binding; Tl+ binding; Mg2+ binding; ATP binding; Cation binding site; Energy transduction......Na,K-ATPase; Mutagenesis; Na+ binding; K+ binding; Tl+ binding; Mg2+ binding; ATP binding; Cation binding site; Energy transduction...

  20. Alpine ski bindings and injuries. Current findings.

    Science.gov (United States)

    Natri, A; Beynnon, B D; Ettlinger, C F; Johnson, R J; Shealy, J E

    1999-07-01

    In spite of the fact that the overall incidence of alpine ski injuries has decreased during the last 25 years, the incidence of serious knee sprains usually involving the anterior cruciate ligament (ACL) has risen dramatically since the late 1970s. This trend runs counter to a dramatic reduction in lower leg injuries that began in the early 1970s and to date has lowered the risk of injury below the knee by almost 90%. One of the primary design objectives of modern ski boots and bindings has been to protect the skier from tibia and ankle fractures. So, in that sense, they have done an excellent job. However, despite advances in equipment design, modern ski bindings have not protected the knee from serious ligament trauma. At the present time, we are unaware of any binding design, settings or function that can protect both the knee and lower extremities from serious ligament sprains. No innovative change in binding design appears to be on the horizon that has the potential to reduce the risk of these severe knee injuries. Indeed, only 1 study has demonstrated a means to help reduce this risk of serious knee sprains, and this study involved education of skiers, not ski equipment. Despite the inability of bindings to reduce the risk of severe knee injuries there can be no doubt that improvement in ski bindings has been the most important factor in the marked reduction in incidence of lower leg and ankle injuries during the last 25 years. The authors strongly endorse the application of present International Standards Organisation (ISO) and American Society for Testing and Materials (ASTM) standards concerning mounting, setting and maintaining modern 'state of the art' bindings.

  1. Dissociation of binding and learning processes.

    Science.gov (United States)

    Moeller, Birte; Frings, Christian

    2017-11-01

    A single encounter of a stimulus together with a response can result in a short-lived association between the stimulus and the response [sometimes called an event file, see Hommel, Müsseler, Aschersleben, & Prinz, (2001) Behavioral and Brain Sciences, 24, 910-926]. The repetition of stimulus-response pairings typically results in longer lasting learning effects indicating stimulus-response associations (e.g., Logan & Etherton, (1994) Journal of Experimental Psychology: Learning, Memory, and Cognition, 20, 1022-1050]. An important question is whether or not what has been described as stimulus-response binding in action control research is actually identical with an early stage of incidental learning (e.g., binding might be seen as single-trial learning). Here, we present evidence that short-lived binding effects can be distinguished from learning of longer lasting stimulus-response associations. In two experiments, participants always responded to centrally presented target letters that were flanked by response irrelevant distractor letters. Experiment 1 varied whether distractors flanked targets on the horizontal or vertical axis. Binding effects were larger for a horizontal than for a vertical distractor-target configuration, while stimulus configuration did not influence incidental learning of longer lasting stimulus-response associations. In Experiment 2, the duration of the interval between response n - 1 and presentation of display n (500 ms vs. 2000 ms) had opposing influences on binding and learning effects. Both experiments indicate that modulating factors influence stimulus-response binding and incidental learning effects in different ways. We conclude that distinct underlying processes should be assumed for binding and incidental learning effects.

  2. Ebola Virus Binding to Tim-1 on T Lymphocytes Induces a Cytokine Storm.

    Science.gov (United States)

    Younan, Patrick; Iampietro, Mathieu; Nishida, Andrew; Ramanathan, Palaniappan; Santos, Rodrigo I; Dutta, Mukta; Lubaki, Ndongala Michel; Koup, Richard A; Katze, Michael G; Bukreyev, Alexander

    2017-09-26

    Ebola virus (EBOV) disease (EVD) results from an exacerbated immunological response that is highlighted by a burst in the production of inflammatory mediators known as a "cytokine storm." Previous reports have suggested that nonspecific activation of T lymphocytes may play a central role in this phenomenon. T-cell immunoglobulin and mucin domain-containing protein 1 (Tim-1) has recently been shown to interact with virion-associated phosphatidylserine to promote infection. Here, we demonstrate the central role of Tim-1 in EBOV pathogenesis, as Tim-1 -/- mice exhibited increased survival rates and reduced disease severity; surprisingly, only a limited decrease in viremia was detected. Tim-1 -/- mice exhibited a modified inflammatory response as evidenced by changes in serum cytokines and activation of T helper subsets. A series of in vitro assays based on the Tim-1 expression profile on T cells demonstrated that despite the apparent absence of detectable viral replication in T lymphocytes, EBOV directly binds to isolated T lymphocytes in a phosphatidylserine-Tim-1-dependent manner. Exposure to EBOV resulted in the rapid development of a CD4 Hi CD3 Low population, non-antigen-specific activation, and cytokine production. Transcriptome and Western blot analysis of EBOV-stimulated CD4 + T cells confirmed the induction of the Tim-1 signaling pathway. Furthermore, comparative analysis of transcriptome data and cytokine/chemokine analysis of supernatants highlight the similarities associated with EBOV-stimulated T cells and the onset of a cytokine storm. Flow cytometry revealed virtually exclusive binding and activation of central memory CD4 + T cells. These findings provide evidence for the role of Tim-1 in the induction of a cytokine storm phenomenon and the pathogenesis of EVD. IMPORTANCE Ebola virus infection is characterized by a massive release of inflammatory mediators, which has come to be known as a cytokine storm. The severity of the cytokine storm is

  3. CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

    Science.gov (United States)

    Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

    2017-09-01

    Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

  4. Protein Binding Capacity of Different Forages Tannin

    Science.gov (United States)

    Yusiati, L. M.; Kurniawati, A.; Hanim, C.; Anas, M. A.

    2018-02-01

    Eight forages of tannin sources(Leucaena leucocephala, Arachis hypogaea, Mimosa pudica, Morus alba L, Swietenia mahagoni, Manihot esculenta, Gliricidia sepium, and Bauhinia purpurea)were evaluated their tannin content and protein binding capacity. The protein binding capacity of tannin were determined using precipitation of bovine serum albumin (BSA). Swietenia mahagonihas higest total tannin level and condensed tannin (CT) compared with other forages (P<0.01). The Leucaena leucocephala has highest hydrolysable tannin (HT) level (P<0.01). The total and condensed tannin content of Swietenia mahagoni were 11.928±0.04 mg/100 mg and 9.241±0.02mg/100mg dry matter (DM) of leaves. The hydrolysable tannin content of Leucaena leucocephala was 5.338±0.03 mg/100 mg DM of leaves. Binding capacity was highest in Swietenia mahagoni and Leucaena leucocephala compared to the other forages (P<0.01). The optimum binding of BSA to tannin in Leucaena leucocephala and Swietenia mahagoniwere1.181±0.44 and 1.217±0.60mg/mg dry matter of leaves. The present study reports that Swietenia mahagoni has highest of tannin content and Leucaena leucocephala and Swietenia mahagoni capacity of protein binding.

  5. Adjustment of legally binding local plans

    DEFF Research Database (Denmark)

    Hvingel, Line Træholt; Aunsborg, Christian; Christensen, Finn Kjær

    2012-01-01

    Traditionally, and by law, new urban areas in Denmark are regulated and planned through legally binding local plans. Recently a tendency has occurred: The municipalities make the legally binding local plans quite open for future adjustment, and they are using a substantial amount of ‘empowerment ...... the considerations of legal rights, the extend of the legal use of empowerment provisions and the combination of the use of legal binding local plans and other legal instruments such as easements and sales agreements.......Traditionally, and by law, new urban areas in Denmark are regulated and planned through legally binding local plans. Recently a tendency has occurred: The municipalities make the legally binding local plans quite open for future adjustment, and they are using a substantial amount of ‘empowerment...... provisions’ which empower the municipalities to later ruling. This way of making plans postpones the actual regulation of an area (i.e. the planning permission) making it an individual ruling for instance at the application of building permits. Case studies show examples of this way of regulating an area...

  6. Assessment of the binding properties of granuloszint

    International Nuclear Information System (INIS)

    Schubiger, P.A.; Hasler, P.H.; Novak-Hofer, I.; Blaeuenstein, P.

    1989-01-01

    123 I-granuloszint (a murine monoclonal antibody - called AK-47 - against NCA-95 glycoprotein of granulocytes) has been proved to be a very convenient and successful radiopharmaceutical for visualizing infectious diseases. For a broad introduction in routine nuclear medicine it was necessary to optimize the labelling method and to determine in vitro exactly those biological and binding parameters which are relevant for an effective application in vivo. Binding to granulocytes has been shown to be specific and saturable (nonspecific binding about 10%) and is not via the Fc part of the antibody. The investigation of the binding properties of 125 I-labelled AK-47 gave the following results: Affinity constant 5x10 8 , 20,000-100,000 epitopes per granulocyte and an immunoreactivity of more than 90%. Labelling with 123 I reduced the immunoreactivity to 40%. The Lindmo method and immunoblotting are used as quality control to check the likely in vivo behaviour of the labelled antibody. There is a good correspondence between the results from the two methods. With our special labelling method and the different in vitro checks we have found a reliable way to control the production and to assure an optimal binding behaviour of 123 I-granuloszint. (orig.)

  7. Assessment of the binding properties of granuloszint

    Energy Technology Data Exchange (ETDEWEB)

    Schubiger, P.A.; Hasler, P.H.; Novak-Hofer, I.; Blaeuenstein, P.

    1989-09-01

    /sup 123/I-granuloszint (a murine monoclonal antibody - called AK-47 - against NCA-95 glycoprotein of granulocytes) has been proved to be a very convenient and successful radiopharmaceutical for visualizing infectious diseases. For a broad introduction in routine nuclear medicine it was necessary to optimize the labelling method and to determine in vitro exactly those biological and binding parameters which are relevant for an effective application in vivo. Binding to granulocytes has been shown to be specific and saturable (nonspecific binding about 10%) and is not via the Fc part of the antibody. The investigation of the binding properties of /sup 125/I-labelled AK-47 gave the following results: Affinity constant 5x10/sup 8/, 20,000-100,000 epitopes per granulocyte and an immunoreactivity of more than 90%. Labelling with /sup 123/I reduced the immunoreactivity to 40%. The Lindmo method and immunoblotting are used as quality control to check the likely in vivo behaviour of the labelled antibody. There is a good correspondence between the results from the two methods. With our special labelling method and the different in vitro checks we have found a reliable way to control the production and to assure an optimal binding behaviour of /sup 123/I-granuloszint. (orig.).

  8. New Mechanisms of Mercury Binding to Peat

    Science.gov (United States)

    Nagy, K. L.; Manceau, A.; Gasper, J. D.; Ryan, J. N.; Aiken, G. R.

    2007-12-01

    Mercury can be immobilized in the aquatic environment by binding to peat, a solid form of natural organic matter. Binding mechanisms can vary in strength and reversibility, and therefore will control concentrations of bioreactive mercury, may explain rates of mercury methylation, and are important for designing approaches to improve water quality using natural wetlands or engineered phytoremediation schemes. In addition, strong binding between mercury and peat is likely to result in the fixation of mercury that ultimately resides in coal. The mechanisms by which aqueous mercury at low concentrations reacts with both dissolved and solid natural organic matter remain incompletely understood, despite recent efforts. We have identified three distinct binding mechanisms of divalent cationic mercury to solid peats from the Florida Everglades using EXAFS spectroscopic data (FAME beamline, European Synchrotron Radiation Facility (ESRF)) obtained on experimental samples as compared to relevant references including mercury-bearing solids and mercury bound to various organic molecules. The proportions of the three molecular configurations vary with Hg concentration, and two new configurations that involve sulfur ligands occur at Hg concentrations up to about 4000 ppm. The binding mechanism at the lowest experimental Hg concentration (60-80 ppm) elucidates published reports on the inhibition of metacinnabar formation in the presence of Hg-bearing solutions and dissolved natural organic matter, and also, the differences in extent of mercury methylation in distinct areas of the Florida Everglades.

  9. Endocytosis of Integrin-Binding Human Picornaviruses

    Directory of Open Access Journals (Sweden)

    Pirjo Merilahti

    2012-01-01

    Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  10. Human serum albumin binding of certain antimalarials

    Science.gov (United States)

    Marković, Olivera S.; Cvijetić, Ilija N.; Zlatović, Mario V.; Opsenica, Igor M.; Konstantinović, Jelena M.; Terzić Jovanović, Nataša V.; Šolaja, Bogdan A.; Verbić, Tatjana Ž.

    2018-03-01

    Interactions between eight in-house synthesized aminoquinolines, along with well-known chloroquine, and human serum albumin (HSA) have been studied by fluorescence spectroscopy. The synthesized aminoquinolines, despite being structurally diverse, were found to be very potent antimalarials. Fluorescence measurements indicate that three compounds having additional thiophene or benzothiophene substructure bind more strongly to HSA than other studied compounds. Competitive binding experiments indicate that these three compounds bind significantly stronger to warfarin compared to diazepam binding site. Fluorescence quenching at three temperatures (20, 25, and 37 °C) was analyzed using classical Stern-Volmer equation, and a static quenching mechanism was proposed. The enthalpy and entropy changes upon sulphur-containing compound-HSA interactions were calculated using Van't Hoff equation. Positive values of enthalpy and entropy changes indicate that non-specific, hydrophobic interactions are the main contributors to HSA-compound interaction. Molecular docking and calculated lipophilicity descriptors indicate the same, pointing out that the increased lipophilicity of sulphur-containing compounds might be a reason for their better binding to HSA. Obtained results might contribute to design of novel derivatives with improved pharmacokinetic properties and drug efficacy.

  11. Binding of anandamide to bovine serum albumin

    DEFF Research Database (Denmark)

    Bojesen, I.N.; Hansen, Harald S.

    2003-01-01

    The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because...... a water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.3) in equilibrium...... that BSA has one high-affinity binding site for anandamide at all four temperatures. The free energy of anandamide binding (¿G) is calculated to -43.05 kJ mol with a large enthalpy (¿H ) contribution of -42.09 kJ mol. Anandamide has vasodilator activity, and the binding to albumin may mediate its transport...

  12. Binding of Fidarestat Stereoisomers with Aldose Reductase

    Directory of Open Access Journals (Sweden)

    Dae-Sil Lee

    2006-11-01

    Full Text Available The stereospecificity in binding to aldose reductase (ALR2 of two fidarestat {6-fluoro-2',5'-dioxospiro[chroman-4,4'-imidazolidine]-2-carboxamide} stereoisomers [(2S,4Sand (2R,4S] has been investigated by means of molecular dynamics simulations using freeenergy integration techniques. The difference in the free energy of binding was found to be2.0 ± 1.7 kJ/mol in favour of the (2S,4S-form, in agreement with the experimentalinhibition data. The relative mobilities of the fidarestats complexed with ALR2 indicate alarger entropic penalty for hydrophobic binding of (2R,4S-fidarestat compared to (2S,4S-fidarestat, partially explaining its lower binding affinity. The two stereoisomers differmainly in the orientation of the carbamoyl moiety with respect to the active site and rotationof the bond joining the carbamoyl substituent to the ring. The detailed structural andenergetic insights obtained from out simulations allow for a better understanding of thefactors determining stereospecific inhibitor-ALR2 binding in the EPF charges model.

  13. DNA binding studies of tartrazine food additive.

    Science.gov (United States)

    Kashanian, Soheila; Zeidali, Sahar Heidary

    2011-07-01

    The interaction of native calf thymus DNA with tartrazine in 10 mM Tris-HCl aqueous solution at neutral pH 7.4 was investigated. Tartrazine is a nitrous derivative and may cause allergic reactions, with a potential of toxicological risk. Also, tartrazine induces oxidative stress and DNA damage. Its DNA binding properties were studied by UV-vis and circular dichroism spectra, competitive binding with Hoechst 33258, and viscosity measurements. Tartrazine molecules bind to DNA via groove mode as illustrated by hyperchromism in the UV absorption band of tartrazine, decrease in Hoechst-DNA solution fluorescence, unchanged viscosity of DNA, and conformational changes such as conversion from B-like to C-like in the circular dichroism spectra of DNA. The binding constants (K(b)) of DNA with tartrazine were calculated at different temperatures. Enthalpy and entropy changes were calculated to be +37 and +213 kJ mol(-1), respectively, according to the Van't Hoff equation, which indicated that the reaction is predominantly entropically driven. Also, tartrazine does not cleave plasmid DNA. Tartrazine interacts with calf thymus DNA via a groove interaction mode with an intrinsic binding constant of 3.75 × 10(4) M(-1).

  14. Binding of ethidium to the nucleosome core particle. 2. Internal and external binding modes

    International Nuclear Information System (INIS)

    McMurray, C.T.; Small, E.W.; van Holde, K.E.

    1991-01-01

    The authors have previously reported that the binding of ethidium bromide to the nucleosome core particle results in a stepwise dissociation of the structure which involves the initial release of one copy each of H2A and H2B. In this report, they have examined the absorbance and fluorescence properties of intercalated and outside bound forms of ethidium bromide. From these properties, they have measured the extent of external, electrostatic binding of the dye versus internal, intercalation binding to the core particle, free from contribution by linker DNA. They have established that dissociation is induced by the intercalation mode of binding to DNA within the core particle DNA, and not by binding to the histones or by nonintercalative binding to DNA. The covalent binding of [ 3 H]-8-azidoethidium to the core particle clearly shows that < 1.0 adduct is formed per histone octamer over a wide range of input ratios. Simultaneously, analyses of steady-state fluorescence enhancement and fluorescence lifetime data from bound ethidium complexes demonstrate extensive intercalation binding. Combined analyses from steady-state fluorescence intensity with equilibrium dialysis or fluorescence lifetime data revealed that dissociation began when ∼14 ethidium molecules are bound by intercalation to each core particle and < 1.0 nonintercalated ion pair was formed per core particle

  15. The interrelationship between ligand binding and self-association of the folate binding protein

    DEFF Research Database (Denmark)

    Holm, Jan; Schou, Christian; Babol, Linnea N.

    2011-01-01

    The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of ...

  16. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models*

    Science.gov (United States)

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-01-01

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. PMID:26912662

  17. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models.

    Science.gov (United States)

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-05-06

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Dissanayake, V.U.; Hughes, J.; Hunter, J.C. (Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge (England))

    1991-07-01

    The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.

  19. Binding of alkylpyridinium chloride surfactants to sodium polystyrene sulfonate

    NARCIS (Netherlands)

    Ishiguro, M.; Koopal, L.K.

    2009-01-01

    Binding of cationic surfactants to anionic polymers is well studied. However, the surfactant binding characteristics at very low concentration near the start of binding and at high concentration, where charge compensation may Occur. are less well known. Therefore, the binding characteristics of

  20. Presence of a highly efficient binding to bacterial contamination can distort data from binding studies

    International Nuclear Information System (INIS)

    Balcar, V.J.

    1990-01-01

    3 HGABA at low concentrations (5-10 nM) was bound by what appeared to be a GABA receptor binding site in bacterial contamination originating from a batch of distilled water. Under experimental conditions similar to those usually employed in 3 HGABA binding studies, the apparent binding displayed a very high specific component and a high efficiency in terms of 3 HGABA bound per mg of protein. The binding was blocked by muscimol but not by isoguvacine, SR95531 and nipecotic acid. These characteristics suggest that the presence of such spurious binding in the experiments using 3H-labeled ligands in brain homogenates may not always be very obvious and, moreover, it can result in subtle, but serious, distortions of data from such studies, which may not be immediately recognized

  1. Receptor binding studies of the living heart

    International Nuclear Information System (INIS)

    Syrota, A.

    1988-01-01

    Receptors form a class of intrinsic membrane proteins (or glycoproteins) defined by the high affinity and specificity with which they bind ligands. Many receptors are associated directly or indirectly with membrane ion channels that open or close after a conformational change of the receptor induced by the binding of the neurotransmitter. Changes in number and/or affinity of cardiac neurotransmitter receptors have been associated with myocardial ischemia and infarction, congestive heart failure, and cardiomyopathy as well as diabetes or thyroid-induced heart muscle disease. These alterations of cardiac receptors have been demonstrated in vitro on membrane homogenates from samples collected mainly during surgery or postmortem. The disadvantage of these in vitro binding techniques is that receptors lose their natural environment and their relationships with the other components of the tissue

  2. The binding of Np to rat bone

    International Nuclear Information System (INIS)

    Ramounet, B.; Taylor, D.M.

    1997-01-01

    Neptunium has been shown to massively deposit in bone, after intravenous or intramuscular injections. Initially, it was uniformly distributed on periosteal and endosteal bone surfaces. The nature of the binding molecules, for this actinide, in the skeleton, has not yet been identified. The aim of this work was to characterize the ligands of neptunium by selective extractions of bone components. The preliminary results displayed the binding of 237 Np(IV) in the organic phase of bone, after intravenous or intramuscular contamination. Further studies are in progress, to quantify the fraction of Np bound to the organic and mineral compartment of bone, and to determine the affinity constant and the turn-over of the binding proteins. (authors)

  3. Dendrimers bind antioxidant polyphenols and cisplatin drug.

    Directory of Open Access Journals (Sweden)

    Amine Abderrezak

    Full Text Available Synthetic polymers of a specific shape and size play major role in drug delivery systems. Dendrimers are unique synthetic macromolecules of nanometer dimensions with a highly branched structure and globular shape with potential applications in gene and drug delivery. We examine the interaction of several dendrimers of different compositions mPEG-PAMAM (G3, mPEG-PAMAM (G4 and PAMAM (G4 with hydrophilic and hydrophobic drugs cisplatin, resveratrol, genistein and curcumin at physiological conditions. FTIR and UV-visible spectroscopic methods as well as molecular modeling were used to analyse drug binding mode, the binding constant and the effects of drug complexation on dendrimer stability and conformation. Structural analysis showed that cisplatin binds dendrimers in hydrophilic mode via Pt cation and polymer terminal NH(2 groups, while curcumin, genistein and resveratrol are located mainly in the cavities binding through both hydrophobic and hydrophilic contacts. The overall binding constants of durg-dendrimers are ranging from 10(2 M(-1 to 10(3 M(-1. The affinity of dendrimer binding was PAMAM-G4>mPEG-PAMAM-G4>mPEG-PAMAM-G3, while the order of drug-polymer stability was curcumin>cisplatin>genistein>resveratrol. Molecular modeling showed larger stability for genisten-PAMAM-G4 (ΔG = -4.75 kcal/mol than curcumin-PAMAM-G4 ((ΔG = -4.53 kcal/mol and resveratrol-PAMAM-G4 ((ΔG = -4.39 kcal/mol. Dendrimers might act as carriers to transport hydrophobic and hydrophilic drugs.

  4. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    Science.gov (United States)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  5. Binding energy of two-dimensional biexcitons

    DEFF Research Database (Denmark)

    Singh, Jai; Birkedal, Dan; Vadim, Lyssenko

    1996-01-01

    Using a model structure for a two-dimensional (2D) biexciton confined in a quantum well, it is shown that the form of the Hamiltonian of the 2D biexciton reduces into that of an exciton. The binding energies and Bohr radii of a 2D biexciton in its various internal energy states are derived...... analytically using the fractional dimension approach. The ratio of the binding energy of a 2D biexciton to that of a 2D exciton is found to be 0.228, which agrees very well with the recent experimental value. The results of our approach are compared with those of earlier theories....

  6. Binding energies of hypernuclei and hypernuclear interactions

    Energy Technology Data Exchange (ETDEWEB)

    Bodmer, A.R. [Argonne National Lab., IL (United States)]|[Univ. of Illinois, Chicago, IL (United States). Dept. of Physics; Murali, S.; Usmani, Q.N. [Jamia Millia Islamia, New Delhi (India). Dept. of Physics

    1996-05-01

    In part 1 the effect of nuclear core dynamics on the binding energies of {Lambda} hypernuclei is discussed in the framework of variational correlated wave functions. In particular, the authors discuss a new rearrangement energy contribution and its effect on the core polarization. In part 2 they consider the interpretation of the {Lambda} single-particle energy in terms of basic {Lambda}-nuclear interactions using a local density approximation based on a Fermi hypernetted chain calculation of the A binding to nuclear matter. To account for the data strongly repulsive 3-body {Lambda}NN forces are required. Also in this framework they discuss core polarization for medium and heavier hypernuclei.

  7. Binding energies of hypernuclei and hypernuclear interactions

    International Nuclear Information System (INIS)

    Bodmer, A.R.; Univ. of Illinois, Chicago, IL; Murali, S.; Usmani, Q.N.

    1996-01-01

    In part 1 the effect of nuclear core dynamics on the binding energies of Λ hypernuclei is discussed in the framework of variational correlated wave functions. In particular, the authors discuss a new rearrangement energy contribution and its effect on the core polarization. In part 2 they consider the interpretation of the Λ single-particle energy in terms of basic Λ-nuclear interactions using a local density approximation based on a Fermi hypernetted chain calculation of the A binding to nuclear matter. To account for the data strongly repulsive 3-body ΛNN forces are required. Also in this framework they discuss core polarization for medium and heavier hypernuclei

  8. Studies on folate binding and a radioassay for serum and whole blood folate using goat milk as binding agent

    International Nuclear Information System (INIS)

    Piyasena, R.D.; Weerasekera, D.A.; Hettiaratchi, N.; Wikramanayake, T.W.; Sri Lanka Univ., Peradeniya Campus. Nuclear Medicine Unit)

    1977-01-01

    Preparations of cow, goat, buffalo, and human milk in addition to pig plasma were tested for folate binding properties. Of these, only pig plasma and goat milk showed sufficient binding to enable use as binding agents in a radioassay for serum and whole blood folate. The binding of folate by cow mild preparations in particular was found to be very poor. (orig.) [de

  9. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Science.gov (United States)

    2011-01-01

    Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of

  10. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Directory of Open Access Journals (Sweden)

    Person Alexandra M

    2011-11-01

    Full Text Available Abstract Background Along with high affinity binding of epibatidine (Kd1≈10 pM to α4β2 nicotinic acetylcholine receptor (nAChR, low affinity binding of epibatidine (Kd2≈1-10 nM to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after

  11. RBPmap: a web server for mapping binding sites of RNA-binding proteins.

    Science.gov (United States)

    Paz, Inbal; Kosti, Idit; Ares, Manuel; Cline, Melissa; Mandel-Gutfreund, Yael

    2014-07-01

    Regulation of gene expression is executed in many cases by RNA-binding proteins (RBPs) that bind to mRNAs as well as to non-coding RNAs. RBPs recognize their RNA target via specific binding sites on the RNA. Predicting the binding sites of RBPs is known to be a major challenge. We present a new webserver, RBPmap, freely accessible through the website http://rbpmap.technion.ac.il/ for accurate prediction and mapping of RBP binding sites. RBPmap has been developed specifically for mapping RBPs in human, mouse and Drosophila melanogaster genomes, though it supports other organisms too. RBPmap enables the users to select motifs from a large database of experimentally defined motifs. In addition, users can provide any motif of interest, given as either a consensus or a PSSM. The algorithm for mapping the motifs is based on a Weighted-Rank approach, which considers the clustering propensity of the binding sites and the overall tendency of regulatory regions to be conserved. In addition, RBPmap incorporates a position-specific background model, designed uniquely for different genomic regions, such as splice sites, 5' and 3' UTRs, non-coding RNA and intergenic regions. RBPmap was tested on high-throughput RNA-binding experiments and was proved to be highly accurate. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Die dogmatiese binding van die prediking

    African Journals Online (AJOL)

    genoemde probleemstelling wat ons homileties na riglyne wil soek, sonder die pretensie dat finale oplossings gevind sal word. Om hierdie probleem te ontleed is dit nodig om eers kortliks 'n begripsbepaling van die begrippe dogma, binding en prediking te maak. Vanuit die begripsbepaling kan ons dan probeer vasstel wat ...

  13. Singular Value Decomposition and Ligand Binding Analysis

    Directory of Open Access Journals (Sweden)

    André Luiz Galo

    2013-01-01

    Full Text Available Singular values decomposition (SVD is one of the most important computations in linear algebra because of its vast application for data analysis. It is particularly useful for resolving problems involving least-squares minimization, the determination of matrix rank, and the solution of certain problems involving Euclidean norms. Such problems arise in the spectral analysis of ligand binding to macromolecule. Here, we present a spectral data analysis method using SVD (SVD analysis and nonlinear fitting to determine the binding characteristics of intercalating drugs to DNA. This methodology reduces noise and identifies distinct spectral species similar to traditional principal component analysis as well as fitting nonlinear binding parameters. We applied SVD analysis to investigate the interaction of actinomycin D and daunomycin with native DNA. This methodology does not require prior knowledge of ligand molar extinction coefficients (free and bound, which potentially limits binding analysis. Data are acquired simply by reconstructing the experimental data and by adjusting the product of deconvoluted matrices and the matrix of model coefficients determined by the Scatchard and McGee and von Hippel equation.

  14. Nucleic acids encoding a cellulose binding domain

    Science.gov (United States)

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1996-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  15. ALG-2, a multifunctional calcium binding protein?

    DEFF Research Database (Denmark)

    Tarabykina, Svetlana; Mollerup, Jens; Winding Gojkovic, P.

    2004-01-01

    ALG-2 was originally discovered as a pro-apoptotic protein in a genetic screen. Due to its ability to bind calcium with high affinity it was postulated to provide a link between the known effect of calcium in programmed cell death and the molecular death execution machinery. This review article...

  16. Tension-induced binding of semiflexible biopolymers

    International Nuclear Information System (INIS)

    Benetatos, Panayotis; Heydt, Alice von der; Zippelius, Annette

    2014-01-01

    We investigate theoretically the effect of polymer tension on the collective behavior of reversibly binding cross-links. For this purpose, we employ a model of two weakly bending wormlike chains aligned in parallel by a tensile force, with a sequence of inter-chain binding sites regularly spaced along the contours. Reversible cross-links attach and detach at the sites with an affinity controlled by a chemical potential. In a mean-field approach, we calculate the free energy of the system and find the emergence of a free-energy barrier which controls the reversible (un)binding. The tension affects the conformational entropy of the chains which competes with the binding energy of the cross-links. This competition gives rise to a sudden increase in the fraction of bound sites as the tension increases. We show that this transition is related to the cross-over between weak and strong localization of a directed polymer in a pinning potential. The cross-over to the strongly bound state can be interpreted as a mechanism for force-stiffening which exceeds the capabilities of single-chain elasticity and thus available only to reversibly cross-linked polymers. (paper)

  17. The Binding Properties of Quechua Suffixes.

    Science.gov (United States)

    Weber, David

    This paper sketches an explicitly non-lexicalist application of grammatical theory to Huallaga (Huanuco) Quechua (HgQ). The advantages of applying binding theory to many suffixes that have previously been treated only as objects of the morphology are demonstrated. After an introduction, section 2 outlines basic assumptions about the nature of HgQ…

  18. Asymmetric Aminalization via Cation-Binding Catalysis

    DEFF Research Database (Denmark)

    Park, Sang Yeon; Liu, Yidong; Oh, Joong Suk

    2018-01-01

    Asymmetric cation-binding catalysis, in principle, can generate "chiral" anionic nucleophiles, where the counter cations are coordinated within chiral environments. Nitrogen-nucleophiles are intrinsically basic, therefore, its use as nucleophiles is often challenging and limiting the scope of the...

  19. Tension-induced binding of semiflexible biopolymers

    Science.gov (United States)

    Benetatos, Panayotis; von der Heydt, Alice; Zippelius, Annette

    2015-03-01

    We investigate theoretically the effect of polymer tension on the collective behaviour of reversible cross-links. We use a model of two parallel-aligned, weakly-bending wormlike chains with a regularly spaced sequence of binding sites subjected to a tensile force. Reversible cross-links attach and detach at the binding sites with an affinity controlled by a chemical potential. In a mean-field approach, we calculate the free energy of the system and we show the emergence of a free energy barrier which controls the reversible (un)binding. The tension affects the conformational entropy of the chains which competes with the binding energy of the cross-links. This competition gives rise to a sudden increase in the fraction of bound sites as the polymer tension increases. The force-induced first-order transition in the number of cross-links implies a sudden force-induced stiffening of the effective stretching modulus of the polymers. This mechanism may be relevant to the formation and stress-induced strengthening of stress fibers in the cytoskeleton. We acknowledge support by the Deutsche Forschungsgemeinschaft (DFG) via grant SFB-937/A1.

  20. Plant ice-binding (antifreeze) proteins

    Science.gov (United States)

    Proteins that determine the temperature at which ice crystals will form in water-based solutions in cells and tissues, that bind to growing ice crystals, thus affecting their size, and that impact ice re-crystallization have been widely-documented and studied in many plant, bacterial, fungal, insect...

  1. The Double Bind: The next Generation

    Science.gov (United States)

    Malcom, Lindsey E.; Malcom, Shirley M.

    2011-01-01

    In this foreword, Shirley Malcom and Lindsey Malcom speak to the history and current status of women of color in science, technology, engineering, and mathematics (STEM) fields. As the author of the seminal report "The Double Bind: The Price of Being a Minority Woman in Science", Shirley Malcom is uniquely poised to give us an insightful…

  2. Binding of cationic surfactants to humic substances

    NARCIS (Netherlands)

    Ishiguro, M.; Tan, W.; Koopal, L.K.

    2007-01-01

    Commercial surfactants are introduced into the environment either through waste products or site-specific contamination. The amphiphilic nature of both surfactants and humic substances (HS) leads to their mutual attraction especially when surfactant and HS are oppositely charged. Binding of the

  3. Overlearned responses hinder S-R binding.

    Science.gov (United States)

    Moeller, Birte; Frings, Christian

    2017-01-01

    Two mechanisms that are important for human action control are the integration of individual action plans (see Hommel, Müsseler, Aschersleben, & Prinz, 2001) and the automatization of overlearned actions to familiar stimuli (see Logan, 1988). In the present study, we analyzed the influence of automatization on action plan integration. Integration with pronunciation responses were compared for response incompatible word and nonword stimuli. Stimulus-response binding effects were observed for nonwords. In contrast, words that automatically triggered an overlearned pronunciation response were not integrated with pronunciation of a different word. That is, automatized response retrieval hindered binding effects regarding the retrieving stimulus and a new response. The results are a first indication of the way that binding and learning processes interact, and might also be a first step to understanding the more complex interdependency of the processes responsible for stimulus-response binding in action control and stimulus-response associations in learning research. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

  4. Nucleotide binding to Na+/K+-ATPase

    Czech Academy of Sciences Publication Activity Database

    Kubala, Martin; Lánský, Zdeněk; Ettrich, R.; Plášek, J.; Teisinger, Jan; Amler, Evžen

    2005-01-01

    Roč. 272, č. S1 (2005), s. 191-191 E-ISSN 1742-4658. [FEBS Congress /30./ and IUBMB Conference /9./. 02.07.2005-07.07.2005, Budapest] Keywords : Na+/K+- ATPase * ATP binding * TNP-ATP Subject RIV: BO - Biophysics

  5. Ada To X-Window Bindings

    Science.gov (United States)

    Souleles, Dean

    1993-01-01

    Ada to X-Window Bindings computer program developed to provide Ada programmers with complete interfaces to Xt Intrinsics and OSF Motif toolkits. Provides "Ada view" of some mostly C-language programming libraries. Package of software written in Ada and C languages.

  6. Alkali binding in hydrated Portland cement paste

    NARCIS (Netherlands)

    Chen, Wei; Brouwers, Jos

    2010-01-01

    The alkali-binding capacity of C–S–H in hydrated Portland cement pastes is addressed in this study. The amount of bound alkalis in C–S–H is computed based on the alkali partition theories firstly proposed by Taylor (1987) and later further developed by Brouwers and Van Eijk (2003). Experimental data

  7. Non-binding relationship between visual features

    Directory of Open Access Journals (Sweden)

    Dragan eRangelov

    2014-10-01

    Full Text Available The answer as to how visual attributes processed in different brain loci at different speeds are bound together to give us our unitary experience of the visual world remains unknown. In this study we investigated whether bound representations arise, as commonly assumed, through physiological interactions between cells in the visual areas. In a focal attentional task in which correct responses from either bound or unbound representations were possible, participants discriminated the colour or orientation of briefly presented single bars. On the assumption that representations of the two attributes are bound, the accuracy of reporting the colour and orientation should co-vary. By contrast, if the attributes are not mandatorily bound, the accuracy of reporting the two attributes should be independent. The results of our psychophysical studies reported here supported the latter, non-binding, relationship between visual features, suggesting that binding does not necessarily occur even under focal attention. We propose a task-contingent binding mechanism, postulating that binding occurs at late, post-perceptual, stages through the intervention of memory.

  8. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

    2016-01-13

    Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

    Directory of Open Access Journals (Sweden)

    Waqasuddin Khan

    Full Text Available Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58.Next, we trained a bidirectional recurrent neural network (BRNN using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72 showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors.

  10. Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes

    DEFF Research Database (Denmark)

    Mandrup, S; Hummel, R; Ravn, S

    1992-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous mod...... have molecularly cloned and characterized the ACBP/DBI gene family in rat. The rat ACBP/DBI gene family comprises one expressed gene and four processed pseudogenes of which one was shown to exist in two allelic forms. The expressed gene is organized into four exons and three introns...

  11. Chromate Binding and Removal by the Molybdate-Binding Protein ModA.

    Science.gov (United States)

    Karpus, Jason; Bosscher, Michael; Ajiboye, Ifedayo; Zhang, Liang; He, Chuan

    2017-04-04

    Effective and cheap methods and techniques for the safe removal of hexavalent chromate from the environment are in increasingly high demand. High concentrations of hexavalent chromate have been shown to have numerous harmful effects on human biology. We show that the E. coli molybdate-binding protein ModA is a genetically encoded tool capable of removing chromate from aqueous solutions. Although previously reported to not bind chromate, we show that ModA binds chromate tightly and is capable of removing chromate to levels well below current US federal standards. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. A simple ligand-binding assay for thyroxine-binding globulin on reusable Sephadex columns

    International Nuclear Information System (INIS)

    Bastomsky, C.H.; Kalloo, H.; Frenkel-Leith, D.B.; McGill Univ., Montreal, Quebec

    1977-01-01

    A method for the assay of thyroxine-binding globulin on reusable Sephadex G-25 columns is described. It depends upon elution by diluted iodothyronine-free serum of protein-bound [ 125 I]thyroxine from the columns under conditions where binding to thyroxine-binding prealbumin and albumin are abolished. It is simple, rapid and precise, and permits determinations inlarge numbers of samples. Values (mg/l; mean +- S.D.) were: normals 31.6+-5.4, hyperthyroid 28.3+-4.8, hypothyroid 40.6+-7.5, oral contraceptives 40.1+-6.8, pregnant 50.3+-5.4, cirrhotics 20.7+-4.3. Concentrations were reduced in serum heated at 56degC, while the uptake of [ 125 I]triiodothyronine was increased. There was a significant negative correlation between thyroxine-binding globulin concentration and triiodothyronine uptake in the heated serum samples and in euthyroid subjects

  13. Multiple binding modes of ibuprofen in human serum albumin identified by absolute binding free energy calculations

    KAUST Repository

    Evoli, Stefania; Mobley, David L.; Guzzi, Rita; Rizzuti, Bruno

    2016-01-01

    experiments, because of the structural adaptability of this protein in accommodating small ligands. In this work, we provide a set of predictions covering the geometry, affinity of binding and protonation state for the pharmaceutically most active form (S

  14. The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

    Directory of Open Access Journals (Sweden)

    Nancy A Stearns

    Full Text Available Antibodies to DNA (anti-DNA are the serological hallmark of systemic lupus erythematosus (SLE and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3, hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

  15. Is there a link between selectivity and binding thermodynamics profiles?

    Science.gov (United States)

    Tarcsay, Ákos; Keserű, György M

    2015-01-01

    Thermodynamics of ligand binding is influenced by the interplay between enthalpy and entropy contributions of the binding event. The impact of these binding free energy components, however, is not limited to the primary target only. Here, we investigate the relationship between binding thermodynamics and selectivity profiles by combining publicly available data from broad off-target assay profiling and the corresponding thermodynamics measurements. Our analysis indicates that compounds binding their primary targets with higher entropy contributions tend to hit more off-targets compared with those ligands that demonstrated enthalpy-driven binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein.

    Science.gov (United States)

    Bertaccini, Edward J; Lindahl, Erik; Sixma, Titia; Trudell, James R

    2008-04-01

    Recent crystal structures of the acetylcholine binding protein (AChBP) have revealed surprisingly small structural alterations upon ligand binding. Here we investigate the extent to which ligand binding may affect receptor dynamics. AChBP is a homologue of the extracellular component of ligand-gated ion channels (LGICs). We have previously used an elastic network normal-mode analysis to propose a gating mechanism for the LGICs and to suggest the effects of various ligands on such motions. However, the difficulties with elastic network methods lie in their inability to account for the modest effects of a small ligand or mutation on ion channel motion. Here, we report the successful application of an elastic network normal mode technique to measure the effects of large ligand binding on receptor dynamics. The present calculations demonstrate a clear alteration in the native symmetric motions of a protein due to the presence of large protein cobratoxin ligands. In particular, normal-mode analysis revealed that cobratoxin binding to this protein significantly dampened the axially symmetric motion of the AChBP that may be associated with channel gating in the full nAChR. The results suggest that alterations in receptor dynamics could be a general feature of ligand binding.

  17. Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

    Directory of Open Access Journals (Sweden)

    Yoko Itakura

    2017-05-01

    Full Text Available Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine. Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations.

  18. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  19. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Directory of Open Access Journals (Sweden)

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

  20. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  1. Quantifying drug-protein binding in vivo

    International Nuclear Information System (INIS)

    Buchholz, B; Bench, G; Keating III, G; Palmblad, M; Vogel, J; Grant, P G; Hillegonds, D

    2004-01-01

    Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS

  2. RNA-Binding Proteins in Plant Immunity

    Directory of Open Access Journals (Sweden)

    Virginia Woloshen

    2011-01-01

    Full Text Available Plant defence responses against pathogen infection are crucial to plant survival. The high degree of regulation of plant immunity occurs both transcriptionally and posttranscriptionally. Once transcribed, target gene RNA must be processed prior to translation. This includes polyadenylation, 5′capping, editing, splicing, and mRNA export. RNA-binding proteins (RBPs have been implicated at each level of RNA processing. Previous research has primarily focused on structural RNA-binding proteins of yeast and mammals; however, more recent work has characterized a number of plant RBPs and revealed their roles in plant immune responses. This paper provides an update on the known functions of RBPs in plant immune response regulation. Future in-depth analysis of RBPs and other related players will unveil the sophisticated regulatory mechanisms of RNA processing during plant immune responses.

  3. Binding of 3H-iloprost to rat gastric mucosa: a pitfall in performing radioligand binding assays

    International Nuclear Information System (INIS)

    Beinborn, M.; Kromer, W.; Staar, U.; Sewing, K.F.

    1985-01-01

    Binding of 3 H-iloprost was studied in a 20,000 x g sediment of the rat gastric mucosa. When pH in both test tubes for total and non-specific binding was kept identical, no displaceable binding of iloprost could be detected. When no care was taken to keep the pH identical in corresponding test tubes of the binding assay, changes in pH simulated specific and displaceable binding of iloprost. Therefore it is concluded that - in contrast to earlier reports - it is not possible to demonstrate specific iloprost binding using the given method

  4. Surface Passivation in Empirical Tight Binding

    OpenAIRE

    He, Yu; Tan, Yaohua; Jiang, Zhengping; Povolotskyi, Michael; Klimeck, Gerhard; Kubis, Tillmann

    2015-01-01

    Empirical Tight Binding (TB) methods are widely used in atomistic device simulations. Existing TB methods to passivate dangling bonds fall into two categories: 1) Method that explicitly includes passivation atoms is limited to passivation with atoms and small molecules only. 2) Method that implicitly incorporates passivation does not distinguish passivation atom types. This work introduces an implicit passivation method that is applicable to any passivation scenario with appropriate parameter...

  5. Oxygen binding to partially nitrosylated hemoglobin.

    Science.gov (United States)

    Fago, Angela; Crumbliss, Alvin L; Hendrich, Michael P; Pearce, Linda L; Peterson, Jim; Henkens, Robert; Bonaventura, Celia

    2013-09-01

    Reactions of nitric oxide (NO) with hemoglobin (Hb) are important elements in protection against nitrosative damage. NO in the vasculature is depleted by the oxidative reaction with oxy Hb or by binding to deoxy Hb to generate partially nitrosylated Hb (Hb-NO). Many aspects of the formation and persistence of Hb-NO are yet to be clarified. In this study, we used a combination of EPR and visible absorption spectroscopy to investigate the interactions of partially nitrosylated Hb with O2. Partially nitrosylated Hb samples had predominantly hexacoordinate NO-heme geometry and resisted oxidation when exposed to O2 in the absence of anionic allosteric effectors. Faster oxidation occurred in the presence of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate (IHP), where the NO-heme derivatives had higher levels of pentacoordinate heme geometry. The anion-dependence of the NO-heme geometry also affected O2 binding equilibria. O2-binding curves of partially nitrosylated Hb in the absence of anions were left-shifted at low saturations, indicating destabilization of the low O2 affinity T-state of the Hb by increasing percentages of NO-heme, much as occurs with increasing levels of CO-heme. Samples containing IHP showed small decreases in O2 affinity, indicating shifts toward the low-affinity T-state and formation of inert α-NO/β-met tetramers. Most remarkably, O2-equilibria in the presence of the physiological effector DPG were essentially unchanged by up to 30% NO-heme in the samples. As will be discussed, under physiological conditions the interactions of Hb with NO provide protection against nitrosative damage without impairing O2 transport by Hb's unoccupied heme sites. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Predicting protein-binding RNA nucleotides with consideration of binding partners.

    Science.gov (United States)

    Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

    2015-06-01

    In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in

  7. Binding Energy and Equilibrium of Compact Objects

    Directory of Open Access Journals (Sweden)

    Germano M.

    2014-04-01

    Full Text Available The theoretical analysis of the existence of a limit mass for compact astronomic ob- jects requires the solution of the Einstein’s equations of g eneral relativity together with an appropriate equation of state. Analytical solutions exi st in some special cases like the spherically symmetric static object without energy sou rces that is here considered. Solutions, i.e. the spacetime metrics, can have a singular m athematical form (the so called Schwarzschild metric due to Hilbert or a nonsingula r form (original work of Schwarzschild. The former predicts a limit mass and, conse quently, the existence of black holes above this limit. Here it is shown that, the origi nal Schwarzschild met- ric permits compact objects, without mass limit, having rea sonable values for central density and pressure. The lack of a limit mass is also demonst rated analytically just imposing reasonable conditions on the energy-matter densi ty, of positivity and decreas- ing with radius. Finally the ratio between proper mass and to tal mass tends to 2 for high values of mass so that the binding energy reaches the lim it m (total mass seen by a distant observer. As it is known the negative binding energ y reduces the gravitational mass of the object; the limit of m for the binding energy provides a mechanism for stable equilibrium of any amount of mass to contrast the gravitatio nal collapse.

  8. What Happened to the IGF Binding Proteins?

    Science.gov (United States)

    Bach, Leon A

    2018-02-01

    Insulinlike growth factor (IGF) binding proteins (IGFBPs) 1 to 6 are high-affinity regulators of IGF activity. They generally inhibit IGF actions by preventing binding to the IGF-I receptor but can also enhance their actions under some conditions. Posttranslational modifications such as glycosylation and phosphorylation modulate IGFBP properties, and IGFBP proteolysis results in IGF release. IGFBPs have more recently been shown to have IGF-independent actions. A number of mechanisms are involved, including modulation of other growth factor pathways, nuclear localization and transcriptional regulation, interaction with the sphingolipid pathway, and binding to non-IGF biomolecules in the extracellular space and matrix, on the cell surface and intracellularly. IGFBPs modulate important biological processes, including cell proliferation, survival, migration, senescence, autophagy, and angiogenesis. Their actions have been implicated in growth, metabolism, cancer, stem cell maintenance and differentiation, and immune regulation. Recent studies have shown that epigenetic mechanisms are involved in the regulation of IGFBP abundance. A more complete understanding of IGFBP biology is necessary to further define their cellular roles and determine their therapeutic potential. Copyright © 2018 Endocrine Society.

  9. Photoaffinity labeling of the oxysterol binding protein

    International Nuclear Information System (INIS)

    Taylor, F.R.; Kandutsch, A.A.; Anzalone, L.; Spencer, T.A.

    1986-01-01

    A cytosolic receptor protein for oxygenated sterols, that is thought to be involved in the regulation of HMG-CoA reductase and cholesterol biosynthesis, can be labeled covalently by the photoactivated affinity compound [5,6- 3 H]-7,7'-azocholestane-3β,25-diol (I). Several other compounds were tested including 25-hydroxycholesta-4,6-dien-3-one, 25-azido-27-norcholest-5-en-3β-ol,3β,25-dihydroxycholest-5-en-7-one and 3β-hydroxycholesta-8(14),9(11)-dien-15-one. However, these sterols either did not bind to the receptor with adequate affinity or did not react covalently with the receptor during photolysis. Compound I binds to the receptor with very high affinity (K/sub d/ = 30 nM). After activation with long wavelength UV, two tritium labeled proteins, M/sub r/ approximately 95K and 65K daltons, are found upon SDS gel electrophoresis. No labeling occurs when the binding reaction is carried out in the presence of a large excess of 25-hydroxycholesterol. It is possible that the smaller polypeptide is a degradation product. Under the reaction conditions investigated so far labeling is relatively inefficient (< 1% of bound sterol). These results are generally consistent with previous information suggesting that the M/sub r/ of the receptor subunit is 97,000. Covalent labeling of the receptor should greatly facilitate its further purification and characterization

  10. Solid-Binding Peptides in Biomedicine.

    Science.gov (United States)

    Care, Andrew; Bergquist, Peter L; Sunna, Anwar

    2017-01-01

    Some peptides are able to bind to inorganic materials such as silica and gold. Over the past decade, Solid-binding peptides (SBPs) have been used increasingly as molecular building blocks in nanobiotechnology. These peptides show selectivity and bind with high affinity to a diverse range of inorganic surfaces e.g. metals, metal oxides, metal compounds, magnetic materials, semiconductors, carbon materials, polymers and minerals. They can be used in applications such as protein purification and synthesis, assembly and the functionalization of nanomaterials. They offer simple and versatile bioconjugation methods that can increase biocompatibility and also direct the immobilization and orientation of nanoscale entities onto solid supports without impeding their functionality. SBPs have been employed in numerous nanobiotechnological applications such as the controlled synthesis of nanomaterials and nanostructures, formation of hybrid biomaterials, immobilization of functional proteins and improved nanomaterial biocompatibility. With advances in nanotechnology, a multitude of novel nanomaterials have been designed and synthesized for diagnostic and therapeutic applications. New approaches have been developed recently to exert a greater control over bioconjugation and eventually, over the optimal and functional display of biomolecules on the surfaces of many types of solid materials. In this chapter we describe SBPs and highlight some selected examples of their potential applications in biomedicine.

  11. Metal ion binding with dehydroannulenes – Plausible two ...

    Indian Academy of Sciences (India)

    WINTEC

    Theoretical investigations have been carried out at B3LYP/6-311++G** level of theory to study the binding ... Alkali metals; dehydroannulenes; binding energy; penetration barrier. 1. .... can be discriminated from larger metal ions by running.

  12. Binding of tritiated corticosterone in brain sections of adrenalectomized rat

    International Nuclear Information System (INIS)

    Sarrieau, A.; Vial, M.; Dussaillant, M.; Rostene, W.; Philibert, P.

    1983-01-01

    A new technique which permits to study the specific binding of tritiated corticosterone in brain sections of adrenalectomized rats is described. Under these conditions, the specific binding of the glucocorticoid represents 60 to 70% of the initial binding. The apparent dissociation constant and the number of binding sites, determined by Scatchard analysis, are in the range of 10 -8 M and 100 fmoles/mg of protein respectively [fr

  13. Binding of Intrinsic and Extrinsic Features in Working Memory

    Science.gov (United States)

    Ecker, Ullrich K. H.; Maybery, Murray; Zimmer, Hubert D.

    2013-01-01

    There is ongoing debate concerning the mechanisms of feature binding in working memory. In particular, there is controversy regarding the extent to which these binding processes are automatic. The present article demonstrates that binding mechanisms differ depending on whether the to-be-integrated features are perceived as forming a coherent…

  14. Cobalamin and its binding protein in rat milk

    DEFF Research Database (Denmark)

    Raaberg, Lasse; Nexø, Ebba; Poulsen, Steen Seier

    1989-01-01

    Cobalamin and its binding protein, haptocorrin, are present in rat milk throughout the lactation period. The concentration of cobalamin is approximately 0.3-times the concentration of the unsaturated binding protein. The concentration of the unsaturated cobalamin-binding protein varies between 18...

  15. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  16. Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site

    International Nuclear Information System (INIS)

    Porter, M.A.; Hartman, F.C.

    1986-01-01

    The ATP-regulatory binding site of phosphoribulokinase was studied using bromoacetylethanolamine phosphate (BrAcNHEtOP). BrAcNHEtOP binds to the active-regulatory binding site of the protein. Following trypsin degradation of the labeled protein, fragments were separated by HPLC and sequenced. (DT)

  17. Atomistic fingerprint of hyaluronan-CD44 binding

    DEFF Research Database (Denmark)

    Vuorio, Joni; Vattulainen, Ilpo; Martinez-Seara, Hector

    2017-01-01

    that hyaluronan can bind CD44 with three topographically different binding modes that in unison define an interaction fingerprint, thus providing a plausible explanation for the disagreement between the earlier studies. Our results confirm that the known crystallographic mode is the strongest of the three binding...

  18. Fractionating the Binding Process: Neuropsychological Evidence from Reversed Search Efficiencies

    Science.gov (United States)

    Humphreys, Glyn W.; Hodsoll, John; Riddoch, M. Jane

    2009-01-01

    The authors present neuropsychological evidence distinguishing binding between form, color, and size (cross-domain binding) and binding between form elements. They contrasted conjunctive search with difficult feature search using control participants and patients with unilateral parietal or fronto/temporal lesions. To rule out effects of task…

  19. A sequential binding mechanism in a PDZ domain

    DEFF Research Database (Denmark)

    Chi, Celestine N; Bach, Anders; Engström, Åke

    2009-01-01

    that ligand binding involves at least a two-step process. By using an ultrarapid continuous-flow mixer, we then detected a hyperbolic dependence of binding rate constants on peptide concentration, corroborating the two-step binding mechanism. Furthermore, we found a similar dependence of the rate constants...

  20. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    International Nuclear Information System (INIS)

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E.

    1991-01-01

    Tritium-labeled α- and β-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10 degrees C, MBP bound α-maltose with 2.7 ± 0.5-fold higher affinity than β-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound α-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound β-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the β-maltodextrin is bound by its reducing end, and, in the other complex, the β-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins

  1. Central Role of Core Binding Factor β2 in Mucosa-Associated Lymphoid Tissue Organogenesis in Mouse.

    Science.gov (United States)

    Nagatake, Takahiro; Fukuyama, Satoshi; Sato, Shintaro; Okura, Hideaki; Tachibana, Masashi; Taniuchi, Ichiro; Ito, Kosei; Shimojou, Michiko; Matsumoto, Naomi; Suzuki, Hidehiko; Kunisawa, Jun; Kiyono, Hiroshi

    2015-01-01

    Mucosa-associated lymphoid tissue (MALT) is a group of secondary and organized lymphoid tissue that develops at different mucosal surfaces. Peyer's patches (PPs), nasopharynx-associated lymphoid tissue (NALT), and tear duct-associated lymphoid tissue (TALT) are representative MALT in the small intestine, nasal cavity, and lacrimal sac, respectively. A recent study has shown that transcriptional regulators of core binding factor (Cbf) β2 and promotor-1-transcribed Runt-related transcription factor 1 (P1-Runx1) are required for the differentiation of CD3-CD4+CD45+ lymphoid tissue inducer (LTi) cells, which initiate and trigger the developmental program of PPs, but the involvement of this pathway in NALT and TALT development remains to be elucidated. Here we report that Cbfβ2 plays an essential role in NALT and TALT development by regulating LTi cell trafficking to the NALT and TALT anlagens. Cbfβ2 was expressed in LTi cells in all three types of MALT examined. Indeed, similar to the previous finding for PPs, we found that Cbfβ2-/- mice lacked NALT and TALT lymphoid structures. However, in contrast to PPs, NALT and TALT developed normally in the absence of P1-Runx1 or other Runx family members such as Runx2 and Runx3. LTi cells for NALT and TALT differentiated normally but did not accumulate in the respective lymphoid tissue anlagens in Cbfβ2-/- mice. These findings demonstrate that Cbfβ2 is a central regulator of the MALT developmental program, but the dependency of Runx proteins on the lymphoid tissue development would differ among PPs, NALT, and TALT.

  2. Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

    Science.gov (United States)

    Chen, Junjie; van Dongen, Mallory A; Merzel, Rachel L; Dougherty, Casey A; Orr, Bradford G; Kanduluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M

    2016-03-14

    Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

  3. Cyclic AMP-Responsive Element-Binding Protein (CREB is Critical in Autoimmunity by Promoting Th17 but Inhibiting Treg Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Xiaohu Wang

    2017-11-01

    Full Text Available The molecular mechanisms that govern differential T cell development into pro-inflammatory Th17 vs. regulatory T (Treg cells remain unclear. Here, we show that selective deletion of CREB in T cells or Th17 cells impaired Th17 cell differentiation in vitro and in vivo, and led to resistance to autoimmune diseases. Mechanistically, CREB, activated by CD3-PKC-ϴ signaling, plays a key role in regulating Th17 cell differentiation, at least in part through directly binding to the Il17-Il17f gene locus. Unexpectedly, although dispensable for FOXP3 expression and for the homeostasis and suppressive function of thymus-derived Treg cells, CREB negatively regulates the survival of TGF-β-induced Treg cells, and deletion of CREB resulted in increased FOXP3+ Treg cells in the intestine and protection in a colitis model. Thus, CREB is critical in autoimmune diseases by promoting Th17 cell and inhibiting de novo Treg cell generation.

  4. LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

    Science.gov (United States)

    Marshall, J C; Shakespear, R A; Odell, W D

    1976-11-01

    Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

  5. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    OpenAIRE

    Bolton, Michael J; Garry, Robert F

    2011-01-01

    Abstract Background The HIV surface glycoprotein gp120 (SU, gp120) and the Plasmodium vivax Duffy binding protein (PvDBP) bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM). Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infectio...

  6. Autoradiographic localization of benzomorphan binding sites in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Crain, B.J.; Kwenjen Chang; McNamara, J.O.; Valdes, F.

    1985-07-17

    The benzomorphan subpopulation of opiate binding sites was labeled by (TH)diprenorphine in the presence of unlabeled ligands selected to quench and delta opiate binding sites. The distribution of benzomorphan binding sites was then localized autoradiographically. The distribution differs from the distributions of , delta and kappa opiate binding and is quite similar to the distribution of US -endorphin immunoreactivity. These observations support the hypothesis, based on biochemical studies in brain membranes, that benzomorphan binding sites may represent the ligand recognition sites of putative epsilon receptors. (Auth.). 34 refs.; 3 figs.

  7. How Arousal Affects Younger and Older Adults' Memory Binding

    Science.gov (United States)

    Nashiro, Kaoru; Mather, Mara

    2009-01-01

    A number of recent studies have shown that associative memory for within-item features is enhanced for emotionally arousing items, whereas arousal-enhanced binding is not seen for associations between distinct items (for a review see Mather, 2007). The costs and benefits of arousal in memory binding have been examined for younger adults but not for older adults. The present experiment examined whether arousal would enhance younger and older adults' within-item and between-item memory binding. The results revealed that arousal improved younger adults' within-item memory binding but not that of older adults. Arousal worsened both groups' between-item memory binding. PMID:21240821

  8. Study of plasma binding of receptor-specific peptides

    OpenAIRE

    Gregor, David

    2008-01-01

    The binding ability of two receptor specific peptides namely 90Y-DOTA-TATE and 111In-DOTA-TATE was studied in therm of interspecies comparison by the method of equilibrium dialysis. This plasma protein binding was different for the chosen animal species (human, rat, rabbit, bovine eventually pork) whereas binding of 90Y-DOTA- TATE was higher than binding of 111In-DOTA-TATE. KEYWORDS: Protein binding, radiofarmaceuticals, equilibrium dialysis, 90Y-DOTA-TATE, 111In- DOTA-TATE

  9. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12 binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

  10. Thyroxine binding to serum thyronine-binding globulin in thyroidectomized adult and normal neonatal rats

    International Nuclear Information System (INIS)

    Young, R.A.; Meyers, B.; Alex, S.; Fang, S.L.; Braverman, L.E.

    1988-01-01

    The amount of tracer [125I]T4 bound to serum thyronine-binding globulin (TBG) was measured by polyacrylamide gel electrophoresis in adult thyroidectomized (TX) rats and normal 1-day to 4-week-old rat puts. Thyroidectomy was associated with the appearance of significant amounts of [125I]T4 binding to serum TBG in lean rats, but not in obese Zucker rats. Treatment of the TX rats in vivo with replacement doses of T4 prevented this increase in TBG binding, but enrichment of serum from TX rats with T4 did not. Significant amounts of tracer [125I]T4 binding to TBG was present in serum from 1- to 3-week-old normal rat pups, but not in 1-day- or 4-week-old pups. There were significantly higher levels of TBG binding of [125I]T4 in serum from 2-week-old rat pups raised in litters of 16 pups compared to those raised in litters of 4 pups. All manipulations that result in the appearance of TBG in rat serum also result in either weight loss or a slowing in the rate of growth, suggesting that the appearance of TBG in rat serum has a nutritional component. This possibility is further supported by the observations that increases in TBG binding of [125I]T4 are not found in obese Zucker rats fed a low protein-high carbohydrate diet for 14 days or fasted for 7 days, or after thyroidectomy, perhaps owing to the large stores of fuel in the obese rat

  11. Human pentraxin 3 binds to the complement regulator c4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Anne Braunschweig

    Full Text Available The long pentraxin 3 (PTX3 is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP. A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.

  12. Antioxidant flavonoids bind human serum albumin

    Science.gov (United States)

    Kanakis, C. D.; Tarantilis, P. A.; Polissiou, M. G.; Diamantoglou, S.; Tajmir-Riahi, H. A.

    2006-10-01

    Human serum albumin (HSA) is a principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. Flavonoids are powerful antioxidants and prevent DNA damage. The antioxidative protections are related to their binding modes to DNA duplex and complexation with free radicals in vivo. However, flavonoids are known to inhibit the activities of several enzymes such as calcium phospholipid-dependent protein kinase, tyrosine protein kinase from rat lung, phosphorylase kinase, phosphatidylinositol 3-kinase and DNA topoisomerases that exhibit the importance of flavonoid-protein interaction. This study was designed to examine the interaction of human serum albumin (HSA) with quercetin (que), kaempferol (kae) and delphinidin (del) in aqueous solution at physiological conditions, using constant protein concentration of 0.25 mM (final) and various drug contents of 1 μM-1 mM. FTIR and UV-vis spectroscopic methods were used to determine the polyphenolic binding mode, the binding constant and the effects of flavonoid complexation on protein secondary structure. The spectroscopic results showed that flavonoids are located along the polypeptide chains through H-bonding interactions with overall affinity constant of Kque = 1.4 × 10 4 M -1, Kkae = 2.6 × 10 5 M -1 and Kdel = 4.71 × 10 5 M -1. The protein secondary structure showed no alterations at low pigment concentration (1 μM), whereas at high flavonoid content (1 mM), major reduction of α-helix from 55% (free HSA) to 42-46% and increase of β-sheet from 15% (free HSA) to 17-19% and β-anti from 7% (free HSA) to 10-20% occurred in the flavonoid-HSA adducts. The major reduction of HSA α-helix is indicative of a partial protein unfolding upon flavonoid interaction.

  13. Metal ion binding to iron oxides

    Science.gov (United States)

    Ponthieu, M.; Juillot, F.; Hiemstra, T.; van Riemsdijk, W. H.; Benedetti, M. F.

    2006-06-01

    The biogeochemistry of trace elements (TE) is largely dependent upon their interaction with heterogeneous ligands including metal oxides and hydrous oxides of iron. The modeling of TE interactions with iron oxides has been pursued using a variety of chemical models. The objective of this work is to show that it is possible to model the adsorption of protons and TE on a crystallized oxide (i.e., goethite) and on an amorphous oxide (HFO) in an identical way. Here, we use the CD-MUSIC approach in combination with valuable and reliable surface spectroscopy information about the nature of surface complexes of the TE. The other objective of this work is to obtain generic parameters to describe the binding of the following elements (Cd, Co, Cu, Ni, Pb, and Zn) onto both iron oxides for the CD-MUSIC approach. The results show that a consistent description of proton and metal ion binding is possible for goethite and HFO with the same set of model parameters. In general a good prediction of almost all the collected experimental data sets corresponding to metal ion binding to HFO is obtained. Moreover, dominant surface species are in agreement with the recently published surface complexes derived from X-ray absorption spectroscopy (XAS) data. Until more detailed information on the structure of the two iron oxides is available, the present option seems a reasonable approximation and can be used to describe complex geochemical systems. To improve our understanding and modeling of multi-component systems we need more data obtained at much lower metal ion to iron oxide ratios in order to be able to account eventually for sites that are not always characterized in spectroscopic studies.

  14. Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

    Science.gov (United States)

    Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

    2015-01-01

    Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

  15. DNS and BIND on IPv6

    CERN Document Server

    Liu, Cricket

    2011-01-01

    If you're preparing to roll out IPv6 on your network, this concise book provides the essentials you need to support this protocol with DNS. You'll learn how DNS was extended to accommodate IPv6 addresses, and how you can configure a BIND name server to run on the network. This book also features methods for troubleshooting problems with IPv6 forward- and reverse-mapping, and techniques for helping islands of IPv6 clients communicate with IPv4 resources. Topics include: DNS and IPv6-Learn the structure and representation of IPv6 addresses, and the syntaxes of AAAA and PTR records in the ip6.a

  16. Binding proteins of somatomedins and their functions

    International Nuclear Information System (INIS)

    Kostelecka, Z.; Blahovec, J.

    1998-01-01

    In this paper the functions of binding proteins are discussed. One variable that provides insulin-like growth factors (IGFs) control at the extracellular level is the presence of high-affinity, soluble insulin-like growth factor proteins (IGFBPs). IGFBP-1 inhibits IGF effect on human osteosarcoma cells. Increased concentration of IGFBP-3 inhibits the proliferation of breast cancer cell line MCF 7 either directly or by competition for IGF receptors. Maybe IGFBPs work as anti-mitogens and IGFs are potential promotors of cancer growth

  17. Defining Starch Binding by Glucan Phosphatases

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2015-01-01

    Starch is a vital energy molecule in plants that has a wide variety of uses in industry, such as feedstock for biomaterial processing and biofuel production. Plants employ a three enzyme cyclic process utilizing kinases, amylases, and phosphatases to degrade starch in a diurnal manner. Starch...... is comprised of the branched glucan amylopectin and the more linear glucan amylose. Our lab has determined the first structures of these glucan phosphatases and we have defined their enzymatic action. Despite this progress, we lacked a means to quickly and efficiently quantify starch binding to glucan...

  18. Apolipoprotein B is a calcium binding protein

    International Nuclear Information System (INIS)

    Dashti, N.; Lee, D.M.; Mok, T.

    1986-01-01

    Human hepatocarcinoma Hep G2 cells were grown in culture medium containing [ 45 Ca 2+ ]. The secreted lipoproteins of d 45 Ca] from the gels showed that the peak of radioactivity corresponded to the apolipoprotein B band. The molar ratio of the incorporated [ 45 Ca 2+ ] and apolipoprotein B was close to unity. No radioactivity was found associated with any other secreted apolipoproteins. To confirm these findings, apolipoprotein B-containing lipoproteins were precipitated with anti-apolipoprotein B and high density lipoproteins were precipitated with anti-apolipoprotein A-I. Only the former precipitate was radioactive. These results suggest that apolipoprotein B is a calcium binding protein

  19. Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization

    DEFF Research Database (Denmark)

    Kalamajski, Sebastian; Aspberg, Anders; Lindblom, Karin

    2009-01-01

    , but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin fragment...... biomineralization activity. We also show that asporin can be expressed in Escherichia coli (Rosetta-gami) with correctly positioned cysteine bridges, and a similar system can possibly be used for the expression of other SLRPs (small LRR proteoglycans/proteins)....

  20. A unique bivalent binding and inhibition mechanism by the yatapoxvirus interleukin 18 binding protein.

    Directory of Open Access Journals (Sweden)

    Brian Krumm

    Full Text Available Interleukin 18 (IL18 is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding

  1. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  2. Development of cholecystokinin binding sites in rat upper gastrointestinal tract

    International Nuclear Information System (INIS)

    Robinson, P.H.; Moran, T.H.; Goldrich, M.; McHugh, P.R.

    1987-01-01

    Autoradiography using 125 I-labeled Bolton Hunter-CCK-33 was used to study the distribution of cholecystokinin binding sites at different stages of development in the rat upper gastrointestinal tract. Cholecystokinin (CCK) binding was present in the distal stomach, esophagus, and gastroduodenal junction in the rat fetus of gestational age of 17 days. In the 20-day fetus, specific binding was found in the gastric mucosa, antral circular muscle, and pyloric sphincter. Mucosal binding declined during postnatal development and had disappeared by day 15. Antral binding declined sharply between day 10 and day 15 and disappeared by day 50. Pyloric muscle binding was present in fetal stomach and persisted in the adult. Pancreatic CCK binding was not observed before day 10. These results suggest that CCK may have a role in the control of gastric emptying and ingestive behavior in the neonatal rat

  3. Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

    Energy Technology Data Exchange (ETDEWEB)

    Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.; Mushrush, Darren J.; Pruitt, Rory N.; Spiller, Benjamin W.; Barbieri, Joseph T.; Lacy, D. Borden (Vanderbilt); (MCW)

    2010-10-19

    Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

  4. Lost time: Bindings do not represent temporal order information.

    Science.gov (United States)

    Moeller, Birte; Frings, Christian

    2018-06-04

    Many accounts of human action control assume bindings between features of stimuli and responses of individual events. One widely accepted assumption about these bindings is that they do not contain temporal-order representations regarding the integrated elements. Even though several theories either explicitly or implicitly include it, this assumption has never been tested directly. One reason for this lack of evidence is likely that typical stimulus-response binding paradigms are inapt for such a test. Adapting a new paradigm of response-response binding to include order switches between response integration and retrieval, we were able to analyze possible representation of order information in bindings for the first time. Binding effects were identical for intact and switched response orders, indicating that bindings indeed include no temporal-order information.

  5. Development of cholecystokinin binding sites in rat upper gastrointestinal tract

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, P.H.; Moran, T.H.; Goldrich, M.; McHugh, P.R.

    1987-04-01

    Autoradiography using /sup 125/I-labeled Bolton Hunter-CCK-33 was used to study the distribution of cholecystokinin binding sites at different stages of development in the rat upper gastrointestinal tract. Cholecystokinin (CCK) binding was present in the distal stomach, esophagus, and gastroduodenal junction in the rat fetus of gestational age of 17 days. In the 20-day fetus, specific binding was found in the gastric mucosa, antral circular muscle, and pyloric sphincter. Mucosal binding declined during postnatal development and had disappeared by day 15. Antral binding declined sharply between day 10 and day 15 and disappeared by day 50. Pyloric muscle binding was present in fetal stomach and persisted in the adult. Pancreatic CCK binding was not observed before day 10. These results suggest that CCK may have a role in the control of gastric emptying and ingestive behavior in the neonatal rat.

  6. Binding of Diphtheria Toxin to Phospholipids in Liposomes

    Science.gov (United States)

    Alving, Carl R.; Iglewski, Barbara H.; Urban, Katharine A.; Moss, Joel; Richards, Roberta L.; Sadoff, Jerald C.

    1980-04-01

    Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine / cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of

  7. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

    Science.gov (United States)

    Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  8. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

    Science.gov (United States)

    Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2016-01-15

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Knowledge-based Fragment Binding Prediction

    Science.gov (United States)

    Tang, Grace W.; Altman, Russ B.

    2014-01-01

    Target-based drug discovery must assess many drug-like compounds for potential activity. Focusing on low-molecular-weight compounds (fragments) can dramatically reduce the chemical search space. However, approaches for determining protein-fragment interactions have limitations. Experimental assays are time-consuming, expensive, and not always applicable. At the same time, computational approaches using physics-based methods have limited accuracy. With increasing high-resolution structural data for protein-ligand complexes, there is now an opportunity for data-driven approaches to fragment binding prediction. We present FragFEATURE, a machine learning approach to predict small molecule fragments preferred by a target protein structure. We first create a knowledge base of protein structural environments annotated with the small molecule substructures they bind. These substructures have low-molecular weight and serve as a proxy for fragments. FragFEATURE then compares the structural environments within a target protein to those in the knowledge base to retrieve statistically preferred fragments. It merges information across diverse ligands with shared substructures to generate predictions. Our results demonstrate FragFEATURE's ability to rediscover fragments corresponding to the ligand bound with 74% precision and 82% recall on average. For many protein targets, it identifies high scoring fragments that are substructures of known inhibitors. FragFEATURE thus predicts fragments that can serve as inputs to fragment-based drug design or serve as refinement criteria for creating target-specific compound libraries for experimental or computational screening. PMID:24762971

  10. Calculation of protein-ligand binding affinities.

    Science.gov (United States)

    Gilson, Michael K; Zhou, Huan-Xiang

    2007-01-01

    Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment.

  11. Specific binding assay technique; standardization of reagent

    International Nuclear Information System (INIS)

    Huggins, K.G.; Roitt, I.M.

    1979-01-01

    The standardization of a labelled constituent, such as anti-IgE, for use in a specific binding assay method is disclosed. A labelled ligand, such as IgE, is standardized against a ligand reference substance, such as WHO standard IgE, to determine the weight of IgE protein represented by the labelled ligand. Anti-light chain antibodies are contacted with varying concentrations of the labelled ligand. The ligand is then contacted with the labelled constituent which is then quantitated in relation to the amount of ligand protein present. The preparation of 131 I-labelled IgE is described. Also disclosed is an improved specific binding assay test method for determining the potency of an allergen extract in serum from an allergic individual. The improvement involved using a parallel model system of a second complex which consisted of anti-light chain antibodies, labelled ligand and the standardized labelled constituent (anti-IgE). The amount of standardized labelled constituent bound to the ligand in the first complex was determined, as described above, and the weight of ligand inhibited by addition of soluble allergen was then used as a measure of the potency of the allergen extract. (author)

  12. Understanding mercury binding on activated carbon

    Energy Technology Data Exchange (ETDEWEB)

    Padak, B.; Wilcox, J. [Stanford University, Stanford, CA (United States)

    2009-10-15

    Understanding the mechanism by which mercury adsorbs on activated carbon is crucial to the design and fabrication of effective capture technologies. In this study, the possible binding mechanism of mercury (Hg) and its species, i.e., HgCl and HgCl{sub 2} on activated carbon is investigated using ab initio-based energetic calculations. The activated carbon surface is modeled by a single graphene layer in which the edge atoms on the upper side are unsaturated in order to simulate the active sites. in some cases, chlorine atoms are placed at the edge sites to examine the effect of chlorine on the binding of Hg, HgCl and HgCl{sub 2}. It has been concluded that both HgCl and HgCl{sub 2} can be adsorbed dissociatively or non-dissociatively. In the case of dissociative adsorption, it is energetically favorable for atomic Hg to desorb and energetically favorable for it to remain on the surface in the Hg{sup 1+} state, HgCl. The Hg{sup 2+}, oxidized compound, HgCl2 was not found to be stable on the surface. The most probable mercury species on the surface was found to be HgCl.

  13. Reflection-Based Python-C++ Bindings

    International Nuclear Information System (INIS)

    Generowicz, Jacek; Lavrijsen, Wim T.L.P.; Marino, Massimo; Mato, Pere

    2004-01-01

    Python is a flexible, powerful, high-level language with excellent interactive and introspective capabilities and a very clean syntax. As such, it can be a very effective tool for driving physics analysis. Python is designed to be extensible in low-level C-like languages, and its use as a scientific steering language has become quite widespread. To this end, existing and custom-written C or C++ libraries are bound to the Python environment as so-called extension modules. A number of tools for easing the process of creating such bindings exist, such as SWIG and Boost. Python. Yet, the process still requires a considerable amount of effort and expertise. The C++ language has few built-in introspective capabilities, but tools such as LCGDict and CINT add this by providing so-called dictionaries: libraries that contain information about the names, entry points, argument types, etc. of other libraries. The reflection information from these dictionaries can be used for the creation of bindings and so the process can be fully automated, as dictionaries are already provided for many end-user libraries for other purposes, such as object persistency. PyLCGDict is a Python extension module that uses LCG dictionaries, as PyROOT uses CINT reflection information, to allow /cwPython users to access C++ libraries with essentially no preparation on the users' behalf. In addition, and in a similar way, PyROOT gives ROOT users access to Python libraries

  14. Electrochemical binding and wiring in battery materials

    Energy Technology Data Exchange (ETDEWEB)

    Pejovnik, S. [National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana (Slovenia); Faculty of Chemistry and Chemical Technology, Askerceva 5, SI-1000 Ljubljana (Slovenia); Dominko, R.; Bele, M.; Gaberscek, M.; Jamnik, J. [National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana (Slovenia)

    2008-10-01

    Binders in battery electrodes not only provide mechanical cohesiveness during battery operation but can also affect the electrode properties via the surface modification. Using atomic force microscopy (AFM), we study the surface structuring of three binders: polyvinylidene fluoride (PVdF), carboxymethyl cellulose (CMC) and gelatin. We try to find correlation between the observed structures and the measured electrochemical charge-discharge characteristics. We further measure the binding ability of gelatin adsorbed from solutions of different pHs. While the best binding ability of gelatin is obtained at pH about 9, the least polarization is observed at pH 12. Both properties are explained based on the observed gelatin structuring as a function of pH. In the second part of this study, gelatin is used as a surface agent that dictates the organization of nanometre-sized carbon black particles around micrometre-sized cathodic active particles. Using microcontact impedance measurements on polished pellets we show that using gelatin-forced carbon black deposition the average electronic resistance around LiMn{sub 2}O{sub 4} particles is decreased by more than two orders of magnitude. We believe that it is this decrease in resistance that improves significantly the rate performance of various cathode materials, such as LiMn{sub 2}O{sub 4} and LiCoO{sub 2}. (author)

  15. Penicillin-binding proteins in Actinobacteria.

    Science.gov (United States)

    Ogawara, Hiroshi

    2015-04-01

    Because some Actinobacteria, especially Streptomyces species, are β-lactam-producing bacteria, they have to have some self-resistant mechanism. The β-lactam biosynthetic gene clusters include genes for β-lactamases and penicillin-binding proteins (PBPs), suggesting that these are involved in self-resistance. However, direct evidence for the involvement of β-lactamases does not exist at the present time. Instead, phylogenetic analysis revealed that PBPs in Streptomyces are distinct in that Streptomyces species have much more PBPs than other Actinobacteria, and that two to three pairs of similar PBPs are present in most Streptomyces species examined. Some of these PBPs bind benzylpenicillin with very low affinity and are highly similar in their amino-acid sequences. Furthermore, other low-affinity PBPs such as SCLAV_4179 in Streptomyces clavuligerus, a β-lactam-producing Actinobacterium, may strengthen further the self-resistance against β-lactams. This review discusses the role of PBPs in resistance to benzylpenicillin in Streptomyces belonging to Actinobacteria.

  16. Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

    Science.gov (United States)

    Oshiro, Satoshi; Honda, Shinya

    2014-04-18

    Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

  17. Palmitate and stearate binding to human serum albumin. Determination of relative binding constants

    DEFF Research Database (Denmark)

    Vorum, H; Fisker, K; Honoré, B

    1997-01-01

    Multiple binding equilibria of two apparently insoluble ligands, palmitate and stearate, to defatted human serum albumin were studied in a 66 mM sodium phosphate buffer (pH 7.4) at 37 degrees C, by determination of dialytic exchange rates of ligands among identical equilibrium solutions. The expe...

  18. Molecular aspects of herbicide binding in chloroplasts = [Molekulaire aspekten van herbicide binding in chloroplasten

    NARCIS (Netherlands)

    Naber, D.

    1989-01-01

    Many weed-controlling agents act by inhibiting the process of photosynthesis. Their mode of action is a displacement of the secondary quinone electron acceptor of photosystem II from its proteinaceous binding environment. This results in a blocking of the electron transport. Consequently

  19. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    Science.gov (United States)

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  20. Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein

    DEFF Research Database (Denmark)

    Rolf, B; Oudenampsen-Krüger, E; Börchers, T

    1995-01-01

    The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated...

  1. RNA binding and replication by the poliovirus RNA polymerase

    International Nuclear Information System (INIS)

    Oberste, M.S.

    1988-01-01

    RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to 32 P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K a for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 x 10 9 M -1 . The polymerase binds to a subgenomic RNAs which contain the 3' end of the genome with a K a similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3' noncoding region

  2. Correcting binding parameters for interacting ligand-lattice systems

    Science.gov (United States)

    Hervy, Jordan; Bicout, Dominique J.

    2017-07-01

    Binding of ligands to macromolecules is central to many functional and regulatory biological processes. Key parameters characterizing ligand-macromolecule interactions are the stoichiometry, inducing the number of ligands per macromolecule binding site, and the dissociation constant, quantifying the ligand-binding site affinity. Both these parameters can be obtained from analyses of classical saturation experiments using the standard binding equation that offers the great advantage of mathematical simplicity but becomes an approximation for situations of interest when a ligand binds and covers more than one single binding site on the macromolecule. Using the framework of car-parking problem with latticelike macromolecules where each ligand can cover simultaneously several consecutive binding sites, we showed that employing the standard analysis leads to underestimation of binding parameters, i.e., ligands appear larger than they actually are and their affinity is also greater than it is. Therefore, we have derived expressions allowing to determine the ligand size and true binding parameters (stoichiometry and dissociation constant) as a function of apparent binding parameters retrieved from standard saturation experiments.

  3. Cation binding at the node of Ranvier: I. Localization of binding sites during development.

    Science.gov (United States)

    Zagoren, J C; Raine, C S; Suzuki, K

    1982-06-17

    Cations are known to bind to the node of Ranvier and the paranodal regions of myelinated fibers. The integrity of these specialized structures is essential for normal conduction. Sites of cation binding can be microscopically identified by the electrondense histochemical reaction product formed by the precipitate of copper sulfate/potassium ferrocyanide. This technique was used to study the distribution of cation binding during normal development of myelinating fibers. Sciatic nerves of C57B1 mice, at 1, 3, 5, 6, 7, 8, 9, 13, 16, 18, 24 and 30 days of age, were prepared for electron microscopy following fixation in phosphate-buffered 2.5% glutaraldehyde and 1% osmic acid, microdissection and incubation in phosphate-buffered 0.1 M cupric sulfate followed by 0.1 M potassium ferrocyanide. Localization of reaction product was studied by light and electron microscopy. By light microscopy, no reaction product was observed prior to 9 days of age. At 13 days, a few nodes and paranodes exhibited reaction product. This increased in frequency and intensity up to 30 days when almost all nodes or paranodes exhibited reaction product. Ultrastructurally, diffuse reaction product was first observed at 3 days of age in the axoplasm of the node, in the paranodal extracellular space of the terminal loops, in the Schwann cell proper and in the terminal loops of Schwann cell cytoplasm. When myelinated axons fulfilled the criteria for mature nodes, reaction product was no longer observed in the Schwann cell cytoplasm, while the intensity of reaction product in the nodal axoplasm and paranodal extracellular space of the terminal loops increased. Reaction product in the latter site appeared to be interrupted by the transverse bands. These results suggest that cation binding accompanies nodal maturity and that the Schwann cell may play a role in production or storage of the cation binding substance during myelinogenesis and development.

  4. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G.; Ribeiro, José M. C.; Andersen, John F.

    2017-07-27

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  5. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone.

    Science.gov (United States)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G; Ribeiro, José M C; Andersen, John F

    2017-09-15

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes , Culex , and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10 R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  6. An Electrostatic Funnel in the GABA-Binding Pathway.

    Directory of Open Access Journals (Sweden)

    Timothy S Carpenter

    2016-04-01

    Full Text Available The γ-aminobutyric acid type A receptor (GABAA-R is a major inhibitory neuroreceptor that is activated by the binding of GABA. The structure of the GABAA-R is well characterized, and many of the binding site residues have been identified. However, most of these residues are obscured behind the C-loop that acts as a cover to the binding site. Thus, the mechanism by which the GABA molecule recognizes the binding site, and the pathway it takes to enter the binding site are both unclear. Through the completion and detailed analysis of 100 short, unbiased, independent molecular dynamics simulations, we have investigated this phenomenon of GABA entering the binding site. In each system, GABA was placed quasi-randomly near the binding site of a GABAA-R homology model, and atomistic simulations were carried out to observe the behavior of the GABA molecules. GABA fully entered the binding site in 19 of the 100 simulations. The pathway taken by these molecules was consistent and non-random; the GABA molecules approach the binding site from below, before passing up behind the C-loop and into the binding site. This binding pathway is driven by long-range electrostatic interactions, whereby the electrostatic field acts as a 'funnel' that sweeps the GABA molecules towards the binding site, at which point more specific atomic interactions take over. These findings define a nuanced mechanism whereby the GABAA-R uses the general zwitterionic features of the GABA molecule to identify a potential ligand some 2 nm away from the binding site.

  7. Gonadal cell surface receptor for plasma retinol-binding protein

    International Nuclear Information System (INIS)

    Krishna Bhat, M.; Cama, H.R.

    1979-01-01

    A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

  8. Oxygen binding to nitric oxide marked hemoglobin

    International Nuclear Information System (INIS)

    Louro, S.R.W.; Ribeiro, P.C.; Bemski, G.

    1979-04-01

    Electron spin resonance spectra of organic phosphate free human hemoglobin marked with nitric oxide at the sixth coordination position of one of the four hemes allow to observe the transition from the tense (T) to the relaxed (R) conformation, as a function of parcial oxygen pressure. The spectra are composites of contributions from α sub(T), α sub(R) and β chains spectra, showing the presence of only two conformations: T and R. In the absence of organic phosphates NO binds to α and β chains with the same probability, but in the presence of phosphates NO combines preferentially with α chains. The dissociation of NO proceeds at least an order of magnitude faster in T than in R configuration. (author) [pt

  9. Surface Passivation in Empirical Tight Binding

    Science.gov (United States)

    He, Yu; Tan, Yaohua; Jiang, Zhengping; Povolotskyi, Michael; Klimeck, Gerhard; Kubis, Tillmann

    2016-03-01

    Empirical Tight Binding (TB) methods are widely used in atomistic device simulations. Existing TB methods to passivate dangling bonds fall into two categories: 1) Method that explicitly includes passivation atoms is limited to passivation with atoms and small molecules only. 2) Method that implicitly incorporates passivation does not distinguish passivation atom types. This work introduces an implicit passivation method that is applicable to any passivation scenario with appropriate parameters. This method is applied to a Si quantum well and a Si ultra-thin body transistor oxidized with SiO2 in several oxidation configurations. Comparison with ab-initio results and experiments verifies the presented method. Oxidation configurations that severely hamper the transistor performance are identified. It is also shown that the commonly used implicit H atom passivation overestimates the transistor performance.

  10. Binding of Serotonin to Lipid Membranes

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Wang, Chunhua; Cruys-Bagger, Nicolaj

    2013-01-01

    Serotonin (5-hydroxytryptamine, 5-HT) is a prevalent neurotransmitter throughout the animal kingdom. It exerts its effect through the specific binding to the serotonin receptor, but recent research has suggested that neural transmission may also be affected by its nonspecific interactions...... with the lipid matrix of the synaptic membrane. However, membrane–5-HT interactions remain controversial and superficially investigated. Fundamental knowledge of this interaction appears vital in discussions of putative roles of 5-HT, and we have addressed this by thermodynamic measurements and molecular...... dynamics (MD) simulations. 5-HT was found to interact strongly with lipid bilayers (partitioning coefficient ∼1200 in mole fraction units), and this is highly unusual for a hydrophilic solute like 5-HT which has a bulk, oil–water partitioning coefficient well below unity. It follows that membrane affinity...

  11. First calculation of the deuteron binding energy

    International Nuclear Information System (INIS)

    Schaegger, B.

    2012-01-01

    No universal constant characterizing the nuclear force has yet been found as for gravity and electromagnetism. The neutron is globally neutral with a zero net charge. The charges contained in a neutron may be separated by the electric field of a nearby proton and therefore being attracted by electrostatic induction in the same way as a rubbed plastic pen attracts small pieces of paper. There is also a magnetic force that may repel the nucleons like magnets in the proper relative orientation. In the deuteron, the heavy hydrogen nucleus, the induced electrostatic attraction is equilibrated by the magnetic repulsion between the opposite and colinear moments of the nucleons. Equilibrium is calculated by minimizing the electromagnetic interaction potential, giving a binding energy of 1.6 MeV, not much lower than the experimental value, 2.2 MeV. No fitting parameter is used: it is a true ab initio calculation

  12. Updating of working memory: lingering bindings.

    Science.gov (United States)

    Oberauer, Klaus; Vockenberg, Kerstin

    2009-05-01

    Three experiments investigated proactive interference and proactive facilitation in a memory-updating paradigm. Participants remembered several letters or spatial patterns, distinguished by their spatial positions, and updated them by new stimuli up to 20 times per trial. Self-paced updating times were shorter when an item previously remembered and then replaced reappeared in the same location than when it reappeared in a different location. This effect demonstrates residual memory for no-longer-relevant bindings of items to locations. The effect increased with the number of items to be remembered. With one exception, updating times did not increase, and recall of final values did not decrease, over successive updating steps, thus providing little evidence for proactive interference building up cumulatively.

  13. A Cationic Smart Copolymer for DNA Binding

    Directory of Open Access Journals (Sweden)

    Tânia Ribeiro

    2017-11-01

    Full Text Available A new block copolymer with a temperature-responsive block and a cationic block was prepared by reversible addition-fragmentation chain transfer (RAFT polymerization, with good control of its size and composition. The first block is composed by di(ethylene glycol methyl ether methacrylate (DEGMA and oligo(ethylene glycol methyl ether methacrylate (OEGMA, with the ratio DEGMA/OEGMA being used to choose the volume phase transition temperature of the polymer in water, tunable from ca. 25 to above 90 °C. The second block, of trimethyl-2-methacroyloxyethylammonium chloride (TMEC, is positively charged at physiological pH values and is used for DNA binding. The coacervate complexes between the block copolymer and a model single strand DNA are characterized by fluorescence correlation spectroscopy and fluorescence spectroscopy. The new materials offer good prospects for biomedical application, for example in controlled gene delivery.

  14. DNA binding and aggregation by carbon nanoparticles

    International Nuclear Information System (INIS)

    An, Hongjie; Liu, Qingdai; Ji, Qiaoli; Jin, Bo

    2010-01-01

    Significant environmental and health risks due to the increasing applications of engineered nanoparticles in medical and industrial activities have been concerned by many communities. The interactions between nanomaterials and genomes have been poorly studied so far. This study examined interactions of DNA with carbon nanoparticles (CNP) using atomic force microscopy (AFM). We experimentally assessed how CNP affect DNA molecule and bacterial growth of Escherichia coli. We found that CNP were bound to the DNA molecules during the DNA replication in vivo. The results revealed that the interaction of DNA with CNP resulted in DNA molecule binding and aggregation both in vivo and in vitro in a dose-dependent manner, and consequently inhabiting the E. coli growth. While this was a preliminary study, our results showed that this nanoparticle may have a significant impact on genomic activities.

  15. DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues.

    Science.gov (United States)

    Ma, Xin; Guo, Jing; Sun, Xiao

    2016-01-01

    DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.

  16. Heavy metals binding properties of esterified lemon

    Energy Technology Data Exchange (ETDEWEB)

    Arslanoglu, Hasan; Altundogan, Hamdi Soner [Department of Chemical Engineering, Firat University, 23279 Elazig (Turkey); Tumen, Fikret, E-mail: ftumen@firat.edu.tr [Department of Chemical Engineering, Firat University, 23279 Elazig (Turkey)

    2009-05-30

    Sorption of Cd{sup 2+}, Cr{sup 3+}, Cu{sup 2+}, Ni{sup 2+}, Pb{sup 2+} and Zn{sup 2+} onto a carboxyl groups-rich material prepared from lemon was investigated in batch systems. The results revealed that the sorption is highly pH dependent. Sorption kinetic data indicated that the equilibrium was achieved in the range of 30-240 min for different metal ions and sorption kinetics followed the pseudo-second-order model for all metals studied. Relative sorption rate of various metal cations was found to be in the general order of Ni{sup 2+} > Cd{sup 2+} > Cu{sup 2+} > Pb{sup 2+} > Zn{sup 2+} > Cr{sup 3+}. The binding characteristics of the sorbent for heavy metal ions were analyzed under various conditions and isotherm data was accurately fitted to the Langmuir equation. The metal binding capacity order calculated from Langmuir isotherm was Pb{sup 2+} > Cu{sup 2+} > Ni{sup 2+} > Cd{sup 2+} > Zn{sup 2+} > Cr{sup 3+}. The mean free energy of metal sorption process calculated from Dubinin-Radushkevich parameter and the Polanyi potential was found to be in the range of 8-11 kJ mol{sup -1} for the metals studied showing that the main mechanism governing the sorption process seems to be ion exchange. The basic thermodynamic parameters of metals ion sorption process were calculated by using the Langmuir constants obtained from equilibration study. The {Delta}G{sup o} and {Delta}H{sup o} values for metals ion sorption on the lemon sorbent showed the process to be spontaneous and exothermic in nature. Relatively low {Delta}H{sup o} values revealed that physical adsorption significantly contributed to the mechanism.

  17. Plant RNA binding proteins for control of RNA virus infection

    Directory of Open Access Journals (Sweden)

    Sung Un eHuh

    2013-12-01

    Full Text Available Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppresses RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering.

  18. Hardware device to physical structure binding and authentication

    Science.gov (United States)

    Hamlet, Jason R.; Stein, David J.; Bauer, Todd M.

    2013-08-20

    Detection and deterrence of device tampering and subversion may be achieved by including a cryptographic fingerprint unit within a hardware device for authenticating a binding of the hardware device and a physical structure. The cryptographic fingerprint unit includes an internal physically unclonable function ("PUF") circuit disposed in or on the hardware device, which generate an internal PUF value. Binding logic is coupled to receive the internal PUF value, as well as an external PUF value associated with the physical structure, and generates a binding PUF value, which represents the binding of the hardware device and the physical structure. The cryptographic fingerprint unit also includes a cryptographic unit that uses the binding PUF value to allow a challenger to authenticate the binding.

  19. Specific binding of beta-endorphin to normal human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chenet, B.; Hollis, V. Jr.; Kang, Y.; Simpkins, C.

    1986-03-05

    Beta-endorphin (BE) exhibits peripheral functions which may not be mediated by interactions with receptors in the brain. Recent studies have demonstrated binding of BE to both opioid and non-opioid receptors on lymphocytes and monocytes. Abood has reported specific binding of /sup 3/H-dihydromorphine in erythrocytes. Using 5 x 10/sup -11/M /sup 125/I-beta-endorphin and 10/sup -5/M unlabeled BE, they have detected 50% specific binding to human erythrocytes. This finding is supported by results from immunoelectron microscopy using rabbit anti-BE antibody and biotinylated secondary antibody with avidin-biotin complexes horseradish peroxidase. Binding is clearly observed and is confined to only one side of the cells. Conclusions: (1) BE binding to human erythrocytes was demonstrated by radioreceptor assay and immunoelectron microscopy, and (2) BE binding sites exist on only one side of the cells.

  20. Partial characterization of GTP-binding proteins in Neurospora

    International Nuclear Information System (INIS)

    Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

    1987-01-01

    Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [ 35 S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [ 35 S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin

  1. [Modification of retinal photoreceptor membranes and Ca ion binding].

    Science.gov (United States)

    Korchagin, V P; Berman, A L; Shukoliukov, S A; Rychkova, M P; Etingof, R N

    1978-10-01

    Calcium binding by modified photoreceptor membranes of cattle retina has been studied. Ca2+-binding the membranes significantly changes after C-phospholipase treatment, displaying the initial growth (less than 65% of lipid phosphorus removed) with subsequent decrease (more than 65% of phosphorus removed). Liposomes of the photoreceptor membranes lipids were found to bind more calcium than do the native photoreceptor membranes. Proteolytic enzymes (papaine, pronase) splitting some rhodopsin fragments do not affect the ability of the membrane to bind Ca2+. The increase of light-induced Ca-binding is observed only after the outer segments preincubation under conditions providing for rhodopsin phosphorylation. This effect was observed also after the splitting of the rhodopsin fragment by papaine. It is concluded that calcium binding in the photoreceptor membranes is mainly due to the phosphate groups of phospholipids.

  2. Conformational Transitions and Convergence of Absolute Binding Free Energy Calculations

    Science.gov (United States)

    Lapelosa, Mauro; Gallicchio, Emilio; Levy, Ronald M.

    2011-01-01

    The Binding Energy Distribution Analysis Method (BEDAM) is employed to compute the standard binding free energies of a series of ligands to a FK506 binding protein (FKBP12) with implicit solvation. Binding free energy estimates are in reasonably good agreement with experimental affinities. The conformations of the complexes identified by the simulations are in good agreement with crystallographic data, which was not used to restrain ligand orientations. The BEDAM method is based on λ -hopping Hamiltonian parallel Replica Exchange (HREM) molecular dynamics conformational sampling, the OPLS-AA/AGBNP2 effective potential, and multi-state free energy estimators (MBAR). Achieving converged and accurate results depends on all of these elements of the calculation. Convergence of the binding free energy is tied to the level of convergence of binding energy distributions at critical intermediate states where bound and unbound states are at equilibrium, and where the rate of binding/unbinding conformational transitions is maximal. This finding mirrors similar observations in the context of order/disorder transitions as for example in protein folding. Insights concerning the physical mechanism of ligand binding and unbinding are obtained. Convergence for the largest FK506 ligand is achieved only after imposing strict conformational restraints, which however require accurate prior structural knowledge of the structure of the complex. The analytical AGBNP2 model is found to underestimate the magnitude of the hydrophobic driving force towards binding in these systems characterized by loosely packed protein-ligand binding interfaces. Rescoring of the binding energies using a numerical surface area model corrects this deficiency. This study illustrates the complex interplay between energy models, exploration of conformational space, and free energy estimators needed to obtain robust estimates from binding free energy calculations. PMID:22368530

  3. Binding Characteristics Of Ivermectin To Blood Cells | Nweke ...

    African Journals Online (AJOL)

    The binding characteristics of Ivermectin were determined using scatchard plots. The percentage binding to platelet rich plasma, white blood cells and red blood cells were 90.00 + 1.00, 96-90 + 1.05 and 46.20 + 1.10 S.D respectively. It was found to bind the highest to white blood cells and the least to red blood cells.

  4. Attenuating illusory binding with TMS of the right parietal cortex

    OpenAIRE

    Esterman, Michael; Verstynen, Timothy; Robertson, Lynn C.

    2007-01-01

    A number of neuroimaging and neuropsychology studies have implicated various regions of parietal cortex as playing a critical role in the binding of color and form into conjunctions. The current study investigates the role of two such regions by examining how parietal transcranial magnetic stimulation (TMS) influences binding errors known as ‘illusory conjunctions.’ Participants made fewer binding errors after 1 Hz rTMS of the right intraparietal sulcus (IPS), while basic perception of featur...

  5. Heterogeneity of Opioid Binding Sites in Guinea Pig Spinal Cord

    Science.gov (United States)

    1984-11-30

    MEDICAL CENTER WILFORD HALL AIR FORCE MEDICAL CENTER Title of Thesis: "Heterogeneity of Opioid Binding Sites in Guinea Pig Spinal Cord" Name of...that the use of any copyrighted material in the dissertation manuscript entitled: "Heterogeneity of Opioid Binding Sites in Guinea Pig Spinal Cord...University of the Health Sciences 11 Abstract Title of Thesis: Heterogenity of Opioid Binding Sites In Guinea Pig Spinal Cord Gary Dean Zarr MAJ/ANC

  6. Rapid identification of DNA-binding proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Nordhoff, E.; Korgsdam, A.-M.; Jørgensen, H.F.

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...... was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA....

  7. Improved Density Functional Tight Binding Potentials for Metalloid Aluminum Clusters

    Science.gov (United States)

    2016-06-01

    unlimited IMPROVED DENSITY-FUNCTIONAL TIGHT BINDING POTENTIALS FOR METALLOID ALUMINUM CLUSTERS by Joon H. Kim June 2016 Thesis Advisor...DATES COVERED Master’s thesis 4. TITLE AND SUBTITLE IMPROVED DENSITY-FUNCTIONAL TIGHT BINDING POTENTIALS FOR METALLOID ALUMINUM CLUSTERS 5. FUNDING...repulsive potentials for use in density-functional tight binding (DFTB) simulations of low-valence aluminum metalloid clusters . These systems are under

  8. Oligosaccharide binding to barley alpha-amylase 1

    DEFF Research Database (Denmark)

    Robert, X.; Haser, R.; Mori, H.

    2005-01-01

    Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough...... in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site...

  9. Mu opioid receptor binding sites in human brain

    International Nuclear Information System (INIS)

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand [ 3 H]DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of [ 3 H]DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas

  10. Penicillin-binding site on the Escherichia coli cell envelope

    International Nuclear Information System (INIS)

    Amaral, L.; Lee, Y.; Schwarz, U.; Lorian, V.

    1986-01-01

    The binding of 35 S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the cell envelope obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin

  11. Thermodynamics of sequence-specific binding of PNA to DNA

    DEFF Research Database (Denmark)

    Ratilainen, T; Holmén, A; Tuite, E

    2000-01-01

    For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes...

  12. DNA Mismatch Binding and Antiproliferative Activity of Rhodium Metalloinsertors

    Science.gov (United States)

    Ernst, Russell J.; Song, Hang; Barton, Jacqueline K.

    2009-01-01

    Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M−1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo. PMID:19175313

  13. Implicit ligand theory for relative binding free energies

    Science.gov (United States)

    Nguyen, Trung Hai; Minh, David D. L.

    2018-03-01

    Implicit ligand theory enables noncovalent binding free energies to be calculated based on an exponential average of the binding potential of mean force (BPMF)—the binding free energy between a flexible ligand and rigid receptor—over a precomputed ensemble of receptor configurations. In the original formalism, receptor configurations were drawn from or reweighted to the apo ensemble. Here we show that BPMFs averaged over a holo ensemble yield binding free energies relative to the reference ligand that specifies the ensemble. When using receptor snapshots from an alchemical simulation with a single ligand, the new statistical estimator outperforms the original.

  14. Binding of ATP by pertussis toxin and isolated toxin subunits

    International Nuclear Information System (INIS)

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L.

    1990-01-01

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [ 3 H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of [ 3 H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [ 3 H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site

  15. Binding of ATP by pertussis toxin and isolated toxin subunits

    Energy Technology Data Exchange (ETDEWEB)

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. (Center for Biologics Evaluation and Research, Bethesda, MD (USA))

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  16. Probing the binding of coumarins and cyclothialidines to DNA gyrase

    DEFF Research Database (Denmark)

    Kampranis, S C; Gormley, N A; Tranter, R

    1999-01-01

    B and coumarin and cyclothialidine drugs and made mutations by site-directed mutagenesis. We used proteolysis as a probe of drug binding to wild-type and mutant proteins. Limited proteolysis of gyrase revealed that binding of these antibiotics is associated with a characteristic proteolytic fingerprint......, suggesting a drug-induced conformational change. The ability of the mutants to bind the drugs was studied by testing their ability to induce the coumarin-associated proteolytic signature and to bind to a novobiocin-affinity column. To analyze further the interaction of the drugs with gyrase, we studied...

  17. The binding cavity of mouse major urinary protein is optimised for a variety of ligand binding modes

    Energy Technology Data Exchange (ETDEWEB)

    Pertinhez, Thelma A.; Ferrari, Elena; Casali, Emanuela [Department of Experimental Medicine, University of Parma, Via Volturno, 39, 43100 Parma (Italy); Patel, Jital A. [Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR (United Kingdom); Spisni, Alberto, E-mail: alberto.spisni@unipr.it [Department of Experimental Medicine, University of Parma, Via Volturno, 39, 43100 Parma (Italy); Smith, Lorna J., E-mail: lorna.smith@chem.ox.ac.uk [Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR (United Kingdom)

    2009-12-25

    {sup 15}N and {sup 1}HN chemical shift data and {sup 15}N relaxation studies have been used to characterise the binding of N-phenyl-naphthylamine (NPN) to mouse major urinary protein (MUP). NPN binds in the {beta}-barrel cavity of MUP, hydrogen bonding to Tyr120 and making extensive non-bonded contacts with hydrophobic side chains. In contrast to the natural pheromone 2-sec-butyl-4,5-dihydrothiazole, NPN binding gives no change to the overall mobility of the protein backbone of MUP. Comparison with 11 different ligands that bind to MUP shows a range of binding modes involving 16 different residues in the {beta}-barrel cavity. These finding justify why MUP is able to adapt to allow for many successful binding partners.

  18. M/sub r/ 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor

    International Nuclear Information System (INIS)

    Moscatelli, D.; Joseph-Silverstein, J.; Manejias, R.; Rifkin, D.B.

    1987-01-01

    A M/sub r/ 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig grain along with the typical M/sub r/ 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The M/sub r/ 25,000 form, like the M/sub r/ 18,000 form was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The M/sub r/ 25,000 guinea pig protein stimulated plasminogen activator production by cultured bovine capillary endothelial cells in a dose-dependent manner at concentration of 0.1-10 ngml, the same range that was effective for guinea pig and human M/sub r/ 18,000 bFGFs. The binding of human 125 I-labeled bFGF to baby hamster kidney cells is inhibited equally by the M/sub r/ 25,000 guinea pig protein and the M/sub r/ 18,000 guinea pig and human bFGFs. Polyclonal antibodies raised against human bFGF recognize both the M/sub r/ 25,000 and 18,000 guinea pig proteins in an immunoblot analysis. In a radioimmunoassay, both the M/sub r/ 25,000 and M/sub r/ 18,000 guinea pig proteins compete equally well with iodinated human bFGF for binding to the anti-human bFGF antibodies. When treated with low concentrations of trypsin, the M/sub r/ 25,000 guinea pig bFGF was converted to a M/sub r/ 18,000 protein. These results show that the two molecules are closely related and suggest that the M/sub r/ 25,000 protein shares substantial homology with the M/sub r/ 18,000 bFGF

  19. Discover binding pathways using the sliding binding-box docking approach: application to binding pathways of oseltamivir to avian influenza H5N1 neuraminidase

    Science.gov (United States)

    Tran, Diem-Trang T.; Le, Ly T.; Truong, Thanh N.

    2013-08-01

    Drug binding and unbinding are transient processes which are hardly observed by experiment and difficult to analyze by computational techniques. In this paper, we employed a cost-effective method called "pathway docking" in which molecular docking was used to screen ligand-receptor binding free energy surface to reveal possible paths of ligand approaching protein binding pocket. A case study was applied on oseltamivir, the key drug against influenza a virus. The equilibrium pathways identified by this method are found to be similar to those identified in prior studies using highly expensive computational approaches.

  20. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  1. Gentamicin binds to the megalin receptor as a competitive inhibitor using the common ligand binding motif of complement type repeats

    DEFF Research Database (Denmark)

    Dagil, Robert; O'Shea, Charlotte; Nykjær, Anders

    2013-01-01

    megalin and investigated its interaction with gentamicin. Using NMR titration data in HADDOCK, we have generated a three-dimensional model describing the complex between megalin and gentamicin. Gentamicin binds to megalin with low affinity and exploits the common ligand binding motif previously described...... to megalin is highly similar to gentamicin binding to calreticulin. We discuss the impact of this novel insight for the future structure-based design of gentamicin antagonists....

  2. Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin.

    Science.gov (United States)

    Rooijakkers, Bart J M; Ikonen, Martina S; Linder, Markus B

    2018-01-01

    Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain's sequence-function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed.

  3. Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin.

    Directory of Open Access Journals (Sweden)

    Bart J M Rooijakkers

    Full Text Available Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain's sequence-function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed.

  4. Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

    Full Text Available As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions. A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC of 0.77 with high precision (94% and high sensitivity (65%. We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA] is available as an on-line server at http://sparks-lab.org.

  5. Effects of heparin on insulin binding and biological activity

    International Nuclear Information System (INIS)

    Kriauciunas, K.M.; Grigorescu, F.; Kahn, C.R.

    1987-01-01

    The effect of heparin, a polyanionic glycosaminoglycan known to alter the function of many proteins, on insulin binding and bioactivity was studied. Cultured human lymphocytes (IM-9) were incubated with varying concentrations of heparin, then extensively washed, and 125 I-labeled insulin binding was measured. Heparin at concentrations used clinically for anticoagulation (1-50 U/ml) inhibited binding in a dose-dependent manner; 50% inhibition of binding occurred with 5-10 U/ml. Scatchard analysis indicated that the decrease in binding was due to a decrease in both the affinity and the apparent number of available insulin receptors. The effect occurred within 10 min at 22 degrees C and persisted even after the cells were extensively washed. Inhibition of insulin binding also occurred when cells were preincubated with heparinized plasma or heparinized serum but not when cells were incubated with normal serum or plasma from blood anticoagulated with EDTA. By contrast, other polyanions and polycations, e.g., poly-L-glutamic acid, poly-L-lysine, succinylated poly-L-lysine, and histone, did not inhibit binding. Heparin also inhibited insulin binding in Epstein-Barr (EB) virus-transformed lymphocytes but had no effect on insulin binding to isolated adipocytes, human erythrocytes, or intact hepatoma cells. When isolated adipocytes were incubated with heparin, there was a dose-dependent inhibition of insulin-stimulated glucose oxidation and, to a lesser extent, of basal glucose oxidation. Although heparin has no effect on insulin binding to intact hepatoma cells, heparin inhibited both insulin binding and insulin-stimulated autophosphorylation in receptors solubilized from these cells

  6. Evolution of Metal(Loid) Binding Sites in Transcriptional Regulators

    Energy Technology Data Exchange (ETDEWEB)

    Ordonez, E.; Thiyagarajan, S.; Cook, J.D.; Stemmler, T.L.; Gil, J.A.; Mateos, L.M.; Rosen, B.P.

    2009-05-22

    Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys{sup 15}, Cys{sup 16}, and Cys{sup 55}. This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

  7. Genetics Home Reference: core binding factor acute myeloid leukemia

    Science.gov (United States)

    ... binding factor acute myeloid leukemia Core binding factor acute myeloid leukemia Printable PDF Open All Close All Enable Javascript ... on PubMed (1 link) PubMed OMIM (1 link) LEUKEMIA, ACUTE MYELOID Sources for This Page Goyama S, Mulloy JC. Molecular ...

  8. Murine alpha1-adrenoceptor subtypes. I. Radioligand binding studies

    NARCIS (Netherlands)

    Yang, M.; Reese, J.; Cotecchia, S.; Michel, M. C.

    1998-01-01

    Alpha1-adrenoceptors were identified in murine tissues by [3H]prazosin saturation binding studies, with a rank order of cerebral cortex > cerebellum > liver > lung > kidney > heart > spleen, with the spleen not exhibiting detectable expression. Competition binding studies were performed with

  9. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  10. Binding of reactive organophosphate by oximes via hydrogen bond

    Indian Academy of Sciences (India)

    In this contribution, the ability of simple oximes to bind a well-known nerve agent simulant (dimethylmethylphosphonate, DMMP) via hydrogen bond is reported. UV/Vis measurements indicate the formation of 1:1 complexes. 1H-, 31P-NMR titrations and T-ROESY experiments confirm that oximes bind the organophosphate ...

  11. The RNA-binding protein repertoire of Arabidopsis thaliana

    KAUST Repository

    Marondedze, Claudius; Thomas, Ludivine; Serano, Natalia Lorena Gorron; Lilley, Kathryn S.; Gehring, Christoph A

    2016-01-01

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently

  12. Cue integration and the perception of action in intentional binding

    DEFF Research Database (Denmark)

    Wolpe, Noham; Haggard, Patrick; Siebner, Hartwig R

    2013-01-01

    'Intentional binding' describes the perceived temporal attraction between a voluntary action and its sensory consequence. Binding has been used in health and disease as an indirect measure of awareness of action or agency, that is, the sense that one controls one's own actions. It has been propos...

  13. Perturbation method for calculating impurity binding energy in an ...

    Indian Academy of Sciences (India)

    Nilanjan Sil

    2017-12-18

    Dec 18, 2017 ... Abstract. In the present paper, we have studied the binding energy of the shallow donor hydrogenic impurity, which is confined in an inhomogeneous cylindrical quantum dot (CQD) of GaAs-AlxGa1−xAs. Perturbation method is used to calculate the binding energy within the framework of effective mass ...

  14. Defining carbohydrate binding of glucan phosphatases via Affinity gel electrophoresis

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2016-01-01

    was to determine a technique to measure carbohydrate binding quickly and efficiently. We established a protocol to reproducibly and quantitatively measure the binding of the enzymes to glucans utilizing Affinity Gel Electrophoresis (AGE). The results show that the various glucan phosphatases possess differing...

  15. Improved assay for measuring heparin binding to bull sperm

    International Nuclear Information System (INIS)

    Miller, D.J.; Ax, R.L.

    1988-01-01

    The binding of heparin to sperm has been used to study capacitation and to rank relative fertility of bulls. Previous binding assays were laborious, used 10 7 sperm per assay point, and required large amounts of radiolabeled heparin. A modified heparin-binding assay is described that used only 5 x 10 4 cells per incubation well and required reduced amounts of [ 3 H] heparin. The assay was performed in 96-well Millititer plates, enabling easy incubation and filtering. Dissociation constants and concentrations of binding sites did not differ if analyzed by Scatchard plots, Woolf plots, or by log-logit transformed weighted nonlinear least squares regression, except in the case of outliers. In such cases, Scatchard analysis was more sensitive to outliers. Nonspecific binding was insignificant using nonlinear logistic fit regression and a proportion graph. The effects were tested of multiple free-thawing of sperm in either a commercial egg yolk extender, 40 mM Tris buffer with 8% glycerol, or 40 mM Tris buffer without glycerol. Freeze-thawing in extender did not affect the dissociation constant or the concentration of binding sites. However, freeze-thawing three times in 40 mM Tris reduced the concentration of binding sites and lowered the dissociation constant (raised the affinity). The inclusion of glycerol in the 40 mM Tris did not significantly affect the estimated dissociation constant or the concentration of binding sites as compared to 40 mM Tris without glycerol

  16. Spectral characterization and DNA binding properties of lanthanide(III)

    African Journals Online (AJOL)

    Spectral data of complexes suggest that the ligand binds metal ion through pyridine- nitrogen, azomethine-nitrogen and amido-oxygen donor atoms. Electrochemical behaviour of metal complexes was investigated by using cyclic voltammetry. The complexes undergo quasi-reversible one electron reduction. The binding ...

  17. Extrapolations of nuclear binding energies from new linear mass relations

    DEFF Research Database (Denmark)

    Hove, D.; Jensen, A. S.; Riisager, K.

    2013-01-01

    We present a method to extrapolate nuclear binding energies from known values for neighboring nuclei. We select four specific mass relations constructed to eliminate smooth variation of the binding energy as function nucleon numbers. The fast odd-even variations are avoided by comparing nuclei...

  18. Biosensors engineered from conditionally stable ligand-binding domains

    Science.gov (United States)

    Church, George M.; Feng, Justin; Mandell, Daniel J.; Baker, David; Fields, Stanley; Jester, Benjamin Ward; Tinberg, Christine Elaine

    2017-09-19

    Disclosed is a biosensor engineered to conditionally respond to the presence of specific small molecules, the biosensors including conditionally stable ligand-binding domains (LBDs) which respond to the presence of specific small molecules, wherein readout of binding is provided by reporter genes or transcription factors (TFs) fused to the LBDs.

  19. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

  20. Binding-Induced Fluorescence of Serotonin Transporter Ligands

    DEFF Research Database (Denmark)

    Wilson, James; Ladefoged, Lucy Kate; Babinchak, Michael

    2014-01-01

    The binding-induced fluorescence of 4-(4-(dimethylamino)-phenyl)-1-methylpyridinium (APP(+)) and two new serotonin transporter (SERT)-binding fluorescent analogues, 1-butyl-4-[4-(1-dimethylamino)phenyl]-pyridinium bromide (BPP(+)) and 1-methyl-4-[4-(1-piperidinyl)phenyl]-pyridinium (PPP(+)), has...