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Sample records for activity collagen synthesis

  1. Collagen-Immobilized Lipases Show Good Activity and Reusability for Butyl Butyrate Synthesis.

    PubMed

    Dewei, Song; Min, Chen; Haiming, Cheng

    2016-11-01

    Candida rugosa lipases were immobilized onto collagen fibers through glutaraldehyde cross-linking method. The immobilization process has been optimized. Under the optimal immobilization conditions, the activity of the collagen-immobilized lipase reached 340 U/g. The activity was recovered of 28.3 % by immobilization. The operational stability of the obtained collagen-immobilized lipase for hydrolysis of olive oil emulsion was determined. The collagen-immobilized lipase showed good tolerance to temperature and pH variations in comparison to free lipase. The collagen-immobilized lipase was also applied as biocatalyst for synthesis of butyl butyrate from butyric acid and 1-butanol in n-hexane. The conversion yield was 94 % at the optimal conditions. Of its initial activity, 64 % was retained after 5 cycles for synthesizing butyl butyrate in n-hexane.

  2. The induction of the collagen capsule synthesis by Trichinella spiralis is closely related to protease-activated receptor 2.

    PubMed

    Park, Mi Kyung; Cho, Min Kyoung; Kang, Shin Ae; Kim, Bo Young; Yu, Hak Sun

    2016-10-30

    The muscle-stage larvae of the parasite Trichinella spiralis have the ability to survive within host muscle tissue by virtue of the formation a nurse cell-parasite complex, which is surrounded by collagen. The formation of the complex is initiated by excretory-secretory (ES) proteins produced by the parasite. To determine the mechanisms underlying collagen capsule formation, we investigated the expression levels of several types of collagen genes and TGF-βI signaling-related genes (Smad2 and Smad3) in muscle cells. Synthesis of type I, IV, and VI collagen, which are major constituents of the collagen capsule, significantly increased during T. spiralis infection. In addition, we found that expression of the protease-activated receptor 2 (PAR2) gene was significantly increased during this period. Expression levels of the collagen genes and TGF-βI, Smad2, and Smad3 were induced by ES proteins and a PAR2 agonist, whereas their enhanced expression levels were reduced by a PAR2 antagonist and serine protease inhibitors. To evaluate the involvement of PAR2 during T. spiralis infection in vivo, we infected wild-type and PAR2 knockout (KO) mice with T. spiralis. Expression levels of type I, IV, and VI collagen genes and TGF-βI signaling-related genes (Smad2 and Smad3) were also decreased in the PAR2 KO mice. Phosphorylation of Smad2/3, which was increased by T. spiralis infection, was significantly diminished in the PAR2 KO mice. In conclusion, ES proteins containing serine protease most likely activate collagen synthesis via PAR2 and TGF-βI signaling, and this event could influence collagen capsule formation.

  3. In vivo determination of arterial collagen synthesis in atherosclerotic rabbits

    SciTech Connect

    Opsahl, W.P.; DeLuca, D.J.; Ehrhart, L.A.

    1986-03-01

    Collagen and non-collagen protein synthesis rates were determined in vivo in tissues from rabbits fed a control or atherogenic diet supplemented with 2% peanut oil and 0.25% cholesterol for 4 months. Rabbits received a bolus intravenous injection of L-(/sup 3/H)-proline (1.0 mCi/kg) and unlabeled L-proline (7 mmoles/kg) in 0.9% NaCl. Plasma proline specific activity decreased only 20% over 5 hr and was similar to the specific activity of free proline in tissues. Thoracic aortas from atherosclerotic rabbits exhibited raised plaques covering at least 75% of the surface. Thoracic intima plus a portion of the media (TIM) was separated from the remaining media plus adventitia (TMA). Dry delipidated weight, total collagen content, and collagen as a percent of dry weight were increased significantly in the TIM of atherosclerotic rabbits. Collagen synthesis rates and collagen synthesis as a percent of total protein synthesis were likewise increased both in the TIM and in the abdominal aortas. No differences from controls either in collagen content or collagen synthesis rates were observed in the TMA, lung or skin. These results demonstrate for the first time in vivo that formation of atherosclerotic plaques is associated with increased rates of collagen synthesis. Furthermore, as previously observed with incubations in vitro, collagen synthesis was elevated to a greater extent than noncollagen protein synthesis in atherosclerotic aortas from rabbits fed cholesterol plus peanut oil.

  4. Pyropia yezoensis peptide promotes collagen synthesis by activating the TGF-β/Smad signaling pathway in the human dermal fibroblast cell line Hs27

    PubMed Central

    Kim, Cho-Rong; Kim, Young-Min; Lee, Min-Kyeong; Kim, In-Hye; Choi, Youn-Hee; Nam, Taek-Jeong

    2017-01-01

    Pyropia yezoensis (P. yezoensis) is a marine algae that exhibits antioxidant, anti-inflammatory, antitumor and anti-aging activities. In this study, we investigated the effects of the P. yezoensis peptide, PYP1-5, on collagen synthesis in the human dermal fibroblast cell line Hs27. Skin aging is related to reduced collagen production and the activities of multiple enzymes, including matrix metalloproteinases (MMPs), which degrade collagen structure in the dermis, and tissue inhibitor of tissue inhibitor of metalloproteinases (TIMPs), which inhibit the action of MMPs. While collagen synthesis is associated with a number of signaling pathways, we examined the increased collagen synthesis via the upregulation of the transforming growth factor-β (TGF-β)/Smad signaling pathway. Using MTS assay, we found that PYP1-5 did not affect cell viability. Moreover, we confirmed that PYP1-5 increased type 1 collagen expression using enzyme-linked immunosorbent assay (ELISA), western blot analysis and quantitative PCR. In addition, we identified changes in various enzymes, as well as the mechanisms behind the PYP1-5-induced collagen synthesis. PYP1-5 decreased the MMP-1 protein and mRNA levels, and increased the TIMP-1 and TIMP-2 protein and mRNA levels. In addition, PYP1-5 activated the TGF-β/Smad signaling pathway, which increased TGF-β1, p-Smad2 and p-Smad3 expression, while inhibiting Smad7, an inhibitor of the TGF-β/Smad pathway. Furthermore, PYP1-5 upregulated transcription factor specificity protein 1 (Sp1) expression, which is reportedly involved in type 1 collagen expression. These findings indicate that PYP1-5 activates the TGF-β/Smad signaling pathway, which subsequently induces collagen synthesis in Hs27 cells. PMID:27878236

  5. Solid-phase synthesis, characterization, and cellular activities of collagen-model nanodiamond-peptide conjugates.

    PubMed

    Knapinska, Anna M; Tokmina-Roszyk, Dorota; Amar, Sabrina; Tokmina-Roszyk, Michal; Mochalin, Vadym N; Gogotsi, Yury; Cosme, Patrick; Terentis, Andrew C; Fields, Gregg B

    2015-05-01

    Nanodiamonds (NDs) have received considerable attention as potential drug delivery vehicles. NDs are small (∼5 nm diameter), can be surface modified in a controllable fashion with a variety of functional groups, and have little observed toxicity in vitro and in vivo. However, most biomedical applications of NDs utilize surface adsorption of biomolecules, as opposed to covalent attachment. Covalent modification provides reliable and reproducible ND-biomolecule ratios, and alleviates concerns over biomolecule desorption prior to delivery. The present study has outlined methods for the efficient solid-phase conjugation of ND to peptides and characterization of ND-peptide conjugates. Utilizing collagen-derived peptides, the ND was found to support or even enhance the cell adhesion and viability activities of the conjugated sequence. Thus, NDs can be incorporated into peptides and proteins in a selective manner, where the presence of the ND could potentially enhance the in vivo activities of the biomolecule it is attached to.

  6. Solid-Phase Synthesis, Characterization, and Cellular Activities of Collagen-Model Nanodiamond-Peptide Conjugates

    PubMed Central

    Knapinska, Anna M.; Tokmina-Roszyk, Dorota; Amar, Sabrina; Tokmina-Roszyk, Michal; Mochalin, Vadym N.; Gogotsi, Yury; Cosme, Patrick; Terentis, Andrew C.; Fields, Gregg B.

    2015-01-01

    Nanodiamonds (NDs) have received considerable attention as potential drug delivery vehicles. NDs are small (~5 nm diameter), can be surface modified in a controllable fashion with a variety of functional groups, and have little observed toxicity in vitro and in vivo. However, most biomedical applications of NDs utilize surface adsorption of biomolecules, as opposed to covalent attachment. Covalent modification provides reliable and reproducible ND–biomolecule ratios, and alleviates concerns over biomolecule desorption prior to delivery. The present study has outlined methods for the efficient solid-phase conjugation of ND to peptides and characterization of ND–peptide conjugates. Utilizing collagen-derived peptides, the ND was found to support or even enhance the cell adhesion and viability activities of the conjugated sequence. Thus, NDs can be incorporated into peptides and proteins in a selective manner, where the presence of the ND could potentially enhance the in vivo activities of the biomolecule it is attached to. PMID:25753561

  7. Release of biologically active TGF-beta from airway smooth muscle cells induces autocrine synthesis of collagen.

    PubMed

    Coutts, A; Chen, G; Stephens, N; Hirst, S; Douglas, D; Eichholtz, T; Khalil, N

    2001-05-01

    In severe or chronic asthma, there is an increase in airway smooth muscle cell (ASMC) mass as well as an increase in connective tissue proteins in the smooth muscle layer of airways. Transforming growth factor-beta (TGF-beta) exists in three isoforms in mammals and is a potent regulator of connective tissue protein synthesis. Using immunohistochemistry, we had previously demonstrated that ASMCs contain large quantities of TGF-beta1-3. In this study, we demonstrate that bovine ASMC-derived TGF-beta associates with the TGF-beta latency binding protein-1 (LTBP-1) expressed by the same cells. The TGF-beta associated with LTBP-1 localizes TGF-beta extracellularly. Furthermore, plasmin, a serine protease, regulates the secretion of a biologically active form of TGF-beta by ASMCs as well as the release of extracellular TGF-beta. The biologically active TGF-beta released by plasmin induces ASMCs to synthesize collagen I in an autocrine manner. The autocrine induction of collagen expression by ASMCs may contribute to the irreversible fibrosis and remodeling seen in the airways of some asthmatics.

  8. Kaempferol inhibits fibroblast collagen synthesis, proliferation and activation in hypertrophic scar via targeting TGF-β receptor type I.

    PubMed

    Li, Hongwei; Yang, Liu; Zhang, Yuebing; Gao, Zhigang

    2016-10-01

    Hypertrophic scar (HPS) formation is a debilitating condition that results in pain, esthetic symptom and loss of tissue function. So far, no satisfactory therapeutic approach has been available for HPS treatment. In this study, we discovered that a natural small molecule, kaempferol, could significantly inhibit HPS formation in a mechanical load-induced mouse model. Our results also demonstrated that kaempferol remarkably attenuated collagen synthesis, proliferation and activation of fibroblasts in vitro and in vivo. Western blot analysis further revealed that kaempferol significantly down-regulated Smad2 and Smad3 phosphorylation in a dose-dependent manner. At last, we found that such bioactivity of kaempferol which resulted from the inhibition of TGF-β1/Smads signaling was induced by the selective binding of kaempferol to TGF-β receptor type I (TGFβRI). These findings suggest that kaempferol could be developed into a promising agent for the treatment of HPS or other fibroproliferative disorders.

  9. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

    SciTech Connect

    Ramp, W.K.; Lenz, L.G.; Galvin, R.J. )

    1991-05-01

    Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of ({sup 3}H)proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of ({sup 3}H)hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of ({sup 3}H)thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.

  10. Liver collagen synthesis in murine schistosomiasis.

    PubMed Central

    Dunn, M A; Rojkind, M; Warren, K S; Hait, P K; Rifas, L; Seifter, S

    1977-01-01

    Collagen synthesis was measured in liver slices obtained from mice with hepatosplenic schistosomiasis. Enlarged fibrotic livers from these mice contained 20 times more collagen than normal. This model of hepatic fibrosis results from an inflammatory granulomatous host response to Schistosoma mansoni ova in portal tracts, rather than from direct lover cell injury as with carbon tetrachloride-induced liver fibrosis. Collagen synthesis, as measured by the formation of labeled protein-bound hydroxyproline, occurred in granulomas isolated from fibrotic livers. Labeled collagen that cochromatographed with type I collagen was extracted with neutral salt solution from liver slices incubated with labeled proline. The free proline pool of the liver was doubled in infected mice; coordinately, liver slices from these animals showed maximal collagen production when the concentration of free proline in the medium was raised to 0.4 mM, the same level measured in the fibrotic livers. Under such conditions, collagen synthesis was at a rate equivalent to the formation of 5.4 nmol of protein-bound hydroxyproline per g liver in 6 h. In comparative incubations in medium containing 0.2 mM proline, fibrotic liver slices produced 16-fold more collagen than normal slices. The proline analogue, L-azetidine 2-carboxylic acid, effectively inhibited synthesis of labeled collagen by fibrotic liver slices. These studies show the synthesis of collagen in a reproducible animal model of the most prevalent form of human liver fibrosis. Difinitition of the controlling factors in this system is of interest for the general problem of fibrosis produced by immunological responses. Images PMID:845255

  11. Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist inhibits collagen synthesis in human hypertrophic scar fibroblasts by targeting Smad3 via miR-145

    SciTech Connect

    Zhu, Hua-Yu; Li, Chao; Zheng, Zhao; Zhou, Qin; Guan, Hao; Su, Lin-Lin; Han, Jun-Tao; Zhu, Xiong-Xiang; Wang, Shu-yue; Li, Jun Hu, Da-Hai

    2015-03-27

    The transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ) functions to regulate cell differentiation and lipid metabolism. Recently, its agonist has been documented to regulate extracellular matrix production in human dermal fibroblasts. This study explored the underlying molecular mechanisms and gene interactions in hypertrophic scar fibroblasts (HSFBs) in vitro. HSFBs were cultured and treated with or without PPAR-γ agonist or antagonist for gene expression. Bioinformatical analysis predicted that miR-145 could target Smad3 expression. Luciferase assay was used to confirm such an interaction. The data showed that PPAR-γ agonist troglitazone suppressed expression of Smad3 and Col1 in HSFBs. PPAR-γ agonist induced miR-145 at the gene transcriptional level, which in turn inhibited Smad3 expression and Col1 level in HSFBs. Furthermore, ELISA data showed that Col1 level in HSFBs was controlled by a feedback regulation mechanism involved in PPAR-γ agonist and antagonist-regulated expression of miR-145 and Smad3 in HSFBs. These findings indicate that PPAR-γ-miR-145-Smad3 axis plays a role in regulation of collagen synthesis in HSFBs. - Highlights: • PPAR-γ agonist inhibits collagen synthesis in HSFBs. • Smad3 and type I collagen expression are decreased by PPAR-γ agonist. • miR-145 expression is increased by PPAR-γ agonist in HSFBs. • Increased miR-145 inhibits collagen synthesis by targeting Smad3. • miR-145 regulates collagen synthesis.

  12. Glutaraldehyde assisted synthesis of collagen derivative modified Fe3+/TiO2 nanocomposite and their enhanced photocatalytic activity

    NASA Astrophysics Data System (ADS)

    Li, Chongyi; Xue, Feng; Ding, Enyong; He, Xiaoling

    2015-11-01

    A unique organic-inorganic hybrid nanocomposite was designed and synthesized by chemically anchoring the cationic collagen-based derivatives onto the surface of Fe3+/TiO2 nanospheres for the significant enhancement in photocatalytic activity under the visible light irradiation. The NMR analysis suggested the successful fabrication of cationic collagen-g-PDMC as grafted materials. In addition, the chemical structures, morphologies and properties of these samples were systematically characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Raman spectrum, ultra violet-visible spectroscopy (UV-vis), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and photoluminescence (PL). And obtained results clearly demonstrated that Fe3+ ions diffusing into TiO2 lattice could be responsible for slightly reducing the average diameter of nanospheres to about 125 nm, promoting phase transition from anatase to rutile to some extent and extending the light harvesting range into visible region markedly. Meanwhile, the achievement that collagen-g-PDMC molecules had been covalently immobilized onto the surface of Fe3+/TiO2 nanoparticles was also well supported by the information acquired. Furthermore, the photocatalytic activities of all the as-prepared products were carefully evaluated by adopting photocatalytic decoloration of methyl orange (MO) solution under the solar direct irradiation, and the sample CFT-3 performed the best in the photocatalytic degradation process, which was mainly attributed to the energetic synergistic effect brought about by Fe3+ ions doping and collagen-g-PDMC molecules immobilized on the surface.

  13. TSLP Is a Potential Initiator of Collagen Synthesis and an Activator of CXCR4/SDF-1 Axis in Keloid Pathogenesis.

    PubMed

    Shin, Jung U; Kim, Seo Hyeong; Kim, Hyeran; Noh, Ji Yeon; Jin, Shan; Park, Chang Ook; Lee, Won Jai; Lee, Dong Won; Lee, Ju Hee; Lee, Kwang Hoon

    2016-02-01

    Recently, thymic stromal lymphopoietin (TSLP), which is well studied in allergic diseases, has been reported in fibrotic diseases, including idiopathic pulmonary fibrosis and atopic dermatitis fibrosis. However, the role of TSLP in keloid is obscure. In this study, we assessed the expression of TSLP in keloid tissue and investigated the possible role of TSLP in keloid pathogenesis. We observed that TSLP expression was increased in keloid tissue compared to normal tissue. Furthermore, TSLP treatment induced increased collagen I and collagen III expression in fibroblasts via transforming growth factor-?; however, there was higher expression in keloid fibroblasts compared to normal fibroblasts. Stromal cell-derived factor-1?, which was recently reported to enhance wound healing through recruiting bone marrow-derived mesenchymal stem cells to the wound area, increased after TSLP treatment in fibroblasts and was primarily expressed in ?-smooth muscle action-positive myofibroblasts in keloid tissue. Furthermore, fibrocytes expressing CXCR4, a stromal cell-derived factor-1? receptor, were significantly increased in keloid tissue compared to normal tissue. Finally, intradermal TSLP injection on BALB/c mice increased stromal cell-derived factor-1? expression and CXCR4(+) fibrocytes infiltration. Our data suggest that TSLP is a potent inducer of collagen and transforming growth factor-? production in keloid fibroblasts. In addition, it might activate the CXCR4/stromal cell-derived factor-1 axis to increase fibrocyte infiltration into the keloid tissue.

  14. A Recombinant Collagen-mRNA Platform for Controllable Protein Synthesis.

    PubMed

    Sun, Liping; Xiong, Yunjing; Bashan, Anat; Zimmerman, Ella; Shulman Daube, Shirley; Peleg, Yoav; Albeck, Shira; Unger, Tamar; Yonath, Hagith; Krupkin, Miri; Matzov, Donna; Yonath, Ada

    2015-07-06

    We have developed a collagen-mRNA platform for controllable protein production that is intended to be less prone to the problems associated with commonly used mRNA therapy as well as with collagen skin-healing procedures. A collagen mimic was constructed according to a recombinant method and was used as scaffold for translating mRNA chains into proteins. Cysteines were genetically inserted into the collagen chain at positions allowing efficient ribosome translation activity while minimizing mRNA misfolding and degradation. Enhanced green fluorescence protein (eGFP) mRNA bound to collagen was successfully translated by cell-free Escherichia coli ribosomes. This system enabled an accurate control of specific protein synthesis by monitoring expression time and level. Luciferase-mRNA was also translated on collagen scaffold by eukaryotic cell extracts. Thus we have demonstrated the feasibility of controllable protein synthesis on collagen scaffolds by ribosomal machinery.

  15. Cyclic tensile strain upregulates collagen synthesis in isolated tendon fascicles

    SciTech Connect

    Screen, Hazel R.C. . E-mail: H.R.C.Screen@qmul.ac.uk; Shelton, Julia C.; Bader, Dan L.; Lee, David A.

    2005-10-21

    Mechanical stimulation has been implicated as an important regulatory factor in tendon homeostasis. In this study, a custom-designed tensile loading system was used to apply controlled mechanical stimulation to isolated tendon fascicles, in order to examine the effects of 5% cyclic tensile strain at 1 Hz on cell proliferation and matrix synthesis. Sample viability and gross structural composition were maintained over a 24 h loading period. Data demonstrated no statistically significant differences in cell proliferation or glycosaminoglycan production, however, collagen synthesis was upregulated with the application of cyclic tensile strain over the 24 h period. Moreover, a greater proportion of the newly synthesised matrix was retained within the sample after loading. These data provide evidence of altered anabolic activity within tendon in response to mechanical stimuli, and suggest the importance of cyclic tensile loading for the maintenance of the collagen hierarchy within tendon.

  16. Activation of hageman factor by collagen

    PubMed Central

    Wilner, G. D.; Nossel, H. L.; LeRoy, E. C.

    1968-01-01

    Purified acid-soluble and insoluble human collagen accelerated the clotting of plateletpoor plasma in silicone-treated tubes. The clot-promoting effect did not appear to be due to thromboplastic activity since the collagen preparations did not activate factor X in the presence of factor VII and calcium. Instead, collagen appeared to accelerate clotting by activating Hageman factor (factor XII) on the basis of the following findings: collagen increased the clot-promoting activity of partially purified Hageman factor but exerted no further effect in the presence of kaolin, a known activator of Hageman factor; clot-promoting eluates were obtained from collagen exposed to normal, hemophilic, or PTC-deficient plasma but not from collagen exposed to Hageman or PTA-deficient plasma. The collagen molecule itself appeared to be required for the clot-promoting activity since digestion with collagenase or thermal denaturation at pH 2.5 (about 35°C) resulted in very marked reduction in clot-promoting activity. Since thermal denaturation is associated with transformation of collagen structure from triple helical to random coil form, it is suggested that the native form of collagen is essential for the ability to activate Hageman factor. Blockage of the free amino groups by treatment with nitrous acid or dinitrofluorobenzene only slightly reduced the clot-promoting activity of collagen. In contrast, since addition of cationic proteins to collagen markedly reduced pro-coagulant activity it is suggested that negatively charged sites on the collagen molecule are critical for Hageman factor activation. This suggestion is supported by the finding that pepsin treatment of collagen, which removes the predominantly negatively charged telopeptides, results in significant decrease in coagulant activity. Esterification of collagen, which neutralizes 80-90% of the free carboxyl groups, reduced coagulant activity by over 90% and it is suggested that the free carboxyl groups of glutamic and

  17. Age-related changes in collagen synthesis and degradation in rat tissues. Importance of degradation of newly synthesized collagen in regulating collagen production.

    PubMed Central

    Mays, P K; McAnulty, R J; Campa, J S; Laurent, G J

    1991-01-01

    During developmental growth, collagens are believed to be continuously deposited into an extracellular matrix which is increasingly stabilized by the formation of covalent cross-links throughout life. However, the age-related changes in rates of synthetic and degradative processes are less well understood. In the present study we measured rates of collagen synthesis in vivo using a flooding dose of unlabelled proline given with [14C]proline and determining production of hydroxy[14C]proline. Degradation of newly synthesized collagen was estimated from the amount of free hydroxy [14C]proline in tissues 30 min after injection. Collagen fractional synthesis rates ranged from about 5%/day in skeletal muscle to 20%/day in hearts of rats aged 1 month. At 15 months of age, collagen fractional synthesis rates had decreased markedly in lung and skin, but in skeletal muscle and heart, rates were unchanged. At 24 months of age, synthesis rates had decreased by at least 10-fold in all tissues, compared with rates at 1 month. The proportion of newly synthesized collagen degraded ranged from 6.4 +/- 0.4% in skin to 61.6 +/- 5.0% in heart at 1 month of age. During aging the proportion degraded increased in all tissues to maximal values at 15 months, ranging from 56 +/- 7% in skin to 96 +/- 1% in heart. These data suggest that there are marked age-related changes in rates of collagen metabolism. They also indicate that synthesis is active even in old animals, where the bulk of collagens produced are destined to be degraded. PMID:2049064

  18. Panax notoginseng stimulates alkaline phosphatase activity, collagen synthesis, and mineralization in osteoblastic MC3T3-E1 cells.

    PubMed

    Ji, Zhe; Cheng, Yizhao; Yuan, Puwei; Dang, Xiaoqian; Guo, Xiong; Wang, Weizhuo

    2015-10-01

    Total Panax notoginseng saponin (PNS) has been extensively used to treat a variety of diseases, such as bone fractures, soft tissue injuries, etc. In this study, mouse calvaria-original osteoblastic MC3T3-E1 cells were cultured in various concentrations of PNS (0.005-5 mg/mL) during the period (1, 5, 14, and 23 d). At the endpoint, the osteogenic capacity of MC3T3-E1 cells was investigated by measuring the alkaline phosphatase (ALP) activity, the deposited calcium, and the expression of osteogenic-related markers, including bone collagen type 1 (Col1) and osteocalcin (OCN). Compared with all groups in each period, the most pronounced effect was observed at the concentration range between 0.05 and 0.5 mg/mL (P < 0.05) and the cell proliferation with PNS treatment was found during the whole osteogenic period. Moreover, cellular ALP activity with PNS was increased during 7, 14, and 21 d and cell mineralization with PNS was enhanced in 14 and 21 d. Furthermore, the differentiation markers Col1 and OCN increased in the PNS-treated cells. Our work suggests that PNS may stimulate the osteogenesis process which contains osteoblastic proliferation, differentiation, and mineralization by increasing cellular ALP activity, extracellular matrix mineralization, and osteoblast-associated molecules in the osteoblasts.

  19. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

    SciTech Connect

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G.

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate

  20. Collagen synthesis by cultured rabbit aortic smooth-muscle cells. Alteration with phenotype.

    PubMed Central

    Ang, A H; Tachas, G; Campbell, J H; Bateman, J F; Campbell, G R

    1990-01-01

    Enzymically isolated rabbit aortic smooth-muscle cells (SMC) in the first few days of primary culture express a 'contractile phenotype', but with time these cells modulate to a 'synthetic phenotype'. Synthetic-state SMC are able to proliferate, and, provided that they undergo fewer than 5 cumulative population doublings, return to the contractile phenotype after reaching confluency [Campbell, Kocher, Skalli, Gabbiani & Campbell (1989) Arteriosclerosis 9, 633-643]. The present study has determined the synthesis of collagen, at the protein and mRNA levels, by cultured SMC as they undergo a change in phenotypic state. The results show that, upon modulating to the synthetic phenotype, SMC synthesized 25-30 times more collagen than did contractile cells. At the same time, non-collagen-protein synthesis increased only 5-6-fold, indicating a specific stimulation of collagen synthesis. Steady-state mRNA levels are also elevated, with alpha 2(I) and alpha 1(III) mRNA levels 30 times and 20 times higher respectively, probably reflecting increased transcriptional activity. Phenotypic modulation was also associated with an alteration in the relative proportions of type I and III collagens synthesized, contractile SMC synthesizing 78.1 +/- 3.6% (mean +/- S.D.) type I collagen and 17.5 +/- 4.7% type III collagen, and synthetic cells synthesizing 90.3 +/- 2.0% type I collagen and 5.8% +/- 1.8% type III collagen. Enrichment of type I collagen was similarly noted at the mRNA level. On return to the contractile state, at confluency, collagen production and the percentage of type I collagen decreased. This further illustrates the close association between the phenotypic state of SMC and their collagen-biosynthetic phenotype. Images Fig. 4. PMID:1689147

  1. Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate.

    PubMed

    Feyen, J H; Petersen, D N; Kream, B E

    1988-04-01

    We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3-100 ng/ml (4-133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.

  2. Stimulation of collagen synthesis in fibroblast cultures by a triterpene extracted from Centella asiatica.

    PubMed

    Maquart, F X; Bellon, G; Gillery, P; Wegrowski, Y; Borel, J P

    1990-01-01

    The drug "Titrated Extract from Centella asiatica" (TECA), used for its stimulating properties on the healing of wounds, is a mixture of 3 terpenes extracted from a tropical plant: asiatic acid (30%, w/w), madecassic acid (30%, w/w) and asiaticoside (40%, w/w). The effects of TECA and its individual components were checked on human foreskin fibroblast monolayer cultures. TECA increased the collagen synthesis in a dose-dependent fashion whereas a simultaneous decrease in the specific activity of neosynthesized collagen was observed. Asiatic acid was found to be the only component responsible for collagen synthesis stimulation. TECA and all three terpenes increased the intracellular free proline pool. This effect was independent of the stimulation of collagen synthesis.

  3. Apigenin induces dermal collagen synthesis via smad2/3 signaling pathway.

    PubMed

    Zhang, Y; Wang, J; Cheng, X; Yi, B; Zhang, X; Li, Q

    2015-04-13

    Decrease in fibroblast-produced collagen has been proven to be the pivotal cause of skin aging, but there is no satisfactory drug which directly increases dermal thickness and collage density. Here we found that a flavonoid natural product, apigenin, could significantly increase collagen synthesis. NIH/3T3 and primary human dermal fibroblasts (HDFs) were incubated with various concentrations of apigenin, with dimethyl sulfoxide (DMSO) serving as the negative control. Real-time reverse-transcription polymerase chain reaction (PCR), Western Blot, and Toluidine blue staining demonstrated that apigenin stimulated type-I and type-III collagen synthesis of fibroblasts on the mRNA and protein levels. Meanwhile, apigenin did not induce expression of alpha smooth muscle actin (α-SMA) in vitro and in vivo, a fibrotic marker in living tissues. Then the production of collagen was confirmed by Masson's trichrome stain, Picrosirius red stain and immunohistochemistry in mouse models. We also clarified that this compound induced collagen synthesis by activating smad2/3 signaling pathway. Taken together, without obvious influence on fibroblasts' apoptosis and viability, apigenin could promote the type-I and type-III collagen synthesis of dermal fibroblasts in vitro and in vivo, thus suggesting that apigenin may serve as a potential agent for esthetic and reconstructive skin rejuvenation.

  4. Apigenin Induces Dermal Collagen Synthesis Via smad2/3 Signaling Pathway

    PubMed Central

    Zhang, Y.; Wang, J.; Cheng, X.; Yi, B.; Zhang, X.; Li, Q.

    2015-01-01

    Decrease in fibroblast-produced collagen has been proven to be the pivotal cause of skin aging, but there is no satisfactory drug which directly increases dermal thickness and collage density. Here we found that a flavonoid natural product, apigenin, could significantly increase collagen synthesis. NIH/3T3 and primary human dermal fibroblasts (HDFs) were incubated with various concentrations of apigenin, with dimethyl sulfoxide (DMSO) serving as the negative control. Real-time reverse-transcription polymerase chain reaction (PCR), Western Blot, and Toluidine blue staining demonstrated that apigenin stimulated type-I and type-III collagen synthesis of fibroblasts on the mRNA and protein levels. Meanwhile, apigenin did not induce expression of alpha smooth muscle actin (α-SMA) in vitro and in vivo, a fibrotic marker in living tissues. Then the production of collagen was confirmed by Masson’s trichrome stain, Picrosirius red stain and immunohistochemistry in mouse models. We also clarified that this compound induced collagen synthesis by activating smad2/3 signaling pathway. Taken together, without obvious influence on fibroblasts’ apoptosis and viability, apigenin could promote the type-I and type-III collagen synthesis of dermal fibroblasts in vitro and in vivo, thus suggesting that apigenin may serve as a potential agent for esthetic and reconstructive skin rejuvenation. PMID:26150153

  5. Collagen degrading activity associated with Mycobacterium species

    PubMed Central

    Masso, F; Paez, A; Varela, E; d Diaz; Zenteno, E; Montano, L

    1999-01-01

    BACKGROUND—The mechanism of Mycobacterium tuberculosis penetration into tissues is poorly understood but it is reasonable to assume that there is a contribution from proteases capable of disrupting the extracellular matrix of the pulmonary epithelium and the blood vessels. A study was undertaken to identify and characterise collagen degrading activity of M tuberculosis.
METHODS—Culture filtrate protein extract (CFPE) was obtained from reference mycobacterial strains and mycobacteria isolated from patients with tuberculosis. The collagen degrading activity of CFPE was determined according to the method of Johnson-Wint using 3H-type I collagen. The enzyme was identified by the Birkedal-Hansen and Taylor method and its molecular mass determined by SDS-PAGE and Sephacryl S-300 gel filtration chromatography using an electroelution purified enzyme.
RESULTS—CFPE from Mycobacterium tuberculosis strain H37Rv showed collagenolytic activity that was four times higher than that of the avirulent strain H37Ra. The 75 kDa enzyme responsible was divalent cation dependent. Other mycobacterial species and those isolated from patients with tuberculosis also had collagen degrading activity.
CONCLUSIONS—Mycobacterium species possess a metalloprotease with collagen degrading activity. The highest enzymatic activity was found in the virulent reference strain H37Rv.

 PMID:10212111

  6. Effects of Panax ginseng extract on human dermal fibroblast proliferation and collagen synthesis.

    PubMed

    Lee, Geum-Young; Park, Kang-Gyun; Namgoong, Sik; Han, Seung-Kyu; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

    2016-03-01

    Current studies of Panax ginseng (or Korean ginseng) have demonstrated that it has various biological effects, including angiogenesis, immunostimulation, antimicrobial and anti-inflammatory effects. Therefore, we hypothesised that P. ginseng may also play an important role in wound healing. However, few studies have been conducted on the wound-healing effects of P. ginseng. Thus, the purpose of this in vitro pilot study was to determine the effects of P. ginseng on the activities of fibroblasts, which are key wound-healing cells. Cultured human dermal fibroblasts were treated with one of six concentrations of P. ginseng: 0, 1, 10 and 100 ng/ml and 1 and 10 µg/ml. Cell proliferation was determined 3 days post-treatment using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, and collagen synthesis was evaluated by the collagen type I carboxy-terminal propeptide method. Cell proliferation levels and collagen synthesis were compared among the groups. The 10 ng/ml to 1 µg/ml P. ginseng treatments significantly increased cell proliferation, and the 1 ng/ml to 1 µg/ml concentrations significantly increased collagen synthesis. The maximum effects for both parameters were observed at 10 ng/ml. P. ginseng stimulated human dermal fibroblast proliferation and collagen synthesis at an optimal concentration of 10 ng/ml.

  7. Synthesis of huaicarbon A/B and their activating effects on platelet glycoprotein VI receptor to mediate collagen-induced platelet aggregation

    PubMed Central

    Yu, Hongli; Chen, Yeqing; Wu, Hao; Wang, Kuilong; Liu, Liping; Zhang, Xingde

    2017-01-01

    Quercetin and rhamnose were efficiently converted into huaicarbon A/B by heating at 250°C for 10-15 min or at 200°C for 25-30 min. With the optimum molar ratio of quercetin/rhamnose (1:3), huaicarbon A and B yields reached 25% and 16% respectively after heating at 250°C, with 55% quercetin conversion. Huaicarbon A/B both promoted washed platelet aggregation dose-dependently, which was antagonized by an inhibitor of glycoprotein VI (GPVI) receptor. Similarly, they both promoted collagen-induced platelet aggregation in platelet-rich plasma in dose-dependent manners. According to the S type dose-response model, EC50 values of huaicarbon A and huaicarbon B were calculated as 33.48 μM and 48.73 μM respectively. They induced intracellular Ca2+ accumulation that was specifically blocked by GPVI antagonist. Huaicarbon A/B enhanced intracellular Ca2+ accumulation and facilitated collagen-induced platelet aggregation, which were blocked by GPVI antagonist. They were conducive to collagen-induced platelet aggregation by activating platelet GPVI receptor. PMID:28337278

  8. Potentiation of collagen synthesis in explants of the rabbit eye by 5 beta-dihydrocortisol

    SciTech Connect

    Southren, A.L.; Hernandez, M.R.; l'Hommedieu, D.; Gordon, G.G.; Weinstein, B.I.

    1986-12-01

    The biologic effect of 5 beta-dihydrocortisol on collagen synthesis was evaluated. The metabolite was found to potentiate subthreshold levels of dexamethasone in increasing /sup 3/H-proline incorporation in cells of the outflow region of the rabbit. Digestion of the tissue with highly purified collagenase indicated that the /sup 3/H-proline was incorporated into collagen type protein. This study demonstrates another biologic activity of 5 beta-dihydrocortisol, a metabolite found to accumulate in cells cultured from trabeculectomy specimens from patients with primary open angle glaucoma.

  9. Novel synthesis and characterization of a collagen-based biopolymer initiated by hydroxyapatite nanoparticles.

    PubMed

    Bhuiyan, D; Jablonsky, M J; Kolesov, I; Middleton, J; Wick, T M; Tannenbaum, R

    2015-03-01

    In this study, we developed a novel synthesis method to create a complex collagen-based biopolymer that promises to possess the necessary material properties for a bone graft substitute. The synthesis was carried out in several steps. In the first step, a ring-opening polymerization reaction initiated by hydroxyapatite nanoparticles was used to polymerize d,l-lactide and glycolide monomers to form poly(lactide-co-glycolide) co-polymer. In the second step, the polymerization product was coupled with succinic anhydride, and subsequently was reacted with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide as the cross-linking agent, in order to activate the co-polymer for collagen attachment. In the third and final step, the activated co-polymer was attached to calf skin collagen type I, in hydrochloric acid/phosphate buffer solution and the precipitated co-polymer with attached collagen was isolated. The synthesis was monitored by proton nuclear magnetic resonance, infrared and Raman spectroscopies, and the products after each step were characterized by thermal and mechanical analysis. Calculations of the relative amounts of the various components, coupled with initial dynamic mechanical analysis testing of the resulting biopolymer, afforded a preliminary assessment of the structure of the complex biomaterial formed by this novel polymerization process.

  10. Topical all-trans retinoic acid stimulates collagen synthesis in vivo.

    PubMed

    Schwartz, E; Cruickshank, F A; Mezick, J A; Kligman, L H

    1991-06-01

    Histochemical and ultrastructural studies demonstrate that topical all-trans retinoic acid (RA) stimulates the deposition of a subepidermal band of collagen in photoaged hairless mice. The aim of this study was to examine the effect of RA treatment on collagen synthesis using biochemical and immunochemical techniques. Albino hairless mice were irradiated three times a week for 10 weeks with four minimal erythema doses of UVB from Westinghouse FS-40 bulbs. In the post-UV period, mice were either nontreated or treated with 0.05% RA or the ethanol-propylene glycol vehicle for up to 10 weeks. Antibodies against the aminopropeptide (AP) of type III procollagen were used in immunofluorescence microscopy and radioimmunoassay techniques. The AP of type III collagen is normally present throughout the dermis and in areas of active collagen synthesis (i.e., the dermal-epidermal junction). In this study, a similar distribution was seen in all untreated and vehicle-treated mice, and in mice treated with RA for 2, 4, and 6 weeks. However, increased staining, in a subepidermal band, was detected in the 8-week RA-treated skin. This region became intensely fluorescent to a depth of 100 mu in the 10-week RA-treated skins. As determined by radioimmunoassay, the content of the AP of type III procollagen increased twofold with 10-week RA treatment. Because the ratio of type I to type III collagens remained constant in treated and untreated skins, it is reasonable to assume that the content of type I collagen increased in proportion to type III collagen in RA-treated skins.

  11. Collagen Synthesis in Tenocytes, Ligament Cells and Chondrocytes Exposed to a Combination of Glucosamine HCl and Chondroitin Sulfate

    PubMed Central

    2007-01-01

    Clinical testing of the nutraceuticals glucosamine (glcN) and chondroitin sulfate (CS) has shown efficacy in providing relief from symptoms in osteoarthritic patients. In vitro and in vivo studies support existence of a synergistic relationship upregulating synthetic activity in chondrocytes. A combination of glcN and CS may also be useful as adjunct therapy in sports-related injuries if similar upregulation of collagen synthesis is elicited in accessory ligament and tendon joint tissue. Collagen and non-collagenous protein (NCP) synthesis in cultures of bovine tenocytes, ligament cells and chondrocytes exposed to glcN + CS were assayed by uptake of radiolabeled proline into collagenase-sensitive material. Assay of radiolabel in hydroxyproline (a specific marker for collagen synthesis) following HPLC isolation confirmed the specificity of the metabolic effect. Synthesis of total collagenase-sensitive material was maximally upregulated at physiologically obtainable doses of glcN + CS. Tissue response followed the sequence ligament cells (+69%) > chondrocytes (+56%) > tenocytes (+22%). Labeled hydroxyproline increased by 132% in ligament cells, 27% in tenocytes and 49% in epitendon cells after a 48 h exposure to 5 μg ml−1 glcN + 4 μg ml−1 CS. Low dose combinations of glcN and CS effectively stimulate in vitro collagen and NCP synthesis by ligament cells, tenocytes and chondrocytes. Hence, therapeutic use following accessory joint tissue trauma may help augment repair processes. PMID:17549239

  12. Photo-active collagen systems with controlled triple helix architecture.

    PubMed

    Tronci, Giuseppe; Russell, Stephen J; Wood, David J

    2013-08-14

    The design of photo-active collagen systems is presented as a basis for establishing biomimetic materials with varied network architecture and programmable macroscopic properties. Following in-house isolation of type I collagen, reaction with vinyl-bearing compounds of varied backbone rigidity, i.e. 4-vinylbenzyl chloride (4VBC) and glycidyl methacrylate (GMA), was carried out. TNBS colorimetric assay, (1)H-NMR and ATR-FTIR confirmed covalent and tunable functionalization of collagen lysines. Depending on the type and extent of functionalization, controlled stability and thermal denaturation of triple helices were observed via circular dichroism (CD), whereby the hydrogen-bonding capability of introduced moieties was shown to play a major role. Full gel formation was observed following photo-activation of functionalized collagen solutions. The presence of a covalent network only slightly affected collagen triple helix conformation (as observed by WAXS and ATR-FTIR), confirming the structural organization of functionalized collagen precursors. Photo-activated hydrogels demonstrated an increased denaturation temperature (DSC) with respect to native collagen, suggesting that the formation of the covalent network successfully stabilized collagen triple helices. Moreover, biocompatibility and mechanical competence of obtained hydrogels were successfully demonstrated under physiologically-relevant conditions. These results demonstrate that this novel synthetic approach enabled the formation of biocompatible collagen systems with defined network architecture and programmable macroscopic properties, which can only partially be obtained with current synthetic methods.

  13. 4-[F-18]fluoroproline: A potential tracer for collagen synthesis. Radiosynthesis and biological evaluation

    SciTech Connect

    Hamacher, K.; Herz, M.; Truckenbrodt, R.

    1996-05-01

    Proline is an important constituent of the structural protein collagen. It has been shown that its fluorinated analogs (2S,4S)- and particularly (2S,4R)-4-fluoroproline are also incorporated into collagen (Gottlieb et al., Biochemistry (1965), 4: 2507). 4-[F-18]fluoroproline is therefore a potential probe for studying abnormal collagen synthesis e. g. in tumors, lung fibrosis and liver cirrhosis. We have evaluated the two diastereomeric forms using a transplantable osteosarcoma in mice as an in vivo model for elevated collagen synthesis, and a MCF 7 mamma carcinoma cell line for monolayer incubation studies.

  14. Collagen-chitosan scaffold modified with Au and Ag nanoparticles: Synthesis and structure

    NASA Astrophysics Data System (ADS)

    Rubina, M. S.; Kamitov, E. E.; Zubavichus, Ya. V.; Peters, G. S.; Naumkin, A. V.; Suzer, S.; Vasil'kov, A. Yu.

    2016-03-01

    Nowadays, the dermal biomimetic scaffolds are widely used in regenerative medicine. Collagen-chitosan scaffold one of these materials possesses antibacterial activity, good compatibility with living tissues and has been already used as a wound-healing material. In this article, collagen-chitosan scaffolds modified with Ag and Au nanoparticles have been synthesized using novel method - the metal-vapor synthesis. The nanocomposite materials are characterized by XPS, TEM, SEM and synchrotron radiation-based X-ray techniques. According to XRD data, the mean size of the nanoparticles (NPs) is 10.5 nm and 20.2 nm in Au-Collagen-Chitosan (Au-CollCh) and Ag-Collagen-Chitosan (Ag-CollCh) scaffolds, respectively in fair agreement with the TEM data. SAXS analysis of the composites reveals an asymmetric size distribution peaked at 10 nm for Au-CollCh and 25 nm for Ag-CollCh indicative of particle's aggregation. According to SEM data, the metal-carrying scaffolds have layered structure and the nanoparticles are rather uniformly distributed on the surface material. XPS data indicate that the metallic nanoparticles are in their unoxidized/neutral states and dominantly stabilized within the chitosan-rich domains.

  15. Design and synthesis of collagen mimetic peptide derivatives for studying triple helix assembly and collagen mimetic peptide-collagen binding interaction

    NASA Astrophysics Data System (ADS)

    Mo, Xiao

    2008-10-01

    Collagen is the principal tensile clement of the extra-cellular matrix in mammals and is the basic scaffold for cells and tissues. Collagen molecules are comprised of homo-trimeric helices (e.g. collagen type II and type III), ABB type hetero-trimeric helices (e.g. collagen type I, type IV, and type V), or ABC type hetero-trimeric helices (e.g. type V). Mimicry of collagen structures can help elucidate collagen triple helical conformation and provide insights into making novel collagen-like biomaterials. Our group previously reported a new physical collagen modification method, which was based on non-covalent interaction between collagen mimetic peptide (CMP: -(Pro-Hyp-Gly) x-) and natural collagen. We hypothesized that CMP binds to collagen through a process involving both strand invasion and triple helix assembly. The aim of this dissertation is to study structural formation and stability of collagen triple helix, and to investigate CMP-collagen binding interactions using two types of CMP derivatives: covalently templated CMP trimer and CMP-nanoparticle conjugates. We demonstrated that covalently templated ABB type CMP hetero-trimers could be prepared by a versatile synthetic strategy involving both solid phase and solution peptide coupling. Our thermal melting studies showed that the templated CMP hetero-trimers formed collagen-like triple helices and their folding kinetics correlated with the amino acid compositions of the individual CMP strands. We also studied the thermal melting behavior and folding kinetics of a templated hetero-trimer complex comprised of CMP and a peptide derived from collagen. This synthetic strategy can be readily extended to synthesize other ABB type hetero-trimers to investigate their local melting behavior and biological activity. We also prepared colloidally stable CMP functionalized gold nanoparticles (Au-CMPs) as a TEM marker for investigating the CMP-collagen interaction. Au-CMP showed preferential binding to collagen fiber's gap

  16. Evidence for a role of collagen synthesis in arterial smooth muscle cell migration.

    PubMed Central

    Rocnik, E F; Chan, B M; Pickering, J G

    1998-01-01

    Migration of smooth muscle cells (SMCs) and collagen synthesis by SMCs are central to the pathophysiology of vascular disease. Both processes can be induced shortly after vascular injury; however, a functional relationship between them has not been established. In this study, we determined if collagen synthesis was required for SMC migration, using ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor of prolyl-4-hydroxylase, and 3,4-DL-dehydroproline (DHP), a proline analogue, which we demonstrate inhibit collagen elaboration by porcine arterial SMCs. SMCs exposed to EDHB or DHP attached normally to collagen- and vitronectin-coated substrates; however, spreading on collagen but not vitronectin was inhibited. SMC migration speed, quantified by digital time-lapse video microscopy, was significantly and reversibly reduced by EDHB and DHP. Flow cytometry revealed that expression of beta1 integrins, through which SMCs interact with collagen, was unaffected by EDHB or DHP. However, both inhibitors prevented normal clustering of beta1 integrins on the surface of SMCs, consistent with a lack of appropriate matrix ligands for integrin engagement. Moreover, there was impaired recruitment of vinculin into focal adhesion complexes of spreading SMCs and disassembly of the smooth muscle alpha-actin-containing cytoskeleton. These findings suggest that de novo collagen synthesis plays a role in SMC migration and implicates a mechanism whereby newly synthesized collagen may be necessary to maintain the transcellular traction system required for effective locomotion. PMID:9576753

  17. Preparation and Biological Activity of New Collagen Composites, Part I: Collagen/Zinc Titanate Nanocomposites.

    PubMed

    Albu, Madalina G; Vladkova, Todorka G; Ivanova, Iliana A; Shalaby, Ahmed S A; Moskova-Doumanova, Veselina S; Staneva, Anna D; Dimitriev, Yanko B; Kostadinova, Anelya S; Topouzova-Hristova, Tanya I

    2016-09-01

    The aim of this investigation was to develop new antimicrobial collagen/zinc titanate (ZnTiO3) biomaterials using a sol-gel cryogenic draying technology in keeping the native collagen activity. Broad-spectrum antimicrobial activity was demonstrated against Firmicutes (Staphylococcus epidermidis, Bacillus cereus, and Candida lusitaniae) and Gracilicutes (Escherichia coli, Salmonella enterica, and Pseudomonas putida) microorganisms. The antimicrobial activity as well as the cytotoxicity were specific for the different test microorganisms (Gram-positive and Gram-negative bacteria and fungi) and model eukaryotic cells (osteosarcoma, fibroblast, and keratinocyte cells), respectively, and both were depending on the ZnTiO3 concentration. Three mechanisms of the antimicrobial action were supposed, including (i) mechanical demolition of the cell wall and membrane by the crystal nanoparticles of the ZnTiO3 entrapped in the collagen matrix, (ii) chelation of its metal ions, and (iii) formation of free oxygen radicals due to the interaction between the microbial cells and antimicrobial agent. It was concluded that the optimal balance between antimicrobial activity and cytotoxicity could be achieved by a variation of the ZnTiO3 concentration. The antifungal and broad-spectrum antibacterial activity of the studied collagen/ZnTiO3 nanocomposites, combined with a low cytotoxicity, makes them a promising anti-infection biomaterial.

  18. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts.

    PubMed

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G

    2012-11-23

    The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

  19. Inhibitory effects of peroxisome proliferator-activated receptor γ agonists on collagen IV production in podocytes.

    PubMed

    Li, Yanjiao; Shen, Yachen; Li, Min; Su, Dongming; Xu, Weifeng; Liang, Xiubin; Li, Rongshan

    2015-07-01

    Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists have beneficial effects on the kidney diseases through preventing microalbuminuria and glomerulosclerosis. However, the mechanisms underlying these effects remain to be fully understood. In this study, we investigate the effects of PPAR-γ agonist, rosiglitazone (Rosi) and pioglitazone (Pio), on collagen IV production in mouse podocytes. The endogenous expression of PPAR-γ was found in the primary podocytes and can be upregulated by Rosi and Pio, respectively, detected by RT-PCR and Western blot. PPAR-γ agonist markedly blunted the increasing of collagen IV expression and extraction in podocytes induced by TGF-β. In contrast, adding PPAR-γ antagonist, GW9662, to podocytes largely prevented the inhibition of collagen IV expression from Pio treatment. Our data also showed that phosphorylation of Smad2/3 enhanced by TGF-β in a time-dependent manner was significantly attenuated by adding Pio. The promoter region of collagen IV gene contains one putative consensus sequence of Smad-binding element (SBE) by promoter analysis, Rosi and Pio significantly ameliorated TGF-β-induced SBE4-luciferase activity. In conclusion, PPAR-γ activation by its agonist, Rosi or Pio, in vitro directly inhibits collagen IV expression and synthesis in primary mouse podocytes. The suppression of collagen IV production was related to the inhibition of TGF-β-driven phosphorylation of Smad2/3 and decreased response activity of SBEs of collagen IV in PPAR-γ agonist-treated mouse podocytes. This represents a novel mechanistic support regarding PPAR-γ agonists as podocyte protective agents.

  20. Supra-molecular assembly of a lumican-derived peptide amphiphile enhances its collagen-stimulating activity.

    PubMed

    Walter, Merlin N M; Dehsorkhi, Ashkan; Hamley, Ian W; Connon, Che J

    2016-02-01

    C16-YEALRVANEVTLN, a peptide amphiphile (PA) incorporating a biologically active amino acid sequence found in lumican, has been examined for its influence upon collagen synthesis by human corneal fibroblasts in vitro, and the roles of supra-molecular assembly and activin receptor-like kinase ALK receptor signaling in this effect were assessed. Cell viability was monitored using the Alamar blue assay, and collagen synthesis was assessed using Sirius red. The role of ALK signaling was studied by receptor inhibition. Cultured human corneal fibroblasts synthesized significantly greater amounts of collagen in the presence of the PA over both 7-day and 21-day periods. The aggregation of the PA to form nanotapes resulted in a notable enhancement in this activity, with an approximately two-fold increase in collagen production per cell. This increase was reduced by the addition of an ALK inhibitor. The data presented reveal a stimulatory effect upon collagen synthesis by the primary cells of the corneal stroma, and demonstrate a direct influence of supra-molecular assembly of the PA upon the cellular response observed. The effects of PA upon fibroblasts were dependent upon ALK receptor function. These findings elucidate the role of self-assembled nanostructures in the biological activity of peptide amphiphiles, and support the potential use of a self-assembling lumican derived PA as a novel biomaterial, intended to promote collagen deposition for wound repair and tissue engineering purposes.

  1. Testosterone delays vascular smooth muscle cell senescence and inhibits collagen synthesis via the Gas6/Axl signaling pathway.

    PubMed

    Chen, Yan-qing; Zhao, Jing; Jin, Cheng-wei; Li, Yi-hui; Tang, Meng-xiong; Wang, Zhi-hao; Zhang, Wei; Zhang, Yun; Li, Li; Zhong, Ming

    2016-06-01

    Testosterone deficiency is associated with a higher incidence of cardiovascular diseases in men. However, its effect on cell senescence, which plays a causal role in vascular aging, remains unclear. Here, we tested the hypothesis that testosterone alleviated vascular smooth muscle cell (VSMC) senescence and collagen synthesis via growth arrest-specific protein 6 (Gas6)/Axl- and Akt/FoxO1a-dependent pathways. Testosterone significantly ameliorated angiotensin II-induced VSMC senescence and collagen overexpression. In addition, testosterone inhibited angiotensin II-induced matrix metalloproteinase-2 (MMP-2) activity, which played a pivotal role in facilitating age-related collagen deposition. Testosterone increased the expression of tissue inhibitor of metalloproteinase-2 but decreased the expression of MMP-2 and membrane type-1 metalloproteinase which contributed to increase MMP-2 activity. The effects on VSMCs senescence and collagen synthesis were mediated by restoration of angiotensin II-induced downregulation of Gas6 and Axl expression and a subsequent reduction of Akt and FoxO1a phosphorylation. The effects of testosterone were reversed by a Gas6 blocker, Axl-Fc, and a specific inhibitor of Axl, R428. Treatment of VSMCs with PI3K inhibitor LY294002 abrogated the downregulating effect of testosterone on MMP-2 activity. Furthermore, when FoxO1a expression was silenced by using a specific siRNA, the inhibitory effect of testosterone on MMP-2 activity was revered as well, that indicated this process was Akt/FoxO1a dependence. Taken together, Gas6/Axl and Akt/FoxO1a were involved in protective effects of testosterone on VSMCs senescence and collagen synthesis. Our results provide a novel mechanism underlying the protective effect of testosterone on vascular aging and may serve as a theoretical basis for testosterone replacement therapy.

  2. STAT6-Dependent Collagen Synthesis in Human Fibroblasts Is Induced by Bovine Milk.

    PubMed

    Kippenberger, Stefan; Zöller, Nadja; Kleemann, Johannes; Müller, Jutta; Kaufmann, Roland; Hofmann, Matthias; Bernd, August; Meissner, Markus; Valesky, Eva

    2015-01-01

    Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold, EC50: 2,5% milk) without toxic effects. Surprisingly, bovine milk was identified as strong inducer of collagen 1A1 synthesis at both, the protein (4-fold, EC50: 0,09% milk) and promoter level. Regarding the underlying molecular pathways, we show functional activation of STAT6 in a p44/42 and p38-dependent manner. More upstream, we identified IGF-1 and insulin as key factors responsible for milk-induced collagen synthesis. These findings show that bovine milk contains bioactive molecules that act on human skin cells. Therefore, it is tempting to test the herein introduced concept in treatment of atrophic skin conditions induced e.g. by UV light or corticosteroids.

  3. Porous Collagen Scaffold Reinforced with Surfaced Activated PLLA Nanoparticles

    PubMed Central

    Xu, Cancan; Lu, Wei; Bian, Shaoquan; Liang, Jie; Fan, Yujiang; Zhang, Xingdong

    2012-01-01

    Porous collagen scaffold is integrated with surface activated PLLA nanoparticles fabricated by lyophilizing and crosslinking via EDC treatment. In order to prepare surface-modified PLLA nanoparticles, PLLA was firstly grafted with poly (acrylic acid) (PAA) through surface-initiated polymerization of acrylic acid. Nanoparticles of average diameter 316 nm and zeta potential −39.88 mV were obtained from the such-treated PLLA by dialysis method. Porous collagen scaffold were fabricated by mixing PLLA nanoparticles with collagen solution, freeze drying, and crosslinking with EDC. SEM observation revealed that nanoparticles were homogeneously dispersed in collagen matrix, forming interconnected porous structure with pore size ranging from 150 to 200 μm, irrespective of the amount of nanoparticles. The porosity of the scaffolds kept almost unchanged with the increment of the nanoparticles, whereas the mechanical property was obviously improved, and the degradation was effectively retarded. In vitro L929 mouse fibroblast cells seeding and culture studies revealed that cells infiltrated into the scaffolds and were distributed homogeneously. Compared with the pure collagen sponge, the number of cells in hybrid scaffolds greatly increased with the increment of incorporated nanoparticles. These results manifested that the surface-activated PLLA nanoparticles effectively reinforced the porous collagen scaffold and promoted the cells penetrating into the scaffold, and proliferation. PMID:22448137

  4. BMP4 Inhibits PDGF-Induced Proliferation and Collagen Synthesis via PKA-Mediated Inhibition of Calpain-2 in Pulmonary Artery Smooth Muscle Cells.

    PubMed

    Cai, PengCheng; Kovacs, Laszlo; Dong, Sam; Wu, Guangyu; Su, Yunchao

    2017-02-24

    In the present study, we investigated the effect of bone morphogenetic protein 4 (BMP4) on PDGF-induced proliferation and collagen synthesis in PASMCs. Normal human PASMCs were incubated with and without PDGF-BB in the absence and presence of BMP4 for 0.5 to 24 h. Then the protein levels of collagen-I, p-Smad2/3, p-Smad1/5, and intracellular active TGFβ1, calpain activity and cell proliferation were measured. The results showed that BMP4 induced an increase in p-Smad1/5 but had no effect on the protein levels of collagen-I, p-Smad2/3, and intracellular active TGFβ1, and calpain activity in PASMCs. Nevertheless, BMP4 attenuated increases in proliferation and protein levels of collagen-I, p-Smad2/3, and intracellular active TGFβ1, and calpain activity in PDGF-BB-treated PASMCs. Moreover, BMP4 increased PKA activity and inhibition of PKA prevented the inhibitory effects of BMP4 on PDGF-BB-induced calpain activation in normal PASMCs. PKA activator forskolin recapitulated the suppressive effect of BMP4 on PDGF-induced calpain activation. Further, BMP4 prevented PDGF-induced decrease in calpain-2 phosphorylation at serine 369 in normal PASMCs. Finally, BMP4 did not attenuate PDGF-induced increases in proliferation, collagen-I protein levels, and calpain activation, and did not induce PKA activation and did not prevent PDGF-induced decrease in calpain-2 phosphorylation at serine 369 in PASMCs from IPAH patients. These data demonstrate that BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in normal PASMCs. The inhibitory effects of BMP4 on PDGF-induced proliferation, collagen synthesis and calpain-2 activation are impaired in PASMCs from PAH patients, which may contribute to pulmonary vascular remodeling in PAH.

  5. Stimulation of DNA and Collagen Synthesis by Autologous Growth Factor in Cultured Fetal Rat Calvaria

    NASA Astrophysics Data System (ADS)

    Canalis, Ernesto; Peck, William A.; Raisz, Lawrence G.

    1980-11-01

    Conditioned medium derived from organ or cell cultures prepared from 19- to 21-day fetal rat calvaria stimulated the incorporation of [3H]proline into collagen and of [3H]thymidine into DNA in organ cultures of the same tissue. Addition of cortisol enhanced the effect on collagen but not on DNA synthesis. These effects appeared to be due to a nondialyzable and heat-stable growth factor.

  6. Type I collagen-mediated synthesis of noble metallic nanoparticles networks and the applications in Surface-Enhanced Raman Scattering and electrochemistry.

    PubMed

    Sun, Yujing; Sun, Lanlan; Zhang, Baohua; Xu, Fugang; Liu, Zhelin; Guo, Cunlan; Zhang, Yue; Li, Zhuang

    2009-08-15

    In this paper, we demonstrated an effective environmentally friendly synthesis route to prepare noble metallic (Au, Ag, Pt and Pd) nanoparticles (NPs) networks mediated by type I collagen in the absence of any seeds or surfactants. In the reactions, type I collagen served as stabilizing agent and assembly template for the synthesized metallic NPs. The hydrophobic interaction between collagen and mica interface as well as the hydrogen bonds between inter- and intra-collagen molecules play important roles in the formation of collagen-metallic NPs networks. The noble metallic NPs networks have many advantages in the applications of Surface-Enhanced Raman Scattering (SERS) and electrochemistry detection. Typically, the as-prepared Ag NPs networks reveal great Raman enhancement activity for 4-ATP, and can even be used to detect low concentration of DNA base, adenine, without any label step. Furthermore, the cyclic voltammograms showed Pt NPs networks have good electrocatalytic ability for the reduction of O(2).

  7. Substance P Inhibits the Collagen Synthesis of Rat Myocardial Fibroblasts Induced by Ang II.

    PubMed

    Yang, Zhiyong; Zhang, Xinzhong; Guo, Naipeng; Li, Bin; Zhao, Sheng

    2016-12-16

    BACKGROUND The aim of this study was to explore the regulating effects of Substance P (SP) on the collagen synthesis of rat myocardial fibroblasts (CFBs) induced by angiotensin II (Ang II) and its potential mechanism. MATERIAL AND METHODS The CFBs of a neonatal SD rat were separately cultured and divided into the control group, Ang II treatment group, and treatment groups with different concentrations of SP, Ang II +; each group was given corresponding treatment respectively. RESULTS Ang II successfully induced the collagen synthesis of CFBs. Compared with the control group, the phosphorylation levels of TGF-β, erk, and smad2/3 were higher (p<0.05). Different concentrations of SP had an effect on Ang II-induced CFBs, reduced the collagen synthesis of CFBs, and increased the expressions of SP receptors, accompanied by lowering TGF-β protein, erk protein phosphorylation level, and smad2/3 protein phosphorylation level (p<0.05). Moreover, the higher the concentrations of SP, the more obvious of an effect it exerted. Treating the Ang II + SP group with aprepitant reduced the inhibiting effects of SP on collagen synthesis. The expression changes of collagen I and collagen III detected by immunocytochemistry were exactly in accordance with the results of qPCR and Western blotting. CONCLUSIONS SP can inhibit collagen synthesis of CFBs after Ang II inducing which may adjust the downstream signaling pathways associated protein including TGF-β, erk and smad2/3. SP can block the progress of myocardial fibrosis and is dose dependent, which is expected to be a promising target for the treatment of myocardial fibrosis.

  8. Substance P Inhibits the Collagen Synthesis of Rat Myocardial Fibroblasts Induced by Ang II

    PubMed Central

    Yang, Zhiyong; Zhang, Xinzhong; Guo, Naipeng; Li, Bin; Zhao, Sheng

    2016-01-01

    Background The aim of this study was to explore the regulating effects of Substance P (SP) on the collagen synthesis of rat myocardial fibroblasts (CFBs) induced by angiotensin II (Ang II) and its potential mechanism. Material/Methods The CFBs of a neonatal SD rat were separately cultured and divided into the control group, Ang II treatment group, and treatment groups with different concentrations of SP, Ang II +; each group was given corresponding treatment respectively. Results Ang II successfully induced the collagen synthesis of CFBs. Compared with the control group, the phosphorylation levels of TGF-β, erk, and smad2/3 were higher (p<0.05). Different concentrations of SP had an effect on Ang II-induced CFBs, reduced the collagen synthesis of CFBs, and increased the expressions of SP receptors, accompanied by lowering TGF-β protein, erk protein phosphorylation level, and smad2/3 protein phosphorylation level (p<0.05). Moreover, the higher the concentrations of SP, the more obvious of an effect it exerted. Treating the Ang II + SP group with aprepitant reduced the inhibiting effects of SP on collagen synthesis. The expression changes of collagen I and collagen III detected by immunocytochemistry were exactly in accordance with the results of qPCR and Western blotting. Conclusions SP can inhibit collagen synthesis of CFBs after Ang II inducing which may adjust the downstream signaling pathways associated protein including TGF-β, erk and smad2/3. SP can block the progress of myocardial fibrosis and is dose dependent, which is expected to be a promising target for the treatment of myocardial fibrosis. PMID:27980320

  9. Regulation of collagen synthesis in human dermal fibroblasts by ascorbic-induced lipid peroxidation

    SciTech Connect

    Geesin, J.C. Johnson and Johnson Consumer Products, Inc., Skillman, NJ ); Gordon, J.S. ); Gordon, J.S. ); Berg, R.A. )

    1991-03-11

    Ascorbic acid has been shown to stimulate collagen synthesis through the induction of lipid peroxidation which leads to increased transcription of the collagen genes. To test the ability of aldehyde products of lipid peroxidation to mediate this effect, the authors treated cultured fibroblasts with 1-200{mu}M of malondialdehyde, acetaldehyde, glyoxal or hexenal in the presence of lipid peroxidation inducing or noninducing concentrations of ascorbic acid. The treatment process involved either pretreatment of cells for 66hrs with either concentration of ascorbate before a 6hr treatment in the presence of ascorbate and the aldehydes, or 6 or 72hr treatment of the cells in the presence of either concentration of ascorbate plus the aldehydes. No effect of any of these aldehydes was seen on ascorbate-stimulated collagen synthesis. Also, pretreatment of fibroblasts for 24hrs with 100nM phorbol myristate acetate (PMA), which produces down regulation of protein kinase C(PKC), failed to alter the ascorbate-stimulation of collagen synthesis. Additionally, the authors tested the ability of benzamide, a poly ACP ribosylation inhibitor, to inhibit the ascorbate response with no specific effect noted. These results do not support the proposed roles for aldehydes, PKC, or poly ADP ribosylation in the mediation of the lipid peroxidation induced stimulation of collagen synthesis.

  10. The promoting effects of geniposidic acid and aucubin in Eucommia ulmoides Oliver leaves on collagen synthesis.

    PubMed

    Li, Y; Sato, T; Metori, K; Koike, K; Che, Q M; Takahashi, S

    1998-12-01

    We have reported that collagen synthesis was stimulated by the administration of a hot water extract from the leaves of Eucommia ulmoides OLIVER, Eucommiaceae (Du-Zhong leaves) in false aged model rats. In this paper, we set out to examine the compounds in Du-Zhong leaves that stimulated collagen synthesis in false aged model rats. In experiment 1, a methanol extract of Du-Zhong leaves also stimulated collagen synthesis in aged model rats. An acetone fraction was derived from the methanol extract by silica gel chromatography in experiment 2. The acetone fraction mainly contained iridoides mono-glycosides such as geniposidic acid and aucubin. The administration of geniposidic acid or aucubin stimulated collagen synthesis in aged model rats in experiments 3 and 4 (significance (p<0.05)). The reported pharmacological effects of Du-Zhong leaves, including healing organs and strengthening bone and muscle, are closely related to collagen metabolism. It appears that geniposidic acid and aucubin are the actual compounds in Du-Zhong which caused the effect in our experiments.

  11. Salamander-derived, human-optimized nAG protein suppresses collagen synthesis and increases collagen degradation in primary human fibroblasts.

    PubMed

    Al-Qattan, Mohammad M; Shier, Medhat K; Abd-Alwahed, Mervat M; Mawlana, Ola H; El-Wetidy, Mohammed S; Bagayawa, Reginald S; Ali, Hebatallah H; Al-Nbaheen, May S; Aldahmash, Abdullah M

    2013-01-01

    Unlike humans, salamanders regrow their amputated limbs. Regeneration depends on the presence of regenerating axons which upregulate the expression of newt anterior gradient (nAG) protein. We had the hypothesis that nAG might have an inhibitory effect on collagen production since excessive collagen production results in scarring, which is a major enemy to regeneration. nAG gene was designed, synthesized, and cloned. The cloned vector was then transfected into primary human fibroblasts. The results showed that the expression of nAG protein in primary human fibroblast cells suppresses the expression of collagen I and III, with or without TGF- β 1 stimulation. This suppression is due to a dual effect of nAG both by decreasing collagen synthesis and by increasing collagen degradation. Furthermore, nAG had an inhibitory effect on proliferation of transfected fibroblasts. It was concluded that nAG suppresses collagen through multiple effects.

  12. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    PubMed Central

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  13. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running.

    PubMed

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-06-28

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d₃-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise.

  14. Synthesis of lipoic acid-peptide conjugates and their effect on collagen and melanogenesis.

    PubMed

    Lu, Chichong; Kim, Bo Mi; Lee, Duckhee; Lee, Min Hee; Kim, Jin Hwa; Pyo, Hyeong-Bae; Chai, Kyu Yun

    2013-11-01

    We report new examples of lipoic acid (LA)-peptide conjugates, their potential as codrugs having anti-melanogenic and anti-aging properties was evaluated. These multifunctional molecules were prepared by linking lipophilic moiety (LA) to the pentapeptide KTTKS. The inhibitory effect of LA-peptide conjugates on melanin synthesis and tyrosinase activity is stronger than that of LA or the pentapeptide alone. Importantly, the conjugates display no cytotoxicity at a high concentration. LA-KTTKS and LA-PEG-KTTKS also inhibit UV-induced matrix metalloproteinase-1 expression up to 49.5% and 69.5% at 0.5 mM, respectively. LA-peptide conjugates stimulate collagen biosynthesis in fibroblasts more efficiently than their parent molecules do. These data suggest that LA-peptide conjugates may have cosmeceutical application as anti-melanogenic and anti-aging agents.

  15. Myocyte-Derived Hsp90 Modulates Collagen Upregulation via Biphasic Activation of STAT-3 in Fibroblasts during Cardiac Hypertrophy.

    PubMed

    Datta, Ritwik; Bansal, Trisha; Rana, Santanu; Datta, Kaberi; Datta Chaudhuri, Ratul; Chawla-Sarkar, Mamta; Sarkar, Sagartirtha

    2017-03-15

    Signal transducer and activator of transcription 3 (STAT-3)-mediated signaling in relation to upregulated collagen expression in fibroblasts during cardiac hypertrophy is well defined. Our recent findings have identified heat shock protein 90 (Hsp90) to be a critical modulator of fibrotic signaling in cardiac fibroblasts in this disease milieu. The present study was therefore intended to analyze the role of Hsp90 in the STAT-3-mediated collagen upregulation process. Our data revealed a significant difference between in vivo and in vitro results, pointing to a possible involvement of myocyte-fibroblast cross talk in this process. Cardiomyocyte-targeted knockdown of Hsp90 in rats (Rattus norvegicus) in which the renal artery was ligated showed downregulated collagen synthesis. Furthermore, the results obtained with cardiac fibroblasts conditioned with Hsp90-inhibited hypertrophied myocyte supernatant pointed toward cardiomyocytes' role in the regulation of collagen expression in fibroblasts during hypertrophy. Our study also revealed a novel signaling mechanism where myocyte-derived Hsp90 orchestrates not only p65-mediated interleukin-6 (IL-6) synthesis but also its release in exosomal vesicles. Such myocyte-derived exosomes and myocyte-secreted IL-6 are responsible in unison for the biphasic activation of STAT-3 signaling in cardiac fibroblasts that culminates in excess collagen synthesis, leading to severely compromised cardiac function during cardiac hypertrophy.

  16. Ultrananocrystalline diamond thin films functionalized with therapeutically active collagen networks.

    SciTech Connect

    Huang, H.; Chen, M.; Bruno, P.; Lam, R.; Robinson, E.; Gruen, D.; Ho, D.; Materials Science Division; Northwestern Univ.

    2009-01-01

    The fabrication of biologically amenable interfaces in medicine bridges translational technologies with their surrounding biological environment. Functionalized nanomaterials catalyze this coalescence through the creation of biomimetic and active substrates upon which a spectrum of therapeutic elements can be delivered to adherent cells to address biomolecular processes in cancer, inflammation, etc. Here, we demonstrate the robust functionalization of ultrananocrystalline diamond (UNCD) with type I collagen and dexamethasone (Dex), an anti-inflammatory drug, to fabricate a hybrid therapeutically active substrate for localized drug delivery. UNCD oxidation coupled with a pH-mediated collagen adsorption process generated a comprehensive interface between the two materials, and subsequent Dex integration, activity, and elution were confirmed through inflammatory gene expression assays. These studies confer a translational relevance to the biofunctionalized UNCD in its role as an active therapeutic network for potent regulation of cellular activity toward applications in nanomedicine.

  17. Photobiomodulation on the proliferation and collagen synthesis of normal human skin fibroblast cells

    NASA Astrophysics Data System (ADS)

    Cheng, Lei; Liu, Timon Cheng-Yi; Chi, Jin-Quan; Li, Yan; Jin, Hua

    2006-01-01

    Background and Objective: Cultured normal human skin fibroblast cells (HSFs) were once used to study the mechanism of the effects of low intensity He-Ne laser irradiation (LHNL) on wound healing. The proliferation and collagen synthesis of HFSs were modulated by LHNL in different papers, respectively, and both of them are studied in this paper. Study Design/Materials and Methods: The dosage was studied for the same radiation time 300s. The proliferation and collagen synthesis were measured by 3-[4,5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay and the spectrophotometric method for the determination of hydroxyproline, respectively. Results: The dose zones were called dose 1, dose 2 and dose 3 from low dose on so that HSF proliferation was inhibited in dose 1 (16, 24 mJ/cm2), and promoted in dose 2 (298, 503, 597mJ/cm2), and the collagen synthesis was inhibited in dose 2 (401, 526 mJ/cm2), and promoted in dose 3 (714, 926, 1539, 1727mJ/cm2), which supports our biological model of photobiomodulation. It was found there is the linear relationship of the effect with dose with dose in each dose zone. Conclusions: The photobiomodulation on the proliferation and collagen synthesis of HSFs might be linearly dose-dependent in limited dosage with radiation time kept constant, which provides a foundation to discuss photobiomodulation on wound healing.

  18. The effects of macrophage-stimulating protein on the migration, proliferation, and collagen synthesis of skin fibroblasts in vitro and in vivo.

    PubMed

    Zhao, Jiajia; Hu, Li; Gong, Niya; Tang, Qingming; Du, Luyang; Chen, Lili

    2015-03-01

    Macrophage-stimulating protein (MSP), an important cytokine with multiple functions, is highly expressed in adipose-derived stem cells-conditioned medium (ASC-CM). ASCs can effectively promote wound healing through paracrine mechanism, suggesting that MSP may play a critical role in wound healing. Through binding to its receptor, RON (Receptuerd'OrigineNantaise, also called macrophage stimulation 1 receptor; MST1R), it can activate epithelial cells and work as an inflammatory mediator. In this study, we found RON was also expressed on dermal fibroblasts and investigated the effects of MSP on proliferation, migration, and collagen synthesis of fibroblasts. With the treatment of different concentrations of MSP (0, 1, 10, 20, 50, and 100 ng/mL) on fibroblasts, proliferation, migration, and collagen synthesis were analyzed by Cell Counting Kit-8 (CCK-8), transwell and real-time polymerase chain reaction. Under the treatment of MSP, the migration, Collagen I, III synthesis, and matrix metalloproteinase-1 (MMP-1) mRNA expression of fibroblasts were upregulated significantly, although there was no effect on fibroblasts proliferation, and the optimal concentration of MSP for migration and collagen synthesis was 10 ng/mL. In the in vivo study, 10 ng/mL MSP was applied to full-thickness skin wound with bacterial cellulose membranes, and this treatment could accelerate the wound healing rate and increased the collagen synthesis of wound sites. This study suggested that MSP appears to promote the migration of fibroblasts, enhances collagen synthesis and remodeling, and effectively improves wound healing.

  19. A thyroid hormone analogue, triiodothyroacetic acid, corrects corticosteroid-downregulated collagen synthesis.

    PubMed

    Yazdanparast, Parviz; Carlsson, Bo; Oikarinen, Aarne; Risteli, Juha; Faergemann, Jan

    2004-05-01

    The aim of this study was to compare the change in collagen synthesis between topical treatments with two doses of triiodothyroacetic acid (TRIAC), a thyroid hormone analogue, and placebo, after pretreatment with topical betamethasone 17-valerate (BM). Eighteen healthy volunteers were pretreated with BM on abdominal skin for 3 days, and were then treated for 14 days with a cream containing TRIAC (0.03% or 0.1%) or a placebo cream. Collagen production was assessed by quantifying the amino terminal propeptides of human type I and type III procollagen (PINP and PIIINP) in fluids from suction-induced blisters on the treated skin. Three days of treatment with BM led to an average reduction of PINP of 70% and of PIIINP of 50%. Seven days after treatment, the median increase in PINP was 230% (p = 0.03) in the Triac 0.03% group, 148% (p = 0.2) in the TRIAC 0.1% and 5% in the placebo group. The median increase in PINP in the skin area from the start of treatment to the end of treatment was 521% (p = 0.06) in the TRIAC 0.03% group, 339% (p = 0.2) in the TRIAC 0.1% group, and 55% in the placebo group (the p values are related to baseline). Seven days after treatment, the median increase in PIIINP was 24% (p = 0.6) in the Triac 0.03% group, 23% (p = 0.6) in the TRIAC 0.1% group, and -12% in the placebo group. The median increase in PIIINP in the skin area from the start of treatment to the end of treatment was 137% (p = 0.7) in the TRIAC 0.03% group, 230% (p = 0.9) in the TRIAC 0.1% group and 58% in the placebo group (the p values are related to baseline). Histologic examinations of sections from punch biopsies taken at the end of the treatment showed more thickened collagen fibers and increased density of PINP-producing dermal fibroblasts in the TRIAC groups compared to the placebo group. The result suggests a potential role for TRIAC-containing cream concomitant with anti-inflammatory topical treatment with potent glucocorticoids to prevent their suppressive activity on

  20. Uniform spatial distribution of collagen fibril radii within tendon implies local activation of pC-collagen at individual fibrils

    NASA Astrophysics Data System (ADS)

    Rutenberg, Andrew D.; Brown, Aidan I.; Kreplak, Laurent

    2016-08-01

    Collagen fibril cross-sectional radii show no systematic variation between the interior and the periphery of fibril bundles, indicating an effectively constant rate of collagen incorporation into fibrils throughout the bundle. Such spatially homogeneous incorporation constrains the extracellular diffusion of collagen precursors from sources at the bundle boundary to sinks at the growing fibrils. With a coarse-grained diffusion equation we determine stringent bounds, using parameters extracted from published experimental measurements of tendon development. From the lack of new fibril formation after birth, we further require that the concentration of diffusing precursors stays below the critical concentration for fibril nucleation. We find that the combination of the diffusive bound, which requires larger concentrations to ensure homogeneous fibril radii, and lack of nucleation, which requires lower concentrations, is only marginally consistent with fully processed collagen using conservative bounds. More realistic bounds may leave no consistent concentrations. Therefore, we propose that unprocessed pC-collagen diffuses from the bundle periphery followed by local C-proteinase activity and subsequent collagen incorporation at each fibril. We suggest that C-proteinase is localized within bundles, at fibril surfaces, during radial fibrillar growth. The much greater critical concentration of pC-collagen, as compared to fully processed collagen, then provides broad consistency between homogeneous fibril radii and the lack of fibril nucleation during fibril growth.

  1. Effect of Collagen Nanotopography on Keloid Fibroblast Proliferation and Matrix Synthesis: Implications for Dermal Wound Healing

    PubMed Central

    Muthusubramaniam, Lalitha; Zaitseva, Tatiana; Paukshto, Michael; Martin, George

    2014-01-01

    Keloids are locally exuberant dermal scars characterized by excessive fibroblast proliferation and matrix accumulation. Although treatment strategies include surgical removal and intralesional steroid injections, an effective regimen is yet to be established due to a high rate of recurrence. The regressing center and growing margin of the keloid have different collagen architecture and also differ in the rate of proliferation. To investigate whether proliferation is responsive to collagen topography, keloid, scar, and dermal fibroblasts were cultured on nanopatterned scaffolds varying in collagen fibril diameter and alignment-small and large diameter, aligned and random fibrils, and compared to cells grown on flat collagen-coated substrates, respectively. Cell morphology, proliferation, and expression of six genes related to proliferation (cyclin D1), phenotype (α-smooth muscle actin), and matrix synthesis (collagens I and III, and matrix metalloproteinase-1 and -2) were measured to evaluate cell response. Fibril alignment was shown to reduce proliferation and matrix synthesis in all three types of fibroblasts. Further, keloid cells were found to be most responsive to nanotopography. PMID:24724556

  2. Collagen-induced platelet activation mainly involves the protein kinase C pathway.

    PubMed Central

    Karniguian, A; Grelac, F; Levy-Toledano, S; Legrand, Y J; Rendu, F

    1990-01-01

    This study analyses early biochemical events in collagen-induced platelet activation. An early metabolic event occurring during the lag phase was the activation of PtdIns(4,5)P2-specific phospholipase C. Phosphatidic acid (PtdOH) formation, phosphorylation of P43 and P20, thromboxane B2 (TXB2) synthesis and platelet secretion began after the lag phase, and were similarly time-dependent, except for TXB2 synthesis, which was delayed. Collagen induced extensive P43 phosphorylation, whereas P20 phosphorylation was weak and always lower than with thrombin. The dose-response curves of P43 phosphorylation and granule secretion were similar, and both reached a peak at 7.5 micrograms of collagen/ml, a dose which induced half-maximal PtdOH and TXB2 formation. Sphingosine, assumed to inhibit protein kinase C, inhibited P43 phosphorylation and secretion in parallel. However, sphingosine was not specific for protein kinase C, since a 15 microM concentration, which did not inhibit P43 phosphorylation, blocked TXB2 synthesis by 50%. Sphingosine did not affect PtdOH formation at all, even at 100 microM, suggesting that collagen itself induced this PtdOH formation, independently of TXB2 generation. The absence of external Ca2+ allowed the cleavage of polyphosphoinositides and the accumulation of InsP3 to occur, but impaired P43 phosphorylation, PtdOH and TXB2 formation, and secretion; these were only restored by adding 0.11 microM-Ca2+. In conclusion, stimulation of platelet membrane receptors for collagen initiates a PtdInsP2-specific phospholipase C activation, which is independent of external Ca2+, and might be the immediate receptor-linked response. A Ca2+ influx is indispensable to the triggering of subsequent platelet responses. This stimulation predominantly involves the protein kinase C pathway associated with secretion, and appears not to be mediated by TXB2, at least during its initial stage. Images Fig. 6. PMID:2163606

  3. Ethinyl oestradiol administration in women suppresses synthesis of collagen in tendon in response to exercise

    PubMed Central

    Hansen, Mette; Koskinen, Satu O; Petersen, Susanne G; Doessing, Simon; Frystyk, Jan; Flyvbjerg, Allan; Westh, Eva; Magnusson, S Peter; Kjaer, Michael; Langberg, Henning

    2008-01-01

    Women are at greater risk than men of sustaining certain kinds of injury and diseases of collagen-rich tissues. To determine whether a high level of oestradiol has an acute influence on collagen synthesis in tendons at rest and in response to exercise, one-legged kicking exercise was performed for 60 min at 67% of maximum power by healthy, young oral contraceptive (OC) users when circulating synthetic (ethinyl) oestradiol was high (n = 11, HE-OC) and compared to similar women who had never used OCs when circulating endogenous oestrogen was low (n = 12, LE-NOC). Interstitial fluid was collected 24 h post-exercise through microdialysis catheters placed anterior to the patellar tendon in both legs and subsequently analysed for the amino-terminal propeptide of type I collagen (PINP), a marker of tendon collagen synthesis. To determine the long-term effect of OC usage, patellar tendon cross-sectional area (CSA) was measured by magnetic resonance imaging (MRI). A lower exercise-induced increase in tendon collagen synthesis was observed in HE-OC than in LE-NOC (ΔPINP (mean ±s.e.m.) 1.5 ± 5.3 versus 24.2 ± 9.4 ng ml−1, P < 0.05). Furthermore, serum and the interstitial peritendinous tissue concentrations of insulin-like growth factor I (IGF-I) and IGF-binding proteins showed a reduced bioavailability in HE-OC compared with results in LE-NOC. No difference in patellar tendon CSA was observed between groups. In conclusion, the selective increase in tendon collagen synthesis in LE-NOC but not HE-OC 24 h post-exercise is consistent with the hypothesis that oestradiol inhibits exercise-induced collagen synthesis in human tendon. The mechanism behind this is either a direct effect of oestradiol, or an indirect effect via a reduction in levels of free IGF-I. However, the data did not indicate any long-term effect on tendon size associated with chronic OC use. PMID:18420709

  4. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  5. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-08

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

  6. Modulation of heat shock protein 90 affects TGF-β-induced collagen synthesis in human dermal fibroblast cells.

    PubMed

    Lee, Sae Bin; Lim, A-Ram; Rah, Dong Kyun; Kim, Kyung Soo; Min, Hyun Jin

    2016-12-01

    Heat shock protein 90 is a chaperone molecule that aids in proper folding of target proteins. Recently, heat shock protein 90 was found to play a role in would healing through regulation of fibroblast functions. The aim of the present study was to investigate the role of heat shock protein 90 in collagen synthesis in human dermal fibroblasts. The effects of transforming growth factor-β, 17-N-allylamino-17-demethoxygeldanamycin, and transfection of heat shock protein 90 were evaluated by real-time PCR, western blot, and immunofluorescence assays. The Smad 2/3 and Akt pathways were evaluated to identify the signaling pathways involved in collagen synthesis. Heat shock protein 90 and collagen levels were compared in keloid and control tissues by immunohistochemical analysis. The expression of collagen was significantly increased after treatment with transforming growth factor-β, while 17-N-allylamino-17-demethoxygeldanamycin inhibited transforming growth factor-β-induced collagen synthesis. Overexpression of heat shock protein 90 itself with or without transforming growth factor-β increased collagen synthesis. These effects were dependent on Smad 2/3 pathway signaling. Finally, expression of heat shock protein 90 was increased in keloid tissue compared with control tissues. Taken together, these results demonstrate that modulation of heat shock protein 90 influences transforming growth factor-β-induced collagen synthesis via regulation of Smad 2/3 phosphorylation.

  7. Mechanical strain and collagen potentiate mitogenic activity of angiotensin II in rat vascular smooth muscle cells.

    PubMed Central

    Sudhir, K; Wilson, E; Chatterjee, K; Ives, H E

    1993-01-01

    The effects of extracellular matrix proteins and mechanical strain on the mitogenic activity of angiotensins I and II (AI and AII) were examined in cultured rat vascular smooth muscle (VSM) cells. VSM cells on various extracellular matrices were exposed to AII (1 microM) for 48 h. On plastic, AII induced only a 1.6-fold increase in [3H]thymidine incorporation, but on fibronectin- or type I collagen-coated plastic, the response to AII was enhanced from two- to fourfold. On a type I collagen-coated silicone elastomer, to which mechanical strain was applied, [3H]thymidine incorporation dramatically increased to a maximum of 53-fold. Dup 753 (10(-5) M) blocked the AII-induced increase in DNA synthesis. AI also increased DNA synthesis in VSM cells, and this response was also enhanced by mechanical strain. Mitogenic activity of AI was blocked by ramiprilat (10(-5) M), indicating that its mitogenic activity was via conversion to AII. The synergy between AII and strain was completely eliminated by neutralizing antibodies to PDGF AB (3 micrograms/ml). Furthermore, the mitogenic effect of AII in unstrained cells was also synergistic with submaximal concentrations of PDGF AB (1 ng/ml). Thus, the synergy between AII and mechanical strain probably results from synergism between AII and PDGF secreted in response to strain. PMID:8254054

  8. High voltage pulsed current in collagen realignment, synthesis, and angiogenesis after Achilles tendon partial rupture

    PubMed Central

    Rampazo, Érika P.; Liebano, Richard E.; Pinfildi, Carlos Eduardo; Folha, Roberta A. C.; Ferreira, Lydia M.

    2016-01-01

    ABSTRACT Objective To verify the efficacy of high voltage pulsed current in collagen realignment and synthesis and in angiogenesis after the partial rupturing of the Achilles tendon in rats. Method Forty male Wistar rats were randomized into four groups of 10 animals each: sham, cathodic stimulation, anodic stimulation, and alternating stimulation. Their Achilles tendons were submitted to direct trauma by a free-falling metal bar. Then, the treatment was administered for six consecutive days after the injury. In the simulation group, the electrodes were positioned on the animal, but the device remained off for 30 minutes. The other groups used a frequency of 120 pps, sensory threshold, and the corresponding polarity. On the seventh day, the tendons were removed and sent for histological slide preparation for birefringence and Picrosirius Red analysis and for blood vessel quantification. Results No significant difference was observed among the groups regarding collagen realignment (types I or III collagen) or quantity of blood vessels. Conclusion High voltage pulsed current for six consecutive days was not effective in collagen realignment, synthesis, or angiogenesis after the partial rupturing of the Achilles tendon in rats. PMID:27556387

  9. Induction of collagen synthesis by ascorbic acid. A possible mechanism.

    PubMed

    Pinnel, S R; Murad, S; Darr, D

    1987-12-01

    L-Ascorbic acid stimulates procollagen synthesis in cultured human skin fibroblasts without appreciably altering noncollagen protein synthesis. The effect is unrelated to intracellular degradation of newly synthesized procollagen. Levels of mRNA for pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III), measured by hybridization with the corresponding cDNA probes, are elevated in the presence of ascorbic acid, whereas the level of mRNA for fibronectin is unchanged. Levels of functional mRNA for procollagen, measured in a cell-free translation assay, are specifically increased in the presence of ascorbic acid. Thus, ascorbic acid appears to control the expression of three different procollagen genes, each of which is located on a separate chromosome. It is proposed that intracellularly accumulated procollagen in ascorbate deficiency may lead to a translational repression of procollagen synthesis. Ascorbic acid may relieve this block by promoting hydroxyproline formation and, consequently, secretion of procollagen from the cell. The increased level of procollagen mRNA under the influence of ascorbic acid may be secondary to increased synthesis of procollagen polypeptides; the control point may be gene transcription or mRNA degradation.

  10. Alveolar type II cell-fibroblast interactions, synthesis and secretion of surfactant and type I collagen.

    PubMed

    Griffin, M; Bhandari, R; Hamilton, G; Chan, Y C; Powell, J T

    1993-06-01

    During alveolar development and alveolar repair close contacts are established between fibroblasts and lung epithelial cells through gaps in the basement membrane. Using co-culture systems we have investigated whether these close contacts influence synthesis and secretion of the principal surfactant apoprotein (SP-A) by cultured rat lung alveolar type II cells and the synthesis and secretion of type I collagen by fibroblasts. The alveolar type II cells remained cuboidal and grew in colonies on fibroblast feeder layers and on Matrigel-coated cell culture inserts but were progressively more flattened on fixed fibroblast monolayers and plastic. Alveolar type II cells cultured on plastic released almost all their SP-A into the medium by 4 days. Alveolar type II cells cultured on viable fibroblasts or Matrigel-coated inserts above fibroblasts accumulated SP-A in the medium at a constant rate for the first 4 days, and probably recycle SP-A by endocytosis. The amount of mRNA for SP-A was very low after 4 days of culture of alveolar type II cells on plastic, Matrigel-coated inserts or fixed fibroblast monolayers: relatively, the amount of mRNA for SP-A was increased 4-fold after culture of alveolar type II cells on viable fibroblasts. Co-culture of alveolar type II cells with confluent human dermal fibroblasts stimulated by 2- to 3-fold the secretion of collagen type I into the culture medium, even after the fibroblasts' growth had been arrested with mitomycin C. Collagen secretion, by fibroblasts, also was stimulated 2-fold by conditioned medium from alveolar type II cells cultured on Matrigel. The amount of mRNA for type I collagen increased only modestly when fibroblasts were cultured in this conditioned medium. This stimulation of type I collagen secretion diminished as the conditioned medium was diluted out, but at high dilutions further stimulation occurred, indicating that a factor that inhibited collagen secretion also was being diluted out. The conditioned medium

  11. The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen

    PubMed Central

    Malcor, Jean-Daniel; Bax, Daniel; Hamaia, Samir W.; Davidenko, Natalia; Best, Serena M.; Cameron, Ruth E.; Farndale, Richard W.; Bihan, Dominique

    2016-01-01

    Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2β1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2β1 and α1β1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons. PMID:26854392

  12. The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen.

    PubMed

    Malcor, Jean-Daniel; Bax, Daniel; Hamaia, Samir W; Davidenko, Natalia; Best, Serena M; Cameron, Ruth E; Farndale, Richard W; Bihan, Dominique

    2016-04-01

    Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2β1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2β1 and α1β1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons.

  13. 96-Well plate assays for measuring collagenase activity using (3)H-acetylated collagen.

    PubMed

    Koshy, P J; Rowan, A D; Life, P F; Cawston, T E

    1999-11-15

    We describe two alternative assays for measuring collagenolytic activity using (3)H-acetylated collagen. Both assays have been developed for the 96-well plate format and measure the amount of radiolabeled collagen fragments released into the supernatant from an insoluble (3)H-acetylated collagen fibril preparation. The first method separates digested solubilized fragments from the intact fibril by sedimentation of the undigested collagen by centrifugation. The second method achieves this separation by filtration of the supernatant through the membrane of a 96-well filtration plate which retains the undigested collagen fibril. Both methods give linear dose- and time-dependent responses of collagenase activity > or = 70% of total collagen lysis. In addition, both assays can be simply modified to measure tissue inhibitors of metalloproteinases (TIMPs) inhibitory activity, which is also linear between 20 and 75% of total collagen lysis with the amount of TIMP added.

  14. Use of collagen hydrolysate as a complex nitrogen source for the synthesis of penicillin by Penicillium chrysogenum.

    PubMed

    Leonhartsberger, S; Lafferty, R M; Korneti, L

    1993-09-01

    Optimal conditions for both biomass formation and penicillin synthesis by a strain of Penicillium chrysogenum were determined when using a collagen-derived nitrogen source. Preliminary investigations were carried out in shaken flask cultures employing a planned experimental program termed the Graeco-Latin square technique (Auden et al., 1967). It was initially determined that up to 30% of a conventional complex nitrogen source such as cottonseed meal could be replaced by the collagen-derived nitrogen source without decreasing the productivity with respect to the penicillin yield. In the pilot scale experiments using a 30 l stirred tank type of bioreactor, higher penicillin yields were obtained when 70% of the conventional complex nitrogen source in the form of cottonseed meal was replaced by the collagen hydrolysate. Furthermore, the maximum rate of penicillin synthesis continued for over a longer period when using collagen hydrolysate as a complex nitrogen source. Penicillin synthesis rates were determined using a linear regression.

  15. Doxazosin Stimulates Galectin-3 Expression and Collagen Synthesis in HL-1 Cardiomyocytes Independent of Protein Kinase C Pathway

    PubMed Central

    Qian, Xiaoqian; Li, Mingyang; Wagner, Mary B.; Chen, Guangping; Song, Xiang

    2016-01-01

    Doxazosin, a drug commonly prescribed for hypertension and prostate disease, increases heart failure risk. However, the underlying mechanism remains unclear. Galectin-3 is an important mediator that plays a pathogenic role in cardiac hypertrophy and heart failure. In the present study, we investigated whether doxazosin could stimulate galectin-3 expression and collagen synthesis in cultured HL-1 cardiomyocytes. We found that doxazosin dose-dependently induced galectin-3 protein expression, with a statistically significant increase in expression with a dose as low as 0.01 μM. Doxazosin upregulated collagen I and α-smooth muscle actin (α-SMA) protein levels and also induced apoptotic protein caspase-3 in HL-1 cardiomyocytes. Although we previously reported that activation of protein kinase C (PKC) stimulates galectin-3 expression, blocking the PKC pathway with the PKC inhibitor chelerythrine did not prevent doxazosin-induced galectin-3 and collagen expression. Consistently, doxazosin treatment did not alter total and phosphorylated PKC. These results suggest that doxazosin-stimulated galectin-3 is independent of PKC pathway. To determine if the α1-adrenergic pathway is involved, we pretreated the cells with the irreversible α-adrenergic receptor blocker phenoxybenzamine and found that doxazosin-stimulated galectin-3 and collagen expression was similar to controls, suggesting that doxazosin acts independently of α1-adrenergic receptor blockade. Collectively, we show a novel effect of doxazosin on cardiomycytes by stimulating heart fibrosis factor galectin-3 expression. The mechanism of action of doxazosin is not mediated through either activation of the PKC pathway or antagonism of α1-adrenergic receptors. PMID:28066244

  16. Type I pro-collagen promoting and anti-collagenase activities of Phyllanthus emblica extract in mouse fibroblasts.

    PubMed

    Chanvorachote, Pithi; Pongrakhananon, Varisa; Luanpitpong, Sudjit; Chanvorachote, Boontarika; Wannachaiyasit, Sumalee; Nimmannit, Ubonthip

    2009-01-01

    As part of an ongoing search for the novel pharmacological activities of Phyllanthus emblica, the present study has shown its type I collagen promoting and anti-collagenase effects on primary mouse fibroblast cells. At a concentration of 0.1 mg/ml, emblica extract significantly increased the type I pro-collagen level up to 1.65-fold, and 6.78-fold greater than that of an untreated control, determined by immunocytochemistry and Western blot analysis, respectively. Emblica extract caused an approximately 7.75-fold greater type I pro-collagen induction compared to the known herbal collagen enhancer asiaticoside at the same treatment concentration (0.1 mg/ml). Moreover, emblica extract inhibited collagenase activity in a dose-dependent manner. Maximal inhibition was observed (78.67 +/- 3.51%) at a concentration of 1 mg/ml. In summary, emblica extract has a promising pharmacological effect that benefits collagen synthesis and protects against its degradation and could be used as a natural anti-aging ingredient.

  17. Synthesis of glycosaminoglycans in differently loaded regions of collagen gels seeded with valvular interstitial cells.

    PubMed

    Gupta, Vishal; Werdenberg, Jennifer A; Blevins, Tracy L; Grande-Allen, K Jane

    2007-01-01

    Cells respond to changes in mechanical strains by varying their production of extracellular matrix macromolecules. Because differences in strain patterns between mitral valve leaflets and chordae tendineae have been linked to different quantities and types of glycosaminoglycans (GAGs), we investigated the effects of various strain conditions on GAG synthesis by valvular interstitial cells (VICs) using an in vitro 3-dimensional tissue-engineering model. VICs from leaflets or chordae were seeded within collagen gels and subjected to uniaxial or biaxial static tension for 1 week. GAGs synthesized within the collagen gels and secreted into the surrounding medium were analyzed using fluorophore-assisted carbohydrate electrophoresis. In constrained conditions, more 4-sulfated GAGs were retained within the collagen gel, whereas more hyaluronan was secreted into the surrounding medium. Selected GAG classes were found in significantly different proportions in collagen gels seeded with leaflet cells versus chordal cells. The only significant difference between uniaxial and biaxial regions was found for 6-sulfated GAGs in the gels seeded with chordal cells (p<0.05). This study suggests how mechanical loading may influence GAG production and localization in the remodeling of the mitral valve and has design implications for engineered tissues.

  18. Roles of epithelial cell-derived periostin in TGF-beta activation, collagen production, and collagen gel elasticity in asthma.

    PubMed

    Sidhu, Sukhvinder S; Yuan, Shaopeng; Innes, Anh L; Kerr, Sheena; Woodruff, Prescott G; Hou, Lydia; Muller, Susan J; Fahy, John V

    2010-08-10

    Periostin is considered to be a matricellular protein with expression typically confined to cells of mesenchymal origin. Here, by using in situ hybridization, we show that periostin is specifically up-regulated in bronchial epithelial cells of asthmatic subjects, and in vitro, we show that periostin protein is basally secreted by airway epithelial cells in response to IL-13 to influence epithelial cell function, epithelial-mesenchymal interactions, and extracellular matrix organization. In primary human bronchial epithelial cells stimulated with periostin and epithelial cells overexpressing periostin, we reveal a function for periostin in stimulating the TGF-beta signaling pathway in a mechanism involving matrix metalloproteinases 2 and 9. Furthermore, conditioned medium from the epithelial cells overexpressing periostin caused TGF-beta-dependent secretion of type 1 collagen by airway fibroblasts. In addition, mixing recombinant periostin with type 1 collagen in solution caused a dramatic increase in the elastic modulus of the collagen gel, indicating that periostin alters collagen fibrillogenesis or cross-linking and leads to stiffening of the matrix. Epithelial cell-derived periostin in asthma has roles in TGF-beta activation and collagen gel elasticity in asthma.

  19. Synthesis, characterization and bioevaluation of drug-collagen hybrid materials for biomedical applications.

    PubMed

    Voicu, Georgeta; Geanaliu-Nicolae, Ruxandra-Elena; Pîrvan, Adrian-Alexandru; Andronescu, Ecaterina; Iordache, Florin

    2016-08-30

    This work presents a study based on the preparation and characterization of drug-collagen hybrid materials. Materials used for obtaining drug-collagen hybrids were collagen type I (Coll) as matrix and fludarabine (F) and epirubicin (E) as hydrophilic active substances. After incorporation of drugs into Coll in different ratios, the obtained hybrid materials (Coll/F and Coll/E) could be used according to our results as potential drug delivery systems in medicine for the topical (local) treatment of cancerous tissues (e.g. the treatment of breast, stomach, lung, colorectal or advanced ovarian cancer). The materials were characterized considering their composition (by XRD, FT-IR and DTA-TG) and their morphology (by SEM). The delivery of drug was assessed by UV-vis. The in vitro citotoxicity demonstrates an antitumoral activity of the obtained hybrid materials and their potential use for biomedical applications as drug delivery systems in tumoral treatments.

  20. Arginine increases insulin-like growth factor-I production and collagen synthesis in osteoblast-like cells.

    PubMed

    Chevalley, T; Rizzoli, R; Manen, D; Caverzasio, J; Bonjour, J P

    1998-08-01

    Protein-energy malnutrition, which is common in elderly patients with osteoporotic hip fractures, is associated with reduced plasma levels of insulin-like growth factor-I (IGF-I). IGF-I is an important regulator of bone metabolism, particularly of osteoblastic bone formation both in vivo and in vitro. Pharmacological doses of arginine (Arg) increase growth hormone (GH) and IGF-I serum levels. Whether amino acids, particularly Arg, can directly modulate the production of IGF-I by osteoblasts is not known. We investigated the effects of increasing concentrations of Arg on IGF-I expression and production, alpha1(I) collagen expression and collagen synthesis, and cell proliferation and cell differentiation, as assessed by alkaline phosphatase (ALP) activity and osteocalcin (OC) release, in confluent mouse osteoblast-like MC3T3-E1 cells. The addition of Arg (7.5-7500 micromol/L, equivalent to 0.1- to 100-fold human plasma concentration) for 48 h increased IGF-I production (adjusted for cell number) in a concentration-dependent manner with a maximum of 2.3 +/- 0.3-fold at 7500 micromol/L Arg [x +/- standard error of the mean (SEM), n = 3 experiments, p < 0.01]. Arg (7.5-7500 micromol/L) increased the percentage of de novo collagen synthesis in a concentration-dependent manner (2.1 +/- 0.4-fold with 7500 micromol/L Arg, p < 0.001) and ALP activity with a maximal stimulation of 144% +/- 13% plateauing at 750 micromol/l Arg (p = 0.002). The steady state level of IGF-I messenger ribonucleic acid (mRNA) and alpha1(I) collagen mRNA (both normalized to cyclophilin mRNA) of cells incubated with Arg at high (100-fold) or low (0.1-fold) human plasma concentrations, was 1.4 +/- 0.2, 1.2 +/- 0.2, and 1.1 +/- 0.2 after 24 h for the 7.5, 1.8, and 0.9 kb IGF-I mRNA transcripts, respectively (n = 3 experiments) and 1.5 +/- 0.2 and 3.1 +/- 0.7 after 24 and 48 h, respectively, for the combined analysis of the 5.6 and 4.7 kb alpha1(I) collagen mRNA transcripts (n = 3 experiments). A

  1. Reinforcement of a porous collagen scaffold with surface-activated PLA fibers.

    PubMed

    Liu, Xi; Huang, Changbin; Feng, Yujie; Liang, Jie; Fan, Yujiang; Gu, Zhongwei; Zhang, Xingdong

    2010-01-01

    A hybrid porous collagen scaffold mechanically reinforced with surface-activated poly(lactic acid) (PLA) fiber was prepared. PLA fibers, 20 mum in diameter and 1 mm in length, were aminolyzed with hexanediamine to introduce free amino groups on the surfaces. After the amino groups were transferred to aldehyde groups by treatment with glutaraldehyde, different amounts (1.5, 3, 5 and 8 mg) of surface-activated PLA fibers were homogeneously mixed with 2 ml type-I collagen solution (pH 2.8, 0.6 wt%). This mixture solution was then freeze-dried and cross-linked to obtain collagen sponges with surface-activated PLA fiber. Scanning electron microscopy observation indicated that the collagen sponges had a highly interconnected porous structure with an average pore size of 170 mum, irrespective of PLA fiber incorporation. The dispersion of surface-activated PLA fibers was homogeneous in collagen sponge, in contrast to unactivated PLA fibers. The compression modulus test results showed that, compared with unactivated PLA fibers, the surface-activated PLA fibers enhanced the resistance of collagen sponge to compression more significantly. Cytotoxicity assay by MTT test showed no cytotoxicity of these collagen sponges. L929 mouse fibroblast cell-culture studies in vitro revealed that the number of L929 cells attached to the collagen sponge with surface-activated PLA fibers, both 6 h and 24 h after seeding, was higher than that in pure collagen sponge and sponge with unactivated PLA fibers. In addition, a better distribution of cells infiltrated in collagen sponge with surface-activated PLA fibers was observed by histological staining. These results indicated that the collagen sponge reinforced with surface-activated PLA fibers is a promising biocompatible scaffold for tissue engineering.

  2. Transdermal nicotine patch enhances type I collagen synthesis in abstinent smokers.

    PubMed

    Sørensen, Lars T; Jorgensen, Lars N; Zillmer, Rikke; Vange, Jakob; Hemmingsen, Ulla; Gottrup, Finn

    2006-01-01

    Cigarette smokers deposit less collagen, expressed as hydroxyproline, in granulation tissue than nonsmokers. We studied the effect of abstinence from smoking and transdermal nicotine patches on deposition of hydroxyproline, proline, type I procollagen, and total proteins. Fifty-four healthy smokers were studied during 10 days of smoking and again from days 10 to 20 following smoking cessation. After the first 10 days of abstinence they were randomized to double-blind treatment with transdermal nicotine patches of 25 mg/day or placebo for a period of 10 days. During this period and during smoking, an expanded polytetrafluoroethylene tube was implanted into the subcutis. Following removal of the implant, total amino acids and peptides were extracted. Hydroxyproline and proline were analyzed by high-pressure liquid chromatography, type I procollagen was analyzed by enzyme-linked immunoassay, and total proteins were determined colorimetrically. In the 39 subjects who complied with the study protocol, abstinence from smoking did not affect the deposition of hydroxyproline, proline, type I procollagen, or total protein in the implants. During abstinence, the type I procollagen level increased by 18% in the transdermal nicotine patches group and decreased by 10% in the placebo group (p<0.05). We conclude that 20 days of abstinence from smoking does not affect collagen deposition in granulation tissue. However, in abstinent smokers, transdermal nicotine patches appears to increase type I collagen synthesis.

  3. In Vitro Oxidation of Collagen Promotes the Formation of Advanced Oxidation Protein Products and the Activation of Human Neutrophils.

    PubMed

    Bochi, Guilherme Vargas; Torbitz, Vanessa Dorneles; de Campos, Luízi Prestes; Sangoi, Manuela Borges; Fernandes, Natieli Flores; Gomes, Patrícia; Moretto, Maria Beatriz; Barbisan, Fernanda; da Cruz, Ivana Beatrice Mânica; Moresco, Rafael Noal

    2016-04-01

    The accumulation of advanced oxidation protein products (AOPPs) has been linked to several pathological conditions. Here, we investigated collagen as a potential source for AOPP formation and determined the effects of hypochlorous acid (HOCl)-treated collagen (collagen-AOPPs) on human neutrophil activity. We also assessed whether alpha-tocopherol could counteract these effects. Exposure to HOCl increased the levels of collagen-AOPPs. Collagen-AOPPs also stimulated the production of AOPPs, nitric oxide (NO), superoxide radicals (O2(-)), and HOCl by neutrophils. Collagen-AOPPs induced apoptosis and decreased the number of viable cells. Alpha-tocopherol prevented the formation of collagen-AOPPs, strongly inhibited the collagen-AOPP-induced production of O2(-) and HOCl, and increased the viability of neutrophils. Our results suggest that collagen is an important protein that interacts with HOCl to form AOPPs, and consequently, collagen-AOPP formation is related to human neutrophil activation and cell death.

  4. Effect of Cold Plasma on Cell Viability and Collagen Synthesis in Cultured Murine Fibroblasts

    NASA Astrophysics Data System (ADS)

    Shi, Xingmin; Cai, Jingfen; Xu, Guimin; Ren, Hongbin; Chen, Sile; Chang, Zhengshi; Liu, Jinren; Huang, Chongya; Zhang, Guanjun; Wu, Xili

    2016-04-01

    An argon atmospheric pressure plasma jet was employed to treat L929 murine fibroblasts cultured in vitro. Experimental results showed that, compared with the control cells, the treatment of fibroblasts with 15 s of plasma led to a significant increase of cell viability and collagen synthesis, while the treatment of 25 s plasma resulted in a remarkable decrease. Exploration of related mechanisms suggested that cold plasma could up-regulate CyclinD1 gene expression and down-regulate p27 gene expression at a low dose, while it could down-regulate CyclinD1 expression and up-regulate p27 expression at a higher dose, thus altering the cell cycle progression, and then affecting cell viability and collagen synthesis of fibroblasts. supported partly by National Natural Science Foundation of China (Nos. 81372076, 51307133 and 51221005), China National Funds for Distinguished Young Scientists (No. 51125029), the Sci-Tech Project of Shaanxi Province of China (No. 2010K16-04), and the Fundamental Research Funds for the Central Universities of China (No. xkjc2013004)

  5. Bioinspired synthesis of hydroxyapatite nanocrystals in the presence of collagen and l-arginine: Candidates for bone regeneration.

    PubMed

    Brasinika, Despoina; Tsigkou, Olga; Tsetsekou, Athena; Missirlis, Yiannis F

    2016-04-01

    This work aims at the bioinspired synthesis of hydroxyapatite (HAp) crystals in the presence of both collagen and l-arginine, in an effort to obtain a homogeneous hybrid material, having a bone-like nanostructure. Collagen (Col) is the most commonly utilized protein in most species of life, while L-arginine (Arg) encourages cell attachment, proliferation, and differentiation on HAp surfaces. Transmission electron microscopy, X-ray diffraction and Fourier transform-infrared spectroscopy were used to analyze surface morphology and structure of nanocrystals obtained under different synthesis conditions. It was shown that collagen and arginine content affect HAp crystallization. Collagen has an inhibition effect since HAp crystal size is reduced with the increase of collagen content. The presence of arginine is crucial as a critical content exists (Ca(2+):Arg = 1:1) under which HAp nanocrystals coexist with brushite. Under the optimum synthesis conditions (HAp/Col weight ratio 70/30 and Ca(2+):Arg molar ratio 1:1) HAp nanoplates of a uniform size (around 10 × 10 nm) were obtained. The biocompatibility of this hybrid powder was assessed using human bone marrow derived mesenchymal stem cells (MSCs). Cell response in terms of MSC attachment (scanning electron microscopy) and viability/proliferation (Alamar Blue) demonstrated a noncytotoxic effect of the new material.

  6. Biomimetic composite microspheres of collagen/chitosan/nano-hydroxyapatite: In-situ synthesis and characterization.

    PubMed

    Teng, Shu-Hua; Liang, Mian-Hui; Wang, Peng; Luo, Yong

    2016-01-01

    The collagen/chitosan/hydroxyapatite (COL/CS/HA) composite microspheres with a good spherical form and a high dispersity were successfully obtained using an in-situ synthesis method. The FT-IR and XRD results revealed that the inorganic phase in the microspheres was crystalline HA containing carbonate ions. The morphology of the composite microspheres was dependent on the HA content, and a more desirable morphology was achieved when 20 wt.% HA was contained. The composite microspheres exhibited a narrow particle distribution, most of which ranged from 5 to 10 μm. In addition, the needle-like HA nano-particles were uniformly distributed in the composite microspheres, and their crystallinity and crystal size decreased with the HA content.

  7. Preparation and biological activity of quaternized carboxymethyl chitosan conjugated with collagen peptide.

    PubMed

    Zhu, Xiaoming; Zhou, Xiaoyu; Yi, Jiayan; Tong, Jun; Wu, Huan; Fan, Lihong

    2014-09-01

    Tissue repair is a spontaneous process which initiated on wounding. If this complex mechanism is disturbed or impaired, the use of biomaterials might increase the chance of successful healing. In this view, a water-soluble chitosan derivative, quaternized carboxymethyl chitosan (QCMC) was prepared and collagen peptides (COPs) were grafted to the backbone by carbodiimide method. The reaction conditions affecting the degree of substitution (DS) were studied including the mass ratio of collagen peptide to QCMC, reaction temperature and reaction time. The hydrogen peroxide-scavenging activity could be different by changing the DS, concentration and molecular weight. MTT assay was used to investigate the cell viability of the derivative. The results indicated that the introduction of collagen peptide into the QCMC improved its hydrogen peroxide-scavenging activity and cell viability with the DS and concentration increased. Therefore, QCMC conjugated with collagen peptides may prove beneficial to the process of the wound-healing.

  8. Properties of collagen gels cross-linked by N-hydroxysuccinimide activated adipic acid deriviate.

    PubMed

    Duan, Lian; Liu, Wentao; Tian, Zhenhua; Li, Conghu; Li, Guoying

    2014-08-01

    In order to improve the properties of collagen gel, N-hydroxysuccinimide activated adipic acid derivative (NHS-AA) was introduced into the formation of collagen fibrils. NHS-AA with different [NHS-AA]/[NH2] ratios (0.1-1.5, calculated by [ester group] of NHS-AA and [NH2] of lysine and hydroxylysine residues of collagen) was added after, simultaneously with or before the formation of collagen fibrils (abbreviated CAF, CSF and CBF, respectively) to obtain different collagen gels. With the same dose of NHS-AA, the cross-linking degree for CAF was lower than those for CSF and CBF. The formation of collagen fibrils was restrained by NHS-AA for CSF and CBF while that for CAF was unaffected. When the dose of NHS-AA increased from 0.1 to 1.5, the water contents of CSF and CBF increased while that of CAF had no obvious change. With lower dose of NHS-AA (0.1), CAF possessed higher value of G' (87.3Pa) and the best thermal stability (47.6°C). As the ratio of [NHS-AA]/[NH2] increased to 1.5, CSF had the maximum value of G' (288.8Pa) and CAF had the best thermal stability (52.9°C). These results showed collagen gels with different properties could be prepared by adding NHS-AA with different adding sequence and dose.

  9. Inhibition of collagen synthesis by select calcium and sodium channel blockers can be mitigated by ascorbic acid and ascorbyl palmitate.

    PubMed

    Ivanov, Vadim; Ivanova, Svetlana; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

    2016-01-01

    Calcium, sodium and potassium channel blockers are widely prescribed medications for a variety of health problems, most frequently for cardiac arrhythmias, hypertension, angina pectoris and other disorders. However, chronic application of channel blockers is associated with numerous side effects, including worsening cardiac pathology. For example, nifedipine, a calcium-channel blocker was found to be associated with increased mortality and increased risk for myocardial infarction. In addition to the side effects mentioned above by different channel blockers, these drugs can cause arterial wall damage, thereby contributing to vascular wall structure destabilization and promoting events facilitating rupture of plaques. Collagen synthesis is regulated by ascorbic acid, which is also essential for its optimum structure as a cofactor in lysine and proline hydroxylation, a precondition for optimum crosslinking of collagen and elastin. Therefore, the main objective in this study was to evaluate effects of various types of channel blockers on intracellular accumulation and cellular functions of ascorbate, specifically in relation to formation and extracellular deposition of major collagen types relevant for vascular function. Effects of select Na- and Ca- channel blockers on collagen synthesis and deposition were evaluated in cultured human dermal fibroblasts and aortic smooth muscle cells by immunoassay. All channel blockers tested demonstrated inhibitory effects on collagen type I deposition to the ECM by fibroblasts, each to a different degree. Ascorbic acid significantly increased collagen I ECM deposition. Nifedipine (50 µM), a representative of channel blockers tested, significantly reduced ascorbic acid and ascorbyl palmitate-dependent ECM deposition of collagen type l and collagen type lV by cultured aortic smooth muscle cells. In addition, nifedipine (50 µM) significantly reduced ascorbate-dependent collagen type l and type lV synthesis by cultured aortic smooth

  10. Inhibition of collagen synthesis by select calcium and sodium channel blockers can be mitigated by ascorbic acid and ascorbyl palmitate

    PubMed Central

    Ivanov, Vadim; Ivanova, Svetlana; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

    2016-01-01

    Calcium, sodium and potassium channel blockers are widely prescribed medications for a variety of health problems, most frequently for cardiac arrhythmias, hypertension, angina pectoris and other disorders. However, chronic application of channel blockers is associated with numerous side effects, including worsening cardiac pathology. For example, nifedipine, a calcium-channel blocker was found to be associated with increased mortality and increased risk for myocardial infarction. In addition to the side effects mentioned above by different channel blockers, these drugs can cause arterial wall damage, thereby contributing to vascular wall structure destabilization and promoting events facilitating rupture of plaques. Collagen synthesis is regulated by ascorbic acid, which is also essential for its optimum structure as a cofactor in lysine and proline hydroxylation, a precondition for optimum crosslinking of collagen and elastin. Therefore, the main objective in this study was to evaluate effects of various types of channel blockers on intracellular accumulation and cellular functions of ascorbate, specifically in relation to formation and extracellular deposition of major collagen types relevant for vascular function. Effects of select Na- and Ca- channel blockers on collagen synthesis and deposition were evaluated in cultured human dermal fibroblasts and aortic smooth muscle cells by immunoassay. All channel blockers tested demonstrated inhibitory effects on collagen type I deposition to the ECM by fibroblasts, each to a different degree. Ascorbic acid significantly increased collagen I ECM deposition. Nifedipine (50 µM), a representative of channel blockers tested, significantly reduced ascorbic acid and ascorbyl palmitate-dependent ECM deposition of collagen type l and collagen type lV by cultured aortic smooth muscle cells. In addition, nifedipine (50 µM) significantly reduced ascorbate-dependent collagen type l and type lV synthesis by cultured aortic smooth

  11. Regulation of migratory activity of human keratinocytes by topography of multiscale collagen-containing nanofibrous matrices.

    PubMed

    Fu, Xiaoling; Xu, Meng; Liu, Jie; Qi, Yanmei; Li, Shaohua; Wang, Hongjun

    2014-02-01

    Nanofibrous matrices hold great promise in skin wound repair partially due to their capability of recapturing the essential attributes of native extracellular matrix (ECM). With regard to limited studies on the effect of nanofibrous matrices on keratinocytes, the present study was aimed to understand how the topographical feature of nanofibrous matrices regulates keratinocyte motility by culturing keratinocytes on polycaprolactone (PCL)/collagen nanofibrous matrices (rough surface with fiber diameters of 331 ± 112 nm) or the matrices coated with a thin layer of collagen gel to form a secondary ultrafine fibrous network (smooth surface with ultrafine fiber diameters of 55 ± 26 nm). It was found that the PCL/collagen nanofibrous matrices alone did not stimulate cell migration, while collagen gel coating could significantly increase cell motility. Further studies demonstrated that the ultrafine fibrous network of collagen gel coating significantly activated integrin β1, Rac1 and Cdc42, facilitated the deposition of laminin-332 (formerly called laminin-5), and promoted the expression of active matrix metalloproteinases (MMPs) (i.e., MMP-2 and 9). Neutralization of integrin β1 activity abrogated the gel coating-induced keratinocyte migration. These findings provide important evidence on the role of topographical features of nanofibrous matrices in regulating the phenotypic alteration of keratinocytes and suggest the possible utility of collagen-containing nanofibrous matrices for skin regeneration especially in re-epithelialization.

  12. Novel synthesis strategy for composite hydrogel of collagen/hydroxyapatite-microsphere originating from conversion of CaCO3 templates.

    PubMed

    Wei, Qingrong; Lu, Jian; Wang, Qiaoying; Fan, Hongsong; Zhang, Xingdong

    2015-03-20

    Inspired by coralline-derived hydroxyapatite, we designed a methodological route to synthesize carbonated-hydroxyapatite microspheres from the conversion of CaCO3 spherulite templates within a collagen matrix under mild conditions and thus constructed the composite hydrogel of collagen/hydroxyapatite-microspheres. Fourier transform infrared spectroscopy (FTIR) and x-ray diffraction (XRD) were employed to confirm the successful generation of the carbonated hydroxyapatite phase originating from CaCO3, and the ratios of calcium to phosphate were tracked over time. Variations in the weight portion of the components in the hybrid gels before and after the phase transformation of the CaCO3 templates were identified via thermogravimetric analysis (TGA). Scanning electron microscopy (SEM) shows these composite hydrogels have a unique multiscale microstructure consisting of a collagen nanofibril network and hydroxyapatite microspheres. The relationship between the hydroxyapatite nanocrystals and the collagen fibrils was revealed by transmission electron microscopy (TEM) in detail, and the selected area electron diffraction (SAED) pattern further confirmed the results of the XRD analyses which show the typical low crystallinity of the generated hydroxyapatite. This smart synthesis strategy achieved the simultaneous construction of microscale hydroxyapatite particles and collagen fibrillar hydrogel, and appears to provide a novel route to explore an advanced functional hydrogel materials with promising potentials for applications in bone tissue engineering and reconstruction medicine.

  13. Novel synthesis strategy for composite hydrogel of collagen/hydroxyapatite-microsphere originating from conversion of CaCO3 templates

    NASA Astrophysics Data System (ADS)

    Wei, Qingrong; Lu, Jian; Wang, Qiaoying; Fan, Hongsong; Zhang, Xingdong

    2015-03-01

    Inspired by coralline-derived hydroxyapatite, we designed a methodological route to synthesize carbonated-hydroxyapatite microspheres from the conversion of CaCO3 spherulite templates within a collagen matrix under mild conditions and thus constructed the composite hydrogel of collagen/hydroxyapatite-microspheres. Fourier transform infrared spectroscopy (FTIR) and x-ray diffraction (XRD) were employed to confirm the successful generation of the carbonated hydroxyapatite phase originating from CaCO3, and the ratios of calcium to phosphate were tracked over time. Variations in the weight portion of the components in the hybrid gels before and after the phase transformation of the CaCO3 templates were identified via thermogravimetric analysis (TGA). Scanning electron microscopy (SEM) shows these composite hydrogels have a unique multiscale microstructure consisting of a collagen nanofibril network and hydroxyapatite microspheres. The relationship between the hydroxyapatite nanocrystals and the collagen fibrils was revealed by transmission electron microscopy (TEM) in detail, and the selected area electron diffraction (SAED) pattern further confirmed the results of the XRD analyses which show the typical low crystallinity of the generated hydroxyapatite. This smart synthesis strategy achieved the simultaneous construction of microscale hydroxyapatite particles and collagen fibrillar hydrogel, and appears to provide a novel route to explore an advanced functional hydrogel materials with promising potentials for applications in bone tissue engineering and reconstruction medicine.

  14. Plasma membrane overgrowth causes fibrotic collagen accumulation and immune activation in Drosophila adipocytes

    PubMed Central

    Zang, Yiran; Wan, Ming; Liu, Min; Ke, Hongmei; Ma, Shuangchun; Liu, Lu-Ping; Ni, Jian-Quan; Carlos Pastor-Pareja, José

    2015-01-01

    Many chronic diseases are associated with fibrotic deposition of Collagen and other matrix proteins. Little is known about the factors that determine preferential onset of fibrosis in particular tissues. Here we show that plasma membrane (PM) overgrowth causes pericellular Collagen accumulation in Drosophila adipocytes. We found that loss of Dynamin and other endocytic components causes pericellular trapping of outgoing Collagen IV due to dramatic cortex expansion when endocytic removal of PM is prevented. Deposits also form in the absence of negative Toll immune regulator Cactus, excess PM being caused in this case by increased secretion. Finally, we show that trimeric Collagen accumulation, downstream of Toll or endocytic defects, activates a tissue damage response. Our work indicates that traffic imbalances and PM topology may contribute to fibrosis. It also places fibrotic deposits both downstream and upstream of immune signaling, consistent with the chronic character of fibrotic diseases. DOI: http://dx.doi.org/10.7554/eLife.07187.001 PMID:26090908

  15. Constitutive Smad signaling and Smad-dependent collagen gene expression in mouse embryonic fibroblasts lacking peroxisome proliferator-activated receptor-{gamma}

    SciTech Connect

    Ghosh, Asish K Wei, Jun; Wu, Minghua; Varga, John

    2008-09-19

    Transforming growth factor-{beta} (TGF-{beta}), a potent inducer of collagen synthesis, is implicated in pathological fibrosis. Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a nuclear hormone receptor that regulates adipogenesis and numerous other biological processes. Here, we demonstrate that collagen gene expression was markedly elevated in mouse embryonic fibroblasts (MEFs) lacking PPAR-{gamma} compared to heterozygous control MEFs. Treatment with the PPAR-{gamma} ligand 15d-PGJ{sub 2} failed to down-regulate collagen gene expression in PPAR-{gamma} null MEFs, whereas reconstitution of these cells with ectopic PPAR-{gamma} resulted in their normalization. Compared to control MEFs, PPAR-{gamma} null MEFs displayed elevated levels of the Type I TGF-{beta} receptor (T{beta}RI), and secreted more TGF-{beta}1 into the media. Furthermore, PPAR-{gamma} null MEFs showed constitutive phosphorylation of cellular Smad2 and Smad3, even in the absence of exogenous TGF-{beta}, which was abrogated by the ALK5 inhibitor SB431542. Constitutive Smad2/3 phosphorylation in PPAR-{gamma} null MEFs was associated with Smad3 binding to its cognate DNA recognition sequences, and interaction with coactivator p300 previously implicated in TGF-{beta} responses. Taken together, these results indicate that loss of PPAR-{gamma} in MEFs is associated with upregulation of collagen synthesis, and activation of intracellular Smad signal transduction, due, at least in part, to autocrine TGF-{beta} stimulation.

  16. The effect of Centella asiatica, vitamins, glycolic acid and their mixtures preparations in stimulating collagen and fibronectin synthesis in cultured human skin fibroblast.

    PubMed

    Hashim, Puziah

    2014-03-01

    Centella asiatica (Linn.) Urban is well known in promoting wound healing and provides significant benefits in skin care and therapeutic products formulation. Glycolic acid and vitamins also play a role in the enhancement of collagen and fibronectin synthesis. Here, we evaluate the specific effect of Centella asiatica (CA), vitamins, glycolic acid and their mixture preparations to stimulate collagen and fibronectin synthesis in cultured human fibroblast cells. The fibroblast cells are incubated with CA, glycolic acid, vitamins and their mixture preparations for 48 h. The cell lysates were analyzed for protein content and collagen synthesis by direct binding enzyme immunoassay. The fibronectin of the cultured supernatant was measured by sandwich enzyme immunoassay. The results showed that CA, glycolic acid, vitamins A, E and C significantly stimulate collagen and fibronectin synthesis in the fibroblast. Addition of glycolic acid and vitamins to CA further increased the levels of collagen and fibronectin synthesis to 8.55 and 23.75 μg/100 μg, respectively. CA, glycolic acid, vitamins A, E, and C, and their mixtures demonstrated stimulatory effect on both extra-cellular matrix synthesis of collagen and fibronectin in in vitro studies on human foreskin fibroblasts, which is beneficial to skin care and therapeutic products formulation.

  17. The anabolic effects of insulin on type II collagen synthesis of Swarm rat chondrosarcoma chondrocytes

    SciTech Connect

    Bembenek, M.E.; Liberti, J.P.

    1984-08-15

    The anabolic effects of insulin on collagen production of freshly isolated Swarm rat chondrosarcoma chondrocytes were investigated. The specific radioactivity of newly synthesized collagen was not increased by insulin, indicating that the hormone has no effect on the specific radioactivity of the aminoacyl tRNA pool. Results of further studies obtained from collagen degradation experiments demonstrated that insulin did not alter the rate of (3H)collagen degradation. Together, these results clearly indicate that insulin stimulates collagen biosynthesis. Polyacrylamide gel analysis of the newly synthesized collagen of both control and insulin-stimulated cells revealed a large-molecular-weight component which migrated with authentic alpha 1(II) collagen and was collagenase-sensitive. Additional studies showed that, although insulin increased the processing and secretion of collagen, the hormone did not cause a shift in the distribution of the extracellular and intracellular collagen pools. Finally, results of studies conducted with the transcriptional inhibitor, actinomycin D, indicated that the anabolic effects of insulin on collagen and non-collagen proteins were mediated at a post-transcriptional site.

  18. Human prostatic acid phosphatase directly stimulates collagen synthesis and alkaline phosphatase content of isolated bone cells

    SciTech Connect

    Ishibe, M.; Rosier, R.N.; Puzas, J.E. )

    1991-10-01

    Human prostatic acid phosphatase (hPAP) directly enhances the differentiated characteristics of isolated bone cells in vitro. This enzyme, when added to cell cultures for 24 h in vitro stimulates collagen synthesis and the production of alkaline phosphatase. The effects are dose dependent, with statistically significant effects occurring from 0.1-100 nM hPAP. Concentrations higher than 100 nM do not evoke greater effects. The maximal effect of hPAP occurs between 12 and 24 h of exposure. The cells stimulated to the greatest degree are osteoprogenitor cells and osteoblasts. Fibroblasts isolated from the same tissue show a lesser sensitivity to hPAP. hPAP has no detectable effect on cell proliferation, as measured by radiolabeled thymidine incorporation or total DNA synthesis. None of the observations reported in this work can be attributed to contaminating proteins in the hPAP preparation. hPAP was radiolabeled with 125I and was used for affinity binding and cross-linking studies. Scatchard analysis of specific binding indicated the presence of 1.0 X 10(5) high affinity binding sites/cell, with a Kd of 6.5 nM. Cross-linking studies demonstrated the presence of one 320-kDa binding complex. The pH profile and kinetic determinations of Km and maximum velocity for hPAP were similar to those previously reported, except for the finding of positive cooperativity of the substrate with the enzyme under the conditions of our assay. We believe that the direct stimulation of bone-forming cells by hPAP may contribute to the sclerotic nature of skeletal bone around sites of neoplastic prostatic metastases and that the effect of the enzyme is probably mediated by a plasma membrane receptor.

  19. Collagen-binding VEGF mimetic peptide: Structure, matrix interaction, and endothelial cell activation

    NASA Astrophysics Data System (ADS)

    Chan, Tania R.

    Long term survival of artificial tissue constructs depends greatly on proper vascularization. In nature, differentiation of endothelial cells and formation of vasculature are directed by dynamic spatio-temporal cues in the extracellular matrix that are difficult to reproduce in vitro. In this dissertation, we present a novel bifunctional peptide that mimics matrix-bound vascular endothelial growth factor (VEGF), which can be used to encode spatially controlled angiogenic signals in collagen-based scaffolds. The peptide, QKCMP, contains a collagen mimetic domain (CMP) that binds to type I collagen by a unique triple helix hybridization mechanism and a VEGF mimetic domain (QK) with pro-angiogenic activity. We demonstrate QKCMP's ability to hybridize with native and heat denatured collagens through a series of binding studies on collagen and gelatin substrates. Circular dichroism experiments show that the peptide retains the triple helical structure vital for collagen binding, and surface plasmon resonance study confirms the molecular interaction between the peptide and collagen strands. Cell culture studies demonstrate QKCMP's ability to induce endothelial cell morphogenesis and network formation as a matrix-bound factor in 2D and 3D collagen scaffolds. We also show that the peptide can be used to spatially modify collagen-based substrates to promote localized endothelial cell activation and network formation. To probe the biological events that govern these angiogenic cellular responses, we investigated the cell signaling pathways activated by collagen-bound QKCMP and determined short and long-term endothelial cell response profiles for p38, ERK1/2, and Akt signal transduction cascades. Finally, we present our efforts to translate the peptide's in vitro bioactivity to an in vivo burn injury animal model. When implanted at the wound site, QKCMP functionalized biodegradable hydrogels induce enhanced neovascularization in the granulation tissue. The results show QKCMP

  20. RB1CC1 protein suppresses type II collagen synthesis in chondrocytes and causes dwarfism.

    PubMed

    Nishimura, Ichiro; Chano, Tokuhiro; Kita, Hiroko; Matsusue, Yoshitaka; Okabe, Hidetoshi

    2011-12-23

    RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations.

  1. The Ratio of Type II Collagen Breakdown to Synthesis and its Relationship with the Progression of Knee Osteoarthritis

    PubMed Central

    Cahue, September; Sharma, Leena; Dunlop, Dorothy; Ionescu, Mirela; Song, Jing; Lobanok, Tatiana; King, Lindsay; Poole, A. Robin

    2007-01-01

    Objective. To examine whether the baseline ratio of a type II collagen breakdown marker to a synthesis marker, or the level of these markers individually, is associated with the likelihood of knee OA progression between baseline and 18 months. Methods. Participants were recruited from community sources and had knee OA. Blood was drawn at baseline. Collagen synthesis was measured by commercial ELISA assay that detects c-propeptide of type II procollagen (CPII). Serum markers of collagenase cleavage of cartilage type II collagen [C2C epitope (COL2−3/4Clong mono) and C1,2C epitope (COL2−3/4Cshort)] were also assayed. Knee radiographs (semi-flexed with fluoro confirmation) were obtained at baseline and 18 months. OA progression was examined using worsening of joint space grade and worsening of Kellgren/Lawrence grade. The relationship between baseline serum markers and subsequent progression was analyzed from logistic regression. Results. Baseline levels of these markers, considered individually, were not associated with a change in the odds of progression. Belonging to the low synthesis tertile was associated with a greater likelihood of progression, approaching significance (adjusted OR 1.86, 95% CI 0.96, 3.63). A greater C2C:CPII ratio and C1,2C:CPII ratio were each associated with an increase in the odds of joint space grade progression, which approached significance (e.g. adjusted OR of C2C:CPII ratio was 3.15, 95% CI 0.91, 10.85). Conclusion. While the degradation markers individually, considered as continuous variables, did not predict OA progression, belonging to the lower synthesis marker tertile and greater degradation/synthesis marker ratios were associated with an elevation in the odds of progression albeit not achieving significance. PMID:17344068

  2. Inhibitory effects of prostaglandin E2 on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma

    PubMed Central

    2014-01-01

    Background Several studies have described an increased cyclooxygenase-2 (COX-2) expression in pancreatic cancer, but the role of COX-2 in tumour development and progression is not clear. The aim of the present study was to examine expression of COX-2 in cancer cells and stromal cells in pancreatic cancer specimens, and to explore the role of PGE2 in pancreatic stellate cell proliferation and collagen synthesis. Methods Immunohistochemistry and immunofluorescence was performed on slides from whole sections of tissue blocks using antibodies against COX-2 and α-smooth muscle actin (αSMA). Pancreatic stellate cells (PSC) were isolated from surgically resected tumour tissue by the outgrowth method. Cells were used between passages 4 and 8. Collagen synthesis was determined by [3H]-proline incorporation, or by enzyme immunoassay measurement of collagen C-peptide. DNA synthesis was measured by incorporation of [3H]-thymidine in DNA. Cyclic AMP (cAMP) was determined by radioimmunoassay. Collagen 1A1 mRNA was determined by RT-qPCR. Results Immunohistochemistry staining showed COX-2 in pancreatic carcinoma cells, but not in stromal cells. All tumours showed positive staining for αSMA in the fibrotic stroma. Cultured PSC expressed COX-2, which could be further induced by interleukin-1β (IL-1β), epidermal growth factor (EGF), thrombin, and PGE2, but not by transforming growth factor-β1 (TGFβ). Indirect coculture with the adenocarcinoma cell line BxPC-3, but not HPAFII or Panc-1, induced COX-2 expression in PSC. Treatment of PSC with PGE2 strongly stimulated cAMP accumulation, mediated by EP2 receptors, and also stimulated phosphorylation of extracellular signal-regulated kinase (ERK). Treatment of PSC with PGE2 or forskolin suppressed both TGFβ-stimulated collagen synthesis and PDGF-stimulated DNA synthesis. Conclusions The present results show that COX-2 is mainly produced in carcinoma cells and suggest that the cancer cells are the main source of PGE2 in pancreatic

  3. Properties of alkali-solubilized collagen solution crosslinked by N-hydroxysuccinimide activated adipic acid

    NASA Astrophysics Data System (ADS)

    Chen, Yihui; Zhang, Min; Liu, Wentao; Li, Guoying

    2011-03-01

    The effect of N-hydroxysuccinimide activated adipic acid (NHS-AA) on the properties of alkali-solubilized collagen solutions was examined. The residual amino group content in crosslinked collagen, determined by trinitrobenzensulfonic acid (TNBS) assay, was decreased with increasing NHS-AA concentration. The results from differential scanning calorimeter (DSC) indicated that the maximum denaturation temperature ( T d) of crosslinked collagen solution was about 4.2°C higher than that of un-crosslinked collagen solution (36.6°C). Moreover, the values of storage modulus ( G'), loss modulus ( G″) and complex viscosity ( η*), obtained by means of dynamic frequency sweeps, were increased as NHS-AA concentration added up to 1.5 mM, and then decreased slightly when further increased NHS-AA concentration. Besides, for collagen solution crosslinked with 1.5 mM NHS-AA, dynamic denaturation temperature ( T dd) was about 1.1°C lower than T d (40.8°C), and the Arrhenius-type time-temperature superposition (TTS) principle was applied to yield the activation energy to be 474.4 kJmol-1.

  4. Contact activation of blood coagulation on a defined kaolin/collagen surface in a microfluidic assay.

    PubMed

    Zhu, Shu; Diamond, Scott L

    2014-12-01

    Generation of active Factor XII (FXIIa) triggers blood clotting on artificial surfaces and may also enhance intravascular thrombosis. We developed a patterned kaolin (0 to 0.3 pg/μm(2))/type 1 collagen fibril surface for controlled microfluidic clotting assays. Perfusion of whole blood (treated only with a low level of 4 μg/mL of the XIIa inhibitor, corn trypsin inhibitor) drove platelet deposition followed by fibrin formation. At venous wall shear rate (100 s(-1)), kaolin accelerated onset of fibrin formation by ~100 sec when compared to collagen alone (250 sec vs. 350 sec), with little effect on platelet deposition. Even with kaolin present, arterial wall shear rate (1000 s(-1)) delayed and suppressed fibrin formation compared to venous wall shear rate. A comparison of surfaces for extrinsic activation (tissue factor TF/collagen) versus contact activation (kaolin/collagen) that each generated equal platelet deposition at 100 s(-1) revealed: (1) TF surfaces promoted much faster fibrin onset (at 100 sec) and more endpoint fibrin at 600 sec at either 100 s(-1) or 1000 s(-1), and (2) kaolin and TF surfaces had a similar sensitivity for reduced fibrin deposition at 1000 s(-1) (compared to fibrin formed at 100 s(-1)) despite differing coagulation triggers. Anti-platelet drugs inhibiting P2Y1, P2Y12, cyclooxygenase-1 or activating IP-receptor or guanylate cyclase reduced platelet and fibrin deposition on kaolin/collagen. Since FXIIa or FXIa inhibition may offer safe antithrombotic therapy, especially for biomaterial thrombosis, these defined collagen/kaolin surfaces may prove useful in drug screening tests or in clinical diagnostic assays of blood under flow conditions.

  5. Fibroblast Activation Protein (FAP) Accelerates Collagen Degradation and Clearance from Lungs in Mice.

    PubMed

    Fan, Ming-Hui; Zhu, Qiang; Li, Hui-Hua; Ra, Hyun-Jeong; Majumdar, Sonali; Gulick, Dexter L; Jerome, Jacob A; Madsen, Daniel H; Christofidou-Solomidou, Melpo; Speicher, David W; Bachovchin, William W; Feghali-Bostwick, Carol; Puré, Ellen

    2016-04-08

    Idiopathic pulmonary fibrosis is a disease characterized by progressive, unrelenting lung scarring, with death from respiratory failure within 2-4 years unless lung transplantation is performed. New effective therapies are clearly needed. Fibroblast activation protein (FAP) is a cell surface-associated serine protease up-regulated in the lungs of patients with idiopathic pulmonary fibrosis as well as in wound healing and cancer. We postulate that FAP is not only a marker of disease but influences the development of pulmonary fibrosis after lung injury. In two different models of pulmonary fibrosis, intratracheal bleomycin instillation and thoracic irradiation, we find increased mortality and increased lung fibrosis in FAP-deficient mice compared with wild-type mice. Lung extracellular matrix analysis reveals accumulation of intermediate-sized collagen fragments in FAP-deficient mouse lungs, consistent within vitrostudies showing that FAP mediates ordered proteolytic processing of matrix metalloproteinase (MMP)-derived collagen cleavage products. FAP-mediated collagen processing leads to increased collagen internalization without altering expression of the endocytic collagen receptor, Endo180. Pharmacologic FAP inhibition decreases collagen internalization as expected. Conversely, restoration of FAP expression in the lungs of FAP-deficient mice decreases lung hydroxyproline content after intratracheal bleomycin to levels comparable with that of wild-type controls. Our findings indicate that FAP participates directly, in concert with MMPs, in collagen catabolism and clearance and is an important factor in resolving scar after injury and restoring lung homeostasis. Our study identifies FAP as a novel endogenous regulator of fibrosis and is the first to show FAP's protective effects in the lung.

  6. Evaluation of cathepsin B activity for degrading collagen IV using a surface plasmon resonance method and circular dichroism spectroscopy.

    PubMed

    Shoji, Atsushi; Kabeya, Mitsutaka; Ishida, Yuuki; Yanagida, Akio; Shibusawa, Yoichi; Sugawara, Masao

    2014-07-01

    Evaluation of cathepsin B activities for degrading collagen IV and heat-denatured collagen IV (gelatin) were performed by surface plasmon resonance (SPR) and circular dichroism (CD) measurements. The optimal pH of cathepsin B activity for degrading each substrate was around 4.0. The ΔRU(15 min), which is a decrease in the SPR signal at 15 min after injection of cathepsin B, was smaller for collagen IV than for heat-denatured collagen IV owing to the presence of triple-helical conformation. An unstable nature of the triple-helical conformation of collagen IV at pH 4.0 was shown by the CD study. Degrading collagen IV by cathepsin B was facilitated owing to a local unwinding of the triple-helical conformation caused by proteolytic cleavage of the non-helical region. The concentration dependence of the initial velocity for degrading collagen IV by cathepsin B at pH 4.0 was biphasic, showing that cathepsin B at low concentration exhibits exopeptidase activity, while the enzyme at high concentration exhibits endopeptidase activity. The kinetic parameters for the exopeptidase activity of cathepsin B toward collagen IV and heat-treated collagen IV were evaluated and discussed in terms of the protease mechanism.

  7. Synthesis and biological applications of collagen-model triple-helical peptides.

    PubMed

    Fields, Gregg B

    2010-03-21

    Triple-helical peptides (THPs) have been utilized as collagen models since the 1960s. The original focus for THP-based research was to unravel the structural determinants of collagen. In the last two decades, virtually all aspects of collagen structural biochemistry have been explored with THP models. More specifically, secondary amino acid analogs have been incorporated into THPs to more fully understand the forces that stabilize triple-helical structure. Heterotrimeric THPs have been utilized to better appreciate the contributions of chain sequence diversity on collagen function. The role of collagen as a cell signaling protein has been dissected using THPs that represent ligands for specific receptors. The mechanisms of collagenolysis have been investigated using THP substrates and inhibitors. Finally, THPs have been developed for biomaterial applications. These aspects of THP-based research are overviewed herein.

  8. Loss of fibulin-4 disrupts collagen synthesis and maturation: implications for pathology resulting from EFEMP2 mutations

    PubMed Central

    Papke, Christina L.; Tsunezumi, Jun; Ringuette, Léa-Jeanne; Nagaoka, Hideaki; Terajima, Masahiko; Yamashiro, Yoshito; Urquhart, Greg; Yamauchi, Mitsuo; Davis, Elaine C.; Yanagisawa, Hiromi

    2015-01-01

    Homozygous recessive mutations in either EFEMP2 (encoding fibulin-4) or FBLN5 (encoding fibulin-5), critical genes for elastogenesis, lead to autosomal recessive cutis laxa types 1B and 1A, respectively. Previously, fibulin-4 was shown to bind lysyl oxidase (LOX), an elastin/collagen cross-linking enzyme, in vitro. Consistently, reported defects in humans with EFEMP2 mutations are more severe and broad in range than those due to FBLN5 mutations and encompass both elastin-rich and collagen-rich tissues. However, the underlying disease mechanism in EFEMP2 mutations has not been fully addressed. Here, we show that fibulin-4 is important for the integrity of aortic collagen in addition to elastin. Smooth muscle-specific Efemp2 loss in mouse (termed SMKO) resulted in altered fibrillar collagen localization with larger, poorly organized fibrils. LOX activity was decreased in Efemp2-null cells, and collagen cross-linking was diminished in SMKO aortas; however, elastin cross-linking was unaffected and the level of mature LOX was maintained to that of wild-type aortas. Proteomic screening identified multiple proteins involved in procollagen processing and maturation as potential fibulin-4-binding partners. We showed that fibulin-4 binds procollagen C-endopeptidase enhancer 1 (Pcolce), which enhances proteolytic cleavage of the procollagen C-terminal propeptide during procollagen processing. Interestingly, however, procollagen cleavage was not affected by the presence or absence of fibulin-4 in vitro. Thus, our data indicate that fibulin-4 serves as a potential scaffolding protein during collagen maturation in the extracellular space. Analysis of collagen in other tissues affected by fibulin-4 loss should further increase our understanding of underlying pathologic mechanisms in patients with EFEMP2 mutations. PMID:26220971

  9. The role of ITAM- and ITIM-coupled receptors in platelet activation by collagen.

    PubMed

    Watson, S P; Asazuma, N; Atkinson, B; Berlanga, O; Best, D; Bobe, R; Jarvis, G; Marshall, S; Snell, D; Stafford, M; Tulasne, D; Wilde, J; Wonerow, P; Frampton, J

    2001-07-01

    The major activation-inducing collagen receptor glycoprotein VI (GPVI) has been cloned within the last two years. It is a member of the Ig superfamily of proteins and is constitutively associated with the ITAM-bearing Fc receptor gamma-chain (FcR gamma-chain). GPVI signals through a pathway that involves several of the proteins used by Fc, B- and T-lymphocyte receptors and which takes place in glycolipid-enriched membrane domains in the plasma membrane known as GEMs. Responses to GPVI are regulated by PECAM-1 (CD31) and possibly other ITIM-bearing receptors. Despite a pivotal role for GPVI, there are important differences between signalling events to collagen and GPVI-specific ligands. This may reflect a role for co-receptors in the response to collagen.

  10. Na/K-ATPase signaling regulates collagen synthesis through microRNA-29b-3p in cardiac fibroblasts.

    PubMed

    Drummond, Christopher A; Hill, Michael C; Shi, Huilin; Fan, Xiaoming; Xie, Jeffrey X; Haller, Steven T; Kennedy, David J; Liu, Jiang; Garrett, Michael R; Xie, Zijian; Cooper, Christopher J; Shapiro, Joseph I; Tian, Jiang

    2016-03-01

    Chronic kidney disease (CKD) is accompanied by cardiac fibrosis, hypertrophy, and dysfunction, which are commonly referred to as uremic cardiomyopathy. Our previous studies found that Na/K-ATPase ligands or 5/6th partial nephrectomy (PNx) induces cardiac fibrosis in rats and mice. The current study used in vitro and in vivo models to explore novel roles for microRNA in this mechanism of cardiac fibrosis formation. To accomplish this, we performed microRNA profiling with RT-qPCR based arrays on cardiac tissue from rats subjected to marinobufagenin (MBG) infusion or PNx. The analysis showed that a series of fibrosis-related microRNAs were dysregulated. Among the dysregulated microRNAs, microRNA (miR)-29b-3p, which directly targets mRNA of collagen, was consistently reduced in both PNx and MBG-infused animals. In vitro experiments demonstrated that treatment of primary cultures of adult rat cardiac fibroblasts with Na/K-ATPase ligands induced significant increases in the fibrosis marker, collagen protein, and mRNA expression compared with controls, whereas miR-29b-3p expression decreased >50%. Transfection of miR-29b-3p mimics into cardiac fibroblasts inhibited cardiotonic steroids-induced collagen synthesis. Moreover, a specific Na/K-ATPase signaling antagonist, pNaKtide, prevented ouabain-induced increases in collagen synthesis and decreases in miR-29b-3p expression in these cells. In conclusion, these data are the first to indicate that signaling through Na/K-ATPase regulates miRNAs and specifically, miR-29b-3p expression both in vivo and in vitro. Additionally, these data indicate that miR-29b-3p expression plays an important role in the formation of cardiac fibrosis in CKD.

  11. Suppression of hedgehog signaling regulates hepatic stellate cell activation and collagen secretion.

    PubMed

    Li, Tao; Leng, Xi-Sheng; Zhu, Ji-Ye; Wang, Gang

    2015-01-01

    Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and transfected into HSC respectively. Suppression of hedgehog signaling were detected by SYBR Green fluorescence quantitative RT-PCR. Effects of hedgehog signaling inhibition on HSC activation and collagen I secretion were analyzed. Hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 were expressed in HSC. siRNA vectors targeting Ihh, Smo and Gli2 were successfully constructed and decreased target gene expression. Suppression of hedgehog signaling significantly decreased the expression of α-SMA in HSC (P<0.01). Collagen type I secretion of HSC were also significantly decreased (P<0.01). In summary, HSC activation and collagen secretion can be regulated by hedgehog signaling. Hedgehog may play a role in the pathogenesis of liver fibrosis.

  12. Regulation of collagen biosynthesis by ascorbic acid: a review.

    PubMed Central

    Pinnell, S. R.

    1985-01-01

    L-ascorbic acid is an essential cofactor for lysyl hydroxylase and prolyl hydroxylase, enzymes essential for collagen biosynthesis. In addition, L-ascorbic acid preferentially stimulates collagen synthesis in a manner which appears unrelated to the effect of L-ascorbic acid on hydroxylation reactions. This reaction is stereospecific and unrelated to intracellular degradation of collagen. The effect apparently occurs at a transcriptional or translational level, since L-ascorbic acid preferentially stimulates collagen-specific mRNA. In addition, it stimulates lysyl hydroxylase activity but inhibits prolyl hydroxylase activity in human skin fibroblasts in culture. PMID:3008449

  13. Sustained Induction of Collagen Synthesis by TGF-β Requires Regulated Intramembrane Proteolysis of CREB3L1

    PubMed Central

    Chen, Qiuyue; Lee, Ching-En; Denard, Bray; Ye, Jin

    2014-01-01

    CREB3L1 (cAMP response element binding protein 3-like 1), a transcription factor synthesized as a membrane-bound precursor and activated through Regulated Intramembrane Proteolysis (RIP), is essential for collagen production by osteoblasts during bone development. Here, we show that TGF-β (transforming growth factor-β), a cytokine known to stimulate production of collagen during wound healing and fibrotic diseases, induces proteolytic activation of CREB3L1 in human A549 cells. This activation results from inhibition of expression of TM4SF20 (transmembrane 4 L6 family member 20), which normally inhibits RIP of CREB3L1. Cleavage of CREB3L1 releases its NH2-terminal domain from membranes, allowing it to enter the nucleus where it binds to Smad4 to activate transcription of genes encoding proteins required for assembly of collagen-containing extracellular matrix. Our findings raise the possibility that inhibition of RIP of CREB3L1 could prevent excess deposition of collagen in certain fibrotic diseases. PMID:25310401

  14. Discoidin Domain Receptor 2 Mediates Collagen-Induced Activation of Membrane-Type 1 Matrix Metalloproteinase in Human Fibroblasts.

    PubMed

    Majkowska, Iwona; Shitomi, Yasuyuki; Ito, Noriko; Gray, Nathanael S; Itoh, Yoshifumi

    2017-03-07

    Membrane-Type 1 Matrix Metalloproteinase (MT1-MMP) is a membrane-bound MMP that is highly expressed in cells with invading capacity including fibroblasts and invasive cancer cell. A potential physiological stimulus for MT1-MMP expression is fibrillar collagen, and it has been shown that it upregulates both MT1-MMP gene and functions in various cell types. However, the mechanisms of collagen-mediated MT1-MMP activation is not clearly understood. In this study we identified discoidin domain receptor 2 (DDR2) as a crucial receptor that mediates this process in human fibroblasts. Knocking down DDR2, but not β1 integrin subunit, a common subunit for all collagen-binding integrins, inhibited collagen-induced activation of proMMP-2 and upregulation of MT1-MMP at the gene and protein level. Interestingly DDR2 knockdown or pharmacological inhibition of DDR2 also inhibited MT1-MMP-dependent cellular degradation of collagen film, suggesting that cell surface collagen degradation by MT1-MMP involves DDR2-mediated collagen signalling. This DDR2-mediated mechanism is only present in non-transformed mesenchymal cells, as collagen-induced MT1-MMP activation in HT1080 fibrosarcoma cells and MT1-MMP function in MDA-MB231 breast cancer cells were not affected by DDR kinase inhibition. DDR2 activation was found to be noticeably more effective when cells were stimulated by collagen without non-helical telopeptides region compared to intact collagen fibrils. Those data suggest that DDR2 is a microenvironmental sensor that regulates fibroblasts migration in collagen-rich environment.

  15. DDR2 plays a role in fibroblast migration independent of adhesion ligand and collagen activated DDR2 tyrosine kinase.

    PubMed

    Herrera-Herrera, Mireya Liliana; Quezada-Calvillo, Roberto

    2012-12-07

    Discoidin domain receptor-2 (DDR2) is a cell surface tyrosine kinase receptor that can be activated by soluble collagen and has been implicated in diverse physiological functions including organism growth and wound repair. In the current studies, we used fibronectin and collagen-coated 2D surfaces and collagen matrices in combination with siRNA technology to investigate the role of DDR2 in a range of fibroblast motile activities. Silencing DDR2 with siRNA inhibited cell spreading and migration, and similar inhibition occurred regardless whether cells were interacting with fibronectin or collagen surfaces. Under the assay conditions used, DDR2 tyrosine kinase activation was not observed unless soluble collagen was added to the incubation medium. Finally silencing DDR2 also inhibited human fibroblast migration in 3D collagen matrices but had no effect on 3D collagen matrix remodeling and contraction. Taken together, our findings suggest that DDR2 is required for normal fibroblast spreading and migration independent of adhesion ligand and collagen activation of DDR2 tyrosine kinase.

  16. cAMP inhibits transforming growth factor-beta-stimulated collagen synthesis via inhibition of extracellular signal-regulated kinase 1/2 and Smad signaling in cardiac fibroblasts.

    PubMed

    Liu, Xiaoqiu; Sun, Shu Qiang; Hassid, Aviv; Ostrom, Rennolds S

    2006-12-01

    Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Cytokines such as transforming growth factor-beta (TGF-beta) play a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and other profibrotic responses, but less is known about pathways that might inhibit fibrosis. Increased cAMP formation inhibits myofibroblast differentiation and collagen production by cardiac fibroblasts, but the mechanism of this inhibition is not known. We sought to characterize the signaling pathways by which cAMP-elevating agents alter collagen expression and myofibroblast differentiation. Treatment with 10 microM forskolin or isoproterenol increased cAMP production and cAMP response element binding protein (CREB) phosphorylation in cardiac fibroblasts and inhibited serum- or TGF-beta-stimulated collagen synthesis by 37% or more. These same cAMP-elevating agents blunted TGF-beta-stimulated expression of collagen I, collagen III, and alpha-smooth muscle actin. Forskolin or isoproterenol treatment blocked the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by TGF-beta despite the fact that these cAMP-elevating agents stimulated ERK1/2 activation on their own. cAMP-elevating agents also attenuated the activation of c-Jun NH(2)-terminal kinase and reduced binding of the transcriptional coactivator CREB-binding protein 1 to transcriptional complexes containing Smad2, Smad3, and Smad4. Pharmacological inhibition of ERK completely blocked TGF-beta-stimulated collagen gene expression, but expression of an active mutant of MEK was additive with TGF-beta treatment. Thus, cAMP-elevating agents inhibit the profibrotic effects of TGF-beta in cardiac fibroblasts largely through inhibiting ERK1/2 phosphorylation but also by reducing Smad-mediated recruitment of transcriptional coactivators.

  17. Asiatic Acid Isolated From Centella Asiatica Inhibits TGF-β1-induced Collagen Expression in Human Keloid Fibroblasts via PPAR-γ Activation

    PubMed Central

    Bian, Difei; Zhang, Jizhou; Wu, Xin; Dou, Yannong; Yang, Yan; Tan, Qian; Xia, Yufeng; Gong, Zhunan; Dai, Yue

    2013-01-01

    Keloids are fibroproliferative disorders characterized by exuberant extracellular matrix deposition and transforming growth factor (TGF)-β/Smad pathway plays a pivotal role in keloid pathogenesis. Centella asiatica extract has been applied in scar management for ages. As one of its major components, asiatic acid (AA) has been recently reported to inhibit liver fibrosis by blocking TGF-β/Smad pathway. However, its effect on keloid remains unknown. In order to investigate the effects of AA on cell proliferation, invasion and collagen synthesis, normal and keloid fibroblasts were exposed to TGF-β1 with or without AA. Relevant experiments including 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2-deoxyuridine (EdU) incorporation assay, Transwell invasion assay, enzyme-linked immunosorbent assay, Western blot, quantitative polymerase chain reaction and RNA interference assay were conducted. As a result, keloid fibroblasts showed higher responsiveness to TGF-β1 stimulation than normal fibroblasts in terms of invasion and collagen synthesis. AA could suppress TGF-β1-induced expression of collagen type I, inhibit Smad 2/3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) expression, while elevate Smad 7 protein level. Noteworthy, the effects of AA on keloid fibroblasts could be abrogated by PPAR-γ antagonist GW9662 and by silencing of PPAR-γ. The present study demonstrated that AA inhibited TGF-β1-induced collagen and PAI-1 expression in keloid fibroblasts through PPAR-γ activation, which suggested that AA was one of the active constituents of C. asiatica responsible for keloid management, and could be included in the arsenal for combating against keloid. PMID:24250248

  18. Collagen degradation and MMP9 activation by Enterococcus faecalis contributes to intestinal anastomotic leak

    PubMed Central

    Shogan, B. D.; Belogortseva, N.; Luong, P. M.; Zaborin, A.; Lax, S.; Bethel, Cindy; Ward, M.; Muldoon, J. P.; Singer, M.; An, G.; Umanskiy, K.; Konda, V.; Shakhsheer, B.; Luo, J.; Klabbers, R.; Hancock, L. E.; Gilbert, J.; Zaborina, O.; Alverdy, J. C.

    2016-01-01

    Even under the most expert care, a properly constructed intestinal anastomosis can fail to heal resulting in leakage of its contents, peritonitis and sepsis. The cause of anastomotic leak remains unknown and its incidence has not changed in decades. Here, we demonstrate that the commensal bacterium Enterococcus faecalis contributes to the pathogenesis of anastomotic leak through its capacity to degrade collagen and to activate tissue matrix metalloprotease-9 (MMP9) in host intestinal tissues. We demonstrate in rats that leaking anastomotic tissues were colonized by E. faecalis strains that showed an increased collagen-degrading activity and also an increased ability to activate host MMP9, both of which contributed to anastomotic leakage. We demonstrate that the E. faecalis genes gelE and sprE were required for E. faecalis-mediated MMP9 activation. Either elimination of E. faecalis strains through direct topical antibiotics applied to rat intestinal tissues or pharmacological suppression of intestinal MMP9 activation prevented anastomotic leak in rats. In contrast, the standard recommended intravenous antibiotics used in patients undergoing colorectal surgery did not eliminate E. faecalis at anastomotic tissues nor did they prevent leak in our rat model. Finally, we show in humans undergoing colon surgery and treated with the standard recommended intravenous antibiotics, that their anastomotic tissues still contained E. faecalis and other bacterial strains with collagen-degrading/MMP9 activity. We suggest that intestinal microbes with the capacity to produce collagenases and to activate host metalloproteinase MMP9 may break down collagen in the gut tissue contributing to anastomotic leak. PMID:25947163

  19. Effect of defective collagen synthesis on epithelial implant interface: lathyritic model in dogs. An experimental preliminary study.

    PubMed

    Cengiz, Murat Inanç; Kirtiloğlu, Tuğrul; Acikgoz, Gökhan; Trisi, Paolo; Wang, Hom-Lay

    2012-04-01

    Peri-implant mucosa is composed of 2 compartments: a marginal junctional epithelium and a zone of connective tissue attachment. Both structures consist mainly of collagen. Lathyrism is characterized by defective collagen synthesis due to inhibition of lysyl oxidase, an enzyme that is essential for interfibrillar collagen cross-linking. The lathyritic agent beta-aminoproprionitrile (β-APN) is considered a suitable agent to disrupt the connective tissue metabolism. Therefore, the purpose of this study was to assess the effect of defective connective tissue metabolism on epithelial implant interface by using β-APN created chronic lathyrism in the canine model. Two 1-year-old male dogs were included in this study. A β-APN dosage of 5 mg/0.4 mL/volume 100 g/body weight was given to the test dog for 10 months, until lathyritic symptoms developed. After this, the mandibular premolar teeth (p2, p3, p4) of both dogs were atraumatically extracted, and the investigators waited 3 months before implants were placed. In the test dog, 3 implants were placed in the left mandible, and 2 implants were placed in the right mandible. In the control dog, 2 implants were placed in the left mandibular premolar site. The dogs were sacrificed 10 months after healing. Peri-implant tissues obtained from the dogs were examined histomorphologically and histopathologically. Bone to implant contact (BIC) values and bone volumes (BV) were lower in the lathyritic group compared to the control group; however, no statistical significance was found. Significant histologic and histomorphometric changes were observed in peri-implant bone, connective tissue, and peri-implant mucosal width between test and control implants. Defective collagen metabolism such as lathyrism may negatively influence the interface between implant and surrounding soft tissue attachment.

  20. Differential effect of water-soluble chitin on collagen synthesis of human bone marrow stem cells and human periodontal ligament stem cells.

    PubMed

    Park, So-Yon; Park, Jung-Chul; Kim, Min-Soo; Lee, Sung-Eun; Kim, Ki-Joon; Jung, Byung-Joo; Park, Wonse; Jeon, Dong-Won; Cho, Kyoo-Sung; Kim, Chang-Sung

    2015-02-01

    Human bone marrow stem cells (hBMSCs) represent a promising regenerative material because of their mutipotency, including their ability to regenerate collagenous soft tissues. We previously found that water-soluble chitin (WSC) enhances the ability of human periodontal ligament stem cells (hPDLSCs) to synthesize collagen tissue. The aim of this study was to determine the effects of WSC on hBMSCs and hPDLSCs for the collagen synthesis both in vitro and in vivo. hBMSCs and hPDLSCs were isolated and expanded with or without 0.3 mg/mL WSC. A series of in vitro and in vivo analyses were performed to evaluate their characteristics as stem cell populations. Then, collagen and hydroxyproline assays were conducted using both in vitro and in vivo assay models, and the real-time polymerase chain reaction was performed to analyze the expression of collagen-related markers. WSC-treated and nontreated hBMSCs and hPDLSCs were transplanted into immunocompromised mice, and histology and immunohistochemistry analyses were conducted after 8 weeks. The in vitro results showed that those cells possessed the characteristics of mesenchymal stem cells. The amount of soluble collagen synthesized was significantly greater in WSC-treated hBMSCs than in the nontreated group; conversely, treatment of hPDLSCs with WSC decreased the formation of soluble collagen. The amount of insoluble collagen synthesized was greater in the WSC-treated groups than in the nontreated groups for both hBMSCs and hPDLSCs. The hydroxyproline contents of the regenerated soluble and insoluble collagens were similar. The expressions of mRNA for collagen types I-V, hyaluronic acid synthase 1 (HAS1), HAS2, and HAS3, and the LOX family were higher in WSC-treated hPDLSCs than in the nontreated group, whereas WSC increased the expression of collagen type III and decreased that of collagen type I in hBMSCs. The histology and immunohistochemistry results revealed that WSC significantly increased the amount of collagen

  1. Role of CD14 and TLR4 in type I, type III collagen expression, synthesis and secretion in LPS-induced normal human skin fibroblasts

    PubMed Central

    Yang, Hongming; Li, Juncong; Wang, Yihe; Hu, Quan

    2015-01-01

    Objectives: The primary aim of this study was to investigate the role of CD14 and TLR4 in type I, type III collagen expression, synthesis and secretion in LPS-induced normal human skin fibroblasts. The secondary aim was to provide theoretical basis for the molecular mechanisms of scar formation induced by LPS. Methods: The normal skin fibroblasts cultured in vitro were randomly divided into four groups: 0.1 μg/mL LPS reference group, CD14 pretreatment + LPS, TLR4 pretreatment + LPS, CD14 and TLR4 pretreatment + LPS. The collagen DNA synthesis was assessed by 3H-proline incorporation method. Real-time Quantitative PCR was used to detect type I, type III collagen mRNA expression. Results: Similar results were revealed for mRNA expression levels. The immunofluorescence staining suggested that type I and type III collagen were expressed in all investigated groups and that the expression was differentially downregulated in groups B, C, D. ELISA demonstrated markedly decreased levels in secreting type I, type III collagens and hydroxyproline in groups B, C, D (P<0.05), and the lowest level was detected in group D (P<0.01). Conclusion: Pretreatment with CD14 or TLR4 alone or their combination can significantly reduce the levels of type I and type III collagen expression, synthesis and secretion, with the most notable reduction detected in case of CD14 and TLR4 combined. We could thus conclude that both CD14 and TLR4 are involved in type I and type III collagen expression, synthesis and secretion in LPS-induced skin fibroblasts. PMID:25932184

  2. Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion.

    PubMed

    Mazur, Anna; Holthoff, Emily; Vadali, Shanthi; Kelly, Thomas; Post, Steven R

    2016-01-01

    Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages.

  3. Water-filtered near-infrared influences collagen synthesis of keloid-fibroblasts in contrast to normal foreskin fibroblasts.

    PubMed

    Zöller, Nadja; König, Anke; Butting, Manuel; Kaufmann, Roland; Bernd, August; Valesky, Eva; Kippenberger, Stefan

    2016-10-01

    Hypertrophic scar development is associated to impaired wound healing, imbalanced fibroblast proliferation and extracellular matrix synthesis. Stigmatization, physical restrictions and high recurrence rates are only some aspects that illustrate the severe influence impaired wound healing can have on patients' life. The treatment of hypertrophic scars especially keloids is still a challenge. In recent years water-filtered near-infrared irradiation (wIRA) composed of near-infrared (NIR) and a thermal component is applied for an increasing penal of clinical purposes. It is described to beneficially influence e.g. wound healing. But discrimination between the thermal and the NIR dependent components of these effects has not been conclusively elucidated. Aim of our study was therefore to investigate the influence of the light fraction on the thermal impact of wIRA irradiation in dermal cells. We concentrated our analysis on morphological properties and collagen synthesis. Foreskin fibroblasts and the keloid fibroblast cell line KF111 were exposed to temperatures between 37°C and 46°C with or without additional irradiation with 360J/cm(2) NIR. Our results show that viability was not influenced by irradiation. Independent of the analysed fibroblast species temperature dependent occurrence of spheric cells could be observed. These morphological changes were clearly counteracted by additional light exposure. Convective heat reduced collagen type I synthesis in both cell species depending on the applied temperature. Co-treatment with NIR significantly reversed this effect in keloid fibroblast cultures treated at 46°C whereas no difference could be observed in the foreskin fibroblasts. The observed influence on collagen type I synthesis was associated to a temperature dependent TGF-β1 secretion reduction. Co-stimulation of keloid cultures with NIR at 46°C completely abolished the temperature dependent TGF-β1 secretion reduction. In foreskin fibroblast cultures co

  4. Colloidal Gold--Collagen Protein Core--Shell Nanoconjugate: One-Step Biomimetic Synthesis, Layer-by-Layer Assembled Film, and Controlled Cell Growth.

    PubMed

    Xing, Ruirui; Jiao, Tifeng; Yan, Linyin; Ma, Guanghui; Liu, Lei; Dai, Luru; Li, Junbai; Möhwald, Helmuth; Yan, Xuehai

    2015-11-11

    The biogenic synthesis of biomolecule-gold nanoconjugates is of key importance for a broad range of biomedical applications. In this work, a one-step, green, and condition-gentle strategy is presented to synthesize stable colloidal gold-collagen core-shell nanoconjugates in an aqueous solution at room temperature, without use of any reducing agents and stabilizing agents. It is discovered that electrostatic binding between gold ions and collagen proteins and concomitant in situ reduction by hydroxyproline residues are critically responsible for the formation of the core-shell nanoconjugates. The film formed by layer-by-layer assembly of such colloidal gold-collagen nanoconjugates can notably improve the mechanical properties and promote cell adhesion, growth, and differentiation. Thus, the colloidal gold-collagen nanoconjugates synthesized by such a straightforward and clean manner, analogous to a biomineralization pathway, provide new alternatives for developing biologically based hybrid biomaterials toward a range of therapeutic and diagnostic applications.

  5. Effect of High Glucose Concentration on Collagen Synthesis and Cholesterol Level in the Phenotypic Modulation of Aortic Cultured Smooth Muscle Cells of Sand Rat (Psammomys obesus)

    PubMed Central

    Bouguerra, S. Aouichat; Benazzoug, Y.; Bekkhoucha, F.; Bourdillon, M. C.

    2004-01-01

    To simulate diabetic conditions, the effects of high glucose concentration on collagen synthesis and cholesterol level in cultured aortic smooth muscle cells of Psammomys were investigated. For collagen biosynthesis, smooth muscle cells (SMCs) were incubated in synthetic proliferative phase and in postconfluent phase with 3H-proline. Cellular cholesterol was determined by enzymatic method. Under high glucose concentration, the results showed morphological modifications characterized by morphometric cellular, nuclear, and nucleolar changes. In biochemical studies, the authors observed an increase of free and esterified cellular cholesterol as well as of total proteins, collagen biosynthesis, and α1 (I+III) and α2 (I) chains of collagen contained in the SMCs and in the extracellular matrix. These results showed the sensitivity of Psammomys aortic SMCs to high glucose concentration and would constitute an interesting cellular model to study atherosclerosis pathogeny in experimental diabetes. PMID:15512791

  6. Fasudil inhibits proliferation and collagen synthesis and induces apoptosis of human fibroblasts derived from urethral scar via the Rho/ROCK signaling pathway

    PubMed Central

    Xu, Ning; Chen, Shao-Hao; Qu, Gen-Yi; Li, Xiao-Dong; Lin, Wen; Xue, Xue-Yi; Lin, Yun-Zhi; Zheng, Qing-Shui; Wei, Yong

    2017-01-01

    Fasudil has shown antifibrotic effects in various fibrotic diseases. However, its effects on human urethral fibroblasts are unknown. This study evaluated the effects of fasudil on cellular proliferation, migration, apoptosis, and collagen synthesis in human fibroblasts derived from urethral scar tissues. Human urethral scar fibroblasts were cultured by explant and incubated for 24 h or 48 h with fasudil (12.5, 25, 50 µmol/L) with or without transforming growth factor β1 (TGF-β1, 10 ng/mL), or left untreated (control). Cell proliferation and migration was determined by MTT assay and Transwell chambers, respectively. Apoptosis was measured by flow cytometry. Levels of α-smooth muscle actin (α-SMA), myosin light-chain phosphatase (MLCP), LIM domain kinase 1 (LIMK1), phospho-cofilin (p-cofilin), collagen I, and collagen III were determined by Western blot. Compared with the control group, TGF-β1 was associated with a significant increase in urethral fibroblast proliferation and migration, and α-SMA, MLCP, LIMK1, p-cofilin, collagen I, and collagen III levels. Compared with the control group, fasudil (with or without TGF-β1), significantly and negatively correlated, in a dose-dependent manner, with the proliferation and migration of urethral fibroblasts, as well as α-SMA, MLCP, LIMK1, p-cofilin, collagen I, and collagen III levels. Moreover, fasudil significantly induced apoptosis of fibroblasts induced by TGF-β1. Higher concentrations of fasudil (50 μmol/L) were associated with greater cell apoptosis without TGF-β1 stimulation compared with the normal control group. Fasudil, with or without TGF-β1 stimulation, may inhibit human urethral fibroblasts proliferation, migration, apoptosis, and collagen synthesis via the Rho/ROCK signaling pathway. PMID:28386357

  7. Modulation of tumor cell stiffness and migration by type IV collagen through direct activation of integrin signaling pathway.

    PubMed

    Chen, Sheng-Yi; Lin, Jo-Shi; Yang, Bei-Chang

    2014-08-01

    Excessive collagen deposition plays a critical role in tumor progression and metastasis. To understand how type IV collagen affects mechanical stiffness and migration, low-collagen-IV-expressing transfectants of B16F10, U118MG, and Huh7 (denoted shCol cells) were established by the lentiviral-mediated delivery of small interfering RNA against type IV-α1 collagen (Col4A1). Although having similar growth rates, shCol cells showed a flatter morphology compared to that of the corresponding controls. Notably, knocking down the Col4A1 gene conferred the cells with higher levels of elasticity and lower motility. Exposure to blocking antibodies against human β1 integrin or α2β1 integrin or the pharmacological inhibition of Src and ERK activity by PP1 and U0126, respectively, effectively reduced cell motility and raised cell stiffness. Reduced Src and ERK activities in shCol cells indicate the involvement of a collagen IV/integrin signaling pathway. The forced expression of β1 integrin significantly stimulated Src and ERK phosphorylation, reduced cell stiffness, and accelerated cell motility. In an experimental metastasis assay using C57BL/6 mice, B16F10 shCol cells formed significantly fewer and smaller lung nodules, confirming the contribution of collagen to metastasis. In summary, the integrin signaling pathway activated in a tumor environment with collagen deposition is responsible for low cell elasticity and high metastatic ability.

  8. Icaritin Inhibits Collagen Degradation-Related Factors and Facilitates Collagen Accumulation in Atherosclerotic Lesions: A Potential Action for Plaque Stabilization

    PubMed Central

    Zhang, Zong-Kang; Li, Jie; Yan, De-Xin; Leung, Wing-Nang; Zhang, Bao-Ting

    2016-01-01

    Most acute coronary syndromes result from rupture of vulnerable atherosclerotic plaques. The collagen content of plaques may critically affect plaque stability. This study tested whether Icaritin (ICT), an intestinal metabolite of Epimedium-derived flavonoids, could alter the collagen synthesis/degradation balance in atherosclerotic lesions. Rabbits were fed with an atherogenic diet for four months. Oral administration of ICT (10 mg·kg−1·day−1) was started after two months of an atherogenic diet and lasted for two months. The collagen degradation-related parameters, including macrophages accumulation, content and activity of interstitial collagenase-1 (MMP-1), and the collagen synthesis-related parameters, including amount and distribution of smooth muscle cells (SMC) and collagen mRNA/protein levels, were evaluated in the aorta. ICT reduced plasma lipid levels, inhibited macrophage accumulation, lowered MMP-1 mRNA and protein expression, and suppressed proteolytic activity of pro-MMP-1 and MMP-1 in the aorta. ICT changed the distribution of the SMCs towards the fibrous cap of lesions without increasing the amount of SMCs. Higher collagen protein content in lesions and aorta homogenates was observed with ICT treatment compared with the atherogenic diet only, without altered collagen mRNA level. These results suggest that ICT could inhibit the collagen degradation-related factors and facilitate collagen accumulation in atherosclerotic lesions, indicating a new potential of ICT in atherosclerotic plaques. PMID:26828485

  9. Icaritin Inhibits Collagen Degradation-Related Factors and Facilitates Collagen Accumulation in Atherosclerotic Lesions: A Potential Action for Plaque Stabilization.

    PubMed

    Zhang, Zong-Kang; Li, Jie; Yan, De-Xin; Leung, Wing-Nang; Zhang, Bao-Ting

    2016-01-28

    Most acute coronary syndromes result from rupture of vulnerable atherosclerotic plaques. The collagen content of plaques may critically affect plaque stability. This study tested whether Icaritin (ICT), an intestinal metabolite of Epimedium-derived flavonoids, could alter the collagen synthesis/degradation balance in atherosclerotic lesions. Rabbits were fed with an atherogenic diet for four months. Oral administration of ICT (10 mg·kg(-1)·day(-1)) was started after two months of an atherogenic diet and lasted for two months. The collagen degradation-related parameters, including macrophages accumulation, content and activity of interstitial collagenase-1 (MMP-1), and the collagen synthesis-related parameters, including amount and distribution of smooth muscle cells (SMC) and collagen mRNA/protein levels, were evaluated in the aorta. ICT reduced plasma lipid levels, inhibited macrophage accumulation, lowered MMP-1 mRNA and protein expression, and suppressed proteolytic activity of pro-MMP-1 and MMP-1 in the aorta. ICT changed the distribution of the SMCs towards the fibrous cap of lesions without increasing the amount of SMCs. Higher collagen protein content in lesions and aorta homogenates was observed with ICT treatment compared with the atherogenic diet only, without altered collagen mRNA level. These results suggest that ICT could inhibit the collagen degradation-related factors and facilitate collagen accumulation in atherosclerotic lesions, indicating a new potential of ICT in atherosclerotic plaques.

  10. Epigallocatechin-3-gallate, a polyphenol component of green tea, suppresses both collagen production and collagenase activity in hepatic stellate cells.

    PubMed

    Nakamuta, Makoto; Higashi, Nobuhiko; Kohjima, Motoyuki; Fukushima, Marie; Ohta, Satoshi; Kotoh, Kazuhiro; Kobayashi, Naoya; Enjoji, Munechika

    2005-10-01

    Catechins such as epigallocatechin-3-gallate (EGCG), epicatechin-3-gallate (ECG), and epigallocatechin (EGC) are polyphenol components of green tea. EGCG is the major component and has been reported to possess a wide range of biological properties including anti-fibrogenic activity. In hepatic fibrosis, activated hepatic stellate cells (HSCs) play a central role. In this study, we investigated the effect of catechins, including EGCG, on collagen production and collagenase activity in rat primary HSCs and activated human HSC-derived TWNT-4 cells. EGCG (50 microM) suppressed type I collagen production in rat HSCs more than ECG (50 microM) did; however, EGC (50 microM) did not show suppressive effects. EGCG also inhibited both collagen production and collagenase activity (active matrix metalloproteinase-1 [MMP-1]) in a dose-dependent manner, but did not affect the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) production in TWNT-4 cells. Real-time PCR unexpectedly revealed that EGCG enhanced the transcription of type I collagen and TIMP-1, but did not affect the transcription of alpha-smooth muscle actin (alpha-SMA), and reduced the transcription MMP-1 in TWNT-4 cells. These findings demonstrated that EGCG inhibited collagen production regardless of enhanced collagen transcription and suppressed collagenase activity, and suggested that EGCG might have therapeutic potential for liver fibrosis.

  11. Activation of nuclear factor κB in colonic mucosa from patients with collagenous and ulcerative colitis

    PubMed Central

    Andresen, L; Jørgensen, V L; Perner, A; Hansen, A; Eugen-Olsen, J; Rask-Madsen, J

    2005-01-01

    Background and aims: Expression of inducible nitric oxide synthase (iNOS) is greatly upregulated in the colonic mucosa of patients with collagenous and ulcerative colitis. As the transcription factor nuclear factor κB (NFκB) is a major inducer of iNOS gene expression, we compared activation and transcriptional activity of NFκB in colonic mucosal biopsies from these patients. Patients: Eight patients with collagenous colitis, six with relapsing ulcerative colitis, and eight with uninflamed bowel were studied. Methods: NFκB DNA binding activity was assessed by electrophoretic mobility shift assay and inhibitor of NFκB (IκB) kinase (IKK) activity by immunocomplex kinase assay. In vivo recruitment of NFκB to the iNOS promoter was determined by chromatin immunoprecipitation analysis and transcriptional activity by NFκB gene expression profiling arrays. Cells showing NFκB activation were identified by immunohistochemistry. Results: In collagenous and ulcerative colitis, as opposed to uninflamed bowel, IKKβ activity and strong NFκB DNA binding gave rise to activation of identical NFκB subunits and recruitment of transcriptionally active p65 to the iNOS promoter. In collagenous colitis, activated NFκB was observed only in epithelial cells while up to 10% of lamina propria macrophages showed activation in ulcerative colitis. Conclusions: In collagenous and ulcerative colitis, colonic mucosal NFκB is activated and recruited to the iNOS promoter in vivo via an IKKβ mediated pathway. As collagenous colitis is not associated with tissue injury, these data challenge the prevailing view that activation of NFκB per se mediates tissue injury. Our results suggest that downstream inflammatory reactions leading to tissue damage originate in lamina propria immune cells, as increased NFκB activity in collagenous colitis was localised solely in epithelial cells, but present also in macrophages in ulcerative colitis. PMID:15753535

  12. Influence of Biodentine® - A Dentine Substitute - On Collagen Type I Synthesis in Pulp Fibroblasts In Vitro

    PubMed Central

    Nikfarjam, Frangis; Beyer, Kim; König, Anke; Hofmann, Matthias; Butting, Manuel; Valesky, Eva; Kippenberger, Stefan; Kaufmann, Roland; Heidemann, Detlef; Bernd, August

    2016-01-01

    Preserving a patient’s own teeth—even in a difficult situation—is nowadays preferable to surgical intervention and therefore promotes development of suitable dental repair materials. Biodentine®, a mineral trioxide aggregate substitute, has been used to replace dentine in a bioactive and biocompatible manner in both the dental crown and the root. The aim of our study was to evaluate the influence of Biodentine® on pulp fibroblasts in vitro. For this study, one to five Biodentine® discs with a diameter of 5.1mm were incubated in DMEM. To obtain Biodentine® suspensions the media were collected and replaced with fresh medium every 24h for 4 days. Primary pulp cells were isolated from freshly extracted wisdom teeth of 20–23 year old patients and incubated with the Biodentine® suspensions. Proliferation, cell morphology, cell integrity and cell viability were monitored. To evaluate the effect of Biodentine® on collagen type I synthesis, the secretion of the N-terminal domain of pro-collagen type I (P1NP) and the release of transforming growth factor-β1 (TGF-β1) were quantified. None of the Biodentine® suspensions tested influenced cell morphology, proliferation or cell integrity. The cell viability varied slightly depending on the suspension used. However, the concentrations of P1NP of all pulp fibroblast cultures treated for 24h with the moderate to high Biodentine® concentration containing suspensions of day 1 were reduced to 5% of the control. Furthermore, a significant TGF-β1 reduction was observed after treatment with these suspensions. It could be shown that Biodentine® is biocompatible. However, dissolved particles of the moderate to high concentrated Biodentine® suspensions 24h after mixing induce a significant reduction of TGF-β1 release and reduce the secretion of collagen type I of primary pulp fibroblasts. PMID:27936042

  13. The EBV oncogene LMP1 protects lymphoma cells from cell death through the collagen-mediated activation of DDR1.

    PubMed

    Cader, Fathima Zumla; Vockerodt, Martina; Bose, Shikha; Nagy, Eszter; Brundler, Marie-Anne; Kearns, Pamela; Murray, Paul G

    2013-12-19

    The malignant Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma are surrounded by a tumor microenvironment that is composed of a variety of cell types, as well as noncellular components such as collagen. Although HRS cells harbor oncogenic Epstein-Barr virus (EBV) in approximately 50% of cases, it is not known if the tumor microenvironment contributes to EBV-driven lymphomagenesis. We show that expression of the EBV-encoded latent membrane protein-1 (LMP1) in primary human germinal center B cells, the presumed progenitors of HRS cells, upregulates discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase activated by collagen. We also show that HRS cells intimately associated with collagen frequently overexpress DDR1 and that short-term exposure to collagen is sufficient to activate DDR1 in Hodgkin lymphoma-derived cell lines. The ectopic expression of DDR1 significantly increased the survival of collagen-treated DG75 Burkitt lymphoma cells, following etoposide treatment. Conversely, knockdown of DDR1 significantly decreased the survival of collagen-treated L428 Hodgkin lymphoma cells in the absence of specific apoptotic stimulus, suggesting that DDR1 also influences baseline survival. Our results identify a hitherto unknown function for collagen in protecting Hodgkin lymphoma cells from apoptosis and suggest an important contribution of the tumor microenvironment in promoting the oncogenic effects of EBV.

  14. Synthesis and characterization of collagen/hyaluronan/chitosan composite sponges for potential biomedical applications.

    PubMed

    Lin, Yen-Chih; Tan, Fa-Jui; Marra, Kacey G; Jan, Shyh-Shyan; Liu, Deng-Cheng

    2009-09-01

    Cells, scaffolds and growth factors are three main components of a tissue-engineered construct. Collagen type I, a major protein of the extracellular matrix (ECM) in mammals, is a suitable scaffold material for regeneration. Another important constituent of the ECM, hyaluronic acid (hyaluronan, HA), has been used for medical purposes due to its hydrogel properties and biodegradability. Chitosan is a linear polysaccharide comprised of beta1- to beta4-linked d-glucosamine residues, and its potential as a biomaterial is based on its cationic nature and high charge density in solution. This study was conducted to evaluate the characteristics of scaffolds composed of different ratios of type I comb collagen and chitosan with added HA in order to obtain the optimum conditions for the manufacture of collagen-hyaluronan-chitosan (Col-HA-Ch; comprising collagen, HA and chitosan mixed in different ratios: 10:1:0, Col10HACh0; 9:1:1, Col9HACh1; 8:1:2, Col8HACh2; 7:1:3, Col7HACh3; 6:1:4, Col6HACh4; and 5:1:5, Col5HACh5) composite porous scaffolds. Microstructural observation of the composite scaffolds was performed using scanning electron microscopy. The mean pore diameters ranged from 120 to 182microm and decreased as the chitosan composition increased. All scaffolds showed high pore interconnectivity. Swelling ratio measurements showed that all specimens could bind 35- to 40-fold of physiological fluid and still maintain their form and stability. For tensile strength, the optimal ratio of collagen and chitosan was 9:1. Thermal stability was investigated using a differential scanning calorimeter and showed that Col5HACh5 and Col6HACh4 were significantly more stable than the other groups. In enzymatic sensitivity, a steady increase in the biostability of the scaffolds was achieved as the chitosan concentration was increased. In biocompatibility testing, the proliferation of the fibroblasts cultured in Co-HA-Ch tri-copolymer scaffolds was high. Overall, we observed the 9

  15. Ventricular remodeling in heart failure: the role of myocardial collagen.

    PubMed

    Janicki, J S; Brower, G L; Henegar, J R; Wang, L

    1995-01-01

    Collagen which is present in the myocardium in relatively small amounts is the most abundant structural protein of the connective tissue network. Its structural organization consists of a complex weave of collagen fibers that surrounds and interconnects myocytes, groups of myocytes, muscle fibers and muscle bundles. The conformation of interstitial fibrillar collagen makes it highly resistant to degradation by all proteinases other than specific collagenases. In hearts with myocardial damage secondary to myocardial infarction, chronic ischemia, inflammation, or cardiomyopathy, a complex sequence of compensatory events occur that eventually result in an adverse left ventricular remodeling. This continual state of remodeling is characterized by persistent collagenase activity, fibrillar collagen degradation, and progressive myocyte loss. The net effect is a shift in the balance between collagen synthesis and degradation which leads to an inadequate fibrillar collagen matrix, progressive ventricular dilatation and sphericalization with wall thinning and eventual congestive heart failure.

  16. Structure-activity relationships and action mechanisms of collagen-like antimicrobial peptides.

    PubMed

    Masuda, Ryo; Dazai, Yui; Mima, Takehiko; Koide, Takaki

    2017-01-01

    An antimicrobial triple-helical peptide, R3, was previously obtained from a collagen-like combinatorial peptide library. In this research, based on structure-activity relationship studies of R3, a more potent peptide, RR4, with increased positive net charge and charge density relative to R3, was developed. RR4 exhibited antimicrobial activity against both Gram-negative and Gram-positive bacterial strains, including multidrug-resistant strains. Its action could be attributed to entry into cells and interactions with intercellular molecules such as DNA/RNA that inhibited cell division rather than increasing bacterial membrane permeability. Furthermore, RR4 exhibited remarkable stability in serum and low cytotoxicity.

  17. In vitro evaluation of cell proliferation and collagen synthesis on titanium following plasma electrolytic oxidation.

    PubMed

    Whiteside, Paul; Matykina, Endzhe; Gough, Julie E; Skeldon, Peter; Thompson, George E

    2010-07-01

    Titania-based coatings produced by plasma electrolytic oxidation are being investigated as bioactive surfaces for titanium implants. In this study, plasma electrolytic oxidation was performed in calcium- and phosphorus-based electrolytes under DC conditions, resulting in coatings of thickness of approximately 8-15 mum. Coating morphologies, microstructures, and compositions were examined by scanning electron microscopy with energy-dispersive X-ray analysis, X-ray diffraction, and electron probe microanalysis. The coatings revealed a cratered morphology, with incorporated calcium and phosphorus species. Proliferation rates of primary human osteoblasts cells on the coatings were up to approximately 37% faster than those for uncoated titanium and 316L stainless steel reference materials. Further, the coatings assisted cell adhesion and generation and anchorage of collagen. The amount of collagen was upto approximately 2.4 times greater than for the reference substrates. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

  18. Synthesis of highly interconnected 3D scaffold from Arothron stellatus skin collagen for tissue engineering application.

    PubMed

    Ramanathan, Giriprasath; Singaravelu, Sivakumar; Raja, M D; Sivagnanam, Uma Tiruchirapalli

    2015-11-01

    The substrate which is avidly used for tissue engineering applications should have good mechanical and biocompatible properties, and all these parameters are often considered as essential for dermal reformation. Highly interconnected three dimensional (3D) wound dressing material with enhanced structural integrity was synthesized from Arothron stellatus fish skin (AsFS) collagen for tissue engineering applications. The synthesized 3D collagen sponge (COL-SPG) was further characterized by different physicochemical methods. The scanning electron microscopy analysis of the material demonstrated that well interconnected pores with homogeneous microstructure on the surface aids higher swelling index and that the material also possessed good mechanical properties with a Young's modulus of 0.89±0.2 MPa. Biocompatibility of the 3D COL-SPG showed 92% growth for both NIH 3T3 fibroblasts and keratinocytes. Overall, the study revealed that synthesized 3D COL-SPG from fish skin will act as a promising wound dressing in skin tissue engineering.

  19. Poly-L-arginine based materials as instructive substrates for fibroblast synthesis of collagen.

    PubMed

    Bygd, Hannah C; Akilbekova, Dana; Muñoz, Adam; Forsmark, Kiva D; Bratlie, Kaitlin M

    2015-09-01

    The interactions of cells and surrounding tissues with biomaterials used in tissue engineering, wound healing, and artificial organs ultimately determine their fate in vivo. We have demonstrated the ability to tune fibroblast responses with the use of varied material chemistries. In particular, we examined cell morphology, cytokine production, and collagen fiber deposition angles in response to a library of arginine-based polymeric materials. The data presented here shows a large range of vascular endothelial growth factor (VEGF) secretion (0.637 ng/10(6) cells/day to 3.25 ng/10(6) cells/day), cell migration (∼15 min < persistence time < 120 min, 0.11 μm/min < speed < 0.23 μm/min), and cell morphology (0.039 < form factor (FF) < 0.107). Collagen orientation, quantified by shape descriptor (D) values that ranges from 0 to 1, representing completely random (D = 0) to aligned (D = 1) fibers, exhibited large variation both in vitro and in vivo (0.167 < D < 0.36 and 0.17 < D < 0.52, respectively). These findings demonstrate the ability to exert a certain level of control over cellular responses with biomaterials and the potential to attain a desired cellular response such as, increased VEGF production or isotropic collagen deposition upon exposure to these materials in wound healing and tissue engineering applications.

  20. Liposomes encapsulating Aloe vera leaf gel extract significantly enhance proliferation and collagen synthesis in human skin cell lines.

    PubMed

    Takahashi, Makoto; Kitamoto, Dai; Asikin, Yonathan; Takara, Kensaku; Wada, Koji

    2009-01-01

    Aloe vela leaf gel extract (AGE) are widely used as cosmetic and pharmaceutical ingredients because of its versatile skin care properties. In order to enhance the bioavailability of AGE, liposomes encapsulating AGE were prepared and examined for their interfacial and biochemical properties. The liposomes prepared from a soybean lecithin (SLP-WHITE, 1.0 wt%) by the Bangham method gave relatively a good trapping efficiency up to the AGE concentration of 0.5 wt%. The stable liposomes were then prepared from 1.0 wt% of SLP-WHITE and different concentrations of AGE by the mechanochemical method using a homogenizer and microfluidizer. The liposomes obtained from 0.25 wt% of AGE were confirmed to be small unilamellar vesicles with a diameter of less than 200 nm, and remained well dispersed for at least two weeks. The obtained liposomes encapsulating AGE were further examined for the effects on proliferation and type I collagen synthesis in normal human neonatal skin fibroblasts, NB1RGB cells. Liposomal AGE clearly showed higher proliferation rate than that of AGE alone. In addition, compared to the control, liposomal AGE significantly increased the collagen synthesis by 23%, while AGE alone showed a small effect. Liposomal AGE was also assayed for the effect on proliferation in normal human epidermal keratinocytes, NHEK(F) cells. Interestingly, liposomal AGE fractions containing 4 and 20 microg/mL of the extract considerably increased the proliferation rate by 77% and 101%, respectively. In contrast, AGE alone fractions containing 4 and 20 microg/mL of the extract increased the rate by 41% and 60%, respectively. Accordingly, the bioavailability and skin care properties of AGE will be significantly enhanced by liposome encapsulation, and the present liposomal AGE should have a great potential as an effective skin care formulation.

  1. Neferine inhibits proliferation and collagen synthesis induced by high glucose in cardiac fibroblasts and reduces cardiac fibrosis in diabetic mice

    PubMed Central

    Liu, Xue; Song, Xiuhui; Lu, Jianjun; Chen, Xueying; Liang, Ershun; Liu, Xiaoqiong; Zhang, Mingxiang; Zhang, Yun; Du, Zhanhui; Zhao, Yuxia

    2016-01-01

    Cardiac fibrosis is a common pathological process accompanying diabetes mellitus. In this report, we studied the effects of neferine (a major bisbenzylisoquinline alkaloid derived from lotus embryos) on cardiac fibrosis induced by diabetes mellitus, as well as the underlying molecular pathways. In vivo, type 1 diabetes mellitus was induced in mice by administering streptozotocin. Diabetic mice were treated with neferine through oral gavage, and cardiac function was assessed using echocardiography. Total collagen deposition was assessed by Masson's trichrome and Picrosirius staining. In vitro, cardiac fibroblasts were cultured in normal or high-glucose medium with or without neferine. Neferine attenuated left ventricular dysfunction and remodeling and reduced collagen deposition in diabetic mice. In vitro, neferine inhibited cardiac fibroblast proliferation, migration, and differentiation into myofibroblasts. In addition, neferine reduced high-glucose-induced collagen production and inhibited TGF-β1-Smad, ERK and p38 MAPK signaling activation in cardiac fibroblasts. These results suggest that neferine may have antifibrogenic effects in diabetes-related cardiac fibrosis. PMID:27533252

  2. Collagen type IV stimulates an increase in intracellular Ca2+ in pancreatic acinar cells via activation of phospholipase C.

    PubMed Central

    Somogyi, L; Lasić, Z; Vukicević, S; Banfić, H

    1994-01-01

    Intracellular Ca2+ responses to extracellular matrix molecules were studied in suspensions of pancreatic acinar cells loaded with Fura-2. Collagen type I, laminin, fibrinogen and fibronectin were unable to raise cytosolic free Ca2+ concentration ([Ca2+]i), whereas collagen type IV, at concentrations from 5 to 50 micrograms/ml, significantly increased it. The effect of collagen type IV was not due to possible contamination with type-I transforming growth factor beta or plasminogen, as neither of these agents was able to increase [Ca2+]i. Using highly specific mass assays, concentrations of inositol lipids, 1,2-diacylglycerol (DAG) and Ins(1,4,5) P3 were measured in pancreatic acinar cells stimulated with collagen type IV. A decrease in the concentrations of PtdIns(4,5) P2 and PtdIns4 P with a concomitant increase in the concentrations of DAG and InsP3 mass were observed, showing that collagen type IV increases [Ca2+]i by activation of phospholipase C. The observed [Ca2+]i signals had two components, the first resulting from Ca2+ release from the intracellular stores, and the second resulting from Ca2+ flux from the extracellular medium through the verapamil-insensitive channels. A tyrosine kinase inhibitor (tyrphostine) was able to block inositol lipid signalling caused by collagen type IV, which together with the insensitivity of this pathway to cholera toxin and pertussis toxin or to preactivation of protein kinase C, the longer duration of the increase in [Ca2+]i and a longer lag period needed for observation of increases in DAG and InsP3 concentration with collagen type IV than with carbachol (50 mM) suggest that activation of phospholipase C by collagen type IV is caused by tyrosine kinase activation. Inositol lipid signalling and increases in [Ca2+]i were also observed with Arg-Gly-Asp (RGD)-containing peptide but not with Arg-Asp-Gly (RDG)-containing peptide. Collagen type IV and RGD-containing peptide, but not carbachol, competed in increasing [Ca2+]i and

  3. Diallylsulfide attenuates excessive collagen production and apoptosis in a rat model of bleomycin induced pulmonary fibrosis through the involvement of protease activated receptor-2

    SciTech Connect

    Kalayarasan, Srinivasan Sriram, Narayanan; Soumyakrishnan, Syamala; Sudhandiran, Ganapasam

    2013-09-01

    Pulmonary fibrosis (PF) can be a devastating lung disease. It is primarily caused by inflammation leading to severe damage of the alveolar epithelial cells. The pathophysiology of PF is not yet been clearly defined, but studying lung parenchymal injury by involving reactive oxygen species (ROS) through the activation of protease activated receptor-2 (PAR-2) may provide promising results. PAR-2 is a G-protein coupled receptor is known to play an important role in the development of PF. In this study, we investigated the inhibitory role of diallylsulfide (DAS) against ROS mediated activation of PAR-2 and collagen production accompanied by epithelial cell apoptosis. Bleomycin induced ROS levels may prompt to induce the expression of PAR-2 as well as extracellular matrix proteins (ECM), such as MMP 2 and 9, collagen specific proteins HSP-47, α-SMA, and cytokines IL-6, and IL-8RA. Importantly DAS treatment effectively decreased the expression of all these proteins. The inhibitory effect of DAS on profibrotic molecules is mediated by blocking the ROS level. To identify apoptotic signaling as a mediator of PF induction, we performed apoptotic protein expression, DNA fragmentation analysis and ultrastructural details of the lung tissue were performed. DAS treatment restored all these changes to near normalcy. In conclusion, treatment of PF bearing rats with DAS results in amelioration of the ROS production, PAR-2 activation, ECM production, collagen synthesis and alveolar epithelial cell apoptosis during bleomycin induction. We attained the first evidence that treatment of DAS decreases the ROS levels and may provide a potential therapeutic effect attenuating bleomycin induced PF. - Highlights: • DAS inhibits PAR-2 activity; bleomycin stimulates PAR-2 activity. • Increase in PAR-2 activity is correlated with pulmonary fibrosis • DAS reduces pro-inflammatory activity linked to facilitating pulmonary fibrosis. • DAS inhibits apoptosis of alveolar epithelial cells.

  4. Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta

    PubMed Central

    Cabral, Wayne A.; Ishikawa, Masaki; Garten, Matthias; Makareeva, Elena N.; Sargent, Brandi M.; Weis, MaryAnn; Barnes, Aileen M.; Webb, Emma A.; Shaw, Nicholas J.; Ala-Kokko, Leena; Lacbawan, Felicitas L.; Högler, Wolfgang; Leikin, Sergey; Blank, Paul S.; Zimmerberg, Joshua; Eyre, David R.; Yamada, Yoshihiko; Marini, Joan C.

    2016-01-01

    Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50–70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes. PMID:27441836

  5. Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation.

    PubMed

    Wang, Su Bin; Jang, Ji Yong; Chae, Yun Hee; Min, Ji Hyun; Baek, Jin Young; Kim, Myunghee; Park, Yunjeong; Hwang, Gwi Seo; Ryu, Jae-Sang; Chang, Tong-Shin

    2015-06-01

    Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47(phox), a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47(phox) and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases.

  6. Desmopressin (DDAVP) improves recruitment of activated platelets to collagen but simultaneously increases platelet endothelial interactions in vitro.

    PubMed

    Calmer, Simone; Ferkau, Annika; Larmann, Jan; Johanning, Kai; Czaja, Eliana; Hagl, Christian; Echtermeyer, Frank; Goudeva, Lilia; Heuft, Hans-Gert; Theilmeier, Gregor

    2014-01-01

    Platelet dysfunction can cause clinically relevant bleeding. Treatment with DDAVP is advocated for this condition. DDAVP increases von Willebrand factor (VWF) on endothelial cells (ECs) and in plasma. VWF could facilitate platelet deposition on subendothelial collagen. VWF also facilitates platelet/EC interactions. Therefore DDAVP could precipitate thromboembolic events. We used a flow chamber model to study in vitro and ex vivo if DDAVP alters recruitment of platelets to EC and collagen. Resting or TRAP-activated platelets and EC were treated individually or simultaneously with 0.4 ng/ml DDAVP. Fluorophor-labeled platelets (10(6)/ml) were resuspended in reconstituted blood and superfused across EC and collagen in an in vitro flow chamber model at arterial shear (320 s(-1)). Adhesion of platelets to the respective surface was recorded fluorescence microscopically and platelet covered area was assessed. TRAP significantly induced adhesiveness of platelets for collagen and EC. DDAVP pretreatment of platelets did not affect adhesiveness of resting or TRAP-activated platelets for collagen or EC. Adhesiveness of resting but not TRAP-activated platelets was induced on DDAVP-treated EC. DDAVP-conditioned EC supernatant contained vWF and significantly increased platelet deposition on collagen. Platelets from patients with clinically suspected platelet dysfunction undergoing aortic valve replacement exhibited decreased platelet deposition on collagen surfaces. In summary, our data confirm that DDAVP can induce release of platelet adhesion promoting factors from EC, which is most likely vWF. DDAVP has no direct effect on platelets. Blood samples from DDAVP-treated patients do not exhibit significantly augmented platelet deposition on collagen ex vivo. This influence of released promoting factors might cause an increase of undesirable interactions of platelets with EC.

  7. Type IV collagen is an activating ligand for the adhesion G protein-coupled receptor GPR126.

    PubMed

    Paavola, Kevin J; Sidik, Harwin; Zuchero, J Bradley; Eckart, Michael; Talbot, William S

    2014-08-12

    GPR126 is an orphan heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) that is essential for the development of diverse organs. We found that type IV collagen, a major constituent of the basement membrane, binds to Gpr126 and activates its signaling function. Type IV collagen stimulated the production of cyclic adenosine monophosphate in rodent Schwann cells, which require Gpr126 activity to differentiate, and in human embryonic kidney (HEK) 293 cells expressing exogenous Gpr126. Type IV collagen specifically bound to the extracellular amino-terminal region of Gpr126 containing the CUB (complement, Uegf, Bmp1) and pentraxin domains. Gpr126 derivatives lacking the entire amino-terminal region were constitutively active, suggesting that this region inhibits signaling and that ligand binding relieves this inhibition to stimulate receptor activity. A new zebrafish mutation that truncates Gpr126 after the CUB and pentraxin domains disrupted development of peripheral nerves and the inner ear. Thus, our findings identify type IV collagen as an activating ligand for GPR126, define its mechanism of activation, and highlight a previously unrecognized signaling function of type IV collagen in basement membranes.

  8. Biological Silicon Stimulates Collagen Type 1 and Osteocalcin Synthesis in Human Osteoblast-Like Cells Through the BMP-2/Smad/RUNX2 Signaling Pathway.

    PubMed

    Dong, Meng; Jiao, Guangjun; Liu, Haichun; Wu, Wenliang; Li, Shangzhi; Wang, Qingshi; Xu, Daxia; Li, Xiaofeng; Liu, Huan; Chen, Yunzhen

    2016-10-01

    Silicon is essential for bone formation. A low-silicon diet leads to bone defects, and numerous animal models have demonstrated that silicon supplementation increases bone mineral density (BMD) and reduces bone fragility. However, the exact mechanism of this action has not been characterized. In this study, we aimed to determine the role of biological silicon in the induction of osteoblast differentiation and the possible underlying mechanism. We examined whether orthosilicic acid promotes collagen type 1 (COL-1) and osteocalcin synthesis through the bone morphogenetic protein-2 (BMP-2)/Smad1/5/runt-related transcription factor 2 (RUNX2) signaling pathway by investigating its effect in vitro at several concentrations on COL-1 and osteocalcin synthesis in human osteosarcoma cell lines (MG-63 and U2-OS). The expression of relevant proteins was detected by Western blotting following exposure to noggin, an inhibitor of BMP-2. In MG-63 cells, immunofluorescence methods were applied to detect changes in the expression of BMP-2, phosphorylated Smad1/5 (P-Smad1/5), and RUNX2. Furthermore, rat bone mesenchymal stem cells (BMSCs) were used to determine the effect of orthosilicic acid on osteogenic differentiation. Exposure to 10 μM orthosilicic acid markedly increased the expression of BMP-2, P-Smad1/5, RUNX2, COL-1, and osteocalcin in osteosarcoma cell lines. Enhanced ALP activity and the formation of mineralized nodules were also observed under these conditions. Furthermore, preconditioning with noggin inhibited the silicon-induced upregulation of P-Smad1/5, RUNX2, and COL-1 expression. In conclusion, the BMP-2/Smad1/5/RUNX2 signaling pathway participates in the silicon-mediated induction of COL-1 and osteocalcin synthesis, and orthosilicic acid promotes the osteogenic differentiation of rat BMSCs.

  9. Formation mechanism and biological activity of novel thiolated human-like collagen iron complex.

    PubMed

    Zhu, Chenhui; Liu, Lingyun; Deng, Jianjun; Ma, Xiaoxuan; Hui, Junfeng; Fan, Daidi

    2016-03-01

    To develop an iron supplement that is effectively absorbed and utilized, thiolated human-like collagen was created to improve the iron binding capacity of human-like collagen. A thiolated human-like collagen-iron complex was prepared in a phosphate buffer, and one mole of thiolated human-like collagen-iron possessed approximately 28.83 moles of iron. The characteristics of thiolated human-like collagen-iron were investigated by ultraviolet-visible absorption spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, and differential scanning calorimetry. The results showed that the thiolated human-like collagen-iron complex retained the secondary structure of human-like collagen and had greater thermodynamic stability than human-like collagen, although interactions between iron ions and human-like collagen occurred during the formation of the complex. In addition, to evaluate the bioavailability of thiolated human-like collagen-iron, an in vitro Caco-2 cell model and an in vivo iron deficiency anemia mouse model were employed. The data demonstrated that the thiolated human-like collagen-iron complex exhibited greater bioavailability and was more easily utilized than FeSO4, ferric ammonium citrate, or ferrous glycinate. These results indicated that the thiolated human-like collagen-iron complex is a potential iron supplement in the biomedical field.

  10. The linker-free covalent attachment of collagen to plasma immersion ion implantation treated polytetrafluoroethylene and subsequent cell-binding activity.

    PubMed

    Bax, Daniel V; McKenzie, David R; Weiss, Anthony S; Bilek, Marcela M M

    2010-03-01

    It is desirable that polymers used for the fabrication of prosthetic implants promote biological functions such as cellular adhesion, differentiation and viability. In this study, we have used plasma immersion ion implantation (PIII) to modify the surface of polytetrafluoroethylene (PTFE), thereby modulating the binding mechanism of collagen. The amount of collagen bound to the polymer surface following PIII-treatment was similar to that bound by non-covalent physisorption. In a manner consistent with previous enzyme and tropoelastin binding data, the collagen bound to the PIII-treated PTFE surface was resistant to sodium dodecyl sulfate (SDS) elution whilst collagen bound to the untreated surface was fully removed. This demonstrates the capability of PIII-treated surfaces to covalently attach collagen without employing chemical linking molecules. Only the collagen bound to the PIII-treated PTFE surface supported human dermal fibroblast attachment and spreading. This indicates that collagen on the PIII-treated surface possesses increased adhesive activity as compared to that on the untreated surface. Cell adhesion was inhibited by EDTA when the collagen was bound to PIII-treated PTFE, as expected for integrin involvement. Additionally this adhesion was sensitive to the conformation of the bound collagen. Increased actin cytoskeletal assembly was observed on cells spreading onto collagen-coated PIII-treated PTFE compared to the collagen-coated untreated PTFE. These data demonstrate the retention of collagen's biological properties following its attachment to PIII-treated PTFE, suggesting advantages for tissue engineering and prosthetic design.

  11. Effects of topical glucocorticoid medication on collagen biosynthesis in the dental pulp.

    PubMed

    Uitto, V J; Antila, R; Ranta, R

    1975-01-01

    The aim of the study was to observe pulpal collagen synthesis in response to trauma and to glucocorticoid medication. The material consisted of 290 rabbit pulps and 76 human premolar pulps. Collagen synthesis was determined by incubating whole pulps in a medium containing [14C]proline, and measuring the formation of [14C]hydroxyproline. The effect of glucocorticoids was studied in vitro using rabbit pulps. Hydrocortisone and dexamethasone inhibited collagen synthesis, whereas prednisolone had no marked effect. Hydrocortisone was found to inhibit the synthesis of [14C]hydroxyproline in neutral salt soluble and insoluble non-dialyzable collagen fractions. [14C]hydroxyproline in the dialyzable fraction was increased, suggesting that hydrocortisone increased collagen degradation. In the human material, premolar pulps were experimentally exposed and then medicated with capping agents. The contralateral teeth were exposed and capped with other capping materials, in some cases they were left as intact controls. The exposure led to an increase in the collagen synthesis as indicated by increased [14C]hydroxyproline formation and elevated protocollagen proline hydroxylase activity in the pulp. This enzyme activity was suppressed in pulps capped with a glucocorticoid paste. In addition, the collagen synthesis rate was lower in pulps treated with another glucocorticoid containing compound, when compared to pulps capped with a calcium hydroxide preparation.

  12. Collagen type I and III synthesis by Tenon's capsule fibroblasts in culture: individual patient characteristics and response to mitomycin C, 5-fluorouracil, and ascorbic acid.

    PubMed Central

    Gross, R L

    1999-01-01

    PURPOSE: This study was performed to better understand the differences between patients in specific components of wound healing as it may pertain to glaucoma filtration surgery, including the use of antimetabolites. METHODS: Human Tenon's capsule fibroblasts were obtained at the time of glaucoma filtering surgery and established in individual cell cultures from 35 glaucoma patients. The dose-response to 5-fluorouracil (5FU) and mitomycin C (MMC) was determined. The individual cell lines were exposed to the antimetabolites and ascorbic acid with measurement of collagen type I and III production by an ELISA-type dot blot assay. These results were then statistically compared to the individual patient characteristics including age, race, previous surgery and medications, and type of glaucoma. RESULTS: 5-FU had little effect on collagen type I and III production or protein synthesis. MMC had an inhibitory effect on collagen secretion and total protein synthesis with increasing concentration. Photomicrographs of the cells after each treatment condition revealed characteristic morphologic changes when compared to controls. There was a large range of collagen type I and III production with correlation between the amounts of each collagen type secreted in response to the antimetabolites. However, there was no correlation with accepted risk factors for filtration failure. CONCLUSION: These antimetabolites act similarly on different cell lines in a nonspecific manner. The results suggest that the increased risk of filtration failure due to age, race, diagnosis, and previous conjunctival surgery is not due to differences in secretion of collagen types I and III by Tenon's capsule fibroblasts. Images FIGURE 3 PMID:10703140

  13. Synthesis, development, characterization and effectiveness of bovine pure platelet gel-collagen-polydioxanone bioactive graft on tendon healing.

    PubMed

    Moshiri, Ali; Oryan, Ahmad; Meimandi-Parizi, Abdolhamid

    2015-06-01

    Bovine platelet gel (BPG) is an accessible and cost-effective source of growth factors which may have a value in tendon regenerative medicine. We produced a collagen implant (CI) as a tendon proper, covered it with polydioxanone (PDS) sheath to simulate paratenon and finally embedded the BPG as an active source of growth factor within the bioimplant to test whether BPG would be able to accelerate and enhance tendon regeneration and repair. After in vitro characterization of the bioactive grafts, the grafts were implanted in rabbit large tendon defect model. Untreated tendons and tendons treated with either CI or CI-PDS were served as controls for the CI-PDS-BPG. The animals were investigated clinically, ultrasonographically and haematologically for 120 days. After euthanasia, dry matter content, water uptake and delivery characteristics and also gross morphological, histopathological and scanning electron microscopic features of the healing tendons were assessed. In vitro, the activated platelets in the scaffold, released their growth factors significantly more than the controls. BPG also increased cell viability, and enhanced cellular differentiation, maturation and proliferation inside the CI-PDS compared with the controls. In vivo, the BPG modulated inflammation, increased quality and rate of fibroplasia and produced a remodelled tendon that had significantly higher collagen content and superior collagen fibril and fibre differentiation than controls. Treatment also significantly improved tendon water uptake and delivery characteristics, animals' serum PDGF level, CI-PDS biocompatibility and biodegradability and reduced peritendinous adhesions, muscle fibrosis and atrophy. BPG was effective on tendon healing and CI-PDS-BPG may be a valuable bioscaffold in tendon reconstructive surgery.

  14. Synthesis, development, characterization and effectiveness of bovine pure platelet gel-collagen-polydioxanone bioactive graft on tendon healing

    PubMed Central

    Moshiri, Ali; Oryan, Ahmad; Meimandi-Parizi, Abdolhamid

    2015-01-01

    Bovine platelet gel (BPG) is an accessible and cost-effective source of growth factors which may have a value in tendon regenerative medicine. We produced a collagen implant (CI) as a tendon proper, covered it with polydioxanone (PDS) sheath to simulate paratenon and finally embedded the BPG as an active source of growth factor within the bioimplant to test whether BPG would be able to accelerate and enhance tendon regeneration and repair. After in vitro characterization of the bioactive grafts, the grafts were implanted in rabbit large tendon defect model. Untreated tendons and tendons treated with either CI or CI-PDS were served as controls for the CI-PDS-BPG. The animals were investigated clinically, ultrasonographically and haematologically for 120 days. After euthanasia, dry matter content, water uptake and delivery characteristics and also gross morphological, histopathological and scanning electron microscopic features of the healing tendons were assessed. In vitro, the activated platelets in the scaffold, released their growth factors significantly more than the controls. BPG also increased cell viability, and enhanced cellular differentiation, maturation and proliferation inside the CI-PDS compared with the controls. In vivo, the BPG modulated inflammation, increased quality and rate of fibroplasia and produced a remodelled tendon that had significantly higher collagen content and superior collagen fibril and fibre differentiation than controls. Treatment also significantly improved tendon water uptake and delivery characteristics, animals’ serum PDGF level, CI-PDS biocompatibility and biodegradability and reduced peritendinous adhesions, muscle fibrosis and atrophy. BPG was effective on tendon healing and CI-PDS-BPG may be a valuable bioscaffold in tendon reconstructive surgery. PMID:25702535

  15. Green Tea and Glycine Modulate the Activity of Metalloproteinases and Collagen in the Tendinitis of the Myotendinous Junction of the Achilles Tendon.

    PubMed

    Vieira, Cristiano Pedrozo; De Oliveira, LetÍCia Prado; Da Ré Guerra, Flávia; Marcondes, Maria Cristina Cintra; Pimentel, Edson Rosa

    2016-07-01

    The myotendinous junction (MTJ) is the weakest element in the muscle-tendon unit of the heel, and thus the most susceptible to injuries. The scarcity of adequate treatments means that tendinitis is a major concern to athletes and other groups who depend on their physical fitness, although green tea and glycine have both been shown to have beneficial effects on the inflammation. The present study investigated the remodeling effects of green tea and glycine in the MTJ of rats with tendinitis. For this, male Wistar rats were divided into five groups: animals without tendinitis and animals with tendinitis; animals with tendinitis supplied with green tea; animals with tendinitis supplied with a glycine diet; animals with tendinitis supplied with a green tea and glycine diet. Tendinitis was induced and the treatment with green tea (700 mg/kg/day) and a 5% glycine diet lasted 7 days. The treatments regulated the activity of metalloproteinases (MMP)-2, -8, and -9, and induced the synthesis of type I collagen, glycosaminoglycans, and non-collagenous proteins. Changes were also noted in the compaction of the collagen molecules and the amount of tenocytes. When combined, green tea and glycine modulated the inflammatory process and induced the synthesis of the elements involved in the post-lesion recovery of the tissue. The data from the MTJ were different when compared with results already published using the whole Achilles tendon. These data indicate that each region of the inflamed tendon can exhibit different responses during the treatment and therefore, modify its extracellular matrix components to facilitate recovery and repair. Anat Rec, 299:918-928, 2016. © 2016 Wiley Periodicals, Inc.

  16. Oral Cnm-positive Streptococcus Mutans Expressing Collagen Binding Activity is a Risk Factor for Cerebral Microbleeds and Cognitive Impairment

    PubMed Central

    Watanabe, Isao; Kuriyama, Nagato; Miyatani, Fumitaro; Nomura, Ryota; Naka, Shuhei; Nakano, Kazuhiko; Ihara, Masafumi; Iwai, Komei; Matsui, Daisuke; Ozaki, Etsuko; Koyama, Teruhide; Nishigaki, Masaru; Yamamoto, Toshiro; Tamura, Aiko; Mizuno, Toshiki; Akazawa, Kentaro; Takada, Akihiro; Takeda, Kazuo; Yamada, Kei; Nakagawa, Masanori; Tanaka, Tokutaro; Kanamura, Narisato; Friedland, Robert P.; Watanabe, Yoshiyuki

    2016-01-01

    Cerebral microbleeds (CMBs) are an important risk factor for stroke and dementia. We have shown that the collagen binding surface Cnm protein expressed on cnm-positive Streptococcus mutans is involved in the development of CMBs. However, whether the collagen binding activity of cnm-positive S. mutans is related to the nature of the CMBs or to cognitive impairment is unclear. Two-hundred seventy nine community residents (70.0 years) were examined for the presence or absence of cnm-positive S. mutans in the saliva by PCR and collagen binding activity, CMBs, and cognitive function were evaluated. Cnm-positive S. mutans was detected more often among subjects with CMBs (p < 0.01) than those without. The risk of CMBs was significantly higher (odds ratio = 14.3) in the group with S. mutans expressing collagen binding activity, as compared to the group without that finding. Deep CMBs were more frequent (67%) and cognitive function was lower among subjects with cnm-positive S. mutans expressing collagen binding activity. This work supports the role of oral health in stroke and dementia and proposes a molecular mechanism for the interaction. PMID:27934941

  17. Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice.

    PubMed

    Liu, Susan B; Ikenaga, Naoki; Peng, Zhen-Wei; Sverdlov, Deanna Y; Greenstein, Andrew; Smith, Victoria; Schuppan, Detlef; Popov, Yury

    2016-04-01

    Collagen stabilization through irreversible cross-linking is thought to promote hepatic fibrosis progression and limit its reversibility. However, the mechanism of this process remains poorly defined. We studied the functional contribution of lysyl oxidase (LOX) to collagen stabilization and hepatic fibrosis progression/reversalin vivousing chronic administration of irreversible LOX inhibitor β-aminopropionitrile (BAPN, or vehicle as control) in C57Bl/6J mice with carbon tetrachloride (CCl4)-induced fibrosis. Fibrotic matrix stability was directly assessed using a stepwise collagen extraction assay and fibrotic septae morphometry. Liver cells and fibrosis were studied by histologic, biochemical methods and quantitative real-time reverse-transcription PCR. During fibrosis progression, BAPN administration suppressed accumulation of cross-linked collagens, and fibrotic septae showed widening and collagen fibrils splitting, reminiscent of remodeling signs observed during fibrosis reversal. LOX inhibition attenuated hepatic stellate cell activation markers and promoted F4/80-positive scar-associated macrophage infiltration without an increase in liver injury. In reversal experiments, BAPN-treated fibrotic mice demonstrated accelerated fibrosis reversal after CCl4withdrawal. Our findings demonstrate for the first time that LOX contributes significantly to collagen stabilization in liver fibrosis, promotes fibrogenic activation of attenuated hepatic stellate cells, and limits fibrosis reversal. Our data support the concept of pharmacologic targeting of LOX pathway to inhibit liver fibrosis and promote its resolution.-Liu, S. B., Ikenaga, N., Peng, Z.-W., Sverdlov, D. Y., Greenstein, A., Smith, V., Schuppan, D., Popov, Y. Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice.

  18. Effect of copper on levels of collagen and alkaline phosphatase activity from chondrocytes in newborn piglets in vitro.

    PubMed

    Yuan, Xue; Wang, Jianguo; Zhu, Xiaoyan; Zhang, Zhigang; Ai, Yongxing; Sun, Guoquan; Wang, Zhe; Liu, Guowen

    2011-12-01

    The effects of different concentrations of copper on collagen content and alkaline phosphatase (AKP) activity from chondrocytes in newborn piglets were measured. Chondrocytes were cultured in media containing 15% fetal calf serum supplemented with 0, 15.6, 31.2, and 62.5 μmol/L copper in a 12-well culture plate. Collagen content and AKP activity from the chondrocyte extracellular matrix increased significantly in the culture media with 15.6, 31.2, and 62.5 μmol/L copper and was the highest at 31.2 μmol/L copper (P < 0.05). Thus, the results indicated that copper could promote AKP activity and collagen production by chondrocytes.

  19. Glatiramer acetate inhibits degradation of collagen II by suppressing the activity of interferon regulatory factor-1.

    PubMed

    Lu, Huading; Zeng, Chun; Zhao, Huiqing; Lian, Liyi; Dai, Yuhu

    2014-06-06

    Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) is considered to be the major one contributing to the process of development of osteoarthritis (OA).Interferon regulatory factor 1 (IRF-1) is an important transcriptional factor accounting for inflammation response induced by TNF-α. The physiological function of IRF-1 in OA is still unknown. In this study, we reported that the expression levels of IRF-1 in OA chondrocytes were significantly higher compared to those in normal chondrocytes, which was reversed by treatment with Glatiramer acetate (GA), a licensed clinical drug for treating patients suffering from multiple sclerosis (MS). We also found that GA is able to attenuate the upregulation of IRF-1 induced by TNF-α. Matrix metalloproteinase13 (MMP-13) is one of the downstream target genes of IRF-1, which can induce the degradation of collagen II. Importantly, our results indicated that GA suppressed the expression of MMP-13 as well as the degradation of collagen II. In addition, GA also suppressed TNF-α-induced production of NO and expression of iNOS. Finally, we found that the inhibition of STAT1 activation played a critical role in the inhibitory effects of GA on the induction of IRF-1 and MMP-13. These data suggest that GA might have a potential effect in therapeutic OA.

  20. Microchimeric fetal cells are recruited to maternal kidney following injury and activate collagen type I transcription.

    PubMed

    Bou-Gharios, George; Amin, Farhana; Hill, Peter; Nakamura, Hiroyuki; Maxwell, Patrick; Fisk, Nicholas M

    2011-01-01

    Fetal cells enter the maternal circulation from the early first trimester of pregnancy, where they persist in tissue decades later. We investigated in mice whether fetal microchimeric cells (FMCs) can be detected in maternal kidney, and whether they play a role in kidney homeostasis. FMCs were identified in vivo in two models: one an adaptive model following unilateral nephrectomy, the other an injury via unilateral renal ischaemia reperfusion. Both models were carried out in mothers that had been mated with transgenic mice expressing luciferase transgene under the control of collagen type I, and had given birth to either 1 or 3 litters. FMCs were detected by Y-probe fluorescent in situ hybridization (FISH) and bioluminescence, and the cell number quantified by real-time polymerase chain reaction. In the adaptive model, the remaining kidney showed more cells by all 3 parameters compared with the nephrectomized kidney, while ischaemia reperfusion resulted in higher levels of FMC participation in injured compared to contralateral kidneys. Bioluminescence showed that FMCs switch on collagen type I transcription implicating mesenchymal lineage cells. After injury, Y-probe in situ hydridization was found mainly in the tubular epithelial network. Finally, we compared FMCs with bone marrow cells and found similar dynamics but altered distribution within the kidney. We conclude that FMCs (1) are long-term sequelae of pregnancy and (2) are recruited to the kidney as a result of injury or adaptation, where they activate the transcriptional machinery of matrix proteins.

  1. Sympathetic Neurotransmitters Modulate Osteoclastogenesis and Osteoclast Activity in the Context of Collagen-Induced Arthritis

    PubMed Central

    Muschter, Dominique; Schäfer, Nicole; Stangl, Hubert; Straub, Rainer H.; Grässel, Susanne

    2015-01-01

    Excessive synovial osteoclastogenesis is a hallmark of rheumatoid arthritis (RA). Concomitantly, local synovial changes comprise neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze if collagen-induced arthritis (CIA) alters bone marrow-derived macrophage (BMM) osteoclastogenesis and osteoclast activity, and how sympathetic neurotransmitters participate in this process. Therefore, BMMs from Dark Agouti rats at different CIA stages were differentiated into osteoclasts in vitro and osteoclast number, cathepsin K activity, matrix resorption and apoptosis were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA) vasoactive intestinal peptide (VIP) and assay-dependent, adenylyl cyclase activator NKH477. We observed modulation of neurotransmitter receptor mRNA expression in CIA osteoclasts without affecting protein level. CIA stage-dependently altered marker gene expression associated with osteoclast differentiation and activity without affecting osteoclast number or activity. Neurotransmitter stimulation modulated osteoclast differentiation, apoptosis and activity. VIP, NA and adenylyl cyclase activator NKH477 inhibited cathepsin K activity and osteoclastogenesis (NKH477, 10-6M NA) whereas ACh mostly acted pro-osteoclastogenic. We conclude that CIA alone does not affect metabolism of in vitro generated osteoclasts whereas stimulation with NA, VIP plus specific activation of adenylyl cyclase induced anti-resorptive effects probably mediated via cAMP signaling. Contrary, we suggest pro-osteoclastogenic and pro-resorptive properties of ACh mediated via muscarinic receptors. PMID:26431344

  2. ACE Inhibitory and Antioxidant Activities of Collagen Hydrolysates from the Ribbon Jellyfish (Chrysaora sp.)

    PubMed Central

    Latiff, Aishah Abd; Gan, Chee-Yuen; Abedin, Md. Zainul; Alias, Abd Karim

    2014-01-01

    Summary Collagen isolated from the ribbon jellyfish (Chrysaora sp.) was hydrolysed using three different proteases (i.e. trypsin, alcalase and Protamex) to obtain bioactive peptides. Angiotensin-I-converting enzyme (ACE) inhibitory activity and antioxidant activities (i.e. ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity) of the peptides were measured and compared, and the effect of the duration of hydrolysis on the bioactivity (ACE inhibitory and antioxidant activities) of peptides was also evaluated. FRAP activity was the highest in Protamex-induced (25–27 mM) and trypsin-induced hydrolysates (24–26 mM) at 7 and 9 h, respectively. Conversely, hydrolysates produced by trypsin for 1 and 3 h showed the highest DPPH radical scavenging activities (94 and 92%, respectively). Trypsin-induced hydrolysates (at 3 h) also showed the highest ACE inhibitory activity (89%). The peptide sequences with the highest activities were identified using tandem mass spectrometry, and the results show that the hydrolysates had a high content of hydrophobic amino acids as well as unique amino acid sequences, which likely contribute to their biological activities. PMID:27904323

  3. Parathyroid hormone blocks the stimulatory effect of insulin-like growth factor-I on collagen synthesis in cultured 21-day fetal rat calvariae

    SciTech Connect

    Kream, B.E.; Petersen, D.N.; Raisz, L.G. )

    1990-01-01

    We examined the interaction of parathyroid hormone (PTH) and recombinant human insulin-like growth factor I (IGF-I) on collagen synthesis in 21-day fetal rat calvariae as assessed by measuring the incorporation of ({sup 3}H)proline into collagenase-digestible protein. After 96 hours of culture, 10 nM PTH antagonized the stimulation of collagen synthesis and partially blocked the increase in dry weight produced by 10 nM IGF-I. The effect of PTH to block IGF-I stimulated collagen synthesis was observed in the central bone of calvariae and was mimicked by forskolin and phorbol 12-myristate 13-acetate, but not by 1,25-dihydroxyvitamin D3, transforming growth factor-alpha or dexamethasone. Our data are consistent with the concept that the direct effect of PTH is to inhibit basal CDP labeling and fully oppose IGF-I stimulated CDP labeling. The finding that this effect of PTH is mimicked by forskolin and PMA suggests that this block in IGF-I stimulation of CDP labeling involves both cAMP and protein kinase C mediated pathways.

  4. Collagen models as a probe in the decay of works of art: synthesis, conformation and immunological studies.

    PubMed

    Zevgiti, Stella; Sakarellos, Constantinos; Sakarellos-Daitsiotis, Maria; Ioakimoglou, Eleni; Panou-Pomonis, Eugenia

    2007-02-01

    Proteinaceous substances such as collagen, casein and albumin have been widely used as binding media in a variety of works of art. Damages of these 'sensitive' materials, mainly caused of the influence of the environment, are responsible for the overall decay of works of art, and their identification is essential to understand ancient technologies, determine the extent of deterioration and help in future restoration and preservation processes. The most commonly used techniques for the identification of proteinaceous binding media are staining techniques, chromatography, spectrometry and immunological methods, although for the latter no systematic studies have been carried out until now. Aiming at contributing to the development of a reliable and reproducible immunoassay for the evaluation of the collagen-based decay of works of art, sequential polypeptides (Pro-X-Gly)n where X represents amino acid residues Val, Lys, Glu and (Hyp-Val-Gly)n were prepared as models of collagen fragments derived from artificially and naturally aged animal collagens. Conformational studies of the polypeptides by CD revealed the occurrence of polyproline II-like structures comparable with those of collagen. Polypeptides and collagen I were administered to animals, and the induced antibodies were used for the immunochemical detection and differentiation of collagen and collagen fragments. The combined application of (i) anti-collagen antibodies, which strongly interact with native collagen, but poorly recognized by artificially aged collagen and (ii) anti-polypeptide antibodies, which do not associate with native collagen, but are strongly recognized by collagen fragments in naturally or artificially aged collagen, is a valuable tool in determining the extent of decay in works of art.

  5. Disturbed synthesis of type II collagen interferes with rate of bone formation and growth and increases bone resorption in transgenic mice.

    PubMed

    Nieminen, Jyrki; Sahlman, Janne; Hirvonen, Teemu; Lapveteläinen, Tuomo; Miettinen, Markku; Arnala, Ilkka; Malluche, Hartmut H; Helminen, Heikki J

    2008-03-01

    Transgenic mice carrying an internally deleted human type II collagen gene (COL2A1) were used to study bone growth and development. This mutation has previously been shown to disturb the development of collagen fibrils in articular cartilage, causing chondrodysplasia and osteoarthritis. Type II collagen expression in bones was investigated with immunohistochemistry. The development and mineralization of the skeleton and anthropometric measurements on bones were evaluated using X-rays and dynamic histomorphometry. Type II collagen was expressed in the cartilage of developing bones. The bones of transgenic mice were smaller compared with the controls. The bone mass remained almost unchanged in transgenic mice after 1 month of age, leading to differences of 47% in trabecular bone volume (P = 0.012) and 40% in trabecular thickness (P < 0.01) at the age of 3 months compared with controls. At the age of 3 months the eroded surface per bone volume was 31% greater in transgenic mice compared with controls (P < 0.05). Trabecular thickness correlated positively with body weight (R = 0.71, P < 0.001). Interestingly, body weight correlated with bone volume in control mice (R = 0.27, P < 0.01), but no correlation was observed in transgenic mice. The disturbed synthesis of cartilage-specific type II collagen in growing transgenic mice retarded bone development, increased bone resorption, and altered tissue properties. This led to a negative net bone balance and small bone size. The results support the idea that an altered synthesis of cartilage-specific molecule(s) can disturb postnatal bone development and growth.

  6. On the correlation between continuum mechanics entities and cell activity in biological soft tissues: assessment of three possible criteria for cell-controlled fibre reorientation in collagen gels and collagenous tissues.

    PubMed

    Kroon, Martin

    2010-05-07

    The biomechanical behaviour of biological cells is of great importance in many physiological processes. One such process is the maintenance of fibrous networks, such as collagenous tissues. The activity of the fibre-producing cells in this type of tissue is very important, and a comprehensive material description needs to incorporate the activity of the cells. In biomechanics, continuum mechanics is often employed to describe deforming solids, and modelling can be much simplified if continuum mechanics entities, such as stress and strain, can be correlated with cell activity. To investigate this, a continuum mechanics framework is employed in which remodelling of a collagen gel is modelled. The remodelling is accomplished by fibroblasts, and the activity of the fibroblasts is linked to the continuum mechanics theory. The constitutive model for the collagen fabric is formulated in terms of a strain energy function, which includes a density function describing the distribution of the collagen fibre orientation. This density function evolves according to an evolution law, where fibroblasts reorient fibres towards the direction of increasing Cauchy stress, elastic deformation, or stiffness. The theoretical framework is applied to experimental results from collagen gels, where gels have undergone remodelling under both biaxial and uniaxial constraint. The analyses indicated that criteria 1 and 2 (Cauchy stress and elastic deformations) are able to predict the collagen fibre distribution after remodelling, whereas criterion 3 (current stiffness) is not. This conclusion is, however, tentative and pertains, strictly speaking, only to fibre remodelling processes, and may not be valid for other types of cell activities.

  7. Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells*

    PubMed Central

    Herchenhan, Andreas; Uhlenbrock, Franziska; Eliasson, Pernilla; Weis, MaryAnn; Eyre, David; Kadler, Karl E.; Magnusson, S. Peter; Kjaer, Michael

    2015-01-01

    Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation. PMID:25979340

  8. Low-Level Light Therapy with 410 nm Light Emitting Diode Suppresses Collagen Synthesis in Human Keloid Fibroblasts: An In Vitro Study

    PubMed Central

    Lee, Hyun Soo; Jung, Soo-Eun; Kim, Sue Kyung; Kim, You-Sun; Sohn, Seonghyang

    2017-01-01

    Background Keloids are characterized by excessive collagen deposition in the dermis, in which transforming growth factor β (TGF-β)/Smad signaling plays an important role. Low-level light therapy (LLLT) is reported as effective in preventing keloids in clinical reports, recently. To date, studies investigating the effect of LLLT on keloid fibroblasts are extremely rare. Objective We investigated the effect of LLLT with blue (410 nm), red (630 nm), and infrared (830 nm) light on the collagen synthesis in keloid fibroblasts. Methods Keloid fibroblasts were isolated from keloid-revision surgery samples and irradiated using 410-, 630-, 830-nm light emitting diode twice, with a 24-hour interval at 10 J/cm2. After irradiation, cells were incubated for 24 and 48 hours and real-time quantitative reverse transcription polymerase chain reaction was performed. Western blot analysis was also performed in 48 hours after last irradiation. The genes and proteins of collagen type I, TGF-β1, Smad3, and Smad7 were analyzed. Results We observed no statistically significant change in the viability of keloid fibroblasts after irradiation. Collagen type I was the only gene whose expression significantly decreased after irradiation at 410 nm when compared to the non-irradiated control. Western blot analysis showed that LLLT at 410 nm lowered the protein levels of collagen type I compared to the control. Conclusion LLLT at 410 nm decreased the expression of collagen type I in keloid fibroblasts and might be effective in preventing keloid formation in their initial stage. PMID:28392641

  9. Stepwise proteolytic activation of type I procollagen to collagen within the secretory pathway of tendon fibroblasts in situ.

    PubMed

    Canty-Laird, Elizabeth G; Lu, Yinhui; Kadler, Karl E

    2012-01-15

    Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo. It was thought that intracellular processing was restricted to the TGN (trans-Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)-Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late embryonic and postnatal rat tail and postnatal mouse tail tendon, C-proteinase activity was detected in TGN and pre-TGN compartments. Preventing activation of the procollagen N- and C-proteinases with the furin inhibitor Dec-RVKR-CMK (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) indicated that only a fraction of intracellular procollagen cleavage was mediated by newly activated proteinases. In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell-matrix interface.

  10. Activated Hepatic Stellate Cells Are Dependent on Self-collagen, Cleaved by Membrane Type 1 Matrix Metalloproteinase for Their Growth

    PubMed Central

    Birukawa, Naoko Kubo; Murase, Kazuyuki; Sato, Yasushi; Kosaka, Akemi; Yoneda, Akihiro; Nishita, Hiroki; Fujita, Ryosuke; Nishimura, Miyuki; Ninomiya, Takafumi; Kajiwara, Keiko; Miyazaki, Miyono; Nakashima, Yusuke; Ota, Sigenori; Murakami, Yuya; Tanaka, Yasunobu; Minomi, Kenjiro; Tamura, Yasuaki; Niitsu, Yoshiro

    2014-01-01

    Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVβ1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVβ1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies. PMID:24867951

  11. Activated hepatic stellate cells are dependent on self-collagen, cleaved by membrane type 1 matrix metalloproteinase for their growth.

    PubMed

    Birukawa, Naoko Kubo; Murase, Kazuyuki; Sato, Yasushi; Kosaka, Akemi; Yoneda, Akihiro; Nishita, Hiroki; Fujita, Ryosuke; Nishimura, Miyuki; Ninomiya, Takafumi; Kajiwara, Keiko; Miyazaki, Miyono; Nakashima, Yusuke; Ota, Sigenori; Murakami, Yuya; Tanaka, Yasunobu; Minomi, Kenjiro; Tamura, Yasuaki; Niitsu, Yoshiro

    2014-07-18

    Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVβ1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVβ1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies.

  12. Distinct determinants on collagen support alpha 2 beta 1 integrin-mediated platelet adhesion and platelet activation.

    PubMed Central

    Santoro, S A; Walsh, J J; Staatz, W D; Baranski, K J

    1991-01-01

    Recent studies have revealed that the sequence of amino acids asp-gly-glu-ala represents an essential determinant of the site within the alpha 1(I)-CB3 fragment of collagen recognized by the alpha 2 beta 1 integrin cell surface collagen receptor (Staatz et al., 1991). Studies employing chemical modifications of collagen amino acid side chains confirm both the essential nature of the acidic side chains of aspartic acid and glutamic acid residues and the nonessentiality of lysine epsilon-amino groups in supporting adhesion mediated by the alpha 2 beta 1 integrin. The approach also indicates the presence of a distinct determinant on collagen separate from the alpha 2 beta 1 recognition site that contains essential lysine side chains and that is necessary for subsequent interactions with the platelet surface that give rise to collagen-induced platelet activation and secretion. The two-step, two-site model for cellular signaling involving both an integrin and a signal-transducing coreceptor suggested by these data may be common to other integrin-mediated processes. PMID:1809397

  13. Electrodeposition of platinum nanoclusters on type I collagen modified electrode and its electrocatalytic activity for methanol oxidation

    NASA Astrophysics Data System (ADS)

    Sun, Yujing; Sun, Lanlan; Xu, Fugang; Guo, Cunlan; Liu, Zhelin; Zhang, Yue; Yang, Tao; Li, Zhuang

    2009-05-01

    We firstly reported a novel polymer matrix fabricated by type I collagen and polymers, and this matrix can be used as nanoreactors for electrodepositing platinum nanoclusters (PNCs). The type I collagen film has a significant effect on the growth of PNCs. The size of the platinum nanoparticles could be readily tuned by adjusting deposition time, potential and the concentration of electrolyte, which have been verified by field-emitted scanning electron microscopy (FE-SEM). Furthermore, cyclic voltammetry (CV) has demonstrated that the as-prepared PNCs can catalyze methanol directly with higher activity than that prepared on PSS/PDDA film, and with better tolerance to poisoning than the commercial E-TEK catalyst. The collagen-polymer matrix can be used as a general reactor to electrodeposit other metal nanostructures.

  14. FKBP65-dependent peptidyl-prolyl isomerase activity potentiates the lysyl hydroxylase 2-driven collagen cross-link switch

    PubMed Central

    Chen, Yulong; Terajima, Masahiko; Banerjee, Priyam; Guo, Houfu; Liu, Xin; Yu, Jiang; Yamauchi, Mitsuo; Kurie, Jonathan M.

    2017-01-01

    Bruck Syndrome is a connective tissue disease associated with inactivating mutations in lysyl hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10-null and –wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde–derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities. PMID:28378777

  15. Pepsin-solubilised collagen (PSC) from Red Sea cucumber (Stichopus japonicus) regulates cell cycle and the fibronectin synthesis in HaCaT cell migration.

    PubMed

    Park, Soo-Yeong; Lim, Hee Kyoung; Lee, Seogjae; Hwang, Hyeong Cheol; Cho, Somi K; Cho, Moonjae

    2012-05-01

    Pepsin-solubilised collagen (PSC) from Red Sea cucumber (Stichopus japonicus) was studied with respect to its wound-healing effects on a human keratinocyte (HaCaT) cell line. Disaggregated collagen fibres were treated with 0.1M NaOH for 24h and digested with pepsin for 72h to reach maximum yield of 26.6%. The results of an in vitro wound-healing test showed that migration of HaCaT cells was 1.5-fold faster on PSC-coated plates than on untreated plates. The migration rate of sea cucumber PSC was similar to that of rat PSC, but five times higher than that of bovine gelatin. HaCaT cells grown on PSC-coated plates revealed increased fibronectin synthesis (6-fold and 3-fold compared to gelatin and rat PSC, respectively). Additionally, sea cucumber PSCs induced HaCaT cell proliferation by decreasing the G1 phase by 5% and maintaining a larger population (8%) of cells in mitosis. Collagen from Red Sea cucumber might be useful as an alternative to mammalian collagen in the nutraceutical and pharmaceutical industries.

  16. Decreased collagen-induced arthritis severity and adaptive immunity in mitogen activated protein kinase kinase 6 -deficient mice

    PubMed Central

    Hammaker, Deepa; Topolewski, Katharyn; Edgar, Meghan; Yoshizawa, Toshio; Fukushima, Akihisa; Boyle, David L.; Firestein, Gary S.

    2011-01-01

    Objective MAPK kinases MKK3 and MKK6 regulate p38 MAPK activation in inflammatory diseases such as rheumatoid arthritis. Previous studies demonstrated that MKK3- or MKK6-deficiency inhibits K/BxN serum-induced arthritis. However, the role of these kinases in adaptive immunity-dependent models of chronic arthritis is not known. The goal of this study was to evaluate MKK3- and MKK6-deficiency in the collagen induced arthritis model. Methods Wildtype, MKK3−/−, and MKK6−/− mice were immunized with bovine type II collagen (CII). Disease activity was evaluated by semiquantitative scoring, histology, and microcomputed tomography. Serum anti-collagen antibody levels were quantified by ELISA. In-vitro T cell cytokine response was measured by flow cytometry and multiplex analysis. Expression of joint cytokines and matrix metalloproteinase was determined by qPCR. Results MKK6-deficiency markedly reduced arthritis severity compared with WT mice, while absence of MKK3 had an intermediate effect. Joint damage was minimal in arthritic MKK6−/− mice and intermediate in MKK3−/− mice compared with wild type mice. MKK6−/− mice had modestly lower levels of pathogenic anti-collagen antibodies than WT or MKK3−/− mice. In vitro T cell assays showed reduced proliferation and IL-17 production by MKK6−/− cells in response to type II collagen. Gene expression of synovial IL-6, matrix metalloproteinases MMP3, and MMP13 was significantly inhibited in MKK6-deficient mice. Conclusion Reduced disease severity in MKK6−/− mice correlated with decreased anti-collagen responses indicating that MKK6 is a crucial regulator of inflammation joint destruction in CIA. MKK6 is a potential therapeutic target in complex diseases involving adaptive immune responses like rheumatoid arthritis. PMID:21953132

  17. Expression of catalytically active Matrix Metalloproteinase-1 in dermal fibroblasts induces collagen fragmentation and functional alterations that resemble aged human skin

    PubMed Central

    Xia, Wei; Hammerberg, Craig; Li, Yong; He, Tianyuan; Quan, Taihao; Voorhees, John J; Fisher, Gary J

    2013-01-01

    Summary Increased expression of matrix metalloproteinase-1 (MMP-1) and reduced production of type I collagen by dermal fibroblasts are prominent features of aged human skin. We have proposed that MMP-1-mediated collagen fibril fragmentation is a key driver of age-related decline of skin function. To investigate this hypothesis, we constructed, characterized, and expressed constitutively active MMP-1 mutant (MMP-1 V94G) in adult human skin in organ culture and fibroblasts in three dimensional collagen lattice cultures. Expression of MMP-1 V94G in young skin in organ culture caused fragmentation and ultrastructural alterations of collagen fibrils similar to those observed in aged human skin in vivo. Expression of MMP-1 V94G in dermal fibroblasts cultured in three-dimensional collagen lattices caused substantial collagen fragmentation, which was markedly reduced by MMP-1 siRNA-mediated knockdown or MMP inhibitor MMI270. Importantly, fibroblasts cultured in MMP-1 V94G-fragmented collagen lattices displayed many alterations observed in fibroblasts in aged human skin, including reduced cytoplasmic area, disassembled actin cytoskeleton, impaired TGF-β pathway, and reduced collagen production. These results support the concept that MMP-1-mediated fragmentation of dermal collagen fibrils alters the morphology and function of dermal fibroblasts, and provide a foundation for understanding specific mechanisms that link collagen fibril fragmentation to age-related decline of fibroblast function. PMID:23601157

  18. Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1).

    PubMed

    Jung, S M; Moroi, M

    2000-03-17

    Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.

  19. The Role of Collagen Charge Clusters in the Modulation of Matrix Metalloproteinase Activity*

    PubMed Central

    Lauer, Janelle L.; Bhowmick, Manishabrata; Tokmina-Roszyk, Dorota; Lin, Yan; Van Doren, Steven R.; Fields, Gregg B.

    2014-01-01

    Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-l-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, kcat/Km values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P23–P23′ subsites of collagenous substrates. PMID:24297171

  20. The role of collagen charge clusters in the modulation of matrix metalloproteinase activity.

    PubMed

    Lauer, Janelle L; Bhowmick, Manishabrata; Tokmina-Roszyk, Dorota; Lin, Yan; Van Doren, Steven R; Fields, Gregg B

    2014-01-24

    Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-L-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, kcat/Km values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P23-P23' subsites of collagenous substrates.

  1. Drosophila type IV collagen mutation associates with immune system activation and intestinal dysfunction.

    PubMed

    Kiss, Márton; Kiss, András A; Radics, Monika; Popovics, Nikoletta; Hermesz, Edit; Csiszár, Katalin; Mink, Mátyás

    2016-01-01

    The basal lamina (BM) contains numerous components with a predominance of type IV collagens. Clinical manifestations associated with mutations of the human COL4A1 gene include perinatal cerebral hemorrhage and porencephaly, hereditary angiopathy, nephropathy, aneurysms and muscle cramps (HANAC), ocular dysgenesis, myopathy, Walker–Warburg syndrome and systemic tissue degeneration. In Drosophila, the phenotype associated with dominant temperature sensitive mutations of col4a1 include severe myopathy resulting from massive degradation of striated muscle fibers, and in the gut, degeneration of circular visceral muscle cells and epithelial cells following detachment from the BM. In order to determine the consequences of altered BMfunctions due to aberrant COL4A1 protein, we have carried out a series of tests using Drosophila DTS-L3 mutants from our allelic series of col4a1 mutations with confirmed degeneration of various cell types and lowest survival rate among the col4a1 mutant lines at restrictive temperature. Results demonstrated epithelial cell degeneration in the gut, shortened gut, enlarged midgut with multiple diverticulae, intestinal dysfunction and shortened life span. Midgut immunohistochemistry analyses confirmed altered expression and distribution of BM components integrin PSI and PSII alpha subunits, laminin gamma 1, and COL4A1 both in larvae and adults. Global gene expression analysis revealed activation of the effector AMP genes of the primary innate immune system including Metchnikowin, Diptericin, Diptericin B, and edin that preceded morphological changes. Attacin::GFP midgut expression pattern further supported these changes. An increase in ROS production and changes in gut bacterial flora were also noted and may have further enhanced an immune response. The phenotypic features of Drosophila col4a1 mutants confirmed an essential role for type IV collagen in maintaining epithelial integrity, gut morphology and intestinal function and suggest that

  2. Modulation of collagen metabolism by the nucleolar protein fibrillarin.

    PubMed

    Lefèvre, F; Garnotel, R; Georges, N; Gillery, P

    2001-11-15

    Metabolic functions of fibroblasts are tightly regulated by the extracellular environment. When cultivated in tridimensional collagen lattices, fibroblasts exhibit a lowered activity of protein synthesis, especially concerning extracellular matrix proteins. We have previously shown that extracellular collagen impaired the processing of ribosomal RNA (rRNA) in nucleoli by generating changes in the expression of nucleolar proteins and a premature degradation of neosynthesized rRNA. In this study, we have investigated whether inhibiting the synthesis of fibrillarin, a major nucleolar protein with decreased expression in collagen lattices, could mimic the effects of extracellular matrix. Monolayer-cultured fibroblasts were transfected with anti-fibrillarin antisense oligodeoxynucleotides, which significantly decreased fibrillarin content. Downregulation of fibrillarin expression inhibited procollagen secretion into the extracellular medium, without altering total collagen production. No changes of pro1(I)collagen mRNA expression or proline hydroxylation were found. A concomitant intracellular retention of collagen and its chaperone protein HSP47 was found, but no effect on the production of other extracellular matrix macromolecules or remodelling enzymes was observed. These data show that collagen processing depends on unknown mechanisms, involving proteins primarily located in the nucleolar compartment with other demonstrated functions, and suggest specific links between nucleolar machinery and extracellular matrix.

  3. Paroxetine alleviates T lymphocyte activation and infiltration to joints of collagen-induced arthritis

    PubMed Central

    Wang, Qingtong; Wang, Longsheng; Wu, Li; Zhang, Mei; Hu, Shanshan; Wang, Rui; Han, Yongsheng; Wu, Yujing; Zhang, Lingling; Wang, Xinming; Sun, Wuyi; Wei, Wei

    2017-01-01

    T cell infiltration to synovial tissue is an early pathogenic mechanism of rheumatoid arthritis (RA). In the present work, we reveal that G protein coupled receptor kinase 2 (GRK2) is abundantly expressed in T cells of collagen-induced arthritis (CIA). A GRK2 inhibitor, paroxetine protects the joints from inflammation and destruction, primarily through inhibition of both CD4+ helper T (Th) cell and CD8+ cytotoxic T (Tc) cell migration to synovial tissue. Meanwhile, paroxetine restores the balance of Th/Tc, effector Th (Theff)/ naïve Th (Thnaive) and effector Tc (Tceff)/ naïve Tc (Tcnaive) to equilibrium by elevating the frequency of Thnaive, Tcnaive and regulatory Th cells; reducing the increased Theff, activated Th and Tceff, having a similar effect as methotrexate (MTX). In addition, both serum and synovial IL-1β, TNF-α and CX3CL1 expression was effectively inhibited in treated rats. In vitro assay confirmed that paroxetine inhibits CX3CL1-induced T cell migration through blocking the activity of GRK2. Among three MAPK families, paroxetine was found to be able to decrease the phosphorylation of ERK. This study elucidates that paroxetine attenuates the symptoms of CIA rats due to its inhibitory effect on T cell activation and infiltration to synovial tissue via suppression of ERK pathway. PMID:28349925

  4. Yeast hydrolysate protects cartilage via stimulation of type II collagen synthesis and suppression of MMP-13 production.

    PubMed

    Lee, Hyun-Sun; Park, So Yeon; Park, Yooheon; Bae, Song Hwan; Suh, Hyung Joo

    2013-09-01

    Type II collagen (COL II) is one of the primary components of hyaline cartilage and plays a key role in maintaining chondrocyte function. COL II is the principal target of destruction, and matrix metalloproteases (MMPs) have a major role in arthritis. In the present study, we investigated the chondroctye protection effects of specific fraction of yeast hydrolysate ((10-30 kDa molecular weight peptides). The mRNA expression of COL II was significantly increased in the YH-treated group compared to the control at concentrations above 50 µg/ml, respectively. The 200 µg/ml YH-treated group (3.43 ± 0.23 µg/ml) showed significantly reduced glycosaminoglycan (GAG) degradation relative to that in the interleukin-1β (IL-1β)-treated control group (4.72 ± 0.05 µg/ml). In the YH-treated group, MMP-13 level was significantly decreased in a dose-dependent manner compared to the IL-1β-treated group without YH treatment. However, MMP-1 and MMP-3 level were not different from that of control. Under the same conditions, we also examined mRNA levels of COL II. The mRNA expression of COL II was significantly higher in the YH-treated group than in the IL-1β-treated control group at concentrations above 100 µg/ml. In conclusion, YH stimulated COL II synthesis and significantly inhibited MMP-13 and GAG degradation caused by IL-1β treatment.

  5. Collagen and gelatin.

    PubMed

    Liu, Dasong; Nikoo, Mehdi; Boran, Gökhan; Zhou, Peng; Regenstein, Joe M

    2015-01-01

    Collagen and gelatin have been widely used in the food, pharmaceutical, and cosmetic industries due to their excellent biocompatibility, easy biodegradability, and weak antigenicity. Fish collagen and gelatin are of renewed interest, owing to the safety and religious concerns of their mammalian counterparts. The structure of collagen has been studied using various modern technologies, and interpretation of the raw data should be done with caution. The structure of collagen may vary with sources and seasons, which may affect its applications and optimal extraction conditions. Numerous studies have investigated the bioactivities and biological effects of collagen, gelatin, and their hydrolysis peptides, using both in vitro and in vivo assay models. In addition to their established nutritional value as a protein source, collagen and collagen-derived products may exert various potential biological activities on cells in the extracellular matrix through the corresponding food-derived peptides after ingestion, and this might justify their applications in dietary supplements and pharmaceutical preparations. Moreover, an increasing number of novel applications have been found for collagen and gelatin. Therefore, this review covers the current understanding of the structure, bioactivities, and biological effects of collagen, gelatin, and gelatin hydrolysates as well as their most recent applications.

  6. Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

    SciTech Connect

    Walker, G.; Bourguignon, L.Y. )

    1990-08-01

    Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

  7. Characterization and antioxidant activities of marine pepsin soluble collagen from the skin of yellow goosefish Lophius litulon

    NASA Astrophysics Data System (ADS)

    Zheng, Bin; Xiang, Xingwei; Zhou, Yufang; Yang, Huicheng; Luo, Hongyu; Liao, Miaofei; Wen, Zhengshun

    2016-06-01

    Characteristics and antioxidant activities of pepsin-soluble collagen (PSC) from yellow goosefish (Lophius litulon) skins were investigated. PSC was characterized as a type I collagen, and its imino acid content was 193 residues/1 000 residues. PSC's denaturation temperature was ~17.56°C and Fourier transform infrared spectra confirmed the presence of triple helices. Solubility analysis showed good solubility at acidic pH (1-6) or low NaCl concentrations (≤2%). PSC showed scavenging activity against hydroxyl radicals and superoxide anions in a concentration-dependent manner. Furthermore, PSC could protect D-galactose-induced skin aging by significantly controlling malondialdehyde formation and improving the activity of superoxide dismutase, glutathione peroxidase, catalase, glutathione, and hydroxyproline. PSC may be a promising antioxidant in appropriate applications.

  8. Construction of multifunctional proteins for tissue engineering: epidermal growth factor with collagen binding and cell adhesive activities.

    PubMed

    Hannachi Imen, Elloumi; Nakamura, Makiko; Mie, Masayasu; Kobatake, Eiry

    2009-01-01

    The development of different techniques based on natural and polymeric scaffolds are useful for the design of different biomimetic materials. These approaches, however, require supplementary steps for the chemical or physical modification of the biomaterial. To avoid such steps, in the present study, we constructed a new multifunctional protein that can be easily immobilized onto hydrophobic surfaces, and at the same time helps enhance specific cell adhesion and proliferation onto collagen substrates. A collagen binding domain was fused to a previously constructed protein, which had an epidermal growth factor fused to a hydrophobic peptide that allows for cell adhesion. The new fusion protein, designated fnCBD-ERE-EGF is produced in Escherichia coli, and its abilities to bind to collagen and promote cell proliferation were investigated. fnCBD-ERE-EGF was shown to keep both collagen binding and cell growth-promoting activities comparable to those of the corresponding unfused proteins. The results obtained in this study also suggest the use of a fnCBD-ERE-EGF as an alternative for the design of multifunctional ECM-bound growth factor based materials.

  9. Antifreeze and cryoprotective activities of ice-binding collagen peptides from pig skin.

    PubMed

    Cao, Hui; Zhao, Ying; Zhu, Yu Bing; Xu, Fei; Yu, Jing Song; Yuan, Min

    2016-03-01

    A novel "hyperactive" ice-binding peptide from porcine collagen was prepared by alkaline protease hydrolysis and a series of column chromatography separations, and then its antifreeze and cryoprotective properties were reported. Using differential scanning calorimetry (DSC), the thermal hysteresis (TH) of ice-binding collagen peptides was closely related to their concentration and crystal fraction. Collagen hydrolysates with maximal TH were obtained by hydrolysis at pH 8.0, DH 15.0%, and 5% alkaline protease at 55°C. After purification by column chromatography, the AP-3 ice-binding collagen peptide (GLLGPLGPRGLL) with 1162.8Da molecular weights exhibited the highest TH (5.28°C), which can be classified as "hyperactive". Recrystallisation and melt-resistance of ice cream were improved by AP-3 ice-binding collagen peptide at 0.2% (w/v) in a similar manner to natural antifreeze proteins. Moreover, the addition of AP-3 collagen peptides in ice cream greatly elevated the glass transition temperature (Tg) to -17.64°C.

  10. Upregulation of Bone Morphogenetic Protein-2 Synthesis and Consequent Collagen II Expression in Leptin-stimulated Human Chondrocytes.

    PubMed

    Chang, Shun-Fu; Hsieh, Rong-Ze; Huang, Kuo-Chin; Chang, Cheng Allen; Chiu, Fang-Yao; Kuo, Hsing-Chun; Chen, Cheng-Nan; Su, Yu-Ping

    2015-01-01

    Bone morphogenetic proteins (BMPs) play positive roles in cartilage development, but they can barely be detected in healthy articular cartilage. However, recent evidence has indicated that BMPs could be detected in osteoarthritic and damaged cartilage and their precise roles have not been well defined. Extremely high amounts of leptin have been reported in obese individuals, which can be associated with osteoarthritis (OA) development. The aim of this study was to investigate whether BMPs could be induced in human primary chondrocytes during leptin-stimulated OA development and the underlying mechanism. We found that expression of BMP-2 mRNA, but not BMP-4, BMP-6, or BMP-7 mRNA, could be increased in human primary chondrocytes under leptin stimulation. Moreover, this BMP-2 induction was mediated through transcription factor-signal transducer and activator of transcription (STAT) 3 activation via JAK2-ERK1/2-induced Ser727-phosphorylation. Of note, histone deacetylases (HDACs) 3 and 4 were both involved in modulating leptin-induced BMP-2 mRNA expression through different pathways: HDAC3, but not HDAC4, associated with STAT3 to form a complex. Our results further demonstrated that the role of BMP-2 induction under leptin stimulation is to increase collagen II expression. The findings in this study provide new insights into the regulatory mechanism of BMP-2 induction in leptin-stimulated chondrocytes and suggest that BMP-2 may play a reparative role in regulating leptin-induced OA development.

  11. Transcript-activated collagen matrix as sustained mRNA delivery system for bone regeneration.

    PubMed

    Badieyan, Zohreh Sadat; Berezhanskyy, Taras; Utzinger, Maximilian; Aneja, Manish Kumar; Emrich, Daniela; Erben, Reinhold; Schüler, Christiane; Altpeter, Philipp; Ferizi, Mehrije; Hasenpusch, Günther; Rudolph, Carsten; Plank, Christian

    2016-10-10

    Transcript therapies using chemically modified messenger RNAs (cmRNAs) are emerging as safe and promising alternatives for gene and recombinant protein therapies. However, their applications have been limited due to transient translation and relatively low stability of cmRNAs compared to DNA. Here we show that vacuum-dried cmRNA-loaded collagen sponges, termed transcript activated matrices (TAMs), can serve as depots for sustained delivery of cmRNA. TAMs provide steady state protein production for up to six days, and substantial residual expression until 11days post transfection. Another advantage of this technology was nearly 100% transfection efficiency as well as low toxicity in vitro. TAMs were stable for at least 6months at room temperature. Human BMP-2-encoding TAMs induced osteogenic differentiation of MC3T3-E1 cells in vitro and bone regeneration in a non-critical rat femoral bone defect model in vivo. In summary, TAMs are a promising tool for bone regeneration and potentially also for other applications in regenerative medicine and tissue engineering.

  12. Activity of physalin F in a collagen-induced arthritis model.

    PubMed

    Brustolim, Daniele; Vasconcelos, Juliana F; Freitas, Luiz Antônio R; Teixeira, Mauro M; Farias, Marcel T; Ribeiro, Yvone M; Tomassini, Therezinha C B; Oliveira, Geraldo G S; Pontes-de-Carvalho, Lain C; Ribeiro-dos-Santos, Ricardo; Soares, Milena B P

    2010-08-27

    The effects of physalin F (1), a steroid derivative purified from Physalis angulata, were investigated in models of collagen-induced arthritis in DBA/1 mice and allergic airway inflammation in BALB/c mice. Oral treatment with 1 or dexamethasone caused a marked decrease in paw edema and joint inflammation when compared to vehicle-treated arthritic mice. In contrast, treatment with 1 had no effect in mice with allergic airway inflammation caused by ovalbumin immunization, whereas dexamethasone significantly reduced the number of inflammatory cells and eosinophils in the broncoalveolar lavage fluid and in lung sections of challenged mice. To further demonstrate that 1 acts through a mechanism different from that of glucocorticoids, a nuclear translocation assay was performed of the glucocorticoid receptor (GR) using COS-7 cells transfected with a plasmid encoding for a yellow fluorescent protein (YFP)-GR fusion protein. Untreated or treated cells with 1 had YFP staining mainly in the cytoplasm, whereas in dexamethasone-treated cells the YFP staining was concentrated in the nuclei. It is concluded that the mechanism of the immunosuppressive activity of physalin F is distinct from that of the glucocorticoids.

  13. Preparation of antibodies to chick-embryo galactosylhydroxylysyl glucosyltransferase and their use for an immunological characterization of the enzyme of collagen synthesis.

    PubMed

    Myllylä, R

    1981-04-14

    Antibodies were prepared against chick-embryo galactosylhydroxylysyl glucosyltransferase and further purified by immunoaffinity chromatography. The antibodies gave a single precipitation line of identity by double immunodiffusion against crude or pure chick-embryo glucosyltransferase. The ability of the antibody to precipitate the transferase was not altered by destroying the secondary structure of the enzyme. The antibody also inhibited the enzyme activity. The degree of inhibition was higher with denatured citrate-soluble rat skin collagen as the substrate than with gelatinized rat skin insoluble collagen or free galactosylhydroxylysine. The cross-reactivity of the glucosyltransferase between different species was low when studied by double immunodiffusion or inhibition kinetics. The antiserum showed no detectable cross-reactivity against other intracellular enzymes of collagen biosynthesis. A line of complete identity was found in double immunodiffusion between the transferases from whole chick embryos and chick embryo tendon, kidney and cartilage. Inhibition by the antiserum of the enzyme from chick embryo tissues synthesizing different collagen types was relatively similar. The data do not support the hypothesis that galactosylhydroxylysyl glucosyltransferase has isoenzymes with markedly different specific activities or immunological properties.

  14. Effect of surfactants on surface activity and rheological properties of type I collagen at air/water interface.

    PubMed

    Kezwoń, Aleksandra; Góral, Ilona; Frączyk, Tomasz; Wojciechowski, Kamil

    2016-12-01

    We describe the effect of three synthetic surfactants (anionic - sodium dodecyl sulfate (SDS), cationic - cetyltrimethylammonium bromide (CTAB) and nonionic - Triton X-100 (TX-100)) on surface properties of the type I calf skin collagen at the air/water interface in acidic solutions (pH 1.8). The protein concentration was fixed at 5×10(-6)molL(-1) and the surfactant concentration was varied in the range 5×10(-6)molL(-1)-1×10(-4)molL(-1), producing the protein/surfactant mixtures with molar ratios of 1:1, 1:2, 1:5, 1:10 and 1:20. An Axisymmetric Drop Shape Analysis (ADSA) method was used to determine the dynamic surface tension and surface dilatational moduli of the mixed adsorption layers. Two spectroscopic techniques: UV-vis spectroscopy and fluorimetry allowed us to determine the effect of the surfactants on the protein structure. The thermodynamic characteristic of the mixtures was studied using isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Modification of the collagen structure by SDS at low surfactant/protein ratios has a positive effect on the mixture's surface activity with only minor deterioration of the rheological properties of the adsorbed layers. The collagen/CTAB mixtures do not show that pronounced improvement in surface activity, while rheological properties are significantly deteriorated. The mixtures with non-ionic TX-100 do not show any synergistic effects in surface activity.

  15. Sequential extracts of human bone show differing collagen synthetic rates.

    PubMed

    Babraj, J; Cuthbertson, D J; Rickhuss, P; Meier-Augenstein, W; Smith, K; Bohé, J; Wolfe, R R; Gibson, J N A; Adams, C; Rennie, M J

    2002-04-01

    Type I collagen is the major bone protein. Little is known quantitatively about human bone collagen synthesis in vivo, despite its importance for the understanding of bone formation and turnover. Our aim was to develop a method that could be used for the physiological and pathophysiological investigation of human bone collagen synthesis. We have carried out preliminary studies in patients undergoing hip replacement and in pigs to validate the use of the flooding dose method using (13)C- or (15)N-labelled proline and we have now refined our techniques to allow them to be used in a normal clinical or physiological setting. The results show that the application of a flooding dose causes bone free-proline labelling to equilibrate with that of blood in pigs and human beings, so that only 150 mg of bone will provide enough sample to prepare and measure the labelling of three fractions of bone collagen (dissolved in NaCl, acetic acid and pepsin/acetic acid) which have the same relative labelling (1.0:0.43:0.1) as measured by GC-combustion-isotope ratio MS. The rates of incorporation were substantially faster than in skeletal muscle samples taken at the same time. The results suggest that different fractions of human bone collagen turnover at markedly higher rates than had been previously considered. This approach should allow us to discover how growth and development, food, activity and drugs affect bone collagen turnover and to measure the effects on it of ageing and bone disease.

  16. Collagen I-induced dendritic cells activation is regulated by TNF-alpha production through down-regulation of IRF4.

    PubMed

    Poudel, Barun; Ki, Hyeon-Hui; Lee, Young-Mi; Kim, Dae-Ki

    2015-03-01

    Previously we have shown that collagen I enhances the maturation and function of dendritic cells (DCs). Inflammatory mediators such as tumour necrosis factor (TNF)- alpha, interleukin (IL)-1 beta and lipopolysaccharide (LPS) are also known to activate DCs. Here we investigated the involvement of TNF-alpha on the collagen I-induced DCs activation. TNF-a neutralization inhibited collagen I-induced IL-12 secretions by DCs. Additionally, we observed suppression of collagen I-induced costimulatory molecules expression along with down-regulation of genes involved in DCs activation pathway. Furthermore, TNF- alpha inhibition upon collagen Istimulation up-regulated the expression of interferon regulatory transcription factor IRF4, when compared to collagen I only treated cells. Collectively, our data demonstrate that collagen I induce TNF- alpha production, which is crucial for the activation and function of DCs, through down-regulation of IRF4, and implicates the importance in development of anti- TNF-alpha therapeutics for several inflammatory diseases.

  17. Effects of salvianolic acid-A on NIH/3T3 fibroblast proliferation, collagen synthesis and gene expression

    PubMed Central

    Liu, Cheng-Hai; Hu, Yi-Yang; Wang, Xiao-Ling; Xu, Lie-Ming; Liu, Ping

    2000-01-01

    AIM: To investigate the mechanisms of salvianolic acid A (SA-A) against liver fibrosis in vitro. METHODS: NIH/3T3 fibroblasts were cultured routinely, and incubated with 10-4 mol/L-10-7 mol/L SA-A for 22 h. The cell viability was assayed by [3H]proline incorporation, cell proliferation by [3H]TdR incorporation, cell collagen synthetic rate was measured with [3H]proline impulse and collagenase digestion method. The total RNA was prepared from the control cells and the drug treated cells respectively, and α (1) I pro-collagen mRNA expression was semi-quantitatively analyzed with RT-PCR. RESULTS: 10-4 mol/L SA-A decreased cell viability and exerted some cytotoxiciy, while 10-5 mol/L-10-7 mol/L SA-A did not affect cell viability, but inhibited cell proliferation significantly, and 10-6 mol/L SA-A had the best effect on cell viability among these concentrations of drugs. 10-5 mol/L-10-6 mol/L SA-A inhibited intracellular collagen synthetic rate, but no significant influence on extracellular collagen secretion. Both 10-5 mol/L and 10-6 mol/L SA-A could decrease α (1) I pro-collagen mRNA expression remarkably. CONCLUSION: SA-A had potent action against liver fibrosis. It inhibited NIH/3T3 fibroblast proliferation, intracellular collagen synthetic rate and type I pro-collagen gene expression, which may be one of the main mechanisms of the drug. PMID:11819598

  18. The relative roles of collagen adhesive receptor DDR2 activation and matrix stiffness on the downregulation of focal adhesion kinase in vascular smooth muscle cells.

    PubMed

    Bhadriraju, Kiran; Chung, Koo-Hyun; Spurlin, Tighe A; Haynes, Ross J; Elliott, John T; Plant, Anne L

    2009-12-01

    Cells within tissues derive mechanical anchorage and specific molecular signals from the insoluble extracellular matrix (ECM) that surrounds them. Understanding the role of different cues that extracellular matrices provide cells is critical for controlling and predicting cell response to scaffolding materials. Using an engineered extracellular matrix of Type I collagen we examined how the stiffness, supramolecular structure, and glycosylation of collagen matrices influence the protein levels of cellular FAK and the activation of myosin II. Our results show that (1) cellular FAK is downregulated on collagen fibrils, but not on a non-fibrillar monolayer of collagen, (2) the downregulation of FAK is independent of the stiffness of the collagen fibrils, and (3) FAK levels are correlated with levels of tyrosine phosphorylation of the collagen adhesion receptor DDR2. Further, siRNA depletion of DDR2 blocks FAK downregulation. Our results suggest that the collagen receptor DDR2 is involved in the regulation of FAK levels in vSMC adhered to Type I collagen matrices, and that regulation of FAK levels in these cells appears to be independent of matrix stiffness.

  19. Inhibitory effect of collagen-derived tripeptides on dipeptidylpeptidase-IV activity.

    PubMed

    Hatanaka, Tadashi; Kawakami, Kayoko; Uraji, Misugi

    2014-12-01

    The collagen tripeptide fragments Gly-Ala-Hyp, Gly-Pro-Ala and Gly-Pro-Hyp were generated by hydrolyzing collagen from pig-skin, cattle-skin, fish-scales and chicken-feet, respectively, with Streptomyces collagenase. Collagenase treatment increased the concentration of tripeptides in the hydrolysates by 13-15% (w/w). Of the three peptides, Gly-Pro-Hyp was a true peptidic inhibitor of dipeptidylpeptidase-IV (DPP-IV), because DPP-IV could not hydrolyze the bond between Pro-Hyp. This tripeptide was a moderately competitive inhibitor (Ki=4.5 mM) of DPP-IV, and its level in the collagen hydrolysates could be greatly increased (4-9% [w/w]) using Streptomyces collagenase.

  20. Collagen in organ development

    NASA Technical Reports Server (NTRS)

    Hardman, P.; Spooner, B. S.

    1992-01-01

    It is important to know whether microgravity will adversely affect developmental processes. Collagens are macromolecular structural components of the extracellular matrix (ECM) which may be altered by perturbations in gravity. Interstitial collagens have been shown to be necessary for normal growth and morphogenesis in some embryonic organs, and in the mouse salivary gland, the biosynthetic pattern of these molecules changes during development. Determination of the effects of microgravity on epithelial organ development must be preceded by crucial ground-based studies. These will define control of normal synthesis, secretion, and deposition of ECM macromolecules and the relationship of these processes to morphogenesis.

  1. Camel milk attenuates the biochemical and morphological features of diabetic nephropathy: inhibition of Smad1 and collagen type IV synthesis.

    PubMed

    Korish, Aida A; Abdel Gader, Abdel Galil; Korashy, Hesham M; Al-Drees, Abdul Majeed; Alhaider, Abdulqader A; Arafah, Maha M

    2015-03-05

    Diabetic nephropathy (DN) is a common microvascular complication of diabetes mellitus (DM) that worsens its morbidity and mortality. There is evidence that camel milk (CM) improves the glycemic control in DM but its effect on the renal complications especially the DN remains unclear. Thus the current study aimed to characterize the effects of CM treatment on streptozotocin (STZ)-induced DN. Using STZ-induced diabetes, we investigated the effect of CM treatment on kidney function, proteinuria, renal Smad1, collagen type IV (Col4), blood glucose, insulin resistance (IR), lipid peroxidation, the antioxidant superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). In addition renal morphology was also examined. The current results showed that rats with untreated diabetes exhibited marked hyperglycemia, IR, high serum urea and creatinine levels, excessive proteinuria, increased renal Smad1 and Col4, glomerular expansion, and extracellular matrix deposition. There was also increased lipid peroxidation products, decreased antioxidant enzyme activity and GSH levels. Camel milk treatment decreased blood glucose, IR, and lipid peroxidation. Superoxide dismutase and CAT expression, CAT activity, and GSH levels were increased. The renoprotective effects of CM were demonstrated by the decreased serum urea and creatinine, proteinuria, Smad1, Col4, and preserved normal tubulo-glomerular morphology. In conclusion, beside its hypoglycemic action, CM attenuates the early changes of DN, decreased renal Smad1 and Col4. This could be attributed to a primary action on the glomerular mesangial cells, or secondarily to the hypoglycemic and antioxidant effects of CM. The protective effects of CM against DN support its use as an adjuvant anti-diabetes therapy.

  2. In situ quantitative evaluation of osteoblastic collagen synthesis under cyclic strain by using second-harmonic-generation microscope

    NASA Astrophysics Data System (ADS)

    Matsubara, Oki; Hase, Eiji; Minamikawa, Takeo; Yasui, Takeshi; Sato, Katsuya

    2016-03-01

    Osteoblast-produced collagen matrix in bone is influenced by the mechanical stimulus from their surroundings. However, it has been still unclear how mechanical stimulus affects collagen production by osteoblasts. Therefore, it is strongly required to investigate the characteristics of osteoblastic bone regenerative tissue engineering. Recently, second-harmonic-generation (SHG) microscope has attracted attention for in situ visualization of collagen fiber because of less invasiveness, unstaining and no fixation, as well as high spatial resolution and 3D imaging. Using SHG microscopy, one can track the temporal dynamics of collagen fiber during the cultured period of the sample. We applied cyclic stretch strain to osteoblasts (MC3T3-E1) by using originally developed cell stretching device. The stimulation time was set to 5min or 3hours with same strain 5% and same frequency 0.5Hz. Cells were seeded onto the PDMS (polydimethylsiloxane) rubber chamber at a density of 50,000 cells/cm2 and cultured in α-MEM with 10% FBS, 1% P/S, 1% Ascorbic acid, 0.2% hydrocortisone and 2% β-Glycerophosphate. SHG imaging was carried out every 7 days. As a result, we confirmed from SHG image that the collagen production was enhanced by the cyclic stretch strain, stretch stimulation time and stretch application term.

  3. Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts

    PubMed Central

    Donejko, Magdalena; Przylipiak, Andrzej; Rysiak, Edyta; Głuszuk, Katarzyna; Surażyński, Arkadiusz

    2014-01-01

    Aim The aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA) on this process. Materials and methods Collagen, [3H]-thymidine incorporation, and prolidase activity were measured in confluent human skin fibroblast cultures that had been treated with 1, 2, and 5 mM caffeine and with caffeine and 500 μg/mL HA. Western immunoblot analysis was performed to evaluate expression of β1-integrin receptor, insulin-like growth factor receptor phospho-Akt protein and mitogen-activated protein kinase (phospho-extracellular signal-regulated kinase). Results Caffeine inhibited collagen biosynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity. Caffeine significantly inhibited the enzyme activity. The addition of HA had no effect on collagen biosynthesis or prolidase activity in fibroblasts incubated with caffeine. Caffeine also had an inhibitory effect on DNA biosynthesis. HA, however, did not have any significant effect on this process. The inhibition of the expression of β1-integrin and insulin-like growth factor receptor in fibroblasts incubated with the caffeine indicates a possible mechanism of inhibition of collagen biosynthesis. Conclusion Caffeine reduces collagen synthesis in human cultured skin fibroblasts. HA did not have any significant protective effect on this process. This is the first study to our knowledge that reports caffeine-induced inhibition of collagen synthesis in human skin fibroblasts. PMID:25342885

  4. Multi-scale mechanical characterization of highly swollen photo-activated collagen hydrogels

    PubMed Central

    Tronci, Giuseppe; Grant, Colin A.; Thomson, Neil H.; Russell, Stephen J.; Wood, David J.

    2015-01-01

    Biological hydrogels have been increasingly sought after as wound dressings or scaffolds for regenerative medicine, owing to their inherent biofunctionality in biological environments. Especially in moist wound healing, the ideal material should absorb large amounts of wound exudate while remaining mechanically competent in situ. Despite their large hydration, however, current biological hydrogels still leave much to be desired in terms of mechanical properties in physiological conditions. To address this challenge, a multi-scale approach is presented for the synthetic design of cyto-compatible collagen hydrogels with tunable mechanical properties (from the nano- up to the macro-scale), uniquely high swelling ratios and retained (more than 70%) triple helical features. Type I collagen was covalently functionalized with three different monomers, i.e. 4-vinylbenzyl chloride, glycidyl methacrylate and methacrylic anhydride, respectively. Backbone rigidity, hydrogen-bonding capability and degree of functionalization (F: 16 ± 12–91 ± 7 mol%) of introduced moieties governed the structure–property relationships in resulting collagen networks, so that the swelling ratio (SR: 707 ± 51–1996 ± 182 wt%), bulk compressive modulus (Ec: 30 ± 7–168 ± 40 kPa) and atomic force microscopy elastic modulus (EAFM: 16 ± 2–387 ± 66 kPa) were readily adjusted. Because of their remarkably high swelling and mechanical properties, these tunable collagen hydrogels may be further exploited for the design of advanced dressings for chronic wound care. PMID:25411409

  5. Multi-scale mechanical characterization of highly swollen photo-activated collagen hydrogels.

    PubMed

    Tronci, Giuseppe; Grant, Colin A; Thomson, Neil H; Russell, Stephen J; Wood, David J

    2015-01-06

    Biological hydrogels have been increasingly sought after as wound dressings or scaffolds for regenerative medicine, owing to their inherent biofunctionality in biological environments. Especially in moist wound healing, the ideal material should absorb large amounts of wound exudate while remaining mechanically competent in situ. Despite their large hydration, however, current biological hydrogels still leave much to be desired in terms of mechanical properties in physiological conditions. To address this challenge, a multi-scale approach is presented for the synthetic design of cyto-compatible collagen hydrogels with tunable mechanical properties (from the nano- up to the macro-scale), uniquely high swelling ratios and retained (more than 70%) triple helical features. Type I collagen was covalently functionalized with three different monomers, i.e. 4-vinylbenzyl chloride, glycidyl methacrylate and methacrylic anhydride, respectively. Backbone rigidity, hydrogen-bonding capability and degree of functionalization (F: 16 ± 12-91 ± 7 mol%) of introduced moieties governed the structure-property relationships in resulting collagen networks, so that the swelling ratio (SR: 707 ± 51-1996 ± 182 wt%), bulk compressive modulus (Ec: 30 ± 7-168 ± 40 kPa) and atomic force microscopy elastic modulus (EAFM: 16 ± 2-387 ± 66 kPa) were readily adjusted. Because of their remarkably high swelling and mechanical properties, these tunable collagen hydrogels may be further exploited for the design of advanced dressings for chronic wound care.

  6. Collagen-biomorphic porous carbon nanofiber monoliths: Biosilicification-assisted sustainable synthesis and application in Li-S battery

    NASA Astrophysics Data System (ADS)

    Hu, Wen; Shen, Tao; Hou, Hongying; Gan, Guoyou; Zheng, Biju; Li, Fengxian; Yi, Jianhong

    2016-12-01

    Monolithic carbon has been synthesized via a sustainable biomimetic route utilizing intrafibrillar silicified collagen sponge as precursor and morphogenesis template. The mineralized silica in the biohybrid prevents collapse of the carbon during pyrolysis. Upon biosilica removal results show that the carbon monoliths inherit the porous fiber structure of the mother collagen. The carbon nanofiber framework facilitates the construction of a high electrical conductive pathway, while the internal spaces developed among the intertwined fibrillar network and pores within nanofiber walls offer room for sulfur storage. The as-obtained carbon-sulfur cathode exhibits an accessible discharge capacity approaching 800mAh g-1 in Li-S battery.

  7. Synthesis of a cyanopeptide-analogue with trypsin activating properties.

    PubMed

    Radau, G; Rauh, D

    2000-04-17

    An efficient synthesis of a peptidic analogue of cyanobacterial metabolites with proposed serine protease inhibitory activity has been developed. Surprisingly, one trypsin activating compound was obtained.

  8. A method for expression and purification of soluble, active Hsp47, a collagen-specific molecular chaperone.

    PubMed

    Thomson, C A; Ananthanarayanan, V S

    2001-10-01

    Hsp47 is regarded as a collagen-specific chaperone with several suggested roles in collagen biosynthesis under normal and disease conditions. We describe here a procedure for the expression and purification of Hsp47 in Escherichia coli using the IMPACT expression system (New England Biolabs) where the guest gene is fused to the adduct, intein, with a chitin-binding domain. Use of this system resulted in relatively high levels of soluble Hsp47 compared to other available protocols, especially when the bacterial cells were induced at 14 degrees C instead of 37 degrees C. The cell lysate was passed through a chitin-Sepharose affinity column and Hsp47 was cleaved from intein using beta-mercaptoethanol. Minor degradation products were subsequently removed using a hydroxylapatite column to yield milligram amounts of pure and active protein suitable for structural studies. Gel electrophoretic analysis of the purified protein indicated the presence of a small proportion of trimeric species when non-reducing conditions were used. The ability to form a trimer may be important for its role as a chaperone. The IMPACT system allows for radiolabelling of purified Hsp47 with (35)S for use in binding experiments. Illustrative data on collagen binding by (35)S-Hsp47 are shown.

  9. Epinephrine--via activation of p38-MAPK--abolishes the effect of aspirin on platelet deposition to collagen.

    PubMed

    Mustonen, P; van Willigen, G; Lassila, R

    2001-12-15

    The mechanism by which epinephrine enhances experimental thrombosis in the presence of aspirin is poorly understood. In this study, we set to explore, in aspirinised platelet-rich plasma (PRP), the effect of epinephrine (100 nmol/l) on platelet deposition to immobilised collagen and the subsequent involvement of several intracellular pathways. Under these experimental conditions, which allow platelet aggregation on top of the collagen-adherent platelets, epinephrine increased platelet deposition by 55-86%. This enhancement could be specifically prohibited by the alpha(2A)-adrenoceptor antagonist, atipamezole, the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580, and the cytosolic phospholipase A(2) (cPLA(2)) inhibitor, mepacrine. The effect of epinephrine coincided with increased phosphorylation of p38MAPK and cPLA(2) and with arachidonic acid (AA) release from platelet membrane. We conclude that epinephrine enhanced platelet deposition on collagen in aspirinised PRP via a mechanism dependent on both free AA in platelet cytosol (released by cPLA(2)) and p38MAPK.

  10. Defective pro alpha 2(I) collagen synthesis in a recessive mutation in mice: a model of human osteogenesis imperfecta.

    PubMed Central

    Chipman, S D; Sweet, H O; McBride, D J; Davisson, M T; Marks, S C; Shuldiner, A R; Wenstrup, R J; Rowe, D W; Shapiro, J R

    1993-01-01

    Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue associated with fractures, osteopenia, and short stature. OI results from mutations affecting the pro alpha 1 or pro alpha 2 gene of type I collagen. We describe a strain of mice with a nonlethal recessively inherited mutation (oim) that results in phenotypic and biochemical features that simulate moderate to severe human OI. The phenotype of homozygous oim mice includes skeletal fractures, limb deformities, generalized osteopenia, and small body size. Their femurs are smaller and demonstrate marked cortical thinning and fewer medullary trabeculae than those of wild-type mice. Breeding studies show the mutation is inherited in most crosses as a single recessive gene on chromosome 6, near the murine Cola-2 gene. Biochemical analysis of skin and bone, as well as isolated dermal fibroblast cultures, demonstrate that alpha 1(I) homotrimeric collagen accumulates in these tissues and is secreted by fibroblasts. Short labeling studies in fibroblasts demonstrate an absence of pro alpha 2(I) collagen chains. Nucleotide sequencing of the cDNA encoding the COOH-propeptide reveals a G deletion at pro alpha 2(I) nucleotide 3983; this results in an alteration of the sequence of the last 48 amino acids. The oim mouse will facilitate the study of type I collagen-related skeletal disease. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8446583

  11. Efficacy of Annona squamosa L in the synthesis of glycosaminoglycans and collagen during wound repair in streptozotocin induced diabetic rats.

    PubMed

    Ponrasu, Thangavel; Suguna, Lonchin

    2014-01-01

    The aim of this work was to find out the effects of Annona squamosa on the formation of glycosaminoglycans and collagen during wound healing in normal and diabetic rats. Diabetes induced rats were segregated into 4 groups, each containing six animals. Groups I and III served as the normal and diabetic control while groups II and IV served as normal and diabetic treated. The animals were treated with 200 μL of Annona squamosa extract topically. The granulation tissues formed were removed on the 8th day and the amount of glycosaminoglycans (GAGs) and collagen formed was evaluated by sequential extraction and SDSPAGE, respectively. Histological evaluation was also carried out using Masson's trichrome stain. In vitro wound healing efficacy of A. squamosa in human dermal fibroblast culture (HDF) was also carried out. The fibroblasts treated with varying concentrations of A. squamosa were examined for proliferation and closure of the wound area and photographed. A. squamosa increased cellular proliferation in HDF culture. The granulation tissues of treated wounds showed increased levels of glycosaminoglycans (P < 0.05) and collagen which were also confirmed by histopathology. The results strongly substantiate the beneficial effects of A. squamosa on the formation of glycosaminoglycans and collagen during wound healing.

  12. Efficacy of Annona squamosa L in the Synthesis of Glycosaminoglycans and Collagen during Wound Repair in Streptozotocin Induced Diabetic Rats

    PubMed Central

    Ponrasu, Thangavel

    2014-01-01

    The aim of this work was to find out the effects of Annona squamosa on the formation of glycosaminoglycans and collagen during wound healing in normal and diabetic rats. Diabetes induced rats were segregated into 4 groups, each containing six animals. Groups I and III served as the normal and diabetic control while groups II and IV served as normal and diabetic treated. The animals were treated with 200 μL of Annona squamosa extract topically. The granulation tissues formed were removed on the 8th day and the amount of glycosaminoglycans (GAGs) and collagen formed was evaluated by sequential extraction and SDSPAGE, respectively. Histological evaluation was also carried out using Masson's trichrome stain. In vitro wound healing efficacy of A. squamosa in human dermal fibroblast culture (HDF) was also carried out. The fibroblasts treated with varying concentrations of A. squamosa were examined for proliferation and closure of the wound area and photographed. A. squamosa increased cellular proliferation in HDF culture. The granulation tissues of treated wounds showed increased levels of glycosaminoglycans (P < 0.05) and collagen which were also confirmed by histopathology. The results strongly substantiate the beneficial effects of A. squamosa on the formation of glycosaminoglycans and collagen during wound healing. PMID:25003104

  13. Bioengineered collagens

    PubMed Central

    Ramshaw, John AM; Werkmeister, Jerome A; Dumsday, Geoff J

    2014-01-01

    Mammalian collagen has been widely used as a biomedical material. Nevertheless, there are still concerns about the variability between preparations, particularly with the possibility that the products may transmit animal-based diseases. Many groups have examined the possible application of bioengineered mammalian collagens. However, translating laboratory studies into large-scale manufacturing has often proved difficult, although certain yeast and plant systems seem effective. Production of full-length mammalian collagens, with the required secondary modification to give proline hydroxylation, has proved difficult in E. coli. However, recently, a new group of collagens, which have the characteristic triple helical structure of collagen, has been identified in bacteria. These proteins are stable without the need for hydroxyproline and are able to be produced and purified from E. coli in high yield. Initial studies indicate that they would be suitable for biomedical applications. PMID:24717980

  14. Identification and Evaluation of Cryoprotective Peptides from Chicken Collagen: Ice-Growth Inhibition Activity Compared to That of Type I Antifreeze Proteins in Sucrose Model Systems.

    PubMed

    Du, Lihui; Betti, Mirko

    2016-06-29

    The ability of chicken collagen peptides to inhibit the growth of ice crystals was evaluated and compared to that of fish antifreeze proteins (AFPs). This ice inhibition activity was assessed using a polarized microscope by measuring ice crystal dimensions in a sucrose model system with and without collagen peptides after seven thermal cycles. The system was stabilized at -25 °C and cycled between -16 and -12 °C. Five candidate peptides with ice inhibition activity were identified using liquid chromatography and tandem mass spectrometry and were then synthesized. Their ice inhibition capacity was compared to that of type I AFPs in a 23% sucrose model system. Specific collagen peptides with certain amino acid sequences reduced the extent of ice growth by approximately 70% at a relatively low concentration (1 mg/mL). These results suggest that specific collagen peptides may act in a noncolligative manner, inhibiting ice crystal growth like type I AFPs, but less efficiently.

  15. Cell-mediated degradation of type IV collagen and gelatin films is dependent on the activation of matrix metalloproteinases.

    PubMed Central

    Atkinson, S J; Ward, R V; Reynolds, J J; Murphy, G

    1992-01-01

    The ability of normal rabbit dermal fibroblasts to degrade films of type IV collagen and gelatin when stimulated by phorbol ester was shown to be dependent on the induction, secretion and activation of 95 kDa gelatinase B and the secretion and activation of 72 kDa gelatinase A and stromelysin. Degradation was inhibited by exogenous human recombinant tissue inhibitor of metalloproteinases-1, specific antibodies to gelatinase and stromelysin and by the reactive-oxygen-metabolite inhibitor catalase. We discuss the various pathways for activation of matrix metalloproteinases in this model system and conclude that, although plasmin may play a key role in the activation of gelatinase B and stromelysin, gelatinase A is activated by a mechanism which has yet to be elucidated. The involvement of oxygen radicals in the direct activation of matrix metalloproteinases in this model is thought to be unlikely. Images Fig. 2. Fig. 3. Fig. 4. PMID:1463464

  16. Characterization of a non-fibrillar-related collagen in the mollusc Haliotis tuberculata and its biological activity on human dermal fibroblasts.

    PubMed

    Fleury, Christophe; Serpentini, Antoine; Kypriotou, Magdalini; Renard, Emmanuelle; Galéra, Philippe; Lebel, Jean-Marc

    2011-10-01

    In invertebrates, members of the collagen family have been found in various phyla. Surprisingly, in mollusc, little is known about such molecules. In this study, we characterize the full-length abalone type IV collagen and we analysed its biological effects on human fibroblast in order to gain insights about this molecule in molluscs and particularly clues about its roles. We screened a cDNA library of Haliotis tuberculata hemocytes. The expression pattern of the transcript is determined using real-time polymerase chain reaction and in situ hybridization. The close identity between α1(IV) C-terminal domain and the vertebrate homologue led us to produce, purify and test in vitro a recombinant protein corresponding to this region using human dermal fibroblasts cell culture. The biological effects were evaluated on proliferation and on differentiation. We found that the 5,334-bp open reading frame transcript encodes a protein of 1,777 amino acids, including an interrupted 1,502-residue collagenous domain and a 232-residue C-terminal non-collagenous domain. The expression pattern of this transcript is mainly found in the mantle and hemocytes. The recombinant protein corresponding α1(IV) C-terminal domain increased fibroblast proliferation by 69% and doubled collagen synthesis produced in primary cultures. This work provides the first complete primary structure of a mollusc non-fibrillar collagen chain and the biological effects of its C-terminal domain on human cells. In this study, we prove that the NC1 domain from a molluscan collagen can improve human fibroblast proliferation as well as differentiation.

  17. Perylenequinones: Isolation, Synthesis, and Biological Activity

    PubMed Central

    Mulrooey, Carol A.; O'Brien, Erin M.; Morgan, Barbara J.

    2013-01-01

    The perylenequinones are a novel class of natural products characterized by pentacyclic conjugated chromophore giving rise to photoactivity. Potentially useful light-activated biological activity, targeting protein kinase C (PKC), has been identified for several of the natural products. Recently discovered new members of this class of compound, as well as several related phenanthroperylenequinones, are reviewed. Natural product modifications that improve biological profiles, and avenues for the total synthesis of analogs, which are not available from the natural product series, are outlined. An overview of structure/function relationships is provided. PMID:24039544

  18. Effect of enzymatic hydrolysis on surface activity and surface rheology of type I collagen.

    PubMed

    Kezwoń, Aleksandra; Chromińska, Ilona; Frączyk, Tomasz; Wojciechowski, Kamil

    2016-01-01

    We describe the adsorption behaviour and rheological properties of a calf skin type I collagen, and of its hydrolysates obtained using a Clostridium histolyticum collagenase (CHC) under moderate conditions (pH 7, 37°C). The effect of CHC concentration (2×10(-9)-2×10(-6)M) and incubation time (35-85min) was studied and optimised to achieve the highest decrease of surface tension and the highest dilational surface viscoelasticity of the adsorbed layers. SDS-PAGE electrophoresis and reverse-phase high performance liquid chromatography (RP-HPLC) were used to characterise the hydrolysis products. The results show that even simple modifications (heat treatment, pH change, partial hydrolysis) of collagen enhances its surface properties, especially in terms of surface dilational elasticity modulus. The use of low enzyme concentration (CHC-to-collagen molar ratio of 4×10(-3)) and short incubation time (<45min) results in moderately hydrolysed products with the highest ability to lower surface tension (γ=53.9mNm(-1)) forming highly elastic adsorbed layers (surface dilational elasticity, E'=74.5mNm(-1)).

  19. Zinc l-pyrrolidone carboxylate inhibits the UVA-induced production of matrix metalloproteinase-1 by in vitro cultured skin fibroblasts, whereas it enhances their collagen synthesis.

    PubMed

    Takino, Y; Okura, F; Kitazawa, M; Iwasaki, K; Tagami, H

    2012-02-01

    Reduced collagen matrix in the dermis constitutes one of the characteristic features of chronologically aged skin, which is further enhanced on the sun-exposed portions of the body by chronic ultraviolet light (UV) irradiation, inducing the unique changes associated with skin photoageing. The zinc salt of l-pyrrolidone carboxylate (Zinc PCA) has long been used as a cosmetic ingredient, because of its astringent and anti-microbial properties. In the present study, by employing cultured normal human dermal fibroblasts, we found that Zinc PCA suppressed UVA-induced activation of activator protein-1 (AP-1) and reduced matrix metalloproteinase-1 production in these cells, which is thought to be involved in collagen degradation in photoaged skin. Moreover, Zinc PCA treatment of the cells increased the expression of an ascorbic acid transporter mRNA, SVCT2, but not SVCT1, resulting in the enhanced production of type I collagen. Based on these in vitro findings, we consider Zinc PCA to be a promising candidate for an anti-skin ageing agent.

  20. Sulfoxide stimulation of chondrogenesis in limb mesenchyme is accompanied by an increase in type II collagen enhancer activity

    SciTech Connect

    Horton, W.E. Jr.; Higginbotham, J.D. )

    1991-05-01

    We have utilized a modification of the limb bud mesenchyme micromass culture system to screen compounds that might stimulate chondrogenesis. Two compounds in the sulfoxide family (methylphenylsulfoxide and p-chlorophenyl methyl sulfoxide) were stimulatory at 10(-2) M and 10(-3) M, respectively; whereas other sulfoxides and organic solvents were not active at these concentrations. In addition, specific growth factors (basic FGF, IGF-I, IGF-II) were not chondroinductive at concentrations that are active in other cell systems. Both sulfoxide compounds stimulated cartilage nodule formation, ({sup 35}S)sulfate incorporation, and activity of the regulatory sequences of the collagen II gene. In contrast, transforming growth factor beta-1 (10 ng/ml) stimulated sulfate incorporation but produced only a diffuse deposition of cartilage matrix and reduced the ability of the cells to utilize the regulatory sequences of the collagen II gene. The sulfoxides appear to promote the differentiation of limb bud cells to chondrocytes and thus exhibit chondroinductive activity.

  1. Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans.

    PubMed

    Simental-Mendía, M; Lara-Arias, J; Álvarez-Lozano, E; Said-Fernández, S; Soto-Domínguez, A; Padilla-Rivas, G R; Martínez-Rodríguez, H G

    2015-12-01

    Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

  2. Collagen structure: new tricks from a very old dog.

    PubMed

    Bella, Jordi

    2016-04-15

    The main features of the triple helical structure of collagen were deduced in the mid-1950s from fibre X-ray diffraction of tendons. Yet, the resulting models only could offer an average description of the molecular conformation. A critical advance came about 20 years later with the chemical synthesis of sufficiently long and homogeneous peptides with collagen-like sequences. The availability of these collagen model peptides resulted in a large number of biochemical, crystallographic and NMR studies that have revolutionized our understanding of collagen structure. High-resolution crystal structures from collagen model peptides have provided a wealth of data on collagen conformational variability, interaction with water, collagen stability or the effects of interruptions. Furthermore, a large increase in the number of structures of collagen model peptides in complex with domains from receptors or collagen-binding proteins has shed light on the mechanisms of collagen recognition. In recent years, collagen biochemistry has escaped the boundaries of natural collagen sequences. Detailed knowledge of collagen structure has opened the field for protein engineers who have used chemical biology approaches to produce hyperstable collagens with unnatural residues, rationally designed collagen heterotrimers, self-assembling collagen peptides, etc. This review summarizes our current understanding of the structure of the collagen triple helical domain (COL×3) and gives an overview of some of the new developments in collagen molecular engineering aiming to produce novel collagen-based materials with superior properties.

  3. Investigation of the effect of stress on the chemistry, metabolism, and biophysics of collagen

    NASA Technical Reports Server (NTRS)

    Houck, J. C.

    1973-01-01

    The research is reported concerning the effect of stress on the chemistry in the connective tissue of the rat. It was found that within a day after administration of cortisol (stress harmone), a significant amount of the insoluble collagen disappeared from the skin. It is concluded that the abrupt catabolism of cutaneous collagen releases peptides. These peptides are rapidly degraded to free amino acids which are active in effecting liver glycogen synthesis, and provide a major energy source to assist the animal in the fight or flight reaction. It is proposed that cutaneous collagen represents a reserve energy pool, which can be mobilized via stress harmones.

  4. Interactions of collagen molecules in the presence of N-hydroxysuccinimide activated adipic acid (NHS-AA) as a crosslinking agent.

    PubMed

    Zhang, Min; Wu, Kun; Li, Guoying

    2011-11-01

    The effect of crosslinking agent on pepsin-soluble bovine collagen solution was examined using N-hydroxysuccinimide activated adipic acid (NHS-AA) as a crosslinker. Electrophoretic patterns indicated that crosslinks formed when NHS-AA was added. A higher polarity level deduced from the changes in the fluorescence emission spectrum of pyrene in the crosslinked collagen solution indicated that the formation of well-ordered aggregates was suppressed. The random aggregation of collagens was also observed by atomic force microscopy (AFM). Furthermore, the association of collagens into fibrils was influenced by crosslinking. Self-assembly was suppressed at 37°C; however, as temperature was increased to 39°C, a small amount of NHS-AA leaded to an improvement in the ability of self-aggregation. Although more random structure was brought about by crosslinking, self-aggregation might still be promoted as temperature was increased, accompanying by the thermal stability improvement of fibrils.

  5. Migration and Proliferative Activity of Mesenchymal Stem Cells in 3D Polylactide Scaffolds Depends on Cell Seeding Technique and Collagen Modification.

    PubMed

    Rodina, A V; Tenchurin, T Kh; Saprykin, V P; Shepelev, A D; Mamagulashvili, V G; Grigor'ev, T E; Lukanina, K I; Orekhov, A S; Moskaleva, E Yu; Chvalun, S N

    2016-11-01

    We analyzed viability of mesenchymal stem cells seeded by static and dynamic methods to highly porous fibrous 3D poly-L-lactide scaffolds with similar physical and chemical properties, but different spatial organization modified with collagen. Standard collagen coating promoted protein adsorption on the scaffold surface and improved adhesive properties of 100 μ-thick scaffolds. Modification of 600-μ scaffolds with collagen under pressure increased proliferative activity of mesenchymal stem cells seeded under static and dynamic (delivery of 100,000 cells in 10 ml medium in a perfusion system at a rate of 1 ml/min) conditions by 47 and 648%, respectively (measured after 120-h culturing by MTT test). Dynamic conditions provide more uniform distribution of collagen on scaffold fibers and promote cell penetration into 3D poly-L-lactide scaffolds with thickness >600 μ.

  6. Endocytic collagen degradation: a novel mechanism involved in protection against liver fibrosis.

    PubMed

    Madsen, Daniel H; Jürgensen, Henrik J; Ingvarsen, Signe; Melander, Maria C; Vainer, Ben; Egerod, Kristoffer L; Hald, Andreas; Rønø, Birgitte; Madsen, Charlotte A; Bugge, Thomas H; Engelholm, Lars H; Behrendt, Niels

    2012-05-01

    Fibrosis of the liver and its end-stage, cirrhosis, represent major health problems worldwide. In these fibrotic conditions, activated fibroblasts and hepatic stellate cells display a net deposition of collagen. This collagen deposition is a major factor leading to liver dysfunction, thus making it crucially important to understand both the collagen synthesis and turnover mechanisms in this condition. Here we show that the endocytic collagen receptor, uPARAP/Endo180, is a major determinant in governing the balance between collagen deposition and degradation. Cirrhotic human livers displayed a marked up-regulation of uPARAP/Endo180 in activated fibroblasts and hepatic stellate cells located close to the collagen deposits. In a hepatic stellate cell line, uPARAP/Endo180 was shown to be active in, and required for, the uptake and intracellular degradation of collagen. To evaluate the functional importance of this collagen receptor in vivo, liver fibrosis was induced in uPARAP/Endo180-deficient mice and littermate wild-type mice by chronic CCl(4) administration. A strong up-regulation of uPARAP/Endo180 was observed in wild-type mice, and a quantitative comparison of collagen deposits in the two groups of mice clearly revealed a fibrosis protective role of uPARAP/Endo180. This effect appeared to directly reflect the activity of the collagen receptor, since no compensatory events were noted when comparing the mRNA expression profiles of the two groups of mice in an array system focused on matrix-degrading components. This function of uPARAP/Endo180 defines a novel role of intracellular collagen turnover in fibrosis protection.

  7. Synthesis and Anticancer Activity of Epipolythiodiketopiperazine Alkaloids

    PubMed Central

    Boyer, Nicolas; Morrison, Karen C.; Kim, Justin; Hergenrother, Paul J.; Movassaghi, Mohammad

    2013-01-01

    The epipolythiodiketopiperazine (ETP) alkaloids are a highly complex class of natural products with potent anticancer activity. Herein, we report the application of a flexible and scalable synthesis, allowing the construction of dozens of ETP derivatives. The evaluation of these compounds against cancer cell lines in culture allows for the first expansive structure–activity relationship (SAR) to be defined for monomeric and dimeric ETP-containing natural products and their synthetic cognates. Many ETP derivatives demonstrate potent anticancer activity across a broad range of cancer cell lines, and kill cancer cellsviainduction of apoptosis. Several traits thatbode well for the translational potential of the ETP class of natural products includeconcise and efficient synthetic access, potent induction of apoptotic cell death, activity against a wide range of cancer types, and a broad tolerance for modifications at multiple sitesthat should facilitate small-molecule drug development, mechanistic studies, and evaluation in vivo. PMID:23914293

  8. Synergistic effect of a novel oxymatrine-baicalin combination against hepatitis B virus replication, α smooth muscle actin expression and type I collagen synthesis in vitro

    PubMed Central

    Cheng, Yang; Ping, Jian; Xu, Huai-Dong; Fu, Hai-Jun; Zhou, Zhao-Hui

    2006-01-01

    AIM: To study the effect of oxymatrine-baicalin combination (OB) against HBV replication in 2.2.15 cells and α smooth muscle actin (α SMA) expression, type I, collagen synthesis in HSC-T6 cells. METHODS: The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to β actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by β actin. RESULTS: In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group (HBsAg, P = 0.043; HBeAg, P = 0.026; respectively); HBV DNA level in the OB group was significantly lower than that in the oxymatrine group (P = 0.041). In HSC-T6 cells the mRNA and protein expression levels of α SMA in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P = 0.013; protein, P = 0.042; respectively); The mRNA and protein expression levels of type I collagen in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P < 0.01; protein, P < 0.01; respectively). CONCLUSION: OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against α SMA expression and typeI collagen synthesis in HSC-T6 cells than oxymatrine in vitro. PMID:16937525

  9. ALCOHOL MODULATION OF MMP AND TIMP EXPRESSION IN THE HEART FAVORS COLLAGEN ACCUMULATION

    PubMed Central

    El Hajj, E.C.; El hajj, M.C.; Voloshenyuk, T.G.; Mouton, A.J.; Khoutorova, E.; Molina, P.E.; Gilpin, N.W.; Gardner, J.D.

    2013-01-01

    Background Chronic alcohol consumption has been shown in human and animal studies to result in collagen accumulation, myocardial fibrosis, and heart failure. Cardiac fibroblasts produce collagen and regulate extracellular matrix (ECM) homeostasis through the synthesis and activity of matrix metalloproteinases (MMP) and tissue inhibitors of MMPs (TIMP), with the balance of MMPs/TIMPs determining the rate of collagen turnover. Dynamic changes of MMP and TIMP expression were reported in alcohol induced hepatic fibrosis; however, the effect of alcohol on MMP/TIMP balance in the heart and cardiac fibroblasts is unknown. We hypothesized that alcohol exposure alters cardiac fibroblast MMP and TIMP expression to promote collagen accumulation in the heart. Methods Cardiac fibroblasts isolated from adult rats were cultured in the presence of alcohol (12.5–200 mM) for 48 hrs. MMP, TIMP, and collagen type I and III expression were assayed by Western blot analysis. Hydroxyproline (HPro) was used as a marker of collagen production. The in vivo cardiac effects of ethanol were determined using rats exposed to ethanol vapor for two weeks, resulting in blood alcohol levels of 150–200 mg/dl. Cardiac collagen volume fraction (CVF), as well as MMP, TIMP and collagen expression, was assessed. Results Ethanol exposed rats exhibited upregulation of TIMP-1, -3 and -4 in the heart, with no significant increases in MMPs. Cardiac fibroblasts exhibited transformation to a profibrotic phenotype following exposure to alcohol. These changes were reflected by increased α-smooth muscle actin and collagen I and III expression, as well as increased collagen secretion. In vivo ethanol exposure also produced fibrosis, indicated by increased CVF and expression of collagens. Conclusion Alcohol exposure modulates cardiac fibroblast MMP/TIMP expression favoring a profile associated with collagen accumulation. Our data suggest that this disrupted MMP/TIMP profile may contribute to the development of

  10. Mechanism for adhesion G protein-coupled receptor GPR56-mediated RhoA activation induced by collagen III stimulation.

    PubMed

    Luo, Rong; Jeong, Sung-Jin; Yang, Annie; Wen, Miaoyun; Saslowsky, David E; Lencer, Wayne I; Araç, Demet; Piao, Xianhua

    2014-01-01

    GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) family. Despite the importance of GPR56 in brain development, where mutations cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria (BFPP), the signaling mechanism(s) remain largely unknown. Like many other adhesion GPCRs, GPR56 is cleaved via a GPCR autoproteolysis-inducing (GAIN) domain into N- and C-terminal fragments (GPR56N and GPR56C); however, the biological significance of this cleavage is elusive. Taking advantage of the recent identification of a GPR56 ligand and the presence of BFPP-associated mutations, we investigated the molecular mechanism of GPR56 signaling. We demonstrate that ligand binding releases GPR56N from the membrane-bound GPR56C and triggers the association of GPR56C with lipid rafts and RhoA activation. Furthermore, one of the BFPP-associated mutations, L640R, does not affect collagen III-induced lipid raft association of GPR56. Instead, it specifically abolishes collagen III-mediated RhoA activation. Together, these findings reveal a novel signaling mechanism that may apply to other members of the adhesion GPCR family.

  11. Computational simulation of platelet deposition and activation: II. Results for Poiseuille flow over collagen.

    PubMed

    Sorensen, E N; Burgreen, G W; Wagner, W R; Antaki, J F

    1999-01-01

    We have previously described the development of a two-dimensional computational model of platelet deposition onto biomaterials from flowing blood (Sorensen et al., Ann. Biomed. Eng. 27:436-448, 1999). The model requires estimation of four parameters to fit it to experimental data: shear-dependent platelet diffusivity and three platelet-deposition-related reaction rate constants. These parameters are estimated for platelet deposition onto a collagen substrate for simple parallel-plate flow of whole blood in both the presence and absence of thrombin. One set of experimental results is used as a benchmark for model-fitting purposes. The "trained" model is then validated by applying it to additional test cases from the literature for parallel-plate Poiseuille flow over collagen at both higher and lower wall shear rates, and in the presence of various anticoagulants. The predicted values agree very well with the experimental results for the training cases, and good reproduction of deposition trends and magnitudes is obtained for the heparin, but not the citrate, validation cases. The model is formulated to be easily extended to synthetic biomaterials, as well as to more complex flows.

  12. One-step synthesis of collagen hybrid gold nanoparticles and formation on Egyptian-like gold-plated archaeological ivory.

    PubMed

    Spadavecchia, Jolanda; Apchain, Emilande; Albéric, Marie; Fontan, Elisabeth; Reiche, Ina

    2014-08-04

    A one-step method is reported to synthesize hybrid gold nanoparticles (AuNPs) by reduction of HAuCl4 in acetic solution in the presence of collagen (Col), dicarboxylic acid-terminated polyethylene glycol (PEG), and cetyltetrammonium bromide (CTAB) mixed with hydoxyapatite (HAP) as surfactants. Such formation process of AuNPs was shown to be responsible for purple stains naturally formed on Egyptianizing archaeological gilded ivories from 8th BC Syria. The understanding of this formation mechanism, which most likely involves a step with hybrid AuNPs, allows the establishing of an authenticity marker of ancient gold-plated ivories.

  13. MAP-kinase activity necessary for TGFbeta1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397.

    PubMed

    Hayashida, Tomoko; Wu, Ming-Hua; Pierce, Amy; Poncelet, Anne-Christine; Varga, John; Schnaper, H William

    2007-12-01

    The signals mediating transforming growth factor beta (TGFbeta)-stimulated kidney fibrogenesis are poorly understood. We previously reported TGFbeta-stimulated, Smad-mediated collagen production by human kidney mesangial cells, and that ERK MAP kinase activity optimizes collagen expression and enhances phosphorylation of the Smad3 linker region. Furthermore, we showed that disrupting cytoskeletal integrity decreases type I collagen production. Focal adhesion kinase (FAK, PTK2) activity could integrate these findings. Adhesion-dependent FAK Y397 phosphorylation was detected basally, whereas FAK Y925 phosphorylation was TGFbeta1-dependent. By immunocytochemistry, TGFbeta1 stimulated the merging of phosphorylated FAK with the ends of thickening stress fibers. Cells cultured on poly-L-lysine (pLL) to promote integrin-independent attachment spread less than those on control substrate and failed to demonstrate focal adhesion (FA) engagement with F-actin. FAK Y397 phosphorylation and ERK activity were also decreased under these conditions. In cells with decreased FAK Y397 phosphorylation from either plating on pLL or overexpressing a FAK Y397F point mutant, serine phosphorylation of the Smad linker region, but not of the C-terminus, was reduced. Y397F and Y925F FAK point mutants inhibited TGFbeta-induced Elk-Gal activity, but only the Y397F mutant inhibited TGFbeta-stimulated collagen-promoter activity. The inhibition by the Y397F mutant or by culture on pLL was prevented by co-transfection of constitutively active ERK MAP kinase kinase (MEK), suggesting that FAK Y397 phosphorylation promotes collagen expression via ERK MAP kinase activity. Finally, Y397 FAK phosphorylation, and both C-terminal and linker-region Smad3 phosphorylation were detected in murine TGFbeta-dependent kidney fibrosis. Together, these data demonstrate adhesion-dependent FAK phosphorylation promoting TGFbeta-induced responses to regulate collagen production.

  14. Betulin Phosphonates; Synthesis, Structure, and Cytotoxic Activity.

    PubMed

    Chrobak, Elwira; Bębenek, Ewa; Kadela-Tomanek, Monika; Latocha, Małgorzata; Jelsch, Christian; Wenger, Emmanuel; Boryczka, Stanisław

    2016-08-26

    Betulin derivatives are a widely studied group of compounds of natural origin due to their wide spectrum of biological activities. This paper describes new betulin derivatives, containing a phosphonate group. The allyl-vinyl isomerization and synthesis of acetylenic derivatives have been reported. Structural identification of products as E and Z isomers has been carried out using ¹H-, (13)C-, (31)P-NMR, and crystallographic analysis. The crystal structure in the orthorhombic space group and analysis of crystal packing contacts for 29-diethoxyphosphoryl-28-cyclopropylpropynoyloxy-lup-20E(29)-en-3β-ol 8a are reported. All new compounds were tested in vitro for their antiproliferative activity against human T47D (breast cancer), SNB-19 (glioblastoma), and C32 (melanoma) cell lines.

  15. A rapid micro-assay method for gelatinolytic activity using tritium-labeled heat-denatured polymeric collagen as a substrate and its application to the detection of enzymes involved in collagen metabolism.

    PubMed

    Sunada, H; Nagai, Y

    1980-06-01

    A rapid micro-assay method for gelatinolytic activitiy has been developed using 3H-labeled heat-denatured polymeric collagen (gelatin) as a substrate to investigate enzymes involved in the post-collagenase catabolism of collagen. The method is based on the incubation of gelatin with enzyme followed by determination of the enzyme digestion products soluble in 67% dioxane. It is sensitive enough to detect microgram levels of gelatin fragments, and can be employed over wide ranges of pH and ionic strength. By applying the method to an embryonic chick skin culture system, three gelatinolytic enzyme fractions which showed high, limited and no caseinolytic activities were demonstrated to be separable by gel chromatography.

  16. Development and application of a new Silent reporter system to quantitate the activity of enhancer elements in the type II Collagen Gene.

    PubMed

    Ito, Kazuo; Shinomura, Tamayuki

    2016-07-01

    Type II collagen is a major component of cartilage, which provide structural stiffness to the tissue. As a sufficient amount of type II collagen is critical for maintaining the biomechanical properties of cartilage, its expression is tightly regulated in chondrocytes. Therefore, it is essential to elucidate in detail the transcriptional mechanism that controls expression of type II collagen, in particular by two enhancer elements we recently discovered. To systematically analyze and compare enhancer activities, we developed a novel reporter assay system that exploits site-specific integration of promoter and enhancer elements to activate a transcriptionally silent reporter gene. Using this system, we found that the enhancer elements have distinct characteristics, with one exhibiting additive effects and the other exhibiting synergistic effects when repeated in tandem.

  17. Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

    PubMed Central

    Weis, MaryAnn; Rai, Jyoti; Hudson, David M.; Dimori, Milena; Zimmerman, Sarah M.; Hogue, William R.; Swain, Frances L.; Burdine, Marie S.; Mackintosh, Samuel G.; Tackett, Alan J.; Suva, Larry J.; Eyre, David R.

    2016-01-01

    Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis. PMID:27119146

  18. Withaferin-A Reduces Type I Collagen Expression In Vitro and Inhibits Development of Myocardial Fibrosis In Vivo

    PubMed Central

    Challa, Azariyas A.; Vukmirovic, Milica; Blackmon, John; Stefanovic, Branko

    2012-01-01

    Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited in vitro activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis. PMID:22900077

  19. Cartilage collagen type II seromarker patterns in axial spondyloarthritis and psoriatic arthritis: associations with disease activity, smoking and HLA-B27.

    PubMed

    Munk, Heidi Lausten; Gudmann, Natasja Staehr; Christensen, Anne Friesgaard; Ejstrup, Leif; Sorensen, Grith Lykke; Loft, Anne Gitte; Bay-Jensen, Anne C; Siebuhr, Anne Sofie; Junker, Peter

    2016-04-01

    The aim of the study was to assess the possible association between type II collagen turnover seromarkers and disease profile in patients with axial spondyloarthritis (SpA) and psoriatic arthritis (PsA). Outpatients with axial SpA (n = 110) or PsA (n = 101) underwent clinical examination including disease activity measures and HLA-B27 typing. The procollagen IIA N-terminal peptide (PIIANP) and a matrix metalloproteinase-generated type II collagen fragment (C2M) were quantified in serum by ELISA. C2M was higher in SpA than in controls, 0.41 versus 0.36 ng/ml (p = 0.004), while PIIANP did not differ between patients and healthy subjects, 2252 versus 2142 ng/ml (p = 0.13). However, DMARD-naïve SpA patients had higher PIIANP, 2461 ng/ml (p = 0.01) and C2M, 0.44 ng/ml (p = 0.0007) levels than controls, and PIIANP correlated with CRP (ρ = 0.34). C2M was lower in SpA smokers, 0.36 ng/ml versus non-smokers, 0.43 ng/ml (p = 0.02), while PIIANP was higher in HLA-B27 positive, 2312 ng/ml versus negative patients, 2021 ng/ml (p = 0.03). In PsA, PIIANP and C2M did not differ between patients and controls, but PIIANP was elevated in patients not receiving DMARDs, 2726 ng/ml. In PsA, PIIANP and C2M did not differ according to smoking and HLA-B27. Cartilage degradation assessed by C2M is increased in SpA irrespective of treatment but not in PsA. Cartilage synthesis reflected by PIIANP is increased in untreated SpA and PsA. PIIANP correlates with CRP in SpA while not in PsA. In DMARD-naïve SpA but not in PsA, HLA-B27 positivity and smoking are associated with a chondro-proliferative metabolic pattern.

  20. Collagenous colitis.

    PubMed Central

    Kingham, J G; Levison, D A; Morson, B C; Dawson, A M

    1986-01-01

    Clinical and pathological aspects of six patients with collagenous colitis are presented. These patients have been observed for between four and 15 years and the evolution of the condition is documented in three (cases 1, 3 and 5). Management and possible pathogenetic mechanisms of this enigmatic condition are discussed. Images Fig. 1 Fig. 2 PMID:3699567

  1. Collagenous gastritis.

    PubMed

    Jin, Xiaoyi; Koike, Tomoyuki; Chiba, Takashi; Kondo, Yutaka; Ara, Nobuyuki; Uno, Kaname; Asano, Naoki; Iijima, Katsunori; Imatani, Akira; Watanabe, Mika; Shirane, Akio; Shimosegawa, Tooru

    2013-09-01

    In the present paper, we report a case of rare collagenous gastritis. The patient was a 25-year-old man who had experienced nausea, abdominal distention and epigastralgia since 2005. Esophagogastroduodenoscopy (EGD) carried out at initial examination by the patient's local doctor revealed an extensively discolored depression from the upper gastric body to the lower gastric body, mainly including the greater curvature, accompanied by residual mucosa with multiple islands and nodularity with a cobblestone appearance. Initial biopsies sampled from the nodules and accompanying atrophic mucosa were diagnosed as chronic gastritis. In August, 2011, the patient was referred to Tohoku University Hospital for observation and treatment. EGD at our hospital showed the same findings as those by the patient's local doctor. Pathological findings included a membranous collagen band in the superficial layer area of the gastric mucosa, which led to a diagnosis of collagenous gastritis. Collagenous gastritis is an extremely rare disease, but it is important to recognize its characteristic endoscopic findings to make a diagnosis.

  2. Collagen-Gelatin Mixtures as Wound Model, and Substrates for VEGF-Mimetic Peptide Binding and Endothelial Cell Activation

    PubMed Central

    Chan, Tania R.; Stahl, Patrick J.; Li, Yang; Yu, S. Michael

    2015-01-01

    In humans, high level of collagen remodeling is seen during normal physiological events such as bone renewal, as well as in pathological conditions, such as arthritis, tumor growth and other chronic wounds. Our lab recently discovered that collagen mimetic peptide (CMP) is able to hybridize with denatured collagens at these collagen remodeling sites with high affinity. Here, we show that the CMP's high binding affinity to denatured collagens can be utilized to deliver angiogenic signals to scaffolds composed of heat-denatured collagens (gelatins). We first demonstrate hybridization between denatured collagens and QKCMP, a CMP with pro-angiogenic QK domain. We show that high levels of QKCMP can be immobilized to a new artificial matrix containing both fibrous type I collagen and heat denatured collagen through triple helix hybridization, and that the QKCMP is able to stimulate early angiogenic response of endothelial cells (ECs). We also show that the QKCMP can bind to excised tissues from burn injuries in cutaneous mouse model, suggesting its potential for promoting neovascularization of burn wounds. PMID:25584990

  3. Adipose tissue macrophages in insulin-resistant subjects are associated with collagen VI and fibrosis and demonstrate alternative activation.

    PubMed

    Spencer, Michael; Yao-Borengasser, Aiwei; Unal, Resat; Rasouli, Neda; Gurley, Catherine M; Zhu, Beibei; Peterson, Charlotte A; Kern, Philip A

    2010-12-01

    Adipose tissue macrophages are associated with insulin resistance and are linked to changes in the extracellular matrix. To better characterize adipose macrophages, the extracellular matrix, and adipocyte-macrophage interactions, gene expression from adipose tissue and the stromal vascular fraction was assessed for markers of inflammation and fibrosis, and macrophages from obese and lean subjects were counted and characterized immunohistochemically. Coculture experiments examined the effects of adipocyte-macrophage interaction. Collagen VI gene expression was associated with insulin sensitivity and CD68 (r = -0.56 and 0.60, P < 0.0001) and with other markers of inflammation and fibrosis. Compared with adipose tissue from lean subjects, adipose tissue from obese subjects contained increased areas of fibrosis, which correlated inversely with insulin sensitivity (r = -0.58, P < 0.02) and positively with macrophage number (r = 0.70, P < 0.01). Although macrophages in crownlike structures (CLS) were more abundant in obese adipose tissue, the majority of macrophages were associated with fibrosis and were not organized in CLS. Macrophages in CLS were predominantly M1, but most other macrophages, particularly those in fibrotic areas, were M2 and also expressed CD150, a marker of M2c macrophages. Coculture of THP-1 macrophages with adipocytes promoted the M2 phenotype, with a lower level of IL-1 expression and a higher ratio of IL-10 to IL-12. Transforming growth factor-β (TGF-β) was more abundant in M2 macrophages and was further increased by coculture with adipocytes. Downstream effectors of TGF-β, such as plasminogen activator inhibitor-1, collagen VI, and phosphorylated Smad, were increased in macrophages and adipocytes. Thus adipose tissue of insulin-resistant humans demonstrated increased fibrosis, M2 macrophage abundance, and TGF-β activity.

  4. Ultraviolet irradiation induces CYR61/CCN1, a mediator of collagen homeostasis, through activation of transcription factor AP-1 in human skin fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Xu, Yiru; He, Tianyuan; Kang, Sewon; Voorhees, John J; Fisher, Gary J

    2010-06-01

    UV irradiation from the sun elevates the production of collagen-degrading matrix metalloproteinases (MMPs) and reduces the production of new collagen. This imbalance of collagen homeostasis impairs the structure and function of the dermal collagenous extracellular matrix (ECM), thereby promoting premature skin aging (photoaging). We report here that aberrant dermal collagen homeostasis in UV-irradiated human skin is mediated in part by a CCN-family member, cysteine-rich protein-61 (CYR61/CCN1). CYR61 is significantly elevated in acutely UV-irradiated human skin in vivo, and UV-irradiated human skin fibroblasts. Knockdown of CYR61 significantly attenuates UV irradiation-induced inhibition of type-I procollagen and upregulation of MMP-1. Determination of CYR61 mRNA and protein indicates that the primary mechanism of CYR61 induction by UV irradiation is transcriptional. Analysis of CYR61 proximal promoter showed that a sequence conforming to the consensus binding site for transcription factor activator protein-1 (AP-1) is required for promoter activity. UV irradiation increased the binding of AP-1-family members c-Jun and c-Fos to this AP-1 site. Furthermore, functional blockade of c-Jun or knockdown of c-Jun significantly reduced the UV irradiation-induced activation of CYR61 promoter and CYR61 gene expression. These data show that CYR61 is transcriptionally regulated by UV irradiation through transcription factor AP-1, and mediates altered collagen homeostasis that occurs in response to UV irradiation in human skin fibroblasts.

  5. Activation of the platelet collagen receptor integrin alpha(2)beta(1): its mechanism and participation in the physiological functions of platelets.

    PubMed

    Jung, S M; Moroi, M

    2000-10-01

    When platelets are stimulated by agonists, integrin alpha(2)beta(1) (GP Ia/IIa), one of the platelet collagen receptors, is activated to forms with high affinities for its ligand collagen. Here we describe our studies to characterize the binding kinetics of the activated integrin forms and the activation mechanism. Under low agonist concentrations, integrin alpha(2)beta(1) is activated through a mechanism involving ADP/ADP receptors; and under high agonist concentrations, multiple signaling pathways are involved in its activation. Such differences in mechanism at low and high agonist concentrations are also suggested in the activation of integrin alpha(IIb)beta(3), the platelet fibrinogen receptor. We describe our flow adhesion studies, from which evidence was obtained about the involvement of integrin alpha(2)beta(1) activation in the physiological function of platelets, adhesion and thrombus formation.

  6. Involvement of H- and N-Ras isoforms in transforming growth factor-{beta}1-induced proliferation and in collagen and fibronectin synthesis

    SciTech Connect

    Martinez-Salgado, Carlos . E-mail: carloms@usal.es; Fuentes-Calvo, Isabel; Garcia-Cenador, Begona; Santos, Eugenio; Lopez-Novoa, Jose M.

    2006-07-01

    Transforming growth factor {beta}1 (TGF-{beta}1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-{beta} and Ras signaling pathways are closely related: TGF-{beta}1 overcomes Ras mitogenic effects and Ras counteracts TGF-{beta} signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-{beta}1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras {sup -/-}/N-ras {sup -/-}) isoforms and from heterozygote mice (H-ras {sup +/-}/N-ras {sup +/-}). ECM synthesis is increased in basal conditions in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts, this increase being higher after stimulation with TGF-{beta}1. TGF-{beta}1-induced fibroblast proliferation is smaller in H-ras {sup -/-}/N-ras {sup -/-} than in H-ras {sup +/-}/N-ras {sup +/-} fibroblasts. Erk activation is decreased in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.

  7. Anti-inflammatory activities of light emitting diode irradiation on collagen-induced arthritis in mice (a secondary publication)

    PubMed Central

    Ohta, Mitsuhiro; Sato, Yusuke; Abiko, Yoshimitsu

    2014-01-01

    Background and aims: Rheumatoid arthritis (RA) is an auto-immune disease afflicting multiple joints of the body, where as a result of the increase in inflammatory cytokines and tissue destructive factors such as matrix metalloproteinase (MMP)-3, deterioration of the bones and cartilages of the joints occurs. The present investigation was carried out to study the anti-inflammatory activities of light emitting diode (LED) irradiation on hind paw inflammation in collagen-induced arthritis (CIA) mice models. Materials and method: RA in the CIA mouse model was induced by immunization of DBA/1J mice with intradermal injections of an emulsion of bovine type II collagen and complete Freund's adjuvant. A total of 20 CIA mice were subdivided into the following groups: control group, CIA group and 2 groups of LED irradiated CIA mice (LED groups) (n=5 per group). The mouse knee joint area in the LED groups (the 570 nm and 940 nm groups) was irradiated with LED energy, three times a week for 500 s per session over 8 weeks at a dose of 5 J/cm2. The hind paw swelling was assessed by the increase in hind paw thickness. The serum levels of the inflammatory cytokines and arthritic factor MMP-3 were determined with an enzyme-linked immunosorbent assay (ELISA). Results: In the LED-570 and LED-940 groups at 4 weeks after arthritis induction, the swelling inhibition index was 18.1±4.9 and 29.3±4.0 respectively. Interleukin (IL)-1β, IL-6 and MMP-3 serum levels were significantly lower in the LED-940 group. Conclusions: LED irradiation, particularly in the near-infrared was effective for inhibition of the inflammatory reactions caused by RA. PMID:25368445

  8. Synthesis of α-collagen fragments and research of their influence on the degree of hydration of a model of epidermis

    PubMed Central

    Grobelna, Beata; Maćkiewicz, Zbigniew

    2013-01-01

    Introduction In recent years the interest into areas of science, such as cosmetology, dermatology, pharmacology or aesthetic medicine has increased significantly. Scientists are more frequently looking for ingredients that affect the skin's condition and slow down the aging process. Practically every year, the scientists discover a number of new chemical substances (both natural and synthetic) that can be potentially used to manufacture cosmetics. Aim To evaluate the influence of selected peptides derived from α-collagen fragments on the degree of hydration of a model of epidermis isolated from a pig. Material and methods The synthesis of selected cosmetic oligopeptides were performed manually, on the solid medium, using procedure of SPPS (solid phase peptide synthesis). Following components: aqua, carbomer, glycerine, phenonip, D-panthenol, dimethicone and triethanolamine were used to prepare a reference hydrogel masks. Both the number of components and the composition of hydrogels have been developed individually for the purposes of this research. For this study the skin from a domestic pig was used. The degree of the skin hydration was measured with the SKINTEST plus camera, which uses the latest semiconductor technology. Results During the study the absorption of hydrogels with peptides was faster than that of the reference hydrogel mask. The combination of hydrophilic properties of the peptide with hydrophobic properties of Palm enabled receiving an amphiphilic structure. Such molecules are considered to be able to penetrate the corneum barrier with the greatest ease. Conclusions The results showed that the modified compounds have contributed to water retention in the cells, thereby increasing the degree of hydration of the biological material. PMID:24278040

  9. Hyperhomocysteinemia accelerates collagen accumulation in the adventitia of balloon-injured rat carotid arteries via angiotensin II type 1 receptor.

    PubMed

    Yao, Dan; Sun, Ning-Ling

    2014-10-27

    Recent studies suggest that hyperhomocysteinemia (HHcy) increases collagen type I accumulation in rat vascular adventitia after balloon injury and that Angiotensin II (Ang II) induces collagen synthesis in vascular adventitial fibroblasts. Reports also indicate that Ang II type1 receptor (AT1R) activation, mediated by homocysteine (Hcy) may contribute to collagen type 1 expression in mouse aortic endothelial cells. However, little is known about the possible mechanisms behind the relationship between Hcy and AT1R in adventitial remodeling. Thus, we investigated whether HHcy induces collagen accumulation via activation of AT1R in the adventitia. Male Sprague-Dawley (SD) rats were randomly divided into a control group and a 1% l-methionine-induced HHcy group. Balloon injury was performed after 12 experimental weeks and animals were sacrificed at 7, 14, and 28 days after injury. Collagen deposition and AT1R expression was measured with Western blot. Serum Hcy, adventitial collagen, and AT1R levels were higher in the HHcy group compared with the control group. Hcy time-dependently induced collagen type 1 and AT1R expression, with the highest induction observed at 48 h. Also, we observed that the AT1R blocker, valsartan, attenuated collagen type 1 and AT1R expression. HHcy exacerbates adventitial remodeling after balloon injury, and the underling mechanisms may be related to AT1R activity.

  10. Detection of active matrix metalloproteinase-3 in serum and fibroblast-like synoviocytes of collagen-induced arthritis mice.

    PubMed

    Lee, Aeju; Choi, Sung-Jae; Park, Kyeongsoon; Park, Jong Woong; Kim, Kwangmeyung; Choi, Kuiwon; Yoon, Soo-Young; Youn, Inchan

    2013-06-19

    The activity of rheumatoid arthritis (RA) correlates with the expression of proteases. Among several proteases, matrix metalloproteinase-3 (MMP-3) is one of the biological markers used to diagnose RA. The active form of MMP-3 is a key enzyme involved in RA-associated destruction of cartilage and bone. Thus, detection of active MMP-3 in serum or in vivo is very important for early diagnosis of RA. In this study, a soluble MMP-3 probe was prepared to monitor RA progression by detecting expression of active MMP-3 in collagen-induced arthritis (CIA) mice in vivo in both serum and fibroblast-like synoviocytes (FLSs). The MMP-3 probe exhibited strong sensitivity to MMP-3 and moderate sensitivity to MMP-7 at nanomolecular concentrations, but was not sensitive to other MMPs such as MMP-2, MMP-9, and MMP-13. In an optical imaging study, the MMP-3 probe produced early and strong NIR fluorescence signals prior to observation of erythema and swelling in CIA mice. The MMP-3 probe was able to rapidly and selectively detect and monitor active MMP-3 in diluted serum from CIA mice. Furthermore, histological data demonstrated that activated FLSs in arthritic knee joints expressed active MMP-3. Together, our results demonstrated that the MMP-3 probe may be useful for detecting active MMP-3 for diagnosis of RA. More importantly, the MMP-3 probe was able to detect active MMP-3 in diluted serum with high sensitivity. Therefore, the MMP-3 probe developed in this study may be a very promising probe, useful as a biomarker for early detection and diagnosis of RA.

  11. Collagen I confers gamma radiation resistance.

    PubMed

    Azorin, E; González-Martínez, P R; Azorin, J

    2012-12-01

    The effect of collagen on the response of somatomammotroph tumor cells (GH3) to gamma, radiation therapy was studied in vitro. After incubating confluent GH3 cell monolayers in a serum-free, maintaining medium, either with or without collagen, the monolayers were irradiated with 137Cs, gamma radiation. Collagen reduces cell mortality via ERK1/2 activation, abolishing gamma radiation, cell death, and promotes cell invasion when acting in synergy with collagen and in association with the, MAPK/ERK1/2 signaling pathway activation. The presence of collagen in somatomammotroph tumors, confers resistance to radiation.

  12. Exosomes from hypoxic endothelial cells have increased collagen crosslinking activity through up-regulation of lysyl oxidase-like 2.

    PubMed

    de Jong, Olivier G; van Balkom, Bas W M; Gremmels, Hendrik; Verhaar, Marianne C

    2016-02-01

    Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations, research on exosome-ECM interactions is limited. Here, we investigate whether the exosome-associated lysyl oxidase family member lysyl oxidase-like 2 (LOXL2) is involved in ECM remodelling. We found that LOXL2 is present on the exterior of endothelial cell (EC)-derived exosomes, placing it in direct vicinity of the ECM. It is up-regulated twofold in EC-derived exosomes cultured under hypoxic conditions. Intact exosomes from hypoxic EC and LOXL2 overexpressing EC show increased activity in a fluorometric lysyl oxidase enzymatic activity assay as well as in a collagen gel contraction assay. Concordantly, knockdown of LOXL2 in exosome-producing EC in both normal and hypoxic conditions reduces activity of exosomes in both assays. Our findings show for the first time that ECM crosslinking by EC-derived exosomes is mediated by LOXL2 under the regulation of hypoxia, and implicate a role for exosomes in hypoxia-regulated focal ECM remodelling, a key process in both fibrosis and wound healing.

  13. Cystamine immobilization on TiO 2 film surfaces and the influence on inhibition of collagen-induced platelet activation

    NASA Astrophysics Data System (ADS)

    Zhou, Yujuan; Weng, Yajun; Zhang, Liping; Jing, Fengjuan; Huang, Nan; Chen, Junying

    2011-12-01

    Poor haemocompatibility is a main issue of artificial cardiovascular materials in clinical application. Nitric oxide (NO), produced by vascular endothelial cells, is a well known inhibitor of platelet adhesion and activation. Thus, NO-releasing biomaterials are beneficial for improving haemocompatibility of blood-contacting biomedical devices. In this paper, a novel method was developed for enhancement of haemocompatibility by exploiting endogenous NO donors. TiO 2 films were firstly synthesized on Si (1 0 0) wafers via unbalanced magnetron sputtering technology, and then polydopamine was grafted on TiO 2 films and used as a linker for further immobilization of cystamine. The obtained surfaces were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis. NO generation is evaluated by saville-griess reagents, and it shows that cystamine immobilized samples are able to catalytically generate NO by decomposing endogenous S-nitrosothiols (RSNO). In vitro platelet adhesion results reveal that cystamine modified surfaces can inhibit collagen-induced platelet activation. ELISA analysis reveals that cGMP in platelets obviously increases on cystamine immobilized surface, which suggests the reducing of platelet activation is through NO/cGMP signal channel. It can be concluded that cystamine immobilized surface shows better blood compatibility by catalyzing NO release from the endogenous NO donor. It may be a promising method for improvement of haemocompatibility of blood-contacting implants.

  14. Jellyfish collagen stimulates production of TNF-α and IL-6 by J774.1 cells through activation of NF-κB and JNK via TLR4 signaling pathway.

    PubMed

    Putra, Agus Budiawan Naro; Nishi, Kosuke; Shiraishi, Ryusuke; Doi, Mikiharu; Sugahara, Takuya

    2014-03-01

    We previously reported that jellyfish collagen stimulates both the acquired and innate immune responses. In the acquired immune response, jellyfish collagen enhanced immunoglobulin production by lymphocytes in vitro and in vivo. Meanwhile, in the innate immune response jellyfish collagen promoted cytokine production and phagocytotic activity of macrophages. The facts that jellyfish collagen plays several potential roles in stimulating cytokine production by macrophages have further attracted us to uncover its mechanisms. We herein describe that the cytokine production-stimulating activity of jellyfish collagen was canceled by a Toll-like receptor 4 (TLR4) inhibitor. Moreover, jellyfish collagen stimulated phosphorylation of inhibitor of κBα (IκBα), promoted the translocation of nucleus factor-κB (NF-κB), and activated c-Jun N-terminal kinase (JNK). A JNK inhibitor also abrogated the cytokine production-stimulating activity of jellyfish collagen. These results suggest that jellyfish collagen may facilitate cytokine production by macrophages through activation of NF-κB and JNK via the TLR4 signaling pathways.

  15. Distinct regions control transcriptional activation of the alpha1(VI) collagen promoter in different tissues of transgenic mice

    PubMed Central

    1996-01-01

    To identify regions involved in tissue specific regulation of transcription of the alpha1(VI) collagen chain, transgenic mice were generated carrying various portions of the gene's 5'-flanking sequence fused to the E. coli beta-galactosidase gene. Analysis of the transgene expression pattern by X-gal staining of embryos revealed that: (a) The proximal 0.6 kb of promoter sequence activated transcription in mesenchymal cells at sites of insertion of superficial muscular aponeurosis into the skin; tendons were also faintly positive. (b) The region between -4.0 and -5.4 kb from the transcription start site was required for activation of the transgene in nerves. It also drove expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (c) The fragment comprised within -6.2 and -7.5 kb was necessary for high level transcription in skeletal muscle and meninges. Positive cells in muscle were mostly mononuclear and probably included connective tissue elements, although staining of myoblasts was not ruled out. This fragment also activated expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (d) beta-Galactosidase staining in vibrissae induced by the sequences -4.0 to -5.4 and -6.2 to -7.5 was not coincident: with the latter sequence labeled nuclei were found mainly in the ventral and posterior quadrant, and, histologically, in the outer layers of mesenchyme surrounding and between the follicles, whereas with the former the remaining quadrants were positive and expressing cells were mostly in the inner layers of the dermal sheath. (e) Other tissues, notably lung, adrenal gland, digestive tract, which produce high amounts of collagen type VI, did not stain for beta-galactosidase. (f) Central nervous system and retina, in which the endogenous gene is inactive, expressed the lacZ transgene in most lines. The data suggest that transcription of alpha1(VI) in different tissues is regulated by distinct sequence

  16. MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix

    SciTech Connect

    Takino, Takahisa; Tsuge, Hisashi; Ozawa, Terumasa; Sato, Hiroshi

    2010-06-11

    Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show here that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21{sup WAF1} and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin {alpha}{sub v}{beta}{sub 3} were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin.

  17. Synthesis of (-)-arctigenin derivatives and their anticancer activity.

    PubMed

    Gui-Rong, Chen; Li-Ping, Cai; De-Qiang, Dou; Ting-Guo, Kang; Hong-Fu, Li; Fu-Rui, Li; Ning, Jiang

    2012-01-01

    The natural dibenzylbutyrolactone type lignanolide (-)-arctigenin, which was prepared from fructus arctii, showed obvious anticancer activity. The synthesis of four new (-)-arctigenin derivatives and their anticancer bioactivities were examined. The structures of the four new synthetic derivatives were elucidated.

  18. Synthesis and in vitro antiproliferative activities of quinoline derivatives.

    PubMed

    Broch, Sidonie; Aboab, Bettina; Anizon, Fabrice; Moreau, Pascale

    2010-04-01

    The synthesis of new di- and trimeric quinoline derivatives is described as well as their in vitro antiproliferative activities toward a human fibroblast primary culture and two human solid cancer cell lines (MCF-7 and PA 1).

  19. Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure.

    PubMed

    Murthy, Shubha; Larson-Casey, Jennifer L; Ryan, Alan J; He, Chao; Kobzik, Lester; Carter, A Brent

    2015-08-01

    Alternative activation of alveolar macrophages is linked to fibrosis following exposure to asbestos. The scavenger receptor, macrophage receptor with collagenous structure (MARCO), provides innate immune defense against inhaled particles and pathogens; however, a receptor for asbestos has not been identified. We hypothesized that MARCO acts as an initial signaling receptor for asbestos, polarizes macrophages to a profibrotic M2 phenotype, and is required for the development of asbestos-induced fibrosis. Compared with normal subjects, alveolar macrophages isolated from patients with asbestosis express higher amounts of MARCO and have greater profibrotic polarization. Arginase 1 (40-fold) and IL-10 (265-fold) were higher in patients. In vivo, the genetic deletion of MARCO attenuated the profibrotic environment and pulmonary fibrosis in mice exposed to chrysotile. Moreover, alveolar macrophages from MARCO(-/-) mice polarize to an M1 phenotype, whereas wild-type mice have higher Ym1 (>3.0-fold) and nearly 7-fold more active TGF-β1 in bronchoalveolar lavage (BAL) fluid (BALF). Arg(432) and Arg(434) in domain V of MARCO are required for the polarization of macrophages to a profibrotic phenotype as mutation of these residues reduced FIZZ1 expression (17-fold) compared with cells expressing MARCO. These observations demonstrate that a macrophage membrane protein regulates the fibrotic response to lung injury and suggest a novel target for therapeutic intervention.

  20. Discoidin domain receptor 2 inhibits fibrillogenesis of collagen type 1.

    PubMed

    Mihai, Cosmin; Iscru, Daniel F; Druhan, Lawrence J; Elton, Terry S; Agarwal, Gunjan

    2006-09-01

    Discoidin domain receptors (DDR1 and DDR2) are widely expressed cell-surface receptors, which bind to and are activated by collagens, including collagen type 1. Activation of DDRs and the resulting downstream signaling is known to regulate the extracellular matrix. However, little is known about how DDRs interact with collagen and its direct impact on collagen regulation. Here, we have established that by binding to collagen, the extracellular domain (ECD) of DDR2 inhibits collagen fibrillogenesis and alters the morphology of collagen type 1 fibers. Our in vitro assays utilized DDR2-Fc fusion proteins, which contain only the ECD of DDR2. Using surface plasmon resonance, we confirmed that further oligomerization of DDR2-Fc (by means of anti-Fc antibody) greatly enhances its binding to immobilized collagen type 1. Collagen turbidity measurements and biochemical assays indicated that DDR2 delays the formation of collagen fibrils. Atomic force microscopy of soluble collagen revealed that a predominately monomeric state of collagen was present with DDR2, while control solutions had an abundance of polymeric collagen. Transmission electron microscopy of collagen fibers, showed that the native periodic banded structure of collagen fibers was weakened and nearly absent in the presence of DDR2. Further, using a cell-based assay we demonstrate that overexpression of full length DDR2 inhibits fibrillogenesis of collagen type 1. Our results demonstrate a novel and important functional role of the DDR2 ECD that may contribute to collagen regulation via modulation of fibrillogenesis.

  1. Integrin alpha1beta1 controls reactive oxygen species synthesis by negatively regulating epidermal growth factor receptor-mediated Rac activation.

    PubMed

    Chen, Xiwu; Abair, Tristin D; Ibanez, Maria R; Su, Yan; Frey, Mark R; Dise, Rebecca S; Polk, D Brent; Singh, Amar B; Harris, Raymond C; Zent, Roy; Pozzi, Ambra

    2007-05-01

    Integrins control many cell functions, including generation of reactive oxygen species (ROS) and regulation of collagen synthesis. Mesangial cells, found in the glomerulus of the kidney, are able to produce large amounts of ROS via the NADPH oxidase. We previously demonstrated that integrin alpha1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by alpha1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin alpha1-null mesangial cells produce excessive ROS. Integrin alpha1-null mesangial cells have constitutively increased basal levels of activated Rac1, which result in its increased translocation to the cell membrane, excessive ROS production, and consequent collagen IV deposition. Basal Rac1 activation is a direct consequence of ligand-independent increased epidermal growth factor receptor (EGFR) phosphorylation in alpha1-null mesangial cells. Thus, our study demonstrates that integrin alpha1beta1-EGFR cross talk is a key step in negatively regulating Rac1 activation, ROS production, and excessive collagen synthesis, which is a hallmark of diseases characterized by irreversible fibrosis.

  2. Measurement of whole body cellular and collagen nitrogen, potassium, and other elements by neutron activation and whole body counting

    SciTech Connect

    James, H.M.; Fabricius, P.J.; Dykes, P.W.

    1987-09-01

    Whole body nitrogen can be measured by neutron activation analysis with an acceptable radiation dose; it is an index of body protein which, in normal subjects, is 65% cellular protein and 35% extracellular connective collagen. Whole body potassium can be measured by whole body counting without irradiating the subject; it is an index of body cell mass. We measured whole body nitrogen, potassium, extracellular water, intracellular water, and fat-folds. The differences between 37 malnourished patients and five normal subjects suggested that the patients had 9 kg less cell mass than normal, but no difference in extracellular mass. Measurements were made on eight patients before and after 14 days of total parenteral nutrition; balance of nitrogen intake and excretion also was measured. The changes were consistent with mean increases of 3 kg of cellular mass and 3 kg of fat with no change of extracellular mass. The accuracy and sensitivity of the whole body measurements need further confirmation for use in patients with changing body composition. Where tissue wasting is largely from the cellular compartment, potassium could be a more sensitive index of wasting than nitrogen. Multielement analysis of nitrogen, potassium, chlorine, and carbon will probably be valuable in elucidating body composition in malnutrition.

  3. Serum- and Growth-Factor-Free Three-Dimensional Culture System Supports Cartilage Tissue Formation by Promoting Collagen Synthesis via Sox9–Col2a1 Interaction

    PubMed Central

    Ahmed, Nazish; Iu, Jonathan; Brown, Chelsea E.; Taylor, Drew Wesley

    2014-01-01

    Objective: One of the factors preventing clinical application of regenerative medicine to degenerative cartilage diseases is a suitable source of cells. Chondrocytes, the only cell type of cartilage, grown in vitro under culture conditions to expand cell numbers lose their phenotype along with the ability to generate hyaline cartilaginous tissue. In this study we determine that a serum- and growth-factor-free three-dimensional (3D) culture system restores the ability of the passaged chondrocytes to form cartilage tissue in vitro, a process that involves sox9. Methods: Bovine articular chondrocytes were passaged twice to allow for cell number expansion (P2) and cultured at high density on 3D collagen-type-II-coated membranes in high glucose content media supplemented with insulin and dexamethasone (SF3D). The cells were characterized after monolayer expansion and following 3D culture by flow cytometry, gene expression, and histology. The early changes in signaling transduction pathways during redifferentiation were characterized. Results: The P2 cells showed a progenitor-like antigen profile of 99% CD44+ and 40% CD105+ and a gene expression profile suggestive of interzone cells. P2 in SF3D expressed chondrogenic genes and accumulated extracellular matrix. Downregulating insulin receptor (IR) with HNMPA-(AM3) or the PI-3/AKT kinase pathway (activated by insulin treatment) with Wortmannin inhibited collagen synthesis. HNMPA-(AM3) reduced expression of Col2, Col11, and IR genes as well as Sox6 and -9. Co-immunoprecipitation and chromatin immunoprecipitation analyses of HNMPA-(AM3)-treated cells showed binding of the coactivators Sox6 and Med12 with Sox9 but reduced Sox9–Col2a1 binding. Conclusions: We describe a novel culture method that allows for increase in the number of chondrocytes and promotes hyaline-like cartilage tissue formation in part by insulin-mediated Sox9–Col2a1 binding. The suitability of the tissue generated via this approach for use in joint

  4. IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kappa B ligand/osteoprotegerin balance.

    PubMed

    Lubberts, Erik; van den Bersselaar, Liduine; Oppers-Walgreen, Birgitte; Schwarzenberger, Paul; Coenen-de Roo, Christina J J; Kolls, Jay K; Joosten, Leo A B; van den Berg, Wim B

    2003-03-01

    IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-kappa B ligand (RANKL)/receptor activator of NF-kappa B/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-kappa B, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.

  5. Type I Collagen and Collagen Mimetics as Angiogenesis Promoting Superpolymers

    SciTech Connect

    Twardowski, T.; Fertala, A.; Orgel, J.P.R.O.; San Antonio, J.D.

    2008-07-18

    Angiogenesis, the development of blood vessels from the pre-existing vasculature, is a key component of embryogenesis and tissue regeneration. Angiogenesis also drives pathologies such as tumor growth and metastasis, and hemangioma development in newborns. On the other hand, promotion of angiogenesis is needed in tissues with vascular insufficiencies, and in bioengineering, to endow tissue substitutes with appropriate microvasculatures. Therefore, much research has focused on defining mechanisms of angiogenesis, and identifying pro- and anti-angiogenic molecules. Type I collagen, the most abundant protein in humans, potently stimulates angiogenesis in vitro and in vivo. Crucial to its angiogenic activity appears to be ligation and possibly clustering of endothelial cell (EC) surface {alpha}1{beta}1/{alpha}2{beta}1 integrin receptors by the GFPGER502-507 sequence of the collagen fibril. However, additional aspects of collagen structure and function that may modulate its angiogenic properties are discussed. Moreover, type I collagen and fibrin, another angiogenic polymer, share several structural features. These observations suggest strategies for creating 'angiogenic superpolymers', including: modifying type I collagen to influence its biological half-life, immunogenicity, and integrin binding capacity; genetically engineering fibrillar collagens to include additional integrin binding sites or angiogenic determinants, and remove unnecessary or deleterious sequences without compromising fibril integrity; and exploring the suitability of poly(ortho ester), PEG-lysine copolymer, tubulin, and cholesteric cuticle as collagen mimetics, and suggesting means of modifying them to display ideal angiogenic properties. The collagenous and collagen mimetic angiogenic superpolymers described here may someday prove useful for many applications in tissue engineering and human medicine.

  6. MT1-MMP and Type II Collagen Specify Skeletal Stem Cells and Their Bone and Cartilage Progeny

    PubMed Central

    Szabova, Ludmila; Yamada, Susan S.; Wimer, Helen; Chrysovergis, Kaliopi; Ingvarsen, Signe; Behrendt, Niels; Engelholm, Lars H.

    2009-01-01

    Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic activity associated with the membrane-type 1 matrix metalloproteinase (MT1-MMP) results in disrupted skeletal development and growth in both cartilage and bone, where MT1-MMP is required for pericellular collagen dissolution. We show here that reconstitution of MT1-MMP activity in the type II collagen–expressing cells of the skeleton rescues not only diminished chondrocyte proliferation, but surprisingly, also results in amelioration of the severe skeletal dysplasia associated with MT1-MMP deficiency through enhanced bone formation. Consistent with this increased bone formation, type II collagen was identified in bone cells and skeletal stem/progenitor cells of wildtype mice. Moreover, bone marrow stromal cells isolated from mice expressing MT1-MMP under the control of the type II collagen promoter in an MT1-MMP–deficient background showed enhanced bone formation in vitro and in vivo compared with cells derived from nontransgenic MT1-MMP–deficient littermates. These observations show that type II collagen is not stringently confined to the chondrocyte but is expressed in skeletal stem/progenitor cells (able to regenerate bone, cartilage, myelosupportive stroma, marrow adipocytes) and in the chondrogenic and osteogenic lineage progeny where collagenolytic activity is a requisite for proper cell and tissue function. PMID:19419317

  7. Abnormal response to physical activity in femurs after heterozygous inactivation of one allele of the Col2a1 gene for type II collagen in mice.

    PubMed

    Nieminen, J; Sahlman, J; Hirvonen, T; Jämsä, T; Tuukkanen, J; Kovanen, V; Kröger, H; Jurvelin, J; Arita, M; Li, S W; Prockop, D J; Hyttinen, M M; Helminen, H J; Lapveteläinen, T; Puustjärvi, K

    2005-08-01

    The objective of this study was to evaluate the influence of heterozygous inactivation of one allele of the type II collagen gene (Col2a1) on biomechanical properties and mineral density of bone under physical loading conditions. C57BL/6-TGN mice with heterozygous knockout (HZK) inactivation of Col2a1 gene and their nontransgenic littermate controls were housed in individual cages with running wheels for 9 and 15 months. The running activity of each mouse was monitored continuously throughout the experiment. Bone mineral density (BMD) of mice femora was measured using dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computerized tomography (pQCT). Biomechanical properties were determined using three-point bending tests. Vertebral bone samples were prepared for quantitative polarized light microscopy and digital densitometry of proteoglycans. The concentration of total collagen and collagen cross-links were analyzed using high-performance liquid chromatograpy (HPLC). The average daily running distance was shorter for the HZK mice between the age of 4 and 15 months as compared with normal runners (P < 0.05). The ultimate breaking force was 14.8% and 23.6% (9 vs. 15 months) lower in HZK-runners than in wild-type runners. BMD of the femur was 6.1% lower in HZK-runners at the age of 9 months (P < 0.05). Physical activity increased cortical BMD in wild-type runners but not in the HZK runners at the age of 9 months. The collagen network of the HZK mice was less organized. There were only minor changes in BMD and mechanical and structural properties between sedentary HZK mice and their wild-type controls. Increased physical activity induced significantly lower bone density, mechanical properties, and organization of collagen fibers in male HZK mice. However, there were no major differences in biomechanical parameters between sedentary HZK and wild-type male mice. This suggests an important guiding role of collagen type II in bone remodelling and

  8. Second harmonic imaging and scoring of collagen in fibrotic tissues

    NASA Astrophysics Data System (ADS)

    Strupler, M.; Pena, A.-M.; Hernest, M.; Tharaux, P.-L.; Martin, J.-L.; Beaurepaire, E.; Schanne-Klein, M.-C.

    2007-04-01

    We compare second harmonic generation (SHG) to histological and immunohistochemical techniques for the visualization and scoring of collagen in biological tissues. We show that SHG microscopy is highly specific for fibrillar collagens and that combined SHG and two-photon excited fluorescence (2PEF) imaging can provide simultaneous three-dimensional visualization of collagen synthesis and assembly sites in transgenic animal models expressing GFP constructs. Finally, we propose several scores for characterizing collagen accumulation based on SHG images and appropriate for different types of collagen distributions. We illustrate the sensitivity of these scores in a murine model of renal fibrosis using a morphological segmentation of the tissue based on endogenous 2PEF signals.

  9. Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells.

    PubMed Central

    Stefanovic, B; Hellerbrand, C; Holcik, M; Briendl, M; Aliebhaber, S; Brenner, D A

    1997-01-01

    The hepatic stellate cell (HSC) is the primary cell responsible for the dramatic increase in the synthesis of type I collagen in the cirrhotic liver. Quiescent HSCs contain a low level of collagen alpha1(I) mRNA, while activated HSCs contain about 60- to 70-fold more of this mRNA. The transcription rate of the collagen alpha1(I) gene is only two fold higher in activated HSCs than in quiescent HSCs. In assays using actinomycin D or 5,6-dichlorobenzimidazole riboside collagen alpha1(I) mRNA has estimated half-lives of 1.5 h in quiescent HSCs and 24 h in activated HSCs. Thus, this 16-fold change in mRNA stability is primarily responsible for the increase in collagen alpha1(I) mRNA steady-state level in activated HSCs. We have identified a novel RNA-protein interaction targeted to the C-rich sequence in the collagen alpha1(I) mRNA 3' untranslated region (UTR). This sequence is localized 24 nucleotides 3' to the stop codon. In transient transfection experiments, mutation of this sequence diminished accumulation of an mRNA transcribed from a collagen alpha1(I) minigene and in stable transfections decreased the half-life of collagen alpha1(I) minigene mRNA. Binding to the collagen alpha1(I) 3' UTR is present in cytoplasmic extracts of activated but not quiescent HSCs. It contains as a subunit alphaCP, which is also found in the complex involved in stabilization of alpha-globin mRNA. The auxiliary factors necessary to promote binding of alphaCP to the collagen 3' UTR are distinct from the factors necessary for binding to the alpha-globin sequence. Since alphaCP is expressed in both quiescent and activated HSCs, these auxiliary factors are responsible for the differentially expressed RNA-protein interaction at the collagen alpha1(I) mRNA 3' UTR. PMID:9271398

  10. Optimal cooperative control synthesis of active displays

    NASA Technical Reports Server (NTRS)

    Garg, S.; Schmidt, D. K.

    1986-01-01

    The utility of augmenting displays to aid the human operator in controlling high order complex systems is well known. Analytical evaluation of various display designs for a simple k/s sup 2 plant in a compensatory tracking task using an optimal Control Model (OCM) of human behavior is carried out. This analysis reveals that significant improvement in performance should be obtained by skillful integration of key information into the display dynamics. The cooperative control synthesis technique previously developed to design pilot-optimal control augmentation is extended to incorporate the simultaneous design of performance enhancing augmented displays. The application of the cooperative control synthesis technique to the design of augmented displays is discussed for the simple k/s sup 2 plant. This technique is intended to provide a systematic approach to design optimally augmented displays tailored for specific tasks.

  11. Optimal cooperative control synthesis of active displays

    NASA Technical Reports Server (NTRS)

    Garg, S.; Schmidt, D. K.

    1985-01-01

    The utility of augmenting displays to aid the human operator in controlling high order complex systems is well known. Analytical evaluations of various display designs for a simple k/s-squared plant in a compensatory tracking task using an Optimal Control Model (OCM) of human behavior is carried out. This analysis reveals that significant improvement in performance should be obtained by skillful integration of key information into the display dynamics. The cooperative control synthesis technique previously developed to design pilot-optimal control augmentation is extended to incorporate the simultaneous design of performance enhancing augmented displays. The application of the cooperative control synthesis technique to the design of augmented displays is discussed for the simple k/s-squared plant. This technique is intended to provide a systematic approach to design optimally augmented displays tailored for specific tasks.

  12. Comparative Study of Collagen versus Synthetic-Based Meniscal Scaffolds in Treating Meniscal Deficiency in Young Active Population

    PubMed Central

    Bulgheroni, Erica; Grassi, Alberto; Campagnolo, Monica; Bulgheroni, Paolo; Mudhigere, Abhishek; Gobbi, Alberto

    2016-01-01

    Purpose: The aim of this study was to compare the effectiveness of 2 different meniscal scaffolds in treating patients with irreparable partial medial meniscal tear and patients complaining of pain in the medial compartment of the knee due to a previous partial medial meniscectomy. Based on previous studies, we hypothesized that both the scaffolds are effective in improving clinical outcomes in these patient populations. Material and Methods: Twenty-eight patients underwent collagen-based medial meniscus implantation (CMI-Menaflex) and 25 with a second-generation scaffold (Actifit). All patients were assessed with Lysholm, Tegner scale, and MRI evaluation—preoperatively, at 6 months, at 12 moths, and followed-up for a minimum of 2 years. Second look arthroscopy and concomitant biopsy were performed in 7 and 12 patients of CMI and Actifit groups, respectively. Results: The CMI group at final follow-up showed improvement in Lysholm score from 58.4 ± 17.3 to 94.5 ± 6.0, while the Actifit group showed improvement from 67.0 ± 15.7 to 90.3 ± 13.1; the improvement was statistically significant in both the groups but intergroup difference was not statistically significant (P = 0.1061). Tegner Activity Scale score improved in both the groups, but intergroup difference was not statistically significant (P = 0.5918). MRI evaluation showed in situ scaffold and no progression of degenerative arthritis in both the groups at final follow-up. Histological evaluation showed more fibrous tissue with blood vessels in the CMI group and the Actift group showed avascular cartilaginous features. Conclusion: Both the scaffolds are effective in improving patients’ symptoms and joint function at short-term follow-up. PMID:26958315

  13. Activation of catalysts for synthesizing methanol from synthesis gas

    DOEpatents

    Blum, David B.; Gelbein, Abraham P.

    1985-01-01

    A method for activating a methanol synthesis catalyst is disclosed. In this method, the catalyst is slurried in an inert liquid and is activated by a reducing gas stream. The activation step occurs in-situ. That is, it is conducted in the same reactor as is the subsequent step of synthesizing methanol from a methanol gas stream catalyzed by the activated catalyst still dispersed in a slurry.

  14. A Novel Functional Role of Collagen Glycosylation

    PubMed Central

    Jürgensen, Henrik J.; Madsen, Daniel H.; Ingvarsen, Signe; Melander, Maria C.; Gårdsvoll, Henrik; Patthy, Laszlo; Engelholm, Lars H.; Behrendt, Niels

    2011-01-01

    Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation. PMID:21768090

  15. Amiloride, protein synthesis, and activation of quiescent cells.

    PubMed

    Lubin, M; Cahn, F; Coutermarsh, B A

    1982-11-01

    Amiloride is known to inhibit both influx of sodium ions and activation of quiescent cells by growth factors. The coincidence of these effects has been cited to support the proposal that influx of sodium ions acts as a mitogenic signal. Although it was noted that amiloride inhibited protein synthesis, this was attributed to an action on transport of amino acids, particularly those coupled to sodium fluxes. We find, however, that amiloride directly inhibits polypeptide synthesis in a reticulocyte lysate. In Swiss 3T3 cells, concentrations of amiloride and of cycloheximide that are nearly matched in their degree of inhibition of protein synthesis, produce about the same degree of inhibition of transit of cells from G0 to S. Inhibition of protein synthesis is sufficient to explain the effect of amiloride on mitogenesis; the drug, therefore, is not suitable for testing the hypothesis that sodium influx is a mitogenic signal.

  16. Expression in SPARC-null mice of collagen type I lacking the globular domain of the α1(I) N-propeptide results in abdominal hernias and loss of dermal collagen.

    PubMed

    Card, Lauren; Henderson, Nikki; Zhang, Yuhua; Bornstein, Paul; Bradshaw, Amy D

    2010-09-01

    The sequence encoding the N-propeptide of collagen I is characterized by significant conservation of amino acids across species; however, the function of the N-propeptide remains poorly defined. Studies in vitro have suggested that one activity of this propeptide might be to act as a feedback inhibitor of collagen I synthesis. To determine whether the N-propeptide contributed to decreased collagen content in SPARC-null mice, mice carrying a deletion of exon 2, which encodes the globular domain of the N-propeptide of collagen I, were crossed to SPARC-null animals. Mice lacking SPARC and expressing collagen I without the globular domain of the N-propeptide were viable and fertile. However, a significant number of animals developed abdominal hernias within the first 2 months of life with an approximate 20% penetrance (~35% of males). The dermis of SPARC-null/exon 2-deleted mice was thinner and contained fewer large collagen fibers in comparison with wild-type or in either single transgenic animal. The average collagen fibril diameter of exon 2-deleted mice did not significantly differ from wild-type mice (WT: 87.9 nm versus exon 2-deleted: 88.2 nm), whereas SPARC-null/exon 2-deleted fibrils were smaller than that of SPARC-null dermis (SPARC-null: 60.2 nm, SPARC-null/exon 2-deleted: 40.8 nm). As measured by hydroxyproline analysis, double transgenic skin biopsies contained significantly less collagen than those of wild-type, those of exon 2-deleted, and those of SPARC-null biopsies. Acetic acid extraction of collagen from skin biopsies revealed an increase in the proportion of soluble collagen in the SPARC-null/exon 2-deleted mice. These results support a function of the N-propeptide of collagen I in facilitating incorporation and stabilization of collagen I into the insoluble ECM and argue against a primary function of the N-propeptide as a negative regulator of collagen synthesis.

  17. Three-dimensional, bioactive, biodegradable, polymer-bioactive glass composite scaffolds with improved mechanical properties support collagen synthesis and mineralization of human osteoblast-like cells in vitro.

    PubMed

    Lu, Helen H; El-Amin, Saadiq F; Scott, Kimberli D; Laurencin, Cato T

    2003-03-01

    In the past decade, tissue engineering-based bone grafting has emerged as a viable alternative to biological and synthetic grafts. The biomaterial component is a critical determinant of the ultimate success of the tissue-engineered graft. Because no single existing material possesses all the necessary properties required in an ideal bone graft, our approach has been to develop a three dimensional (3-D), porous composite of polylactide-co-glycolide (PLAGA) and 45S5 bioactive glass (BG) that is biodegradable, bioactive, and suitable as a scaffold for bone tissue engineering (PLAGA-BG composite). The objectives of this study were to examine the mechanical properties of a PLAGA-BG matrix, to evaluate the response of human osteoblast-like cells to the PLAGA-BG composite, and to evaluate the ability of the composite to form a surface calcium phosphate layer in vitro. Structural and mechanical properties of PLAGA-BG were measured, and the formation of a surface calcium phosphate layer was evaluated by surface analysis methods. The growth and differentiation of human osteoblast-like cells on PLAGA-BG were also examined. A hypothesis was that the combination of PLAGA with BG would result in a biocompatible and bioactive composite, capable of supporting osteoblast adhesion, growth and differentiation, with mechanical properties superior to PLAGA alone. The addition of bioactive glass granules to the PLAGA matrix resulted in a structure with higher compressive modulus than PLAGA alone. Moreover, the PLAGA-BA composite was found to be a bioactive material, as it formed surface calcium phosphate deposits in a simulated body fluid (SBF), and in the presence of cells and serum proteins. The composite supported osteoblast-like morphology, stained positively for alkaline phosphatase, and supported higher levels of Type I collagen synthesis than tissue culture polystyrene controls. We have successfully developed a degradable, porous, polymer bioactive glass composite possessing

  18. Synthesis and antitumour activity of 4-aminoquinazoline derivatives

    NASA Astrophysics Data System (ADS)

    Lipunova, G. N.; Nosova, E. V.; Charushin, V. N.; Chupakhin, O. N.

    2016-07-01

    Pieces of data on the synthesis and antitumour activity of 4-aminoquinazolines are summarized and analyzed. Key methods for the synthesis of these compounds are considered, primarily cyclocondensation of carboxylic acid derivatives, as well as the oxidation of quinazolines and the cyclization of disubstituted thioureas. Improvements of synthetic schemes for erlotinib, gefitinib and lapatinib, which are the best-known pharmaceuticals based on compounds of the title class, are also considered. Synthetic strategies and biological activities for new 4-aminoquinazoline derivatives that are EGFR-tyrosine kinase inhibitors, multiactive compounds, and labelled compounds for use as positron emission tomography (PET) imaging agents are discussed. The bibliography includes 263 references.

  19. Catalytic Activation of Nitrogen Dioxide for Selective Synthesis of Nitroorganics

    DTIC Science & Technology

    2015-01-15

    AFRL-OSR-VA-TR-2015-0035 Catalytic activation of nitrogen dioxide for selective synthesis SETH BROWN UNIVERSITY OF NOTRE DAME DU LAC Final Report 01...8-98) v Prescribed by ANSI Std. Z39.18 12-01-2015 Final 15 Aug 2011 - 14 Aug 2014 Catalytic activation of nitrogen dioxide for selective synthesis...reductive elimination of the nitroarene has not. Nitrogen dioxide can be used as a source of the nitro group in reactions with arylboronic acids or their

  20. Structure-function analysis of peroxidasin provides insight into the mechanism of collagen IV crosslinking.

    PubMed

    Lázár, Enikő; Péterfi, Zalán; Sirokmány, Gábor; Kovács, Hajnal A; Klement, Eva; Medzihradszky, Katalin F; Geiszt, Miklós

    2015-06-01

    Basement membranes provide structural support and convey regulatory signals to cells in diverse tissues. Assembly of collagen IV into a sheet-like network is a fundamental mechanism during the formation of basement membranes. Peroxidasin (PXDN) was recently described to catalyze crosslinking of collagen IV through the formation of sulfilimine bonds. Despite the significance of this pathway in tissue genesis, our understanding of PXDN function is far from complete. In this work we demonstrate that collagen IV crosslinking is a physiological function of mammalian PXDN. Moreover, we carried out structure-function analysis of PXDN to gain a better insight into its role in collagen IV synthesis. We identify conserved cysteines in PXDN that mediate the oligomerization of the protein into a trimeric complex. We also demonstrate that oligomerization is not an absolute requirement for enzymatic activity, but optimal collagen IV coupling is only catalyzed by the PXDN trimers. Localization experiments of different PXDN mutants in two different cell models revealed that PXDN oligomers, but not monomers, adhere on the cell surface in "hot spots," which represent previously unknown locations of collagen IV crosslinking.

  1. Pyrophosphate-condensing activity linked to nucleic acid synthesis.

    PubMed Central

    Volloch, V Z; Rits, S; Tumerman, L

    1979-01-01

    In some preparations of DNA dependent RNA polymerase a new enzymatic activity has been found which catalyzes the condensation of two pyrophosphate molecules, liberated in the process of RNA synthesis, to one molecule of orthophosphate and one molecule of Mg (or Mn) - chelate complex with trimetaphosphate. This activity can also cooperate with DNA-polymerase, on condition that both enzymes originate from the same cells. These results point to two general conclusions. First, energy is conserved in the overall process of nucleic acid synthesis and turnover, so that the process does not require an energy influx from the cell's general resources. Second, the synthesis of nucleic acids is catalyzed by a complex enzyme system which contains at least two separate enzymes, one responsible for nucleic acid polymerization and the other for energy conservation via pyrophosphate condensation. Images PMID:88040

  2. Activation of formylmethanofurna synthesis in cell extracts of Methanobacterium thermoautotrophicum

    SciTech Connect

    Bobik, T.A.; Wolfe, R.S. )

    1989-03-01

    In cell extracts of Methanobacterium thermoautotrophicum, formylmethanofuran (formyl-MFR) synthesis (an essential CO{sub 2} fixation reaction that is an early step in CO{sub 2} reduction to methane) is subject to a complex activation that involves a heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine O{sup 3}-phosphate (CoM-S-S-HTP). In this paper we report that titanium(III) citrate, a low-potential reducing agent, stimulated CO{sub 2} reduction to methane and activated formyl-MFR synthesis in cell extracts. Titanium(III) citrate functioned as the sole source of electrons for formyl-MFR synthesis and enabled this reaction to occur independently of CoM-S-S-HTP. In addition, CoM-S-S-HTP was found to activate an unknown electron carrier that reduced metronidazole. The activation of formyl-MFR synthesis by CoM-S-S-HTP may involve the activation of a low-potential electron carrier.

  3. Optimal cooperative control synthesis of active displays

    NASA Technical Reports Server (NTRS)

    Garg, S.; Schmidt, D. K.

    1985-01-01

    A technique is developed that is intended to provide a systematic approach to synthesizing display augmentation for optimal manual control in complex, closed-loop tasks. A cooperative control synthesis technique, previously developed to design pilot-optimal control augmentation for the plant, is extended to incorporate the simultaneous design of performance enhancing displays. The technique utilizes an optimal control model of the man in the loop. It is applied to the design of a quickening control law for a display and a simple K/s(2) plant, and then to an F-15 type aircraft in a multi-channel task. Utilizing the closed loop modeling and analysis procedures, the results from the display design algorithm are evaluated and an analytical validation is performed. Experimental validation is recommended for future efforts.

  4. Optimal cooperative control synthesis of active displays

    NASA Technical Reports Server (NTRS)

    Gary, Sanjay; Schmidt, David K.

    1987-01-01

    A technique is developed that is intended to provide a systematic approach to synthesizing display augmentation for optimal manual control in complex, closed-loop tasks. A cooperative control synthesis technique, previously developed to design pilot-optimal control augmentation for the plant, is extended to incorporate the simultaneous design of performance enhancing displays. The technique utilizes an optimal control model of the man in the loop. It is applied to the design of a quickening control law for a display and a simple K/(s squared) plant, and then to an F-15 type aircraft in a multichannel task. Utilizing the closed-loop modeling and analysis procedures, the results from the display design algorithm are evaluated and an analytical validation is performed. Experimental validation is recommended for future efforts.

  5. Quantity change in collagen following 830-nm diode laser welding

    NASA Astrophysics Data System (ADS)

    Tang, Jing; O'Callaghan, David; Rouy, Simone; Godlewski, Guilhem; Prudhomme, Michel

    1996-12-01

    The actual mechanism for production of laser welding of tissue is presently unknown, but collagen plays an important role is tissue welded after laser irradiance. The quantity change in collagen extracted from the abdominal aorta of Wistar rats after tissue welding using an 830 nm diode laser was investigated. The collagen contents following repeated pepsin digestion after acetic acid extraction were determined with Sircol collagen assay. Compared with untreated aorta, the collagen content of the treated vessel was obvious decreased immediately after laser irradiation and following an initial increase on day 3, there was a peak at day 10. The results suggest that a part of collagen molecules is denatured by the heat of laser. There is an effect of stimulating collagen synthesis after laser welding with parameters used in this study.

  6. Motualevic Acids and Analogs: Synthesis and Antimicrobial Structure Activity Relationships

    PubMed Central

    Cheruku, Pradeep; Keffer, Jessica L.; Dogo-Isonagie, Cajetan; Bewley, Carole A.

    2010-01-01

    Synthesis of the marine natural products motualevic acids A, E, and analogs in which modifications have been made to the ω-brominated lipid (E)-14,14-dibromotetra-deca-2,13-dienoic acid or amino acid unit are reported, together with antimicrobial activities against Staphylococcus aureus, methicillin-resistant S. aureus, Enterococcus faecium, and vancomycin-resistant Enterococcus. PMID:20538459

  7. Synthesis and antimicrobial activity of some novel dicationic sulphonophanes.

    PubMed

    Rajakumar, Perumal; Satheeshkumar, Chinnadurai; Mohanraj, Gunasekaran; Mathivanan, Narayanasamy

    2011-07-01

    The synthesis of some novel imidazole-based dicationic sulfonophanes incorporating various spacer units is described. All the sulphonophanes exhibit good antibacterial and antifungal activity against five bacterial strains Bacillus subtilis, Staphylococcus aureus, Vibrio cholera, Escherichia coli, Proteus vulgaris and human pathogenic fungus Candida albicans.

  8. Chitosan nanoparticles enhance the antibacterial activity of chlorhexidine in collagen membranes used for periapical guided tissue regeneration.

    PubMed

    Barreras, Uriel Soto; Méndez, Fernando Torres; Martínez, Rita Elizabeth Martínez; Valencia, Carolina Samano; Rodríguez, Panfilo Raymundo Martinez; Rodríguez, Juan Pablo Loyola

    2016-01-01

    Endodontic failure is mainly associated with the persistence of microbial infection in the root canal system and/or the periradicular area. Microorganisms and their toxins located in the root canal system may trigger apical periodontitis and tissue destruction. Tissue regeneration in periapical surgery by using membrane barriers has shown an improved healing and bone closure. However, bacterial membrane contamination is a main reason of failure. In this in vitro study, different brands of chlorhexidine, a combination of chitosan nanoparticles containing chlorhexidine were tested against Enterococcus faecalis on agar plate's cultures and infected collagen membranes. Our results indicated that chitosan nanoparticles acted synergistically with chlorhexidine, inhibiting and eliminating significantly a greater amount of colony former units in both BHI-agar cultures and infected collagen membranes. These results suggested that chitosan nanoparticles could be used to improve regenerative procedures in periapical surgery.

  9. Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells.

    PubMed

    Lee, Hak Joo; Mariappan, Meenalakshmi M; Feliers, Denis; Cavaglieri, Rita C; Sataranatarajan, Kavithalakshmi; Abboud, Hanna E; Choudhury, Goutam Ghosh; Kasinath, Balakuntalam S

    2012-02-10

    Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

  10. Curcuminoids extract, hydrolyzed collagen and green tea extract synergically inhibit inflammatory and catabolic mediator's synthesis by normal bovine and osteoarthritic human chondrocytes in monolayer.

    PubMed

    Comblain, Fanny; Sanchez, Christelle; Lesponne, Isabelle; Balligand, Marc; Serisier, Samuel; Henrotin, Yves

    2015-01-01

    The main objective of this study was to assess the in vitro effects of curcuminoids extract, hydrolyzed collagen and green tea extract in normal bovine chondrocytes and osteoarthritic human chondrocytes cultured in monolayer. This study also investigated the synergic or additive effects of these compounds. Enzymatically isolated primary bovine or human chondrocytes were cultured in monolayer until confluence and then incubated for 24 hours or 48 hours in the absence or in the presence of interleukin-1β and with or without curcuminoids extract, hydrolyzed collagen or green tea extract, added alone or in combination, at different concentrations. Cell viability was neither affected by these compounds, nor by interleukin 1β. In the absence of interleukin-1β, compounds did not significantly affect bovine chondrocytes metabolism. In human chondrocytes and in the absence of interleukin 1β, curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract significantly inhibited matrix metalloproteinase-3 production. In interleukin-1β-stimulated bovine chondrocytes, interleukin-6, inducible nitric oxide synthase, cyclooxygenase2, matrix metalloproteinase 3, a disintegrin and metalloproteinase with thrombospondin type I motifs 4 and a disintegrin and metalloproteinase with thrombospondin type I motifs 5 expressions were decreased by curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract. The combination of the three compounds was significantly more efficient to inhibit interleukin-1β stimulated matrix metalloproteinase-3 expression than curcuminoids extract alone. In interleukin-1β-stimulated human chondrocytes, nitric oxide, interleukin-6 and matrix metalloproteinase 3 productions were significantly reduced by curcuminoids extract alone or in combination with hydrolyzed collagen and green tea extract. These findings indicate that a mixture of curcuminoids extract, hydrolyzed collagen and green tea

  11. Adopting the principles of collagen biomineralization for intrafibrillar infiltration of yttria-stabilized zirconia into three-dimensional collagen scaffolds

    PubMed Central

    Zhou, Bin; Niu, Li-na; Shi, Wei; Zhang, Wei; Arola, Dwayne D.; Breschi, Lorenzo; Mao, Jing; Pashley, David H.

    2014-01-01

    In this paper, we report a process for generating collagen-yttria-stabilized amorphous zirconia hybrid scaffolds by introducing acetylacetone-inhibited zirconia precursor nanodroplets into a poly(allylamine)-coated collagen matrix. This polyelectrolyte coating triggers intrafibrillar condensation of the precursors into amorphous zirconia, which is subsequently transformed into tetragonal yttria-stabilized zirconia after calcination. Our findings represent a new paradigm in the synthesis of non-naturally occurring collagen-based hybrid scaffolds under alcoholic mineralizing conditions. PMID:25477773

  12. SPARC regulates collagen interaction with cardiac fibroblast cell surfaces.

    PubMed

    Harris, Brett S; Zhang, Yuhua; Card, Lauren; Rivera, Lee B; Brekken, Rolf A; Bradshaw, Amy D

    2011-09-01

    Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [(3)H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α(1)(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.

  13. Synthesis of actively adjustable springs by antagonistic redundant actuation

    NASA Technical Reports Server (NTRS)

    Yi, Byung-Ju; Freeman, Robert A.

    1992-01-01

    A methodology for active spring generation is presented based on antagonistic redundant actuation. Antagonistic properties are characterized using an effective system stiffness. 'Antagonistic stiffness' is generated by preloading a closed-chain (parallel) linkage system. Internal load distribution is investigated along with the necessary conditions for spring synthesis. The performance and stability of a proposed active spring are shown by simulation, and applications are discussed.

  14. Activated matrix metalloproteinase and disrupted myocardial collagen matrix in increased sympathetic activity following stimulation of dorsal medulla in the vagotomized feline model.

    PubMed

    Cheng, Ching-Chang; Tung, Kwong-Chung; Fu, Yun-Ching; Gong, Chi-Li; Chen, Ying-Tsung; Lin, Nai-Nu; Lin, James A; Chiu, Yung-Tsung

    2008-02-29

    Sympathetic hyperactivation in many kinds of neurocardiogenic injury can result in obvious heart failure. We generated a vagotomized feline model in which sympathetic hyperactivation was induced by electrical stimulation of dorsal medulla (ESDM) of brain stem to investigate the relationship between disruption of extracellular collagen matrix (ECM) and activation of matrix metalloproteinases (MMPs) in myocardium in the sympathetic hyperactivity. Mean blood pressure, heart rate and plasma norepinephrine were all significantly increased from baseline to a peak at 5 min after ESDM. Echocardiographic study showed significant left ventricular dilatation and hypokinesia (ejection fraction: from 87.7 +/- 6.3% to 39.4 +/- 7.8%) from baseline to 180 mm after ESDM. Histopathological finding revealed significant overstretching or spring-like disappearance and disruption of ECM. MMP-2 expression was significantly increased in left ventricular myocardium as compared to sham. These results suggest that ESDM-induced sympathetic hyperactivity causes the expression of MMP-2 that disrupts myocardial ECM, contributing to the development of cardiac dysfunction.

  15. Synthesis and activities of antitumor agents.

    PubMed

    Suami, T; Machinami, T; Hisamatsu, T

    1979-03-01

    N-(2-Chloroethyl)-N-nitrosocarbamoyl derivatives of glycosylamines have been prepared. Six N-(2-chloroethyl)-N-nitrosoureas, including three disaccharide derivatives, were submitted to a determination of antitumor activity. All the compounds tested exhibited strong antitumor activity against leukemia L1210 in mice.

  16. Collagen-mediated hemostasis.

    PubMed

    Manon-Jensen, T; Kjeld, N G; Karsdal, M A

    2016-03-01

    Collagens mediate essential hemostasis by maintaining the integrity and stability of the vascular wall. Imbalanced turnover of collagens by uncontrolled formation and/or degradation may result in pathologic conditions such as fibrosis. Thickening of the vessel wall because of accumulation of collagens may lead to arterial occlusion or thrombosis. Thinning of the wall because of collagen degradation or deficiency may lead to rupture of the vessel wall or aneurysm. Preventing excessive hemorrhage or thrombosis relies on collagen-mediated actions. Von Willebrand factor, integrins and glycoprotein VI, as well as clotting factors, can bind collagen to restore normal hemostasis after trauma. This review outlines the essential roles of collagens in mediating hemostasis, with a focus on collagens types I, III, IV, VI, XV, and XVIII.

  17. Biomedical applications of collagens.

    PubMed

    Ramshaw, John A M

    2016-05-01

    Collagen-based biomedical materials have developed into important, clinically effective materials used in a range of devices that have gained wide acceptance. These devices come with collagen in various formats, including those based on stabilized natural tissues, those that are based on extracted and purified collagens, and designed composite, biosynthetic materials. Further knowledge on the structure and function of collagens has led to on-going developments and improvements. Among these developments has been the production of recombinant collagen materials that are well defined and are disease free. Most recently, a group of bacterial, non-animal collagens has emerged that may provide an excellent, novel source of collagen for use in biomaterials and other applications. These newer collagens are discussed in detail. They can be modified to direct their function, and they can be fabricated into various formats, including films and sponges, while solutions can also be adapted for use in surface coating technologies.

  18. Imaging Prostate Cancer Microenvironment by Collagen Hybridization

    DTIC Science & Technology

    2013-10-01

    Dyn. 237, 2607−2621. (3) von der Mark, H., von der Mark, K., and Gay , S. (1976) Study of differential collagen synthesis during development of the...GFP encoding plasmid DNA through electrostatic interactions and enhance gene transfection (adapted from Ref. [52]). www.sciencedirect.com Current

  19. Synthesis and Activity of Biomimetic Biofilm Disruptors

    PubMed Central

    2013-01-01

    Biofilms are often associated with human bacterial infections, and the natural tolerance of biofilms to antibiotics challenges treatment. Compounds with antibiofilm activity could become useful adjuncts to antibiotic therapy. We used norspermidine, a natural trigger for biofilm disassembly in the developmental cycle of Bacillus subtilis, to develop guanidine and biguanide compounds with up to 20-fold increased potency in preventing biofilm formation and breaking down existing biofilms. These compounds also were active against pathogenic Staphylococcus aureus. An integrated approach involving structure–activity relationships, protonation constants, and crystal structure data on a focused synthetic library revealed that precise spacing of positively charged groups and the total charge at physiological pH distinguish potent biofilm inhibitors. PMID:23406351

  20. Synthesis and antimalarial activity of prodigiosenes.

    PubMed

    Marchal, Estelle; Smithen, Deborah A; Uddin, Md Imam; Robertson, Andrew W; Jakeman, David L; Mollard, Vanessa; Goodman, Christopher D; MacDougall, Kristopher S; McFarland, Sherri A; McFadden, Geoffrey I; Thompson, Alison

    2014-06-28

    Several analogues of the natural compound prodigiosin with modified A- and C-rings were synthesised as were some of their tin, cobalt, boron and zinc complexes. The antimalarial activity of these prodigiosenes was evaluated in vitro using the 3D7 Plasmodium falciparum strain. The presence of a nitrogen atom in the A-ring is needed for antimalarial activity but the presence of an alkyl group at the β'-position of the C-ring seems detrimental. Dibutyl tin complexes exhibit IC50 values mostly in the nanomolar range with equal or improved activity compared to the free-base prodigiosene ligand, despite the fact that the general toxicity of such tin complexes is demonstrably lower than that of the free-bases.

  1. Synthesis and trypanocidal activity of novel benzimidazole derivatives.

    PubMed

    Velázquez-López, José Miguel; Hernández-Campos, Alicia; Yépez-Mulia, Lilián; Téllez-Valencia, Alfredo; Flores-Carrillo, Paulina; Nieto-Meneses, Rocío; Castillo, Rafael

    2016-09-01

    The present work reports the synthesis and biological activity of a series of 14 benzimidazole derivatives designed to act on the enzyme triosephosphate isomerase of Trypanosoma cruzi (TcTIM). This enzyme is involved in the metabolism of glucose, the only source of energy for the parasite. In this study, we found four compounds that inhibit TcTIM moderately and lack inhibitory activity against human TIM (HsTIM). In vitro studies against T. cruzi epimastigotes showed two compounds that were more active than the reference drug nifurtimox, and these presented a low cytotoxic effect in mouse macrophages (J744 cell line).

  2. Synthesis and fungicidal activity of a series of novel aryloxylepidines.

    PubMed

    Kirby, N V; Daeuble, J F; Davis, L N; Hannum, A C; Hellwig, K; Lawler, L K; Parker, M H; Pieczko, M E

    2001-09-01

    A series of novel (hetero) aryloxylepidine derivatives was devised as hybrid structures of the phenoxyquinoline and phenethoxyquin(az)oline fungicides. Synthesis of these targets required the development of several new routes to derivatised 4-hydroxymethylquinolines, and subsequent coupling with phenols or haloarenes. The aryloxylepidines generally showed moderate broad-spectrum fungicidal activity across several diseases of cereals. Substitution of the quinoline ring with chlorine at the 7- and/or 5-positions gave molecules with high levels of protectant activity against Erysiphe graminis f sp tritici (powdery mildew of wheat), but this did not improve the level of fungicidal activity against other diseases. In vitro activity against mitochondrial electron transport complex I (MET) derived from Ustilago maydis showed that 8-fluorolepidine analogues were moderately active at this target site, while the more fungicidally active 7- and 5,7-substituted compounds were inactive. This indicates that MET is not the primary target of these highly active powdery mildewicides.

  3. Synthesis of active nitroguaiacol ether derivatives of streptomycin.

    PubMed Central

    Abad, J P; Amils, R

    1990-01-01

    The synthesis, purification, and biological properties of nitroguaiacol ether derivatives of streptomycin and their corresponding radioactive reduced products were examined. These derivatives are biologically active against gram-positive and gram-negative eubacteria and they are also photoreactive because of the presence of the nitroguaiacol group in the molecule. We demonstrated that these derivatives can be used as streptomycin analogs in photoaffinity labeling of the macromolecular structures related to the mode of action of the antibiotic. Images PMID:2127172

  4. [Autoimmune mechanisms toward type I collagen during parodontitis].

    PubMed

    2014-04-01

    The article presents original data about autoimmune mechanisms according to the severity of the process during the chronic generalized Parodontitis. The medical examination of 179 patients with different forms of Parodontitis demonstrated that during Parodontitis the synthesis and re-synthesis of type I Collagen is negatively affected and as a result, type I Collagen concentration in the blood serum raises. In addition, the selection of antigen reactive lymphocytes increases toward Type I Collagen, which in turn boosts their quantity in blood and also stimulates the creation of auto-antibodies toward Type I Collagen. This is reflected by the increased quantity of auto-antibodies in the blood serum. The intensity of these processes amplifies as the Parodontitis inflammation becomes more severe. These results demonstrate that the autoimmune process develops toward Type I Collagen during Parodontitis and its intensity reflects the severity of the pathological processes in Parodontitis.

  5. Hydrogen sulfide alleviates myocardial collagen remodeling in association with inhibition of TGF-β/Smad signaling pathway in spontaneously hypertensive rats.

    PubMed

    Sun, Lili; Jin, Hongfang; Sun, Lujing; Chen, Siyao; Huang, Yaqian; Liu, Jia; Li, Zhenzhen; Zhao, Manman; Sun, Yan; Tang, Chaoshu; Zhao, Bin; Du, Junbao

    2015-01-20

    The study was designed to explore the role and possible mechanisms of hydrogen sulfide (H2S) in the regulation of myocardial collagen remodeling in spontaneously hypertensive rats (SHRs). We treated nine-week-old male SHRs and age- and sex-matched Wistar-Kyoto rats (WKYs) with NaHS (90 μmol/kg(-1)·day(-1)) for 9 wks. At 18 wks, plasma H2S, tail arterial pressure, morphology of the heart, myocardial ultrastructure and collagen volume fraction (CVF), myocardial expressions of collagen I and III protein and procollagen I and III mRNA, transforming growth factor-β1 (TGF-β1), TGF-β type I receptor (TβR-I), type II receptor (TβR-II), p-Smad2 and 3, matrix metalloproteinase (MMP)-13 and tissue inhibitors of MMP (TIMP)-1 proteins were determined. TGF-β1-stimulated cultured cardiac fibroblasts (CFs) were used to further study the mechanisms. The results showed that compared with WKYs, SHRs showed a reduced plasma H2S, elevated tail artery pressure and increased myocardial collagen, TGF-β1, TβR-II, p-Smad2 and p-Smad3 expressions. However, NaHS markedly decreased tail artery pressure and inhibited myocardial collagen, TGF-β1, TβR-II, p-Smad2 and p-Smad3 protein expressions, but H2S had no effect on the expressions of MMP-13 and TIMP-1. Hydralazine reduced blood pressure but had no effect on myocardial collagen, MMP-13 and TIMP-1 expressions and TGF-β1/Smad signaling pathway. H2S prevented activation of the TGF-β1/Smad signaling pathway and abnormal collagen synthesis in CFs. In conclusion, the results suggested that H2S could prevent myocardial collagen remodeling in SHR. The mechanism might be associated with inhibition of collagen synthesis via TGF-β1/Smad signaling pathway.

  6. Impaired intestinal wound healing in Fhl2-deficient mice is due to disturbed collagen metabolism

    SciTech Connect

    Kirfel, Jutta Pantelis, Dimitrios; Kabba, Mustapha; Kahl, Philip; Roeper, Anke; Kalff, Joerg C.; Buettner, Reinhard

    2008-12-10

    Four and one half LIM domain protein FHL2 participates in many cellular processes involved in tissue repair such as regulation of gene expression, cytoarchitecture, cell adhesion, migration and signal transduction. The repair process after wounding is initiated by the release of peptides and bioactive lipids. These molecules induce synthesis and deposition of a provisional extracellular matrix. We showed previously that sphingosine-1-phosphate (S1P) triggers a signal transduction cascade mediating nuclear translocation of FHL2 in response to activation of the RhoA GTPase. Our present study shows that FHL2 is an important signal transducer influencing the outcome of intestinal anastomotic healing. Early wound healing is accompanied by reconstitution and remodelling of the extracellular matrix and collagen is primarily responsible for wound strength. Our results show that impaired intestinal wound healing in Fhl2-deficient mice is due to disturbed collagen III metabolism. Impaired collagen III synthesis reduced the mechanical stability of the anastomoses and led to lower bursting pressure in Fhl2-deficient mice after surgery. Our data confirm that FHL2 is an important factor regulating collagen expression in the early phase of wound healing, and thereby is critically involved in the physiologic process of anastomosis healing after bowel surgery and thus may represent a new therapeutic target.

  7. ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis.

    PubMed

    Aragoneses-Fenoll, L; Montes-Casado, M; Ojeda, G; Acosta, Y Y; Herranz, J; Martínez, S; Blanco-Aparicio, C; Criado, G; Pastor, J; Dianzani, U; Portolés, P; Rojo, J M

    2016-04-15

    Class IA phosphoinositide 3-kinases (PI3Ks) are essential to function of normal and tumor cells, and to modulate immune responses. T lymphocytes express high levels of p110α and p110δ class IA PI3K. Whereas the functioning of PI3K p110δ in immune and autoimmune reactions is well established, the role of p110α is less well understood. Here, a novel dual p110α/δ inhibitor (ETP-46321) and highly specific p110α (A66) or p110δ (IC87114) inhibitors have been compared concerning T cell activation in vitro, as well as the effect on responses to protein antigen and collagen-induced arthritis in vivo. In vitro activation of naive CD4(+) T lymphocytes by anti-CD3 and anti-CD28 was inhibited more effectively by the p110δ inhibitor than by the p110α inhibitor as measured by cytokine secretion (IL-2, IL-10, and IFN-γ), T-bet expression and NFAT activation. In activated CD4(+) T cells re-stimulated through CD3 and ICOS, IC87114 inhibited Akt and Erk activation, and the secretion of IL-2, IL-4, IL-17A, and IFN-γ better than A66. The p110α/δ inhibitor ETP-46321, or p110α plus p110δ inhibitors also inhibited IL-21 secretion by differentiated CD4(+) T follicular (Tfh) or IL-17-producing (Th17) helper cells. In vivo, therapeutic administration of ETP-46321 significantly inhibited responses to protein antigen as well as collagen-induced arthritis, as measured by antigen-specific antibody responses, secretion of IL-10, IL-17A or IFN-γ, or clinical symptoms. Hence, p110α as well as p110δ Class IA PI3Ks are important to immune regulation; inhibition of both subunits may be an effective therapeutic approach in inflammatory autoimmune diseases like rheumatoid arthritis.

  8. Matrine induces the apoptosis of fibroblast-like synoviocytes derived from rats with collagen-induced arthritis by suppressing the activation of the JAK/STAT signaling pathway

    PubMed Central

    Yang, Yongsheng; Dong, Qiumei; Li, Rongheng

    2017-01-01

    The induction of apoptosis-resistant rheumatoid synovial tissue cells has been related to constitutively active Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling in rheumatoid arthritis (RA). The excessive proliferation and inherent resistance to apoptosis of fibroblast-like synoviocytes (FLS) is an important mechanism by which RA originates. However, the effects of matrine on FLS in RA is unclear. The present study aimed to investigate the mechanism of action of matrine in a rat model of collagen-induced arthritis (CIA). The CIA model was established using bovine type II collagen. FLS were isolated from control and CIA rats, cultured in vitro, and confirmed to harbor fibroblast-like characteristics. After treatment of FLS with varying concentrations of matrine, the JAK2 inhibitor AG490, or a combination of both drugs, cell proliferation, apoptosis rate, expression of apoptotic markers and the activation of the JAK/STAT pathway were assessed. Additionally, CIA rats were administered either matrine or methotrexate by oral gavage to examine the effects of therapeutic intervention on arthritis pathogenesis. The arthritis index (AI) was measured and ankle joint structure was analyzed histologically to determine the severity of CIA. Furthermore, expression levels of apoptotic markers and members of the JAK/STAT family were also examined in vivo. Compared with the CIA group, matrine reduced AI and improved ankle pathology. Matrine also inhibited FLS proliferation, induced G0/G1 cell cycle arrest, and increased the rate of apoptosis in vitro. The effects of matrine on apoptosis induction were further confirmed by observations that Bcl-2 levels were decreased, whereas Bax and caspase-3 levels were increased in the matrine-treated synovial tissues and FLS. Finally, matrine treatment also diminished the phosphorylation, and hence activation of JAK2, STAT1 and STAT3. Our results suggest that matrine induces the apop-tosis of FLS from rats

  9. Development of a gene-activated scaffold platform for tissue engineering applications using chitosan-pDNA nanoparticles on collagen-based scaffolds.

    PubMed

    Raftery, Rosanne M; Tierney, Erica G; Curtin, Caroline M; Cryan, Sally-Ann; O'Brien, Fergal J

    2015-07-28

    Biomaterial scaffolds that support cell infiltration and tissue formation can also function as platforms for the delivery of therapeutics such as drugs, proteins, and genes. As burst release of supraphysiological quantities of recombinant proteins can result in adverse side effects, the objective of this study was to explore the potential of a series of collagen-based scaffolds, developed in our laboratory, as gene-activated scaffold platforms with potential in a range of tissue engineering applications. The potential of chitosan, a biocompatible material derived from the shells of crustaceans, as a gene delivery vector was assessed using mesenchymal stem cells (MSCs). A transfection efficiency of >45% is reported which is similar to what is achieved with polyethyleneimine (PEI), a non-viral gold standard vector, without causing cytotoxic side effects. When the optimised chitosan nanoparticles were incorporated into a series of collagen-based scaffolds, sustained transgene expression from MSCs seeded on the scaffolds was maintained for up to 28days and interestingly the composition of the scaffold had an effect on transfection efficiency. These results demonstrate that by simply varying the scaffold composition and the gene (or combinations thereof) chosen; the system has potential for a myriad of therapeutic applications.

  10. Enhanced silver nanoparticle synthesis by optimization of nitrate reductase activity.

    PubMed

    Vaidyanathan, Ramanathan; Gopalram, Shubaash; Kalishwaralal, Kalimuthu; Deepak, Venkataraman; Pandian, Sureshbabu Ram Kumar; Gurunathan, Sangiliyandi

    2010-01-01

    Nanostructure materials are attracting a great deal of attention because of their potential for achieving specific processes and selectivity, especially in biological and pharmaceutical applications. The generation of silver nanoparticles using optimized nitrate reductase for the reduction of Ag(+) with the retention of enzymatic activity in the complex is being reported. This report involves the optimization of enzyme activity to bring about enhanced nanoparticle synthesis. Response surface methodology and central composite rotary design (CCRD) were employed to optimize a fermentation medium for the production of nitrate reductase by Bacillus licheniformis at pH 8. The four variables involved in the study of nitrate reductase were Glucose, Peptone, Yeast extract and KNO(3). Glucose had a significant effect on nitrate reductase production. The optimized medium containing (%) Glucose: 1.5, Peptone: 1, Yeast extract: 0.35 and KNO(3): 0.35 resulted in a nitrate reductase activity of 452.206 U/ml which is same as that of the central level. The medium A (showing least nitrate reductase activity) and the medium B (showing maximum nitrate reductase activity) were compared for the synthesis. Spectrophotometric analysis revealed that the particles exhibited a peak at 431 nm and the A(431) for the medium B was 2-fold greater than that of the medium A. The particles were also characterized using TEM. The particles synthesized using the optimized enzyme activity ranged from 10 to 80 nm and therefore can be extended to various medicinal applications.

  11. Prospects and limitations of the rational engineering of fibrillar collagens

    PubMed Central

    Majsterek, Ireneusz; McAdams, Erin; Adachi, Eijiro; Dhume, Shirish T.; Fertala, Andrzej

    2003-01-01

    Recombinant collagens are attractive proteins for a number of biomedical applications. To date, significant progress was made in the large-scale production of nonmodified recombinant collagens; however, engineering of novel collagen-like proteins according to customized specifications has not been addressed. Herein we investigated the possibility of rational engineering of collagen-like proteins with specifically assigned characteristics. We have genetically engineered two DNA constructs encoding multi-D4 collagens defined as collagen-like proteins, consisting primarily of a tandem of the collagen II D4 periods that correspond to the biologically active region. We have also attempted to decrease enzymatic degradation of novel collagen by mutating a matrix metalloproteinase 1 cleavage site present in the D4 period. We demonstrated that the recombinant collagen α-chains consisting predominantly of the D4 period but lacking most of the other D periods found in native collagen fold into a typical collagen triple helix, and the novel procollagens are correctly processed by procollagen N-proteinase and procollagen C-proteinase. The nonmutated multi-D4 collagen had a normal melting point of 41°C and a similar carbohydrate content as that of control. In contrast, the mutant multi-D4 collagen had a markedly lower thermostability of 36°C and a significantly higher carbohydrate content. Both collagens were cleaved at multiple sites by matrix metalloproteinase 1, but the rate of hydrolysis of the mutant multi-D4 collagen was lower. These results provide a basis for the rational engineering of collagenous proteins and identifying any undesirable consequences of altering the collagenous amino acid sequences. PMID:12931004

  12. Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes.

    PubMed

    Solursh, M; Jensen, K L; Reiter, R S; Schmid, T M; Linsenmayer, T F

    1986-09-01

    We have examined whether the production of hypertrophic cartilage matrix reflecting a late stage in the development of chondrocytes which participate in endochondral bone formation, is the result of cell lineage, environmental influence, or both. We have compared the ability of cultured limb mesenchyme and mesectoderm to synthesize type X collagen, a marker highly selective for hypertrophic cartilage. High density cultures of limb mesenchyme from stage 23 and 24 chick embryos contain many cells that react positively for type II collagen by immunohistochemistry, but only a few of these initiate type X collagen synthesis. When limb mesenchyme cells are cultured in or on hydrated collagen gels or in agarose (conditions previously shown to promote chondrogenesis in low density cultures), almost all initiate synthesis of both collagen types. Similarly, collagen gel cultures of limb mesenchyme from stage 17 embryos synthesize type II collagen and with some additional delay type X collagen. However, cytochalasin D treatment of subconfluent cultures on plastic substrates, another treatment known to promote chondrogenesis, induces the production of type II collagen, but not type X collagen. These results demonstrate that the appearance of type X collagen in limb cartilage is environmentally regulated. Mesectodermal cells from the maxillary process of stages 24 and 28 chick embryos were cultured in or on hydrated collagen gels. Such cells initiate synthesis of type II collagen, and eventually type X collagen. Some cells contain only type II collagen and some contain both types II and X collagen. On the other hand, cultures of mandibular processes from stage 29 embryos contain chondrocytes with both collagen types and a larger overall number of chondrogenic foci than the maxillary process cultures. Since the maxillary process does not produce cartilage in situ and the mandibular process forms Meckel's cartilage which does not hypertrophy in situ, environmental influences

  13. Leopard syndrome associated with hyperelastic skin: analysis of collagen metabolism in cultured skin fibroblasts.

    PubMed

    Ohkura, T; Ohnishi, Y; Kawada, A; Tajima, S; Ishibashi, A; Ono, K

    1999-01-01

    We present a patient with Leopard syndrome and hyperelastic skin. Biochemical analysis using cultured skin fibroblasts showed normal type III and V collagen synthesis, lysyl hydroxylation level of type I procollagen and processing of pro-alpha(1) and alpha(2)(I). Our results suggest that molecular defects of hyperelasticity in Leopard syndrome are not related to abnormal collagen metabolism, although not all steps of collagen synthesis have been investigated.

  14. Aqua mediated synthesis of bio-active compounds.

    PubMed

    Panda, Siva S

    2013-05-01

    Recently the aqueous medium has attracted the interest of organic chemists, and many. Moreover, in the past 20 years, the drug-discovery process has undergone extraordinary changes, and high-throughput biological screening of potential drug candidates has led to an ever-increasing demand for novel drug-like compounds. Noteworthy advantages were observed during the course of study on aqua mediated synthesis of compounds of medicinal importance. The established advantages of water as a solvent for reactions are, water is the most abundant and available resource on the planet and many biochemical processes occur in aqueous medium. This review will focus on describing new developments in the application of water in medicinal chemistry for the synthesis of bio-active compounds possessing various biological properties.

  15. Synthesis and biological activity of homoarginine-containing opioid peptides.

    PubMed

    Izdebski, Jan; Kunce, Danuta; Schiller, Peter W; Chung, Nga N; Gers, Tomasz; Zelman, Monika; Grabek, Monika

    2007-01-01

    Two tris-alkoxycarbonyl homoarginine derivatives, Boc-Har{omega,omega'-[Z(2Br)]2}-OH and Boc-Har{omega,omega'-[Z(2Cl)]2}-OH, were prepared by guanidinylation of Boc-Lys-OH, and used for the synthesis of neo-endorphins and dynorphins. The results were compared with that obtained in the synthesis in which Boc-Lys(Fmoc)-OH was incorporated into the peptide chain, and after removing Fmoc protection, the resulting peptide-resin was guanidinylated with N,N'-[Z(2Br)]2- or N,N'-[Z(2Cl)]2-S-methylisourea. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. The results indicated that replacement of Arg by Har may be a good avenue for the design of biologically active peptides with increased resistance to degradation by trypsin-like enzymes.

  16. Temporal variation in the deposition of different types of collagen within a porous biomaterial implant.

    PubMed

    White, Jacinta F; Werkmeister, Jerome A; Bisucci, Teresa; Darby, Ian A; Ramshaw, John A M

    2014-10-01

    The deposition of new collagen in association with a medical implant has been studied using expanded polytetrafluoroethylene vascular replacement samples implanted subcutaneously in sheep, for up to 28 days. New type I collagen mRNA synthesis was followed by in situ hybridization, while the accumulation of new collagen types III, V, VI, XII, and XIV was followed by immunohistochemistry. All the collagen detected in the pores of the implant were newly deposited at various times after implantation and were not due to any pre-existing dermal collagen that may have been present around the implant. Collagen deposition was seen initially surrounding the implant and, with time, was seen to infiltrate within its pores. In situ hybridization showed that the majority of infiltrating cells had switched on mRNA that coded for type I collagen production. Histology showed that cellular infiltration increased with time, accompanied by increasing collagen deposition. The deposition of different collagen types happened at different rates. The type V and VI collagens preceded the major interstitial collagens in the newly deposited tissue, although at longer time points, detection of type V collagen appeared to decrease. After disruption of the interstitial collagens with enzyme, the "masked" type V collagen was clearly still visible by immunohistochemistry. Little type XII collagen could be seen within the porous mesh, although it was seen in the surrounding tissues. By contrast, type XIV was seen throughout the porous structure of the implanted mesh, with less being visible outside the material where type XII was more abundant.

  17. Design, Synthesis, and Antibacterial Activities of Novel Heterocyclic Arylsulphonamide Derivatives.

    PubMed

    Singh, Anuradha; Srivastava, Ritika; Singh, Ramendra K

    2017-02-13

    Design, synthesis, and antibacterial activities of a series of arylsulphonamide derivatives as probable peptide deformylase (PDF) inhibitors have been discussed. Compounds have been designed following Lipinski's rule and after docking into the active site of PDF protein (PDB code: 1G2A) synthesized later on. Furthermore, to assess their antibacterial activity, screening of the compound was done in vitro conditions against Gram-positive and Gram-negative bacterial strains. In silico, studies revealed these compounds as potential antibacterial agents and this fact was also supported by their prominent scoring functions. Antibacterial results indicated that these molecules possessed a significant activity against Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, and Escherichia coli with MIC values ranging from 0.06 to 0.29 μM. TOPKAT results showed that high LD50 values and the compounds were assumed non-carcinogenic when various animal models were studied computationally.

  18. Enigmatic insight into collagen

    PubMed Central

    Deshmukh, Shrutal Narendra; Dive, Alka M; Moharil, Rohit; Munde, Prashant

    2016-01-01

    Collagen is a unique, triple helical molecule which forms the major part of extracellular matrix. It is the most abundant protein in the human body, representing 30% of its dry weight. It is the fibrous structural protein that makes up the white fibers (collagen fibers) of skin, tendons, bones, cartilage and all other connective tissues. Collagens are not only essential for the mechanical resistance and resilience of multicellular organisms, but are also signaling molecules defining cellular shape and behavior. The human body has at least 16 types of collagen, but the most prominent types are I, II and III. Collagens are produced by several cell types and are distinguishable by their molecular compositions, morphologic characteristics, distribution, functions and pathogenesis. This is the major fibrous glycoprotein present in the extracellular matrix and in connective tissue and helps in maintaining the structural integrity of these tissues. It has a triple helical structure. Various studies have proved that mutations that modify folding of the triple helix result in identifiable genetic disorders. Collagen diseases share certain similarities with autoimmune diseases, because autoantibodies specific to each collagen disease are produced. Therefore, this review highlights the role of collagen in normal health and also the disorders associated with structural and functional defects in collagen. PMID:27601823

  19. Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

    SciTech Connect

    Wang, Xianwei Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L.

    2012-03-15

    Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac fibroblast

  20. Activation of Melanin Synthesis in Alternaria infectoria by Antifungal Drugs

    PubMed Central

    Fernandes, Chantal; Prados-Rosales, Rafael; Silva, Branca M. A.; Nakouzi-Naranjo, Antonio; Zuzarte, Mónica; Chatterjee, Subhasish; Stark, Ruth E.; Casadevall, Arturo

    2015-01-01

    The importance of Alternaria species fungi to human health ranges from their role as etiological agents of serious infections with poor prognoses in immunosuppressed individuals to their association with respiratory allergic diseases. The present work focuses on Alternaria infectoria, which was used as a model organism of the genus, and was designed to unravel melanin production in response to antifungals. After we characterized the pigment produced by A. infectoria, we studied the dynamics of 1,8-dihydroxynaphthalene (DHN)-melanin production during growth, the degree of melanization in response to antifungals, and how melanization affected susceptibility to several classes of therapeutic drugs. We demonstrate that A. infectoria increased melanin deposition in cell walls in response to nikkomycin Z, caspofungin, and itraconazole but not in response to fluconazole or amphotericin B. These results indicate that A. infectoria activates DHN-melanin synthesis in response to certain antifungal drugs, possibly as a protective mechanism against these drugs. Inhibition of DHN-melanin synthesis by pyroquilon resulted in a lower minimum effective concentration (MEC) of caspofungin and enhanced morphological changes (increased hyphal balloon size), characterized by thinner and less organized A. infectoria cell walls. In summary, A. infectoria synthesizes melanin in response to certain antifungal drugs, and its susceptibility is influenced by melanization, suggesting the therapeutic potential of drug combinations that affect melanin synthesis. PMID:26711773

  1. Activation of Melanin Synthesis in Alternaria infectoria by Antifungal Drugs.

    PubMed

    Fernandes, Chantal; Prados-Rosales, Rafael; Silva, Branca M A; Nakouzi-Naranjo, Antonio; Zuzarte, Mónica; Chatterjee, Subhasish; Stark, Ruth E; Casadevall, Arturo; Gonçalves, Teresa

    2015-12-28

    The importance of Alternaria species fungi to human health ranges from their role as etiological agents of serious infections with poor prognoses in immunosuppressed individuals to their association with respiratory allergic diseases. The present work focuses on Alternaria infectoria, which was used as a model organism of the genus, and was designed to unravel melanin production in response to antifungals. After we characterized the pigment produced by A. infectoria, we studied the dynamics of 1,8-dihydroxynaphthalene (DHN)-melanin production during growth, the degree of melanization in response to antifungals, and how melanization affected susceptibility to several classes of therapeutic drugs. We demonstrate that A. infectoria increased melanin deposition in cell walls in response to nikkomycin Z, caspofungin, and itraconazole but not in response to fluconazole or amphotericin B. These results indicate that A. infectoria activates DHN-melanin synthesis in response to certain antifungal drugs, possibly as a protective mechanism against these drugs. Inhibition of DHN-melanin synthesis by pyroquilon resulted in a lower minimum effective concentration (MEC) of caspofungin and enhanced morphological changes (increased hyphal balloon size), characterized by thinner and less organized A. infectoria cell walls. In summary, A. infectoria synthesizes melanin in response to certain antifungal drugs, and its susceptibility is influenced by melanization, suggesting the therapeutic potential of drug combinations that affect melanin synthesis.

  2. Effect of polystyrene and polyether imide cell culture inserts with different roughness on chondrocyte metabolic activity and gene expression profiles of aggrecan and collagen.

    PubMed

    König, Josephine; Kohl, Benjamin; Kratz, Karl; Jung, Friedrich; Lendlein, Andreas; Ertel, Wolfgang; Schulze-Tanzil, Gundula

    2013-01-01

    In vitro cultured autologous chondrocytes can be used for implantation to support cartilage repair. For this purpose, a very small number of autologous cells harvested from a biopsy have to be expanded in monolayer culture. Commercially available polymer surfaces lead to chondrocyte dedifferentiation. Hence, the demanding need for optimized polymers and surface topologies supporting chondrocytes' differentiated phenotypes in vitro arises. In this study we explored the effect of tailored cell culture plate inserts prepared from polystyrene (PS) and polyether imide (PEI) exhibiting three different roughness levels (R0, RI, RII) on chondrocyte morphology, metabolism and gene expression profile. As a control, commercially available tissue culture plastic (TCP) dishes were included. Primary porcine articular chondrocytes were seeded on tailored PS and PEI inserts with three different roughness levels. The metabolic activity of the chondrocytes was determined after 24 hours using alamar blue assay. Chondrocyte gene expression profiles (aggrecan, type I and type II collagen) were monitored after 48 hours using Real Time Detection (RTD)-PCR. Chondrocytes cultured on PS and PEI surfaces formed cell clusters after 24 and 48 hours, which was not observed on TCP. The metabolic activity of chondrocytes cultured on PS was lower than of chondrocytes cultured on PEI, but also lower than on TCP. Gene expression analyses revealed an elevated expression of cartilage-specific aggrecan and an impaired expression of both collagen types by chondrocytes on PS and PEI compared with TCP. In summary, PEI is a biocompatible biomaterial suitable for chondrocyte culturing, which can be further chemically functionalized for generating specific surface interactions or covalent binding of biomolecules.

  3. Platelets activated by collagen through the immunoreceptor tyrosine-based activation motif in the Fc receptor gamma-chain play a pivotal role in the development of myocardial ischemia-reperfusion injury.

    PubMed

    Takaya, Norihide; Katoh, Youichi; Iwabuchi, Kazuhisa; Hayashi, Ichiro; Konishi, Hakuoh; Itoh, Seigo; Okumura, Ko; Ra, Chisei; Nagaoka, Isao; Daida, Hiroyuki

    2005-12-01

    Platelet activation and the formation of platelet microaggregates in coronary vessels play pivotal roles in myocardial ischemia and reperfusion injury. The Fc receptor gamma-chain (FcR gamma) is coexpressed with glycoprotein (GP) VI, forming a platelet collagen receptor, and the activation of platelets by collagen is closely coupled with tyrosine phosphorylation of the FcRgamma. To examine the functional significance of platelet FcR gamma/GPVI complex in the early phase of myocardial ischemia and reperfusion injury in mice, we performed coronary occlusion and reperfusion experiments using wild type mice and FcRgamma-deficient (FcRgamma(-/-)) mice that lack GPVI. The infarct size was significantly smaller in FcRgamma(-/-) mice subjected to occlusion and reperfusion of the coronary artery than in control FcR gamma(+/+) mice. Twenty-four hours after the reperfusion, electron microscopy of the injured tissue showed substantially more platelet aggregation and occlusive platelet microthrombi in the capillaries of the damaged areas of the wild type mice than in those of the FcR gamma(-/-) mice. Platelet Syk was scarcely activated in the FcR gamma(-/-) mice after myocardial ischemia and reperfusion, but significantly activated in the FcR gamma(+/+) mice. CD11b expression on neutrophils was elevated after myocardial ischemia and reperfusion in both mouse groups, whereas myeloperoxidase activity in the injured areas was significantly lower in the FcRgamma(-/-) mice than in the FcRgamma(+/+) mice. These results suggest that the collagen-induced activation of platelets through the FcR gamma plays a pivotal role in the extension of myocardial ischemia-reperfusion injury. FcRgamma and GPVI may be important therapeutic targets for myocardial ischemia-reperfusion injury.

  4. Synthesis and biological activity of novel deoxycholic acid derivatives.

    PubMed

    Popadyuk, Irina I; Markov, Andrey V; Salomatina, Oksana V; Logashenko, Evgeniya B; Shernyukov, Andrey V; Zenkova, Marina A; Salakhutdinov, Nariman F

    2015-08-01

    We report the synthesis and biological activity of new semi-synthetic derivatives of naturally occurring deoxycholic acid (DCA) bearing 2-cyano-3-oxo-1-ene, 3-oxo-1(2)-ene or 3-oxo-4(5)-ene moieties in ring A and 12-oxo or 12-oxo-9(11)-ene moieties in ring C. Bioassays using murine macrophage-like cells and tumour cells show that the presence of the 9(11)-double bond associated with the increased polarity of ring A or with isoxazole ring joined to ring A, improves the ability of the compounds to inhibit cancer cell growth.

  5. Synthesis and antiviral activity of azoles obtained from carbohydrates.

    PubMed

    Barradas, José Sebastián; Errea, María Inés; D'Accorso, Norma B; Sepúlveda, Claudia S; Talarico, Laura B; Damonte, Elsa B

    2008-09-22

    Herein we describe the synthesis of 1,2,4-triazolyl-3-thione;1,3,4-oxadiazole, and imidazo[2,1-b]thiazole derivatives from carbohydrates. The antiviral activity of these compounds was tested against Dengue and Junin virus (the etiological agent of Argentine hemorrhagic fever). The 3-(p-bromobenzoyl)-5-(1,2-O-isopropylidene-3-O-methyl-alpha-d-xylofuranos-5-ulos-5-yl)imidazo[2,1-b]thiazole was able to inhibit the replication of both viruses in Vero cells at concentration significantly lower than the CC(50).

  6. Synthesis and biological activity of fluorescent neonicotinoid insecticide thiamethoxam.

    PubMed

    Taillebois, Emiliane; Langlois, Paul; Cunha, Thomas; Seraphin, Denis; Thany, Steeve H

    2014-08-01

    Here, we describe the synthesis of two new fluorescent derivatives of thiamethoxam and compared their toxicity on aphid Acyrthosiphon pisum and their mode of action on insect nicotinic acetylcholine receptors expressed on the sixth abdominal ganglion. The compound 3 with two 2-chlorothiazole moieties was found to be more toxic using toxicological bioassays 24 h and 48 h after exposure while compound 4 appeared more active using cockroach ganglionic depolarization. Interestingly, thiamethoxam appeared more effective than component 3 and 4, respectively. Our results demonstrated that component 3 and 4 act as agonists of insect nicotinic acetylcholine receptors.

  7. Optimal placement of active elements in control augmented structural synthesis

    NASA Technical Reports Server (NTRS)

    Sepulveda, A. E.; Jin, I. M.; Schmit, L. A., Jr.

    1992-01-01

    A methodology for structural/control synthesis is presented in which the optimal location of active members is treated in terms of (0,1) variables. Structural member sizes, control gains and (0,1) placement variables are treated simultaneously as design variables. Optimization is carried out by generating and solving a sequence of explicit approximate problems using a branch and bound strategy. Intermediate design variable and intermediate response quantity concepts are used to enhance the quality of the approximate design problems. Numerical results for example problems are presented to illustrate the efficacy of the design procedure set forth.

  8. Ultrasound-assisted Micro-emulsion Synthesis of a Highly Active Nano-particle Catalyst

    DTIC Science & Technology

    2010-03-01

    Ultrasound-assisted Micro -emulsion Synthesis of a Highly Active Nano -particle Catalyst by Rongzhong Jiang and Charles Rong ARL-TR-5114...ARL-TR-5114 March 2010 Ultrasound-assisted Micro -emulsion Synthesis of a Highly Active Nano -particle Catalyst Rongzhong Jiang and...TYPE DRI 3. DATES COVERED (From - To) 2009 to 2010 4. TITLE AND SUBTITLE Ultrasound-assisted Micro -emulsion Synthesis of a Highly Active Nano

  9. Galactosylated collagen matrix enhanced in vitro maturation of human embryonic stem cell-derived hepatocyte-like cells.

    PubMed

    Ghodsizadeh, Arefeh; Hosseinkhani, Hossein; Piryaei, Abbas; Pournasr, Behshad; Najarasl, Mostafa; Hiraoka, Yosuke; Baharvand, Hossein

    2014-05-01

    Due to their important biomedical applications, functional human embryonic stem cell-derived hepatocyte-like cells (hESC-HLCs) are an attractive topic in the field of stem cell differentiation. Here, we have initially differentiated hESCs into functional hepatic endoderm (HE) and continued the differentiation by replating them onto galactosylated collagen (GC) and collagen matrices. The differentiation of hESC-HE cells into HLCs on GC substrate showed significant up-regulation of hepatic-specific genes such as ALB, HNF4α, CYP3A4, G6P, and ASGR1. There was more albumin secretion and urea synthesis, as well as more cytochrome p450 activity, in differentiated HLCs on GC compared to the collagen-coated substrate. These results suggested that GC substrate has the potential to be used for in vitro maturation of hESC-HLCs.

  10. Antifungal activity of silver nanoparticles obtained by green synthesis.

    PubMed

    Mallmann, Eduardo José J; Cunha, Francisco Afrânio; Castro, Bruno N M F; Maciel, Auberson Martins; Menezes, Everardo Albuquerque; Fechine, Pierre Basílio Almeida

    2015-01-01

    Silver nanoparticles (AgNPs) are metal structures at the nanoscale. AgNPs have exhibited antimicrobial activities against fungi and bacteria; however synthesis of AgNPs can generate toxic waste during the reaction process. Accordingly, new routes using non-toxic compounds have been researched. The proposal of the present study was to synthesize AgNPs using ribose as a reducing agent and sodium dodecyl sulfate (SDS) as a stabilizer. The antifungal activity of these particles against C. albicans and C. tropicalis was also evaluated. Stable nanoparticles 12.5 ± 4.9 nm (mean ± SD) in size were obtained, which showed high activity against Candida spp. and could represent an alternative for fungal infection treatment.

  11. Synthesis and Antimicrobial Activity of Silver-Doped Hydroxyapatite Nanoparticles

    PubMed Central

    Ciobanu, Carmen Steluta; Iconaru, Simona Liliana; Chifiriuc, Mariana Carmen; Costescu, Adrian; Le Coustumer, Philippe; Predoi, Daniela

    2013-01-01

    The synthesis of nanosized particles of Ag-doped hydroxyapatite with antibacterial properties is of great interest for the development of new biomedical applications. The aim of this study was the evaluation of Ca10−xAgx(PO4)6(OH)2 nanoparticles (Ag:HAp-NPs) for their antibacterial and antifungal activity. Resistance to antimicrobial agents by pathogenic bacteria has emerged in the recent years and became a major health problem. Here, we report a method for synthesizing Ag doped nanocrystalline hydroxyapatite. A silver-doped nanocrystalline hydroxyapatite was synthesized at 100°C in deionised water. Also, in this paper Ag:HAp-NPs are evaluated for their antimicrobial activity against Gram-positive and Gram-negative bacteria and fungal strains. The specific antimicrobial activity revealed by the qualitative assay is demonstrating that our compounds are interacting differently with the microbial targets, probably due to the differences in the microbial wall structures. PMID:23509801

  12. ANTIFUNGAL ACTIVITY OF SILVER NANOPARTICLES OBTAINED BY GREEN SYNTHESIS

    PubMed Central

    MALLMANN, Eduardo José J.; CUNHA, Francisco Afrânio; CASTRO, Bruno N.M.F.; MACIEL, Auberson Martins; MENEZES, Everardo Albuquerque; FECHINE, Pierre Basílio Almeida

    2015-01-01

    Silver nanoparticles (AgNPs) are metal structures at the nanoscale. AgNPs have exhibited antimicrobial activities against fungi and bacteria; however synthesis of AgNPs can generate toxic waste during the reaction process. Accordingly, new routes using non-toxic compounds have been researched. The proposal of the present study was to synthesize AgNPs using ribose as a reducing agent and sodium dodecyl sulfate (SDS) as a stabilizer. The antifungal activity of these particles against C. albicans and C. tropicalis was also evaluated. Stable nanoparticles 12.5 ± 4.9 nm (mean ± SD) in size were obtained, which showed high activity against Candida spp. and could represent an alternative for fungal infection treatment. PMID:25923897

  13. The exercise-induced biochemical milieu enhances collagen content and tensile strength of engineered ligaments.

    PubMed

    West, Daniel W D; Lee-Barthel, Ann; McIntyre, Todd; Shamim, Baubak; Lee, Cassandra A; Baar, Keith

    2015-10-15

    Exercise stimulates a dramatic change in the concentration of circulating hormones, such as growth hormone (GH), but the biological functions of this response are unclear. Pharmacological GH administration stimulates collagen synthesis; however, whether the post-exercise systemic milieu has a similar action is unknown. We aimed to determine whether the collagen content and tensile strength of tissue-engineered ligaments is enhanced by serum obtained post-exercise. Primary cells from a human anterior cruciate ligament (ACL) were used to engineer ligament constructs in vitro. Blood obtained from 12 healthy young men 15 min after resistance exercise contained GH concentrations that were ∼7-fold greater than resting serum (P < 0.001), whereas IGF-1 was not elevated at this time point (P = 0.21 vs. rest). Ligament constructs were treated for 7 days with medium supplemented with serum obtained at rest (RestTx) or 15 min post-exercise (ExTx), before tensile testing and collagen content analysis. Compared with RestTx, ExTx enhanced collagen content (+19%; 181 ± 33 vs. 215 ± 40 μg per construct P = 0.001) and ligament mechanical properties - maximal tensile load (+17%, P = 0.03 vs. RestTx) and ultimate tensile strength (+10%, P = 0.15 vs. RestTx). In a separate set of engineered ligaments, recombinant IGF-1, but not GH, enhanced collagen content and mechanics. Bioassays in 2D culture revealed that acute treatment with post-exercise serum activated mTORC1 and ERK1/2. In conclusion, the post-exercise biochemical milieu, but not recombinant GH, enhances collagen content and tensile strength of engineered ligaments, in association with mTORC1 and ERK1/2 activation.

  14. Preparation of (3H)collagen for studies of the biologic fate of xenogenic collagen implants in vivo

    SciTech Connect

    McPherson, J.M.; Sawamura, S.J.; Conti, A.

    1986-06-01

    Reduction of a commercially available, pepsin-solubilized, bovine dermal collagen (Vitrogen 100) with sodium (3H)borohydride provided radiolabeled collagen preparations with specific activities ranging from 7.1-12.0 muCi/mg collagen. These specific activities were 2-3 times greater than those obtained by reduction of intact rat tail tendon collagen under similar conditions. The alpha, beta, and higher aggregate components of type I collagen were radiolabeled as well as the alpha component of a small amount of type III collagen present in the samples. Fractionation of cyanogen bromide peptides showed that alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB3,5 were the predominant peptides labeled by this procedure. Amino acid analysis indicated that the majority of the radioactivity was in reducible cross-links, precursors of these cross-links, and in hexosyllysine residues. Reconstitution experiments comparing this radiolabeled collagen with nonlabeled collagen showed them to be indistinguishable. Bacterial collagenase digestion of this reconstituted fibrillar collagen in both a lightly cross-linked (glutaraldehyde 0.0075%) and noncross-linked form provided evidence that digestion of labeled and nonlabeled collagens proceeded at similar rates. Thus, labeling did not change the properties of the collagen. Cross-linking made the preparation refractory to proteolytic degradation. Injection of fibrillar collagen preparations, spiked with radiolabeled collagen, into the guinea pig dermis followed by quantitation of the amount of radioactivity recovered from implant sites as a function of time, indicated that the lightly cross-linked samples also were more resistant to degradation in vivo than the noncross-linked preparation. The half-life of noncross-linked collagen was about 4 days while that of the cross-linked collagen was about 25 days.

  15. Transforming growth factor-β1 induces type II collagen and aggrecan expression via activation of extracellular signal-regulated kinase 1/2 and Smad2/3 signaling pathways.

    PubMed

    Zhu, Yanhui; Tao, Hairong; Jin, Chen; Liu, Yonzhang; Lu, Xiongwei; Hu, Xiaopeng; Wang, Xiang

    2015-10-01

    Transforming growth factor (TGF)‑β regulates the anabolic metabolism of articular cartilage and prevents cartilage degradation. TGF‑β1 influences cellular proliferation, differentiation and the extracellular matrix through activation of the extracellular signal‑regulated kinase (ERK)1/2 and Smad2/3 signaling pathways. However, it has remained to be fully elucidated precisely how the ERK1/2 and Smad2/3 signaling pathways mediate anabolic processes of articular cartilage. The present study investigated how ERK1/2 and Smad2/3 signaling mediate TGF‑β1‑stimulated type II collagen and aggrecan expression in rat chondrocytes. The results confirmed that TGF‑β1 stimulates type II collagen and aggrecan expression in rat chondrocytes, and furthermore, that the ERK1/2 and Smad2/3 signaling pathways were activated by TGF‑β1. Conversely, the TGF‑β receptor I (ALK5) kinase inhibitor SB525334 significantly impaired TGF‑β1‑induced type II collagen and aggrecan expression, coinciding with a reduction of ERK1/2 and Smad3 phosphorylation. In addition, TGF‑β1‑induced type II collagen and aggrecan expression were significantly suppressed by ERK1/2 inhibitor PD98059. Similarly, TGF‑β1‑stimulated type II collagen and aggrecan expression were decreased in the presence of a Smad3 phosphorylation inhibitor SIS3. Therefore, the present study demonstrated that the ERK1/2 and Smad2/3 signaling pathways regulate type II collagen and aggrecan expression in rat chondrocytes.

  16. Synthesis and SAR studies of praziquantel derivatives with activity against Schistosoma japonicum.

    PubMed

    Wang, Wen-Long; Song, Li-Jun; Chen, Xia; Yin, Xu-Ren; Fan, Wen-Hua; Wang, Gu-Ping; Yu, Chuan-Xin; Feng, Bainian

    2013-07-31

    The synthesis and structure-activity relationship (SAR) studies of praziquantel derivatives with activity against adult Schistosoma japonicum are described. Several of them showed better worm killing activity than praziquantel and could serve as leads for further optimization.

  17. Synthesis, characteristics and antimicrobial activity of ZnO nanoparticles

    NASA Astrophysics Data System (ADS)

    Janaki, A. Chinnammal; Sailatha, E.; Gunasekaran, S.

    2015-06-01

    The utilization of various plant resources for the bio synthesis of metallic nano particles is called green technology and it does not utilize any harmful protocols. Present study focuses on the green synthesis of ZnO nano particles by Zinc Carbonate and utilizing the bio-components of powder extract of dry ginger rhizome (Zingiber officinale). The ZnO nano crystallites of average size range of 23-26 nm have been synthesized by rapid, simple and eco friendly method. Zinc oxide nano particles were characterized by using X-ray diffraction (XRD), Scanning Electron Microscope (SEM), Energy Dispersive X-ray spectroscopy (EDX). FTIR spectra confirmed the adsorption of surfactant molecules at the surface of ZnO nanoparticles and the presence of ZnO bonding. Antimicrobial activity of ZnO nano particles was done by well diffusion method against pathogenic organisms like Klebsiella pneumonia, Staphylococcus aureus and Candida albicans and Penicillium notatum. It is observed that the ZnO synthesized in the process has the efficient antimicrobial activity.

  18. Synthesis, characteristics and antimicrobial activity of ZnO nanoparticles.

    PubMed

    Janaki, A Chinnammal; Sailatha, E; Gunasekaran, S

    2015-06-05

    The utilization of various plant resources for the bio synthesis of metallic nano particles is called green technology and it does not utilize any harmful protocols. Present study focuses on the green synthesis of ZnO nano particles by Zinc Carbonate and utilizing the bio-components of powder extract of dry ginger rhizome (Zingiber officinale). The ZnO nano crystallites of average size range of 23-26 nm have been synthesized by rapid, simple and eco friendly method. Zinc oxide nano particles were characterized by using X-ray diffraction (XRD), Scanning Electron Microscope (SEM), Energy Dispersive X-ray spectroscopy (EDX). FTIR spectra confirmed the adsorption of surfactant molecules at the surface of ZnO nanoparticles and the presence of ZnO bonding. Antimicrobial activity of ZnO nano particles was done by well diffusion method against pathogenic organisms like Klebsiella pneumonia, Staphylococcus aureus and Candida albicans and Penicillium notatum. It is observed that the ZnO synthesized in the process has the efficient antimicrobial activity.

  19. Silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity.

    PubMed

    Panacek, Ales; Kvítek, Libor; Prucek, Robert; Kolar, Milan; Vecerova, Renata; Pizúrova, Nadezda; Sharma, Virender K; Nevecna, Tat'jana; Zboril, Radek

    2006-08-24

    A one-step simple synthesis of silver colloid nanoparticles with controllable sizes is presented. In this synthesis, reduction of [Ag(NH(3))(2)](+) complex cation by four saccharides was performed. Four saccharides were used: two monosaccharides (glucose and galactose) and two disaccharides (maltose and lactose). The syntheses performed at various ammonia concentrations (0.005-0.20 mol L(-1)) and pH conditions (11.5-13.0) produced a wide range of particle sizes (25-450 nm) with narrow size distributions, especially at the lowest ammonia concentrations. The average size, size distribution, morphology, and structure of particles were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), and UV/Visible absorption spectrophotometry. The influence of the saccharide structure (monosacharides versus disaccharides) on the size of silver particles is briefly discussed. The reduction of [Ag(NH(3))(2)](+) by maltose produced silver particles with a narrow size distribution with an average size of 25 nm, which showed high antimicrobial and bactericidal activity against Gram-positive and Gram-negative bacteria, including highly multiresistant strains such as methicillin-resistant Staphylococcus aureus. Antibacterial activity of silver nanoparticles was found to be dependent on the size of silver particles. A very low concentration of silver (as low as 1.69 mug/mL Ag) gave antibacterial performance.

  20. Effect of Allium sativum and fish collagen on the proteolytic and angiotensin-I converting enzyme-inhibitory activities in cheese and yogurt.

    PubMed

    Shori, A B; Baba, A S; Keow, J N

    2012-12-15

    There is an increasing demand of functional foods in developed countries. Yogurt plays an important role in the management of blood pressure. Several bioactive peptides isolated from Allium sativum or fish collagen have shown antihypertensive activity. Thus, in the present study the effects of A. sativum and/or Fish Collagen (FC) on proteolysis and ACE inhibitory activity in yogurt (0, 7 and 14 day) and cheese (0, 14 and 28 day) were investigated. Proteolytic activities were the highest on day 7 of refrigerated storage in A. sativum-FC-yogurt (337.0 +/- 5.3 microg g(-1)) followed by FC-yogurt (275.3 +/- 2.0 microg g(-1)), A. sativum-yogurt (245.8 +/- 4.2 microg g(-1)) and plain-yogurt (40.4 +/- 1.2 microg g(-1)). On the other hand, proteolytic activities in cheese ripening were the highest (p < 0.05) on day 14 of storage for plain and A. sativum-cheeses (411.4 +/- 4.3 and 528.7 +/- 1.6 microg g(-1), respectively). However, the presence of FC increased the proteolysis to the highest level on day 28 of storage for FC- and A. sativum-FC cheeses (641.2 +/- 0.1 and 1128.4 +/- 4.5 microg g(-1), respectively). In addition, plain- and A. sativum-yogurts with or without FC showed maximal inhibition of ACE on day 7 of storage. Fresh plain- and A. sativum-cheeses showed ACE inhibition (72.3 +/- 7.8 and 50.4 +/- 1.6 % respectively), the presence of FC in both type of cheeses reduced the ACE inhibition to 62.9 +/- 0.8 and 44.5 +/- 5.0%, respectively. However, refrigerated storage increased ACE inhibition in cheeses (p < 0.05 on day 28) in the presence of FC more than in the absence. In conclusion, the presence of FC in A. sativum-yogurt or cheese enhanced the proteolytic activity. Thus, it has potential in the development of an effective dietary strategy for hypertension associated cardiovascular diseases.

  1. Characterization of riboflavin-modified dentin collagen matrix.

    PubMed

    Fawzy, A; Nitisusanta, L; Iqbal, K; Daood, U; Beng, L T; Neo, J

    2012-11-01

    Crosslinking is considered a possible approach to increasing the mechanical and structural stability and biodegradation resistance of the dentin collagen matrix. The aim of this study was to investigate the mechanical and chemical variations and collagen degradation resistance associated with crosslinking of the dentin collagen matrix with UVA-activated riboflavin. Dentin collagen matrix specimens were treated with 0.1 and 1% riboflavin for 2 min and photo-activated with 7 mW/cm(2) UVA (368 nm) for 2 min. The structural change of the dentin collagen network with collagenase exposure was investigated by AFM and SEM at different time-points. The variations in surface/bulk mechanical properties and biodegradation resistance were characterized by nano-indentation, conventional mechanical testing, and hydroxyproline liberation at different time-points. Chemical changes associated with riboflavin/collagen-matrix interaction were analyzed by micro-Raman spectroscopy. UVA-activated riboflavin increased the mechanical properties, mechanical stability, and biodegradation resistance of the dentin collagen matrix. Higher collagen-network structural resistance against collagenolytic challenges was found with crosslinking. micro-Raman spectroscopy showed a strong dependency, in both intensity and wave-number, of certain Raman bands (1242-1667 cm(-1)) with crosslinking indicating the collagen/riboflavin interactions. UVA-activated riboflavin (1%) more efficiently crosslinked the dentin collagen matrix within a relatively clinically acceptable time-frame compared with 0.1% riboflavin.

  2. Synthesis of nano-sized amorphous boron powders through active dilution self-propagating high-temperature synthesis method

    SciTech Connect

    Wang, Jilin; Gu, Yunle; Li, Zili; Wang, Weimin; Fu, Zhengyi

    2013-06-01

    Graphical abstract: Nano-sized amorphous boron powders were synthesized by active dilution self-propagating high-temperature synthesis (SHS) method. The effects of endothermic reaction rate, the possible chemical reaction mechanism and active dilution model for synthesis of the product were also discussed. Highlights: ► Nano-sized amorphous boron powders were synthesized by active dilution self-propagating high-temperature synthesis method. ► The morphology, particle size and purity of the samples could be effectively controlled via changing the endothermic rate. ► The diluter KBH{sub 4} played an important role in active dilution synthesis of amorphous nano-sized boron powders. ► The active dilution method could be further popularized and become a common approach to prepare various inorganic materials. - Abstract: Nano-sized amorphous boron powders were synthesized by active dilution self-propagating high-temperature synthesis (SHS) method at temperatures ranging from 700 °C to 850 °C in a SHS furnace using Mg, B{sub 2}O{sub 3} and KBH{sub 4} as raw materials. Samples were characterized by X-ray powder diffraction (XRD), Laser particle size analyzer, Fourier transform infrared spectra (FTIR), X-ray energy dispersive spectroscopy (EDX), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and high-resolution transmission TEM (HRTEM). The boron powders demonstrated an average particle size of 50 nm with a purity of 95.64 wt.%. The diluter KBH{sub 4} played an important role in the active dilution synthesis of amorphous nano-sized boron powders. The effects of endothermic reaction rate, the possible chemical reaction mechanism and active dilution model for synthesis of the product were also discussed.

  3. Synthesis and antibacterial activity of of silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Maliszewska, I.; Sadowski, Z.

    2009-01-01

    Silver nanoparticles have been known to have inhibitory and bactericidal effects but the antimicrobial mechanism have not been clearly revealed. Here, we report on the synthesis of metallic nanoparticles of silver using wild strains of Penicillium isolated from environment. Kinetics of the formation of nanosilver was monitored using the UV-Vis. TEM micrographs showed the formation of silver nanoparticles in the range 10-100 nm. Obtained Ag nanoparticles were evaluated for their antimicrobial activity against the gram-positive and gram-negative bacteria. As results, Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were effectively inhibited. Nanosilver is a promising candidate for development of future antibacterial therapies because of its wide spectrum of activity.

  4. Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1.

    PubMed

    Xu, Huifang; Raynal, Nicolas; Stathopoulos, Stavros; Myllyharju, Johanna; Farndale, Richard W; Leitinger, Birgit

    2011-01-01

    The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I-III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.

  5. Influence of external uniaxial cyclic strain on oriented fibroblast-seeded collagen gels.

    PubMed

    Berry, Catherine C; Shelton, Julia C; Bader, Dan L; Lee, David A

    2003-08-01

    This study investigates the influence of cyclic tensile strain, applied to fully contracted fibroblast-seeded collagen constructs. The constructs were preloaded to either 2 or 10 mN. The preloaded constructs were subsequently subjected to a further 10% cyclic strain (0-10%) at 1 Hz, using a triangular waveform, or were cultured in the preloaded state. In all cases cellular viability was maintained during the conditioning period. Cell proliferation was enhanced by the application of cyclic strain within constructs preloaded to both 2 and 10 mN. Collagen synthesis was enhanced by cyclic strain within constructs preloaded at 2 mN only. The profile of matrix metalloproteinase (MMP) expression, determined by zymography, was broadly similar in constructs preloaded at 2 mN with or without the application of cyclic strain. By contrast, constructs preloaded at 10 mN and subjected to cyclic strain expressed enhanced levels of staining for latent MMP-1, latent MMP-9, and both latent and active MMP-2, when compared with the other conditioning regimens. The structural stiffness of constructs preloaded at 2 mN and subjected to cyclic strain was enhanced compared with control specimens, reflecting the increase in collagen synthesis. By contrast, the initial failure loads for cyclically strained constructs preloaded at 10 mN were reduced, potentially because of enhanced catabolic activity.

  6. Control of Collagen Triple Helix Stability by Phosphorylation.

    PubMed

    Acevedo-Jake, Amanda M; Ngo, Daniel H; Hartgerink, Jeffrey D

    2017-03-10

    The phosphorylation of the collagen triple helix plays an important role in collagen synthesis, assembly, signaling, and immune response, although no reports detailing the effect this modification has on the structure and stability of the triple helix exist. Here we investigate the changes in stability and structure resulting from the phosphorylation of collagen. Additionally, the formation of pairwise interactions between phosphorylated residues and lysine is examined. In all tested cases, phosphorylation increases helix stability. When charged-pair interactions are possible, stabilization via phosphorylation can play a very large role, resulting inasmuch as a 13.0 °C increase in triple helix stability. Two-dimensional NMR and molecular modeling are used to study the local structure of the triple helix. Our results suggest a mechanism of action for phosphorylation in the regulation of collagen and also expand upon our understanding of pairwise amino acid stabilization of the collagen triple helix.

  7. Plasma clot-promoting effect of collagen in relation to collagen-platelet interaction

    SciTech Connect

    Gentry, P.A.; Schneider, M.D.; Miller, J.K.

    1981-01-01

    The hemostatic function of several acid-soluble collagen preparations and a fibrillar-form collagen preparation have been compared. Pepsin-treated acid-soluble collagen isolated from burro and horse aortic tissue and acid-soluble colagen isolated from human umbilical cord readily promoted platelet aggregation, but failed to activate the coagulation mechanism even after prolonged incubation with plasma at 37 C. By contrast, fibrillar-form collagen isolated from burro aorta was both an efficient stimulant for the induction of platelet aggregation and a potent clot-promoting agent. Similar results were found for all the collagen preparations irrespective of whether the studies were conducted with sheep or with burro plasma. Heat denaturation studies showed that the hemostatic functon of the fibrillar-form colagen was dependent on an intact tirple-helical structure.

  8. Synthesis of Cu/CNTs nanocomposites for antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Singhal, Sunil Kumar; Lal, Maneet; Lata; Ranjan Kabi, Soumya; Behari Mathur, Rakesh

    2012-12-01

    We report a facile method for the synthesis of Cu/multi-walled carbon nanotubes (CNTs) composite powder employing a chemical reduction method followed by high-energy ball milling involving the use of sodium borohydride as a reducing agent and copper sulphate as the precursor material. Control of oxidation of Cu nanoparticles (CuNPs) is a key factor in the synthesis of Cu/CNTs nanocomposites via chemical reduction methods and other methods. To overcome this problem we have applied a new facile rapid synthesis method using a combination of molecular-level mixing followed by high-energy ball milling to produce mostly CuNPs. X-ray diffraction results indicated the presence of mostly CuNPs in composite powder. Scanning electron microscopy and high resolution transmission electron microscopy (HRTEM) was used to ascertain the dispersion of CNTs in Cu matrix. Most of the CuNPs synthesized in the present work had a particle size ranging from 20-50 nm as revealed by HRTEM characterization. Moreover, the CNTs were also found to be homogeneously dispersed in Cu matrix. The Cu/CNTs nanocomposite has a wide range of applications from fuel cells to electronic chip components. In the present work we have investigated the antimicrobial activity of Cu powder and varying concentrations of Cu/CNTs nanocomposite against gram negative Providencia sp. bacteria, and gram positive Bacillus sp. bacteria. These findings suggest that Cu/CNTs nanocomposite can be used in antibacterial controlling systems and as an effective growth inhibitor in the case of various microorganisms.

  9. Co-Culture of Tumor Spheroids and Fibroblasts in a Collagen Matrix-Incorporated Microfluidic Chip Mimics Reciprocal Activation in Solid Tumor Microenvironment

    PubMed Central

    Jeong, Su-Yeong; Lee, Ji-Hyun; Shin, Yoojin; Chung, Seok; Kuh, Hyo-Jeong

    2016-01-01

    Multicellular 3D culture and interaction with stromal components are considered essential elements in establishing a ‘more clinically relevant’ tumor model. Matrix-embedded 3D cultures using a microfluidic chip platform can recapitulate the microscale interaction within tumor microenvironments. As a major component of tumor microenvironment, cancer-associated fibroblasts (CAFs) play a role in cancer progression and drug resistance. Here, we present a microfluidic chip-based tumor tissue culture model that integrates 3D tumor spheroids (TSs) with CAF in proximity within a hydrogel scaffold. HT-29 human colorectal carcinoma cells grew into 3D TSs and the growth was stimulated when co-cultured with fibroblasts as shown by 1.5-folds increase of % changes in diameter over 5 days. TS cultured for 6 days showed a reduced expression of Ki-67 along with increased expression of fibronectin when co-cultured with fibroblasts compared to mono-cultured TSs. Fibroblasts were activated under co-culture conditions, as demonstrated by increases in α-SMA expression and migratory activity. When exposed to paclitaxel, a survival advantage was observed in TSs co-cultured with activated fibroblasts. Overall, we demonstrated the reciprocal interaction between TSs and fibroblasts in our 7-channel microfluidic chip. The co-culture of 3D TS-CAF in a collagen matrix-incorporated microfluidic chip may be useful to study the tumor microenvironment and for evaluation of drug screening and evaluation. PMID:27391808

  10. Digital active control law synthesis for aeroservoelastic systems

    NASA Technical Reports Server (NTRS)

    Mukhopadhyay, Vivekananda

    1988-01-01

    This paper presents a formulation for synthesis of digital active control laws for aeroservoelastic systems, which are typically modeled by large order equations in order to accurately represent the rigid and flexible body modes, unsteady aerodynamic forces, actuator dynamics, and gust spectra. The control law is expected to satisfy multiple design requirements on the dynamic loads, responses, actuator deflection and rate limitations, as well as maintain certain stability margins, yet should be simple enough to be implemented by an onboard digital microprocessor. The synthesis procedure minimizes a linear quadratic Gaussian type cost function, by updating selected free parameters of the control law, while satisfying a set of inequality constraints on the design loads, responses and stability margins. A stable classical control law or an estimator based full or reduced order control law can be used as an initial design starting point. The gradients of the cost function and the constraints, with respect to the digital control law design variables are derived analytically, to facilitate rapid convergence. Selected design responses can be treated as constraints instead of lumping them into the cost function, in order to satisfy individual root-mean-square load and response limitations. Constraints are also imposed on the minimum singular value requirements for stability robustness improvement.

  11. Photocatalytically active titanium dioxide nanopowders: Synthesis, photoactivity and magnetic separation

    NASA Astrophysics Data System (ADS)

    Nikkanen, J.-P.; Heinonen, S.; Huttunen Saarivirta, E.; Honkanen, M.; Levänen, E.

    2013-12-01

    Two approaches were used to obtain nanocrystalline titanium dioxide (TiO2) photocatalyst powders. Firstly, low-temperature synthesis method and secondly liquid flame spraying. The structural properties of the produced powders were determined with X-ray diffraction, transmission electron microscopy and nitrogen adsorption tests. The photocatalytic properties of the powders were studied with methylene blue (MB) discoloration tests. After discolorations tests, TiO2 was coagulated with magnetite particles using FeCl3·6 H2O at a fixed pH value. Magnetic separation of coagulated TiO2 and magnetite was carried out by a permanent magnet. The obtained results showed that the particle size of the powders synthesized at low-temperature was very small and the specific surface area high. The phase content of the powder was also shown to depend greatly on the acidity of the synthesis solution. Powder synthesized by liquid flame spraying was mixture of anatase and rutile phases with essentially larger particle size and lower specific surface area than those of low-temperature synthesized powders. The MB discoloration test showed that photocatalytic activity depends on the phase structure as well as the specific surface area of the synthesized TiO2 powder. The magnetic separation of TiO2-magnetite coagulate from solution proved to be efficient around pH:8.

  12. Human IgG1 monoclonal antibody against human collagen 17 noncollagenous 16A domain induces blisters via complement activation in experimental bullous pemphigoid model.

    PubMed

    Li, Qiang; Ujiie, Hideyuki; Shibaki, Akihiko; Wang, Gang; Moriuchi, Reine; Qiao, Hong-jiang; Morioka, Hiroshi; Shinkuma, Satoru; Natsuga, Ken; Long, Heather A; Nishie, Wataru; Shimizu, Hiroshi

    2010-12-15

    Bullous pemphigoid (BP) is an autoimmune blistering disease caused by IgG autoantibodies targeting the noncollagenous 16A (NC16A) domain of human collagen 17 (hCOL17), which triggers blister formation via complement activation. Previous in vitro analysis demonstrated that IgG1 autoantibodies showed much stronger pathogenic activity than IgG4 autoantibodies; however, the exact pathogenic role of IgG1 autoantibodies has not been fully demonstrated in vivo. We constructed a recombinant IgG1 mAb against hCOL17 NC16A from BP patients. In COL17-humanized mice, this mAb effectively reproduced a BP phenotype that included subepidermal blisters, deposition of IgG1, C1q and C3, neutrophil infiltration, and mast cell degranulation. Subsequently, alanine substitutions at various C1q binding sites were separately introduced to the Fc region of the IgG1 mAb. Among these mutated mAbs, the one that was mutated at the P331 residue completely failed to activate the complement in vitro and drastically lost pathogenic activity in COL17-humanized mice. These findings indicate that P331 is a key residue required for complement activation and that IgG1-dependent complement activation is essential for blister formation in BP. This study is, to our knowledge, the first direct evidence that IgG1 Abs to hCOL17 NC16A can induce blister formation in vivo, and it raises the possibility that IgG1 mAbs with Fc modification may be used to block pathogenic epitopes in autoimmune diseases.

  13. Immunostimulation effect of jellyfish collagen.

    PubMed

    Sugahara, Takuya; Ueno, Masashi; Goto, Yoko; Shiraishi, Ryusuke; Doi, Mikiharu; Akiyama, Koichi; Yamauchi, Satoshi

    2006-09-01

    Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 degrees C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles' tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.

  14. TGF-β2 promotes RPE cell invasion into a collagen gel by mediating urokinase-type plasminogen activator (uPA) expression.

    PubMed

    Sugioka, Koji; Kodama, Aya; Okada, Kiyotaka; Iwata, Mihoko; Yoshida, Koji; Kusaka, Shunji; Matsumoto, Chota; Kaji, Hiroshi; Shimomura, Yoshikazu

    2013-10-01

    Transforming growth factor-beta (TGF-β) is one of the main epithelial-mesenchymal transition (EMT)-inducing factors. In general, TGF-β-induced EMT promotes cell migration and invasion. TGF-β also acts as a potent regulator of pericellular proteolysis by regulating the expression and secretion of plasminogen activators. Urokinase-type plasminogen activator (uPA) is a serine protease that binds to its cell surface receptor (uPAR) with high affinity. uPA binding to uPAR stimulates uPAR's interaction with transmembrane proteins, such as integrins, to regulate cytoskeletal reorganization and cell migration, differentiation and proliferation. However, the influence of TGF-β and the uPA/uPAR system on EMT in retinal pigment epithelial (RPE) cells is still unclear. The purpose of this study was to determine the effect of TGF-β2, which is the predominant isoform in the retina, and the uPA/uPAR system on RPE cells. In this study, we first examined the effect of TGF-β2 and/or the inhibitor of uPA (u-PA-STOP(®)) on the proliferation of a human retinal pigment epithelial cell line (ARPE-19 cells). Treatment with TGF-β2 or u-PA-STOP(®) suppressed cell proliferation. Combination treatment of TGF-β2 and u-PA-STOP(®) enhanced cell growth suppression. Furthermore, western blot analysis, fibrin zymography and real-time reverse transcription PCR showed that that TGF-β2 induced EMT in ARPE-19 cells and that the expression of uPA and uPAR expression was up-regulated during EMT. The TGF-β inhibitor SB431542 suppressed TGF-β2-stimulated uPA expression and secretion but did not suppress uPAR expression. Furthermore, we seeded ARPE-19 cells onto Transwell chambers and allowed them to invade the collagen matrix in the presence of TGF-β2 alone or with TGF-β2 and u-PA-STOP(®). TGF-β2 treatment induced ARPE-19 cell invasion into the collagen gel. Treatment with a combination of TGF-β2 and the uPA inhibitor strongly inhibited ARPE-19 cell invasion compared with treatment with

  15. Activated platelets signal chemokine synthesis by human monocytes.

    PubMed Central

    Weyrich, A S; Elstad, M R; McEver, R P; McIntyre, T M; Moore, K L; Morrissey, J H; Prescott, S M; Zimmerman, G A

    1996-01-01

    Human blood monocytes adhere rapidly and for prolonged periods to activated platelets that display P-selectin, an adhesion protein that recognizes a specific ligand on leukocytes, P-selectin glycoprotein-1. We previously demonstrated that P-selectin regulates expression and secretion of cytokines by stimulated monocytes when it is presented in a purified, immobilized form or by transfected cells. Here we show that thrombin-activated platelets induce the expression and secretion of monocyte chemotactic protein-1 and IL-8 by monocytes. Enhanced monokine synthesis requires engagement of P-selectin glycoprotein-1 on the leukocyte by P-selectin on the platelet. Secretion of the chemokines is not, however, directly signaled by P-selectin; instead, tethering of the monocytes by P-selectin is required for their activation by RANTES (regulated upon activation normal T cell expressed presumed secreted), a platelet chemokine not previously known to induce immediate-early gene products in monocytes. Adhesion of monocytes to activated platelets results in nuclear translocation of p65 (RelA), a component of the NF-kappaB family of transcription factors that binds kappaB sequences in the regulatory regions of monocyte chemotactic protein-1, IL-8, and other immediate-early genes. However, expression of tissue factor, a coagulation protein that also has a kappaB sequence in the 5' regulatory region of its gene, is not induced in monocytes adherent to activated platelets. Thus, contact of monocytes with activated platelets differentially affects the expression of monocyte products. These experiments suggest that activated platelets regulate chemokine secretion by monocytes in inflammatory lesions in vivo and provide a model for the study of gene regulation in cell-cell interactions. PMID:8617886

  16. The adaptor molecule signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is essential in mechanisms involving the Fyn tyrosine kinase for induction and progression of collagen-induced arthritis.

    PubMed

    Zhong, Ming-Chao; Veillette, André

    2013-11-01

    Signaling lymphocytic activation molecule-associated protein (SAP) is an Src homology 2 domain-only adaptor involved in multiple immune cell functions. It has also been linked to immunodeficiencies and autoimmune diseases, such as systemic lupus erythematosus. Here, we examined the role and mechanism of action of SAP in autoimmunity using a mouse model of autoimmune arthritis, collagen-induced arthritis (CIA). We found that SAP was essential for development of CIA in response to collagen immunization. It was also required for production of collagen-specific antibodies, which play a key role in disease pathogenesis. These effects required SAP expression in T cells, not in B cells. In mice immunized with a high dose of collagen, the activity of SAP was nearly independent of its ability to bind the protein tyrosine kinase Fyn and correlated with the capacity of SAP to promote full differentiation of follicular T helper (TFH) cells. However, with a lower dose of collagen, the role of SAP was more dependent on Fyn binding, suggesting that additional mechanisms other than TFH cell differentiation were involved. Further studies suggested that this might be due to a role of the SAP-Fyn interaction in natural killer T cell development through the ability of SAP-Fyn to promote Vav-1 activation. We also found that removal of SAP expression during progression of CIA attenuated disease severity. However, it had no effect on disease when CIA was clinically established. Together, these results indicate that SAP plays an essential role in CIA because of Fyn-independent and Fyn-dependent effects on TFH cells and, possibly, other T cell types.

  17. Synthesis, antimicrobial, and antiproliferative activities of substituted phenylfuranylnicotinamidines

    PubMed Central

    Youssef, Magdy M; Arafa, Reem K; Ismail, Mohamed A

    2016-01-01

    This research work deals with the design and synthesis of a series of substituted phenylfuranylnicotinamidines 4a–i. Facile preparation of the target compounds was achieved by Suzuki coupling-based synthesis of the nitrile precursors 3a–i, followed by their conversion to the corresponding nicotinamidines 4a–i utilizing LiN(TMS)2. The antimicrobial activities of the newly synthesized nicotinamidine derivatives were evaluated against the Gram-negative bacterial strains Escherichia coli and Pseudomonas aeruginosa as well as the Gram-positive bacterial strains Staphylococcus aureus and Bacillus megaterium. The minimum inhibitory concentration values of nicotinamidines against all tested microorganisms were in the range of 10–20 μM. In specific, compounds 4a and 4b showed excellent minimum inhibitory concentration values of 10 μM against Staphylococcus aureus bacterial strain and were similar to ampicillin as an antibacterial reference. On the other hand, selected nicotinamidine derivatives were biologically screened for their cytotoxic activities against a panel of 60 cell lines representing nine types of human cancer at a single high dose at National Cancer Institute, Bethesda, MD, USA. Nicotinamidines showing promising activities were further assessed in a five-dose screening assay to determine their compound concentration causing 50% growth inhibition of tested cell (GI50), compound concentration causing 100% growth inhibition of tested cell (TGI), and compound concentration causing 50% lethality of tested cell (LC50) values. Structure-activity relationship studies demonstrated that the activity of members of this series can be modulated from cytostatic to cytotoxic based on the substitution pattern/nature on the terminal phenyl ring. The most active compound was found to be 4e displaying a submicromolar GI50 value of 0.83 μM, with TGI and LC50 values of 2.51 and 100 μM, respectively. Finally, the possible underlying mechanism of action of this series of

  18. Synthesis and biological activity of salinomycin conjugates with floxuridine.

    PubMed

    Huczyński, Adam; Antoszczak, Michał; Kleczewska, Natalia; Lewandowska, Marta; Maj, Ewa; Stefańska, Joanna; Wietrzyk, Joanna; Janczak, Jan; Celewicz, Lech

    2015-03-26

    As part of our program to develop anticancer agents, we have synthesized new compounds, which are conjugates between well-known anticancer drug, floxuridine and salinomycin which is able to selectivity kill cancer stem cells. The conjugates were obtained in two ways i.e. by copper(I) catalysed click Huisgen cycloaddition reaction performed between 3'-azido-2',3'-dideoxy-5-fluorouridine and salinomycin propargyl amide, and by the ester synthesis starting from salinomycin and floxuridine under mild condition. The compounds obtained were characterized by spectroscopic methods and evaluated for their in vitro cytotoxicity against seven human cancer cell lines as well as antibacterial activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE). The conjugate obtained by esterification reaction showed a significantly higher antiproliferative activity against the drug-resistant cancer cells and lower toxicity than those of salinomycin and floxuridine towards normal cells, as well as standard anticancer drugs, such as cisplatin and doxorubicin. The conjugate compound revealed also moderate activity against MRSA and MRSE bacterial strains. Very high activity of floxuridine and 5-fluorouracil against MRSA and MRSE has been also observed.

  19. Synthesis and DPPH radical scavenging activity of prenylated phenol derivatives.

    PubMed

    Osorio, Mauricio; Aravena, Jacqueline; Vergara, Alejandra; Taborga, Lautaro; Baeza, Evelyn; Catalán, Karen; González, Cesar; Carvajal, Marcela; Carrasco, Héctor; Espinoza, Luis

    2012-01-06

    The synthesis of twenty six prenylated phenols derivatives is reported. These compounds were obtained under mild conditions via Electrophilic Aromatic Substitution (EAS) coupling reactions between phenol derivatives containing electron-donor subtituents and 3-methyl-2-buten-1-ol using BF(3)×OEt(2). Dialkylations were also produced with this method. The formation of a chroman ring by intramolecular cyclization between a sp2 carbon from the prenyl group with the hydroxyl substituent in the ortho position occurred with some phenols. All the synthesized compounds were evaluated as antioxidants according to a DPPH radical scavenging activity assay. IC(50) values of five synthesized compounds indicated they were as good antioxidants as Trolox™.

  20. Inhibitors of Angiogenesis in Cancer Therapy - Synthesis and Biological Activity.

    PubMed

    Gensicka, Monika; Głowacka, Agnieszka; Dzierzbicka, Krystyna; Cholewinski, Grzegorz

    2015-01-01

    Angiogenesis is the process of formation of new capillaries from preexisting blood vessels. Angiogenesis is involved in normal physiological processes, and plays an important role in tumor invasion and development of metastases. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis. VEGF is a mitogen for vascular endothelial cells and stimulates their proliferation. By inhibiting the biological activity of VEGF, and then signal cascades with neutralizing VEGF antibodies and signal inhibitors, may negatively regulate the growth and metastasis. Anti-angiogenesis therapy is less toxic than chemotherapy. Angiogenesis is a multistep and multifactorial process, and therefore, can be blocked at different levels. In this review article, the authors present the synthesis of novel inhibitors of angiogenesis, together with the results of biological tests in vitro, and in some cases, state trials.

  1. The effect of vascular endothelial growth factor on aggrecan and type II collagen expression in rat articular chondrocytes.

    PubMed

    Chen, Xuan-Yin; Hao, Ya-Rong; Wang, Zhe; Zhou, Jian-Lin; Jia, Qi-Xue; Qiu, Bo

    2012-11-01

    The expression of vascular endothelial growth factor (VEGF) directly correlates with the Mankin score and the degree of cartilage destruction. The biological activity of VEGF on articular cartilage remains unknown, so this study was performed to investigate the effect of VEGF on aggrecan and type II collagen expression in vitro. We carried out this study at the Center Laboratory of Renmin Hospital at Wuhan University. Rat articular chondrocytes were cultured in a monolayer. Then, the experiment was divided into 4 groups: group A (control group), without any disposal; group B, treated with 10 ng/ml VEGF; group C, treated with 10 ng/ml IL-1β; and group D, treated with 10 ng/ml VEGF + 10 ng/ml IL-1β. After 48 h, messenger RNA (mRNA) expression of aggrecan and type II collagen was evaluated by real-time polymerase chain reaction (real-Time PCR), and protein expression of aggrecan and type II collagen was detected by Western blotting. VEGF was found to significantly inhibit the expression of aggrecan and type II collagen at the gene and protein levels. These findings suggest that VEGF may result in degeneration of articular cartilage by inhibiting the synthesis and expression of aggrecan and type II collagen.

  2. Collagen vascular disease

    MedlinePlus

    ... developed these disorders were previously said to have "connective tissue" or "collagen vascular" disease. We now have names ... be used. These include as undifferentiated systemic rheumatic (connective tissue) diseases or overlap syndromes. Images Dermatomyositis, heliotrope eyelids ...

  3. A novel functional role of collagen glycosylation: interaction with the endocytic collagen receptor uparap/ENDO180.

    PubMed

    Jürgensen, Henrik J; Madsen, Daniel H; Ingvarsen, Signe; Melander, Maria C; Gårdsvoll, Henrik; Patthy, Laszlo; Engelholm, Lars H; Behrendt, Niels

    2011-09-16

    Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation.

  4. Nanomechanics of collagen microfibrils

    PubMed Central

    Vesentini, Simone; Redaelli, Alberto; Gautieri, Alfonso

    2013-01-01

    Summary Collagen constitutes one third of the human proteome, providing mechanical stability, elasticity and strength to organisms and is thus the prime construction material in biology. Collagen is also the dominating material in the extracellular matrix where its stiffness controls cell differentiation, growth and pathology. We use atomistic-based hierarchical multiscale modeling to describe this complex biological material from the bottom up. This includes the use and development of large-scale computational modeling tools to investigate several aspects related to collagen-based tissues, including source of visco-elasticity and deformation mechanisms at the nanoscale level. The key innovation of this research is that until now, collagen materials have primarily been described at macroscopic scales, without explicitly understanding the mechanical contributions at the molecular and fibrillar levels. The major impact of this research will be the development of fundamental models of collagenous tissues, important to the design of new scaffolding biomaterials for regenerative medicine as well as for the understanding of collagen-related diseases. PMID:23885342

  5. Human recombinant type I collagen produced in plants.

    PubMed

    Shoseyov, Oded; Posen, Yehudit; Grynspan, Frida

    2013-07-01

    As a central element of the extracellular matrix, collagen is intimately involved in tissue development, remodeling, and repair and confers high tensile strength to tissues. Numerous medical applications, particularly, wound healing, cell therapy, bone reconstruction, and cosmetic technologies, rely on its supportive and healing qualities. Its synthesis and assembly require a multitude of genes and post-translational modifications, where even minor deviations can be deleterious or even fatal. Historically, collagen was always extracted from animal and human cadaver sources, but bare risk of contamination and allergenicity and was subjected to harsh purification conditions resulting in irreversible modifications impeding its biofunctionality. In parallel, the highly complex and stringent post-translational processing of collagen, prerequisite of its viability and proper functioning, sets significant limitations on recombinant expression systems. A tobacco plant expression platform has been recruited to effectively express human collagen, along with three modifying enzymes, critical to collagen maturation. The plant extracted recombinant human collagen type I forms thermally stable helical structures, fibrillates, and demonstrates bioactivity resembling that of native collagen. Deployment of the highly versatile plant-based biofactory can be leveraged toward mass, rapid, and low-cost production of a wide variety of recombinant proteins. As in the case of collagen, proper planning can bypass plant-related limitations, to yield products structurally and functionally identical to their native counterparts.

  6. Sol-gel assisted fabrication of collagen hydrolysate composite scaffold: a novel therapeutic alternative to the traditional collagen scaffold.

    PubMed

    Ramadass, Satiesh Kumar; Perumal, Sathiamurthi; Gopinath, Arun; Nisal, Anuya; Subramanian, Saravanan; Madhan, Balaraman

    2014-09-10

    Collagen is one of the most widely used biomaterial for various biomedical applications. In this Research Article, we present a novel approach of using collagen hydrolysate, smaller fragments of collagen, as an alternative to traditionally used collagen scaffold. Collagen hydrolysate composite scaffold (CHCS) was fabricated with sol-gel transition procedure using tetraethoxysilane as the silica precursor. CHCS exhibits porous morphology with pore sizes varying between 380 and 780 μm. Incorporation of silica conferred CHCS with controlled biodegradation and better water uptake capacity. Notably, 3T3 fibroblast proliferation was seen to be significantly better under CHCS treatment when compared to treatment with collagen scaffold. Additionally, CHCS showed excellent antimicrobial activity against the wound pathogens Staphylococcus aureus, Bacillus subtilis, and Escherichia coli due to the inherited antimicrobial activity of collagen hydrolysate. In vivo wound healing experiments with full thickness excision wounds in rat model demonstrated that wounds treated with CHCS showed accelerated healing when compared to wounds treated with collagen scaffold. These findings indicate that the CHCS scaffold from collagen fragments would be an effective and affordable alternative to the traditionally used collagen structural biomaterials.

  7. Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor.

    PubMed

    An, Bo; Abbonante, Vittorio; Xu, Huifang; Gavriilidou, Despoina; Yoshizumi, Ayumi; Bihan, Dominique; Farndale, Richard W; Kaplan, David L; Balduini, Alessandra; Leitinger, Birgit; Brodsky, Barbara

    2016-02-26

    A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities.

  8. Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor*

    PubMed Central

    An, Bo; Abbonante, Vittorio; Xu, Huifang; Gavriilidou, Despoina; Yoshizumi, Ayumi; Bihan, Dominique; Farndale, Richard W.; Kaplan, David L.; Balduini, Alessandra; Leitinger, Birgit; Brodsky, Barbara

    2016-01-01

    A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities. PMID:26702058

  9. Synthesis and fungicidal activity of ethaboxam against Oomycetes.

    PubMed

    Kim, Dal-Soo; Chun, Sam-Jae; Jeon, Jae-Jin; Lee, Sang-Who; Joe, Goon-Ho

    2004-10-01

    This study describes the chemical synthesis and intrinsic fungicidal activity of ethaboxam [(RS)-N-(alpha-cyano-2-thenyl)-4-ethyl-2-(ethylamino)-1,3-thiazole-5-carboxamide], a new Oomycetes fungicide. In in vitro tests, ethaboxam showed inhibitory activity against isolates of Phytophthora and some Pythium spp, with MIC values ranging from 0.1 to 0.5 mg litre(-1) for nine isolates of Phytophthora infestans (Montagne) de Bary and from 1.0 to 5.0 mg litre(-1) for eight isolates of Phytophthora capsici Leonian. In tests to determine time and concentration for complete inactivation of each pathogen (five isolates of P infestans and five isolates of P capsici), ethaboxam inactivated all isolates of P infestans within 48h at 10 mg litre(-1) and those of P capsici within 96 h at 10 mg litre(-1). Ethaboxam effectively suppressed development of tomato late blight caused by P infestans and pepper Phytophthora blight caused by P capsici in the studies conducted to determine its preventive, curative, persistent and systemic activity. These results show that ethaboxam has desirable fungicidal characteristics as an Oomycetes fungicide.

  10. Resonant active sites in catalytic ammonia synthesis: A structural model

    NASA Astrophysics Data System (ADS)

    Cholach, Alexander R.; Bryliakova, Anna A.; Matveev, Andrey V.; Bulgakov, Nikolai N.

    2016-03-01

    Adsorption sites Mn consisted of n adjacent atoms M, each bound to the adsorbed species, are considered within a realistic model. The sum of bonds Σ lost by atoms in a site in comparison with the bulk atoms was used for evaluation of the local surface imperfection, while the reaction enthalpy at that site was used as a measure of activity. The comparative study of Mn sites (n = 1-5) at basal planes of Pt, Rh, Ir, Fe, Re and Ru with respect to heat of N2 dissociative adsorption QN and heat of Nad + Had → NHad reaction QNH was performed using semi-empirical calculations. Linear QN(Σ) increase and QNH(Σ) decrease allowed to specify the resonant Σ for each surface in catalytic ammonia synthesis at equilibrium Nad coverage. Optimal Σ are realizable for Ru2, Re2 and Ir4 only, whereas other centers meet steric inhibition or unreal crystal structure. Relative activity of the most active sites in proportion 5.0 × 10- 5: 4.5 × 10- 3: 1: 2.5: 3.0: 1080: 2270 for a sequence of Pt4, Rh4, Fe4(fcc), Ir4, Fe2-5(bcc), Ru2, Re2, respectively, is in agreement with relevant experimental data. Similar approach can be applied to other adsorption or catalytic processes exhibiting structure sensitivity.

  11. Collagen catabolism through Coll2-1 and Coll2-1NO2 and myeloperoxidase activity in marathon runners.

    PubMed

    Henrotin, Yves; Labasse, Alain; Franck, Thierry; Bosseloir, Alain; Bury, Thierry; Deberg, Michelle

    2013-12-01

    To determine the influence of marathon on the serum levels of two markers of cartilage degradation, Coll2-1 and its nitrated form, Coll2-1NO2, and of a marker of neutrophils activation, the myeloperoxidase (MPO). Coll2-1, Coll2-1NO2, total and active MPO were measured in 98 marathon runners without joint pain and with an average age of 47 years. Sera were taken at rest right before the departure and within 30 min after the marathon. The subjects were submitted to a questionnaire concerning their physical activity and their life style. The levels of Coll2-1, Coll2-1NO2 and active MPO were not affected by age, body mass index, sex or performance. The levels of total MPO were higher in female than in male (p < 0.05), but were not affected by the other parameters. After the marathon, Coll2-1 and Coll2-1NO2 concentrations were slightly but systematically decreased. The total and active MPO concentrations were increased by 2 to 3-fold in comparison to the pre-marathon values (p < 0.001 for total and active MPO). The active MPO/total MPO ratio was significantly enhanced after the marathon (p < 0.001). The variation of total MPO during the marathon was negatively correlated with the training time per week (r = -0.34; p = 0.009). The serum levels of Coll2-1 and Coll2-1NO2 were slightly decreased by marathon, indicating that intensive running could reduce cartilage catabolism. Furthermore, Coll2-1NO2 was not correlated with the total and active MPO indicating that Coll2-1 nitration did not result of a systemic oxidative phenomenon but reflects local changes.

  12. Temperature-sensitive polymer-conjugated IFN-gamma induces the expression of IDO mRNA and activity by fibroblasts populated in collagen gel (FPCG).

    PubMed

    Sarkhosh, Kourosh; Tredget, Edward E; Uludag, Hasan; Kilani, Ruhangiz T; Karami, Ali; Li, Yunyuan; Iwashina, Takashi; Ghahary, Aziz

    2004-10-01

    Indoleamine 2,3-dioxygenase (IDO) is an intracellular tryptophan-catabolizing enzyme possessing various immunosuppressive properties. Here, we report the use of this enzyme to suppress the proliferation of peripheral blood mononuclear cells (PBMC) co-cultured with IDO-expressing fibroblasts of an allogeneic skin substitute in vitro. Fetal foreskin fibroblasts populated within collagen gel (FPCG) were treated with interferon-gamma (IFN-gamma) conjugated with a temperature-sensitive polymer to induce the expression of IDO mRNA and protein. SDS-PAGE showed successful conjugation of IFN-gamma with the temperature-sensitive polymer. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by the measurement of kynurenine levels. The results of Northern blot analysis showed an induction of IDO mRNA expression when treated with polymer-conjugated IFN-gamma. Kynurenine levels, as a measure of IDO bioactivity, were significantly higher in IFN-gamma-treated fibroblasts than in controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA in FPCG treated with polymer-conjugated IFN-gamma was significantly longer than in those treated with free (non-conjugated) IFN-gamma (P < 0.001). IFN-gamma radiolabeling showed a prolonged retention of IFN-gamma within collagen gel in its polymer-conjugated form, compared to its free form. Presence of IDO protein in FPCG was demonstrated by Western analysis even 16 days after removal of the conditioned medium (containing released IFN-gamma). To demonstrate the immunosuppressive effects of IDO on the proliferation of PBMC, IDO-expressing FPCG treated with polymer-conjugated IFN-gamma were co-cultured with PBMC for a period of 5 days. The results showed a significant reduction in proliferation of PBMC co-cultured with IFN-gamma-treated IDO-expressing fibroblasts, compared to those co-cultured with non-IDO-expressing fibroblasts (P < 0.001). The addition of an IDO inhibitor (1-methyl

  13. Turnover Rates of Hepatic Collagen and Circulating Collagen-Associated Proteins in Humans with Chronic Liver Disease

    PubMed Central

    Li, Kelvin; Gatmaitan, Michelle; Luo, Flora; Cattin, Jerome; Nakamura, Corelle; Holmes, William E.; Angel, Thomas E.; Peters, Marion G.; Turner, Scott M.; Hellerstein, Marc K.

    2015-01-01

    Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of 2H2O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2–0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates. PMID:25909381

  14. Purification, characterization, cDNA cloning and in vitro expression of a serine proteinase from the intestinal tract of sea cucumber (Stichopus japonicus) with collagen degradation activity.

    PubMed

    Yan, Long-Jie; Zhan, Chun-Lan; Cai, Qiu-Feng; Weng, Ling; Du, Cui-Hong; Liu, Guang-Ming; Su, Wen-Jin; Cao, Min-Jie

    2014-05-21

    Sea cucumber (Stichopus japonicus) autolysis during transportation and processing is a major problem and the specific proteinases responsible for autolysis have not yet been identified. In the present study, a 34 kDa serine proteinase (SP) was isolated to high purity from sea cucumber intestinal tract by a series of column chromatographies. Peptide mass fingerprinting revealed that six peptide fragments were identical to a proprotein convertase subtilisin/kexin type 9 preproprotein from sea cucumber A. japonicus. The enzyme hydrolyzed gelatin effectively at pH 6.0-9.0 and 35-40 °C, and the enzyme activity was strongly inhibited by SP inhibitors. Sea cucumber collagen was hydrolyzed significantly by purified SP at 37 °C and more gradually at 4 °C, suggesting that SP may be involved in autolysis. In addition, the SP gene that codes for 377 amino acid residues was cloned into an E. coli expression vector and expressed in vitro. A polyclonal antibody against rSP was prepared and found to react specifically against both rSP and endogenous SP, which may prove useful for future studies on the physiological functions of SP.

  15. The Synthesis and Study of Azole Carboxamide Nucleosides as Agents Active Against RNA Viruses.

    DTIC Science & Technology

    1986-09-15

    5012 62770A 62770A8,1. AH 355 11. TITLE (Include Security Classification) The Synthesis and Study of Azole Carboxamide Nucleosides as Agents Active...broad-spectrum antiviral agent has stimulated a great deal of effort toward the chemical synthesis of nucleosides of other azole heterocycles. During the...4 II. Chemistry and Discussion . . .. .. . 6 1. Synthesis of Certain 5’-Substituted Derivatives of Ribavirin and Tiazofurin . . .. . 6 2

  16. Glycosaminoglycan sulodexide inhibition of MMP-9 gelatinase secretion and activity: possible pharmacological role against collagen degradation in vascular chronic diseases.

    PubMed

    Mannello, Ferdinando; Medda, Virginia; Ligi, Daniela; Raffetto, Joseph D

    2013-05-01

    We evaluated the effects of the glycosaminoglycan sulodexide (SDX; antithrombotic/profibrinolytic drug) on the activity and release of matrix metalloproteinases (MMPs) in human blood. This was a prospective non-randomized study, analyzing by zymography and ELISA the in vitro effects of SDX on pro-enzyme, complexed, and active MMP forms in plasma and serum from 60 healthy donors, and in U-937 leukemia cell line. The levels and zymographic profile of MMP-2 did not show significant changes among samples and during SDX treatments. However, pro- and complexed forms of MMP-9 were strongly affected by SDX treatment (P<0.001), with significant decrease of MMP-9 secretion from white blood cells in a dose-dependent fashion (P<0.0001), without any displacement of MMP prodomains. The mechanism of reduced release of MMP-9 forms from leukocytes and inhibition of proteolytic activity due to SDX treatment may support the hypothesis that drugs based upon inhibitors of MMP-9 activity may provide a therapeutic tool for the underlying pathological destruction of extracellular matrix, and offering novel pharmacologic applications for chronic inflammatory vascular diseases, including varicose vein and chronic venous diseases associated with enhanced MMP activation in blood and limbs.

  17. Abnormal Collagen Metabolism in Cultured Skin Fibroblasts from Patients with Duchenne Muscular Dystrophy

    NASA Astrophysics Data System (ADS)

    Rodemann, H. Peter; Bayreuther, Klaus

    1984-08-01

    Total collagen synthesis is decreased by about 29% (P < 0.01) in skin fibroblasts established in vitro from male patients with Duchenne muscular dystrophy (DMD) as compared with that in normal male skin fibroblasts in vitro. The reduction in collagen synthesis is associated with an approximately 2-fold increase in collagen degradation in DMD fibroblasts. Correlated to these alterations in the metabolism of collagen, DMD fibroblasts express a significantly higher hydroxyproline/proline ratio (DMD: 1.36-1.45; P < 0.01) than do normal fibroblasts (controls: 0.86-0.89). The increased hydroxylation of proline residues of collagen (composed of type I and type III) could be the cause for the enhanced degradation of collagen in DMD fibroblasts.

  18. Synthesis and characterization of collagen grafted poly(hydroxybutyrate-valerate) (PHBV) scaffold for loading of bovine serum albumin capped silver (Ag/BSA) nanoparticles in the potential use of tissue engineering application.

    PubMed

    Bakare, Rotimi A; Bhan, Chandra; Raghavan, Dharmaraj

    2014-01-13

    The objective of this study is to synthesize and characterize collagen grafted poly(3-hydroxylbutyrate-co-3-hydroxylvalerate) (PHBV) film for loading of BSA capped silver (Ag/BSA) nanoparticles. Thermal radical copolymerization and aminolysis methods were used to functionalize macroporous PHBV, followed by collagen grafting so as to formulate collagen-g-poly(hydroxyethylmethyl acrylate)-g-poly(3-hydroxylbutyrate-co-3-hydroxylvalerate) [collagen-g-PHEMA-g-PHBV] and collagen-g-aminated-poly(3-hydroxylbutyrate-co-3-hydroxylvalerate) [collagen-g-NH2-PHBV] films, respectively. Spectroscopic (FTIR, XPS), physical (SEM), and thermal (TGA) techniques were used to characterize the functionalized PHBV films. The amount of collagen present on grafted PHBV film was quantified by the Bradford method. The Ag/BSA nanoparticles were then loaded on collagen grafted and untreated PHBV films, and the nanoparticles loading were determined by atomic absorption spectrometry. The amount of nanoparticles loaded on collagen grafted PHBV film was found to be significantly greater than that on the untreated PHBV film. The nanoparticles loaded PHBV film can potentially serve as a scaffold to promote the growth of bone cells while inhibiting the bacterial growth.

  19. Integrin-dependent Control of Translation: Engagement of Integrin αIIbβ3 Regulates Synthesis of Proteins in Activated Human Platelets

    PubMed Central

    Pabla, Ravinder; Weyrich, Andrew S.; Dixon, Dan A.; Bray, Paul F.; McIntyre, Thomas M.; Prescott, Stephen M.; Zimmerman, Guy A.

    1999-01-01

    Integrins are widely expressed plasma membrane adhesion molecules that tether cells to matrix proteins and to one another in cell–cell interactions. Integrins also transmit outside-in signals that regulate functional responses of cells, and are known to influence gene expression by regulating transcription. In previous studies we found that platelets, which are naturally occurring anucleate cytoplasts, translate preformed mRNA transcripts when they are activated by outside-in signals. Using strategies that interrupt engagement of integrin αIIbβ3 by fibrinogen and platelets deficient in this integrin, we found that αIIbβ3 regulates the synthesis of B cell lymphoma 3 (Bcl-3) when platelet aggregation is induced by thrombin. We also found that synthesis of Bcl-3, which occurs via a specialized translation control pathway regulated by mammalian target of rapamycin (mTOR), is induced when platelets adhere to immobilized fibrinogen in the absence of thrombin and when integrin αIIbβ3 is engaged by a conformation-altering antibody against integrin αIIbβ3. Thus, outside-in signals delivered by integrin αIIbβ3 are required for translation of Bcl-3 in thrombin-stimulated aggregated platelets and are sufficient to induce translation of this marker protein in the absence of thrombin. Engagement of integrin α2β1 by collagen also triggered synthesis of Bcl-3. Thus, control of translation may be a general mechanism by which surface adhesion molecules regulate gene expression. PMID:9885253

  20. Spatio-temporal modification of collagen scaffolds mediated by triple helical propensity.

    PubMed

    Wang, Allen Y; Foss, Catherine A; Leong, Shirley; Mo, Xiao; Pomper, Martin G; Yu, Seungju M

    2008-07-01

    Functionalized collagen that incorporates exogenous compounds may offer new and improved biomaterials applications, especially in drug-delivery, multifunctional implants, and tissue engineering. To that end, we developed a specific and reversible collagen modification technique utilizing associative chain interactions between synthetic collagen mimetic peptide (CMP) [(ProHypGly) chi; Hyp = hydroxyproline] and type I collagen. Here we show temperature-dependent collagen binding and subsequent release of a series of CMPs with varying chain lengths indicating a triple helical propensity driven binding mechanism. The binding took place when melted, single-strand CMPs were allowed to fold while in contact with reconstituted type I collagens. The binding affinity is highly specific to collagen as labeled CMP bound to nanometer scale periodic positions on type I collagen fibers and could be used to selectively image collagens in ex vivo human liver tissue. When heated to physiological temperature, bound CMPs discharged from the collagen at a sustained rate that correlated with CMP's triple helical propensity, suggesting that sustainability is mediated by dynamic collagen-CMP interactions. We also report on the spatially defined modification of collagen film with linear and multi-arm poly(ethylene glycol)-CMP conjugates; at 37 degrees C, these PEG-CMP conjugates exhibited temporary cell repelling activity lasting up to 9 days. These results demonstrate new opportunities for targeting pathologic collagens for diagnostic or therapeutic applications and for fabricating multifunctional collagen coatings and scaffolds that can temporally and spatially control the behavior of cells associated with the collagen matrices.

  1. Synthesis of novel bisindolylmethane Schiff bases and their antibacterial activity.

    PubMed

    Imran, Syahrul; Taha, Muhammad; Ismail, Nor Hadiani; Khan, Khalid Mohammed; Naz, Farzana; Hussain, Memona; Tauseef, Saima

    2014-08-06

    In an effort to develop new antibacterial drugs, some novel bisindolylmethane derivatives containing Schiff base moieties were prepared and screened for their antibacterial activity. The synthesis of the bisindolylmethane Schiff base derivatives 3-26 was carried out in three steps. First, the nitro group of 3,3'-((4-nitrophenyl)-methylene)bis(1H-indole) (1) was reduced to give the amino substituted bisindolylmethane 2 without affecting the unsaturation of the bisindolylmethane moiety using nickel boride in situ generated. Reduction of compound 1 using various catalysts showed that combination of sodium borohydride and nickel acetate provides the highest yield for compound 2. Bisindolylmethane Schiff base derivatives were synthesized by coupling various benzaldehydes with amino substituted bisindolylmethane 2. All synthesized compounds were characterized by various spectroscopic methods. The bisindolylmethane Schiff base derivatives were evaluated against selected Gram-positive and Gram-negative bacterial strains. Derivatives having halogen and nitro substituent display weak to moderate antibacterial activity against Salmonella typhi, S. paratyphi A and S. paratyphi B.

  2. Synthesis of marmycin A and investigation into its cellular activity

    NASA Astrophysics Data System (ADS)

    Cañeque, Tatiana; Gomes, Filipe; Mai, Trang Thi; Maestri, Giovanni; Malacria, Max; Rodriguez, Raphaël

    2015-09-01

    Anthracyclines such as doxorubicin are used extensively in the treatment of cancers. Anthraquinone-related angucyclines also exhibit antiproliferative properties and have been proposed to operate via similar mechanisms, including direct genome targeting. Here, we report the chemical synthesis of marmycin A and the study of its cellular activity. The aromatic core was constructed by means of a one-pot multistep reaction comprising a regioselective Diels-Alder cycloaddition, and the complex sugar backbone was introduced through a copper-catalysed Ullmann cross-coupling, followed by a challenging Friedel-Crafts cyclization. Remarkably, fluorescence microscopy revealed that marmycin A does not target the nucleus but instead accumulates in lysosomes, thereby promoting cell death independently of genome targeting. Furthermore, a synthetic dimer of marmycin A and the lysosome-targeting agent artesunate exhibited a synergistic activity against the invasive MDA-MB-231 cancer cell line. These findings shed light on the elusive pathways through which anthraquinone derivatives act in cells, pointing towards unanticipated biological and therapeutic applications.

  3. Synthesis and photocatalytic activity of titania microspheres with hierarchical structures

    SciTech Connect

    Cheng, Qian-Qian; Cao, Ying; Yang, Lin; Zhang, Pei-Pei; Wang, Kui; Wang, Hua-Jie

    2011-03-15

    Research highlights: {yields} Lauryl alcohol directs the formation of titania with hierarchical structures. {yields} Hierarchical structures endow the higher specific area to titania. {yields} Titania had a higher photocatalytic activity than commercial Degussa P25. {yields} The synthesis process of the target product is low-cost. -- Abstract: A combined sol-gel and solvothermal process was introduced to fabricate the titania microspheres with hierarchical structures by using lauryl alcohol as the structure-directing agent. Scanning electron microscope, high-resolution transmission electron microscope, Fourier transform infrared spectrograph and powder X-ray powder diffraction indicated that the molar ratio of lauryl alcohol, water and tetra-n-butyl titanate was the key factor for the formation of the mono-dispersed titania with anatase phase and the optimal ratio was 1.2:4:1. The diameter of the end-product was 523 {+-} 74 nm and it was composed of smaller nanoparticles with about 6.8 nm size in diameter. Photocatalytic activity of the end-product was investigated by employing Rhodamine B and Methylene blue as the model compounds. The target microspheres exhibited the higher photocatalytic efficiency compared with commercial Degussa P25 titania and this result might be due to the hierarchical structures of microspheres according to the analysis of Brunauer-Emmett-Teller specific surface areas.

  4. The discoidin domain receptor DDR2 is a receptor for type X collagen.

    PubMed

    Leitinger, Birgit; Kwan, Alvin P L

    2006-08-01

    During endochondral ossification, collagen X is deposited in the hypertrophic zone of the growth plate. Our previous results have shown that collagen X is capable of interacting directly with chondrocytes, primarily via integrin alpha2beta1. In this study, we determined whether collagen X could also interact with the non-integrin collagen receptors, discoidin domain receptors (DDRs), DDR1 or DDR2. The widely expressed DDRs are receptor tyrosine kinases that are activated by a number of different collagen types. Collagen X was found to be a much better ligand for DDR2 than for DDR1. Collagen X bound to the DDR2 extracellular domain with high affinity and stimulated DDR2 autophosphorylation, the first step in transmembrane signalling. Expression of DDR2 in the epiphyseal plate was confirmed by RT-PCR and immunohistochemistry. The spatial expression of DDR2 in the hypertrophic zone of the growth plate is consistent with a physiological interaction of DDR2 with collagen X. Surprisingly, the discoidin domain of DDR2, which fully contains the binding sites for the fibrillar collagens I and II, was not sufficient for collagen X binding. The nature of the DDR2 binding site(s) within collagen X was further analysed. In addition to a collagenous domain, collagen X contains a C-terminal NC1 domain. DDR2 was found to recognise the triple-helical region of collagen X as well as the NC1 domain. Binding to the collagenous region was dependent on the triple-helical conformation. DDR2 autophosphorylation was induced by the collagen X triple-helical region but not the NC1 domain, indicating that the triple-helical region of collagen X contains a specific DDR2 binding site that is capable of receptor activation. Our study is the first to describe a non-fibrillar collagen ligand for DDR2 and will form the basis for further studies into the biological function of collagen X during endochondral ossification.

  5. CCK1 and CCK2 Receptors Are Expressed on Pancreatic Stellate Cells and Induce Collagen Production

    PubMed Central

    Berna, Marc J.; Seiz, Oliver; Nast, Jan Friso; Benten, Daniel; Bläker, Michael; Koch, Johannes; Lohse, Ansgar W.; Pace, Andrea

    2010-01-01

    The gastrointestinal hormone cholecystokinin (CCK) can induce acute pancreatitis in rodents through its action on acinar cells. Treatment with CCK, in combination with other agents, represents the most commonly used model to induce experimental chronic pancreatitis. Pancreatic stellate cells (PSC) are responsible for pancreatic fibrosis and therefore play a predominant role in the genesis of chronic pancreatitis. However, it is not known whether PSC express CCK receptors. Using real time PCR techniques, we demonstrate that CCK1 and CCK2 receptors are expressed on rat PSC. Interestingly both CCK and gastrin significantly induced type I collagen synthesis. Moreover, both inhibit proliferation. These effects are comparable with TGF-β-stimulated PSC. Furthermore, the natural agonists CCK and gastrin induce activation of pro-fibrogenic pathways Akt, ERK, and Src. Using specific CCK1 and CCK2 receptor (CCK2R) inhibitors, we found that Akt activation is mainly mediated by CCK2R. Akt activation by CCK and gastrin could be inhibited by the PI3K inhibitor wortmannin. Activation of ERK and the downstream target Elk-1 could be inhibited by the MEK inhibitor U0126. These data suggest that CCK and gastrin have direct activating effects on PSC, are able to induce collagen synthesis in these cells, and therefore appear to be important regulators of pancreatic fibrogenesis. Furthermore, similar to TGF-β, both CCK and gastrin inhibit proliferation in PSC. PMID:20843811

  6. Development of multifunctional collagen scaffolds directed by collagen mimetic peptides

    NASA Astrophysics Data System (ADS)

    Wang, Yi-Lan (Allen)

    Collagen is widely used for soft tissue replacement and tissue engineering scaffold. Functionalized collagen may offer new and improved applications for collagen-based biomaterials. But passively adsorbed molecules readily diffuse out from collagen matrix, and conventional chemical reactions on collagen are difficult to control and may compromise the biochemical feature of natural collagen. Hence, the aim of this dissertation is to develop a new physical collagen modification method through the non-covalent immobilization of collagen mimetic peptides (CMPs) and CMP derivatives on collagen scaffolds, thereby evading the drawbacks of passive and chemical modifications. Most of the research on CMPs over the past three decades has focused on synthesizing CMPs and understanding the effects of amino acid sequence on the peptide structural stability. Although few attempts have been made to develop biomaterials based on pure CMP, CMP has never used in complex with natural collagen. We demonstrate that CMPs with varying chain lengths have strong propensity to associate with natural 2-D and 3-D collagen substrates. We also show that CMPs can recognize and bind to reconstituted type I collagen fibers as well as collagens of ex vivo human liver tissue. The practical use of CMPs conjugated with linear and multi-arm poly(ethylene glycol)s allows to control cell organization in 2-D collagen substrates. Our cell adhesion studies suggest that under certain conditions (e.g. high incubation temperature, small CMP size), the bound CMP derivatives can be released from the collagen matrix, which may provide new opportunities for manipulating cell behavior especially by dynamically controlling the amount of signaling molecules in the collagen matrix. Polyanionic charged CMP was synthesized to modulate tubulogenesis of endothelial cells by attracting VEGF with 3-D collagen gel and a new PEG hydrogel using bifunctional CMP conjugates was synthesized as physico-chemical crosslinkers for

  7. Exosite Interactions Impact Matrix Metalloproteinase Collagen Specificities*

    PubMed Central

    Robichaud, Trista K.; Steffensen, Bjorn; Fields, Gregg B.

    2011-01-01

    Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I–III collagen sequences located N- or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769–783 from type I–III collagens, the second inserted α1(II) collagen residues 763–768 N-terminal to the consensus sequence, and the third inserted α1(II) collagen residues 784–792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased kcat/Km and kcat for MMP-1. MMP-13 showed the opposite behavior with a decreased kcat/Km and kcat and a greatly improved Km in response to the C-terminal residues. Insertion of the N-terminal residues enhanced kcat/Km and kcat for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced Km and dramatically decreased kcat, resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs. PMID:21896477

  8. Relationship of lipogenic enzyme activities to the rate of rat liver fatty acid synthesis

    SciTech Connect

    Nelson, G.; Kelley, D.; Schmidt, P.; Virk, S.; Serrato, C.

    1986-05-01

    The mechanism by which diet regulates liver lipogenesis is unclear. Here the authors report how dietary alterations effect the activities of key enzymes of fatty acid (FA) synthesis. Male Sprague-Dawley rats, 400-500 g, were fasted for 48h and then refed a fat-free, high carbohydrate (HC) diet (75% cal. from sucrose) for 0,3,9,24 and 48h, or refed a HC diet for 48h, then fed a high-fat (HF) diet (44% cal. from corn oil) for 3,9,24 and 48h. The FA synthesis rate and the activities of acetyl CoA carboxylase (AC), fatty acid synthase (FAS), ATP citrate lyase (CL), and glucose 6-phosphate dehydrogenase (G6PDH) were determined in the livers. FA synthesis was assayed with /sup 3/H/sub 2/O, enzyme activities were measured spectrophotometrically except for AC which was assayed with /sup 14/C-bicarbonate. There was no change in the activity of AC during fasting or on the HC diet. Fasting decreased the rate of FA synthesis by 25% and the activities of FAS and CL by 50%; refeeding the HC diet induced parallel changes in FA synthesis and the activities of FAS, CL, and G6PDH. After 9h on the HF diet, FA synthesis had decreased sharply, AC activity increased significantly while no changes were detected in the other activities. Subsequently FA synthesis did not change while the activities of the enzymes decreased slowly. These enzymes did not appear to regulate FA synthesis during inhibition of lipogenesis, but FAS, CL or G6PDH may be rate limiting in the induction phase. Other key factors may regulate FA synthesis during dietary alterations.

  9. LARP6 Meets Collagen mRNA: Specific Regulation of Type I Collagen Expression

    PubMed Central

    Zhang, Yujie; Stefanovic, Branko

    2016-01-01

    Type I collagen is the most abundant structural protein in all vertebrates, but its constitutive rate of synthesis is low due to long half-life of the protein (60–70 days). However, several hundred fold increased production of type I collagen is often seen in reparative or reactive fibrosis. The mechanism which is responsible for this dramatic upregulation is complex, including multiple levels of regulation. However, posttranscriptional regulation evidently plays a predominant role. Posttranscriptional regulation comprises processing, transport, stabilization and translation of mRNAs and is executed by RNA binding proteins. There are about 800 RNA binding proteins, but only one, La ribonucleoprotein domain family member 6 (LARP6), is specifically involved in type I collagen regulation. In the 5′untranslated region (5’UTR) of mRNAs encoding for type I and type III collagens there is an evolutionally conserved stem-loop (SL) structure; this structure is not found in any other mRNA, including any other collagen mRNA. LARP6 binds to the 5′SL in sequence specific manner to regulate stability of collagen mRNAs and their translatability. Here, we will review current understanding of how is LARP6 involved in posttranscriptional regulation of collagen mRNAs. We will also discuss how other proteins recruited by LARP6, including nonmuscle myosin, vimentin, serine threonine kinase receptor associated protein (STRAP), 25 kD FK506 binding protein (FKBP25) and RNA helicase A (RHA), contribute to this process. PMID:27011170

  10. Multicomponent synthesis of acylated short peptoids with antifungal activity against plant pathogens.

    PubMed

    Galetti, Matías D; Cirigliano, Adriana M; Cabrera, Gabriela M; Ramírez, Javier A

    2012-02-01

    In this article, we describe the synthesis of a small library of short peptoids composed of four glycine residues and acylated with a fatty acid that showed a remarkable in vitro activity against two fungal plant pathogens. Their straightforward synthesis implied two consecutive Ugi reactions and can be efficiently extended to the construction of highly diverse libraries.

  11. Development of interleukin-1 receptor antagonist mutants with enhanced antagonistic activity in vitro and improved therapeutic efficacy in collagen-induced arthritis.

    PubMed

    Dahlén, Eva; Barchan, Karin; Herrlander, Daniel; Höjman, Patrik; Karlsson, Marie; Ljung, Lill; Andersson, Mats; Bäckman, Eva; Hager, Ann-Christin Malmborg; Walse, Björn; Joosten, Leo; van den Berg, Wim

    2008-04-01

    Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring inhibitor of the pro-inflammatory interleukin-1-mediated activation of the interleukin-1 receptor (IL-1R). Although wild-type IL-1Ra is used for treatment of inflammatory diseases, its effect is moderate and/or short-lived. The objective of this study was to generate IL-1Ra mutants with enhanced antagonistic activity for potential therapeutic use. Using a directed evolution approach in which libraries of IL-1Ra gene mutants were generated and screened in functional assays, mutants with desired properties were identified. Initially, diversity was introduced into the IL-1Ra using random mutagenesis. Mutations resulting in enhanced antagonistic activity were identified by screening in a reporter cell assay. To further enhance the antagonistic activity, selected mutations were recombined using the DNA recombination technology Fragment-INduced Diversity (FIND). Following three rounds of FIND recombination, several mutants with up to nine times enhanced antagonistic activity (mean IC50 +/- SEM value: 0.78 +/- 0.050 vs. 6.8 +/- 1.1 ng/ml for mutant and wild-type, respectively) were identified. Sequence analysis identified the mutations D47N, E52R and E90Y as being most important for this effect, however, the mutations P38Y, H54R, Q129L and M136N further enhanced the antagonistic function. Analysis of identified mutations in protein models based on the crystal structure of the IL-1Ra/IL-1R complex suggested that mutations found to enhance the antagonistic activity had a stabilizing effect on the IL-1Ra mutants or increased the affinity for the IL-1R. Finally, the therapeutic effect of one mutant was compared to that of wild-type IL-1Ra in collagen-induced arthritis in mice. Indeed, the enhanced antagonistic effect of the mutants observed in vitro was also seen in vivo. In conclusion, these results demonstrate that directed evolution of IL-1Ra is an effective means of generating highly potent therapeutic

  12. Effect of kiwifruit juice on beef collagen.

    PubMed

    Sugiyama, Sumi; Hirota, Aya; Okada, Chikako; Yorita, Taeko; Sato, Kenji; Ohtsuki, Kozo

    2005-02-01

    The objective of this study is to clarify the difference in susceptibility to protease digestion by kiwifruit juice between collagen domains under different conditions. In addition, the effect of pre-treatment with kiwifruit juice on collagen in meat during cooking processes was examined. Kiwifruit juice can degrade denatured collagen, but it can not cleave the triple helical domain of collagen. Thus, kiwifruit juice does not have collagenase activity. On the other hand, the cross-linked subunits of acid-soluble collagen were converted to monomeric subunits by kiwifruit juice treatment at acidic pH, suggesting that the globular domains, in which cross-links preferentially occur, can be degraded by kiwifruit juice. The pre-treatment with kiwifruit juice significantly decreased the shear force of connective tissue in comparison with other pre-treatments without protease activity, but inversely increased the liberation of collagen-related peptides in the outer solution by heating processes at 50 and 70 degrees C or by a shorter heating time at 100 degrees C. This can be explained by the protease-mediated degradation of globular domains. However, this effect was not observed with a prolonged heating period at 100 degrees C, and the liberation of collagen-related peptides by pre-treatment with kiwifruit juice at 100 degrees C was less than that at 70 degrees C for all heating periods. Thus, it can be suggested that the pre-treatment with kiwifruit juice might be useful in meat softening under vacuum-cooking and grilling, but not under stewing.

  13. Derivatives of diphosphonic acids: synthesis and biological activity

    NASA Astrophysics Data System (ADS)

    Zolotukhina, M. M.; Krutikov, V. I.; Lavrent'ev, A. N.

    1993-07-01

    The scientific-technical and patent literature on the synthesis of derivatives of diphosphonic acids is surveyed. Various methods of synthesis of diphosphonate, phosphonylphosphinyl, and phosphonophosphate compounds are described. The principal aspects of the use of the above compounds in medicine, biochemistry, and agriculture are examined. The bibliography includes 174 references.

  14. Comparative studies of type X collagen expression in normal and rachitic chicken epiphyseal cartilage

    PubMed Central

    1989-01-01

    The levels of type X collagen in mineralizing normal chicken epiphyses and nonmineralizing rachitic chicken tibial epiphyses were measured and compared. Qualitative immunoperoxidase studies with anti-chick type X collagen monoclonal antibodies on sections from normal and rachitic cartilage demonstrated that the type X collagen levels in rachitic growth plates are reduced. Northern hybridization of mRNA and biosynthetic studies have confirmed that type X collagen synthesis in rickets is also decreased. In hypocalcemic rickets, the level of type X collagen mRNA is reduced by 80% whereas the level of type X collagen mRNA is only reduced by 50% in normocalcemic rickets. These observations provide additional evidence that type X collagen is involved in the process of cartilage mineralization and also suggest that the partial recovery of type X collagen synthesis in normocalcemic rickets may be related to the elevated plasma concentration of calcium. Calcium concentration may therefore play an important role in the control of type X collagen synthesis. PMID:2477383

  15. Collagen fibrils: nanoscale ropes.

    PubMed

    Bozec, Laurent; van der Heijden, Gert; Horton, Michael

    2007-01-01

    The formation of collagen fibrils from staggered repeats of individual molecules has become "accepted" wisdom. However, for over thirty years now, such a model has failed to resolve several structural and functional questions. In a novel approach, it was found, using atomic force microscopy, that tendon collagen fibrils are composed of subcomponents in a spiral disposition-that is, their structure is similar to that of macroscale ropes. Consequently, this arrangement was modeled and confirmed using elastic rod theory. This work provides new insight into collagen fibril structure and will have wide application-from the design of scaffolds for tissue engineering and a better understanding of pathogenesis of diseases of bone and tendon, to the conservation of irreplaceable parchment-based museum exhibits.

  16. Tenascin-x deficiency mimics ehlers-danlos syndrome in mice through alteration of collagen deposition

    SciTech Connect

    Mao, J.R.; Taylor, G.; Dean, W.B.; Wagner, D.R.; Afzal, V.; Lotz, J.C.; Rubin, E.M.; Bristow, J.

    2002-03-01

    Tenascin-X is a large extracellular matrix protein of unknown function1-3. Tenascin-X deficiency in humans is associated with Ehlers-Danlos syndrome4,5, a generalized connective tissue disorder resulting from altered metabolism of the fibrillar collagens6. Because TNXB is the first Ehlers-Danlos syndrome gene that does not encode a fibrillar collagen or collagen-modifying enzyme7-14, we suggested that tenascin-X might regulate collagen synthesis or deposition15. To test this hypothesis, we inactivated Tnxb in mice. Tnxb-/- mice showed progressive skin hyperextensibility, similar to individuals with Ehlers-Danlos syndrome. Biomechanical testing confirmed increased deformability and reduced tensile strength of their skin. The skin of Tnxb-/- mice was histologically normal, but its collagen content was significantly reduced. At the ultrastructural level, collagen fibrils of Tnxb-/- mice were of normal size and shape, but the density of fibrils in their skin was reduced, commensurate with the reduction in collagen content. Studies of cultured dermal fibroblasts showed that although synthesis of collagen I by Tnxb-/- and wildtype cells was similar, Tnxb-/- fibroblasts failed to deposit collagen I into cell-associated matrix. This study confirms a causative role for TNXB in human Ehlers-Danlos syndrome and suggests that tenascin-X is an essential regulator of collagen deposition by dermal fibroblasts.

  17. Diastereoselective Synthesis of Biologically Active Cyclopenta[b]indoles.

    PubMed

    Santos, Marilia S; Fernandes, Daniara C; Rodrigues, Manoel T; Regiani, Thais; Andricopulo, Adriano D; Ruiz, Ana Lúcia T G; Vendramini-Costa, Débora B; de Carvalho, João E; Eberlin, Marcos N; Coelho, Fernando

    2016-08-05

    The cyclopenta[b]indole motif is present in several natural and synthetic biologically active compounds, being directly responsible for the biological effects some of them present. We described herein a three step sequence for the synthesis of cyclopenta[b]indoles with a great structural diversity. The method is based on an oxidative Michael addition of suitable indoles on the double bond of Morita-Baylis-Hillman adducts mediated by a hypervalent iodine reagent (IBX) to form β-ketoesters, which were chemoselectively reduced with NaBH4 in THF to give the corresponding β-hydroxy-esters. The diastereoisomeric mixture was then treated with a catalytic amount of triflic acid (20 mol %) to give cyclopenta[b]indoles with overall yields ranging from 8 to 73% (for 2 steps). The acid-catalyzed cyclization step gave the required heterocycles, via an intramolecular Friedel-Crafts reaction, with high diastereoselectivity, where only the trans product was observed. A mechanistic study monitored by ESI-(+)-MS was also conducted to collect evidence about the mechanism of this reaction. The new molecules herein synthesized were also evaluated against a panel of human cancer cells demonstrating a promising antitumoral profile.

  18. Highly bacterial resistant silver nanoparticles: synthesis and antibacterial activities

    NASA Astrophysics Data System (ADS)

    Chudasama, Bhupendra; Vala, Anjana K.; Andhariya, Nidhi; Mehta, R. V.; Upadhyay, R. V.

    2010-06-01

    In this article, we describe a simple one-pot rapid synthesis route to produce uniform silver nanoparticles by thermal reduction of AgNO3 using oleylamine as reducing and capping agent. To enhance the dispersal ability of as-synthesized hydrophobic silver nanoparticles in water, while maintaining their unique properties, a facile phase transfer mechanism has been developed using biocompatible block co-polymer pluronic F-127. Formation of silver nanoparticles is confirmed by X-ray diffraction (XRD), transmission electron microscopy (TEM) and UV-vis spectroscopy. Hydrodynamic size and its distribution are obtained from dynamic light scattering (DLS). Hydrodynamic size and size distribution of as-synthesized and phase transferred silver nanoparticles are 8.2 ± 1.5 nm (σ = 18.3%) and 31.1 ± 4.5 nm (σ = 14.5%), respectively. Antimicrobial activities of hydrophilic silver nanoparticles is tested against two Gram positive ( Bacillus megaterium and Staphylococcus aureus), and three Gram negative ( Escherichia coli, Proteus vulgaris and Shigella sonnei) bacteria. Minimum inhibitory concentration (MIC) values obtained in the present study for the tested microorganisms are found much better than those reported for commercially available antibacterial agents.

  19. Indenopyrazole oxime ethers: synthesis and β1-adrenergic blocking activity.

    PubMed

    Angelone, Tommaso; Caruso, Anna; Rochais, Christophe; Caputo, Angela Maria; Cerra, Maria Carmela; Dallemagne, Patrick; Filice, Elisabetta; Genest, David; Pasqua, Teresa; Puoci, Francesco; Saturnino, Carmela; Sinicropi, Maria Stefania; El-Kashef, Hussein

    2015-03-06

    This paper reports the synthesis and cardiac activity of new β-blockers derived from (Z/E)-indeno[1,2-c]pyrazol-4(1H)-one oximes (5a,b). The latter compounds were allowed to react with epichlorohydrin, followed by reacting the oxiranyl derivatives formed (6a,b) with some aliphatic amines to give the target compounds (Z/E)-1-phenyl-1H-indeno[1,2-c]pyrazol-4-one O-((2-hydroxy-3-(substituted amino)propyl)oxime (7a-c) and (Z/E)-1-methyl-1H-indeno[1,2-c]pyrazol-4-one O-((2-hydroxy-3-(substituted amino)propyl)oxime (8a-c). These final products 7a-c and 8a-c were evaluated for their ability to modulate the cardiac performance of a prototype mammalian heart. The results showed that, out of these molecules tested, 7b elicits a more potent depressant effect on contractility and relaxation, and competitively antagonizes β1-adrenergic receptors.

  20. Synthesis and photocatalytic activity of poly(triazine imide).

    PubMed

    Ham, Yeilin; Maeda, Kazuhiko; Cha, Dongkyu; Takanabe, Kazuhiro; Domen, Kazunari

    2013-01-01

    Poly(triazine imide) was synthesized with incorporation of Li(+) and Cl(-) ions (PTI/Li(+)Cl(-)) to form a carbon nitride derivative. The synthesis of this material by the temperature-induced condensation of dicyandiamide was examined both in a eutectic mixture of LiCl-KCl and without KCl. On the basis of X-ray diffraction measurements of the synthesized materials, we suggest that a stoichiometric amount of LiCl is necessary to obtain the PTI/Li(+)Cl(-) phase without requiring the presence of KCl at 873 K. PTI/Li(+)Cl(-) with modification by either Pt or CoO(x) as cocatalyst photocatalytically produced H(2) or O(2), respectively, from water. The production of H(2) or O(2) from water indicates that the valence and conduction bands of PTI/Li(+)Cl(-) were properly located to achieve overall water splitting. The treatment of PTI/Li(+)Cl(-) with [Pt(NH(3))(4)](2+) cations enabled the deposition of Pt through ion exchange, demonstrating photocatalytic activity for H(2) evolution, while treatment with [PtCl(6)](2-) anions resulted in no Pt deposition. This was most likely because of the preferential exchange between Li(+) ions and [Pt(NH(3))(4)](2+) cations.

  1. Periodic knee injections of collagen tripeptide delay cartilage degeneration in rabbit experimental osteoarthritis

    PubMed Central

    2013-01-01

    Introduction Collagen peptides have been reported to possess various biological activities for various cell types. The purposes of this study were, first, to examine the therapeutic effects of collagen tripeptide (Ctp) in rabbit osteoarthritis and, second, to explore a synergetic effect with hyaluronan (HA). Methods Osteoarthritis was induced by anterior cruciate ligament transection of the right knee in 72 Japanese white rabbits and they were divided into four groups (control, Ctp, HA and Ctp/HA). Each material was injected weekly into the knee, and knee joint samples were collected 5, 10 and 15 weeks after surgery. Macroscopic and histomorphological analyses of cartilage were conducted. Expression of type II collagen and matrix metalloproteinase-13 was also analyzed immunohistochemically. A Tukey's honestly significant difference test was used to evaluate the statistical significance of difference in the macroscopic, histological and immnohistochemical results. Results All treatment groups exhibited slightly higher resistance to the progression of osteoarthritis than the control group macroscopically 15 weeks after surgery. Histologically, intra-articular injection of Ctp significantly reduced cartilage degradation 10 weeks after surgery, and Ctp/HA significantly reduced it 5 weeks after surgery in comparison with the control. Immunohistochemically, both Ctp-treated and Ctp/HA-treated groups had significantly increased type II collagen-positive chondrocytes at the fifth week after the surgery, although the numbers of matrix metalloproteinase-13-positive chondrocytes were not affected. Conclusion Periodical injections of Ctp and Ctp/HA delayed progression of cartilage degeneration of early osteoarthritis induced by anterior cruciate ligament transection in rabbits. This effect appears to be exerted by promotion of type II collagen synthesis predominantly. PMID:23433227

  2. Structure and function of collagen types

    SciTech Connect

    Mayne, R.; Burgeson, R.E.

    1987-01-01

    This book contains 10 chapters. Some of the chapter titles are: The Classical Collagens: Types I, II, and III; Type IV Collagen; Type IX Collagen; and Analysis of Collagen Structure by Molecular Biology Techniques.

  3. [The genetics of collagen diseases].

    PubMed

    Kaplan, J; Maroteaux, P; Frezal, J

    1986-01-01

    Heritable disorders of collagen include Ehler-Danlos syndromes (11 types are actually known), Larsen syndrome and osteogenesis imperfecta. Their clinical, genetic and biochemical features are reviewed. Marfan syndrome is closely related to heritable disorders of collagen.

  4. Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)-bioglass/chitosan-collagen composite scaffolds: a bone tissue engineering applications.

    PubMed

    Pon-On, Weeraphat; Charoenphandhu, Narattaphol; Teerapornpuntakit, Jarinthorn; Thongbunchoo, Jirawan; Krishnamra, Nateetip; Tang, I-Ming

    2014-05-01

    In the present study, composite scaffolds made with different weight ratios (0.5:1, 1:1 and 2:1) of bioactive glass (15Ca:80Si:5P) (BG)/polyvinyl alcohol (PVA) (PVABG) and chitosan (Chi)/collagen (Col) (ChiCol) were prepared by three mechanical freeze-thaw followed by freeze-drying to obtain the porous scaffolds. The mechanical properties and the in vitro biocompatibility of the composite scaffolds to simulated body fluid (SBF) and to rat osteoblast-like UMR-106 cells were investigated. The results from the studies indicated that the porosity and compressive strength were controlled by the weight ratio of PVABG:ChiCol. The highest compressive modulus of the composites made was 214.64 MPa which was for the 1:1 weight ratio PVABG:ChiCol. Mineralization study in SBF showed the formation of apatite crystals on the PVABG:ChiCol surface after 7 days of incubation. In vitro cell availability and proliferation tests confirmed the osteoblast attachment and growth on the PVABG:ChiCol surface. MTT and ALP tests on the 1:1 weight ratio PVABG:ChiCol composite indicated that the UMR-106 cells were viable. Alkaline phosphatase activity was found to increase with increasing culturing time. In addition, we showed the potential of PVABG:ChiCol drug delivery through PBS solution studies. 81.14% of BSA loading had been achieved and controlled release for over four weeks was observed. Our results indicated that the PVABG:ChiCol composites, especially the 1:1 weight ratio composite exhibited significantly improved mechanical, mineral deposition, biological properties and controlled release. This made them potential candidates for bone tissue engineering applications.

  5. Contribution of the collagen binding activity (VWF:CB) in the range of tests for the diagnosis and classification of von Willebrand disease.

    PubMed

    Ferhat-Hamida, Meriem Yasmine; Boukerb, Houda; Hariti, Ghania

    2015-01-01

    Von willebrand disease (VWD) is a common inherited bleeding disorder. The diagnosis may need a large panel of tests that differ in term of sensibility and specificity, and because of the effect of multifactorial modifiers (genetic or environmental); there is difficulty in defining diagnostic limits. We performed a panel of tests on 19 patients suffering from recurrent bleeding, to diagnose and classify VWD subtypes, by introducing the von Willebrand factor (VWF) collagen binding test (VWF:CB), then comparing the results with the activity of VWF risticetin cofactor (VWF:RCo) and multimer pattern. We considered 30% limit rate of VWF, as described by many authors, to make the diagnosis of VWD. The diagnosis of type 1 of VWD has been confirmed in 7patients, subtype 2A in 2 patients, subtype 2M in 2 patients and type 3 in 2 patients. We also defined a new group of 6 patients named "uncertain type 1" that didn't fill into the type 1 diagnostic criterion. The comparison between VWF:CB and VWF:RCo showed good correlation for all types of VWD except for type 2 while comparison between VWF:CB and multimer pattern showed good concordance for all types of VWD diagnosed. In conclusion, VWF:CB can be a good alternative to VWF:RCo for the diagnosis of quantitative deficiencies of VWF. It can also replace the multimer pattern study. However, the introduction of VWF:CB didn't help in the diagnosis of the "uncertain type 1" group of patients and cannot be a replacement for qualitative defect.

  6. Synthesis and antibacterial activity evaluation of two androgen derivatives.

    PubMed

    Lauro, Figueroa-Valverde; Francisco, Díaz-Cedillo; Elodia, García-Cervera; Eduardo, Pool-Gómez; Maria, López-Ramos; Marcela, Rosas-Nexticapa; Lenin, Hau-Heredia; Bety, Sarabia-Alcocer

    2015-01-01

    In this study two androgen derivatives were synthesized using several strategies; the first stage an aza-steroid derivative (3) was developed by the reaction of a testosterone derivative (1) with thiourea (2) in presence of hydrogen chloride. The second step, involves the synthesis of an amino-steroid derivative (4) by the reaction of 1 with 2 using boric acid as catalyst. The third stage was achieved by the preparation of an aminoaza-androgen derivative (6) by the reaction of 3 with ethylenediamine using boric acid as catalyst. In addition, the compound 6 was made reacting with dihydrotestosterone to form a new androgen derivative (7) in presence of boric acid. The following step was achieved by the reaction of 7 with chloroacetyl chloride to synthesize an azetidinone-androgen derivative (8) using triethylamine as catalyst. Additionally, a thiourea-androgen derivative (9) was synthetized by the reaction of 4 with dihydrotestosterone using boric acid as catalyst. Finally, the compound 9 was made reacting with chloroacetyl chloride in presence of triethylamine to synthesize a new azetidinone-androgen derivative (10). On the other hand, antibacterial activity of compounds synthesized was evaluated on Gram negative (Escherichia coli and Vibrio cholerae) and Gram positive (Staphylococos aureus) bacteria. The results indicate that only the compound 3 and 8 decrease the growth bacterial of E. coli and V. cholerae. Nevertheless, growth bacterial of S. aureus was not inhibited by these compounds. These data indicate that antibacterial activity exerted by the compounds 3 and 8 depend of their structure chemical in comparison with the controls and other androgen derivatives that are involved in this study.

  7. Phthalides: Distribution in Nature, Chemical Reactivity, Synthesis, and Biological Activity.

    PubMed

    León, Alejandra; Del-Ángel, Mayela; Ávila, José Luis; Delgado, Guillermo

    2017-01-01

    oxidation, reduction, addition, elimination, and cycloaddition reactions, and treatments with Lewis acids of (Z)-ligustilide have afforded linear dimers. Some intramolecular condensations and differentiated cyclizations of the dimeric phthalides have been carried out, providing evidences for the particular chemical reactivity of these compounds.Several structural modifications of phthalides have been carried out subjecting them to microbial transformations by different species of bacteria, fungi and algae, and these included resolutions of racemic mixtures and oxidations, among others.The [π4s + π2s] and [π2s + π2s] cycloadditions of (Z)-ligustilide for the synthesis of dimeric phthalides have been reported, and different approaches involving cyclizations, Alder-Rickert reactions, Sharpless asymmetric hydroxylations, or Grignard additions have been used for the synthesis of monomeric phthalides. The use of phthalides as building blocks for divergent oriented synthesis has been proven.Many of the naturally occurring phthalides display different biological activities including antibacterial, antifungal, insecticidal, cytotoxic, and anti-inflammatory effects, among many others, with a considerable recent research on the topic. In the case of compounds isolated from the Apiaceae, the bioactivities correlate with the traditional medicinal uses of the natural sources. Some monomeric phthalides have shown their ability to attenuate certain neurological diseases, including stroke, Alzheimer's and Parkinson's diseases.The present contribution covers the distribution of phthalides in nature and the findings in the structural diversity, chemical reactivity, biotransformations, syntheses, and bioactivity of natural and semisynthetic phthalides.

  8. Human collagen Krox up-regulates type I collagen expression in normal and scleroderma fibroblasts through interaction with Sp1 and Sp3 transcription factors.

    PubMed

    Kypriotou, Magdalini; Beauchef, Gallic; Chadjichristos, Christos; Widom, Russell; Renard, Emmanuelle; Jimenez, Sergio A; Korn, Joseph; Maquart, François-Xavier; Oddos, Thierry; Von Stetten, Otto; Pujol, Jean-Pierre; Galéra, Philippe

    2007-11-02

    Despite several investigations, the transcriptional mechanisms that regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. We have investigated the role of hc-Krox transcription factor on type I collagen expression by human dermal fibroblasts. hc-Krox exerted a stimulating effect on type I collagen protein synthesis and enhanced the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in foreskin fibroblasts (FF), adult normal fibroblasts (ANF), and scleroderma fibroblasts (SF). Forced hc-Krox expression was found to up-regulate COL1A1 transcription through a -112/-61-bp sequence in FF, ANF, and SF. Knockdown of hc-Krox by short interfering RNA and decoy strategies confirmed the transactivating effect of hc-Krox and decreased substantially COL1A1 transcription levels in all fibro-blast types. The -112/-61-bp sequence bound specifically hc-Krox but also Sp1 and CBF. Attempts to elucidate the potential interactions between hc-Krox, Sp1, and Sp3 revealed that all of them co-immunoprecipitate from FF cellular extracts when a c-Krox antibody was used and bind to the COL1A1 promoter in chromatin immunoprecipitation assays. Moreover, hc-Krox DNA binding activity to its COL1A1-responsive element is increased in SF, cells producing higher amounts of type I collagen compared with ANF and FF. These data suggest that the regulation of COL1A1 gene transcription in human dermal fibroblasts involves a complex machinery that implicates at least three transcription proteins, hc-Krox, Sp1, and Sp3, which could act in concert to up-regulate COL1A1 transcriptional activity and provide evidence for a pro-fibrotic role of hc-Krox.

  9. A concise synthesis of optically active solanacol, the germination stimulant for seeds of root parasitic weeds.

    PubMed

    Kumagai, Hiroshi; Fujiwara, Mami; Kuse, Masaki; Takikawa, Hirosato

    2015-01-01

    Solanacol, isolated from tobacco (Nicotiana tabacum L.), is a germination stimulant for seeds of root parasitic weeds. A concise synthesis of optically active solanacol has been achieved by employing enzymatic resolution as a key step.

  10. MICROWAVE-ASSISTED GREENER SYNTHESIS OF PHARMACEUTICALLY ACTIVE HETEROCYCLES UNDER BENIGN CONDITIONS

    EPA Science Inventory

    Green chemistry is a rapidly developing new field that provides us a proactive avenue for the sustainable development of future science and technologies. Environmentally benign protocols have been developed for the synthesis of various pharmaceutically active heterocycles namely ...

  11. Isolation, biological activity, synthesis, and medicinal chemistry of the pederin/mycalamide family of natural products.

    PubMed

    Mosey, R Adam; Floreancig, Paul E

    2012-09-01

    This review highlights the broad range of science that has arisen from the isolation of pederin, the mycalamides, theopederins, and onnamides, and psymberin. Specific topics include structure determination, biological activity, synthesis, and analog preparation and analysis.

  12. "Click" synthesis of small molecule probes for activity-based fingerprinting of matrix metalloproteases.

    PubMed

    Wang, Jun; Uttamchandani, Mahesh; Li, Junqi; Hu, Mingyu; Yao, Shao Q

    2006-09-28

    By using "Click Chemistry", we achieved the facile synthesis of various affinity-based hydroxamate probes that enable generation of activity-based fingerprints of a variety of metalloproteases, including matrix metalloproteases (MMPs), in proteomics experiments.

  13. The Non-phagocytic Route of Collagen Uptake

    PubMed Central

    Madsen, Daniel H.; Ingvarsen, Signe; Jürgensen, Henrik J.; Melander, Maria C.; Kjøller, Lars; Moyer, Amanda; Honoré, Christian; Madsen, Charlotte A.; Garred, Peter; Burgdorf, Sven; Bugge, Thomas H.; Behrendt, Niels; Engelholm, Lars H.

    2011-01-01

    The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen. PMID:21652704

  14. Platelet collagen receptor integrin alpha2beta1 activation involves differential participation of ADP-receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change.

    PubMed

    Jung, S M; Moroi, M

    2001-06-01

    In agonist-induced platelet activation, the collagen platelet receptor integrin alpha2beta1 is activated to high-affinity states through ADP involvement [Jung, S.M. & Moroi, M. (2000) J. Biol. Chem. 275, 8016-8026]. Here we determined the ADP-receptor subtypes involved and their relative contributions to alpha2beta1 activation (assessed by soluble-collagen binding) using the P2Y12 antagonist AR-C69931MX and P2Y1 antagonists adenosine 3',5'-diphosphate (Ado(3,5)PP) and adenosine 3'-phosphate 5'-phosphosulfate (AdoPPS). All three inhibited alpha2beta1 activation induced by low or high ADP, low thrombin, or low collagen-related peptide (CRP) concentrations; however, AR-C69931MX was markedly more inhibitory than the P2Y1 antagonists, suggesting the greater contribution of P2Y12. Inhibition patterns by various combinations of AR-C69931MX, AdoPPS, and wortmannin suggested that P2Y1 and P2Y12 mediate alpha2beta1 activation through different pathways, with possible involvement of phosphoinositide 3-kinase in both. Low concentrations of the acetoxy-methyl derivative of 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (calcium chelator) markedly decreased alpha2beta1 activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca2+ level (fluorimetric method) and alpha2beta1 activation (soluble-collagen binding) in the same platelet preparation indicated that alpha2beta1 activation via ADP receptors was independent of intracellular Ca2+ release. Our data indicate that integrin alpha2beta1 activation by ADP occurs through an inside-out signaling mechanism involving differential contributions by P2Y1 and P2Y12 wherein each contributes to some portion of the activation, with the stronger contribution of P2Y12. Furthermore, intracellular Ca2+ increase is not directly related to integrin alpha2beta1 activation, meaning that it is separate from the calcium mobilization pathways that these two ADP receptors are involved in.

  15. Collagen hydrolysate based collagen/hydroxyapatite composite materials

    NASA Astrophysics Data System (ADS)

    Ficai, Anton; Albu, Madalina Georgiana; Birsan, Mihaela; Sonmez, Maria; Ficai, Denisa; Trandafir, Viorica; Andronescu, Ecaterina

    2013-04-01

    The aim of this study was to study the influence of collagen hydrolysate (HAS) on the formation of ternary collagen-hydrolysate/hydroxyapatite composite materials (COLL-HAS/HA). During the precipitation process of HA, a large amount of brushite is resulted at pH = 7 but, practically pure HA is obtained at pH ⩾ 8. The FTIR data reveal the duplication of the most important collagen absorption bands due to the presence of the collagen hydrolysate. The presence of collagen hydrolysate is beneficial for the management of bone and joint disorders such as osteoarthritis and osteoporosis.

  16. Role of Flightless-I (Drosophila) homolog in the transcription activation of type I collagen gene mediated by transforming growth factor beta

    SciTech Connect

    Lim, Mi-Sun; Jeong, Kwang Won

    2014-11-21

    Highlights: • FLII activates TGFβ-mediated expression of COL1A2 gene. • TGFβ induces the association of FLII with SMAD3 and BRG1 in A549 cells. • FLII is required for the recruitment of SWI/SNF complex and chromatin accessibility to COL1A2 promoter. - Abstract: Flightless-I (Drosophila) homolog (FLII) is a nuclear receptor coactivator that is known to interact with other transcriptional regulators such as the SWI/SNF complex, an ATP-dependent chromatin-remodeling complex, at the promoter or enhancer region of estrogen receptor (ER)-α target genes. However, little is known about the role of FLII during transcription initiation in the transforming growth factor beta (TGFβ)/SMAD-dependent signaling pathway. Here, we demonstrate that FLII functions as a coactivator in the expression of type I collagen gene induced by TGFβ in A549 cells. FLII activates the reporter gene driven by COL1A2 promoter in a dose-dependent manner. Co-expression of GRIP1, CARM1, or p300 did not show any synergistic activation of transcription. Furthermore, the level of COL1A2 expression correlated with the endogenous level of FLII mRNA level. Depletion of FLII resulted in a reduction of TGFβ-induced expression of COL1A2 gene. In contrast, over-expression of FLII caused an increase in the endogenous expression of COL1A2. We also showed that FLII is associated with Brahma-related gene 1 (BRG1) as well as SMAD in A549 cells. Notably, the recruitment of BRG1 to the COL1A2 promoter region was decreased in FLII-depleted A549 cells, suggesting that FLII is required for TGFβ-induced chromatin remodeling, which is carried out by the SWI/SNF complex. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments revealed that depletion of FLII caused a reduction in chromatin accessibility at the COL1A2 promoter. These results suggest that FLII plays a critical role in TGFβ/SMAD-mediated transcription of the COL1A2 gene

  17. Collagen-type specificity of glycoprotein VI as a determinant of platelet adhesion.

    PubMed

    Jung, Stephanie M; Takemura, Yukitoshi; Imamura, Yasutada; Hayashi, Toshihiko; Adachi, Eijiro; Moroi, Masaaki

    2008-02-01

    Of the two physiologically important platelet collagen receptors, glycoprotein (GP) VI is the receptor responsible for platelet activation. However, its reactivities towards different types of vascular collagen have not been directly and quantitatively analysed with collagen preparations of defined composition, although the other major platelet collagen receptor integrin alpha(2)beta(1) was shown to react with collagen types I-VI and VIII under either static or flow conditions. We analysed the collagen type specificity of GPVI binding to identify the physiological contribution of the various vascular collagens and how platelet reactivity towards the various collagens may be affected by fibril size. We used two methods to analyse the binding of recombinant GPVI (GPVI-Fc(2)) to different types of bovine collagen: binding to collagen microparticles in suspension and binding to immobilized collagen. GPVI-Fc(2) bound to type I-III collagens that can form large fibrils, but not to type V that only forms small fibrils. The apparent GPVI binding to types IV and V could be ascribed to type I collagen that was a contaminant in each of these preparations. Kinetic analyses of the binding data showed that type III collagen fibrils have both a higher Kd and Bmax than types I and II. Flow adhesion studies demonstrated that type III collagen supports the formation of larger platelet aggregates than type I. Our present results suggest that the physiological importance of type III collagen is to induce thrombus formation. Furthermore, these studies indicate that GPVI mainly binds to collagen types that can form large collagen fibrils.

  18. Laccase Catalyzed Synthesis of Iodinated Phenolic Compounds with Antifungal Activity

    PubMed Central

    Ihssen, Julian; Schubert, Mark; Thöny-Meyer, Linda; Richter, Michael

    2014-01-01

    Iodine is a well known antimicrobial compound. Laccase, an oxidoreductase which couples the one electron oxidation of diverse phenolic and non-phenolic substrates to the reduction of oxygen to water, is capable of oxidizing unreactive iodide to reactive iodine. We have shown previously that laccase-iodide treatment of spruce wood results in a wash-out resistant antimicrobial surface. In this study, we investigated whether phenolic compounds such as vanillin, which resembles sub-structures of softwood lignin, can be directly iodinated by reacting with laccase and iodide, resulting in compounds with antifungal activity. HPLC-MS analysis showed that vanillin was converted to iodovanillin by laccase catalysis at an excess of potassium iodide. No conversion of vanillin occurred in the absence of enzyme. The addition of redox mediators in catalytic concentrations increased the rate of iodide oxidation ten-fold and the yield of iodovanillin by 50%. Iodinated phenolic products were also detected when o-vanillin, ethyl vanillin, acetovanillone and methyl vanillate were incubated with laccase and iodide. At an increased educt concentration of 0.1 M an almost one to one molar ratio of iodide to vanillin could be used without compromising conversion rate, and the insoluble iodovanillin product could be recovered by simple centrifugation. The novel enzymatic synthesis procedure fulfills key criteria of green chemistry. Biocatalytically produced iodovanillin and iodo-ethyl vanillin had significant growth inhibitory effects on several wood degrading fungal species. For Trametes versicolor, a species causing white rot of wood, almost complete growth inhibition and a partial biocidal effect was observed on agar plates. Enzymatic tests indicated that the iodinated compounds acted as enzyme responsive, antimicrobial materials. PMID:24594755

  19. The synthesis and photocatalytic activity of ZnSe microspheres

    NASA Astrophysics Data System (ADS)

    Cao, Huaqiang; Xiao, Yujiang; Zhang, Sichun

    2011-01-01

    This paper reports the synthesis of semiconductor ZnSe microspheres composed of nanoparticles via a solvothermal route between the organic molecule selenophene (C4H4 Se) and ZnCl2 without adding any surfactant. The ZnSe microspheres were characterized by x-ray diffraction (XRD), Raman spectroscopy, scanning electron microscopy (SEM), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), specific surface area measurement, and photoluminescence (PL) spectra. A strong and broad blue PL emission at 443 nm in wavelength (~2.79 eV in photon energy) is attributed to the near-band-edge (NBE) emission of ZnSe, while the 530 nm peak is a defect-related (DL) emission. The photocatalytic activity of the as-prepared ZnSe microspheres was evaluated by photodegradation of methyl orange (MO) dye under ultraviolet (UV) light and visible light irradiation. The degradations of MO reach 94% or 95.1%, close to 100%, in the presence of the as-synthesized ZnSe microspheres or commercial ZnSe powder after 7 or 10 h under UV irradiation, respectively. Meanwhile the degradations of MO reach 94.3% or 60.6% in the presence of the as-synthesized ZnSe microspheres or commercial ZnSe powder after 12 h, respectively. The degradation rate of ZnSe microspheres is twice that of ZnSe commercial powder under UV light irradiation, and three times under visible light irradiation. The degradation process of MO dye on ZnSe microspheres under UV or visible light is also discussed.

  20. The synthesis and photocatalytic activity of ZnSe microspheres.

    PubMed

    Cao, Huaqiang; Xiao, Yujiang; Zhang, Sichun

    2011-01-07

    This paper reports the synthesis of semiconductor ZnSe microspheres composed of nanoparticles via a solvothermal route between the organic molecule selenophene (C(4)H(4) Se) and ZnCl(2) without adding any surfactant. The ZnSe microspheres were characterized by x-ray diffraction (XRD), Raman spectroscopy, scanning electron microscopy (SEM), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), specific surface area measurement, and photoluminescence (PL) spectra. A strong and broad blue PL emission at 443 nm in wavelength (∼2.79 eV in photon energy) is attributed to the near-band-edge (NBE) emission of ZnSe, while the 530 nm peak is a defect-related (DL) emission. The photocatalytic activity of the as-prepared ZnSe microspheres was evaluated by photodegradation of methyl orange (MO) dye under ultraviolet (UV) light and visible light irradiation. The degradations of MO reach 94% or 95.1%, close to 100%, in the presence of the as-synthesized ZnSe microspheres or commercial ZnSe powder after 7 or 10 h under UV irradiation, respectively. Meanwhile the degradations of MO reach 94.3% or 60.6% in the presence of the as-synthesized ZnSe microspheres or commercial ZnSe powder after 12 h, respectively. The degradation rate of ZnSe microspheres is twice that of ZnSe commercial powder under UV light irradiation, and three times under visible light irradiation. The degradation process of MO dye on ZnSe microspheres under UV or visible light is also discussed.

  1. Hypoxia-inducible factor-1 (HIF-1) but not HIF-2 is essential for hypoxic induction of collagen prolyl 4-hydroxylases in primary newborn mouse epiphyseal growth plate chondrocytes.

    PubMed

    Aro, Ellinoora; Khatri, Richa; Gerard-O'Riley, Rita; Mangiavini, Laura; Myllyharju, Johanna; Schipani, Ernestina

    2012-10-26

    Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are α(2)β(2) tetramers with three isoforms of the catalytic α subunit, yielding C-P4Hs of types I-III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1α, HIF-2α, or the von Hippel-Lindau protein. The data obtained showed that C-P4H α(I) and α(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment.

  2. Modulation of cell attachment and collagen production of anterior cruciate ligament cells via submicron grooves/ridges structures with different cell affinity.

    PubMed

    Wang, Peng-Yuan; Wu, Tsung-Han; Chao, Pen-Hsiu Grace; Kuo, Wei-Hsuan; Wang, Meng-Jiy; Hsu, Cheng-Che; Tsai, Wei-Bor

    2013-01-01

    This study aimed to investigate the effects of submicron-grooved topography and surface cell affinity on the attachment, proliferation and collagen synthesis of anterior cruciate ligament (ACL) cells. Two grooved polystyrene (PS) surfaces (equal groove/ridge width of 800 nm) with a groove depth of 100 or 700 nm were fabricated and modified by oxygen plasma treatment, dopamine deposition and conjugation of RGD-containing peptides to enhance cell affinity. The elongation and alignment of ACL cells was enhanced by grooved structures with increasing groove depths regardless of surface chemistry. On the other hand, cell spreading and proliferation mainly depended on surface chemistry, in accordance with surface cell affinity: O(2) plasma < dopamine deposition < RGD conjugation. The synthesis of type I collagen was the highest by the ACL cells cultured on the 700 nm grooved surface conjugated with RGD peptides, indicating that both surface grooved topography and chemistry play a role in modulating collagen production of ACL cells. Furthermore, the type I collagen deposited on the 700 nm PS surface was aligned with grooves/ridges. Our results indicated that both ligand presentation and cell alignment are important in the physiological activities of ACL fibroblasts. Such information is critical for design of biomaterials for ACL tissue engineering.

  3. Possible association of elevated serum collagen type IV level with skin sclerosis in systemic sclerosis.

    PubMed

    Motegi, Sei-Ichiro; Sekiguchi, Akiko; Fujiwara, Chisako; Toki, Sayaka; Ishikawa, Osamu

    2016-08-29

    Collagen type IV is the primary collagen in the basement membranes around blood vessels and in the dermoepidermal junction in the skin. Perivascular collagen type IV is synthesized by endothelial cells and pericytes, and contributes to the homeostasis and remodeling of blood vessels. It has been well recognized that elevated serum collagen type IV levels are associated with the liver fibrosis. The objective was to examine serum collagen type IV levels and their clinical associations in patients with systemic sclerosis (SSc), and to examine the expression of collagen type IV in the fibrotic skin in SSc. Serum collagen type IV levels in SSc patients and diffuse cutaneous type SSc patients were significantly higher than those in healthy individuals. Serum collagen type IV levels were positively correlated with modified Rodnan total skin score. Serum collagen type IV levels in early stage (disease duration ≤3 years) diffuse cutaneous SSc patients were significantly elevated. Serum collagen type IV levels in SSc patients with digital ulcers (DU) were significantly elevated. In immunohistochemical staining, the expression of collagen type IV around dermal small vessels in the affected skin was reduced compared with those of normal individuals. These results suggest that elevated serum collagen type IV levels may be associated with the skin sclerosis in the early stage of SSc. The measurement of serum collagen type IV levels in SSc patients may be useful as a disease activity marker in skin sclerosis and DU.

  4. "Miswak" Based Green Synthesis of Silver Nanoparticles: Evaluation and Comparison of Their Microbicidal Activities with the Chemical Synthesis.

    PubMed

    Shaik, Mohammed Rafi; Albalawi, Ghadeer H; Khan, Shams Tabrez; Khan, Merajuddin; Adil, Syed Farooq; Kuniyil, Mufsir; Al-Warthan, Abdulrahman; Siddiqui, Mohammed Rafiq H; Alkhathlan, Hamad Z; Khan, Mujeeb

    2016-11-06

    Microbicidal potential of silver nanoparticles (Ag-NPs) can be drastically improved by improving their solubility or wettability in the aqueous medium. In the present study, we report the synthesis of both green and chemical synthesis of Ag-NPs, and evaluate the effect of the dispersion qualities of as-prepared Ag-NPs from both methods on their antimicrobial activities. The green synthesis of Ag-NPs is carried out by using an aqueous solution of readily available Salvadora persica L. root extract (RE) as a bioreductant. The formation of highly crystalline Ag-NPs was established by various analytical and microscopic techniques. The rich phenolic contents of S. persica L. RE (Miswak) not only promoted the reduction and formation of NPs but they also facilitated the stabilization of the Ag-NPs, which was established by Fourier transform infrared spectroscopy (FT-IR) analysis. Furthermore, the influence of the volume of the RE on the size and the dispersion qualities of the NPs was also evaluated. It was revealed that with increasing the volume of RE the size of the NPs was deteriorated, whereas at lower concentrations of RE smaller size and less aggregated NPs were obtained. During this study, the antimicrobial activities of both chemically and green synthesized Ag-NPs, along with the aqueous RE of S. persica L., were evaluated against various microorganisms. It was observed that the green synthesized Ag-NPs exhibit comparable or slightly higher antibacterial activities than the chemically obtained Ag-NPs.

  5. Synthesis and structure-activity relationships of potent antitumor active quinoline and naphthyridine derivatives.

    PubMed

    Srivastava, Sanjay K; Jha, Amrita; Agarwal, Shiv K; Mukherjee, Rama; Burman, Anand C

    2007-11-01

    The disease of cancer has been ranked second after cardiovascular diseases and plant-derived molecules have played an important role for the treatment of cancer. Nine cytotoxic plant-derived molecules such as vinblastine, vincristine, navelbine, etoposide, teniposide, taxol, taxotere, topotecan and irinotecan have been approved as anticancer drugs. Recently, epothilones are being emerging as future potential anti-tumor agents. However, targeted cancer therapy has now been rapidly expanding and small organic molecules are being exploited for this purpose. Amongst target specific small organic molecules, quinazoline was found as one of the most successful chemical class in cancer chemotherapy as three drugs namely Gefitinib, Erlotinib and Canertinib belong to this series. Now, quinazoline related chemical classes such as quinolines and naphthyridines are being exploited in cancer chemotherapy and a number of molecules such as compounds EKB-569 (52), HKI-272 (78) and SNS-595 (127a) are in different phases of clinical trials. This review presents the synthesis of quinolines and naphthyridines derivatives, screened for anticancer activity since year 2000. The synthesis of most potent derivatives in each prototype has been delineated. A brief structure activity relationship for each prototype has also been discussed. It has been observed that aniline group at C-4, aminoacrylamide substituents at C-6, cyano group at C-3 and alkoxy groups at C-7 in the quinoline ring play an important role for optimal activity. While aminopyrrolidine functionality at C-7, 2'-thiazolyl at N-1 and carboxy group at C-3 in 1,8-naphthyridine ring are essential for eliciting the cytotoxicity. This review would help the medicinal chemist to design and synthesize molecules for targeted cancer chemotherapy.

  6. Collagen fibril diameter and alignment promote the quiescent keratocyte phenotype

    PubMed Central

    Muthusubramaniam, Lalitha; Peng, Lily; Zaitseva, Tatiana; Paukshto, Michael; Martin, George R.; Desai, Tejal

    2011-01-01

    In this study, we investigated how matrix nanotopography affects corneal fibroblast phenotype and matrix synthesis. To this end, corneal fibroblasts isolated from bovine corneas were grown on collagen nanofiber scaffolds of different diameters and alignment – 30 nm aligned fibrils (30A), 300 nm or larger aligned fibrils (300A), and 30 nm nonaligned fibrils (30NA) in comparison to collagen coated flat glass substrates (FC). Cell morphology was visualized using confocal microscopy. Quantitative PCR was used to measure expression levels of six target genes: the corneal crystallin - transketolase (TKT), the myofibroblast marker - α-smooth muscle actin (SMA), and four matrix proteins - collagen 1 (COL1), collagen 3 (COL3), fibronectin (FN) and biglycan. It was found that SMA expression was down-regulated and TKT expression was increased on all three collagen nanofiber substrates, compared to the FC control substrates. However, COL3 and biglycan expression was also significantly increased on 300A, compared to the FC substrates. Thus matrix nanotopography down-regulates the fibrotic phenotype, promotes formation of the quiescent keratocyte phenotype and influences matrix synthesis. These results have significant implications for the engineering of corneal replacements and for promoting regenerative healing of the cornea after disease and/or injury. PMID:22213336

  7. Collagen-binding Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) of Gram-positive Bacteria Inhibit Complement Activation via the Classical Pathway*

    PubMed Central

    Kang, Mingsong; Ko, Ya-Ping; Liang, Xiaowen; Ross, Caná L.; Liu, Qing; Murray, Barbara E.; Höök, Magnus

    2013-01-01

    Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r2C1s2 was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens. PMID:23720782

  8. Collagen Prolyl Hydroxylases are Essential for Breast Cancer Metastasis

    PubMed Central

    Gilkes, Daniele M.; Chaturvedi, Pallavi; Bajpai, Saumendra; Wong, Carmen Chak-Lui; Wei, Hong; Pitcairn, Stephen; Hubbi, Maimon E.; Wirtz, Denis; Semenza, Gregg L.

    2013-01-01

    Metastasis is the leading cause of death among patients with breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies for cancer patients. Intratumoral hypoxia is found in the majority of breast cancers and is associated with an increased risk of metastasis and patient mortality. Here we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced invasion and metastasis to lymph nodes and lungs. Lastly, we establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies, and demonstrate that ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, decreases tumor fibrosis and metastasis in a mouse model of breast cancer. PMID:23539444

  9. Diversity-oriented synthesis and cytotoxic activity evaluation of biaryl-containing macrocycles.

    PubMed

    Pérez-Labrada, Karell; Cruz-Mendoza, Marco A; Chávez-Riveros, Alejandra; Hernández-Vázquez, Eduardo; Torroba, Tomás; Miranda, Luis D

    2017-03-03

    Synthesis of biaryl-containing macrocycles has been carried out through a four-step approach comprising two Ugi four component reactions and a Suzuki-Miyaura macrocyclization. This protocol allowed the synthesis of 12- and 14-membered macrocycles. Cytotoxic activity evaluation showed that some of the molecules were effective against leukemia, glioblastoma and lung cancer cell lines (IC50 = 4.0, 5.9 and 7.6, respectively).

  10. The rate of synthesis and decomposition of tissue proteins in hypokinesia and increased muscular activity

    NASA Technical Reports Server (NTRS)

    Fedorov, I. V.; Chernyy, A. V.; Fedorov, A. I.

    1978-01-01

    During hypokinesia and physical loading (swimming) of rats, the radioactivity of skeletal muscle, liver, kidney, heart, and blood proteins was determined after administration of radioactive amino acids. Tissue protein synthesis decreased during hypokinesia, and decomposition increased. Both synthesis and decomposition increased during physical loading, but anabolic processes predominated in the total tissue balance. The weights of the animals decreased in hypokinesia and increased during increased muscle activity.

  11. Phlorizin, an Active Ingredient of Eleutherococcus senticosus, Increases Proliferative Potential of Keratinocytes with Inhibition of MiR135b and Increased Expression of Type IV Collagen.

    PubMed

    Choi, Hye-Ryung; Nam, Kyung-Mi; Lee, Hyun-Sun; Yang, Seung-Hye; Kim, Young-Soo; Lee, Jongsung; Date, Akira; Toyama, Kazumi; Park, Kyoung-Chan

    2016-01-01

    E. senticosus extract (ESE), known as antioxidant, has diverse pharmacologic effects. It is also used as an antiaging agent for the skin and phlorizin (PZ) is identified as a main ingredient. In this study, the effects of PZ on epidermal stem cells were investigated. Cultured normal human keratinocytes and skin equivalents are used to test whether PZ affects proliferative potential of keratinocytes and how it regulates these effects. Skin equivalents (SEs) were treated with ESE and the results showed that the epidermis became slightly thickened on addition of 0.002% ESE. The staining intensity of p63 as well as proliferating cell nuclear antigen (PCNA) is increased, and integrin α6 was upregulated. Analysis of ESE confirmed that PZ is the main ingredient. When SEs were treated with PZ, similar findings were observed. In particular, the expression of integrin α6, integrin β1, and type IV collagen was increased. Levels of mRNA for type IV collagen were increased and levels of miR135b were downregulated. All these findings suggested that PZ can affect the proliferative potential of epidermal cells in part by microenvironment changes via miR135b downregulation and following increased expression of type IV collagen.

  12. Phlorizin, an Active Ingredient of Eleutherococcus senticosus, Increases Proliferative Potential of Keratinocytes with Inhibition of MiR135b and Increased Expression of Type IV Collagen

    PubMed Central

    Choi, Hye-Ryung; Nam, Kyung-Mi; Lee, Hyun-Sun; Yang, Seung-Hye; Kim, Young-Soo; Lee, Jongsung; Date, Akira; Toyama, Kazumi; Park, Kyoung-Chan

    2016-01-01

    E. senticosus extract (ESE), known as antioxidant, has diverse pharmacologic effects. It is also used as an antiaging agent for the skin and phlorizin (PZ) is identified as a main ingredient. In this study, the effects of PZ on epidermal stem cells were investigated. Cultured normal human keratinocytes and skin equivalents are used to test whether PZ affects proliferative potential of keratinocytes and how it regulates these effects. Skin equivalents (SEs) were treated with ESE and the results showed that the epidermis became slightly thickened on addition of 0.002% ESE. The staining intensity of p63 as well as proliferating cell nuclear antigen (PCNA) is increased, and integrin α6 was upregulated. Analysis of ESE confirmed that PZ is the main ingredient. When SEs were treated with PZ, similar findings were observed. In particular, the expression of integrin α6, integrin β1, and type IV collagen was increased. Levels of mRNA for type IV collagen were increased and levels of miR135b were downregulated. All these findings suggested that PZ can affect the proliferative potential of epidermal cells in part by microenvironment changes via miR135b downregulation and following increased expression of type IV collagen. PMID:27042261

  13. Platelets interact with soluble and insoluble collagens through characteristically different reactions.

    PubMed

    Jung, S M; Moroi, M

    1998-06-12

    Platelet interaction with soluble and insoluble collagens was characterized through binding studies. In contrast to resting platelets, cells reacted with activators, TS2/16 (integrin alpha2 beta1-activating antibody), thrombin, collagen-related peptide, or ADP, exhibited specific soluble collagen binding that is Mg2+-dependent, but inhibited by prostaglandin I2, Ca2+, and Gi9 (anti-integrin alpha2 beta1 antibody). Each platelet has 1500-3500 soluble collagen binding sites, with a dissociation constant of 3. 5-9 x 10(-8) M. This is the first study to show the specific binding of soluble collagen to platelets; our data strongly suggest that the receptor is integrin alpha2 beta1 after it becomes activated upon platelet activation. These results suggest that activation of platelets transforms integrin alpha2 beta1 to a state with higher affinity binding sites for soluble collagen. The soluble collagen-platelet interaction was compared with the platelet interaction with fibrillar collagen, which has until now not been demonstrated to bind specifically to platelets. Here, we demonstrated specific, biphasic fibrillar collagen binding. One phase is rapid and metal ion-independent, and accounts for most of the binding. The other phase is slow and Mg2+-dependent. The characteristic differences in the specific bindings of soluble and fibrous collagens demonstrate the different contributions of two different collagen receptors.

  14. Potency of Fish Collagen as a Scaffold for Regenerative Medicine

    PubMed Central

    Yamamoto, Kohei; Yanagiguchi, Kajiro

    2014-01-01

    Cells, growth factors, and scaffold are the crucial factors for tissue engineering. Recently, scaffolds consisting of natural polymers, such as collagen and gelatin, bioabsorbable synthetic polymers, such as polylactic acid and polyglycolic acid, and inorganic materials, such as hydroxyapatite, as well as composite materials have been rapidly developed. In particular, collagen is the most promising material for tissue engineering due to its biocompatibility and biodegradability. Collagen contains specific cell adhesion domains, including the arginine-glycine-aspartic acid (RGD) motif. After the integrin receptor on the cell surface binds to the RGD motif on the collagen molecule, cell adhesion is actively induced. This interaction contributes to the promotion of cell growth and differentiation and the regulation of various cell functions. However, it is difficult to use a pure collagen scaffold as a tissue engineering material due to its low mechanical strength. In order to make up for this disadvantage, collagen scaffolds are often modified using a cross-linker, such as gamma irradiation and carbodiimide. Taking into account the possibility of zoonosis, a variety of recent reports have been documented using fish collagen scaffolds. We herein review the potency of fish collagen scaffolds as well as associated problems to be addressed for use in regenerative medicine. PMID:24982861

  15. Altered Matrix Metalloproteinase-2 and -9 Expression/Activity Links Placental Ischemia and Anti-angiogenic sFlt-1 to Uteroplacental and Vascular Remodeling and Collagen Deposition in Hypertensive Pregnancy

    PubMed Central

    Li, Wei; Mata, Karina M.; Mazzuca, Marc Q.; Khalil, Raouf A.

    2014-01-01

    Preeclampsia is a complication of pregnancy manifested as maternal hypertension and often fetal growth restriction. Placental ischemia could be an initiating event, but the linking mechanisms leading to hypertension and growth restriction are unclear. We have shown an upregulation of matrix metalloproteinases (MMPs) during normal pregnancy (Norm-Preg). To test the role of MMPs in hypertensive-pregnancy (HTN-Preg), maternal and fetal parameters, MMPs expression, activity and distribution, and collagen and elastin content were measured in uterus, placenta and aorta of Norm-Preg rats and in rat model of reduced uteroplacental perfusion pressure (RUPP). Maternal blood pressure was higher, and uterine, placental and aortic weight, and the litter size and pup weight were less in RUPP than Norm-Preg rats. Western blots and gelatin zymography revealed decreases in amount and gelatinase activity of MMP-2 and MMP-9 in uterus, placenta and aorta of RUPP compared with Norm-Preg rats. Immunohistochemistry confirmed reduced MMPs in uterus, placenta and aortic media of RUPP rats. Collagen, but not elastin, was more abundant in uterus, placenta and aorta of RUPP than Norm-Preg rats. The anti-angiogenic factor soluble fms-like tyrosine kinase-1 (sFlt-1) decreased MMPs in uterus, placenta and aorta of Norm-Preg rats, and vascular endothelial growth factor (VEGF) reversed the decreases in MMPs in tissues of RUPP rats. Thus placental ischemia and anti-angiogenic sFlt-1 decrease uterine, placental and vascular MMP-2 and MMP-9, leading to increased uteroplacental and vascular collagen, and growth-restrictive remodeling in HTN-Preg. Angiogenic factors and MMP activators may reverse the decrease in MMPs and enhance growth-permissive remodeling in preeclampsia. PMID:24704473

  16. Fibrillar, fibril-associated and basement membrane collagens of the arterial wall: architecture, elasticity and remodeling under stress.

    PubMed

    Osidak, M S; Osidak, E O; Akhmanova, M A; Domogatsky, S P; Domogatskaya, A S

    2015-01-01

    The ability of a human artery to pass through 150 million liters of blood sustaining 2 billion pulsations of blood pressure with minor deterioration depends on unique construction of the arterial wall. Viscoelastic properties of this construction enable to re-seal the occuring damages apparently without direct immediate participance of the constituent cells. Collagen structures are considered to be the elements that determine the mechanoelastic properties of the wall in parallel with elastin responsible for elasticity and resilience. Collagen scaffold architecture is the function-dependent dynamic arrangement of a dozen different collagen types composing three distinct interacting forms inside the extracellular matrix of the wall. Tightly packed molecules of collagen types I, III, V provide high tensile strength along collagen fibrils but toughness of the collagen scaffold as a whole depends on molecular bonds between distinct fibrils. Apart of other macromolecules in the extracellular matrix (ECM), collagen-specific interlinks involve microfilaments of collagen type VI, meshwork-organized collagen type VIII, and FACIT collagen type XIV. Basement membrane collagen types IV, XV, XVIII and cell-associated collagen XIII enable transmission of mechanical signals between cells and whole artery matrix. Collagen scaffold undergoes continuous remodeling by decomposition promoted with MMPs and reconstitution from newly produced collagen molecules. Pulsatile stress-strain load modulates both collagen synthesis and MMP-dependent collagen degradation. In this way the ECM structure becomes adoptive to mechanical challenges. The mechanoelastic properties of the arterial wall are changed in atherosclerosis concomitantly with collagen turnover both type-specific and dependent on the structure. Improving the feedback could be another approach to restore sufficient blood circulation.

  17. Tunability of collagen matrix mechanical properties via multiple modes of mineralization

    PubMed Central

    Smith, Lester J.; Deymier, Alix C.; Boyle, John J.; Li, Zhen; Linderman, Stephen W.; Pasteris, Jill D.; Xia, Younan; Genin, Guy M.; Thomopoulos, Stavros

    2016-01-01

    Functionally graded, mineralized collagen tissues exist at soft-to-hard material attachments throughout the body. However, the details of how collagen and hydroxyapatite mineral (HA) interact are not fully understood, hampering efforts to develop tissue-engineered constructs that can assist with repair of injuries at the attachments of soft tissues to bone. In this study, spatial control of mineralization was achieved in collagen matrices using simulated body fluids (SBFs). Based upon previous observations of poor bonding between reconstituted collagen and HA deposited using SBF, we hypothesized that mineralizing collagen in the presence of fetuin (which inhibits surface mineralization) would lead to more mineral deposition within the scaffold and therefore a greater increase in stiffness and toughness compared with collagen mineralized without fetuin. We tested this hypothesis through integrated synthesis, mechanical testing and modelling of graded, mineralized reconstituted collagen constructs. Results supported the hypothesis, and further suggested that mineralization on the interior of reconstituted collagen constructs, as promoted by fetuin, led to superior bonding between HA and collagen. The results provide us guidance for the development of mineralized collagen scaffolds, with implications for bone and tendon-to-bone tissue engineering. PMID:26855755

  18. Collagen XII Contributes to Epicardial and Connective Tissues in the Zebrafish Heart during Ontogenesis and Regeneration

    PubMed Central

    Marro, Jan; Pfefferli, Catherine; de Preux Charles, Anne-Sophie; Bise, Thomas

    2016-01-01

    Zebrafish heart regeneration depends on cardiac cell proliferation, epicardium activation and transient reparative tissue deposition. The contribution and the regulation of specific collagen types during the regenerative process, however, remain poorly characterized. Here, we identified that the non-fibrillar type XII collagen, which serves as a matrix-bridging component, is expressed in the epicardium of the zebrafish heart, and is boosted after cryoinjury-induced ventricular damage. During heart regeneration, an intense deposition of Collagen XII covers the outer epicardial cap and the interstitial reparative tissue. Analysis of the activated epicardium and fibroblast markers revealed a heterogeneous cellular origin of Collagen XII. Interestingly, this matrix-bridging collagen co-localized with fibrillar type I collagen and several glycoproteins in the post-injury zone, suggesting its role in tissue cohesion. Using SB431542, a selective inhibitor of the TGF-β receptor, we showed that while the inhibitor treatment did not affect the expression of collagen 12 and collagen 1a2 in the epicardium, it completely suppressed the induction of both genes in the fibrotic tissue. This suggests that distinct mechanisms might regulate collagen expression in the outer heart layer and the inner injury zone. On the basis of this study, we postulate that the TGF-β signaling pathway induces and coordinates formation of a transient collagenous network that comprises fibril-forming Collagen I and fiber-associated Collagen XII, both of which contribute to the reparative matrix of the regenerating zebrafish heart. PMID:27783651

  19. Stabilization of collagen through bioconversion: An insight in protein-protein interaction.

    PubMed

    Usharani, Nagarajan; Jayakumar, Gladstone Christopher; Kanth, Swarna Vinodh; Rao, Jonnalagadda Raghava

    2014-08-01

    Collagen is a natural protein, which is used as a vital biomaterial in tissue engineering. The major concern about native collagen is lack of its thermal stability and weak resistance to proteolytic degradation. In this scenario, the crosslinking compounds used for stabilization of collagen are mostly of chemical nature and exhibit toxicity. The enzyme mediated crosslinking of collagen provides a novel alternative, nontoxic method for stabilization. In this study, aldehyde forming enzyme (AFE) is used in the bioconversion of hydroxylmethyl groups of collagen to formyl groups that results in the formation of peptidyl aldehyde. The resulted peptidyl aldehyde interacts with bipolar ions of basic amino acid residues of collagen. Further interaction leads to the formation of conjugated double bonds (aldol condensation involving the aldehyde group of peptidyl aldehyde) within the collagen. The enzyme modified collagen matrices have shown an increase in the denaturation temperature, when compared with native collagen. Enzyme modified collagen membranes exhibit resistance toward collagenolytic activity. Moreover, they exhibited a nontoxic nature. The catalytic activity of AFE on collagen as a substrate establishes an efficient modification, which enhances the structural stability of collagen. This finds new avenues in the context of protein-protein stabilization and discovers paramount application in tissue engineering.