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Optimizing antibody immobilization strategies for the construction of protein microarrays 总被引:11,自引:0,他引:11
Peluso P Wilson DS Do D Tran H Venkatasubbaiah M Quincy D Heidecker B Poindexter K Tolani N Phelan M Witte K Jung LS Wagner P Nock S 《Analytical biochemistry》2003,312(2):113-124
Antibody microarrays have the potential to revolutionize protein expression profiling. The intensity of specific signal produced on a feature of such an array is related to the amount of analyte that is captured from the biological mixture by the immobilized antibody (the "capture agent"). This in turn is a function of the surface density and fractional activity of the capture agents. Here we investigate how these two factors are affected by the orientation of the capture agents on the surface. We compare randomly versus specifically oriented capture agents based on both full-sized antibodies and Fab' fragments. Each comparison was performed using three different antibodies and two types of streptavidin-coated monolayer surfaces. The specific orientation of capture agents consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvements over surfaces with randomly oriented capture agents. Surface plasmon resonance revealed a dense monolayer of Fab' fragments that are on average 90% active when specifically oriented. Randomly attached Fab's could not be packed at such a high density and generally also had a lower specific activity. These results emphasize the importance of attaching proteins to surfaces such that their binding sites are oriented toward the solution phase. 相似文献
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Polyana C Tizioto Jeremy F Taylor Jared E Decker Caio F Gromboni Mauricio A Mudadu Robert D Schnabel Luiz L Coutinho Gerson B Mour?o Priscila SN Oliveira Marcela M Souza James M Reecy Renata T Nassu Flavia A Bressani Patricia Tholon Tad S Sonstegard Mauricio M Alencar Rymer R Tullio Ana RA Nogueira Luciana CA Regitano 《遗传、选种与进化》2015,47(1)
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Pillinger MH Marjanovic N Kim SY Scher JU Izmirly P Tolani S Dinsell V Lee YC Blaser MJ Abramson SB 《The Journal of biological chemistry》2005,280(11):9973-9979
Because matrix metalloproteinases (MMPs) play roles in inflammatory tissue injury, we asked whether MMP secretion by gastric epithelial cells may contribute to gastric injury in response to signals involved in Helicobacter pylori-induced inflammation and/or cyclooxygenase inhibition. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and epidermal growth factor (EGF) stimulated gastric cell MMP-1 secretion, indicating that MMP-1 secretion occurs in inflammatory as well as non-inflammatory situations. MMP-1 secretion required activation of the MAPK Erk and subsequent protein synthesis but was down-regulated by the alternate MAPK, p38. In contrast, secretion of MMP-13 was stimulated by TNF-alpha/IL-1beta but not EGF and was Erk-independent and mediated by p38. MMP-13 secretion was more rapid (peak, 6 h) than MMP-1 (peak > or =30 h) and only partly depended on protein synthesis, suggesting initial release of a pre-existing MMP-13 pool. Therefore, MMP-1 and MMP-13 secretion are differentially regulated by MAPKs. MMP-1 secretion was regulated by E prostaglandins (PGEs) in an Erk-dependent manner. PGEs enhanced Erk activation and MMP-1 secretion in response to EGF but inhibited Erk and MMP-1 when TNF-alpha and IL-1beta were the stimuli, indicating that the effects of PGEs on gastric cell responses are context-dependent. These data show that secretion of MMPs is differentially regulated by MAPKs and suggest mechanisms through which H. pylori infection and/or cyclooxygenase inhibition may induce epithelial cell signaling to contribute to gastric ulcerogenesis. 相似文献
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The aim of this study was to determine the first effect of lead on microbial activity in soil. The study was carried out in the soil samples from four different radish (Raphanus sativus L. var. radicula, Brassicaceae) fields along the highway in a district (Kadirli, Osmaniye) of the Eastern Mediterranean Region, Turkey. After the calculation of Pb contents, the Pb amounts of the soil samples were brought up to 50 and 100 mg Pb kg?1 by treatment with Pb(NO 3 ) 2 , and the samples for the carbon and the nitrogen mineralization were incubated under controlled conditions (28°C, constant moist). The carbon mineralization was determined by a CO 2 respiration method for 30 days. The nitrogen mineralization was observed in vitro for 6 weeks. The untreated group was statistically different from the 50 and 100 mg Pb kg?1 treatments in the aspect of the C(CO 2 ) outlet during mineralization (P ≤ 0.05), but difference between the 50 and 100 mg Pb kg?1 treatments was not significant. NH 4 -N and NO 3 -N contents of each soil were shown differences between across treatments. Based on these results, it is possible to conclude that the addition of 50 and 100 mg Pb kg?1 provided a toxic effect threshold for the microbial activity into 30 days. 相似文献
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Twelve fungi namelyAlternaria alternata, Aspergillus flavus, A niger, A ochraceus, Actinomucor repens, Capnodoium spp., Curvularia lunata, Fusarium
pallidoroseum, F solani, F verticillioides, Penicillium citrinum and Rhizopus stolonifer were recorded from samples ofAegle marmelos, Aesculus indica, Buchanania lanzan andPinus gerardiana. In case ofPrunus amygdalus only Rstolonifer was recorded. A significant variation in pattern of mycoflora incidence was observed in terms of source and season. Fungal
infestation in most of the substrates was found to be highest during monsoon. Aflatoxins were the most common mycotoxins elaborated
by different isolates ofA flavus obtained fromA marmelos, B lanzan andP gerardiana. The amount of aflatoxins produced by the toxigenic isolates ofA flavus was in the range of traces to 0.9–26.0 μg/ml inA marmelos, 0.8–17.5 μg/ml inP gerardiana and 0.65–13.2 μg/ml inB lanzan. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi. Aflatoxins were detected
almost in all the samples analyzed for mycotoxin contamination. However, traces of zearalenone were detected inA marmelos. The concentration of aflatoxin B1 was in the range of 0.13–0.75 μg/g inA marmelos, 0.09–0.60 μg/g inP gerardiana and 0.01–0.20 ug/g inB lanzan. Mycotoxins were not detected inAesculus indica andPrunus amygdalus. 相似文献
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Walking humansconserve mechanical and, presumably, metabolic energy with an invertedpendulum-like exchange of gravitational potential energy and horizontalkinetic energy. Walking in simulated reduced gravity involves arelatively high metabolic cost, suggesting that the inverted-pendulummechanism is disrupted because of a mismatch of potential and kineticenergy. We tested this hypothesis by measuring the fluctuations andexchange of mechanical energy of the center of mass at differentcombinations of velocity and simulated reduced gravity. Subjects walkedwith smaller fluctuations in horizontal velocity in lower gravity, suchthat the ratio of horizontal kinetic to gravitational potential energyfluctuations remained constant over a fourfold change in gravity. Theamount of exchange, or percent recovery, at 1.00 m/s was notsignificantly different at 1.00, 0.75, and 0.50 G (average 64.4%),although it decreased to 48% at 0.25 G. As a result, the amount ofwork performed on the center of mass does not explain the relatively high metabolic cost of walking in simulated reduced gravity. 相似文献
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