An inhibitor of collagenase from human amniotic fluid. Purification, characterization and action on metalloproteinases.
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ABSTRACT:
SUBMITTER: Murphy G
PROVIDER: S-EPMC1162867 | biostudies-other | 1981 Apr
REPOSITORIES: biostudies-other
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Project description:Amniotic fluid-derived products are a promising resource for cell therapy and tissue engineering due to their anti-inflammatory, angiogenic, and antifibrotic properties. Human amniotic fluid (hAF) has been used in medical applications such as wound healing, skin disorders, and ophthalmic conditions. Recently, we demonstrated that hAF is an effective treatment for myocardial ischemia-reperfusion injury in adult rats. However, the protein composition of full-term acellular hAF has remained poorly characterized. To uncover the biologically active components underlying hAF's cardioprotective effects, we conducted a global proteomic analysis of hAF collected from six patients at full-term cesarean sections. Previously shown to improve cardiac function in ischemic rats, these samples were analyzed by using tandem mass spectrometry. We identified 657 proteins, including 148 unique to the deep learning platform Inferys. Bioinformatic analysis revealed that these proteins are involved in immunity, inflammatory responses, cell adhesion, and apoptotic signaling pathways. In addition, these proteins were highly modified, with methylation and deamidation being the most abundant modifications. This study represents the first mass-spectrometry-based characterization of full-term, acellular hAF, suggesting that hAF offers a wide array of immune-modulating proteins working together to provide robust cardioprotection and a valuable treatment for ischemia-reperfusion injury.
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Project description:Pulmonary surfactant (PS) reduces surface tension at the air-liquid interface in the alveolar epithelium of the lung, which is required for breathing and for the pulmonary maturity of the developing foetus. However, the origin of PS had never been thoroughly investigated, although it was assumed to be secreted from the foetal developing lung. Human amniotic membrane (hAM), particularly its epithelial cell layer, composes the amniotic sac enclosing the amniotic fluid. In this study, we therefore aimed to investigate a potential contribution of the cellular components of the hAM to pulmonary surfactant found in amniotic fluid. We identified that cells within the native membrane contain lamellar bodies and express all four surfactant proteins as well as ABCA3. Lipidomic profiling by nanoESI - MS/MS revealed the presence of the essential lipid species as found in PS. Also, the biophysical activity of conditioned cell culture supernatant obtained from hAM was tested with captive bubble surfactometry. hAM supernatant showed the ability to reduce surface tension, similar to human PS obtained from bronchoalveolar lavage. This means that hAM produces the essential PS-associated components and can therefore contribute as second potential source of PS in amniotic fluid aside from the foetal lung.
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Project description:Mycotoxin exposure assessments through biomonitoring studies, based on the analysis of amniotic fluid, provides useful information about potential exposure of mothers and fetuses to ubiquitous toxic metabolites that are routinely found in food and the environment. In this study, amniotic fluid samples (n = 86) were collected via abdominal amniocentesis at 15-22 weeks of gestation from pregnant women with a high risk of chromosomal anomalies or genetic fetal defects detected during 1st trimester prenatal screening. These samples were analyzed for the presence of the most typical Aspergillus, Penicillium and Fusarium mycotoxins, with a focus on aflatoxins, ochratoxins and trichothecenes, using the LC-FLD/DAD method. The results showed that the toxin was present in over 75% of all the tested samples and in 73% of amniotic fluid samples from fetuses with genetic defects. The most frequently identified toxins were nivalenol (33.7%) ranging from <LOQ to 4037.6 ng/mL, and aflatoxins (31.4%), including aflatoxin G1, ranging from <LOQ to 0.4 ng/mL. Ochratoxin A and deoxynivalenol were identified in 26.7% and 27.9% of samples, respectively. Bearing in mind the above, the detection of mycotoxin levels in amniotic fluid is useful for the estimation of overall risk characterization with an attempt to link the occurrence of fetal abnormalities with exposure to mycotoxins in utero.
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Project description:1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32mug of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5-8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25 degrees C, producing the two characteristic products TC(A)((3/4)) and TC(B)((1/4)). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25 degrees C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37 degrees C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the alpha-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37 degrees C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins alpha(2)-macroglobulin and beta(1)-anti-collagenase both inhibited the enzyme, but alpha(1)-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.
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Project description:On purification, human fibroblast collagenase breaks down into two major forms (Mr22,000 and Mr 27,000) and one minor form (Mr 25,000). The most likely mechanism is autolysis, although the presence of contaminating enzymes cannot be excluded. From N-terminal sequencing studies, the 22,000-Mr fragment contains the active site; differential binding to concanavalin A shows the 25,000-Mr fragment is a glycosylated form of the 22,000-Mr fragment. These low-Mr forms can be separated by Zn2+-chelate chromatography. An activity profile of this column, combined with data from substrate gels, indicates no activity against collagen in the 22,000-Mr and 25,000-Mr forms, but rather, activity casein and gelatin. The 27,000-Mr form has no activity. The 22,000/25,000-Mr form can act as an activator for collagenase in a similar way to that reported for stromelysin. The activity of the 22,000/25,000-Mr form is not inhibited by the tissue inhibitor of metalloproteinases (TIMP). The 27,000-Mr C-terminal part of the collagenase molecule therefore appears to be important in maintaining the substrate-specificity of the enzyme, and also plays a role in the binding of TIMP.
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Project description:Fetal wounds repair by regeneration rather than wound healing and the environment is dominated by amniotic fluid. We are looking at early transcriptional regulation of keratinocytes cultured in amniotic fluid in vitro. Keratinocytes were isolated and expanded to passage three after which they were starved in DMEM for 12h then cultured for 24h in human amniotic fluid (50%), fcs (50%) or DMEM alone for another 24h. N=2, pooled replicates per CEL-file.
2021-08-25 | GSE182704 | GEO
Project description:Amniotic fluid-derived products are a promising resource for cell therapy and tissue engineering due to their anti-inflammatory, angiogenic, and antifibrotic properties. Human amniotic fluid (hAF) has been used in medical applications such as wound healing, skin disorders, and ophthalmic conditions. Recently, we demonstrated that hAF is an effective treatment for myocardial ischemia-reperfusion injury in adult rats. However, the protein composition of full-term acellular hAF has remained poorly characterized. To uncover the biologically active components underlying hAF’s cardioprotective effects, we conducted a global proteomic analysis of hAF collected from six patients at full-term cesarean sections. Previously shown to improve cardiac function in ischemic rats, these samples were analyzed by using tandem mass spectrometry. We identified 657 proteins, including 148 unique to the deep learning platform Inferys. Bioinformatic analysis revealed that these proteins are involved in immunity, inflammatory responses, cell adhesion, and apoptotic signaling pathways. In addition, these proteins were highly modified, with methylation and deamidation being the most abundant modifications. This study represents the first mass-spectrometry-based characterization of full-term, acellular hAF, suggesting that hAF offers a wide array of immune-modulating proteins working together to provide robust cardioprotection and a valuable treatment for ischemia-reperfusion injury.
2025-05-07 | PXD053775 | Pride
Project description:BackgroundMesenchymal stem cell-derived conditioned medium (MSC-CM) has emerged as a promising cell-free tool for restoring degenerative diseases and treating traumatic injuries. The present study describes the effect of selenium as a reactive oxygen species (ROS) scavenger and its additive effect with basic fibroblast growth factor (bFGF) on in vitro expansion of amniotic fluid (AF)-MSCs and the paracrine actions of AF-MSC-CM as well as the associated cellular and molecular mechanisms.MethodsIn this study, we obtained CM from human AF-MSCs cultured with selenium. The stemness of selenium-treated AF-MSCs was evaluated by cell growth and differentiation potential. Human fibroblasts were treated with AF-MSC-CM and analyzed for cell signaling changes. For in vivo wound healing assay, ICR mice with a full-thickness skin wound were used.ResultsSelenium played a critical role in in vitro expansion of AF-MSCs through activation of the AKT-ERK1/2, Smad2, and Stat3 signaling pathways along with inactivation of GSK3β. When administered together with bFGF, it showed remarkable effect in inhibiting ROS accumulation and preserving their multipotency. Proliferation and migration of human dermal fibroblasts and in vivo wound healing were improved in the CMs derived from AF-MSCs exposed to selenium and bFGF, which was caused by the Smad2, AKT-MEK1/2-ERK, and NFκB signaling triggered by the paracrine factors of AF-MSCs, such as TGF-β, VEGF, and IL-6. Our results suggest the following: (a) supplementation of selenium in AF-MSC culture contributes to in vitro expansion and preservation of multipotency, (b) ROS accumulation causes progressive losses in proliferative and differentiation potential, (c) the separate activities of bFGF and selenium in MSCs exert an additive effect when used together, and (d) the additive combination improves the therapeutic effects of AF-MSC-derived CMs on tissue repair and regeneration.ConclusionAntioxidants, such as selenium, should be considered as an essential supplement for eliciting the paracrine effects of MSC-CMs.
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Project description:Despite increasing interest in human amniotic fluid cells, very little is known about the regulation and function of p53 in this cell type. In this study, we show that undifferentiated human amniotic fluid cells express p53, yet at lower levels than in cancer cells. The p53 protein in amniotic fluid cells is mainly localized in the nuclei, however, its antiproliferative activity is compromised in these cells. Igf2, a maternal imprinted gene, and c-jun, a proto-oncogene, are regulated by p53 in these cells. DNA damage leads to an increase in p53 abundance in human amniotic fluid cells and to transcriptional activation of its target genes. Interestingly, cell differentiation toward the neural lineage leads to p53 induction as differentiation progresses.
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