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Epithelial Function and Airway Responsiveness
Published in Alastair G. Stewart, AIRWAY WALL REMODELLING in ASTHMA, 2020
Roy G. Goldie, Janet M. H. Preuss
In the presence of the cyclooxygenase inhibitor indomethacin, removal of guinea pig tracheal epithelium still caused a 150-fold increase in sensitivity to SP. In the presence of phosphoramidon, this was dramatically reduced to 18-fold, suggesting that the residual potency increase was due to the effect of a nonprostanoid inhibitory substance.173
Stimulus-Secretion Coupling: Intracellular Proteins and Nucleotides
Published in Stephen W. Carmichael, Susan L. Stoddard, The Adrenal Medulla 1986 - 1988, 2017
Stephen W. Carmichael, Susan L. Stoddard
Mundy, Hermann and Strittmatter (1987) noted that chromaffin cells have metal-dependent endoproteinases in both the plasma membrane and the soluble fraction of homogenized cells. They demonstrated that the metal-dependent endoproteinases in these two subcellular fractions can be differentiated by selective inhibitors. In both intact and permeabilized cells, the inhibitor phosphoramidon did not block exocytosis and did not inhibit the soluble proteinases but did inhibit the plasma membrane metalloproteinase. Because soluble metalloproteinase activity is inhibited by other proteinase inhibitors at concentrations that block exocytosis, a soluble metalloproteinase, but not the plasma membrane one, appears to be required in exocytosis.
Proinflammatory Peptides in Relation to Other Inflammatory Mediators
Published in Sami I. Said, Proinflammatory and Antiinflammatory Peptides, 2020
Pierangelo Geppetti, Costanza Emanueli, Michela Figini, Domenico Regoli
In the case of the kinin/tachykinin pathway stimuli such as lowering of the pH, antigenic stimulation or chemical injury are required in order to promote tissue kinin accumulation, which causes tachykinin release that results in increased vascular permeability and bronchoconstriction. Recent studies (64) in mice in which the gene encoding for NEP was disrupted by homologous recombination [NEP(—/—)] indicate that activation of the kinin-tachykinin inflammatory pathway may occur even in the absence of any obvious proinflammatory stimulus. NEP(—/—) mice showed baseline levels of plasma extravasation in the trachea, urinary bladder, pancreas, and various areas of the gut that were higher than those measured in their wild-type controls (64). Studies with tachykinin and kinin receptor antagonists provided information regarding the mechanism that causes increased inflammation in NEP(—/—) mice. Thus, icatibant, a B2 receptor antagonist abolished and SR 140333, an NK1 receptor antagonist, markedly reduced the increased baseline levels of plasma extravasation in NEP(—/—) mice (64). Similar findings were obtained if plasma extravasation was measured 10–15 min after the pharmacological blockade of NEP with phosphoramidon (64). These observations suggested that in mice, (a) kinins are continuously formed from their precursors, possibly by constitutively activated kallikreins; (b) NEP is a major factor controlling kinin levels, and the absence of NEP leads to uncontrolled inflammation mediated by kinins; and (c) the inflammatory response caused by uncontrolled kinin levels is mediated mainly by neurogenic inflammatory mechanisms. The study of the role of peptidases in the regulation of inflammation under baseline conditions might provide new insights into the mechanisms of initiation and termination of inflammatory responses, particularly at the vascular level.
Meeting report: 2nd workshop of the peptide ADME discussion group
Published in Xenobiotica, 2021
Anders Sonesson, Inga Bjørnsdottir, Jesper Kammersgaard Christensen
In contrast to small molecules, the proteolytic cleavage is the predominant metabolisation process of peptides. Special attention needs to be paid on the peptidase neprilysin (NEP, neutral endopeptidase). This metallopeptidase is expressed on neutrophils, thus highly active in blood (Antczak et al. 2001). In addition, the cleavage pattern of this peptidase is broad covering many different peptide hormones – more than 50 different natural substrates are described (Bayes-Genis et al. 2016). We could show within an in vivo imaging study in xenograft-tumour bearing mice, that cleavage of a radioactive labelled peptidic radiotracer by neprilysin leads to a low tumour uptake. The performance of the tracer could be significantly improved by co-administration of phosphoramidon, a well-known inhibitor of neprilysin. By establishing a screening assay for neprilysin degradation, a straight-forward peptide optimization could be performed to enhance the stability of the peptidic radiotracer, and hence lead to increased tumour uptake of the tracer. A systematic investigation of the neprilysin cleavage pattern was started to improve the medicinal chemistry optimization process.
STAT1 participates in the induction of substance P expression in airway epithelial cells by respiratory syncytial virus
Published in Experimental Lung Research, 2020
Yu-Long Luo, Sheng Wang, Zhi-Xin Fang, Yi-Chu Nie, Li-Ting Zhang, Chu-Qin Huang, Li Long, Ke-Fang Lai
BEAS-2B cells were inoculated with RSV virus solutions with multiplicity of infection (MOI) values of 1, 0.1, and 0.01, or RSV virus solutions (MOI = 1) that were heat-inactivated (at 65 °C) or UV-inactivated. The culture supernatant from uninfected Hep-2 cells was used as mock. After 2 h of adsorption, the solutions were discarded, and fresh culture medium was added for continuing culture. If samples were used to measure the SP protein level, protease inhibitor cocktail (v:v 1:400) (Sigma, P1860) and 10 μM of the NEP inhibitor phosphoramidon (Amresco, J585) were simultaneously added to the culture medium. IFN-α (20 ng/ml) (Genscript, Z03003), STAT1 inhibitor fludarabine (1 μM) (Selleck, S1491), recombinant RSV A2 G protein (2, 10 μg/ml) (Sino Biological, 11070-V08H), and fractalkine (10, 50 ng/ml) (Prospec, CHM-235) were added to the corresponding treatment groups, and the culture supernatant was collected for testing after 48 h or 72 h of treatment. Human bronchial epithelial cells (LONZA, CC-2541) monolayer cultured in Bronchial Epithelial Cell Growth Medium (BEGMTM, LONZA, CC3170) was differentiated at the air-liquid interface for 21 days. Then the cell culture was inoculated with mock, RSV (MOI = 1) or fludarabine (1 μM) in Bronchial Epithelial Cell Growth Basal Medium (BEBMTM, LONZA, CC3171) for 48 hours to test the effect of STAT1 in RSV infection-induced SP expression.
Potent neutralizing monoclonal antibodies against Ebola virus isolated from vaccinated donors
Published in mAbs, 2020
Pengfei Fan, Xiangyang Chi, Guodong Liu, Guanying Zhang, Zhengshan Chen, Yujiao Liu, Ting Fang, Jianmin Li, Logan Banadyga, Shihua He, Changming Yu, Xiangguo Qiu, Wei Chen
To generate GPcl, GP∆Muc was digested by thermolysin as previously described with a minor modification.27,44 Thermolysin (T7902; Sigma-Aldrich) was added to a solution of 2 mg/mL GP∆Muc in PBS at a final concentration of 0.5 mg/mL and incubated for 1 h at 37°C. The reaction was stopped by phosphoramidon at a final concentration of 0.5 mM, and then the buffer was exchanged for PBS using an Amicon Ultra 0.5 mL-50 kDa cutoff centrifugal filter unit (UFC5050; Merck). The concentrated solution was filtered through 0.20-μm microfilters (SLLGR04NL; Merck) and purified on a Superdex 200 Increase 10/300 GL gel column (28990944; GE Healthcare).