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31.
Antibodies Recognising the GABAA /Benzodiazepine Receptor Including Its Regulatory Sites 总被引:7,自引:4,他引:3
F. Anne Stephenson Stefano O. Casalotti Cleanthi Mamalaki Eric A. Barnard 《Journal of neurochemistry》1986,46(3):854-861
Polyclonal antibodies have been raised against the GABA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X-100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I-labelled gamma-aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immunoprecipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl-beta-carboline-3-carboxylate binding, the [35S]t-butylbicyclophosphorothionate binding (TBPS), the barbiturate-enhanced [3H]flunitrazepam binding, and the GABA-enhanced [3H]flunitrazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the alpha-subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators. 相似文献
32.
Sariqa Wagley Claudia Hemsley Rachael Thomas Madeleine G. Moule Muthita Vanaporn Clio Andreae Matthew Robinson Stan Goldman Brendan W. Wren Clive S. Butler Richard W. Titball 《Journal of bacteriology》2014,196(2):407-416
The twin arginine translocation (Tat) system in bacteria is responsible for transporting folded proteins across the cytoplasmic membrane, and in some bacteria, Tat-exported substrates have been linked to virulence. We report here that the Tat machinery is present in Burkholderia pseudomallei, B. mallei, and B. thailandensis, and we show that the system is essential for aerobic but not anaerobic growth. Switching off of the Tat system in B. thailandensis grown anaerobically resulted in filamentous bacteria, and bacteria showed increased sensitivity to some β-lactam antibiotics. In Galleria mellonella and zebrafish infection models, the Tat conditional mutant was attenuated. The aerobic growth-restricted phenotype indicates that Tat substrates may play a functional role in oxygen-dependent energy conservation. In other bacteria, aerobic growth restriction in Tat mutants has been attributed to the inability to translocate PetA, the Rieske iron-sulfur protein which forms part of the quinol-cytochrome c oxidoreductase complex. Here, we show that PetA is not responsible for aerobic growth restriction in B. thailandensis. However, we have identified an operon encoding 2 proteins of unknown function (BTH_I2176 and BTH_I2175) that play a role in aerobic growth restriction, and we present evidence that BTH_I2176 is Tat translocated. 相似文献
33.
Nefeli Slavi Clio Rubinos Leping Li Caterina Sellitto Thomas W. White Richard Mathias Miduturu Srinivas 《The Journal of biological chemistry》2014,289(47):32694-32702
Maintenance of adequate levels of glutathione (GSH) in the lens nucleus is critical for protection of lens proteins from the effects of oxidative stress and for lens transparency. How GSH is transported to the nucleus is unknown. We show that GSH diffuses to the nucleus from the outer cortex, where a high concentration of the anti-oxidant is established by synthesis or uptake, via the network of gap junctions. Using electrophysiological measurements, we found that channels formed by Cx46 and Cx50, the two connexin isoforms expressed in the lens, were moderately cation-selective (PNa/PCl ∼5 for Cx46 and ∼3 for Cx50). Single channel permeation of the larger GSH anion was low but detectable (PNa/PGSH ∼12 for Cx46 and ∼8 for Cx50), whereas permeation of divalent anion glutathione disulfide (GSSG) was undetectable. Measurement of GSH levels in the lenses from connexin knock-out (KO) mice indicated Cx46, and not Cx50, is necessary for transport of GSH to the core. Levels of GSH in the nucleus were markedly reduced in Cx46 KO, whereas they were unaffected by Cx50 KO. We also show that GSH delivery to the nucleus is not dependent on the lens microcirculation, which is believed to be responsible for extracellular transport of other nutrients to membrane transporters in the core. These results indicate that glutathione diffuses from cortical fiber cells to the nucleus via gap junction channels formed by Cx46. We present a model of GSH diffusion from outer cells to inner fiber cells through gap junctions. 相似文献
34.
Ratti C Sobolevsky S Calabrese F Andris C Reades J Martino M Claxton R Strogatz SH 《PloS one》2010,5(12):e14248
Do regional boundaries defined by governments respect the more natural ways that people interact across space? This paper proposes a novel, fine-grained approach to regional delineation, based on analyzing networks of billions of individual human transactions. Given a geographical area and some measure of the strength of links between its inhabitants, we show how to partition the area into smaller, non-overlapping regions while minimizing the disruption to each person''s links. We tested our method on the largest non-Internet human network, inferred from a large telecommunications database in Great Britain. Our partitioning algorithm yields geographically cohesive regions that correspond remarkably well with administrative regions, while unveiling unexpected spatial structures that had previously only been hypothesized in the literature. We also quantify the effects of partitioning, showing for instance that the effects of a possible secession of Wales from Great Britain would be twice as disruptive for the human network than that of Scotland. 相似文献
35.
Stefanie Deinhardt-Emmer Sarah Bttcher Clio Hring Liane Giebeler Andreas Henke Roland Zell Johannes Jungwirth Paul M. Jordan Oliver Werz Franziska Hornung Christian Brandt Mike Marquet Alexander S. Mosig Mathias W. Pletz Michael Schacke Jürgen Rdel Regine Heller Sandor Nietzsche Bettina Lffler Christina Ehrhardt 《Journal of virology》2021,95(10)
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37.
MAb 4C5 is a cell impermeable, anti-HSP90 murine monoclonal antibody, originally produced using hybridoma technology. We have previously shown that mAb 4C5 specifically recognizes both the α- and to a lesser extent the β-isoform of HSP90. Additionally, in vitro and in vivo studies revealed that by selectively inhibiting the function of cell-surface HSP90, mAb 4C5 significantly impairs cancer cell invasion and metastasis. Here we describe the reconstitution of mAb 4C5 into a mouse-human chimera. More importantly we report that mAb 4C5 and consequently its chimeric counterpart are completely devoid of heavy chain and consist only of a functional kappa light chain dimer. The chimeric antibody is shown to retain the original antibody's specificity and functional properties. Thus it is capable of inhibiting the function of surface HSP90, leading to reduced cancer cell invasion in vitro. Finally, we present in vivo evidence showing that the chimeric 4C5 significantly inhibits the metastatic deposit formation of MDA-MB-453 cells into the lungs of SCID mice. These data suggest that a chimeric kappa light chain antibody could be potentially used as an anti-cancer agent, thereby introducing a novel type of antibody fragment, with reduced possible adverse immunogenic effects, into cancer therapeutics. 相似文献
38.
Sanne Boessenkool Kristian Hanghøj Heidi M. Nistelberger Clio Der Sarkissian Agata T. Gondek Ludovic Orlando James H. Barrett Bastiaan Star 《Molecular ecology resources》2017,17(4):742-751
The feasibility of genome‐scale studies from archaeological material remains critically dependent on the ability to access endogenous, authentic DNA. In the majority of cases, this represents a few per cent of the DNA extract, at most. A number of specific pre‐extraction protocols for bone powder aimed to improve ancient DNA recovery before library amplification have recently been developed. Here, we test the effects of combining two of such protocols, a bleach wash and a predigestion step, on 12 bone samples of Atlantic cod and domestic horse aged 750–1350 cal. years before present. Using high‐throughput sequencing, we show that combined together, bleach wash and predigestion consistently yield DNA libraries with higher endogenous content than either of these methods alone. Additionally, the molecular complexity of these libraries is improved and endogenous DNA templates show larger size distributions. Other library characteristics, such as DNA damage profiles or the composition of microbial communities, are little affected by the pre‐extraction protocols. Application of the combined protocol presented in this study will facilitate the genetic analysis of an increasing number of ancient remains and will reduce the cost of whole‐genome sequencing. 相似文献
39.
Mamalaki A Gritzapis AD Kretsovali A Belimezi M Papamatheakis J Perez SA Papamichail M Baxevanis CN 《Cancer immunology, immunotherapy : CII》2003,52(8):513-522
To broaden the applicability of adoptive cellular immunotherapy against HER-2/neu overexpressing human cancers, we constructed a chimeric scFv/gamma gene composed of the variable regions of a HER-2/neu specific monoclonal antibody (mAb) joined to the signaling gamma-chain of the Fc(epsilon)RI receptor. The scFv(anti-HER-2/neu)/gamma chimeric gene was successfully expressed as functional surface receptor in the MD.45 cytolytic T-cell (CTL) hybridoma (MD.45-HER/gamma). Expression of the chimeric protein triggered IL-2 and IFN-gamma secretion in vitro upon encountering cell surface HER-2/neu and mediated non-major-histocompatibility-complex (MHC)-restricted HER-2/neu-specific target cell lysis. We also examined the in vivo activity of the MD.45-HER/gamma transduced cells. Severe combined immunodeficiency disease (SCID) mice that were given HER-2/neu positive (+) human tumor cell lines had significantly increased survival compared to mice treated with saline only, or with MD.45 cells transduced with a control anti-trinitrophenyl (anti-TNP) chimeric receptor gene (MD.45-TNP/gamma). These results demonstrate the feasibility of redirecting MD.45 CTL to react in vitro and in vivo with a variety of HER-2/neu(+) tumor cells by our gene transduction protocol. Moreover, they open the possibility of using the same chimeric gene for transducing primary lymphocytes and thus allowing adoptive immunotherapy against HER-2/neu(+) cancers. 相似文献
40.
Psaridi-Linardaki L Mamalaki A Remoundos M Tzartos SJ 《The Journal of biological chemistry》2002,277(30):26980-26986
The N-terminal extracellular domain (amino acids 1-210; halpha-(1-210)) of the alpha subunit of the human muscle nicotinic acetylcholine receptor (AChR), bearing the binding sites for cholinergic ligands and the main immunogenic region, the major target for anti-AChR antibodies in patients with myasthenia gravis, was expressed in the yeast, Pichia pastoris. The recombinant protein was water-soluble and glycosylated, and fast protein liquid chromatography analysis showed it to be a monomer. halpha-(1-210) bound (125)I-alpha-bungarotoxin with a high affinity (K(d) = 5.1 +/- 2.4 nm), and this binding was blocked by unlabeled d-tubocurarine and gallamine (K(i) approximately 7.5 mm). Interestingly, (125)I-alpha-bungarotoxin binding was markedly impaired by in vitro deglycosylation of halpha-(1-210). Several monoclonal antibodies that show partial or strict conformation-dependent binding to the AChR were able to bind to halpha-(1-210), as did antibodies from a large proportion of myasthenic patients. These results suggest that the extracellular domain of the human AChR alpha subunit expressed in P. pastoris has an apparently near native conformation. The correct folding of the recombinant protein, together with its relatively high expression yield, makes it suitable for structural studies on the nicotinic acetylcholine receptor and for use as an autoantigen in myasthenia gravis studies. 相似文献