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International Journal of Molecular... Aug 2020The aim of this study was to determine the concentration of galectin-3, PINP and PIIINP in patients with metabolic syndrome (MS) and atrial fibrillation (AF) with an...
The aim of this study was to determine the concentration of galectin-3, PINP and PIIINP in patients with metabolic syndrome (MS) and atrial fibrillation (AF) with an assessment of the relationship with severity of left atrium fibrosis. A total of 480 subjects were included in the case-control study: MS patients ( = 337), 176 of whom had AF, 72 patients with AF without MS and 71 healthy subjects. Galectin-3, PINP and PIIINP blood concentrations and metabolic parameters were compared with the severity of left atrium fibrosis, measured by CARTO3. Galectin-3 in AF and MS patients is higher than in MS without AF and in healthy subjects (10.3 (4.8-15.4), 5.1 (4.3-8.8), 3.2 (2.4-4.2) ng/mL, < 0.0001). Galectin-3 serum concentration in AF patients with MS is higher than in patients without MS: 10.3 (4.8-15.4), 6.8 (5.2-8.1) ng/mL, = 0.0001. PINP and PIIINP concentration were higher in patients with AF and MS than in MS without AF: 3499.1 (2299.2-4567.3), 2130.9 (1425.3-2861.8) pg/mL, < 0.0001, 94.9 (64.8-123.5), 57.6 (40.5-86.9) ng/mL, < 0.0001. Galectin-3 correlates with PINP (r = 0.496, < 0.001) and PIIINP concentration ( = 0.451, < 0.0001). The correlation between galectin-3, PINP and the severity of left atrium fibrosis was found (r = 0.410, < 0.001; = 0.623, < 0.001). Galectin-3 higher than 12.6 ng/mL increased the risk of AF more than five-fold. High galectin-3, PINP and PIIINP concentrations were associated with heart remodeling in MS patients and increased the risk of AF.
Topics: Adult; Atrial Fibrillation; Biomarkers; Female; Fibrosis; Galectin 3; Humans; Male; Metabolic Syndrome; Middle Aged; Peptide Fragments; Procollagen; ROC Curve; Risk Factors
PubMed: 32784491
DOI: 10.3390/ijms21165689 -
Analytical and Bioanalytical Chemistry Jul 2023Recombinant collagen production, especially using yeasts as expression systems, could represent a promising alternative over traditional extractive methods from animal...
Development of a rapid, efficient, and reusable magnetic bead-based immunocapture system for recombinant human procollagen type II isolation from yeast fermentation broth.
Recombinant collagen production, especially using yeasts as expression systems, could represent a promising alternative over traditional extractive methods from animal sources, offering controllable, scalable, and high-quality products. Monitoring the efficiency and efficacy of procollagen/collagen expression, especially in the initial fermentation phases, can be difficult and time consuming, as biological matrices necessitate purification and commonly used analytical methods are only partially informative. We propose a straightforward, efficient, and reusable immunocapture system able to specifically isolate human procollagen type II from fermentation broths and to release it in few experimental steps. A recovered sample allows for a detailed characterization providing information on structural identity and integrity, which can strongly support the monitoring of fermentation processes. The immunocapture system relies on the use of protein A-coated magnetic beads which have been functionalized and cross-linked with a human anti-procollagen II antibody (average immobilization yield of 97.7%) to create a stable and reusable support for the specific procollagen fishing. We set up the binding and release conditions ensuring specific and reproducible binding with a synthetic procollagen antigen. The absence of non-specific interaction with the support and binding specificity was demonstrated, and the latter was also confirmed by a peptide mapping epitope study in reversed-phase liquid chromatography high-resolution mass spectrometry (RP-LC-HRMS). The bio-activated support proved to be reusable and stable over 21 days from the initial use. Finally, the system was successfully tested on a raw yeast fermentation sample to provide a proof of concept of the applicability within recombinant collagen production.
Topics: Animals; Humans; Collagen Type II; Saccharomyces cerevisiae; Fermentation; Collagen; Procollagen; Magnetic Phenomena
PubMed: 37246979
DOI: 10.1007/s00216-023-04752-1 -
European Journal of Biochemistry Oct 1979A combination of dodecylsulphate/polyacrylamide gel electrophoresis and fluorography has been used to quantify the synthesis of type I and type III collagens by...
A combination of dodecylsulphate/polyacrylamide gel electrophoresis and fluorography has been used to quantify the synthesis of type I and type III collagens by periodontal ligament in situ and periodontal-ligament fibroblasts in vitro. The separation of 14C-labelled collagen alpha chains was achieved by introducing an interrupted reduction step, and the total radioactivity in the alpha-chain bands related to the fluorographic response by a series of standard curves. From these curves an accurate assessment of the relative amounts of type I and III collagen synthesized could be made. The same system also allowed the synthesis and processing of the respective procollagens to be analyzed. For the study in vivo, 200-g male rats were injected with 2 mCi [14C]glycine and killed 0.5-6 h later. Periodontal ligament was dissected from the mandibular molars and the newly-synthesized collagens extracted with 0.45 M sodium chloride. In the study in vitro, confluent monkey periodontal-ligament fibroblasts were cultured in the presence of [14C]proline and [14C]glycine. Analysis of labelled collagens showed a rapid conversion of type I procollagen to collagen but type III collagen was recovered as a procollagen intermediate both in vitro and in vivo. Analysis of duplicate samples after pepsin digestion showed type III collagen synthesis to comprise 15% of the total collagen synthesized in vivo and 20% in early subcultures in vitro. However, the proportion of type III synthesized by the fibroblasts decreased on subculturing. The data demonstrate that fibroblasts in vitro retain the basic characteristics of collagen synthesis and procollagen processing found in vivo, but the overall phenotypic expression of the cells is not stable in culture.
Topics: Animals; Collagen; Kinetics; Male; Pepsin A; Peptide Fragments; Periodontal Ligament; Procollagen; Rats
PubMed: 389626
DOI: 10.1111/j.1432-1033.1979.tb04200.x -
Scientific Reports Mar 2022Bone morphogenetic protein 1 (BMP1) belongs to the astacin/BMP1/tolloid-like family of zinc metalloproteinases, which play a fundamental role in the development and...
Bone morphogenetic protein 1 (BMP1) belongs to the astacin/BMP1/tolloid-like family of zinc metalloproteinases, which play a fundamental role in the development and formation of extracellular matrix (ECM). BMP1 mediates the cleavage of carboxyl terminal (C-term) propeptides from procollagens, a crucial step in fibrillar collagen fiber formation. Blocking BMP1 by small molecule or antibody inhibitors has been linked to anti-fibrotic activity in the preclinical models of skin, kidney and liver fibrosis. Therefore, we reason that BMP1 may be important for the pathogenesis of lung fibrosis and BMP1 could be a potential therapeutic target for progressive fibrotic disease such as idiopathic pulmonary fibrosis (IPF). Here, we observed the increased expression of BMP1 in both human IPF lungs and mouse fibrotic lungs induced by bleomycin. Furthermore, we developed an inducible Bmp1 conditional knockout (cKO) mouse strain. We found that Bmp1 deletion does not protect mice from lung fibrosis triggered by bleomycin. Moreover, we found no significant impact of BMP1 deficiency upon C-term propeptide of type I procollagen (CICP) production in the fibrotic mouse lungs. Based on these results, we propose that BMP1 is not required for lung fibrosis in mice and BMP1 may not be considered a candidate therapeutic target for IPF.
Topics: Animals; Bleomycin; Bone Morphogenetic Protein 1; Extracellular Matrix; Idiopathic Pulmonary Fibrosis; Mice; Procollagen
PubMed: 35361882
DOI: 10.1038/s41598-022-09557-3 -
The American Journal of Pathology Jul 1990The authors have determined the cell types producing alpha 1 (I), alpha 2 (I), alpha 1 (III), and alpha 1 (IV) procollagen gene transcripts in adult human liver by in...
The authors have determined the cell types producing alpha 1 (I), alpha 2 (I), alpha 1 (III), and alpha 1 (IV) procollagen gene transcripts in adult human liver by in situ hybridization with [35S]-labeled RNA probes. The liver specimens comprised a total of 20 biopsies with normal histology and biopsies with fibrosis or cirrhosis at different clinical stages and of heterogeneous origins. In normal liver, procollagen type I, III, and IV transcripts were detected in stromal and vascular mesenchymal cells of portal tracts and central veins, as well as in some perisinusoidal cells of the lobule. In fibrotic liver, increased levels of these procollagen mRNAs were observed in the same locations, and particularly enhanced in stromal cells of fibrotic septa and portal tracts, as well as in perisinusoidal cells. Expression of alpha 1 (IV) procollagen RNA was additionally found in some vascular endothelial and bile duct epithelial cells. Although previously suggested as the major source of liver collagens, hepatocytes showed no significant procollagen transcript levels in any of our samples. Thus, procollagen synthesis does not appear to be a function of hepatocytes, but rather of mesenchymal, endothelial, and bile duct epithelial cells in adult human liver. These findings may have implications for the development of specifically targeted antifibrotic therapies.
Topics: Adult; Aged; Humans; Liver; Liver Cirrhosis; Middle Aged; Procollagen; RNA, Messenger; Transcription, Genetic
PubMed: 2372043
DOI: No ID Found -
The Journals of Gerontology. Series A,... Jun 2021Circulating levels of procollagen type III N-terminal peptide (P3NP) may reflect increased fibrosis of skeletal muscle and other tissues with aging. Herein, we tested if...
BACKGROUND
Circulating levels of procollagen type III N-terminal peptide (P3NP) may reflect increased fibrosis of skeletal muscle and other tissues with aging. Herein, we tested if P3NP was associated with baseline and 7-year change in physical function.
METHOD
Participants (n = 400) were from the Long Life Family Study, a study of exceptional familial longevity. Plasma P3NP concentration was measured using a sandwich enzyme-linked immunosorbent assay (inter-assay coefficient of variation <5.5%). At baseline and 7-year follow-up visits, physical function was measured using the Short Physical Performance Battery (SPPB score 0-12), which consists of gait speed, balance, and chair-rise tests. Grip strength was measured using a handheld dynamometer. The association between log-transformed P3NP and physical function was examined using generalized estimating equations adjusted for familial relatedness, age, sex, height, weight, lifestyle characteristics, liver function, kidney function, lung function, and chronic disease prevalence.
RESULTS
Participants were aged 73.1 ± 15.2 years (range: 39-104), 54% female, had body mass index of 26.6 ± 4.3 kg/m2, and gait speeds of 1.0 ± 0.3 m/s. One standard deviation higher log-transformed P3NP was related to worse baseline SPPB score (β = -0.9points), gait speed (β = -0.05m/s), chair-rises per-second (β = -0.46chair-rises/10 seconds), and grip strength (β = -2.0kg; all p < .001). Higher P3NP was also associated with greater declines in gait speed (β = -1.41, p < .001) and transitioning to being unable to perform chair-rises (β = 0.41, p < .001) after 7 years.
CONCLUSION
Plasma P3NP may be a strong, novel biomarker of current and future physical function. Future research is needed to extend our findings to other cohorts and determine mechanisms underlying these associations.
Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Female; Geriatric Assessment; Humans; Longevity; Male; Middle Aged; Peptide Fragments; Physical Functional Performance; Procollagen
PubMed: 32794566
DOI: 10.1093/gerona/glaa197 -
The Journal of Biological Chemistry May 1983Procollagen IV was isolated from culture media of the mouse endodermal cell line PF-HR9. Some of the triple helical procollagen IV molecules were associated at their NH2...
Procollagen IV was isolated from culture media of the mouse endodermal cell line PF-HR9. Some of the triple helical procollagen IV molecules were associated at their NH2 ends to tetramers which were identified by electron microscopy, velocity sedimentation, and electrophoresis. The formation of these tetramers in cell cultures and from isolated procollagen IV molecules was investigated. After an initial noncovalent association, which is reversible, disulfide bonds form between molecules. Even alkylated molecules form disulfide-linked tetramers when exposed to a mixture of reduced and oxidized glutathione. This reaction requires an adequate concentration of procollagen and is not facilitated by added laminin, Ca2+, or Mg2+ ions. Cystine, as a normal constituent of cell culture media, interferes in tetramer assembly, presumably by forming mixed disulfides. Tetramers formed normally and under the influence of glutathione are similar, but probably not identical, and resemble those isolated from fragmented basement membranes. We conclude that the NH2 ends of procollagen IV molecules can associate into tetramers without the help of other molecules and that disulfide bridges subsequently stabilize the association in various ways.
Topics: Animals; Calcium; Cell Line; Endoderm; Female; Glutathione; Glycoproteins; Laminin; Macromolecular Substances; Magnesium; Mice; Microscopy, Electron; Pregnancy; Procollagen
PubMed: 6853554
DOI: No ID Found -
The Journal of Investigative Dermatology Sep 1987Regulation of collagen gene expression was studied in keloids and fibroblast cultures established from keloid biopsies from 9 patients. The collagen concentration in...
Regulation of collagen gene expression was studied in keloids and fibroblast cultures established from keloid biopsies from 9 patients. The collagen concentration in keloid tissue was not different from that in normal skin. The activities of 2 enzymes catalyzing intracellular collagen biosynthesis, prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT) were significantly elevated in the keloids, the mean increase in the former enzyme being 5-fold and in the latter 3-fold with respect to the controls. The mean procollagen production rate in the keloid fibroblasts was at the control level, with only 1 keloid cell line showing a procollagen synthesis rate higher than the mean value + 2 SD of the controls. The mean PH and GGT activities of the keloid fibroblasts were not elevated, but PH activity in 2 cell lines and GGT activity in 1 cell line were higher than the mean + 2 SD for the controls. Cellular type I, III, IV, and V procollagen mRNAs were measured by slot blot hybridization using specific human cDNA clones for the various collagen types. The amounts of type I, III, and V procollagen mRNAs corresponded to the ratios in which these collagen types are produced by fibroblasts. No synthesis of type IV procollagen mRNA by keloid fibroblasts was observed. The total amount of type I and III procollagen mRNAs correlated significantly (p less than 0.01) with the procollagen synthesis rate measured after radioactive labeling of the cells in the keloid and control fibroblasts, indicating that collagen production in these cells is mainly controlled by regulating the final steady state levels of collagen mRNA. The results suggest that fibroblasts isolated from keloids often synthesize normal amounts of collagen.
Topics: Cells, Cultured; Collagen; Fibroblasts; Gene Expression Regulation; Humans; Keloid; Procollagen; RNA, Messenger; Skin
PubMed: 3624897
DOI: 10.1111/1523-1747.ep12471056 -
Proceedings of the National Academy of... Nov 1980Interest in cell-associated collagens led others to isolate A and B collagen chains, also known as type V collagen, from many tissues, but only after pepsin cleavage....
Interest in cell-associated collagens led others to isolate A and B collagen chains, also known as type V collagen, from many tissues, but only after pepsin cleavage. Soluble precursors of these chains are synthesized in vitro by crop, a chicken embryo muscle tissue, and are converted by at least two processing steps from procollagens via intermediates to final forms which are large than the pepsin-derived A and B chains. Heterotrimeric, disulfide-bridged procollagen molecules corresponding to B2A exist. Components were separated by ion exchange chromatography, velocity sedimentation, and electrophoresis, and the relationships between them were established by sequential radioactive labeling and comparison of peptides generated by protease cleavage.
Topics: Animals; Chick Embryo; Disulfides; Pepsin A; Peptide Fragments; Procollagen; Solubility
PubMed: 6779280
DOI: 10.1073/pnas.77.11.6434 -
The Journal of Biological Chemistry Jul 1986Cultured human articular and costal chondrocytes were used as a model system to examine the effects of recombinant gamma-interferon (IFN-gamma) on synthesis of...
Cultured human articular and costal chondrocytes were used as a model system to examine the effects of recombinant gamma-interferon (IFN-gamma) on synthesis of procollagens, the steady state levels of types I and II procollagen mRNAs, and the expression of major histocompatibility complex class II (Ia-like) antigens on the cell surface. Adult articular chondrocytes synthesized mainly type II collagen during weeks 1-3 of primary culture, whereas types I and III collagens were also produced after longer incubation and predominated after the first subculture. Juvenile costal chondrocytes synthesized no detectable alpha 2(I) collagen chains until after week 1 of primary culture; type II collagen was the predominant species even after weeks of culture. The relative amounts of types I and II collagens synthesized were reflected in the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. In articular chondrocytes, the levels of alpha 1(I) procollagen mRNA were disproportionately low (alpha 1(I)/alpha 2(I) less than 1.0) compared with costal chondrocytes (alpha 1 (I)/alpha 2(I) approximately 2). Recombinant IFN-gamma (0.1-100 units/ml) inhibited synthesis of type II as well as types I and III collagens associated with suppression of the levels of alpha 1(I), alpha 2(I), and alpha 1(II) procollagen mRNAs. IFN-gamma suppressed the levels of alpha 1(I) and alpha 1(II) procollagen mRNAs to a greater extent than alpha 2(I) procollagen mRNA in articular but not in costal chondrocytes. Human leukocyte interferon (IFN-alpha) at 1000 units/ml suppressed collagen synthesis and procollagen mRNA levels to a similar extent as IFN-gamma at 1.0 unit/ml. In addition, IFN-gamma but not IFN-alpha induced the expression of HLA-DR antigens on intact cells. The lymphokine IFN-gamma could, therefore, have a role in suppressing cartilage matrix synthesis in vivo under conditions in which the chondrocytes are in proximity to T lymphocytes and their products.
Topics: Cartilage; Cartilage, Articular; Cells, Cultured; Collagen; Fibroblasts; Humans; Interferon-gamma; Male; Nucleic Acid Hybridization; Procollagen; RNA, Messenger; Recombinant Proteins; Ribs; Skin; Transcription, Genetic
PubMed: 3087985
DOI: No ID Found