Removal of adsorbed elastase from partially digested elastin.
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ABSTRACT:
SUBMITTER: Fromageot HP
PROVIDER: S-EPMC1172896 | biostudies-other | 1976 Jun
REPOSITORIES: biostudies-other
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Project description:Stimulating or maintaining the proliferative capacity of postnatal mammalian cardiomyocytes is a major challenge to cardiac regeneration. Previously, it is found that fetal cardiac extracellular matrix (ECM) can promote neonatal rat cardiomyocyte proliferation in vitro better than neonatal or adult ECM. It is hypothesized that partial digestion of adult ECM (PD-ECM) would liberate less crosslinked components that promote cardiomyocyte proliferation, similar to fetal ECM. Neonatal rat cardiac cells are seeded onto substrates coated with adult rat cardiac ECM that has been solubilized in pepsin-HCl for 1, 3, 6, 12, 24, or 48 h. Cardiomyocyte proliferation and fold-change in numbers from 1 to 5 d are highest on 1 and 3 h PD-ECM compared to other conditions. Sarcomeres tend to mature on 24 and 48 h PD-ECM where low proliferation is observed. 3 h PD-ECM is primarily composed of Fibrillin-1, Fibrinogen, and Laminins while 48 h PD-ECM is dominated by Collagen I. Our results suggest that adult ECM retains regenerative cues that may be masked by more abundant, mature ECM components. PD-ECM provides a simple yet powerful approach to promoting cardiomyocyte proliferation.
| S-EPMC4504755 | biostudies-literature
Project description:Many fundamental cellular and extracellular processes in the body are mediated by enzymes. At the single molecule level, enzyme activity is influenced by mechanical forces. However, the effects of mechanical forces on the kinetics of enzymatic reactions in complex tissues with intact extracellular matrix (ECM) have not been identified. Here we report that physiologically relevant macroscopic mechanical forces modify enzyme activity at the molecular level in the ECM of the lung parenchyma. Porcine pancreatic elastase (PPE), which binds to and digests elastin, was fluorescently conjugated (f-PPE) and fluorescent recovery after photobleach was used to evaluate the binding kinetics of f-PPE in the alveolar walls of normal mouse lungs. Fluorescent recovery after photobleach indicated that the dissociation rate constant (k(off)) for f-PPE was significantly larger in stretched than in relaxed alveolar walls with a linear relation between k(off) and macroscopic strain. Using a network model of the parenchyma, a linear relation was also found between k(off) and microscopic strain on elastin fibers. Further, the binding pattern of f-PPE suggested that binding sites on elastin unfold with strain. The increased overall reaction rate also resulted in stronger structural breakdown at the level of alveolar walls, as well as accelerated decay of stiffness and decreased failure stress of the ECM at the macroscopic scale. These results suggest an important role for the coupling between mechanical forces and enzyme activity in ECM breakdown and remodeling in development, and during diseases such as pulmonary emphysema or vascular aneurysm. Our findings may also have broader implications because in vivo, enzyme activity in nearly all cellular and extracellular processes takes place in the presence of mechanical forces.
| S-EPMC2965944 | biostudies-literature
Project description:BackgroundDuring the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin.MethodsMonoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra- and inter-assay variation. The NE specificity was demonstrated using elastin cleaved in vitro with matrix metalloproteinases (MMPs), cathepsin G (CatG), NE and intact elastin. Clinical relevance was assessed by measuring levels of NE-generated elastin fragments in serum of patients diagnosed with idiopathic pulmonary fibrosis (IPF, n = 10) or lung cancer (n = 40).ResultsAnalyte recovery of EL-NE for human serum was between 85% and 104%, the analyte was stable for four freeze/thaw cycles and after 24 h storage at 4°C. EL-NE was specific for NE-degraded elastin. Levels of NE-generated elastin fragments for elastin incubated in the presence of NE were 900% to 4700% higher than those seen with CatG or MMP incubation or in intact elastin. Serum levels of NE-generated elastin fragments were significantly increased in patients with IPF (137%, p = 0.002) and in patients with lung cancer (510%, p < 0.001) compared with age- and sex-matched controls.ConclusionsThe EL-NE assay was specific for NE-degraded elastin. The EL-NE assay was able to specifically quantify NE-degraded elastin in serum. Serum levels of NE-degraded elastin might be used to detect excessive lung tissue degradation in lung cancer and IPF.
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Project description:A microassay for elastase activity has been designed. Microtubes are coated with soluble elastin substrate by passive adsorption to the tube walls. On contact with enzymes in test samples, radioactivity is released into the sample buffer, which is then transferred to vials containing scintillation fluid without further processing. Porcine pancreatic elastase (EC 3.4.21.11) is easily detected in concentrations down to 8 micrograms/litre. The interassay coefficient of variation (CV) was below 10% for values above 50 micrograms/litre. The assay is rapid, precise, economical and sensitive.
| S-EPMC1153386 | biostudies-other
Project description:We have investigated the effect of human lung elastin on the inhibition of human leucocyte elastase by human alpha 1-proteinase inhibitor and bronchial inhibitor. Elastin was unable to dissociate the elastase-inhibitor complexes during the 150 min of the elastolysis reaction. When elastase was added to mixtures of elastin and alpha 1-proteinase inhibitor, it was fully bound to the latter. The competition between elastin and bronchial inhibitor was also in favour of the latter, but a 1.5 molar excess of inhibitor over elastase was required to achieve total binding of the enzyme. About 25% of elastin-bound elastase was found to be resistant to the inhibitory effect of alpha 1-proteinase inhibitor. The major isoenzyme and the mixture of the three minor isoenzymes of elastase exhibited similar behaviour. By contrast, bronchial inhibitor was as efficient in inhibiting the elastin-bound elastase as it was in inhibiting the free enzyme. This inhibitor was also able to inhibit fully the fraction of elastin-bound elastase that was resistant to alpha 1-proteinase inhibitor. We also describe a rapid procedure for the isolation of gram quantities of alpha 1-proteinase inhibitor.
| S-EPMC1147125 | biostudies-other
Project description:BackgroundElastic fibers are composed primarily of the protein elastin and they provide reversible elasticity to the large arteries. Degradation of elastic fibers is a common histopathology in aortic aneurysms. Pentagalloyl glucose (PGG) has been shown to bind elastin and stabilize elastic fibers in some in vitro studies and in vivo models of abdominal aortic aneurysms, however its effects on native arteries are not well described.ObjectivePerform detailed studies of the biomechanical effects of PGG on native arteries and the preventative capabilities of PGG for elastin degraded arteries.MethodsWe treated mouse carotid arteries with PGG, elastase (ELA), and PGG+ELA and compared the wall structure, solid mechanics, and fluid transport properties to untreated (UNT) arteries.ResultsWe found that PGG alone decreased compliance compared to UNT arteries, but did not affect any other structural or biomechanical measures. Mild (30 sec) ELA treatment caused collapse and fragmentation of the elastic lamellae, plastic deformation, decreased compliance, increased modulus, and increased hydraulic conductance of the arterial wall compared to UNT. PGG+ELA treatment partially protected from all of these changes, in particular the plastic deformation. PGG mechanical protection varied considerably across PGG+ELA samples and appeared to correlate with the structural changes.ConclusionsOur results provide important considerations for the effects of PGG on native arteries and a baseline for further biomechanical studies on preventative elastic fiber stabilization.
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Project description:Copper-based catalysts gain activity through the presence of poorly coordinated Cu atoms and incomplete oxidation at the surface. The catalytic mechanisms can in principle be observed by controlled dosing of reactants to single-crystal substrates. However, the interconnected influences of surface defects, partial oxidation, and adsorbate coverage present a large matrix of conditions that have not been fully explored in the literature. We recently characterized oxygen and carbon monoxide coadsorption on Cu(111), a nominally defect-free surface, and now extend our study to the stepped surface Cu(211). Temperature-programmed desorption of CO adsorbed to bare metal surfaces confirms that two sites dominate desorption from a saturated layer: atop terrace atoms of local (111) character and atop step edge atoms with CO bound more strongly to the latter. At low coverage, discrete CO resonances in reflection adsorption infrared spectra can be assigned to these sites: 2077 cm-1 for extended (111) terraces, 2093 cm-1 for step sites, and additional kink-adsorbed molecules at 2110 cm-1. With increasing coverage, in contrast to Cu(111), the infrared spectral features on Cu(211) evolve and shift as a consequence of dipole-dipole coupling between differentially occupied types of sites. Auger electron spectroscopy shows that exposure to background O2 oxidizes the (211) surface at a rate nearly 1 order of magnitude greater than (111); we argue that the resulting surface is stoichiometric Cu2O, as previously found for Cu(111). This oxide binds CO less strongly than the bare metal and the underlying crystal cut continues to influence the adsorption sites available to CO. On oxidized (111) terraces, broad absorption peaks at 2115-2120 cm-1; on oxidized Cu(211), CO adsorbed to step sites appears as a resolved secondary peak at 2144 cm-1. This suite of spectroscopic signatures, obtained under carefully controlled conditions, will help to determine the origin and fate of adsorbed species in future studies of reaction mechanisms on copper.
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Project description:Swine wastewater contains high concentrations of organic compounds, nutrients (nitrogen and phosphorus), heavy metals, and residual antibiotics, amongst others, that have negative impacts on the water environment. The main aim of this work was to remove nutrients from anaerobically digested swine wastewater using an intermittent cycle extended aeration system (ICEAS). The effects of operational parameters such as cycle time, organic loading rate, C/N ratio, and aeration/mixing ratio on the pollutant removal efficiencies of ICEAS were studied and compared with the performance of a conventional sequencing batch reactor (SBR). The following optimal conditions were obtained: cycle time, 6 h; organic loading rate, 0.86 kg COD m-3 day-1; C/N ratio, 2.49-2.82; and aeration/mixing ratio, 1.57. The pH was maintained in the range of 6.0-8.0. The total organic carbon (TOC), total nitrogen (TN), ammonium (NH4 +), total phosphorus (TP), and color removal efficiencies of ICEAS were higher than those of the conventional SBR, with removal efficiencies of 95.22, 88.29, 97.69, 85.81, and 97.84%, respectively, compared to 94.34, 81.16, 94.15, 77.94, and 96.95%, respectively, observed in the SBR. TOC, TN, NH4 +, TP, and the color removal efficiencies of ICEAS were higher by 0.88, 7.13, 3.54, 7.87, and 0.95%, respectively, than the conventional SBR. The good results from this study show that ICEAS is a promising technology for the removal of organic contaminants and nutrients from anaerobically digested swine wastewater and that the effluent water quality meets the Vietnamese discharge standard (QCVN 62-MT:2016/BTNMT) for swine wastewater effluents.
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Project description:Intact, thioglycollate-stimulated murine macrophages cultured on an insoluble [3H]-elastin substratum progressively hydrolysed the elastin. Cell lysates had little activity. We compared the effect of various proteinase inhibitors on elastinolysis by either live cells or cell-free, elastase-rich conditioned medium. Only known inhibitors of macrophage elastase blocked the activity of elastase-rich cell-conditioned medium whereas inhibitors of cathepsin B also suppressed intact cell activity. Serum proteinase inhibitors blocked cell-derived soluble elastase activity but not intact cell elastolytic activity. We also observed that plasminogen added to the cell cultures markedly increased elastinolysis by live macrophages or cell-free elastase-rich medium. Purified plasmin alone had no measurable effect on native elastin. Additional experiments indicated that the plasmin enhancement was due to elastin-dependent activation of latent macrophage elastase. These results indicate that live macrophage elastinolysis is a co-operative process involving multiple proteinases, especially a cysteine proteinase(s) and elastase. Plasmin may be a physiological activator of latent macrophage elastase.
| S-EPMC1144235 | biostudies-other
Project description:TFIIS stimulates RNA cleavage by RNA polymerase II and promotes the resolution of backtracking events. TFIIS acts in the chromatin context, but its contribution to the chromatin landscape has not yet been investigated. Co-transcriptional chromatin alterations include subtle changes in nucleosome positioning, like those expected to be elicited by TFIIS, which are elusive to detect. The most popular method to map nucleosomes involves intensive chromatin digestion by micrococcal nuclease (MNase). Maps based on these exhaustively digested samples miss any MNase-sensitive nucleosomes caused by transcription. In contrast, partial digestion approaches preserve such nucleosomes, but introduce noise due to MNase sequence preferences. A systematic way of correcting this bias for massively parallel sequencing experiments is still missing.To investigate the contribution of TFIIS to the chromatin landscape, we developed a refined nucleosome-mapping method in Saccharomyces cerevisiae. Based on partial MNase digestion and a sequence-bias correction derived from naked DNA cleavage, the refined method efficiently mapped nucleosomes in promoter regions rich in MNase-sensitive structures. The naked DNA correction was also important for mapping gene body nucleosomes, particularly in those genes whose core promoters contain a canonical TATA element. With this improved method, we analyzed the global nucleosomal changes caused by lack of TFIIS. We detected a general increase in nucleosomal fuzziness and more restricted changes in nucleosome occupancy, which concentrated in some gene categories. The TATA-containing genes were preferentially associated with decreased occupancy in gene bodies, whereas the TATA-like genes did so with increased fuzziness. The detected chromatin alterations correlated with functional defects in nascent transcription, as revealed by genomic run-on experiments.The combination of partial MNase digestion and naked DNA correction of the sequence bias is a precise nucleosomal mapping method that does not exclude MNase-sensitive nucleosomes. This method is useful for detecting subtle alterations in nucleosome positioning produced by lack of TFIIS. Their analysis revealed that TFIIS generally contributed to nucleosome positioning in both gene promoters and bodies. The independent effect of lack of TFIIS on nucleosome occupancy and fuzziness supports the existence of alternative chromatin dynamics during transcription elongation.
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