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41.
K C Anderson J A Roach J F Daley S F Schlossman L M Nadler 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(10):3612-3618
B cell-enriched preparations were prepared from human peripheral blood and lymphoid tissues by the depletion of T cells and monocytes. Only B cells by virtue of their staining with anti-B1 conjugated to fluorescein were additionally examined. Dual fluorescence staining and flow cytometric analysis demonstrated that the majority of "resting" human peripheral blood and splenic B cells co-express the B cell-restricted B1 and B2 antigens and lack B5, a B cell-restricted activation antigen, and interleukin 2 receptor (IL 2R). In contrast, nearly 2/3 and 1/3 of B1+ cells isolated from lymph node expressed IL 2R and B5 antigens, respectively. When B1+ B cells from peripheral blood and spleen were "activated" by anti-Ig, they lost the B2 antigen and acquired the B5 and/or IL 2R antigens. 2/3 of B1+ cells strongly expressed IL 2R, and up to 1/2 of B1+ cells co-expressed B5. Delineation of increased numbers of B1+ cells that co-express B5 and/or IL 2R within lymphoid tissues obtained from patients with diseases characterized by "activated" B cells provides in vivo confirmation that these phenotypic changes correlate with B cell activation. We believe that the identification and isolation of these and similar subsets of cells defined by differing cell surface phenotypes should further our understanding both of normal B cell activation and the pathophysiology of B cell disease states. 相似文献
42.
Xiaobing Xia Vincent L. G. Postis Moazur Rahman Gareth S. A. Wright Peter C. J. Roach Sarah E. Deacon 《Molecular membrane biology》2013,30(8):691-701
The toxic metalloid arsenic is an abundant element and most organisms possess transport systems involved in its detoxification. One such family of arsenite transporters, the ACR3 family, is widespread in fungi and bacteria. To gain a better understanding of the molecular mechanism of arsenic transport, we report here the expression and characterization of a family member, So_ACR3, from the bacterium Shewanella oneidensis MR-1. Surprisingly, expression of this transporter in the arsenic-hypersensitive Escherichia coli strain AW3110 conferred resistance to arsenate, but not to arsenite. Purification of a C-terminally His-tagged form of the protein allowed the binding of putative permeants to be directly tested: arsenate but not arsenite quenched its intrinsic fluorescence in a concentration-dependent fashion. Fourier transform infrared spectroscopy showed that the purified protein was predominantly α-helical. A mutant bearing a single cysteine residue at position 3 retained the ability to confer arsenate resistance, and was accessible to membrane impermeant thiol reagents in intact cells. In conjunction with successful C-terminal tagging with oligohistidine, this finding is consistent with the experimentally-determined topology of the homologous human apical sodium-dependent bile acid transporter, namely 7 transmembrane helices and a periplasmic N-terminus, although the presence of additional transmembrane segments cannot be excluded. Mutation to alanine of the conserved residue proline 190, in the fourth putative transmembrane region, abrogated the ability of the transporter to confer arsenic resistance, but did not prevent arsenate binding. An apparently increased thermal stability is consistent with the mutant being unable to undergo the conformational transitions required for permeant translocation. 相似文献
43.
The amyloid-beta rise and gamma-secretase inhibitor potency depend on the level of substrate expression 总被引:2,自引:0,他引:2
Burton CR Meredith JE Barten DM Goldstein ME Krause CM Kieras CJ Sisk L Iben LG Polson C Thompson MW Lin XA Corsa J Fiedler T Pierdomenico M Cao Y Roach AH Cantone JL Ford MJ Drexler DM Olson RE Yang MG Bergstrom CP McElhone KE Bronson JJ Macor JE Blat Y Grafstrom RH Stern AM Seiffert DA Zaczek R Albright CF Toyn JH 《The Journal of biological chemistry》2008,283(34):22992-23003
The amyloid-beta (Abeta) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-beta precursor protein (APP) through consecutive proteolytic cleavages by beta-site APP-cleaving enzyme and gamma-secretase. Unexpectedly gamma-secretase inhibitors can increase the secretion of Abeta peptides under some circumstances. This "Abeta rise" phenomenon, the same inhibitor causing an increase in Abeta at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Abeta rise depends on the beta-secretase-derived C-terminal fragment of APP (betaCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Abeta, known as "p3," formed by alpha-secretase cleavage, did not exhibit a rise. In addition to the Abeta rise, low betaCTF or C99 expression decreased gamma-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, gamma-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Abeta under conditions of low substrate expression. The Abeta rise was also observed in rat brain after dosing with the gamma-secretase inhibitor BMS-299897. The Abeta rise and potency shift are therefore relevant factors in the development of gamma-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Abeta rise, including the "incomplete processing" and endocytic models, are discussed. 相似文献
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Guillaume Allorent Ryutaro Tokutsu Thomas Roach Graham Peers Pierre Cardol Jacqueline Girard-Bascou Daphné Seigneurin-Berny Dimitris Petroutsos Marcel Kuntz Cécile Breyton Fabrice Franck Francis-André Wollman Krishna K. Niyogi Anja Krieger-Liszkay Jun Minagawa Giovanni Finazzi 《The Plant cell》2013,25(2):545-557
Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II. 相似文献
46.
Katy Morgan Roach Stephen Mark Duffy William Coward Carol Feghali-Bostwick Heike Wulff Peter Bradding 《PloS one》2013,8(12)
Background
Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that KCa3.1 K+ channel-dependent cell processes contribute to IPF pathophysiology.Methods
KCa3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of KCa3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct KCa3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]).Results
Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed KCa3.1 channel mRNA and protein. KCa3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. KCa3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. KCa3.1 was expressed strongly in IPF tissue. KCa3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility in vitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca2+.Conclusions
KCa3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking KCa3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic. 相似文献47.
Hua Jiang Yueqing Xie Andrew Burnette John Roach Steven L. Giardina Toby T. Hecht Stephen P. Creekmore Gautam Mitra Jianwei Zhu 《Applied microbiology and biotechnology》2013,97(2):621-632
Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion—toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the VH or VL. Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, VL and VH PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins—MR1-1 and HA22. 相似文献
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50.
DANIEL TRAN TAKASHI KADONO MARIA LIA MOLAS RAFIK ERRAKHI JOËL BRIAND BERNADETTE BILIGUI TOMONORI KAWANO FRANÇOIS BOUTEAU 《Plant, cell & environment》2013,36(3):569-578
Ozone (O3) is an air pollutant with an impact increasingly important in our industrialized world. It affects human health and productivity in various crops. We provide the evidences that treatment of Arabidopsis thaliana with O3 results in ascorbate‐derived oxalic acid production. Using cultured cells of A. thaliana as a model, here we further showed that oxalic acid induces activation of anion channels that trigger depolarization of the cell, increase in cytosolic Ca2+ concentration, generation of reactive oxygen species and cell death. We confirmed that O3 reacts with ascorbate in the culture, thus resulting in production of oxalic acid and this could be part of the O3‐induced signalling pathways that trigger programmed cell death. 相似文献