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Sample records for human extracellular superoxide

  1. Extracellular superoxide dismutase is present in secretory vesicles of human neutrophils and released upon stimulation.

    PubMed

    Iversen, Marie B; Gottfredsen, Randi H; Larsen, Ulrike G; Enghild, Jan J; Praetorius, Jeppe; Borregaard, Niels; Petersen, Steen V

    2016-08-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme present in the extracellular matrix (ECM), where it provides protection against oxidative degradation of matrix constituents including type I collagen and hyaluronan. The enzyme is known to associate with macrophages and polymorphonuclear leukocytes (neutrophils) and increasing evidence supports a role for EC-SOD in the development of an inflammatory response. Here we show that human EC-SOD is present at the cell surface of isolated neutrophils as well as stored within secretory vesicles. Interestingly, we find that EC-SOD mRNA is absent throughout neutrophil maturation indicating that the protein is synthesized by other cells and subsequently endocytosed by the neutrophil. When secretory vesicles were mobilized by neutrophil stimulation using formyl-methionyl-leucyl-phenylalanine (fMLF) or phorbol 12-myristate 13-acetate (PMA), the protein was released into the extracellular space and found to associate with DNA released from stimulated cells. The functional consequences were evaluated by the use of neutrophils isolated from wild-type and EC-SOD KO mice, and showed that EC-SOD release significantly reduce the level of superoxide in the extracellular space, but does not affect the capacity to generate neutrophil extracellular traps (NETs). Consequently, our data signifies that EC-SOD released from activated neutrophils affects the redox conditions of the extracellular space and may offer protection against highly reactive oxygen species such as hydroxyl radicals otherwise generated as a result of respiratory burst activity of activated neutrophils.

  2. Cloning, Purification, and Characterization of Recombinant Human Extracellular Superoxide Dismutase in SF9 Insect Cells.

    PubMed

    Shrestha, Pravesh; Yun, Ji-Hye; Kim, Woo Taek; Kim, Tae-Yoon; Lee, Weontae

    2016-03-01

    A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1-240) and truncated (residues 19-240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.

  3. Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product

    SciTech Connect

    Tibell, L.; Hjalmarsson, K.; Edlund, T.; Skogman, G.; Engstroem, A.; Marklund, S.L.

    1987-10-01

    A complementary DNA clone from human placenta, encoding human extracellular superoxide dismutase, has recently been isolated and characterized. An expression plasmid, based on the EC-SOD complementary DNA, was transfected into Chinese hamster ovary cells (CHO-K1). The transfected cells secreted human EC-SOD to the culture medium. The secreted recombinant (r) EC-SOD was isolated in high yield with a three-step procedure beginning with immobilized monoclonal anti-EC-SOD antibodies. The properties of the rEC-SOD were compared with native (n) EC-SOD isolated from human umbilical cords. The specific activities and amino-terminal amino acid sequences were identical. The amino acid compositions were virtually identical and very similar to the composition deduced from the complementary DNA sequence. Both rEC-SOD and nEC-SOD contained 4 Cu and 4 Zn atoms per molecule, and the presence of Zn in EC-SOD is thus now established. The rEC-SOD produced is type C, since its affinity for heparin-Sepharose was identical to that of nEC-SOD type C. Both enzymes bound to concanavalin A, lentil lectin, and wheat germ lectin and are thus glycoproteins. rEC-SOD and nEC-SOD seem to have the same subunit structure and composition as analyzed by polyacrylamide gel electrophoresis and gel chromatography.

  4. Heparin-affinity patterns and composition of extracellular superoxide dismutase in human plasma and tissues.

    PubMed

    Sandström, J; Karlsson, K; Edlund, T; Marklund, S L

    1993-09-15

    The tetrameric extracellular superoxide dismutase (EC-SOD) in human tissues and plasma has previously been found to be heterogenous with regard to heparin affinity and could be divided into at least three classes: A, lacking heparin affinity; B, with weak affinity; and C, with strong affinity. Using rigorous extraction conditions and an extensive set of anti-proteolytic agents, tissue EC-SOD is now shown to be almost exclusively of native homotetrameric C-class. Plasma EC-SOD on the other hand is shown to be mainly composed of a complex mixture of heterotetramers with modifications probably residing in the C-terminal heparin-binding domain. Proteolytic truncations appear to be a major cause of this heterogeneity. The findings suggest that, since 99% of the EC-SOD in the human body exists in the extravascular space of tissue, EC-SOD is primarily synthesized in tissues and secreted as homotetrameric native EC-SOD C. This tissue EC-SOD C should exist almost completely sequestered by heparin sulphate proteoglycans. C-terminal modifications subsequently occurring in the EC-SOD C would weaken the binding to heparan sulphate proteoglycan, facilitate entrance to the vasculature through capillaries and lymph flow, and finally result in the heterogeneous plasma EC-SOD pattern. With the new extraction and analysis procedure, the tissue content of EC-SOD is found to be higher than previously reported. It is found, for example, when compared with Mn-SOD, to be higher in umbilical cord and uterus, about equal in placenta and testis and as high as that of CuZn-SOD in umbilical cord. The findings suggest that the protection level against superoxide radicals provided by EC-SOD in the tissue interstitial space, given the small distribution volume, is not much less prominent than that bestowed on the intracellular space by CuZn-SOD and Mn-SOD.

  5. Extracellular superoxide dismutase increased the therapeutic potential of human mesenchymal stromal cells in radiation pulmonary fibrosis.

    PubMed

    Wei, Li; Zhang, Jing; Yang, Zai-Liang; You, Hua

    2017-05-01

    Pulmonary fibrosis induced by irradiation is a significant problem of radiotherapy in cancer patients. Extracellular superoxide dismutase (SOD3) is found to be predominantly and highly expressed in the extracellular matrix of lung and plays a pivotal role against oxidative damage. Early administration of mesenchymal stromal cells (MSCs) has been demonstrated to reduce fibrosis of damaged lung. However, injection of MSCs at a later stage would be involved in fibrosis development. The present study aimed to determine whether injection of human umbilical cord-derived MSCs (UC-MSCs) over-expressing SOD3 at the established fibrosis stage would have beneficial effects in a mice model of radiation pulmonary fibrosis. Herein, pulmonary fibrosis in mice was induced using Cobalt-60 ((60)Co) irradiator with 20 Gy, followed by intravenous injection of UC-MSCs, transduced or not to express SOD3 at 2 h (early delivery) and 60 day (late delivery) post-irradiation, respectively. Our results demonstrated that the early administration of UC-MSCs could attenuate the microscopic damage, reduce collagen deposition, inhibit (myo)fibroblast proliferation, reduce inflammatory cell infiltration, protect alveolar type II (AE2) cell injury, prevent oxidative stress and increase antioxidant status, and reduce pro-fibrotic cytokine level in serum. Furthermore, the early treatment with SOD3-infected UC-MSCs resulted in better improvement. However, we failed to observe the therapeutic effects of UC-MSCs, transduced to express SOD3, during established fibrosis. Altogether, our results demonstrated that the early treatment with UC-MSCs alone significantly reduced radiation pulmonary fibrosis in mice through paracrine effects, with further improvement by administration of SOD3-infected UC-MSCs, suggesting that SOD3-infected UC-MSCs may be a potential cell-based gene therapy to treat clinical radiation pulmonary fibrosis. Copyright © 2017 International Society for Cellular Therapy. Published by

  6. Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken

    PubMed Central

    Byun, Sung June; Ji, Mi-Ran; Jang, Ye-Jin; Hwang, A-In; Chung, Hee Kyoung; Kim, Jeom Sun; Kim, Kyung-Woon; Chung, Hak-Jae; Yang, Byoung-Chul; Jeon, Iksoo; Park, Jin-Ki; Yoo, Jae Gyu; Kim, Tae-Yoon

    2013-01-01

    Extracellular superoxide dismutase (EC-SOD) is a metalloprotein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white. [BMB Reports 2013; 46(8): 404-409] PMID:23977988

  7. Hydrogen peroxide induce modifications of human extracellular superoxide dismutase that results in enzyme inhibition.

    PubMed

    Gottfredsen, Randi H; Larsen, Ulrike G; Enghild, Jan J; Petersen, Steen V

    2013-01-01

    Superoxide dismutase (EC-SOD) controls the level of superoxide in the extracellular space by catalyzing the dismutation of superoxide into hydrogen peroxide and molecular oxygen. In addition, the enzyme reacts with hydrogen peroxide in a peroxidase reaction which is known to disrupt enzymatic activity. Here, we show that the peroxidase reaction supports a site-specific bond cleavage. Analyses by peptide mapping and mass spectrometry shows that oxidation of Pro112 supports the cleavage of the Pro112-His113 peptide bond. Substitution of Ala for Pro112 did not inhibit fragmentation, indicating that the oxidative fragmentation at this position is dictated by spatial organization and not by side-chain specificity. The major part of EC-SOD inhibited by the peroxidase reaction was not fragmented but found to encompass oxidations of histidine residues involved in the coordination of copper (His98 and His163). These oxidations are likely to support the dissociation of copper from the active site and thus loss of enzymatic activity. Homologous modifications have also been described for the intracellular isozyme, Cu/Zn-SOD, reflecting the almost identical structures of the active site within these enzymes. We speculate that the inactivation of EC-SOD by peroxidase activity plays a role in regulating SOD activity in vivo, as even low levels of superoxide will allow for the peroxidase reaction to occur.

  8. Effects of oxidative stress on expression of extracellular superoxide dismutase, CuZn-superoxide dismutase and Mn-superoxide dismutase in human dermal fibroblasts.

    PubMed

    Strålin, P; Marklund, S L

    1994-03-01

    To determine the effect of oxidative stress on expression of extracellular superoxide dismutase (EC-SOD), CuZn-SOD and Mn-SOD, two fibroblast lines were exposed for periods of up to 4 days to a wide concentration range of oxidizing agents: xanthine oxidase plus hypoxanthine, paraquat, pyrogallol, alpha-naphthoflavone, hydroquinone, catechol, Fe2+ ions, Cu2+ ions, buthionine sulphoximine, diethylmaleate, t-butyl hydroperoxide, cumene hydroperoxide, selenite, citiolone and high oxygen partial pressure. The cell lines were cultured both under serum starvation and at a serum concentration that permitted growth. Under no condition was there any evidence of EC-SOD induction. Instead, the agents uniformly, dose-dependently and continuously reduced EC-SOD expression. We interpret the effect to be due to toxicity. Enhancement of the protection against oxidative stress by addition of CuZn-SOD, catalase and low concentrations of selenite did not influence the expression of any of the SOD isoenzymes. Removal of EC-SOD from cell surfaces by heparin also did not influence SOD expression. Mn-SOD was moderately induced by high doses of the first 11 oxidants. Apart from reduction at high toxic doses, there were no significant effects on the CuZn-SOD activity by any of the treatments. Thus EC-SOD, previously shown to be profoundly influenced by inflammatory cytokines, was not induced by its substrate or other oxidants. In a similar fashion, Mn-SOD, previously shown to be greatly induced and depressed by cytokines, was only moderately influenced by oxidants. We suggest that the regulation of these SOD isoenzymes in mammalian tissues primarily occurs in a manner co-ordinated by cytokines, rather than as a response of individual cells to oxidants.

  9. Expression, subcellular localization, and enzyme activity of a recombinant human extra-cellular superoxide dismutase in tobacco (Nicotiana benthamiana L.).

    PubMed

    Park, Ki Youl; Kim, Eun Yu; Lee, Weontae; Kim, Tae-Yoon; Kim, Woo Taek

    2016-03-01

    Human extracellular superoxide dismutase (hEC-SOD) is an enzyme that scavenges reactive oxygen species (ROS). Because of its antioxidant activity, hEC-SOD has been used as a therapeutic protein to treat skin disease and arthritis in mammalian systems. In this study, codon-optimized hEC-SOD was expressed in tobacco (Nicotiana benthamiana L.) via a plant-based transient protein expression system. Plant expression binary vectors containing full-length hEC-SOD (f-hEC-SOD) and modified hEC-SOD (m-hEC-SOD), in which the signal peptide and heparin-binding domain were deleted, were constructed for the cytosolic-, endoplasmic reticulum (ER)-, and chloroplast-localizations in tobacco leaf mesophyll cells. The results demonstrated that f-hEC-SOD was more efficiently expressed in the cytosolic fractions than in the ER or chloroplasts of tobacco cells. Our data further indicated that differently localized f-hEC-SOD and m-hEC-SOD displayed SOD enzyme activities, suggesting that the hEC-SODs expressed by plants may be functionally active. The f-hEC-SOD was expressed up to 3.8% of the total leaf soluble protein and the expression yield was calculated to be 313.7 μg f-hEC-SOD per g fresh weight of leaf. Overall, our results reveal that it was possible to express catalytically active hEC-SODs by means of a transient plant expression system in tobacco leaf cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Extracellular superoxide dismutase of boar seminal plasma.

    PubMed

    Kowalowka, M; Wysocki, P; Fraser, L; Strzezek, J

    2008-08-01

    Superoxide dismutase (SOD) is an enzymatic component of the antioxidant defense system that protects spermatozoa by catalysing the dismutation of superoxide anions to hydrogen peroxide and oxygen. Age and season effects on SOD activity in the seminal plasma were measured in boars at the onset of 8 months through a 35-month period. It was found that age-related changes in SOD activity in the seminal plasma were markedly higher in boars less than 2 years of age. However, it appeared that SOD activity was established at the early sexual maturity age (8-12 months). There were variations in SOD activity throughout the season, being significantly higher in spring and autumn than in summer. A secretory extracellular form of SOD (EC-SOD) was purified to homogeneity (350-fold) from boar seminal plasma, using a three-step purification protocol (affinity chromatography followed by ion exchange and ceramic hydroxyapatite chromatography). The molecular properties and specificity of SOD (molecular mass, isoelectric point, optimum pH, thermostability and susceptibility to inhibitors) confirmed that the purified enzyme is an extracellular form of Cu/Zn-superoxide dismutase occurring in boar seminal plasma. The results of this study indicate that EC-SOD is an important antioxidant enzyme of boar seminal plasma, which plays an important physiological role in counteracting oxidative stress in spermatozoa.

  11. Role of extracellular superoxide dismutase in hypertension.

    PubMed

    Gongora, Maria Carolina; Qin, Zhenyu; Laude, Karine; Kim, Ha Won; McCann, Louise; Folz, J Rodney; Dikalov, Sergey; Fukai, Tohru; Harrison, David G

    2006-09-01

    We previously found that angiotensin II-induced hypertension increases vascular extracellular superoxide dismutase (ecSOD), and proposed that this is a compensatory mechanism that blunts the hypertensive response and preserves endothelium-dependent vasodilatation. To test this hypothesis, we studied ecSOD-deficient mice. ecSOD(-/-) and C57Blk/6 mice had similar blood pressure at baseline; however, the hypertension caused by angiotensin II was greater in ecSOD(-/-) compared with wild-type mice (168 versus 147 mm Hg, respectively; P<0.01). In keeping with this, angiotensin II increased superoxide and reduced endothelium-dependent vasodilatation in small mesenteric arterioles to a greater extent in ecSOD(-/-) than in wild-type mice. In contrast to these findings in resistance vessels, angiotensin II paradoxically improved endothelium-dependent vasodilatation, reduced intracellular and extracellular superoxide, and increased NO production in aortas of ecSOD(-/-) mice. Whereas aortic expression of endothelial NO synthase, Cu/ZnSOD, and MnSOD were not altered in ecSOD(-/-) mice, the activity of Cu/ZnSOD was increased by 80% after angiotensin II infusion. This was associated with a concomitant increase in expression of the copper chaperone for Cu/ZnSOD in the aorta but not in the mesenteric arteries. Moreover, the angiotensin II-induced increase in aortic reduced nicotinamide-adenine dinucleotide phosphate oxidase activity was diminished in ecSOD(-/-) mice as compared with controls. Thus, during angiotensin II infusion, ecSOD reduces hypertension, minimizes vascular superoxide production, and preserves endothelial function in resistance arterioles. We also identified novel compensatory mechanisms involving upregulation of copper chaperone for Cu/ZnSOD, increased Cu/ZnSOD activity, and decreased reduced nicotinamide-adenine dinucleotide phosphate oxidase activity in larger vessels. These compensatory mechanisms preserve large vessel function when ecSOD is absent in

  12. Lung Extracellular Superoxide Dismutase Overexpression Lessens Bleomycin-Induced Pulmonary Hypertension and Vascular Remodeling

    PubMed Central

    Van Rheen, Zachary; Fattman, Cheryl; Domarski, Shannon; Majka, Susan; Klemm, Dwight; Stenmark, Kurt R.; Nozik-Grayck, Eva

    2011-01-01

    Interstitial lung disease is a devastating disease in humans that can be further complicated by the development of secondary pulmonary hypertension. Accumulating evidence indicates that the oxidant superoxide can contribute to the pathogenesis of both interstitial lung disease and pulmonary hypertension. We used a model of pulmonary hypertension secondary to bleomycin-induced pulmonary fibrosis to test the hypothesis that an imbalance in extracellular superoxide and its antioxidant defense, extracellular superoxide dismutase, will promote pulmonary vascular remodeling and pulmonary hypertension. We exposed transgenic mice overexpressing lung extracellular superoxide dismutase and wild-type littermates to a single dose of intratracheal bleomycin, and evaluated the mice weekly for up to 35 days. We assessed pulmonary vascular remodeling and the expression of several genes critical to lung fibrosis, as well as pulmonary hypertension and mortality. The overexpression of extracellular superoxide dismutase protected against late remodeling within the medial, adventitial, and intimal layers of the vessel wall after the administration of bleomycin, and attenuated pulmonary hypertension at the same late time point. The overexpression of extracellular superoxide dismutase also blocked the early up-regulation of two key genes in the lung known to be critical in pulmonary fibrosis and vascular remodeling, the transcription factor early growth response–1 and transforming growth factor–β. The overexpression of extracellular superoxide dismutase attenuated late pulmonary hypertension and significantly improved survival after exposure to bleomycin. These data indicate an important role for an extracellular oxidant/antioxidant imbalance in the pathogenesis of pulmonary vascular remodeling associated with secondary pulmonary hypertension attributable to bleomycin-induced lung fibrosis. PMID:20539010

  13. Extracellular Superoxide Dismutase: Growth Promoter or Tumor Suppressor?

    PubMed Central

    Laukkanen, Mikko O.

    2016-01-01

    Extracellular superoxide dismutase (SOD3) gene transfer to tissue damage results in increased healing, increased cell proliferation, decreased apoptosis, and decreased inflammatory cell infiltration. At molecular level, in vivo SOD3 overexpression reduces superoxide anion (O2−) concentration and increases mitogen kinase activation suggesting that SOD3 could have life-supporting characteristics. The hypothesis is further strengthened by the observations showing significantly increased mortality in conditional knockout mice. However, in cancer SOD3 has been shown to either increase or decrease cell proliferation and survival depending on the model system used, indicating that SOD3-derived growth mechanisms are not completely understood. In this paper, the author reviews the main discoveries in SOD3-dependent growth regulation and signal transduction. PMID:27293512

  14. Extracellular superoxide anion production contributes to the virulence of Xanthomonas oryzae pv. oryzae.

    PubMed

    Li, Xin; Pang, Xinyue; Zhi, Dejuan; Wang, Jinsheng; Li, Minquan; Li, Hongyu

    2009-02-01

    Endogenous superoxide anion production was determined by electron spin resonance in wild-type strains and avrXa7 mutants of Xanthomonas oryzae pv. oryzae. The localization of superoxide anion was carried out in the intra- and extra-cellular fractions. Results showed the presence of superoxide anion in multi-locations of X. oryzae pv. oryzae cells. The extracellular fraction was the major location of superoxide anion production. Furthermore, a positive relationship was shown between the levels of endogenous superoxide anion and the virulence of strains. These indubitable results suggested that the superoxide anion contributes to the virulence of X. oryzae pv. oryzae.

  15. Phenotypes of mice lacking extracellular superoxide dismutase and copper- and zinc-containing superoxide dismutase.

    PubMed

    Sentman, Marie-Louise; Granström, Micael; Jakobson, Håkan; Reaume, Andrew; Basu, Samar; Marklund, Stefan L

    2006-03-17

    Mice lacking the secreted extracellular superoxide dismutase (EC-SOD) or the cytosolic copper- and zinc-containing SOD (CuZn-SOD) show relatively mild phenotypes. To explore the possibility that the isoenzymes have partly overlapping functions, single and double knockout mice were examined. The absence of EC-SOD was found to be without effect on the lifespan of mice, and the reduced lifespan of CuZn-SOD knockouts was not further shortened by EC-SOD deficiency. The urinary excretion of isoprostanes was increased in CuZn-SOD knockout mice, and plasma thiobarbituric acid-reactive substances levels were elevated in EC-SOD knockout mice. These oxidant stress markers showed potentiated increases in the absence of both isoenzymes. Other alterations were mainly found in CuZn-SOD knockout mice, such as halved glutathione peroxidase activity in the tissues examined and increased glutathione and iron in the liver. There were no changes in tissue content of the alternative superoxide scavenger ascorbate, but there was a 25% reduction in ascorbate in blood plasma in mice lacking CuZn-SOD. No increase was found in the urinary excretion of the terminal metabolites of NO, nitrite, and nitrate in any of the genotypes. In conclusion, apart from the increases in the global urinary and plasma oxidant stress markers, our phenotype studies revealed no other evidence that the copper- and zinc-containing SOD isoenzymes have overlapping roles.

  16. Pharmacological induction of vascular extracellular superoxide dismutase expression in vivo.

    PubMed

    Oppermann, Marc; Balz, Vera; Adams, Volker; Dao, Vu Thao-Vi; Bas, Murat; Suvorava, Tatsiana; Kojda, Georg

    2009-07-01

    Pentaerythritol tetranitrate (PETN) treatment reduces progression of atherosclerosis and endothelial dysfunction and decreases oxidation of low-density lipoprotein (LDL) in rabbits. These effects are associated with decreased vascular superoxide production, but the underlying molecular mechanisms remain unknown. Previous studies demonstrated that endogenous nitric oxide could regulate the expression of extracellular superoxide dismutase (ecSOD) in conductance vessels in vivo. We investigated the effect of PETN and overexpression of endothelial nitric oxide synthase (eNOS(++)) on the expression and activity of ecSOD. C57BL/6 mice were randomized to receive placebo or increasing doses of PETN for 4 weeks and eNOS(++) mice with a several fold higher endothelial-specific eNOS expression were generated. The expression of ecSOD was determined in the lung and aortic tissue by real-time PCR and Western blot. The ecSOD activity was measured using inhibition of cytochrome C reduction. There was no effect of PETN treatment or eNOS overexpression on ecSOD mRNA in the lung tissue, whereas ecSOD protein expression increased from 2.5-fold to 3.6-fold (P < 0.05) by 6 mg PETN/kg body weight (BW)/day and 60 mg PETN/kg BW/day, respectively. A similar increase was found in aortic homogenates. eNOS(++) lung cytosols showed an increase of ecSOD protein level of 142 +/- 10.5% as compared with transgene-negative littermates (P < 0.05), which was abolished by N(omega)-nitro-L-arginine treatment. In each animal group, the increase of ecSOD expression was paralleled by an increase of ecSOD activity. Increased expression and activity of microvascular ecSOD are likely induced by increased bioavailability of vascular nitric oxide. Up-regulation of vascular ecSOD may contribute to the reported antioxidative and anti-atherosclerotic effects of PETN.

  17. Pharmacological induction of vascular extracellular superoxide dismutase expression in vivo

    PubMed Central

    Oppermann, Marc; Balz, Vera; Adams, Volker; Thao-Vi Dao, Vu; Bas, Murat; Suvorava, Tatsiana; Kojda, Georg

    2009-01-01

    Pentaerythritol tetranitrate (PETN) treatment reduces progression of atherosclerosis and endothelial dysfunction and decreases oxidation of low-density lipoprotein (LDL) in rabbits. These effects are associated with decreased vascular superoxide production, but the underlying molecular mechanisms remain unknown. Previous studies demonstrated that endogenous nitric oxide could regulate the expression of extracellular superoxide dismutase (ecSOD) in conductance vessels in vivo. We investigated the effect of PETN and overexpression of endothelial nitric oxide synthase (eNOS++) on the expression and activity of ecSOD. C57BL/6 mice were randomized to receive placebo or increasing doses of PETN for 4 weeks and eNOS++ mice with a several fold higher endothelial-specific eNOS expression were generated. The expression of ecSOD was determined in the lung and aortic tissue by real-time PCR and Western blot. The ecSOD activity was measured using inhibition of cytochrome C reduction. There was no effect of PETN treatment or eNOS overexpression on ecSOD mRNA in the lung tissue, whereas ecSOD protein expression increased from 2.5-fold to 3.6-fold (P < 0.05) by 6 mg PETN/kg body weight (BW)/day and 60 mg PETN/kg BW/day, respectively. A similar increase was found in aortic homogenates. eNOS++ lung cytosols showed an increase of ecSOD protein level of 142 ± 10.5% as compared with transgene-negative littermates (P < 0.05), which was abolished by Nω-nitro-L-arginine treatment. In each animal group, the increase of ecSOD expression was paralleled by an increase of ecSOD activity. Increased expression and activity of microvascular ecSOD are likely induced by increased bioavailability of vascular nitric oxide. Up-regulation of vascular ecSOD may contribute to the reported antioxidative and anti-atherosclerotic effects of PETN. PMID:19320775

  18. The Influence of Extracellular Superoxide on Iron Redox Chemistry and Bioavailability to Aquatic Microorganisms

    PubMed Central

    Rose, Andrew L.

    2012-01-01

    Superoxide, the one-electron reduced form of dioxygen, is produced in the extracellular milieu of aquatic microbes through a range of abiotic chemical processes and also by microbes themselves. Due to its ability to promote both oxidative and reductive reactions, superoxide may have a profound impact on the redox state of iron, potentially influencing iron solubility, complex speciation, and bioavailability. The interplay between iron, superoxide, and oxygen may also produce a cascade of other highly reactive transients in oxygenated natural waters. For microbes, the overall effect of reactions between superoxide and iron may be deleterious or beneficial, depending on the organism and its chemical environment. Here I critically discuss recent advances in understanding: (i) sources of extracellular superoxide in natural waters, with a particular emphasis on microbial generation; (ii) the chemistry of reactions between superoxide and iron; and (iii) the influence of these processes on iron bioavailability and microbial iron nutrition. PMID:22514548

  19. Superoxide Induces Neutrophil Extracellular Trap Formation in a TLR-4 and NOX-Dependent Mechanism

    PubMed Central

    Al-Khafaji, Ahmed B; Tohme, Samer; Yazdani, Hamza Obaid; Miller, David; Huang, Hai; Tsung, Allan

    2016-01-01

    Neutrophils constitute the early innate immune response to perceived infectious and sterile threats. Neutrophil extracellular traps (NETs) are a novel mechanism to counter pathogenic invasion and sequelae of ischemia, including cell death and oxidative stress. Superoxide is a radical intermediate of oxygen metabolism produced by parenchymal and nonparenchymal hepatic cells, and is a hallmark of oxidative stress after liver ischemia-reperfusion (I/R). While extracellular superoxide recruits neutrophils to the liver and initiates sterile inflammatory injury, it is unknown whether superoxide induces the formation of NETs. We hypothesize that superoxide induces NET formation through a signaling cascade involving Toll-like receptor 4 (TLR-4) and neutrophil NADPH oxidase (NOX). We treated neutrophils with extracellular superoxide and observed NET DNA release, histone H3 citrullination and increased levels of MPO-DNA complexes occurring in a TLR-4–dependent manner. Inhibition of superoxide generation by allopurinol and inhibition of NOX by diphenyleneiodonium prevented NET formation. When mice were subjected to warm liver I/R, we found significant NET formation associated with liver necrosis and increased serum ALT in TLR-4 WT but not TLR-4 KO mice. To reduce circulating superoxide, we pretreated mice undergoing I/R with allopurinol and N-acetylcysteine, which resulted in decreased NETs and ameliorated liver injury. Our study demonstrates a requirement for TLR-4 and NOX in superoxide-induced NETs, and suggests involvement of superoxide-induced NETs in pathophysiologic settings. PMID:27453505

  20. Molecular cloning of an Onchocerca volvulus extracellular Cu-Zn superoxide dismutase.

    PubMed Central

    James, E R; McLean, D C; Perler, F

    1994-01-01

    Onchocerca volvulus, a human parasitic nematode, is the third leading cause of preventable blindness worldwide. This study describes the molecular cloning of a novel superoxide dismutase (SOD) from the parasite. This putative O. volvulus extracellular SOD (OvEcSOD) is 628 nucleotides (nt) long, including a 22-nt 5' spliced leader (SL1) and a portion encoding an N-terminal hydrophobic 42-amino-acid signal peptide. The remainder of the cDNA shares 71% identity with an O. volvulus cytosolic SOD sequence and is 3 nt longer. All residues involved in metal ion binding, active site formation, folding, and dimer formation in SODs are conserved. Data indicate the OvEcSOD and O. volvulus cytosolic SOD are separate gene products and that the OvEcSOD appears to possess the characteristics of a membrane-bound or secreted enzyme which may be involved in the parasite defense against phagocyte-generated reactive oxygen species. Images PMID:8300230

  1. N-Glycosylation is essential for the secretion of extracellular superoxide dismutase.

    PubMed

    Ota, Fumi; Kizuka, Yasuhiko; Kitazume, Shinobu; Adachi, Tetsuo; Taniguchi, Naoyuki

    2016-10-01

    Extracellular superoxide dismutase (EC-SOD or SOD3) protects against various oxidative stress-related diseases by scavenging reactive superoxides in the extracellular space. It is the only SOD isozyme that is secreted and glycosylated (at asparagine 89). However, the physiological roles of its glycosylation are poorly understood. In this study, we found that the glycosylation site on EC-SOD is well conserved and that a glycosylation-deficient EC-SOD mutant retains its enzymatic activity, but is not secreted. This impairment in secretion may, in part, be due to the ability of the mutants to form unusual higher order oligomers. Our findings reveal that the glycan modification is a key regulator of EC-SOD secretion and contributes to the understanding of the roles of glycans in EC-SOD-related diseases.

  2. Effect of exogenous phenols on superoxide production by extracellular peroxidase from wheat seedling roots.

    PubMed

    Chasov, A V; Minibayeva, F V

    2009-07-01

    Competitive and complimentary relationships of various peroxidase substrates were studied to elucidate the enzymatic mechanisms underlying production of reactive oxygen species in plant cell apoplast. Dianisidine peroxidase released from wheat seedling roots was inhibited by ferulate and coniferol, while ferulic and coniferyl peroxidases were activated by o-dianisidine. Both ferulate and coniferol, when added together with hydrogen peroxide, stimulated superoxide production by extracellular peroxidase. We suggest that substrate-substrate activation of extracellular peroxidases is important for stress-induced oxidative burst in plant cells.

  3. Irradiation Enhances Hippocampus-Dependent Cognition in Mice Deficient in Extracellular Superoxide Dismutase

    PubMed Central

    Raber, Jacob; Villasana, Laura; Rosenberg, Jenna; Zou, Yani; Huang, Ting Ting; Fike, John R.

    2009-01-01

    The effects of ionizing irradiation on the brain are associated with oxidative stress. While oxidative stress following irradiation is generally viewed as detrimental for hippocampal function, it might have beneficial effects as part of an adaptive or preconditioning response to a subsequent challenge. Here we show that in contrast to what is seen in wild-type mice, irradiation enhances hippocampus-dependent cognitive measures in mice lacking extracellular superoxide dismutase. These outcomes were associated with genotype-dependent effects on measures of oxidative stress. When cortices and hippocampi were analyzed for nitrotyrosine formation as an index of oxidative stress, the levels were chronically elevated in mice lacking extracellular superoxide dismutase. However, irradiation caused a greater increase in nitrotyrosine levels in wild-type mice than mice lacking extracellular superoxide dismutase. These paradoxical genotype-dependent effects of irradiation on measures of oxidative stress and cognitive function underscore potential beneficial effects associated with chronic oxidative stress if it exists prior to a secondary insult such as irradiation. PMID:20020436

  4. Extracellular Production and Degradation of Superoxide in the Coral Stylophora pistillata and Cultured Symbiodinium

    PubMed Central

    Saragosti, Eldad; Tchernov, Dan; Katsir, Adi; Shaked, Yeala

    2010-01-01

    Background Reactive oxygen species (ROS) are thought to play a major role in cell death pathways and bleaching in scleractinian corals. Direct measurements of ROS in corals are conspicuously in short supply, partly due to inherent problems with ROS quantification in cellular systems. Methodology/Principal Findings In this study we characterized the dynamics of the reactive oxygen species superoxide anion radical (O2−) in the external milieu of the coral Stylophora pistillata. Using a sensitive, rapid and selective chemiluminesence-based technique, we measured extracellular superoxide production and detoxification activity of symbiont (non-bleached) and aposymbiont (bleached) corals, and of cultured Symbiodinium (from clades A and C). Bleached and non-bleached Stylophora fragments were found to produce superoxide at comparable rates of 10−11–10−9 mol O2− mg protein−1 min−1 in the dark. In the light, a two-fold enhancement in O2− production rates was observed in non-bleached corals, but not in bleached corals. Cultured Symbiodinium produced superoxide in the dark at a rate of . Light was found to markedly enhance O2− production. The NADPH Oxidase inhibitor Diphenyleneiodonium chloride (DPI) strongly inhibited O2− production by corals (and more moderately by algae), possibly suggesting an involvement of NADPH Oxidase in the process. An extracellular O2− detoxifying activity was found for bleached and non-bleached Stylophora but not for Symbiodinium. The O2− detoxifying activity was partially characterized and found to resemble that of the enzyme superoxide dismutase (SOD). Conclusions/Significance The findings of substantial extracellular O2− production as well as extracellular O2− detoxifying activity may shed light on the chemical interactions between the symbiont and its host and between the coral and its environment. Superoxide production by Symbiodinium possibly implies that algal bearing corals are more susceptible to an internal

  5. A common theme in extracellular fluids of beetles: extracellular superoxide dismutases crucial for balancing ROS in response to microbial challenge

    PubMed Central

    Gretscher, René R.; Streicher, Priska E.; Strauß, Anja S.; Wielsch, Natalie; Stock, Magdalena; Wang, Ding; Boland, Wilhelm; Burse, Antje

    2016-01-01

    Extracellular Cu/Zn superoxide dismutases (SODs) are critical for balancing the level of reactive oxygen species in the extracellular matrix of eukaryotes. In the present study we have detected constitutive SOD activity in the haemolymph and defensive secretions of different leaf beetle species. Exemplarily, we have chosen the mustard leaf beetle, Phaedon cochleariae, as representative model organism to investigate the role of extracellular SODs in antimicrobial defence. Qualitative and quantitative proteome analyses resulted in the identification of two extracellular Cu/Zn SODs in the haemolymph and one in the defensive secretions of juvenile P. cochleariae. Furthermore, quantitative expression studies indicated fat body tissue and defensive glands as the main synthesis sites of these SODs. Silencing of the two SODs revealed one of them, PcSOD3.1, as the only relevant enzyme facilitating SOD activity in haemolymph and defensive secretions in vivo. Upon challenge with the entomopathogenic fungus, Metarhizium anisopliae, PcSOD3.1-deficient larvae exhibited a significantly higher mortality compared to other SOD-silenced groups. Hence, our results serve as a basis for further research on SOD regulated host-pathogen interactions. In defensive secretions PcSOD3.1-silencing affected neither deterrent production nor activity against fungal growth. Instead, we propose another antifungal mechanism based on MRJP/yellow proteins in the defensive exudates. PMID:27068683

  6. A common theme in extracellular fluids of beetles: extracellular superoxide dismutases crucial for balancing ROS in response to microbial challenge.

    PubMed

    Gretscher, René R; Streicher, Priska E; Strauß, Anja S; Wielsch, Natalie; Stock, Magdalena; Wang, Ding; Boland, Wilhelm; Burse, Antje

    2016-04-12

    Extracellular Cu/Zn superoxide dismutases (SODs) are critical for balancing the level of reactive oxygen species in the extracellular matrix of eukaryotes. In the present study we have detected constitutive SOD activity in the haemolymph and defensive secretions of different leaf beetle species. Exemplarily, we have chosen the mustard leaf beetle, Phaedon cochleariae, as representative model organism to investigate the role of extracellular SODs in antimicrobial defence. Qualitative and quantitative proteome analyses resulted in the identification of two extracellular Cu/Zn SODs in the haemolymph and one in the defensive secretions of juvenile P. cochleariae. Furthermore, quantitative expression studies indicated fat body tissue and defensive glands as the main synthesis sites of these SODs. Silencing of the two SODs revealed one of them, PcSOD3.1, as the only relevant enzyme facilitating SOD activity in haemolymph and defensive secretions in vivo. Upon challenge with the entomopathogenic fungus, Metarhizium anisopliae, PcSOD3.1-deficient larvae exhibited a significantly higher mortality compared to other SOD-silenced groups. Hence, our results serve as a basis for further research on SOD regulated host-pathogen interactions. In defensive secretions PcSOD3.1-silencing affected neither deterrent production nor activity against fungal growth. Instead, we propose another antifungal mechanism based on MRJP/yellow proteins in the defensive exudates.

  7. Involvement of Extracellular Cu/Zn Superoxide Dismutase in Cotton Fiber Primary and Secondary Cell Wall Biosynthesis

    USDA-ARS?s Scientific Manuscript database

    Extracellular Cu/Zn superoxide dismutases (CSDs) that catalyze the conversion of superoxide to hydrogen peroxide have been suggested to be involved in lignification of secondary walls in spinach, pine and aspen. In cotton fibers, hydrogen peroxide was proposed to be involved in the induction of seco...

  8. In vitro inhibition of superoxide anion production and superoxide dismutase activity by zinc in human spermatozoa.

    PubMed

    Gavella, M; Lipovac, V; Vucić, M; Sverko, V

    1999-08-01

    The in vitro effect of zinc on superoxide anion (O2-) generation and on SOD-like activity in spermatozoa of infertile men was investigated. The formation of superoxide anion was stimulated by NADPH and the level of superoxide anion was measured by the reduction of ferricytochrome c. Both Percoll-isolated (n = 14) and washed spermatozoa (n = 14) exposed to 1 mmol/L zinc (60 min, 37 degrees C), released less (p < 0.002 and p < 0.04, respectively) superoxide anions than did zinc-untreated spermatozoa. These results implicate a possible role for zinc as a scavenger of excessive superoxide anions produced by defective spermatozoa in semen after ejaculation. Additionally, zinc was found to dose-dependently inhibit superoxide dismutase (SOD)-like activity of spermatozoa in vitro. The inhibition of SOD-like activity by an equal concentration of zinc (1 mmol/L) was less pronounced in oligospermic (p < 0.002; n = 16) and asthenozoospermic (p < 0.0005; n = 20) than in normozoospermic samples (p < 0.0001; n = 20). This differential ability of zinc to inhibit SOD-like activity may be relevant to the physiological function of spermatozoa in fertilization. The evidence that zinc may elicit an inhibition of both superoxide anion production and SOD-like activity in human spermatozoa, indicate the existence of novel, zinc-related mechanism(s) involved in the oxidative events occurring after ejaculation, with a possible modulatory effect on germ cell function.

  9. Extracellular superoxide production, viability and redox poise in response to desiccation in recalcitrant Castanea sativa seeds.

    PubMed

    Roach, Thomas; Beckett, Richard P; Minibayeva, Farida V; Colville, Louise; Whitaker, Claire; Chen, Hongying; Bailly, Christophe; Kranner, Ilse

    2010-01-01

    Reactive oxygen species (ROS) are implicated in seed death following dehydration in desiccation-intolerant 'recalcitrant' seeds. However, it is unknown if and how ROS are produced in the apoplast and if they play a role in stress signalling during desiccation. We studied intracellular damage and extracellular superoxide (O(2)(.-)) production upon desiccation in Castanea sativa seeds, mechanisms of O(2)(.-) production and the effect of exogenously supplied ROS. A transient increase in extracellular O(2)(.-) production by the embryonic axes preceded significant desiccation-induced viability loss. Thereafter, progressively more oxidizing intracellular conditions, as indicated by a significant shift in glutathione half-cell reduction potential, accompanied cell and axis death, coinciding with the disruption of nuclear membranes. Most hydrogen peroxide (H(2)O(2))-dependent O(2)(.-) production was found in a cell wall fraction that contained extracellular peroxidases (ECPOX) with molecular masses of approximately 50 kDa. Cinnamic acid was identified as a potential reductant required for ECPOX-mediated O(2)(.-) production. H(2)O(2), applied exogenously to mimic the transient ROS burst at the onset of desiccation, counteracted viability loss of sub-lethally desiccation-stressed seeds and of excised embryonic axes grown in tissue culture. Hence, extracellular ROS produced by embryonic axes appear to be important signalling components involved in wound response, regeneration and growth.

  10. Extracellular superoxide dismutase is necessary to maintain renal blood flow during sepsis development.

    PubMed

    Constantino, Larissa; Galant, Letícia Selinger; Vuolo, Francieli; Guarido, Karla Lorena; Kist, Luiza Wilges; de Oliveira, Giovanna Medeiros Tavares; Pasquali, Matheus Augusto de Bittencourt; de Souza, Cláudio Teodoro; da Silva-Santos, José Eduardo; Bogo, Maurício Reis; Moreira, José Cláudio Fonseca; Ritter, Cristiane; Dal-Pizzol, Felipe

    2017-12-01

    Extracellular superoxide dismutase (ECSOD) protects nitric oxide (NO) bioavailability by decreasing superoxide levels and preventing peroxynitrite generation, which is important in maintaining renal blood flow and in preventing acute kidney injury. However, the profile of ECSOD expression after sepsis is not fully understood. Therefore, we intended to evaluate the content and gene expression of superoxide dismutase (SOD) isoforms in the renal artery and their relation to renal blood flow. Sepsis was induced in Wistar rats by caecal ligation and perforation. Several times after sepsis induction, renal blood flow (12, 24 and 48 h); the renal arterial content of SOD isoforms, nitrotyrosine, endothelial and inducible nitric oxide synthase (e-NOS and i-NOS), and phosphorylated vasodilator-stimulated phosphoprotein (pVASP); and SOD activity (3, 6 and 12 h) were measured. The influence of a SOD inhibitor was also evaluated. An increase in ECSOD content was associated with decreased 3-nitrotyrosine levels. These events were associated with an increase in pVASP content and maintenance of renal blood flow. Moreover, previous treatment with a SOD inhibitor increased nitrotyrosine content and reduced renal blood flow. ECSOD appears to have a major role in decreasing peroxynitrite formation in the renal artery during the early stages of sepsis development, and its application can be important in renal blood flow control and maintenance during septic insult.

  11. Extracellular superoxide dismutase deficiency impairs wound healing in advanced age by reducing neovascularization and fibroblast function

    PubMed Central

    Fujiwara, Toshihiro; Duscher, Dominik; Rustad, Kristine C.; Kosaraju, Revanth; Rodrigues, Melanie; Whittam, Alexander J.; Januszyk, Michael; Maan, Zeshaan N.; Gurtner, Geoffrey C.

    2016-01-01

    Advanced age is characterized by impairments in wound healing, and evidence is accumulating that this may be due in part to a concomitant increase in oxidative stress. Extended exposure to reactive oxygen species (ROS) is thought to lead to cellular dysfunction and organismal death via the destructive oxidation of intra-cellular proteins, lipids and nucleic acids. Extracellular superoxide dismutase (ecSOD/SOD3) is a prime antioxidant enzyme in the extracellular space that eliminates ROS. Here, we demonstrate that reduced SOD3 levels contribute to healing impairments in aged mice. These impairments include delayed wound closure, reduced neovascularization, impaired fibroblast proliferation and increased neutrophil recruitment. We further establish that SOD3 KO and aged fibroblasts both display reduced production of TGF-β1, leading to decreased differentiation of fibroblasts into myofibroblasts. Taken together, these results suggest that wound healing impairments in ageing are associated with increased levels of ROS, decreased SOD3 expression and impaired extracellular oxidative stress regulation. Our results identify SOD3 as a possible target to correct age-related cellular dysfunction in wound healing. PMID:26663425

  12. Extracellular superoxide production associated with secondary root growth following desiccation of Pisum sativum seedlings.

    PubMed

    Roach, Thomas; Kranner, Ilse

    2011-10-15

    The seedling stage is arguably the most vulnerable phase in the plant life cycle, where the young establishing plant is extremely sensitive to environmental stresses such as drought. Here, the production of superoxide (O(2)(-)), a molecule involved in stress signaling, was measured in response to desiccation of Pisum sativum L. seedlings. Following desiccation that was sufficient to kill the radicle meristem, viability could be retained by seedlings that grew secondary roots. Upon rehydration, secondary roots formed in a region that had displayed intense extracellular O(2)(-)production on desiccation. Treating partially desiccated seedlings with hydrogen peroxide (H(2)O(2)) prevented viability loss. In summary, reactive oxygen species (ROS) appear to participate in the signaling required for secondary root formation following desiccation stress of P. sativum seedlings.

  13. Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

    PubMed

    Song, Hyun-Ouk; Ryu, Jae-Sook

    2013-08-01

    Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2 (.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.

  14. Ras Oncogene-Mediated Progressive Silencing of Extracellular Superoxide Dismutase in Tumorigenesis

    PubMed Central

    Cammarota, Francesca; de Vita, Gabriella; Salvatore, Marco; Laukkanen, Mikko O.

    2015-01-01

    Extracellular superoxide dismutase (SOD3) is a secreted enzyme that uses superoxide anion as a substrate in a dismutase reaction that results in the formation of hydrogen peroxide. Both of these reactive oxygen species affect growth signaling in cells. Although SOD3 has growth-supporting characteristics, the expression of SOD3 is downregulated in epithelial cancer cells. In the current work, we studied the mechanisms regulating SOD3 expression in vitro using thyroid cell models representing different stages of thyroid cancer. We demonstrate that a low level of RAS activation increases SOD3 mRNA synthesis that then gradually decreases with increasing levels of RAS activation and the decreasing degree of differentiation of the cancer cells. Our data indicate that SOD3 regulation can be divided into two classes. The first class involves RAS–driven reversible regulation of SOD3 expression that can be mediated by the following mechanisms: RAS GTPase regulatory genes that are responsible for SOD3 self-regulation; RAS-stimulated p38 MAPK activation; and RAS-activated increased expression of the mir21 microRNA, which inversely correlates with sod3 mRNA expression. The second class involves permanent silencing of SOD3 mediated by epigenetic DNA methylation in cells that represent more advanced cancers. Therefore, the work suggests that SOD3 belongs to the group of ras oncogene-silenced genes. PMID:26550576

  15. Extracellular haem peroxidases mediate Mn(II) oxidation in a marine Roseobacter bacterium via superoxide production.

    PubMed

    Andeer, Peter F; Learman, Deric R; McIlvin, Matt; Dunn, James A; Hansel, Colleen M

    2015-10-01

    Manganese (Mn) oxides are among the strongest sorbents and oxidants in environmental systems. A number of biotic and abiotic pathways induce the oxidation of Mn(II) to Mn oxides. Here, we use a combination of proteomic analyses and activity assays, to identify the enzyme(s) responsible for extracellular superoxide-mediated Mn oxide formation by a bacterium within the ubiquitous Roseobacter clade. We show that animal haem peroxidases (AHPs) located on the outer membrane and within the secretome are responsible for Mn(II) oxidation. These novel peroxidases have previously been implicated in direct Mn(II) oxidation by phylogenetically diverse bacteria. Yet, we show that in this Roseobacter species, AHPs mediate Mn(II) oxidation not through a direct reaction but by producing superoxide and likely also by degrading hydrogen peroxide. These findings point to a eukaryotic-like oscillatory oxidative-peroxidative enzymatic cycle by these AHPs that leads to Mn oxide formation by this organism. AHP expression appears unaffected by Mn(II), yet the large energetic investment required to produce and secrete these enzymes points to an as yet unknown physiological function. These findings are further evidence that bacterial peroxidases and secreted enzymes, in general, are unappreciated controls on the cycling of metals and reactive oxygen species (ROS), and by extension carbon, in natural systems.

  16. Homeobox, Wnt, and Fibroblast Growth Factor Signaling is Augmented During Alveogenesis in Mice Lacking Superoxide Dismutase 3, Extracellular.

    PubMed

    Thimraj, Tania A; Birru, Rahel L; Mitra, Ankita; Schulz, Holger; Leikauf, George D; Ganguly, Koustav

    2017-04-01

    Superoxide dismutase 3, extracellular (SOD3) polymorphisms have been implicated in reduced pulmonary function development and altered risk for chronic obstructive pulmonary disease. We previously reported that gene-targeted Sod3-/- mice have impaired lung function and human SOD3 variants are associated with reduced pulmonary function in children. Reduced lung SOD3 levels were reported in mice with lower lung function with the greatest difference occurring during alveogenesis phase [postnatal (P) days 14-28]. Interactions between homeobox (HOX), wingless-type MMTV integration site member (WNT), and fibroblast growth factor (FGF) signaling govern complex developmental processes in several organs. A subset of HOX family members, HOXA5 and HOXB5, is expressed in the developing lung. Therefore, in this study we assessed the transcript expression of these family members and their downstream targets in Sod3-/- mice during alveogenesis (P14). In the lung of Sod3-/- mice, Hoxa5 and Hoxb5 increased. These transcription factors regulate WNT gene expression and were accompanied by increases in their downstream targets Wnt2 and Wnt5A, canonical and noncanonical WNT members, respectively. The WNT signaling target, lymphoid enhancer binding factor 1 (Lef1), also increased along with its downstream targets Fgf2, Fgf7, and Fgf10 in the lungs of Sod3-/- mice. Due to limited knowledge on the role of FGF2 in lung development, we further examined FGF2 protein and found increased levels in the bronchial and alveolar type II epithelial cells of Sod3-/- mice compared to age-matched controls. Thus, our findings suggest that deficient management of extracellular superoxide can lead to altered lung developmental signaling during alveogenesis in mice.

  17. Extracellular Superoxide Dismutase Inhibits Innate Immune Responses and Clearance of an Intracellular Bacterial Infection

    PubMed Central

    Break, Timothy J.; Jun, Sujung; Indramohan, Mohanalaxmi; Carr, Karen D.; Sieve, Amy N.; Dory, Ladislav; Berg, Rance E.

    2012-01-01

    Reactive oxygen and nitrogen species (ROS/RNS) play important roles during immune responses to bacterial pathogens. Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of ROS/RNS and contributes to tissue protection during inflammatory insults. The participation of ecSOD in immune responses seems therefore intuitive, yet is poorly understood. In the present study, we utilized mice with varying levels of ecSOD activity to investigate the involvement of this enzyme in immune responses against Listeria monocytogenes. Surprisingly, our data demonstrate that, despite enhanced neutrophil recruitment to the liver, ecSOD activity negatively impacted host survival and bacterial clearance. Increased ecSOD activity was accompanied by decreased co-localization of neutrophils with bacteria, as well as increased neutrophil apoptosis, which reduced overall and neutrophil-specific TNF-α production. Liver leukocytes from mice lacking ecSOD produced equivalent nitric oxide (NO·) when compared to mice expressing ecSOD. However, during infection, there were higher levels of peroxynitrite (NO3·−) in livers from mice lacking ecSOD compared to mice expressing ecSOD. Neutrophil depletion studies revealed that high levels of ecSOD activity resulted in neutrophils with limited protective capacity, whereas neutrophils from mice lacking ecSOD provided superior protection compared to neutrophils from wild-type mice. Taken together, our data demonstrate that ecSOD activity reduces innate immune responses during bacterial infection and provides a potential target for therapeutic intervention. PMID:22393157

  18. Superoxide radicals increase transforming growth factor-{beta}1 and collagen release from human lung fibroblasts via cellular influx through chloride channels

    SciTech Connect

    Qi Shufan Hartog, Gertjan J.M. den; Bast, Aalt

    2009-05-15

    Reactive oxygen species (ROS) have been implicated in the pathogenesis of fibrosis. However, it remains unclear which ROS is the major cause. We hypothesize that superoxide elicits specific toxicity to human lung fibroblasts and plays an important role in the development of pulmonary fibrosis. In this study, superoxide generated from xanthine and xanthine oxidase activated lung fibroblasts by increasing the release of TGF-{beta}1 and collagen. This was associated with increased levels of intracellular superoxide. SOD and tempol, by scavenging respectively extracellular and intracellular superoxide, prevented the activation of fibroblasts induced by exposure to exogenous superoxide, whereas catalase did not. Moreover, hydrogen peroxide did not activate fibroblasts. Apparently, superoxide rather than hydrogen peroxide is involved in the regulation of TGF-{beta}1 and collagen release in lung fibroblasts. The chloride channel blocker, DIDS, inhibited the increase of intracellular superoxide levels induced by exogenous superoxide and consequently prevented the activation of fibroblasts. This suggests that the cellular influx of superoxide through chloride channels is essential for superoxide-induced activation of fibroblasts. ERK1/2 and p38 MAPKs are involved in the intracellular pathway leading to superoxide-induced fibroblasts activation. Superoxide possesses until now undiscovered specific pro-fibrotic properties in human lung fibroblasts. This takes place via the cellular influx of superoxide through chloride channels rather than via the formation of hydrogen peroxide.

  19. Temperature and Light Effects on Extracellular Superoxide Production by Algal and Bacterial Symbionts in Corals: Implications for Coral Bleaching

    NASA Astrophysics Data System (ADS)

    Brighi, C.; Diaz, J. M.; Apprill, A.; Hansel, C. M.

    2014-12-01

    Increased surface seawater temperature due to global warming is one of the main causes of coral bleaching, a phenomenon in which corals lose their photosynthetic algae. Light and temperature induced production of superoxide and other reactive oxygen species (ROS) by these symbiotic algae has been implicated in the breakdown of their symbiotic association with the coral host and subsequent coral bleaching. Nevertheless, a direct link between Symbiodinium ROS production and coral bleaching has not been demonstrated. In fact, given the abundance and diversity of microorganisms within the coral holobiont, the concentration and fluxes of ROS within corals may involve several microbial sources and sinks. Here, we explore the role of increased light and temperature on superoxide production by coral-derived cultures of Symbiodinium algae and Oceanospirillales bacteria of the genus Endozoicomonas, which are globally common and abundant associates of corals. Using a high sensitivity chemiluminescent technique, we find that heat stress (exposure to 34°C vs. 23°C for 2hr or 24hr) has no significant effect on extracellular superoxide production by Symbiodinium isolates within clades B and C, regardless of the level of light exposure. Exposure to high light, however, increased superoxide production by these organisms at both 34°C and 23°C. On the other hand, extracellular superoxide production by Endozoicomonas bacteria tested under the same conditions was stimulated by the combined effects of thermal and light stress. The results of this research suggest that the sources and physical triggers for biological superoxide production within corals are more complex than currently assumed. Thus, further investigations into the biological processes controlling ROS dynamics within corals are required to improve our understanding of the mechanisms underpinning coral bleaching and to aid in the development of mitigation strategies.

  20. Extracellular superoxide dismutase is important for hippocampal neurogenesis and preservation of cognitive functions after irradiation.

    PubMed

    Zou, Yani; Corniola, Rikki; Leu, David; Khan, Aslam; Sahbaie, Peyman; Chakraborti, Ayanabha; Clark, David J; Fike, John R; Huang, Ting-Ting

    2012-12-26

    Cranial irradiation is widely used in cancer therapy, but it often causes cognitive defects in cancer survivors. Oxidative stress is considered a major cause of tissue injury from irradiation. However, in an earlier study mice deficient in the antioxidant enzyme extracellular superoxide dismutase (EC-SOD KO) showed reduced sensitivity to radiation-induced defects in hippocampal functions. To further dissect the role of EC-SOD in neurogenesis and in response to irradiation, we generated a bigenic EC-SOD mouse model (OE mice) that expressed high levels of EC-SOD in mature neurons in an otherwise EC-SOD-deficient environment. EC-SOD deficiency was associated with reduced progenitor cell proliferation in the subgranular zone of dentate gyrus in KO and OE mice. However, high levels of EC-SOD in the granule cell layer supported normal maturation of newborn neurons in OE mice. Following irradiation, wild-type mice showed reduced hippocampal neurogenesis, reduced dendritic spine densities, and defects in cognitive functions. OE and KO mice, on the other hand, were largely unaffected, and the mice performed normally in neurocognitive tests. Although the resulting hippocampal-related functions were similar in OE and KO mice following cranial irradiation, molecular analyses suggested that they may be governed by different mechanisms: whereas neurotrophic factors may influence radiation responses in OE mice, dendritic maintenance may be important in the KO environment. Taken together, our data suggest that EC-SOD plays an important role in all stages of hippocampal neurogenesis and its associated cognitive functions, and that high-level EC-SOD may provide protection against irradiation-related defects in hippocampal functions.

  1. Effects of Altered Levels of Extracellular Superoxide Dismutase and Irradiation on Hippocampal Neurogenesis in Female Mice

    SciTech Connect

    Zou, Yani; Leu, David; Chui, Jennifer; Fike, John R.; Huang, Ting-Ting

    2013-11-15

    Purpose: Altered levels of extracellular superoxide dismutase (EC-SOD) and cranial irradiation have been shown to affect hippocampal neurogenesis. However, previous studies were only conducted in male mice, and it was not clear if there was a difference between males and females. Therefore, female mice were studied and the results compared with those generated in male mice from an earlier study. Methods and Materials: Female wild-type, EC-SOD-null (KO), and EC-SOD bigenic mice with neuronal-specific expression of EC-SOD (OE) were subjected to a single dose of 5-Gy gamma rays to the head at 8 weeks of age. Progenitor cell proliferation, differentiation, and long-term survival of newborn neurons were determined. Results: Similar to results from male mice, EC-SOD deficiency and irradiation both resulted in significant reductions in mature newborn neurons in female mice. EC-SOD deficiency reduced long-term survival of newborn neurons whereas irradiation reduced progenitor cell proliferation. Overexpression of EC-SOD corrected the negative impacts from EC-SOD deficiency and irradiation and normalized the production of newborn neurons in OE mice. Expression of neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 were significantly reduced by irradiation in wild-type mice, but the levels were not changed in KO and OE mice even though both cohorts started out with a lower baseline level. Conclusion: In terms of hippocampal neurogenesis, EC-SOD deficiency and irradiation have the same overall effects in males and females at the age the studies were conducted.

  2. Ferric human neuroglobin scavenges superoxide to form oxy adduct.

    PubMed

    Yamashita, Taku; Hafsi, Leila; Masuda, Eri; Tsujino, Hirofumi; Uno, Tadayuki

    2014-01-01

    Neuroglobin (Ngb) is the third member of the vertebrate globin family, and the structure was solved as a typical globin fold with a b-type heme. Although it has been proposed that Ngb could be involved in neuroprotection against oxidative stress, the protective mechanism has not been fully identified yet. In order to clarify functions under hypoxic condition, in this study, we focused on the scavenger activity of human Ngb (hNgb) against superoxide. The activity of hNgb for superoxide was evaluated to be 7.4 µM for IC50, the half maximal inhibitory concentration. The result indicates that hNgb can be an anti-oxidant, and the value was almost the same as that of ascorbic acid. In addition, we characterized oxidation states of a heme iron in superoxide-treated hNgb with spectroscopic measurements. Superoxide-treated hNgb in the ferric form was readily converted to the oxygenated ferrous form, and the result suggested that ferric hNgb could scavenge superoxide by change of an oxidation state in a heme iron. Moreover, mutational experiments were performed, and the each variant mutated at 46 and 55 positions suggested a disulfide bond between Cys46 and Cys55 could be essential to be sensors for oxidative stress with the direct binding of superoxide. As a consequence, we concluded that redox changes of the heme iron and the disulfide bond could regulate neuroprotective functions of hNgb, and it suggests that hNgb can afford protection against hypoxic and ischemic stress in the brain.

  3. Superoxide dismutase protects Escherichia coli against killing by human serum.

    PubMed

    McManus, D C; Josephy, P D

    1995-02-20

    To assess the role of superoxide dismutase in protecting Escherichia coli from killing by human serum and neutrophils, we constructed isogenic, smooth-lipopolysaccharide K-12 strains, either sod wild-type, delta sodA, or delta sodA delta sodB. The delta sodA delta sodB strain was killed by serum much more readily than either the wild-type or delta sodA strain. After allowing for this serum sensitivity difference, the delta sodA delta sodB strain also showed increased susceptibility to phagocytic killing by human neutrophils. These results indicate that superoxide dismutase protects E. coli from killing by serum (complement system) and by human neutrophils, possibly by a role in maintaining bacterial membrane structure.

  4. Cell wall peroxidases in the liverwort Dumortiera hirsuta are responsible for extracellular superoxide production, and can display tyrosinase activity.

    PubMed

    Li, Jackson L Y; Sulaiman, Mariam; Beckett, Richard P; Minibayeva, Farida V

    2010-04-01

    In our earlier work, we showed that the liverwort Dumortiera hirsuta produces an extracellular oxidative burst of superoxide radicals during rehydration following desiccation stress. The oxidative burst is a common early response of organisms to biotic and abiotic stresses, with suggested roles in signal transduction, formation of protective substances such as suberin, melanin and lignin and defense against pathogens. To discover which enzymes are responsible for the extracellular superoxide production, we isolated apoplastic fractions from D. hirsuta, surveyed for the presence of potential redox enzymes, and performed non-denaturing polyacrylamide gel electrophoresis activity stains. Various isoforms of peroxidase (EC 1.11.1.7) and tyrosinase (o-diphenolase) (EC 1.10.3.1) were present at significant levels in the apoplast. In-gel activity staining revealed that some peroxidases isoforms could produce superoxide, while tryosinases could readily metabolize 3,4-dihydroxy phenyl l-alanine (l-dopa) into melanins. Interestingly, some peroxidase isoforms could oxidize the native tyrosinase substrate l-dopa at significant levels, even in the absence of hydrogen peroxide, while others could do so only in the presence of hydrogen peroxide. In D. hirsuta, peroxidases may play an important role in melanin formation. Possible functions for these diverse oxidases in liverwort biology are discussed.

  5. Superoxide generation from human polymorphonuclear leukocytes by liposome-encapsulated hemoglobin.

    PubMed

    Abe, H; Ikebuchi, K; Niwa, K; Inanami, O; Kuwabara, M; Fujihara, M; Hirayama, J; Ikeda, H

    2001-07-01

    We investigated the interactions between liposome-encapsulated hemoglobin (Neo Red Cells: NRC) and human polymorphonuclear leukocytes as assessed by superoxide generation. NRC triggered superoxide generation from neutrophils in a dose-dependent manner. Empty liposomes also induced superoxide production of neutrophils. Superoxide generation of neutrophils induced by phorbol myristate acetate (PMA) was delayed but intensified both by NRC and empty liposomes. The intensity of superoxide generation induced by NRC was smaller than that by the empty liposomes. As NRC contained superoxide dismutase (SOD) that was copurified with hemoglobin from red blood cells and its activity remained, SOD contained in NRC may partially eliminate superoxide.

  6. Rutin inhibits proliferation, attenuates superoxide production and decreases adhesion and migration of human cancerous cells.

    PubMed

    Ben Sghaier, Mohamed; Pagano, Alessandra; Mousslim, Mohamed; Ammari, Youssef; Kovacic, Hervé; Luis, José

    2016-12-01

    Lung and colorectal cancer are the principal causes of death in the world. Rutin, an active flavonoid compound, is known for possessing a wide range of biological activities. In this study, we examined the effect of rutin on the viability, superoxide anion production, adhesion and migration of human lung (A549) and colon (HT29 and Caco-2) cancer cell lines. In order to control the harmlessness of the tested concentrations of rutin, the viability of cancer cell lines was assessed using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. ROS generation was measured by lucigenin chemiluminescence detecting superoxide ions. To investigate the effect of rutin on the behavior of human lung and colon cancer cell lines, we performed adhesion assays, using various purified extracellular matrix (ECM) proteins. Finally, in vitro cell migration assays were explored using modified Boyden chambers. The viability of cancerous cells was inhibited by rutin. It also significantly attenuated the superoxide production in HT29 cells. In addition, rutin affected adhesion and migration of A549 and HT29 cell. These findings indicate that rutin, a natural molecule, might have potential as anticancer agent against lung and colorectal carcinogenesis.

  7. Effect of hypoxia mimetic cobalt chloride on the expression of extracellular-superoxide dismutase in retinal pericytes.

    PubMed

    Adachi, Tetsuo; Aida, Kazunari; Nishihara, Hiroko; Kamiya, Tetsuro; Hara, Hirokazu

    2011-01-01

    The initial clinical stage of diabetic retinopathy (DR) is characterized by the development of intraretinal microvascular abnormalities. The increased formation of reactive oxygen species (ROS) is thought to be a key event in the pathogenesis of DR. Extracellular-superoxide dismutase (EC-SOD) is an anti-inflammatory enzyme that is distributed mainly in vascular cells and protects cells from ROS by scavenging superoxide anion. Treatment with cobalt chloride (CoCl(2)) decreased the expression of EC-SOD but not other SOD isozymes in pericytes accompanied with an increase of intracellular ROS production. Pre-treatment with N-acetylcysteine (NAC) significantly suppressed the ROS production and down-regulation of EC-SOD. We observed the activation of caspase-3 and DNA fragmentation as signs of apoptotic process by CoCl(2) treatment. In addition, these phenomena were significantly inhibited by pre-treatment with NAC. EC-SOD enhancer 4-phenyl butyric acid also suppressed the caspase-3 activation. It is known that the presence of a high level of EC-SOD throughout the vessel walls might have an important protective role against superoxide in the vascular system. The decrease in EC-SOD expression accompanied with elevation of ROS level in pericytes under hypoxia might induce and/or promote the ROS-triggered apoptosis of pericytes and the development of pathogenesis in DR.

  8. Preclinical trials of human erythrocyte superoxide dismutase injection.

    PubMed

    Zhou, C; Fang, Y; Jiang, D; Yang, S; Lu, X; Sui, J; Li, P; Ren, J

    2000-07-01

    To assess the quality of human erythrocyte superoxide dismutase (SOD) injection reaching the official standard for its clinical uses. Human erythrocyte SOD injection prepared by McCord-Fridovich's method without column chromatography but with some modifications was used in preclinical trials, to observe the general pharmacology and pharmacodynamics of the product. The quality of human erythrocyte SOD injection conformed to the official standard of a biological product, which was found to be non-toxic and did not have any effects on the central and autonomic nervous systems as well as cardiovascular and respiratory systems. The efficacy of anti-inflammation and promotion of immuno-regulation especially on carrageenan and adjuvant-induced polyarthritis were shown in animals. Human erythrocyte SOD injection is appropriate for prophylactic and therapeutic uses in clinical trials.

  9. Reduction of extracellular superoxide dismutase activity by decapeptide derived from FGF-receptor.

    PubMed

    Willems, J; Noppe, W; Slegers, H

    1995-11-01

    Several synthetic decapeptides containing an HAV tripeptide motif were tested for their ability to modulate the enzymatic activity of rat extracellular SOD, an enzyme which also contains an HAV motif. Out of nine decapeptides that were tested, only a FGF-receptor derived peptide was active as a negative modulator of enzyme activity. These results strengthen the thesis that HAV motifs are not only involved in homophilic interactions and suggest that soluble FGF-receptor molecules might moderate the activity of extracellular SOD.

  10. Differential effects of superoxide dismutase and superoxide dismutase/catalase mimetics on human breast cancer cells.

    PubMed

    Shah, Manisha H; Liu, Guei-Sheung; Thompson, Erik W; Dusting, Gregory J; Peshavariya, Hitesh M

    2015-04-01

    Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H2O2) have been implicated in development and progression of breast cancer. In the present study, we have evaluated the effects of the superoxide dismutase (SOD) mimetic MnTmPyP and the SOD/catalase mimetic EUK 134 on superoxide and H2O2 formation as well as proliferation, adhesion, and migration of MCF-7 and MDA-MB-231 cells. Superoxide and H2O2 production was examined using dihydroethidium and Amplex red assays, respectively. Cell viability and adhesion were measured using a tetrazolium-based MTT assay. Cell proliferation was determined using trypan blue assay. Cell cycle progression was analyzed using flow cytometry. Clonal expansion of a single cell was performed using a colony formation assay. Cell migration was measured using transwell migration assay. Dual luciferase assay was used to determine NF-κB reporter activity. EUK 134 effectively reduced both superoxide and H2O2, whereas MnTmPyP removed superoxide but enhanced H2O2 formation. EUK 134 effectively attenuated viability, proliferation, clonal expansion, adhesion, and migration of MCF-7 and MDA-MB-231 cells. In contrast, MnTmPyP only reduced clonal expansion of MCF-7 and MDA-MB-231 cells but had no effect on adhesion and cell cycle progression. Tumor necrosis factor-alpha-induced NF-κB activity was reduced by EUK 134, whereas MnTmPyP enhanced this activity. These data indicate that the SOD mimetic MnTmPyP and the SOD/catalase mimetic EUK 134 exert differential effects on breast cancer cell growth. Inhibition of H2O2 signaling using EUK 134-like compound might be a promising approach to breast cancer therapy.

  11. Cu,Zn Superoxide Dismutase is a Peroxisomal Enzyme in Human Fibroblast and Hepatoma Cells

    NASA Astrophysics Data System (ADS)

    Keller, Gilbert-Andre; Warner, Thomas G.; Steimer, Kathelyn S.; Hallewell, Robert A.

    1991-08-01

    The intracellular localization of Cu,Zn superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been examined by immunofluorescence using four monoclonal anti-Cu,Zn superoxide dismutase antibodies raised against a recombinant human Cu,Zn superoxide dismutase derivative produced and purified from Escherichia coli. Colocalization with catalase, a peroxisomal matrix enzyme, was used to demonstrate the peroxisomal localization of Cu,Zn superoxide dismutase in human fibroblasts and hepatoma cells. In the fibroblasts of Zellweger syndrome patients, the enzyme is not transported to the peroxisomal ghosts but, like catalase, remains in the cytoplasm. In addition, immunocryoelectron microscopy of yeast cells expressing human Cu,Zn superoxide dismutase showed that the enzyme is translocated to the peroxisomes.

  12. Quinone Reduction by the Na+-Translocating NADH Dehydrogenase Promotes Extracellular Superoxide Production in Vibrio cholerae▿ †

    PubMed Central

    Lin, Po-Chi; Türk, Karin; Häse, Claudia C.; Fritz, Günter; Steuber, Julia

    2007-01-01

    The pathogenicity of Vibrio cholerae is influenced by sodium ions which are actively extruded from the cell by the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR). To study the function of the Na+-NQR in the respiratory chain of V. cholerae, we examined the formation of organic radicals and superoxide in a wild-type strain and a mutant strain lacking the Na+-NQR. Upon reduction with NADH, an organic radical was detected in native membranes by electron paramagnetic resonance spectroscopy which was assigned to ubisemiquinones generated by the Na+-NQR. The radical concentration increased from 0.2 mM at 0.08 mM Na+ to 0.4 mM at 14.7 mM Na+, indicating that the concentration of the coupling cation influences the redox state of the quinone pool in V. cholerae membranes. During respiration, V. cholerae cells produced extracellular superoxide with a specific activity of 10.2 nmol min−1 mg−1 in the wild type compared to 3.1 nmol min−1 mg−1 in the NQR deletion strain. Raising the Na+ concentration from 0.1 to 5 mM increased the rate of superoxide formation in the wild-type V. cholerae strain by at least 70%. Rates of respiratory H2O2 formation by wild-type V. cholerae cells (30.9 nmol min−1 mg−1) were threefold higher than rates observed with the mutant strain lacking the Na+-NQR (9.7 nmol min−1 mg−1). Our study shows that environmental Na+ could stimulate ubisemiquinone formation by the Na+-NQR and hereby enhance the production of reactive oxygen species formed during the autoxidation of reduced quinones. PMID:17322313

  13. Royal Jelly Constituents Increase the Expression of Extracellular Superoxide Dismutase through Histone Acetylation in Monocytic THP-1 Cells.

    PubMed

    Makino, Junya; Ogasawara, Rie; Kamiya, Tetsuro; Hara, Hirokazu; Mitsugi, Yukari; Yamaguchi, Eiji; Itoh, Akichika; Adachi, Tetsuo

    2016-04-22

    Extracellular superoxide dismutase (EC-SOD) is one of the main SOD isozymes and plays an important role in the prevention of cardiovascular diseases by accelerating the dismutation reaction of superoxide. Royal jelly includes 10-hydroxy-2-decenoic acid (10H2DA, 2), which regulates the expression of various types of genes in epigenetics through the effects of histone deacetylase (HDAC) antagonism. The expression of EC-SOD was previously reported to be regulated epigenetically through histone acetylation in THP-1 cells. Therefore, we herein evaluated the effects of the royal jelly constituents 10-hydroxydecanoic acid (10HDA, 1), sebacic acid (SA, 3), and 4-hydroperoxy-2-decenoic acid ethyl ester (4-HPO-DAEE, 4), which is a derivative of 2, on the expression of EC-SOD in THP-1 cells. The treatment with 1 mM 1, 2, or 3 or 100 μM 4 increased EC-SOD expression and histone H3 and H4 acetylation levels. Moreover, the enrichment of acetylated histone H4 was observed in the proximal promoter region of EC-SOD and was caused by the partial promotion of ERK phosphorylation (only 4) and inhibition of HDAC activities, but not by the expression of HDACs. Overall, 4 exerted stronger effects than 1, 2, or 3 and has potential as a candidate or lead compound against atherosclerosis.

  14. Transcriptional regulation of extracellular copper zinc superoxide dismutase from white shrimp Litopenaeus vannamei following Vibrio alginolyticus and WSSV infection.

    PubMed

    Tian, Jianxiao; Chen, Juan; Jiang, Dan; Liao, Shaoan; Wang, Anli

    2011-01-01

    The cDNA encoding an extracellular copper zinc superoxide dismutase (LvECSOD) was cloned from the hepatopancreas of white shrimp Litopenaeus vannamei. It consisted of 915 bp nucleotides with an open reading frame corresponding to a deduced protein of 178 amino acids. The LvECSOD contains a putative signal peptide of 16 amino acids, two potential N-linked glycosylation sites (N(115)GTA and N(135)ITG) and a copper zinc superoxide dismutase family signature sequence (G(162)NAGaRvACctI(173)). It was found that four copper binding sites, four zinc binding sites and two cysteines involving in the formation of the disulfide bridge were conserved in the protein. LvECSOD shared 33-58% identity to ECSODs from other organisms. Expression analysis revealed that LvECSOD mRNA was widely distributed in all the tissues examined. When the shrimp challenged with Vibrio alginolyticus or white spot syndrome virus (WSSV), expression of LvECSOD mRNA in the hepatopancreas and hemocytes was mediated responsively. Our results suggested that LvECSOD was implicated in the immune response induced by V. alginolyticus and WSSV.

  15. Analysis of Extracellular Superoxide Dismutase and Akt in Ascending Aortic Aneurysm With Tricuspid or Bicuspid Aortic Valve

    PubMed Central

    Arcucci, A.; Ruocco, M.R.; Albano, F.; Granato, G.; Romano, V.; Corso, G.; Bancone, C.; De Vendittis, E.; Corte, A. Della

    2014-01-01

    Ascending aortic aneurysm (AsAA) is a consequence of medial degeneration (MD), deriving from apoptotic loss of smooth muscle cells (SMC) and fragmentation of elastin and collagen fibers. Alterations of extracellular matrix structure and protein composition, typical of medial degeneration, can modulate intracellular pathways. In this study we examined the relevance of extracellular superoxide dismutase (SOD3) and Akt in AsAA pathogenesis, evaluating their tissue distribution and protein levels in ascending aortic tissues from controls (n=6), patients affected by AsAA associated to tricuspid aortic valve (TAV, n=9) or bicuspid aortic valve (BAV, n=9). The results showed a significant reduction of SOD3, phospho-Akt and Akt protein levels in AsAA tissues from patients with BAV, compared to controls, whereas the differences observed between controls and patients with TAV were not significant. The decreased levels of SOD3 and Akt in BAV aortic tissues are associated with decreased Erk1/Erk2 phosphorylation and MMP-9 levels increase. The authors suggest a role of decreased SOD3 protein levels in the progression of AsAA with BAV and a link between ECM modifications of aortic media layer and impaired Erk1/Erk2 and Akt signaling in the late stages of the aortopathy associated with BAV. PMID:25308842

  16. Different influences of extracellular and intracellular superoxide on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries.

    PubMed

    Tawa, Masashi; Shimosato, Takashi; Iwasaki, Hirotaka; Imamura, Takeshi; Okamura, Tomio

    2015-02-01

    Superoxide production is increased in diseased blood vessels, which is considered to lead to impairment of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway. To investigate the respective influence of extracellular and intracellular superoxide on vascular function through the NO/sGC/cGMP pathway, mechanical responses of rat external iliac arteries without endothelium were studied under exposure to a superoxide-generating agent, pyrogallol, or menadione. Exposure to pyrogallol impaired the relaxation induced by acidified NaNO2 (exogenous NO) but not that by nitroglycerin (organic nitrate), BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator), or 8-Br-cGMP (cGMP analog). Superoxide dismutase (SOD) and tempol restored the impaired relaxation by acidified NaNO2. Superoxide production in the bathing solution, but not in artery segments, was significantly increased by exposure to pyrogallol, which was abolished in the presence of SOD or tempol. However, exposure to menadione impaired the relaxant response to acidified NaNO2, nitroglycerin, or BAY 41-2272, whereas it augmented that to BAY 60-2770. Also, this exposure had no effect on the 8-Br-cGMP-induced vasorelxation. Superoxide production in artery segments was dramatically enhanced by exposure to menadione, whereas that in the bathing solution was not affected. This increase in vascular superoxide production was normalized by tempol but not by SOD. These findings suggest that extracellular superoxide reacts with NO only outside the cell, whereas intracellular superoxide not only scavenges NO inside the cell but also shifts the sGC redox equilibrium.

  17. Superoxide radical generation and Mn- and Cu-Zn superoxide dismutases activities in human leukemic cells.

    PubMed

    Kato, Masahiko; Minakami, Hisanori; Kuroiwa, Minoru; Kobayashi, Yasuyuki; Oshima, Shigeru; Kozawa, Kunihisa; Morikawa, Akihiro; Kimura, Hirokazu

    2003-03-01

    Mn- and Cu-Zn superoxide dismutase (SOD) activities and generation of superoxide radicals (O(2) (-)) were assessed in leukemic cells from 10 patients with acute myeloid or monocytic leukemia (AML) and 10 patients with acute lymphoblastic leukemia (ALL), using a sensitive, specific chemiluminescence method. Leukemic cells were classified according to the French-American-British classification. M4 AML cells from two patients produced some O(2) (-) upon stimulation with opsonized zymosan (OZ), phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (FMLP), but less than normal granulocytes or monocytes. M5b AML cells from one patient produced as much O(2) (-) in response to these stimulants as normal monocytes. No O(2) (-) generation was induced in other types of leukemic cells. Total SOD activity in AML cells was significantly greater in normal granulocytes, but was only half of the activity in ALL cells. Mn-SOD in AML cells was very low or undetectable. These results suggest that except in M5b cells, decreased O(2) (-) production may contribute to susceptibility to infections in AML patients. Decreased Mn-SOD activity in AML cells may predispose them to oxidative stress. Copyright 2002 John Wiley & Sons, Ltd.

  18. Increasing Superoxide Production and the Labile Iron Pool in Tumor Cells may Sensitize Them to Extracellular Ascorbate.

    PubMed

    McCarty, Mark Frederick; Contreras, Francisco

    2014-01-01

    Low millimolar concentrations of ascorbate are capable of inflicting lethal damage on a high proportion of cancer cells lines, yet leave non-transformed cell lines unscathed. Extracellular generation of hydrogen peroxide, reflecting reduction of molecular oxygen by ascorbate, has been shown to mediate this effect. Although some cancer cell lines express low catalase activity, this cannot fully explain the selective sensitivity of cancer cells to hydrogen peroxide. Ranzato and colleagues have presented evidence for a plausible new explanation of this sensitivity - a high proportion of cancers, via NADPH oxidase complexes or dysfunctional mitochondria, produce elevated amounts of superoxide. This superoxide, via a transition metal-catalyzed transfer of an electron to the hydrogen peroxide produced by ascorbate, can generate deadly hydroxyl radical (Haber-Weiss reaction). It thus can be predicted that concurrent measures which somewhat selectively boost superoxide production in cancers will enhance their sensitivity to i.v. ascorbate therapy. One way to achieve this is to increase the provision of substrate to cancer mitochondria. Measures which inhibit the constitutive hypoxia-inducible factor-1 (HIF-1) activity in cancers (such as salsalate and mTORC1 inhibitors, or an improvement of tumor oxygenation), or that inhibit the HIF-1-inducible pyruvate dehydrogenase kinase (such as dichloroacetate), can be expected to increase pyruvate oxidation. A ketogenic diet should provide more lipid substrate for tumor mitochondria. The cancer-killing activity of 42°C hyperthermia is to some degree contingent on an increase in oxidative stress, likely of mitochondrial origin; reports that hydrogen peroxide synergizes with hyperthermia in killing cancer cells suggest that hyperthermia and i.v. ascorbate could potentiate each other's efficacy. A concurrent enhancement of tumor oxygenation might improve results by decreasing HIF-1 activity while increasing the interaction of ascorbic

  19. Bioengineering Human Myocardium on Native Extracellular Matrix

    PubMed Central

    Guyette, Jacques P.; Charest, Jonathan M; Mills, Robert W; Jank, Bernhard J.; Moser, Philipp T.; Gilpin, Sarah E.; Gershlak, Joshua R.; Okamoto, Tatsuya; Gonzalez, Gabriel; Milan, David J.; Gaudette, Glenn R.; Ott, Harald C.

    2015-01-01

    Rationale More than 25 million individuals suffer from heart failure worldwide, with nearly 4,000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only about 2,500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. Objective The objective of this study is to translate previous work to human scale and clinically relevant cells, for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human iPS-derived cardiac myocytes. Methods and Results To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiac myocytes derived from non-transgenic human induced pluripotent stem cells (iPSCs) and generated tissues of increasing three-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole heart scaffolds with human iPSC-derived cardiac myocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue, showed electrical conductivity, left ventricular pressure development, and metabolic function. Conclusions Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human iPS-derived cardiac myocytes, and enable

  20. Hypochlorous acid regulates neutrophil extracellular trap release in humans.

    PubMed

    Palmer, L J; Cooper, P R; Ling, M R; Wright, H J; Huissoon, A; Chapple, I L C

    2012-02-01

    Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti-microbial strategy regulated non-exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine.

  1. The effects of aging on pulmonary oxidative damage, protein nitration, and extracellular superoxide dismutase down-regulation during systemic inflammation.

    PubMed

    Starr, Marlene E; Ueda, Junji; Yamamoto, Shoji; Evers, B Mark; Saito, Hiroshi

    2011-01-15

    Systemic inflammatory response syndrome (SIRS), a serious clinical condition characterized by whole-body inflammation, is particularly threatening for elderly patients, who suffer much higher mortality rates than the young. A major pathological consequence of SIRS is acute lung injury caused by neutrophil-mediated oxidative damage. Previously, we reported an increase in protein tyrosine nitration (a marker of oxidative/nitrosative damage) and a decrease in the antioxidant enzyme extracellular superoxide dismutase (EC-SOD) in the lungs of young mice during endotoxemia-induced SIRS. Here we demonstrate that during endotoxemia, down-regulation of EC-SOD is significantly more profound and prolonged, whereas up-regulation of iNOS is augmented, in aged compared to young mice. Aged mice also showed 2.5-fold higher protein nitration levels, compared to young mice, with particularly strong nitration in the pulmonary vascular endothelium during SIRS. Additionally, by two-dimensional gel electrophoresis, Western blotting, and mass spectrometry, we identified proteins that show increased tyrosine nitration in age- and SIRS-dependent manners; these proteins (profilin-1, transgelin-2, LASP 1, tropomyosin, and myosin) include components of the actin cytoskeleton responsible for maintaining pulmonary vascular permeability. Reduced EC-SOD in combination with increased oxidative/nitrosative damage and altered cytoskeletal protein function due to tyrosine nitration may contribute to augmented lung injury in the aged with SIRS.

  2. Extracellular Superoxide Dismutase Expression in Papillary Thyroid Cancer Mesenchymal Stem/Stromal Cells Modulates Cancer Cell Growth and Migration

    PubMed Central

    Parascandolo, Alessia; Rappa, Francesca; Cappello, Francesco; Kim, Jaehyup; Cantu, David A.; Chen, Herbert; Mazzoccoli, Gianluigi; Hematti, Peiman; Castellone, Maria Domenica; Salvatore, Marco; Laukkanen, Mikko O.

    2017-01-01

    Tumor stroma-secreted growth factors, cytokines, and reactive oxygen species (ROS) influence tumor development from early stages to the metastasis phase. Previous studies have demonstrated downregulation of ROS-producing extracellular superoxide dismutase (SOD3) in thyroid cancer cell lines although according to recent data, the expression of SOD3 at physiological levels stimulates normal and cancer cell proliferation. Therefore, to analyze the expression of SOD3 in tumor stroma, we characterized stromal cells from the thyroid. We report mutually exclusive desmoplasia and inflammation in papillary and follicular thyroid cancers and the presence of multipotent mesenchymal stem/stromal cells (MSCs) in non-carcinogenic thyroids and papillary thyroid cancer (PTC). The phenotypic and differentiation characteristics of Thyroid MSCs and PTC MSCs were comparable with bone marrow MSCs. A molecular level analysis showed increased FIBROBLAST ACTIVATING PROTEIN, COLLAGEN 1 TYPE A1, TENASCIN, and SOD3 expression in PTC MSCs compared to Thyroid MSCs, suggesting the presence of MSCs with a fibrotic fingerprint in papillary thyroid cancer tumors and the autocrine-paracrine conversion of SOD3 expression, which was enhanced by cancer cells. Stromal SOD3 had a stimulatory effect on cancer cell growth and an inhibitory effect on cancer cell migration, thus indicating that SOD3 might be a novel player in thyroid tumor stroma. PMID:28216675

  3. Cytokines directly induce degranulation and superoxide production from human eosinophils.

    PubMed

    Horie, S; Gleich, G J; Kita, H

    1996-08-01

    Cytokines are implicated in allergic diseases and can modulate effector functions of eosinophils stimulated by another agonist. However, little is known about the capacity of cytokines to directly trigger eosinophil degranulation. We attempted to determine whether cytokines can directly induce degranulation and superoxide production from eosinophils. Eosinophils from normal donors were incubated with various cytokines in albumin-coated tissue culture plates for 4 hours. To quantitate degranulation, the amounts of eosinophil-derived neurotoxin in supernatants were measured by radioimmunoassay. In addition, superoxide production was measured by superoxide dismutase-inhibitable reduction of cytochrome c. IL-5, IL-3, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor- alpha, and RANTES all induced eosinophil degranulation. Granulocyte-macrophage colony-stimulating factor was the most potent and induced eosinophil-derived neurotoxin release comparable to that induced by secretory IgA beads, one of the most potent secretagogues for eosinophils. In addition, IL-5 and tumor necrosis factor- alpha were synergistic in their induction of eosinophil degranulation. In contrast, IL-1, IL-8, interferon- gamma, and macrophage inflammatory protein-1 alpha did not induce degranulation. Finally, IL-5, IL-3, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor- alpha, but not RANTES, also induced superoxide production from eosinophils. Certain cytokines directly induce eosinophil degranulation and superoxide production in vitro. Therefore these cytokines may be important in the release of toxic granule proteins from eosinophils in allergic diseases.

  4. Naloxone inhibits superoxide but not enzyme release by human neutrophils

    SciTech Connect

    Simpkins, C.; Alailima, S.; Tate, E.

    1986-03-01

    The release of toxic oxygen metabolites and enzymes by phagocytic cells is thought to play a role in the multisystemic tissue injury of sepsis. Naloxone protects septic animals. We have found that at concentrations administered to animals (10/sup -7/ to 10/sup -4/M), naloxone inhibited (p < .001) the release of superoxide (O/sub 2//sup -/) by human neutrophils (HN), stimulated with N-formyl methionyl leucyl phenylalanine (FMLP). Naloxone had no effect on cell viability. Maximum inhibition was 65% of the total O/sub 2//sup -/ released (13.1 nMoles/8 min/320,000 cells). FMLP-stimulated release of beta-glucoronidase or lysozyme was not altered by naloxone. Naloxone had no effect on the binding of /sup 3/H FMLP to HN. Using /sup 3/H naloxone and various concentrations of unlabeled naloxone higher affinity (K/sub D/ = 12nM) and lower affinity (K/sub D/ = 4.7 x 10/sup -5/) binding sites were detected. The K/sub D/ of the low affinity site corresponded to the ED/sub 50/ for naloxone inhibition of O/sub 2//sup -/ (1 x 10/sup -5/M). Binding to this low affinity site was decreased by (+) naloxone, beta-endorphin and N acetyl beta-endorphin, but not by leu-enkephalin, thyrotropin releasing factor, prostaglandin D/sub 2/ or E/sub 2/. Conclusions: (1) naloxone inhibits FMLP-stimulated O/sub 2/ but not enzyme release, (2) this inhibition is not due to alteration of FMLP receptor binding, (3) naloxone may act via a low affinity binding site which is ligand specific, and (4) a higher affinity receptor is present on HN.

  5. Extracellular Superoxide Dismutase Ameliorates Skeletal Muscle Abnormalities, Cachexia and Exercise Intolerance in Mice with Congestive Heart Failure

    PubMed Central

    Okutsu, Mitsuharu; Call, Jarrod A.; Lira, Vitor A.; Zhang, Mei; Donet, Jean A.; French, Brent A.; Martin, Kyle S.; Peirce-Cottler, Shayn M.; Rembold, Christopher M.; Annex, Brian H.; Yan, Zhen

    2014-01-01

    Background Congestive heart failure (CHF) is a leading cause of morbidity and mortality, and oxidative stress has been implicated in the pathogenesis of cachexia (muscle wasting) and the hallmark symptom, exercise intolerance. We have previously shown that a nitric oxide (NO)-dependent antioxidant defense renders oxidative skeletal muscle resistant to catabolic wasting. Here, we aimed to identify and determine the functional role of the NO-inducible antioxidant enzyme(s) in protection against cardiac cachexia and exercise intolerance in CHF. Methods and Results We demonstrated that systemic administration of endogenous nitric oxide donor S-Nitrosoglutathione in mice blocked the reduction of extracellular superoxide dismutase (EcSOD) protein expression, the induction of MAFbx/Atrogin-1 mRNA expression and muscle atrophy induced by glucocorticoid. We further showed that endogenous EcSOD, expressed primarily by type IId/x and IIa myofibers and enriched at endothelial cells, is induced by exercise training. Muscle-specific overexpression of EcSOD by somatic gene transfer or transgenesis [muscle creatine kinase (MCK)-EcSOD] in mice significantly attenuated muscle atrophy. Importantly, when crossbred into a mouse genetic model of CHF [α-myosin heavy chain (MHC)-calsequestrin] MCK-EcSOD transgenic mice had significant attenuation of cachexia with preserved whole body muscle strength and endurance capacity in the absence of reduced heart failure. Enhanced EcSOD expression significantly ameliorated CHF-induced oxidative stress, MAFbx/Atrogin-1 mRNA expression, loss of mitochondria and vascular rarefaction in skeletal muscle. Conclusions EcSOD plays an important antioxidant defense function in skeletal muscle against cardiac cachexia and exercise intolerance in CHF. PMID:24523418

  6. Extracellular histones disarrange vasoactive mediators release through a COX-NOS interaction in human endothelial cells.

    PubMed

    Pérez-Cremades, Daniel; Bueno-Betí, Carlos; García-Giménez, José Luis; Ibañez-Cabellos, José Santiago; Hermenegildo, Carlos; Pallardó, Federico V; Novella, Susana

    2017-02-28

    Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone-mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose-dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX-1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX-2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial-dependent mediators through an up-regulation in COX-2-PGIS-PGI2 pathway which involves a COX-2-dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone-mediated pathologies.

  7. Homeostasis of Extracellular ATP in Human Erythrocytes*

    PubMed Central

    Montalbetti, Nicolas; Leal Denis, Maria F.; Pignataro, Omar P.; Kobatake, Eiry; Lazarowski, Eduardo R.; Schwarzbaum, Pablo J.

    2011-01-01

    We explored the intra- and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferin-luciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through β-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin + papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide (10Panx1) decreased [ATP]e by 75–84%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of ∼1 pmol × (106 cells)−1. A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol × (106 cells)−1, followed by a slower exponential decay (t½ = 3.7 min) to a constant value of 1.3 pmol × (106 cells)−1. Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (∼28 fmol × (106 cells min)−1), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood. PMID:21921036

  8. Relationship of superoxide production to cytoplasmic free calcium in human monocytes.

    PubMed Central

    Scully, S P; Segel, G B; Lichtman, M A

    1986-01-01

    Calcium has been proposed as an intracellular second messenger for activation of secretion, phagocytosis, and the oxidative burst of neutrophils. We have examined the role of calcium in human monocyte activation. Concanavalin A (Con A)-stimulated monocytes displayed an increment in cytoplasmic ionized calcium at 31 +/- 6 s and the onset of superoxide production at 61 +/- 9 s. The increase in cytoplasmic calcium invariably preceded the onset of superoxide production. If the external calcium concentration was reduced to less than 28 nM by the addition of 10 mM EGTA, superoxide production was not diminished at 5 min; however, superoxide production decreased thereafter. The Con A-evoked increment in cytoplasmic ionized calcium was blunted upon the addition of EGTA and decreased further with time. Both the production of superoxide and the Con A-evoked increment in cytoplasmic ionized calcium displayed a 50% inhibition after 15 min of calcium depletion and were completely inhibited after 60 min. Total cell calcium fell from 0.7 to 0.5 fmol/cell, and the basal level of ionized calcium fell from 83 to 30 nM after 60 min. Histidine, a strong chelator of divalent cations other than calcium and magnesium, had no effect on monocyte superoxide production or on ionized calcium concentrations, indicating that EGTA inhibition was due to cell calcium depletion. In calcium-depleted cells, Con A did not evoke superoxide production until calcium was restored to the incubation medium. The restoration of calcium to Con A-treated, calcium-depleted monocytes permitted a rapid rise in the cytoplasmic ionized calcium, and the production of superoxide within 9 s. These data suggest that an increase in ionized cytoplasmic calcium is necessary for the activation of monocyte superoxide production by Con A. The rise in ionized calcium in response to Con A results, in part, from an internal redistribution of calcium, which is sufficient to permit superoxide generation. PMID:3007579

  9. Altered Thiol Chemistry in Human Amyotrophic Lateral Sclerosis-linked Mutants of Superoxide Dismutase 1*

    PubMed Central

    Solsona, Carles; Kahn, Thomas B.; Badilla, Carmen L.; Álvarez-Zaldiernas, Cristina; Blasi, Juan; Fernandez, Julio M.; Alegre-Cebollada, Jorge

    2014-01-01

    Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS. PMID:25096579

  10. Extracellular superoxide dismutase in cultured astrocytes: decrease in cell-surface activity and increase in medium activity by lipopolysaccharide-stimulation.

    PubMed

    Iitsuka, Ichiro; Motoyoshi-Yamashiro, Akiko; Moriyama, Mitsuaki; Kannan-Hayashi, Yukiko; Fujimoto, Yuka; Takano, Katsura; Murakami, Koji; Yoneda, Yukio; Nakamura, Yoichi

    2012-10-01

    Under pathological conditions such as ischemia/reperfusion, a large amount of superoxide anion (O(2) (-)) is produced and released in brain. Among three isozymes of superoxide dismutase (SOD), extracellular (EC)-SOD, known to be excreted outside cells and bound to extracellular matrix, should play a role to detoxify O(2) (-) in extracellular space; however, a little is known about EC-SOD in brain. In order to evaluate the SOD activity in extracellular space of CNS as direct as possible, we attempted to measure the cell-surface SOD activity on primary cultured rat brain cells by the inhibition of color development of a water-soluble tetrazolium due to O(2) (-) generation by xanthine oxidase/hypoxanthine added into extracellular medium of intact cells. The cell-surface SOD activity on cultured neuron and microglia was below the detection limit; however, that on cultured astrocyte was high enough to measure. By means of RT-PCR, all mRNA of three isozymes of SOD could be detected in the three types of the cells examined; however, the semi-quantitative analysis revealed that the level of EC-SOD mRNA in astrocytes was significantly higher than that in neurons and microglia. When astrocytes were stimulated with lipopolysaccharide (LPS) for 12-24 h, the cell-surface SOD activity decreased to a half, whereas the activity recovered after 36-48 h. The decrease in the activity was dependent on the LPS concentration. On the other hand, the SOD activity in the medium increased by the LPS-stimulation in a dose dependent manner; suggesting that the SOD protein localized on cell-surface, probably EC-SOD, was released into the medium. These results suggest that EC-SOD of astrocyte play a role for detoxification of extracellular O(2) (-) and the regulation of EC-SOD in astrocytes may contribute to the defensive mechanism against oxidative stress in brain.

  11. Copper, Zinc Superoxide Dismutase is Primarily a Cytosolic Protein in Human Cells

    NASA Astrophysics Data System (ADS)

    Crapo, James D.; Oury, Tim; Rabouille, Catherine; Slot, Jan W.; Chang, Ling-Yi

    1992-11-01

    The intracellular localization of human copper, zinc superoxide dismutase (Cu,Zn-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) was evaluated by using EM immunocytochemistry and both isolated human cell lines and human tissues. Eight monoclonal antibodies raised against either native or recombinant human Cu,Zn-SOD and two polyclonal antibodies raised against either native or recombinant human Cu,Zn-SOD were used. Fixation with 2% paraformaldehyde/0.2% glutaraldehyde was found necessary to preserve normal distribution of the protein. Monoclonal antibodies were less effective than polyclonal antibodies in recognizing the antigen after adequate fixation of tissue. Cu,Zn-SOD was found widely distributed in the cell cytosol and in the cell nucleus, consistent with it being a soluble cytosolic protein. Mitochondria and secretory compartments did not label for this protein. In human cells, peroxisomes showed a labeling density slightly less than that of cytoplasm.

  12. Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

    SciTech Connect

    Weslake, R.J.; Chesney, S.L.; Petkau, A.; Friesen, A.D.

    1986-05-15

    Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.

  13. Luminol-amplified chemiluminescence detects mainly superoxide anion produced by human neutrophils

    PubMed Central

    Bedouhène, Samia; Moulti-Mati, Farida; Hurtado-Nedelec, Margarita; Dang, Pham My-Chan; El-Benna, Jamel

    2017-01-01

    Reactive oxygen species (ROS) are produced by numerous biological systems and by several phagocytes such as neutrophils and macrophages. ROS include mostly superoxide anion, hydrogen peroxide, singlet oxygen and hydroxyl radical, which are involved in a variety of biological processes such as immunity, inflammation, apoptosis and cell signaling. Thus, there is a need for a sensitive and reliable method to measure ROS. The luminol-amplified chemiluminescence technique is widely used to measure ROS production by neutrophils; however, it is unclear which ROS species are detected by this technique. In this study, we show that Xanthine/Xanthine oxidase (XXO), a known superoxide-producing system, stimulated a luminol-amplified chemiluminescence in the absence of horseradish peroxidase (HRPO), while the presence of HRPO enhanced the response. Both reactions were inhibited by superoxide dismutase (SOD), but not by catalase, confirming that superoxide anion, and not hydrogen peroxide, is the species oxidizing luminol to produce chemiluminescence. Glucose/Glucose oxidase (GGO), a known hydrogen peroxide-producing system, did not induce luminol-amplified chemiluminescence in the absence of HRPO; however, addition of HRPO resulted in a chemiluminescence response, which was inhibited by catalase, but not by SOD. Myeloperoxidase (MPO), isolated from human neutrophils, was also able to enhance the superoxide- and hydrogen peroxide-dependent luminol-amplified chemiluminescence. The production of ROS by stimulated human neutrophils was detected by luminol-amplified chemiluminescence, which was only partially inhibited by SOD and catalase. Interestingly, adding HRPO to stimulated neutrophils increased the luminol-amplified chemiluminescence, which was strongly inhibited by SOD, but not by catalase. These results show that (a) luminol-amplified chemiluminescence is able to detect superoxide anion in the absence of peroxidases, but not hydrogen peroxide; (b) in the presence of

  14. Nitration and Inactivation of Manganese Superoxide Dismutase in Chronic Rejection of Human Renal Allografts

    NASA Astrophysics Data System (ADS)

    MacMillan-Crow, L. A.; Crow, John P.; Kerby, Jeffrey D.; Beckman, Joseph S.; Thompson, John A.

    1996-10-01

    Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 μ M) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.

  15. Extracellular RNAs: development as biomarkers of human disease

    PubMed Central

    Quinn, Joseph F.; Patel, Tushar; Wong, David; Das, Saumya; Freedman, Jane E.; Laurent, Louise C.; Carter, Bob S.; Hochberg, Fred; Keuren-Jensen, Kendall Van; Huentelman, Matt; Spetzler, Robert; Kalani, M. Yashar S.; Arango, Jorge; Adelson, P. David; Weiner, Howard L.; Gandhi, Roopali; Goilav, Beatrice; Putterman, Chaim; Saugstad, Julie A.

    2015-01-01

    Ten ongoing studies designed to test the possibility that extracellular RNAs may serve as biomarkers in human disease are described. These studies, funded by the NIH Common Fund Extracellular RNA Communication Program, examine diverse extracellular body fluids, including plasma, serum, urine and cerebrospinal fluid. The disorders studied include hepatic and gastric cancer, cardiovascular disease, chronic kidney disease, neurodegenerative disease, brain tumours, intracranial haemorrhage, multiple sclerosis and placental disorders. Progress to date and the plans for future studies are outlined. PMID:26320940

  16. Loss of extracellular superoxide dismutase leads to acute lung damage in the presence of ambient air: a potential mechanism underlying adult respiratory distress syndrome.

    PubMed

    Gongora, Maria Carolina; Lob, Heinrich E; Landmesser, Ulf; Guzik, Tomasz J; Martin, W David; Ozumi, Kiyoski; Wall, Susan M; Wilson, David Scott; Murthy, Niren; Gravanis, Michael; Fukai, Tohru; Harrison, David G

    2008-10-01

    The extracellular superoxide dismutase 3 (SOD3) is highly expressed in both blood vessels and lungs. In different models of pulmonary injury, SOD3 is reduced; however, it is unclear whether this contributes to lung injury. To study the role of acute SOD3 reduction in lung injury, the SOD3 gene was deleted in adult mice by using the Cre-Lox technology. Acute reduction of SOD3 led to a fivefold increase in lung superoxide, marked inflammatory cell infiltration, a threefold increase in the arterial-alveolar gradient, respiratory acidosis, histological changes similar to those observed in adult respiratory distress syndrome, and 85% mortality. Treatment with the SOD mimetic MnTBAP and intranasal administration of SOD-containing polyketal microparticles reduced mortality, prevented the histological alterations, and reduced lung superoxide levels. To understand how mice with the SOD3 embryonic deletion survived without lung injury, gene array analysis was performed. These data demonstrated the up-regulation of 37 genes and down-regulation of nine genes, including those involved in cell signaling, inflammation, and gene transcription in SOD3-/- mice compared with either mice with acute SOD3 reduction or wild-type controls. These studies show that SOD3 is essential for survival in the presence of ambient oxygen and that acute loss of this enzyme can lead to severe lung damage. Strategies either to prevent SOD3 inactivation or to augment its levels might prove useful in the treatment of acute lung injury.

  17. Discriminative protection against hydroxyl and superoxide anion radicals by quercetin in human leucocytes in vitro.

    PubMed

    Wilms, Lonneke C; Kleinjans, Jos C S; Moonen, Edwin J C; Briedé, Jacob J

    2008-03-01

    Antioxidants play a vital role in the cellular protection against oxidative damage. Quercetin is a well-investigated antioxidant and known to be able to protect against cellular oxidative DNA damage. In this study, we tried to relate the protection by quercetin pre-treatment against oxidative DNA damage in human leucocytes in vitro to the interaction of quercetin in solution with hydroxyl and superoxide anion radicals as measured by electron spin resonance (ESR) spectrometry, using DMPO as a spin trap. Further, scavenging capacity of quercetin-treated leucocytes in vitro was evaluated by ESR spectrometry. Quercetin appears capable of protecting human leucocytes against oxidative DNA damage caused by hydrogen peroxide in a dose-dependent manner. The protection of leucocytes against superoxides is ambiguous. Incubation concentrations of quercetin (1, 10, and 50 microM) reduced levels of superoxide-induced oxidative DNA damage, while at 100 microM the amount of damage was increased. These results are supported by ESR-findings on quercetin in solution, also showing a prooxidant effect at 100 microM. ESR spectroscopy showed rate constant values for the reaction kinetics of quercetin in lowering iron-dependent hydroxyl radical formation and NADH-dependent superoxide anion formation of respectively 3.2 x 10(12)M(-1)s(-1) and 1.1 x 10(4)M(-1)s(-1). This shows that quercetin is a more potent inhibitor of hydroxyl radical formation than a scavenger of superoxide anions.

  18. Extracellular NAD+ regulates intracellular calcium levels and induces activation of human granulocytes

    PubMed Central

    2005-01-01

    β-NAD+e (extracellular β-NAD+), present at nanomolar levels in human plasma, has been implicated in the regulation of [Ca2+]i (the intracellular calcium concentration) in various cell types, including blood cells, by means of different mechanisms. Here, we demonstrate that micromolar NAD+e (both the α and the β extracellular NAD+ forms) induces a sustained [Ca2+]i increase in human granulocytes by triggering the following cascade of causally related events: (i) activation of adenylate cyclase and overproduction of cAMP; (ii) activation of protein kinase A; (iii) stimulation of ADP-ribosyl cyclase activity and consequent overproduction of cADP-ribose, a universal Ca2+ mobilizer; and (iv) influx of extracellular Ca2+. The NAD+e-triggered [Ca2+]i elevation translates into granulocyte activation, i.e. superoxide and nitric oxide generation, and enhanced chemotaxis in response to 0.1–10 μM NAD+e. Thus extracellular β-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation. PMID:16225456

  19. HIV-1 Nef regulates the release of superoxide anions from human macrophages

    PubMed Central

    2005-01-01

    The NADPH oxidase enzymatic complex participates in the oxidative burst by producing ROS (reactive oxygen species). Altered levels of ROS production may have pathogenetic implications due to the loss of some innate immune functions such as oxidative burst and phagocytosis. Considering that HIV-1 Nef protein plays a primary role in AIDS pathogenesis, by affecting the immune system, we sought to dissect possible effects of Nef on the release of superoxide anions. We show here that the inducible expression of Nef in human phagocytic cells modulates the superoxide release in a biphasic manner. In particular, an early Nef-induced increase of the superoxide release was followed by a dramatic decrease starting from 10 h after the Nef induction. This was observed whatever the presence of cell activators such as GM-CSF (granulocyte/macrophage colony-stimulating factor) or fMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine). Whereas the early increase in superoxide release is probably the result of the already described Nef-dependent activation of PAK-2 (p21-activated kinase 2)–Rac2, we were interested in investigating the mechanisms underlying the late inhibition of superoxide release observed originally. In this regard, we individuated at least three independent requirements for the Nef-induced blockade of superoxide release: (i) the active protein synthesis; (ii) both the membrane localization and the interaction with endocytotic machinery of Nef; and (iii) the release of soluble factor(s). Moreover, we observed that IL-10 (interleukin-10) inhibits superoxide release, whereas its depletion restored NADPH oxidase activity. We propose that the cell membrane-to-lysosome Nef transit leads to the synthesis and release of soluble factor(s) and, among them, IL-10 might significantly contribute to the inhibition of NAPDH oxidase activity. PMID:15847608

  20. Radiation resistance and the CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase activities of seven human cell lines.

    PubMed

    Marklund, S L; Westman, N G; Roos, G; Carlsson, J

    1984-10-01

    CuZn superoxide dismutase, Mn superoxide dismutase, catalase, and glutathione peroxidase form the primary enzymic defense against toxic oxygen reduction metabolites in cells. To test the importance of these protective enzymes in the cellular radiation response, the enzymic activities of seven different human cell lines were determined in parallel with their clonogenic survival characteristics. A positive correlation between the content of glutathione peroxidase in cell lines and their extrapolation numbers (n) and quasithreshold doses (Dq) was detected. Between the cellular contents of the other enzymes and D0, n, and Dq no positive correlations could be established. An interesting finding was a very high Mn superoxide dismutase content in a malignant mesothelioma cell line P7, which had an extremely high D0, 5.0 Gy.

  1. Diverse human extracellular RNAs are widely detected in human plasma

    PubMed Central

    Freedman, Jane E.; Gerstein, Mark; Mick, Eric; Rozowsky, Joel; Levy, Daniel; Kitchen, Robert; Das, Saumya; Shah, Ravi; Danielson, Kirsty; Beaulieu, Lea; Navarro, Fabio C. P.; Wang, Yaoyu; Galeev, Timur R.; Holman, Alex; Kwong, Raymond Y.; Murthy, Venkatesh; Tanriverdi, Selim E.; Koupenova, Milka; Mikhalev, Ekaterina; Tanriverdi, Kahraman

    2016-01-01

    There is growing appreciation for the importance of non-protein-coding genes in development and disease. Although much is known about microRNAs, limitations in bioinformatic analyses of RNA sequencing have precluded broad assessment of other forms of small-RNAs in humans. By analysing sequencing data from plasma-derived RNA from 40 individuals, here we identified over a thousand human extracellular RNAs including microRNAs, piwi-interacting RNA (piRNA), and small nucleolar RNAs. Using a targeted quantitative PCR with reverse transcription approach in an additional 2,763 individuals, we characterized almost 500 of the most abundant extracellular transcripts including microRNAs, piRNAs and small nucleolar RNAs. The presence in plasma of many non-microRNA small-RNAs was confirmed in an independent cohort. We present comprehensive data to demonstrate the broad and consistent detection of diverse classes of circulating non-cellular small-RNAs from a large population. PMID:27112789

  2. Novel mechanisms for superoxide-scavenging activity of human manganese superoxide dismutase determined by the K68 key acetylation site.

    PubMed

    Lu, Jiaqi; Cheng, Kuoyuan; Zhang, Bo; Xu, Huan; Cao, Yuanzhao; Guo, Fei; Feng, Xudong; Xia, Qing

    2015-08-01

    Superoxide is the primary reactive oxygen species generated in the mitochondria. Manganese superoxide dismutase (SOD2) is the major enzymatic superoxide scavenger present in the mitochondrial matrix and one of the most crucial reactive oxygen species-scavenging enzymes in the cell. SOD2 is activated by sirtuin 3 (SIRT3) through NAD(+)-dependent deacetylation. However, the exact acetylation sites of SOD2 are ambiguous and the mechanisms underlying the deacetylation-mediated SOD2 activation largely remain unknown. We are the first to characterize SOD2 mutants of the acetylation sites by investigating the relative enzymatic activity, structures, and electrostatic potential of SOD2 in this study. These SOD2 mutations affected the superoxide-scavenging activity in vitro and in HEK293T cells. The lysine 68 (K68) site is the most important acetylation site contributing to SOD2 activation and plays a role in cell survival after paraquat treatment. The molecular basis underlying the regulation of SOD2 activity by K68 was investigated in detail. Molecular dynamics simulations revealed that K68 mutations induced a conformational shift of residues located in the active center of SOD2 and altered the charge distribution on the SOD2 surface. Thus, the entry of the superoxide anion into the coordinated core of SOD2 was inhibited. Our results provide a novel mechanistic insight, whereby SOD2 acetylation affects the structure and charge distribution of SOD2, its tetramerization, and p53-SOD2 interactions of SOD2 in the mitochondria, which may play a role in nuclear-mitochondrial communication during aging.

  3. Involvement of superoxide radical in extracellular ferric reduction by iron-deficient bean roots. [Phadeolus vulgaris L. var Prelude

    SciTech Connect

    Cakmak, I.; van de Wetering, D.A.M.; Marschner, H.; Bienfait, H.F.

    1987-09-01

    The recent proposal of Tipton and Thowsen that iron-deficient plants reduce ferric chelates in cell walls by a system dependent on the leakage of malate from root cells was tested. Results are presented showing that this mechanism could not be responsible for the high rates of ferric reduction shown by roots of iron-deficient bean (Phaseolus vulgaris L. var Prelude) plants. The role of O/sub 2/ in the reduction of ferric chelates by roots of iron-deficient bean plants was also tested. The rate of Fe(III) reduction was the same in the presence and in the absence of O/sub 2/. However, in the presence of O/sub 2/ the reaction was partially inhibited by superoxide dismutase (SOD), which indicates a role for the superoxide radical, O/sub 2//sup -/, as a facultative intermediate electron carrier. The inhibition by SOD increased with substrate pH and with decrease in concentration of the ferrous scavenger bathophenanthroline-disulfonate. The results are consistent with a mechanism for transmembrane electron in which a flavin or quinone is the final electron carrier in the plasma membrane. The results are discussed in relation to the ecological importance that O/sub 2//sup -/ may have in the acquisition of ferric iron by dicotyledonous plants.

  4. Effect of stilbene derivative on superoxide generation and enzyme release from human neutrophils in vitro

    PubMed Central

    Pečivová, Jana; Harmatha, Juraj; Sviteková, Klára; Nosáľ, Radomír

    2012-01-01

    Neutrophils represent the body′s primary line of defense against invading pathogens. They most rapidly reach the site of injury or infection, liberate antimicrobial proteins, proteases and produce reactive oxygen species. Prolonged or excessive liberation of these very effective and toxic substances could intensify the inflammatory process and enhance tissue damage in many diseases, such as allergies, infections and rheumatoid arthritis. Pterostilbene belongs to stilbenoids, structural analogues of resveratrol, which act as natural protective agents in defending the plant against viral and microbial attack. It possesses anticancerous, antidiabetic and anti-inflammatory properties. The study provides new information on the effect of pterostilbene [0.01–100 µmol/l] on superoxide generation in and myeloperoxidase (MPO) release from azurophil granules of isolated human neutrophils. PMA [1µmol/l], which activates NADPH-oxidase via protein kinase C, was used for stimulation of neutrophils Unstimulated cells showed neither superoxide generation nor myelopereoxidase release after preincubation with the drug studied. Pterostilbene dose dependently decreased superoxide generation in and MPO release from stimulated human neutrophils, however a significant decrease was recorded only in the concentration 100 µmol/l. The effect of pterostilbene was more pronounced on superoxide generation in comparison to MPO release. Our results suggest that the effect of pterostilbene may prove beneficial in controlling inflammation. PMID:23118590

  5. Manganese superoxide dismutase from human pathogen Clostridium difficile.

    PubMed

    Li, Wei; Wang, Hongfei; Lei, Cheng; Ying, Tianlei; Tan, Xiangshi

    2015-05-01

    Clostridium difficile is a human pathogen that causes severe antibiotic-associated Clostridium difficile infection (CDI). Herein the MnSODcd from C. difficile was cloned, expressed in Escherichia Coli,and characterized by X-ray crystallography, UV/Vis and EPR spectroscopy, and activity assay, et al. The crystal structure of MnSODcd (2.32 Å) reveals a manganese coordination geometry of distorted trigonal bipyramidal, with His111, His197 and Asp193 providing the equatorial ligands and with His56 and a hydroxide or water forming the axial ligands. The catalytic activity of MnSODcd (8,600 U/mg) can be effectively inhibited by 2-methoxyestradiol with an IC50 of 75 μM. The affinity investigation between 2-methoxyestradiol and MnSODcd by ITC indicated a binding constant of 8.6 μM with enthalpy changes (ΔH = -4.08 ± 0.03 kcal/mol, ΔS = 9.53 ± 0.02 cal/mol/deg). An inhibitory mechanism of MnSODcd by 2-methoxyestradiol was probed and proposed based on molecular docking models and gel filtration analysis. The 2-methoxyestradiol may bind MnSODcd to interfere with the cross-linking between the two active sites of the dimer enzyme, compromising the SOD activity. These results provide valuable insight into the rational design of MnSODcd inhibitors for potential therapeutics for CDI.

  6. Dose dependent and divergent effects of superoxide anion on cell death, proliferation, and migration of activated human hepatic stellate cells

    PubMed Central

    Novo, E; Marra, F; Zamara, E; Bonzo, L Valfrè di; Caligiuri, A; Cannito, S; Antonaci, C; Colombatto, S; Pinzani, M; Parola, M

    2006-01-01

    Background and aim Activated myofibroblast‐like cells, originating from hepatic stellate cells (HSC/MFs) or other cellular sources, play a key profibrogenic role in chronic liver diseases (CLDs) that, as suggested by studies in animal models or rat HSC/MFs, may be modulated by reactive oxygen intermediates (ROI). In this study, human HSC/MFs, exposed to different levels of superoxide anion (O2•−) and, for comparison, hydrogen peroxide (H2O2), were analysed in terms of cytotoxicity, proliferative response, and migration. Methods Cultured human HSC/MFs were exposed to controlled O2•− generation by hypoxanthine/xanthine oxidase systems or to a range of H2O2 concentrations. Induction of cell death, proliferation, and migration were investigated using morphology, molecular biology, and biochemical techniques. Results Human HSC/MFs were shown to be extremely resistant to induction of cell death by O2•− and only high rates of O2•− generation induced either necrotic or apoptotic cell death. Non‐cytotoxic low levels of O2•−, able to upregulate procollagen type I expression (but not tissue inhibitor of metalloproteinase 1 and 2), stimulated migration of human HSC/MFs in a Ras/extracellular regulated kinase (ERK) dependent, antioxidant sensitive way, without affecting basal or platelet derived growth factor (PDGF) stimulated cell proliferation. Non‐cytotoxic levels of H2O2 did not affect Ras/ERK or proliferative response. A high rate of O2•− generation or elevated levels of H2O2 induced cytoskeletal alterations, block in motility, and inhibition of PDGF dependent DNA synthesis. Conclusions Low non‐cytotoxic levels of extracellularly generated O2•− may stimulate selected profibrogenic responses in human HSC/MFs without affecting proliferation. PMID:16041064

  7. Preliminary neutron diffraction analysis of challenging human manganese superoxide dismutase crystals.

    PubMed

    Azadmanesh, Jahaun; Trickel, Scott R; Weiss, Kevin L; Coates, Leighton; Borgstahl, Gloria E O

    2017-04-01

    Superoxide dismutases (SODs) are enzymes that protect against oxidative stress by dismutation of superoxide into oxygen and hydrogen peroxide through cyclic reduction and oxidation of the active-site metal. The complete enzymatic mechanisms of SODs are unknown since data on the positions of hydrogen are limited. Here, methods are presented for large crystal growth and neutron data collection of human manganese SOD (MnSOD) using perdeuteration and the MaNDi beamline at Oak Ridge National Laboratory. The crystal from which the human MnSOD data set was obtained is the crystal with the largest unit-cell edge (240 Å) from which data have been collected via neutron diffraction to sufficient resolution (2.30 Å) where hydrogen positions can be observed.

  8. Preliminary neutron diffraction analysis of challenging human manganese superoxide dismutase crystals

    PubMed Central

    Azadmanesh, Jahaun; Trickel, Scott R.; Borgstahl, Gloria E. O.

    2017-01-01

    Superoxide dismutases (SODs) are enzymes that protect against oxidative stress by dismutation of superoxide into oxygen and hydrogen peroxide through cyclic reduction and oxidation of the active-site metal. The complete enzymatic mechanisms of SODs are unknown since data on the positions of hydrogen are limited. Here, methods are presented for large crystal growth and neutron data collection of human manganese SOD (MnSOD) using perdeuteration and the MaNDi beamline at Oak Ridge National Laboratory. The crystal from which the human MnSOD data set was obtained is the crystal with the largest unit-cell edge (240 Å) from which data have been collected via neutron diffraction to sufficient resolution (2.30 Å) where hydrogen positions can be observed. PMID:28368283

  9. Methamphetamine toxicity is attenuated in mice that overexpress human manganese superoxide dismutase.

    PubMed

    Maragos, W F; Jakel, R; Chesnut, D; Pocernich, C B; Butterfield, D A; St Clair, D; Cass, W A

    2000-09-29

    We have investigated methamphetamine (MA) toxicity in transgenic mice that overexpress the human form of mitochondrial manganese superoxide dismutase (MnSOD). Our results reveal a significant reduction in the long-term depletion of striatal dopamine and protein oxidation following repeated administration of MA in transgenic vs. non-transgenic littermates. These findings support the notion that ROS contribute to MA-induced brain damage and suggest that mitochondria may play an important role in this form of neurodegeneration.

  10. Macrophage Stimulating Protein (MSP) evokes superoxide anion production by human macrophages of different origin

    PubMed Central

    Brunelleschi, Sandra; Penengo, Lorenza; Lavagno, Luisa; Santoro, Claudio; Colangelo, Donato; Viano, Ilario; Gaudino, Giovanni

    2001-01-01

    Macrophage Stimulating Protein (MSP), a serum factor related to Hepatocyte Growth Factor, was originally discovered to stimulate chemotaxis of murine resident peritoneal macrophages. MSP is the ligand for Ron, a member of the Met subfamily of tyrosine kinase receptors. The effects of MSP on human macrophages and the role played in human pathophysiology have long been elusive.We show here that human recombinant MSP (hrMSP) evokes a dose-dependent superoxide anion production in human alveolar and peritoneal macrophages as well as in monocyte-derived macrophages, but not in circulating human monocytes. Consistently, the mature Ron protein is expressed by the MSP responsive cells but not by the unresponsive monocytes. The respiratory burst evoked by hrMSP is quantitatively higher than the one induced by N-formylmethionyl-leucyl-phenylalanine and similar to phorbol myristate acetate-evoked one.To investigate the mechanisms involved in NADPH oxidase activation, leading to superoxide anion production, different signal transduction inhibitors were used. By using the non selective tyrosine kinase inhibitor genistein, the selective c-Src inhibitor PP1, the tyrosine phosphatase inhibitor sodium orthovanadate, the phosphatidylinositol 3-kinase inhibitor wortmannin, the p38 inhibitor SB203580, the MEK inhibitor PD098059, we demonstrate that hrMSP-evoked superoxide production is mediated by tyrosine kinase activity, requires the activation of Src but not of PI 3-kinase. We also show that MAP kinase and p38 signalling pathways are involved.These results clearly indicate that hrMSP induces the respiratory burst in human macrophages but not in monocytes, suggesting for the MSP/Ron complex a role of activator as well as of possible marker for human mature macrophages. PMID:11704649

  11. Amelioration of salt-induced vascular dysfunction in mesenteric arteries of Dahl salt-sensitive rats by missense mutation of extracellular superoxide dismutase

    PubMed Central

    Beyer, Andreas M.; Raffai, Gabor; Weinberg, Brian D.; Fredrich, Katherine; Rodgers, Matthew S.; Geurts, Aron M.; Jacob, Howard J.; Dwinell, Melinda R.

    2013-01-01

    Superoxide dismutase (SOD) enzymes, including extracellular SOD (ecSOD), are important for scavenging superoxide radicals (O2·−) in the vasculature. This study investigated vascular control in rats [SS-Sod3m1Mcwi (ecSODE124D)] with a missense mutation that alters a single amino acid (E124D) of ecSOD that produces a malfunctioning protein in the salt-sensitive (Dahl SS) genetic background. We hypothesized that this mutation would exacerbate endothelial dysfunction due to elevated vascular O2·− levels in SS, even under normal salt (NS; 0.4% NaCl) conditions. Aortas of ecSODE124D rats fed standard rodent chow showed enhanced sensitivity to phenylephrine and reduced relaxation to acetylcholine (ACh) vs. SS rats. Endothelium-dependent dilation to ACh was unaffected by the mutation in small mesenteric arteries of ecSODE124D rats fed NS diet, and mesenteric arteries of ecSODE124D rats were protected from endothelial dysfunction during short-term (3–5 days) high-salt (HS; 4% NaCl) diet. ACh-induced dilation of mesenteric arteries of ecSODE124D rats and SS rats fed NS diet was inhibited by NG-nitro-l-arginine methyl ester and/or by H2O2 scavenging with polyethylene glycol-catalase at higher concentrations of ACh. Total SOD activity was significantly higher in ecSODE124D rats vs. SS controls fed HS diet, most likely reflecting a compensatory response to loss of a functional ecSOD isoform. These findings indicate that, contrary to its effect in the aorta, this missense mutation of ecSOD in the SS rat genome has no negative effect on vascular function in small resistance arteries, but instead protects against salt-induced endothelial dysfunction, most likely via compensatory mechanisms involving an increase in total SOD activity. PMID:24322611

  12. Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa.

    PubMed

    Cocchia, N; Pasolini, M P; Mancini, R; Petrazzuolo, O; Cristofaro, I; Rosapane, I; Sica, A; Tortora, G; Lorizio, R; Paraggio, G; Mancini, A

    2011-04-15

    New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H(2)O(2) and O(2) production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: native semen (control 1); native semen diluted 1:3 with Kenney semen extender (control 2); spermatozoa diluted after centrifugation in extender without (control 3) or with SOD at 25 IU/ml (experimental 1) or 50 IU/ml (experimental 2). Each sample was analyzed for motility, viability and acrosome status, immediately after semen preparation and again after storage at 5 °C for 24 h, 48 h and 7 2h. Acrosome integrity was evaluated by Chlortetracycline (CTC) and Fluorescent-labeled peanut lectin agglutinin (PNA-FITC conjugated staining). A proteomic approach of quantifying extracellular signal regulated kinase (ERK) was also evaluated as an indirect indicator of oxidative stress. In all samples sperm progressive motility and sperm acrosomal integrity showed a significant reduction between fresh and cooled spermatozoa at 24 h, 48 h and 72 h. Quality parameters of sperm were significantly higher (Progressive Motility P < 0.01; Viability P < 0.001) in aliquots supplemented with SOD. ERK phosphorylation was statistically higher (P < 0.01) in aliquots without SOD. The Authors concluded that addition of SOD to semen extenders improves the quality of chilled equine semen and reduces ERK activation.

  13. HPLC-based monitoring of products formed from hydroethidine-based fluorogenic probes – The ultimate approach for intra-and extracellular superoxide detection

    PubMed Central

    Kalyanaraman, Balaraman; Dranka, Brian P.; Hardy, Micael; Michalski, Radoslaw; Zielonka, Jacek

    2013-01-01

    Background Nearly ten years ago, we demonstrated that superoxide radical anion (O2−̣) reacts with the hydroethidine dye (HE, also known as dihydroethidium, DHE) to form a diagnostic marker product, 2-hydroxyethidium (2-OH-E+). This particular product is not derived from reacting HE with other biologically relevant oxidants (hydrogen peroxide, hydroxyl radical, or peroxynitrite). This discovery negated the longstanding view that O2−̣reacts with HE to form the other oxidation product, ethidium (E+). It became clear that due to the overlapping fluorescence spectra of E+ and 2-OH-E+, fluorescence-based techniques using the “red fluorescence” are not suitable for detecting and measuring O2−̣in cells using HE or other structurally analogous fluorogenic probes (MitoSOX™ Red or hydropropidine). However, using HPLC-based assays, 2-OH-E+ and analogous hydroxylated products can be easily detected and quickly separated from other oxidation products. Scope of review The principles discussed in this chapter are generally applicable in free radical biology and medicine, redox biology, and clinical and translational research. The assays developed here could be used to discover new and targeted inhibitors for various superoxide-producing enzymes, including NADPH oxidase (NOX) isoforms. Major conclusions HPLC-based approaches using site-specific HE-based fluorogenic probes are eminently suitable for monitoring O2−̣in intra-and extracellular compartments and in mitochondria. The use of fluorescence-microscopic methods should be avoided because of spectral overlapping characteristics of O2−̣-derived marker product and other, non-specific oxidized fluorescent products formed from these probes. General significance Methodologies and site-specific fluorescent probes described in this review can be suitably employed to delineate oxy radical dependent mechanisms in cells under physiological and pathological conditions. PMID:23668959

  14. Myeloid zinc finger (MZF)-like, Kruppel-like and Ets families of transcription factors determine the cell-specific expression of mouse extracellular superoxide dismutase.

    PubMed Central

    Zelko, Igor N; Folz, Rodney J

    2003-01-01

    Extracellular superoxide dismutase (EC-SOD or SOD3) is an important protective enzyme against the toxicity of superoxide radicals that are produced under both physiological and pathophysiological conditions. We have isolated and characterized over 11 kb of the mouse EC-SOD gene and its 5'- and 3'-flanking regions. The gene consists of two exons, with the entire coding region located within exon 2. In order to study the mechanism of cell-specific gene regulation for mouse EC-SOD, we characterized 2500 bp of its 5'-flanking region using cultured cells derived from mouse lung fibroblasts (MLg), kidney medulla (mIMCD3) and hepatocytes (Hepa 1-6). Real-time PCR showed that basal expression of EC-SOD was considerably higher in MLg cells compared with the other cell types. Reporter-gene assays revealed that the proximal promoter region was sufficient to support this high expression in MLg cells. Although no obvious TATA box was identified, our results show that a highly purine-rich region from -208 to +104 contains active binding sites for both the Kruppel-like and Ets families of transcription factors. Using electrophoretic mobility shift, DNase footprinting and reporter gene assays, we identified myeloid zinc finger 1 and gut-enriched Kruppel-like-factor-like nuclear transcription factors as repressors of EC-SOD expression, whereas nuclear transcription factors from the Ets family, such as Elf-1 and GA-binding protein alpha and beta, were potent activators of EC-SOD transcription. We propose a model that highlights competition between Ets activators and Kruppel-like repressors within the proximal promoter region that determines the level of EC-SOD expression in a particular cell type. PMID:12374566

  15. Extracellular Ca2+ regulates the respiratory burst of human neutrophils.

    PubMed

    Bei, L; Hu, T; Qian, Z M; Shen, X

    1998-09-16

    The role of extracellular calcium in the activation of respiratory burst in human neutrophils was studied by using the receptor agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the activator of protein kinase C phorbol myristate acetate (PMA). The level of intracellular free calcium was measured by using both cell suspensions and single cells in the presence and absence of extracellular calcium. The Ca2+-ATPase inhibitor, thapsigargin, was used to activate higher Ca2+ influx, while a novel calcium channel blocker, panax notoginseng saponins (PNGS) was used to block the Ca2+ entry from extracellular space during the responding period of cells. It was found that about two-thirds of the activation of respiratory burst initiated by the receptor agonist were attributed to the Ca2+ influx under normal physiological conditions. The higher Ca2+ influx resulted in tremendous enhancement of the intensity of respiratory burst initiated by fMLP and marked acceleration of the onset of the respiratory burst stimulated by PMA. It is evident that both intra- and extracellular Ca2+ are required for full activation of the respiratory burst of human neutrophils, and the Ca2+ influx from extracellular space plays an important role either in generation of reactive oxygen metabolites or in activation of protein kinase C.

  16. Downregulation of high-isoelectric-point extracellular superoxide dismutase mediates alterations in the metabolism of reactive oxygen species and developmental disturbances in hybrid aspen.

    PubMed

    Srivastava, Vaibhav; Schinkel, Helga; Witzell, Johanna; Hertzberg, Magnus; Torp, Mikaela; Srivastava, Manoj Kumar; Karpinska, Barbara; Melzer, Michael; Wingsle, Gunnar

    2007-01-01

    Transgenic hybrid aspen (Populus tremula L. x P. tremuloides Michx.) plants expressing a high-isoelectric-point superoxide dismutase (hipI-SOD) gene in antisense orientation were generated to investigate its function. Immunolocalization studies showed the enzyme to be localized extracellularly, in the secondary cell wall of xylem vessels and phloem fibers. The antisense lines of hipI-SOD exhibited a distinct phenotype; growth rate was reduced, stems were thinner and leaves smaller than in wild-type (WT) plants. The abundance of hipI-SOD was reduced in the bark and xylem of plants from these antisense lines. The vascular tissue of transgenic lines became lignified earlier than in WT plants and also showed an increased accumulation of reactive oxygen species (ROS). Xylem fibers and vessels were shorter and thinner in the transgenic lines than in WT plants. The total phenolic content was enhanced in the antisense lines. Furthermore, microarray analysis indicated that several enzymes involved in cell signaling, lignin biosynthesis and stress responses were upregulated in apical vascular tissues of transgenic plants. The upregulation of selected genes involved in lignin biosynthesis was also verified by real-time PCR. The results suggest that, in the transgenic plants, a premature transition into maturation occurs and the process is discussed in terms of the effects of increased accumulation of ROS due to reduced expression of hipI-SOD during development and differentiation.

  17. The involvement of superoxide and iron ions in the NADPH-dependent lipid peroxidation in human placental mitochondria.

    PubMed

    Klimek, J

    1988-01-19

    Incubation of human term placental mitochondria with Fe2+ and a NADPH-generating system initiated high levels of lipid peroxidation, as measured by the production of malondialdehyde. Malondialdehyde formation was accompanied by a corresponding decrease of the unsaturated fatty acid content. This NADPH-dependent lipid peroxidation was strongly inhibited by superoxide dismutase and singlet oxygen scavengers, markedly stimulated by paraquat, but was not affected by hydroxyl radical scavengers. Catalase enhanced the production of malondialdehyde by placental mitochondria. The effects of catalase and hydroxyl radical scavengers suggest that the initiation of NADPH-dependent lipid peroxidation is not dependent upon the hydroxyl radical produced via an iron-catalyzed Fenton reaction. These studies provide evidence that hydrogen peroxide strongly inhibits NADPH-dependent mitochondrial lipid peroxidation. The inhibitory effect of superoxide dismutase and stimulatory effect of paraquat, which was abolished by the addition of superoxide dismutase, suggests that superoxide may promote NADPH-dependent lipid peroxidation in human placental mitochondria.

  18. HPLC-based monitoring of products formed from hydroethidine-based fluorogenic probes--the ultimate approach for intra- and extracellular superoxide detection.

    PubMed

    Kalyanaraman, Balaraman; Dranka, Brian P; Hardy, Micael; Michalski, Radoslaw; Zielonka, Jacek

    2014-02-01

    Nearly ten years ago, we demonstrated that superoxide radical anion (O2⋅¯) reacts with the hydroethidine dye (HE, also known as dihydroethidium, DHE) to form a diagnostic marker product, 2-hydroxyethidium (2-OH-E(+)). This particular product is not derived from reacting HE with other biologically relevant oxidants (hydrogen peroxide, hydroxyl radical, or peroxynitrite). This discovery negated the longstanding view that O2⋅¯ reacts with HE to form the other oxidation product, ethidium (E(+)). It became clear that due to the overlapping fluorescence spectra of E(+) and 2-OH-E(+), fluorescence-based techniques using the "red fluorescence" are not suitable for detecting and measuring O2⋅¯ in cells using HE or other structurally analogous fluorogenic probes (MitoSOX(TM) Red or hydropropidine). However, using HPLC-based assays, 2-OH-E(+) and analogous hydroxylated products can be easily detected and quickly separated from other oxidation products. The principles discussed in this chapter are generally applicable in free radical biology and medicine, redox biology, and clinical and translational research. The assays developed here could be used to discover new and targeted inhibitors for various superoxide-producing enzymes, including NADPH oxidase (NOX) isoforms. HPLC-based approaches using site-specific HE-based fluorogenic probes are eminently suitable for monitoring O2⋅¯ in intra- and extracellular compartments and in mitochondria. The use of fluorescence-microscopic methods should be avoided because of spectral overlapping characteristics of O2⋅¯-derived marker product and other, non-specific oxidized fluorescent products formed from these probes. Methodologies and site-specific fluorescent probes described in this review can be suitably employed to delineate oxy radical dependent mechanisms in cells under physiological and pathological conditions. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros

  19. Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes.

    PubMed Central

    Ganz, T

    1987-01-01

    Human polymorphonuclear leukocytes (PMN) contain three antimicrobial and cytotoxic peptides which belong to a family of mammalian granulocyte peptides named defensins. To determine their potential availability for extracellular microbicidal or cytotoxic events, we quantified the extracellular release of defensins after stimulation of human PMN with phorbol myristate acetate and opsonized zymosan. As determined by enzyme immunoassay and confirmed by polyacrylamide gel electrophoresis and densitometry, 10(6) human PMN contained 4 to 5 micrograms of defensins. After stimulation with a high concentration of phorbol myristate acetate (1 microgram/ml), about 8% of PMN defensins were found in the media. Release of defensins correlated best with the release of azurophil granule marker beta-glucuronidase or elastase and poorly with the release of either the specific granule marker lactoferrin or cytoplasmic lactate dehydrogenase. Phagocytosis of opsonized zymosan resulted in the extracellular release of less than 3% of PMN defensins. The factors responsible for less release of defensins into media relative to the release of other azurophil granule proteins may include heterogeneity of azurophil granules and the affinity of defensins for cellular surfaces and opsonized particles. In vivo, defensins are most likely to reach effective microbicidal or cytotoxic concentrations in PMN-rich exudates (pus), in confined environments of the phagolysosomes, or in intercellular clefts between PMN and their targets. PMID:3643886

  20. Superoxide dismutase and the resistance of Escherichia coli to phagocytic killing by human neutrophils.

    PubMed Central

    Papp-Szabò, E; Sutherland, C L; Josephy, P D

    1993-01-01

    Transformation of Escherichia coli K-12-derived strains with a plasmid carrying the genetic determinants for synthesis of lipopolysaccharide O antigen by Shigella dysenteriae allows the construction of phenotypically smooth derivatives. We show that such E. coli K-12 derivatives are highly resistant to killing by human serum. Isogenic wild-type and sodB mutant (Fe superoxide dismutase-deficient) strains were constructed. The results of experiments on phagocytic killing of these strains by human neutrophils are reported. We observed no difference between the sensitivities of wild-type and sodB mutant strains to phagocytic killing, in contrast to the results reported by other researchers who used species other than E. coli or strains other than K-12. Images PMID:8454348

  1. Possible involvement of an extracellular superoxide dismutase (SodA) as a radical scavenger in poly(cis-1,4-isoprene) degradation.

    PubMed

    Schulte, Carina; Arenskötter, Matthias; Berekaa, Mahmoud M; Arenskötter, Quyen; Priefert, Horst; Steinbüchel, Alexander

    2008-12-01

    Gordonia westfalica Kb1 and Gordonia polyisoprenivorans VH2 induce the formation of an extracellular superoxide dismutase (SOD) during poly(cis-1,4-isoprene) degradation. To investigate the function of this enzyme in G. polyisoprenivorans VH2, the sodA gene was disrupted. The mutants exhibited reduced growth in liquid mineral salt media containing poly(cis-1,4-isoprene) as the sole carbon and energy source, and no SOD activity was detectable in the supernatants of the cultures. Growth experiments revealed that SodA activity is required for optimal growth on poly(cis-1,4-isoprene), whereas this enzyme has no effect on aerobic growth in the presence of water-soluble substrates like succinate, acetate, and propionate. This was detected by activity staining, and proof of expression was by antibody detection of SOD. When SodA from G. westfalica Kb1 was heterologously expressed in the sodA sodB double mutant Escherichia coli QC779, the recombinant mutant exhibited increased resistance to paraquat, thereby indicating the functionality of the G. westfalica Kb1 SodA and indirectly protection of G. westfalica cells by SodA from oxidative damage. Both sodA from G. polyisoprenivorans VH2 and sodA from G. westfalica Kb1 coded for polypeptides comprising 209 amino acids and having approximately 90% and 70% identical amino acids, respectively, to the SodA from Mycobacterium smegmatis strain MC(2) 155 and Micrococcus luteus NCTC 2665. As revealed by activity staining experiments with the wild type and the disruption mutant of G. polyisoprenivorans, this bacterium harbors only one active SOD belonging to the manganese family. The N-terminal sequences of the extracellular SodA proteins of both Gordonia species showed no evidence of leader peptides for the mature proteins, like the intracellular SodA protein of G. polyisoprenivorans VH2, which was purified under native conditions from the cells. In G. westfalica Kb1 and G. polyisoprenivorans VH2, SodA probably provides protection

  2. Possible Involvement of an Extracellular Superoxide Dismutase (SodA) as a Radical Scavenger in Poly(cis-1,4-Isoprene) Degradation▿

    PubMed Central

    Schulte, Carina; Arenskötter, Matthias; Berekaa, Mahmoud M.; Arenskötter, Quyen; Priefert, Horst; Steinbüchel, Alexander

    2008-01-01

    Gordonia westfalica Kb1 and Gordonia polyisoprenivorans VH2 induce the formation of an extracellular superoxide dismutase (SOD) during poly(cis-1,4-isoprene) degradation. To investigate the function of this enzyme in G. polyisoprenivorans VH2, the sodA gene was disrupted. The mutants exhibited reduced growth in liquid mineral salt media containing poly(cis-1,4-isoprene) as the sole carbon and energy source, and no SOD activity was detectable in the supernatants of the cultures. Growth experiments revealed that SodA activity is required for optimal growth on poly(cis-1,4-isoprene), whereas this enzyme has no effect on aerobic growth in the presence of water-soluble substrates like succinate, acetate, and propionate. This was detected by activity staining, and proof of expression was by antibody detection of SOD. When SodA from G. westfalica Kb1 was heterologously expressed in the sodA sodB double mutant Escherichia coli QC779, the recombinant mutant exhibited increased resistance to paraquat, thereby indicating the functionality of the G. westfalica Kb1 SodA and indirectly protection of G. westfalica cells by SodA from oxidative damage. Both sodA from G. polyisoprenivorans VH2 and sodA from G. westfalica Kb1 coded for polypeptides comprising 209 amino acids and having approximately 90% and 70% identical amino acids, respectively, to the SodA from Mycobacterium smegmatis strain MC2 155 and Micrococcus luteus NCTC 2665. As revealed by activity staining experiments with the wild type and the disruption mutant of G. polyisoprenivorans, this bacterium harbors only one active SOD belonging to the manganese family. The N-terminal sequences of the extracellular SodA proteins of both Gordonia species showed no evidence of leader peptides for the mature proteins, like the intracellular SodA protein of G. polyisoprenivorans VH2, which was purified under native conditions from the cells. In G. westfalica Kb1 and G. polyisoprenivorans VH2, SodA probably provides protection

  3. Significant association between SNPs in the superoxide dismutase 3, extracellular (SOD3) gene and resistance to Aeromonas hydrophila in the freshwater mussel Hyriopsis cumingii.

    PubMed

    Wang, G L; Li, X L; Li, J L

    2013-12-01

    Extracellular superoxide dismutase (SOD3) is a major antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). In this study, the SOD3 gene was identified and characterized from the freshwater mussel Hyriopsis cumingii (Hc-SOD3). The cDNA sequence consists of 763 bp, encoding a protein of 208 amino acids. The amino acid sequence possesses two CuZnSOD signature sequences, and amino acids required for binding of Cu (His-93, -95, -110 and -169) and Zn (His-110, -118, -129 and Asp-132) were conserved in Hc-SOD3. The Hc-SOD3 genomic sequence was 9165 bp in length, containing four exons and three introns. Eighteen single nucleotide polymorphisms were detected in the Hc-SOD3 gene from resistant stock (RS) and susceptible stock (SS) of H. cumingii to Aeromonas hydrophila. The genotype and allele distribution were examined in resistant and susceptible stocks. Among them, a C/G substitution at the g.7994C>G locus and G/C substitution at the g.8087G>C locus were significantly associated with resistance/susceptibility of H. cumingii to A. hydrophila, both in genotype (P = 0.017, P = 0.004 respectively) and allele frequency (P = 0.021, P = 0.006 respectively). Linkage disequilibrium analysis revealed that g.7994C>G, g.8001A>G, g.8035G>A, g.8087G>C and g.8191T>A were in linkage disequilibrium. The results suggest that the two polymorphic loci, g.7994C>G and g.8087G>C, could be potential genetic markers for future molecular selection of strains that are resistant to diseases.

  4. Crystal structure of nitrated human manganese superoxide dismutase: mechanism of inactivation.

    PubMed

    Quint, Patrick; Reutzel, Robbie; Mikulski, Rose; McKenna, Robert; Silverman, David N

    2006-02-01

    A cellular consequence of the reaction of superoxide and nitric oxide is enhanced peroxynitrite levels. Reaction of peroxynitrite with manganese superoxide dismutase (MnSOD) causes nitration of the active-site residue Tyr34 and nearly complete inhibition of catalysis. We report the crystal structures at 2.4 A resolution of human MnSOD nitrated by peroxynitrite and the unmodified MnSOD. A comparison of these structures showed no significant conformational changes of active-site residues or solvent displacement. The side chain of 3-nitrotyrosine 34 had a single conformation that extended toward the manganese with O1 of the nitro group within hydrogen-bonding distance (3.1 A) of Nepsilon2 of the second-shell ligand Gln143. Also, nitration of Tyr34 caused a weakening, as evidenced by the lengthening, of a hydrogen bond between its phenolic OH and Gln143, part of an extensive hydrogen-bond network in the active site. Inhibition of catalysis can be attributed to a steric effect of 3-nitrotyrosine 34 that impedes substrate access and binding, and alteration of the hydrogen-bond network that supports proton transfer in catalysis. It is also possible that an electrostatic effect of the nitro group has altered the finely tuned redox potential necessary for efficient catalysis, although the redox potential of nitrated MnSOD has not been measured.

  5. Human red cells scavenge extracellular hydrogen peroxide and inhibit formation of hypochlorous acid and hydroxyl radical.

    PubMed Central

    Winterbourn, C C; Stern, A

    1987-01-01

    The ability of intact human red cells to scavenge extracellularly generated H2O2 and O2-, and to prevent formation of hydroxyl radicals and hypochlorous acid has been examined. Red cells inhibited oxidation of ferrocytochrome c by H2O2. Cells treated with aminotriazole no longer inhibited, indicating that protection was almost entirely due to intracellular catalase. Contribution by the GSH system was slight, and apparent only with low H2O2 concentrations when catalase was inhibited by aminotriazole. The cells were about a quarter as efficient at inhibiting cytochrome c oxidation as an equivalent concentration of purified catalase. No inhibition of O2(-)-dependent reduction of ferricytochrome c or nitroblue tetrazolium was observed, although extracted red cell superoxide dismutase inhibited nitroblue tetrazolium reduction at one fortieth the concentration of that in the cells. Red cells efficiently inhibited deoxyribose oxidation by hydroxyl radicals generated from H2O2, O2- and Fe(EDTA), and myeloperoxidase-dependent oxidation of methionine to methionine sulfoxide by stimulated neutrophils. Most of the red cell inhibition of hydroxyl radical production, and all the inhibition of methionine oxidation, was prevented by blocking intracellular catalase with aminotriazole. Thus red cells are able to efficiently scavenge H2O2, but not O2-, produced in their environment, and to inhibit formation of hydroxyl radicals and hypochlorous acid. They may therefore have an important role in extracellular antioxidant defense. PMID:2824562

  6. Wolbachia endosymbionts induce neutrophil extracellular trap formation in human onchocerciasis

    PubMed Central

    Tamarozzi, Francesca; Turner, Joseph D.; Pionnier, Nicolas; Midgley, Angela; Guimaraes, Ana F.; Johnston, Kelly L.; Edwards, Steven W.; Taylor, Mark J.

    2016-01-01

    The endosymbiotic bacteria, Wolbachia, induce neutrophilic responses to the human helminth pathogen Onchocerca volvulus. The formation of Neutrophil Extracellular Traps (NETs), has been implicated in anti-microbial defence, but has not been identified in human helminth infection. Here, we demonstrate NETs formation in human onchocerciasis. Extracellular NETs and neutrophils were visualised around O. volvulus in nodules excised from untreated patients but not in nodules from patients treated with the anti-Wolbachia drug, doxycycline. Whole Wolbachia or microspheres coated with a synthetic Wolbachia lipopeptide (WoLP) of the major nematode Wolbachia TLR2/6 ligand, peptidoglycan associated lipoprotein, induced NETosis in human neutrophils in vitro. TLR6 dependency of Wolbachia and WoLP NETosis was demonstrated using purified neutrophils from TLR6 deficient mice. Thus, we demonstrate for the first time that NETosis occurs during natural human helminth infection and demonstrate a mechanism of NETosis induction via Wolbachia endobacteria and direct ligation of Wolbachia lipoprotein by neutrophil TLR2/6. PMID:27752109

  7. Alteration of cellular phenotype and responses to oxidative stress by manganese superoxide dismutase and a superoxide dismutase mimic in RWPE-2 human prostate adenocarcinoma cells.

    PubMed

    Zhong, Weixiong; Yan, Tao; Webber, Mukta M; Oberley, Terry D

    2004-06-01

    To study biologic effects of increased manganese superoxide dismutase (MnSOD) on cell behavior, we overexpressed MnSOD in a human prostate cancer cell line RWPE-2 by cDNA transfection. Stable transfectants of MnSOD showed a two- to threefold increase in MnSOD protein and enzymatic activity and a decrease in growth rate with prolonged cell population doubling times. Western blot analysis showed a 1.5- to twofold increase in the cyclin-dependent kinase inhibitor p21(Waf1) in MnSOD transfectants. Overexpression of MnSOD resulted in a seven- to eightfold increase in reduced glutathione (GSH), 18- to 26-fold increase in oxidized glutathione (GSSG), and a two- to threefold decrease in the ratio of GSH to GSSG. MnSOD-overexpressing cells showed an increase in sensitivity to the cytotoxicity of buthionine sulfoximine, a glutathione-depleting agent, and vitamin C, but a decrease in sensitivity to sodium selenite. Treatment with a superoxide dismutase (SOD) mimic MnTMPyP resulted in similar effects of MnSOD overexpression on cell responses to vitamin C and selenium. These data demonstrate that overexpression of MnSOD or treatment with SOD mimics can result in antioxidant or prooxidant effects in cells, depending on the presence of other antioxidants and prooxidants. MnSOD also has redox regulatory effects on cell growth and gene expression. These findings suggest that MnSOD and SOD mimics have the potential for cancer prevention or treatment.

  8. Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

    PubMed Central

    Denis, María Florencia Leal; Incicco, J. Jeremías; Espelt, María Victoria; Verstraeten, Sandra V.; Pignataro, Omar P.; Lazarowski, Eduardo R.; Schwarzbaum, Pablo J.

    2014-01-01

    SUMMARY Background The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). Methods Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. Results Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10 μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. Conclusions MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. General Significance Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation. PMID:23742824

  9. Inhibitory effects of N-acetylcysteine on superoxide anion generation in human polymorphonuclear leukocytes.

    PubMed

    Villagrasa, V; Cortijo, J; Martí-Cabrera, M; Ortiz, J L; Berto, L; Esteras, A; Bruseghini, L; Morcillo, E J

    1997-05-01

    It has been suggested that reactive oxygen species released by activated polymorphonuclear leukocytes (PMN) in man is one mechanism of tissue injury. Therapeutic action aimed at increasing antioxidant defence mechanisms is still a clinical challenge. This study examines the activity of N-acetylcysteine, a known antioxidant, in the protection of PMN exposed in-vitro to the chemoattractant peptide fMet-Leu-Phe (FMLP), the protein kinase C activator phorbol myristate acetate or the lipid peroxidation promoter t-butyl hydroperoxide. FMLP (3-300 nM) and phorbol myristate acetate (160 pm-160 nM) induced concentration-related superoxide anion generation. Pre-treatment with N-acetylcysteine (33-333 microM) resulted in concentration-related inhibition of superoxide production induced by FMLP (30 nM) or phorbol myristate acetate (16 nM);-log IC50 values were 3.97 +/- 0.07 and 3.91 +/- 0.10, respectively. Changes in intracellular calcium ion concentration ([Ca2+]i) induced by FMLP (30 nM) were studied in fura-2-loaded human PMN. FMLP produced a transient calcium response, i.e. a peak followed by decay to a residual value above baseline. N-Acetylcysteine (333 microM) did not affect either basal [Ca2+]i values or changes in [Ca2+]i values after treatment with FMLP. Activation by phorbol myristate acetate caused a reduction in glutathione levels from 5.94 +/- 0.86 (control) to 1.84 +/- 0.51 nmol/3 x 10(6) cells (P < 0.05 compared with control). Pre-treatment with N-acetylcysteine (333 microM) fully reversed the reduction in glutathione levels induced by phorbol myristate acetate (4.83 +/- 0.68 nmol/3 x 10(6) cells; P > 0.05 compared with control). Exposure to t-butyl hydroperoxide (0.5 mM, 30 min) markedly increased malondialdehyde levels (from 0.03 +/- 0.02 to 0.73 +/- 0.07 nmol/10(6) cells), and index of lipid peroxidation. Malondialdehyde levels were significantly reduced in PMN treated with N-acetylcysteine (333 microM; 0.55 +/- 0.04 nmol/10(6) cells; P < 0.05 compared with

  10. An extracellular proteasome releases endostatin from human collagen XVIII.

    PubMed

    Reiss-Pistilli, Maria L V; Schuppan, Detlef; Barroso, Madalena M S; Assunção-Miranda, Iranaia; Farias, Shirley; Lery, Letícia; Bauer, Michael; Juliano, Luiz; Juliano, Maria A; Coelho-Sampaio, Tatiana

    2017-02-01

    Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.

  11. Vitamin B12 protects against superoxide-induced cell injury in human aortic endothelial cells

    PubMed Central

    Moreira, Edward S.; Brasch, Nicola E.; Yun, June

    2011-01-01

    Superoxide (O2•−) is implicated in inflammatory states including arteriosclerosis and ischemia-reperfusion injury. Cobalamin (Cbl) supplementation is beneficial for treating many inflammatory diseases and also provides protection in oxidative-stress-associated pathologies. Reduced Cbl reacts with O2•− at rates approaching that of superoxide dismutase (SOD), suggesting a plausible mechanism for its anti-inflammatory properties. Elevated homocysteine (Hcy) is an independent risk factor for vascular disease and endothelial dysfunction. Hcy increases O2•− levels in human aortic endothelial cells (HAEC). Here, we explore protective effects of Cbl in HAEC exposed to various O2•− sources, including increased Hcy levels. Hcy increased O2•− levels (1.6-fold) in HAEC, concomitant with a 20% reduction in cell viability and a 1.5-fold increase in apoptotic death. Pre-treatment of HAEC with physiologically relevant concentrations of cyanocobalamin (CNCbl) (10 – 50 nM) prevented Hcy-induced increases in O2•− and cell death. CNCbl inhibited both Hcy and rotenone- induced mitochondrial O2•− production. Similarly, HAEC challenged with paraquat showed a 1.5-fold increase in O2•− levels and a 30% decrease in cell viability, both of which were prevented with CNCbl (10 nM) pre-treatment. CNCbl also attenuated elevated O2•− levels following exposure of cells to a Cu/Zn-SOD inhibitor. Our data suggest that Cbl acts as an efficient intracellular O2•− scavenger. PMID:21672628

  12. The therapeutic effect of extracellular superoxide dismutase (EC-SOD) mouse embryonic fibroblast (MEF) on collagen-induced arthritis (CIA) mice.

    PubMed

    Yu, Dong Hoon; Kim, Myoung Ok; Kim, Sung Hyun; Shin, Mi Jung; Kim, Bong Soo; Kim, Hei Jung; Lee, Sang Ryeul; Lee, Sang Gyu; Yoo, Seung-Ah; Kim, Wan Uk; Hyun, Byung Hwa; Park, Young Sik; Kim, Tae Yoon; Ryoo, Zae Young

    2008-01-01

    Rheumatoid arthritis is a chronic inflammatory disease. The generation of reactive oxygen species (ROS) within an inflamed joint has been suggested as playing a significant pathogenic role. Extracellular superoxide dismutase (EC-SOD) is a major scavenger enzyme of ROS, which has received growing attention for its therapeutic potential. To investigate the therapeutic effect of EC-SOD in mice with collagen-induced arthritis (CIA), we used mouse embryonic fibroblast (MEF) of transgenic mice that overexpresses EC-SOD on the skin by using hK14 promoter. DBA/1 mice that had been treated with bovine type II collagen were administrated subcutaneous injections of EC-SOD transgenic MEF (each at 1.4 x 10(60 cells) on days 28, 35, and 42 after primary immunization. To test EC-SOD activity, blood samples were collected in each group on day 49. The EC-SOD activity was nearly 1.5-fold higher in the transgenic MEF-treated group than in the nontransgenic MEF-treated group (p < 0.05). The severity of arthritis in mice was scored in a double-blind manner, with each paw being assigned a separate clinical score. The severity of arthritis in EC-SOD transgenic MEF-treated mice was significantly suppressed in the arthritic clinical score (p < 0.05). To investigate the alteration of cytokine levels, ELISA was used to measure blood samples. Levels of IL-1beta and TNF-alpha were reduced in the transgenic MEF-treated group (p < 0.05). Abnormalities of the joints were examined by H&E staining. There were no signs of inflammation except for mild hyperplasia of the synovium in the transgenic MEF-treated group. The proliferation of CII-specific T cells was lower in the transgenic MEF-treated mice than in those in the other groups. The transfer of EC-SOD transgenic MEF has shown a therapeutic effect in CIA mice and this approach may be a safer and more effective form of therapy for rheumatoid arthritis.

  13. Human serum albumin nanoparticles for efficient delivery of Cu, Zn superoxide dismutase gene

    PubMed Central

    Mo, Yun; Barnett, Micheal E.; Takemoto, Dolores; Davidson, Harriet

    2007-01-01

    Purpose To assess the potential of human serum albumin nanoparticles (HSA NP) as a nonviral vector for ocular delivery of Cu, Zn superoxide dismutase (SOD1) gene. Methods Cu, Zn superoxide dismutase (SOD1) gene-encapsulated nanoparticles (NP) were developed using human serum albumin (HSA), an endogenous protein, by a desolvation-crosslinking method. The pSOD-loaded HSA NP was evaluated for in vitro release characteristics, stability against DNase I and vitreous humor degradation, cytotoxicity, cellular uptake mechanisms, in vitro transfection efficiency, and in vivo gene expression. In vitro studies employed cultured human retinal pigment epithelial (ARPE-19) cells and in vivo studies employed a mouse model. For cell uptake analysis, fluorescein isothiocyanate (FITC)-labeled human serum albumin (HSA) was used. Results Plasmid containing SOD1 gene was encapsulated in HSA by a desolvation-crosslinking method. Gene-loaded HSA NP has a mean size of 120 nm, zeta potential of -44 mV, and plasmid encapsulation efficiency of 84%. At high crosslinking degree, HSA NP sustained the in vitro release of plasmid over 6 days, and stabilized plasmid DNA against DNase I and vitreous humor degradation. No cytotoxicity was observed in ARPE 19 cells treated with blank HSA NP at concentrations up to 5 mg/ml for 96 h. Cellular uptake of HSA NP was via receptor-mediated endocytosis that involves primarily caveolae-pathways. Confocal analysis indicated rapid endo/lysosomal escape of HSA NP. Further, confocal studies indicated that HSA readily enters the cell nucleus. In vitro, pSOD-HSA NP resulted in more than 80% transfection efficiency in ARPE-19 cells, which was 5 fold higher than Lipofectamine. HSA NP-transfected cells exhibited enhanced SOD1 activity that was 5 fold higher than untreated cells, indicating the overexpression of the functional gene. Intravitreal injection of HSA NP to the mouse eye at a dose of 130 ng of plasmid produced detectable level of fusion protein expression at

  14. Comparative transcriptomic analysis of human and Drosophila extracellular vesicles

    PubMed Central

    Lefebvre, Fabio Alexis; Benoit Bouvrette, Louis Philip; Perras, Lilyanne; Blanchet-Cohen, Alexis; Garnier, Delphine; Rak, Janusz; Lécuyer, Éric

    2016-01-01

    Extracellular vesicles (EVs) are membrane-enclosed nanoparticles containing specific repertoires of genetic material. In mammals, EVs can mediate the horizontal transfer of various cargos and signaling molecules, notably miRNA and mRNA species. Whether this form of intercellular communication prevails in other metazoans remains unclear. Here, we report the first parallel comparative morphologic and transcriptomic characterization of EVs from Drosophila and human cellular models. Electronic microscopy revealed that human and Drosophila cells release similar EVs with diameters ranging from 30 to 200 nm, which contain complex populations of transcripts. RNA-seq identified abundant ribosomal RNAs, related pseudogenes and retrotransposons in human and Drosophila EVs. Vault RNAs and Y RNAs abounded in human samples, whereas small nucleolar RNAs involved in pseudouridylation were most prevalent in Drosophila EVs. Numerous mRNAs were identified, largely consisting of exonic sequences displaying full-length read coverage and enriched for translation and electronic transport chain functions. By analogy with human systems, these sizeable similarities suggest that EVs could potentially enable RNA-mediated intercellular communication in Drosophila. PMID:27282340

  15. Scavenging of superoxide anions by lecithinized superoxide dismutase in HL-60 cells.

    PubMed

    Ishihara, Tsutomu; Shibui, Misaki; Hoshi, Takaya; Mizushima, Tohru

    2016-01-01

    Superoxide dismutase covalently bound to four lecithin molecules (PC-SOD) has been found to have beneficial therapeutic effects in animal models of various diseases. However, the mechanism underlying these improved therapeutic effects has not yet been elucidated. It has previously been shown that PC-SOD localizes on the plasma membrane and in the lysosomes of cells. In this study, we evaluated the superoxide anion-scavenging activity of PC-SOD in HL-60 human promyelocytic leukemia cells. Compared to SOD, PC-SOD had only 17% scavenging activity in cell-free systems. Nevertheless, by analyzing enzyme activities in cell suspensions containing PC-SOD or SOD, PC-SOD and SOD showed almost equal activity for scavenging extracellular superoxide anions produced by HL-60 cells. Furthermore, the activity for scavenging extracellular superoxide anions increased with increased amount of PC-SOD on the plasma membrane. Moreover, PC-SOD exhibited no obvious inhibitory effect on the scavenging of intracellular superoxide anions. These results suggested that the association of PC-SOD with the plasma membrane plays a key role in its beneficial therapeutic effects. Thus, this finding may provide a rationale for selecting target diseases for PC-SOD treatment.

  16. In vitro preparation of iron-substituted human manganese superoxide dismutase: possible toxic properties for mitochondria.

    PubMed

    Yamakura, Fumiyuki; Kobayashi, Kazuo; Furukawa, Satoshi; Suzuki, Yasunori

    2007-08-01

    We prepared an iron-substituted form of recombinant human manganese superoxide dismutase (MnSOD) by using guanidine hydrochloride for the first time as a model of iron-misincorporated MnSOD, the formation of which has been reported by M. Yang et al. upon disruption of mitochondrial metal homeostasis in yeast (Yang et al. 2006, EMBO J. 25, 1775-1783). The iron-substituted enzyme contained 0.79 g atoms of Fe/mol of subunits and had a specific activity of 80 units/mg protein/g atom of Fe/mol of subunit, which was less than 3% of the activity of the purified MnSOD. Fe-substituted MnSOD (Fe-MnSOD) showed the same absorption spectrum as that of bacterial Fe-MnSODs reported, a similar pH-dependent change of the enzymatic activity, and a similar electron paramagnetic resonance spectrum. Fe-MnSOD showed more thermal stability than native MnSOD. The Fe-substituted enzyme showed a hydrogen-peroxide-mediated radical-generating activity, which was monitored by a cation radical of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) formation similar to that of Cu,ZnSOD, but native human MnSOD and FeSOD showed no radical-generation ability. This evidence suggests that a substitution of Mn to Fe in human MnSOD in mitochondria may produce a disadvantage for oxidative stress in three ways: loss of the enzymatic activity, increase of stability, and gain of radical-generating ability.

  17. Release of gelatinase and superoxide from human mononuclear phagocytes in response to particulate Tamm Horsfall protein.

    PubMed Central

    Thomas, D. B.; Davies, M.; Williams, J. D.

    1993-01-01

    This study describes the in vitro activation of human mononuclear phagocytes by particulate Tamm Horsfall protein (THP). Peripheral blood monocytes phagocytosed THP particles with the accompanying release of superoxide radicals, N-acetyl-beta-D-glucosaminidase, and neutral metalloproteinase. Immunoprecipitation and substrate gel analysis identified the neutral proteinase as a 95-kd gelatinase. A comparison with other particulate ligands highlighted the specificity of the response to THP and showed that the magnitude of the response was comparable with that obtained with lipopolysaccharide (100 micrograms/ml). Parallel studies using peritoneal macrophages resulted in a similar pattern of enzyme release and reactive oxygen species synthesis. THP has been implicated in the pathogenesis of tubulointerstitial nephritis associated with reflux nephropathy. The present study indicates that an inflammatory response initiated by a neutrophil-THP interaction may be extended into a chronic phase via the activation of mononuclear phagocytes. The subsequent release of reactive oxygen metabolites and proteinases may contribute to the tissue damage and fibrosis associated with chronic immune-mediated tubulointerstitial nephritis. Images Figure 4 Figure 5 Figure 6 PMID:8380953

  18. Reduction of Hexavalent Chromium by Human Cytochrome b5: Generation of Hydroxyl Radical and Superoxide

    PubMed Central

    Borthiry, Griselda R.; Antholine, William E.; Kalyanaraman, B.; Myers, Judith M.; Myers, Charles R.

    2007-01-01

    The reduction of hexavalent chromium, Cr(VI), can generate reactive Cr intermediates and various types of oxidative stress. The potential role of human microsomal enzymes in free radical generation was examined using reconstituted proteoliposomes (PLs) containing purified cytochrome b5 and NADPH:P450 reductase. Under aerobic conditions, the PLs reduced Cr(VI) to Cr(V) which was confirmed by ESR using isotopically pure 53Cr(VI). When 5-Diethoxyphos-phoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO•) adduct was observed as well as a smaller signal for the superoxide (O2•−) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 μM). NADPH, Cr(VI), O2 and the PLs were all required for significant HO• generation. Superoxide dismutase eliminated the O2• − adduct and resulted in a 30% increase in the HO• adduct. Catalase largely diminished the HO• adduct signal indicating its dependence on H2O2. Some sources of catalase were found to have Cr(VI)-reducing contaminants which could confound results, but a source of catalase free of these contaminants was used for these studies. Exogenous H2O2 was not needed, indicating that it was generated by the PLs. Adding exogenous H2O2, however, did increase the amount of DEPMPO/HO• adduct. The inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO, and experiments with dimethylsulfoxide (DMSO) plus the spin trap α-phenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO•. Quantification of the various species over time was consistent with a stoichiometric excess of HO• relative to the net amount of Cr(VI) reduced. This also represents the first demonstration of a role for cytochrome b5 in the generation of HO•. Overall, the simultaneous generation of Cr(V) and H2O2 by the PLs and the resulting generation of HO• at low Cr

  19. Mechanism of the Reaction of Human Manganese Superoxide Dismutase with Peroxynitrite: Nitration of Critical Tyrosine 34.

    PubMed

    Demicheli, Verónica; Moreno, Diego M; Jara, Gabriel E; Lima, Analía; Carballal, Sebastián; Ríos, Natalia; Batthyany, Carlos; Ferrer-Sueta, Gerardo; Quijano, Celia; Estrı́n, Darío A; Martí, Marcelo A; Radi, Rafael

    2016-06-21

    Human Mn-containing superoxide dismutase (hMnSOD) is a mitochondrial enzyme that metabolizes superoxide radical (O2(•-)). O2(•-) reacts at diffusional rates with nitric oxide to yield a potent nitrating species, peroxynitrite anion (ONOO(-)). MnSOD is nitrated and inactivated in vivo, with active site Tyr34 as the key oxidatively modified residue. We previously reported a k of ∼1.0 × 10(5) M(-1) s(-1) for the reaction of hMnSOD with ONOO(-) by direct stopped-flow spectroscopy and the critical role of Mn in the nitration process. In this study, we further established the mechanism of the reaction of hMnSOD with ONOO(-), including the necessary re-examination of the second-order rate constant by an independent method and the delineation of the microscopic steps that lead to the regio-specific nitration of Tyr34. The redetermination of k was performed by competition kinetics utilizing coumarin boronic acid, which reacts with ONOO(-) at a rate of ∼1 × 10(6) M(-1) s(-1) to yield the fluorescence product, 7-hydroxycoumarin. Time-resolved fluorescence studies in the presence of increasing concentrations of hMnSOD provided a k of ∼1.0 × 10(5) M(-1) s(-1), fully consistent with the direct method. Proteomic analysis indicated that ONOO(-), but not other nitrating agents, mediates the selective modification of active site Tyr34. Hybrid quantum-classical (quantum mechanics/molecular mechanics) simulations supported a series of steps that involve the initial reaction of ONOO(-) with Mn(III) to yield Mn(IV) and intermediates that ultimately culminate in 3-nitroTyr34. The data reported herein provide a kinetic and mechanistic basis for rationalizing how MnSOD constitutes an intramitochondrial target for ONOO(-) and the microscopic events, with atomic level resolution, that lead to selective and efficient nitration of critical Tyr34.

  20. Sources and Functions of Extracellular Small RNAs in Human Circulation.

    PubMed

    Fritz, Joëlle V; Heintz-Buschart, Anna; Ghosal, Anubrata; Wampach, Linda; Etheridge, Alton; Galas, David; Wilmes, Paul

    2016-07-17

    Various biotypes of endogenous small RNAs (sRNAs) have been detected in human circulation, including microRNAs, transfer RNAs, ribosomal RNA, and yRNA fragments. These extracellular sRNAs (ex-sRNAs) are packaged and secreted by many different cell types. Ex-sRNAs exhibit differences in abundance in several disease states and have, therefore, been proposed for use as effective biomarkers. Furthermore, exosome-borne ex-sRNAs have been reported to elicit physiological responses in acceptor cells. Exogenous ex-sRNAs derived from diet (most prominently from plants) and microorganisms have also been reported in human blood. Essential issues that remain to be conclusively addressed concern the (a) presence and sources of exogenous ex-sRNAs in human bodily fluids, (b) detection and measurement of ex-sRNAs in human circulation, (c) selectivity of ex-sRNA export and import, (d) sensitivity and specificity of ex-sRNA delivery to cellular targets, and (e) cell-, tissue-, organ-, and organism-wide impacts of ex-sRNA-mediated cell-to-cell communication. We survey the present state of knowledge of most of these issues in this review.

  1. Sources and functions of extracellular small RNAs in human circulation

    PubMed Central

    Fritz, Joëlle V.; Heintz-Buschart, Anna; Ghosal, Anubrata; Wampach, Linda; Etheridge, Alton; Galas, David; Wilmes, Paul

    2017-01-01

    Various biotypes of endogenous small RNAs (sRNAs) have been detected in human circulation including microRNAs, tRNA, rRNA and yRNA fragments. These extracellular sRNAs (ex-sRNAs) are packaged and secreted by many different cell types. Ex-sRNAs exhibit differences in abundance in several disease contexts and have therefore been proposed as well-suited biomarkers. Furthermore, exosome-borne ex-sRNAs have been reported to elicit physiological responses in receiving cells. Albeit controversial, exogenous ex-sRNAs derived from plants and microorganisms have also been described in human blood. Essential questions which remain to be conclusively addressed in the field concern the (i) presence and mechanistic sources of exogenous ex-sRNA in human bodily fluids, (ii) detection and measurement of ex-sRNA in human circulation, (iii) selectivity of ex-sRNA export and import, (iv) sensitivity and specificity of ex-sRNA delivery to cellular targets, and (v) cell-, tissue-, organ- and organism-wide impacts of ex-sRNAs. We will survey the present state of knowledge of most of these questions in this review. PMID:27215587

  2. Growth hormone augments superoxide anion secretion of human neutrophils by binding to the prolactin receptor.

    PubMed Central

    Fu, Y K; Arkins, S; Fuh, G; Cunningham, B C; Wells, J A; Fong, S; Cronin, M J; Dantzer, R; Kelley, K W

    1992-01-01

    Recombinant human growth hormone (HuGH) and human prolactin (HuPRL), but not GH of bovine or porcine origin, prime human neutrophils for enhanced superoxide anion (O2-) secretion. Since HuGH, but not GH of other species, effectively binds to the HuPRL receptor (HuPRL-R), we used a group of HuGH variants created by site-directed mutagenesis to identify the receptor on human neutrophils responsible for HuGH priming. A monoclonal antibody (MAb) directed against the HuPRL-R completely abrogated O2- secretion by neutrophils incubated with either HuGH or HuPRL, whereas a MAb to the HuGH-R had no effect. The HuGH variant K172A/F176A, which has reduced affinity for both the HuGH-binding protein (BP) and the HuPRL-BP, was unable to prime human neutrophils. This indicates that priming is initiated by a ligand-receptor interaction, the affinity of which is near that defined for receptors for PRL and GH. Another HuGH variant, K168A/E174A, which has relatively low affinity for the HuPRL-BP but slightly increased affinity for the HuGH-BP, had much reduced ability to prime neutrophils. In contrast, HuGH variant E56D/R64M, which has a similar affinity as wild-type HuGH for the HuPRL-BP but a lower affinity for the HuGH-BP, primed neutrophils as effectively as the wild-type HuGH. Finally, binding of HuGH to the HuPRL-BP but not to the HuGH-BP has been shown to be zinc dependent, and priming of neutrophils by HuGH was also responsive to zinc. Collectively, these data directly couple the binding of HuGH to the HuPRL-R with one aspect of functional activation of human target cells. Images PMID:1310696

  3. Cationic liposomes evoke proinflammatory mediator release and neutrophil extracellular traps (NETs) toward human neutrophils.

    PubMed

    Hwang, Tsong-Long; Hsu, Ching-Yun; Aljuffali, Ibrahim A; Chen, Chun-Han; Chang, Yuan-Ting; Fang, Jia-You

    2015-04-01

    Cationic liposomes are widely used as nanocarriers for therapeutic and diagnostic purposes. The cationic components of liposomes can induce inflammatory responses. This study examined the effect of cationic liposomes on human neutrophil activation. Cetyltrimethylammonium bromide (CTAB) or soyaethyl morpholinium ethosulfate (SME) was incorporated into liposomes as the cationic additive. The liposomes' cytotoxicity and their induction of proinflammatory mediators, intracellular calcium, and neutrophil extracellular traps (NETs) were investigated. The interaction of the liposomes with the plasma membrane triggered the stimulation of neutrophils. CTAB liposomes induced complete leakage of lactate dehydrogenase (LDH) at all concentrations tested, whereas SME liposomes released LDH in a concentration-dependent manner. CTAB liposomes proved to more effectively activate neutrophils compared with SME liposomes, as indicated by increased superoxide anion and elastase levels. Calcium influx increased 9-fold after treatment with CTAB liposomes. This influx was not changed by SME liposomes compared with the untreated control. Scanning electron microscopy (SEM) and immunofluorescence images indicated the presence of NETs after treatment with cationic liposomes. NETs could be quickly formed, within minutes, after CTAB liposomal treatment. In contrast to this result, NET formation was slowly and gradually increased by SME liposomes, within 4h. Based on the data presented here, it is important to consider the toxicity of cationic liposomes during administration in the body. This is the first report providing evidence of NET production induced by cationic liposomes. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Epigenetic regulation of human buccal mucosa mitochondrial superoxide dismutase gene expression by diet.

    PubMed

    Thaler, Roman; Karlic, Heidrun; Rust, Petra; Haslberger, Alexander G

    2009-03-01

    The impact of nutrition on the epigenetic machinery has increasingly attracted interest. The aim of the present study was to demonstrate the effects of various diets on methylation and gene expression. The antioxidative enzyme mitochondrial superoxide dismutase (MnSOD) was chosen as the model system because epigenetic regulation has been previously shown in cell lines for this gene. Promoter methylation and gene expression of MnSOD in buccal swabs from three sample groups were analysed. The three groups included: (1) forty vegetarians (aged 20-30 years); (2) age-matched omnivores; (3) elderly omnivores (aged>85 years). A 3-fold increase in the expression of the MnSOD gene was associated with decreased CpG methylation of the analysed promoter region in the vegetarian group compared with the age-matched omnivores group. Expression and promoter methylation of the MnSOD gene in elderly omnivores showed no significant differences compared with younger omnivores. In accordance with previous findings in various tissues, DNA global methylation was found to be significantly higher (30 %) in buccal swabs of younger subjects (independent of the diet), than in those of elderly omnivores. In the control experiment which was designed to verify the findings of the human buccal swab studies, the Caco-2 cell line was treated with zebularine. Results of the control study showed a 6-fold increase of MnSOD expression, an approximately 40 % decreased methylation of specified CpG in the MnSOD promoter and a 50 % reduction of global DNA methylation. These results indicate that diet affects the epigenetic regulation of human MnSOD.

  5. Human keratinocytes synthesize and secrete the extracellular matrix protein, thrombospondin.

    PubMed

    Wikner, N E; Dixit, V M; Frazier, W A; Clark, R A

    1987-02-01

    Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

  6. Apoptosis induced by selenomethionine and methioninase is superoxide-mediated and p53-dependent in human prostate cancer cells

    PubMed Central

    Zhao, Rui; Domann, Frederick E.

    2006-01-01

    Selenomethionine (SeMet) is the chemical form or major component of selenium used for cancer chemoprevention in several clinical trials. However, evidence from experimental studies indicates that SeMet has weaker anticancer effects than most other forms of selenium. Recent studies showed that the anticancer activity of SeMet can be enhanced by methioninase (METase), indicating that SeMet metabolites are responsible for its anticancer activity. In the present study, we demonstrated that wild-type p53-expressing LNCaP human prostate cancer cells were more sensitive to co-treatment with SeMet and METase than p53-null PC3 human prostate cancer cells. SeMet and METase co-treatment significantly increased levels of superoxide and apoptosis in LNCaP cells. Co-treatment with SeMet and METase resulted in increased levels of phosphorylated p53 (serine15), total p53, Bax, and p21Waf1 proteins. LNCaP cells treated with SeMet and METase also showed p53 translocation to mitochondria, decreased mitochondrial membrane potential, cytochrome c release into the cytosol, and activation of caspase 9. The effects of SeMet and METase were suppressed by pre-treatment with a synthetic superoxide dismutase mimic or by knockdown of p53 via RNA interference. Reexpression of wild-type p53 in PC3 cells resulted in increases in superoxide production, apoptosis, and caspase 9 activity, and a decrease in mitochondrial membrane potential following co-treatment with SeMet and METase. Our study demonstrates that apoptosis induced by SeMet plus METase is superoxide-mediated and p53-dependent via mitochondrial pathway(s). These results suggest that superoxide and p53 may play a role in cancer chemoprevention by selenium. PMID:17172431

  7. Modulation of Human Cardiac TRPM7 Current by Extracellular Acidic pH Depends upon Extracellular Concentrations of Divalent Cations

    PubMed Central

    Mačianskienė, Regina; Almanaitytė, Mantė; Jekabsone, Aistė; Mubagwa, Kanigula

    2017-01-01

    TRPM7 channels participate in a variety of physiological/pathological processes. TRPM7 currents are modulated by protons but opposing effects of external pH (pHo) (potentiation vs inhibition) have been reported. TRPM7 has been less studied in human cardiomyocytes than in heart-derived non-cardiomyocyte cells. We used the whole-cell patch-clamp technique on isolated human atrial cardiomyocytes to investigate the impact of an acidic pHo on the TRPM7 current. With voltage-dependent and other ion channels inhibited, cardiomyocytes were challenged with external acidification in either the presence or the absence of extracellular divalent cations. TRPM7 outward and inward currents were increased by acidic pHo in extracellular medium containing Ca2+ and Mg2+, but suppressed by acidic pHo in the absence of extracellular Ca2+ and Mg2+. The potentiating effect in the presence of extracellular divalents occurred at pHo below 6 and was voltage-dependent. The inhibitory effect in the absence of extracellular divalents was already marked at pHo of 6 and was practically voltage-independent. TRPM7 current density was higher in cardiomyocytes from patients with history of coronary vascular disease and the difference compared to cardiomyocytes from patients without history of myocardial ischemia increased with acidic pHo. We demonstrate that proton-induced modification of TRPM7 currents depends on the presence of extracellular Ca2+ and Mg2+. Variability of the TRPM7 current density in human cardiomyocytes is related to the clinical history, being higher in atrial fibrillation and in ischemic cardiomyopathy. PMID:28129376

  8. Superoxide anion production by human spermatozoa as a part of the ionophore-induced acrosome reaction process.

    PubMed

    Griveau, J F; Renard, P; Le Lannou, D

    1995-04-01

    The involvement of superoxide anion (O2o-) in human sperm capacitation and/or acrosome reaction was investigated. Addition of superoxide dismutase (SOD) to the medium at the beginning of the capacitation process or 15 min before induction of the acrosome reaction, decreased the level of ionophore-induced acrosome reaction. Hyperactivation was unaffected by the presence of SOD during the capacitation process. Addition of calcium ionophore to the sperm suspension increased production of O2o- by the spermatozoa by four to five-fold and induced the acrosome reaction. In the presence of SOD, superoxide anion could not be detected in the medium and the rate of induced-acrosome reaction was decreased greatly. The presence of an inhibitor of protein kinase C inhibited the production of O2o- in the medium and reduced the induced-acrosome reaction. The production of O2o- and the acrosome reaction were also increased by exposure of spermatozoa to 12-myristate 13-acetate phorbol ester, a specific activator of protein kinase C. While the level of spontaneous acrosome reaction was not increased by the direct addition of O2o- to the medium, its presence induced the release of unesterified fatty acids from membrane phospholipids. These findings suggest that the production of O2o- by spermatozoa could be involved in the ionophore-induced acrosome reaction, possibly through the de-esterification of membrane phospholipids. However, this production of superoxide anion is not sufficient on its own to induce the acrosome reaction.

  9. Dietary fish oil decreases superoxide generation by human neutrophils: relation to cyclooxygenase pathway and lysosomal enzyme release.

    PubMed

    Luostarinen, R; Saldeen, T

    1996-09-01

    12 volunteers with slightly elevated serum triglyceride levels were given 30 ml fish oil (5.4 g eicosapentaenoic acid and 3.2 g docosahexaenoic acid) daily for 4 weeks. The percentage of eicosapentaenoic acid increased (P < 0.01) and the percentage of linoleic (P < 0.05) and arachidonic acid (P < 0.01) decreased in neutrophil phospholipids. Superoxide generation by neutrophils initiated by phorbol myristate acetate decreased significantly from 48.6 +/- 8.8 to 34.7 +/- 11.1 nmol/10 min/400,000 cells (means +/- SD, P < 0.01, n = 11). Treatment of the cells with the cyclooxygenase inhibitor indomethacin had no significant influence on the decrease in superoxide generation, indicating that cyclooxygenase products were not involved in this effect of fish oil. Neutrophil elastase release did not change significantly, suggesting that neutrophil lysosomal enzyme release and superoxide generation may be under separate control. In conclusion, dietary fish oil decreased superoxide generation by human neutrophils without involvement of the cyclooxygenase pathway and without altering neutrophil lysosomal enzyme release. Dietary fish oil could have beneficial effects in pathological conditions with activated neutrophils, such as ischaemic heart disease.

  10. β-eudesmol, a sesquiterpene from Teucrium ramosissimum, inhibits superoxide production, proliferation, adhesion and migration of human tumor cell.

    PubMed

    Ben Sghaier, Mohamed; Mousslim, Mohamed; Pagano, Alessandra; Ammari, Youssef; Luis, José; Kovacic, Hervé

    2016-09-01

    Reactive oxygen species are well-known mediators of various biological responses. Recently, new homologues of the catalytic subunit of NADPH oxidase have been discovered in non phagocytic cells. These new homologues (Nox1-Nox5) produce low levels of superoxides compared to the phagocytic homologue Nox2/gp91phox. In this study we examined the effect of β-eudesmol, a sesquiterpenoid alcohol isolated from Teucrium ramosissimum leaves, on proliferation, superoxide anion production, adhesion and migration of human lung (A549) and colon (HT29 and Caco-2) cancer cell lines. Proliferation of tumor cells was inhibited by β-eudesmol. It also significantly inhibited superoxide production in A549 cells. Furthermore, β-eudesmol inhibited adhesion and migration of A549 and HT29 cell. These results demonstrate that β-eudesmol may be a novel anticancer agent for the treatment of lung and colon cancer by different ways: by inhibition of superoxide production or by blocking proliferation, adhesion and migration.

  11. Superoxide produced in the matrix of mitochondria enhances methylmercury toxicity in human neuroblastoma cells.

    PubMed

    Mailloux, Ryan J; Yumvihoze, Emmanuel; Chan, Hing Man

    2015-12-15

    The mechanism of intracellular metabolism of methylmercury (MeHg) is not fully known. It has been shown that superoxide (O2(-)), the proximal reactive oxygen species (ROS) generated by mitochondria, is responsible for MeHg demethylation. Here, we investigated the impact of different mitochondrial respiratory inhibitors, namely rotenone and antimycin A, on the O2(-)mediated degradation of MeHg in human neuroblastoma cells SH-K-SN. We also utilized paraquat (PQ) which generates O2(-) in the mitochondrial matrix. We found that the cleavage of the carbon-metal bond in MeHg was highly dependent on the topology of O2(-) production by mitochondria. Both rotenone and PQ, which increase O2(-) in the mitochondrial matrix at a dose-dependent manner, enhanced the conversion of MeHg to inorganic mercury (iHg). Surprisingly, antimycin A, which prompts emission of O2(-) into the intermembrane space, did not have the same effect even though antimycin A induced a dose dependent increase in O2(-) emission. Rotenone and PQ also enhanced the toxicity of sub-toxic doses (0.1 μM) MeHg which correlated with the accumulation of iHg in mitochondria and depletion of mitochondrial protein thiols. Taken together, our results demonstrate that MeHg degradation is mediated by mitochondrial O2(-), specifically within the matrix of mitochondria when O2(-) is in adequate supply. Our results also show that O2(-) amplifies MeHg toxicity specifically through its conversion to iHg and subsequent interaction with protein cysteine thiols (R-SH). The implications of our findings in mercury neurotoxicity are discussed herein.

  12. Enthalpic Barriers Dominate the Folding and Unfolding of the Human Cu,Zn Superoxide Dismutase Monomer

    PubMed Central

    Kayatekin, Can; Cohen, Noah R.; Matthews, C. Robert

    2013-01-01

    The rate-limiting step in the formation of the native dimeric state of human Cu, Zn superoxide dismutase (SOD1) is a very slow monomer folding reaction that governs the lifetime of its unfolded state. Mutations at dozens of sites in SOD1 are known to cause a fatal motor neuron disease, amyotrophic lateral sclerosis, and recent experiments implicate the unfolded state as a source of soluble oligomers and histologically observable aggregates thought to be responsible for toxicity. To determine the thermodynamic properties of the transition state ensemble (TSE) limiting the folding of this high contact order β-sandwich motif, a combined thermal/urea denaturation thermodynamic/kinetic analysis was performed. The barriers to folding and unfolding are dominated by the activation enthalpy at 298 K and neutral pH; the activation entropy is favorable and reduces the barrier height for both reactions. The absence of secondary structure formation or large-scale chain collapse prior to crossing the barrier for folding led to the conclusion that dehydration of nonpolar surfaces in the TSE is responsible for the large and positive activation enthalpy. Although the activation entropy favors the folding reaction, the transition from the unfolded state to the native state is entropically disfavored at 298 K. The opposing entropic contributions to the free energies of the TSE and the native state during folding provide insights into structural properties of the TSE. The results also imply a crucial role for water in governing the productive folding reaction and enhancing the propensity for the aggregation of SOD1. PMID:22999954

  13. Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis.

    PubMed

    Mejía, Susana P; Cano, Luz E; López, Juan A; Hernandez, Orville; González, Ángel

    2015-05-01

    Neutrophils play an important role as effector cells and contribute to the resistance of the host against microbial pathogens. Neutrophils are able to produce extracellular traps (NETs) in response to medically important fungi, including Aspergillus spp., Candida albicans and Cryptococcus gattii. However, NET production in response to Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) did not alter NET production against conidia, it partially suppressed NET formation against P. brasiliensis yeasts. Cytochalasin D or IFN-γ did not affect the production of NETs against the fungus. Additionally, a mutant strain of P. brasiliensis with reduced expression of an alternative oxidase induced significantly higher levels of NETs in comparison with the WT strain. Finally, c.f.u. quantification of P. brasiliensis showed no significant differences when neutrophils were treated with DPI, DNase I or cytochalasin D as compared with untreated cells. These data establish that NET formation by human neutrophils appears to be either dependent or independent of reactive oxygen species production, correlating with the fungal morphotype used for stimulation. However, this mechanism was ineffective in killing the fungus.

  14. Muscadine grape skin extract reverts snail-mediated epithelial mesenchymal transition via superoxide species in human prostate cancer cells

    PubMed Central

    2014-01-01

    Background Snail transcription factor can induce epithelial-mesenchymal transition (EMT), associated with decreased cell adhesion-associated molecules like E-cadherin, increased mesenchymal markers like vimentin, leading to increased motility, invasion and metastasis. Muscadine grape skin extract (MSKE) has been shown to inhibit prostate cancer cell growth and induce apoptosis without affecting normal prostate epithelial cells. We investigated novel molecular mechanisms by which Snail promotes EMT in prostate cancer cells via Reactive Oxygen Species (ROS) and whether it can be antagonized by MSKE. Methods ARCaP and LNCaP cells overexpressing Snail were utilized to examine levels of reactive oxygen species (ROS), specifically, superoxide, in vitro using Dihydroethidium (DHE) or HydroCy3 dyes. Mitosox staining was performed to determine whether the source of ROS was mitochondrial in origin. We also investigated the effect of Muscadine grape skin extract (MSKE) on EMT marker expression by western blot analysis. Migration and cell viability using MTS proliferation assay was performed following MSKE treatments. Results Snail overexpression in ARCaP and LNCaP cells was associated with increased concentration of mitochondrial superoxide, in vitro. Interestingly, MSKE decreased superoxide levels in ARCaP and LNCaP cells. Additionally, MSKE and Superoxide Dismutase (SOD) reverted EMT as evidenced by decreased vimentin levels and re-induction of E-cadherin expression in ARCaP-Snail cells after 3 days, concomitant with reduced cell migration. MSKE also decreased Stat-3 activity in ARCaP-Snail cells. Conclusions This study shows that superoxide species may play a role in Snail transcription factor-mediated EMT. Therefore, therapeutic targeting of Snail with various antioxidants such as MSKE may prove beneficial in abrogating EMT and ROS-mediated tumor progression in human prostate cancer. PMID:24617993

  15. Muscadine grape skin extract reverts snail-mediated epithelial mesenchymal transition via superoxide species in human prostate cancer cells.

    PubMed

    Burton, Liza J; Barnett, Petrina; Smith, Basil; Arnold, Rebecca S; Hudson, Tamaro; Kundu, Kousik; Murthy, Niren; Odero-Marah, Valerie A

    2014-03-12

    Snail transcription factor can induce epithelial-mesenchymal transition (EMT), associated with decreased cell adhesion-associated molecules like E-cadherin, increased mesenchymal markers like vimentin, leading to increased motility, invasion and metastasis. Muscadine grape skin extract (MSKE) has been shown to inhibit prostate cancer cell growth and induce apoptosis without affecting normal prostate epithelial cells. We investigated novel molecular mechanisms by which Snail promotes EMT in prostate cancer cells via Reactive Oxygen Species (ROS) and whether it can be antagonized by MSKE. ARCaP and LNCaP cells overexpressing Snail were utilized to examine levels of reactive oxygen species (ROS), specifically, superoxide, in vitro using Dihydroethidium (DHE) or HydroCy3 dyes. Mitosox staining was performed to determine whether the source of ROS was mitochondrial in origin. We also investigated the effect of Muscadine grape skin extract (MSKE) on EMT marker expression by western blot analysis. Migration and cell viability using MTS proliferation assay was performed following MSKE treatments. Snail overexpression in ARCaP and LNCaP cells was associated with increased concentration of mitochondrial superoxide, in vitro. Interestingly, MSKE decreased superoxide levels in ARCaP and LNCaP cells. Additionally, MSKE and Superoxide Dismutase (SOD) reverted EMT as evidenced by decreased vimentin levels and re-induction of E-cadherin expression in ARCaP-Snail cells after 3 days, concomitant with reduced cell migration. MSKE also decreased Stat-3 activity in ARCaP-Snail cells. This study shows that superoxide species may play a role in Snail transcription factor-mediated EMT. Therefore, therapeutic targeting of Snail with various antioxidants such as MSKE may prove beneficial in abrogating EMT and ROS-mediated tumor progression in human prostate cancer.

  16. Cupric Ions Induce the Oxidation and Trigger the Aggregation of Human Superoxide Dismutase 1

    PubMed Central

    Li, Cheng; Xu, Wen-Chang; Xie, Zhen-Sheng; Pan, Kai; Hu, Jiao; Chen, Jie; Pang, Dai-Wen; Yang, Fu-Quan; Liang, Yi

    2013-01-01

    Background Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1. Methodology/Principal Findings As revealed by 90° light scattering, dynamic light scattering, SDS-PAGE, and atomic force microscopy, free cupric ions in solution not only induce the oxidation of either apo A4V or Zn2-A4V and trigger the oligomerization and aggregation of oxidized A4V under copper-mediated oxidative conditions, but also trigger the aggregation of non-oxidized form of such a pathogenic mutant. As evidenced by mass spectrometry and SDS-PAGE, Cys-111 is a primary target for oxidative modification of pathological human SOD1 mutant A4V by either excess Cu2+ or hydrogen peroxide. The results from isothermal titration calorimetry show that A4V possesses two sets of independent binding sites for Cu2+: a moderate-affinity site (106 M-1) and a high-affinity site (108 M-1). Furthermore, Cu2+ binds to wild-type SOD1 oxidized by hydrogen peroxide in a way similar to A4V, triggering the aggregation of such an oxidized form. Conclusions/Significance We demonstrate that excess cupric ions induce the oxidation and trigger the aggregation of A4V SOD1, and suggest that Cu2+ plays a key role in the mechanism of aggregation of both A4V and oxidized wild-type SOD1. A plausible model for how pathological SOD1 mutants aggregate in ALS-affected motor neurons with the disruption of copper homeostasis has been provided. PMID:23755211

  17. Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis.

    PubMed

    Perry, J Jefferson P; Hearn, Amy S; Cabelli, Diane E; Nick, Harry S; Tainer, John A; Silverman, David N

    2009-04-21

    Superoxide dismutase (SOD) enzymes are critical in controlling levels of reactive oxygen species (ROS) that are linked to aging, cancer, and neurodegenerative disease. Superoxide (O(2)(*-)) produced during respiration is removed by the product of the SOD2 gene, the homotetrameric manganese superoxide dismutase (MnSOD). Here, we examine the structural and catalytic roles of the highly conserved active-site residue Tyr34, based upon structure-function studies of MnSOD enzymes with mutations at this site. Substitution of Tyr34 with five different amino acids retained the active-site protein structure and assembly but caused a substantial decrease in the catalytic rate constant for the reduction of superoxide. The rate constant for formation of the product inhibition complex also decreases but to a much lesser extent, resulting in a net increase in the level of product inhibited form of the mutant enzymes. Comparisons of crystal structures and catalytic rates also suggest that one mutation, Y34V, interrupts the hydrogen-bonded network, which is associated with a rapid dissociation of the product-inhibited complex. Notably, with three of the Tyr34 mutants, we also observe an intermediate in catalysis, which has not been reported previously. Thus, these mutants establish a means of trapping a catalytic intermediate that promises to help elucidate the mechanism of catalysis.

  18. Involvement of superoxide generated by NADPH oxidase in the shedding of procoagulant vesicles from human monocytic cells exposed to bupivacaine.

    PubMed

    Azma, Toshiharu; Ogawa, Saori; Nishioka, Akira; Kinoshita, Hiroyuki; Kawahito, Shinji; Nagasaka, Hiroshi; Matsumoto, Nobuyuki

    2017-08-17

    It is known that a variety of sized procoagulant vesicles that express tissue factor are released from several types of cells including monocytes by mechanisms related to the induction of apoptosis, while it has not yet been evaluated whether superoxide is involved in the production of such vesicles. Here, we report that a local anesthetic bupivacaine induces apoptosis in human monocytic cells THP-1 within a short observation period, where the shedding of procoagulant vesicles is associated. The property as procoagulant vesicles was evaluated using flow cytometry by the binding of FITC-conjugated fibrinogen to vesicles in the presence of fresh frozen plasma and the suppression of this binding by heparin. Bupivacaine (1 mg/ml) increased the apoptotic cells and procoagulant vesicles. LY294002 (100 µM), that inhibits the recruiting of intracellular component of NADPH oxidase to construct the activated form of this enzyme complex, or superoxide dismutase (1500 unit/ml) suppressed bupivacaine-provoked induction of apoptosis and the increase of procoagulant vesicles. We suggest that this simple experimental system is useful to explore the molecular mechanisms of action of superoxide in the shedding of procoagulant vesicles from human monocytic cells.

  19. Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps.

    PubMed

    Ávila, Eva E; Salaiza, Norma; Pulido, Julieta; Rodríguez, Mayra C; Díaz-Godínez, César; Laclette, Juan P; Becker, Ingeborg; Carrero, Julio C

    2016-01-01

    Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica.

  20. Entamoeba histolytica Trophozoites and Lipopeptidophosphoglycan Trigger Human Neutrophil Extracellular Traps

    PubMed Central

    Ávila, Eva E.; Rodríguez, Mayra C.; Díaz-Godínez, César; Laclette, Juan P.; Becker, Ingeborg; Carrero, Julio C.

    2016-01-01

    Neutrophil defense mechanisms include phagocytosis, degranulation and the formation of extracellular traps (NET). These networks of DNA are triggered by several immune and microbial factors, representing a defense strategy to prevent microbial spread by trapping/killing pathogens. This may be important against Entamoeba histolytica, since its large size hinders its phagocytosis. The aim of this study was to determine whether E. histolytica and their lipopeptidophosphoglycan (EhLPPG) induce the formation of NETs and the outcome of their interaction with the parasite. Our data show that live amoebae and EhLPPG, but not fixed trophozoites, induced NET formation in a time and dose dependent manner, starting at 5 min of co-incubation. Although immunofluorescence studies showed that the NETs contain cathelicidin LL-37 in close proximity to amoebae, the trophozoite growth was only affected when ethylene glycol tetra-acetic acid (EGTA) was present during contact with NETs, suggesting that the activity of enzymes requiring calcium, such as DNases, may be important for amoeba survival. In conclusion, E. histolytica trophozoites and EhLPPG induce in vitro formation of human NETs, which did not affect the parasite growth unless a chelating agent was present. These results suggest that NETs may be an important factor of the innate immune response during infection with E. histolytica. PMID:27415627

  1. TRK-530 inhibits accumulation of superoxide anions derived from human polymorphonuclear leukocytes and bone resorption induced by activated osteoclasts.

    PubMed

    Tanahashi, M; Funaba, Y; Tateishi, A; Kawabe, N; Nakadate-Matsushita, T

    1998-03-01

    TRK-530, a newly synthesized bisphosphonate, was assessed for its effects on the accumulation of superoxide anions derived from human formyl-methionyl-leucyl-phenylalanine-stimulated polymorphonuclear leukocytes (PMN), and for its effects on bone resorption using a pit formation assay. TRK-530 concentration-dependently inhibited superoxide accumulation derived from PMN and osteoclast pit formation stimulated by 1,25-dihydroxyvitamin D3. Incadronate and risedronate had a strong inhibitory effect on pit formation, but no antioxidative activity. These data suggest that the anti-bone resorption activities of TRK-530 are possibly unrelated to its antioxidant properties. However, it is difficult to conclude at present which mechanisms play the most important role in the anti-bone resorption activities of TRK-530.

  2. Superoxide dismutase activity of the naturally occurring human serum albumin-copper complex without hydroxyl radical formation.

    PubMed

    Kato, Ryunosuke; Akiyama, Matofusa; Kawakami, Hiroyoshi; Komatsu, Teruyuki

    2014-01-01

    The superoxide radical anion (O2(.-)) is biologically toxic and contributes to the pathogenesis of various diseases. Here we describe the superoxide dismutase (SOD) activity of human serum albumin (HSA) complexed with a single Cu(II) ion at the N-terminal end (HSA-Cu complex). The structure of this naturally occurring copper-coordinated blood serum protein has been characterized by several physicochemical measurements. The O2(.-) dismutation ability of the HSA-Cu (1:1) complex is almost the same as that of the well-known SOD mimics, such as Mn(III) -tetrakis(N-methylpyridinium)porphyrin. Interestingly, the HSA-Cu complex does not induce a subsequent Fenton reaction to produce the hydroxyl radical (OH(.)), which is one of the most harmful reactive oxygen species.

  3. Superoxide-dependent uptake of vitamin C in human glioma cells.

    PubMed

    Rodríguez, Federico S; Salazar, Katterine A; Jara, Nery A; García-Robles, María A; Pérez, Fernando; Ferrada, Luciano E; Martínez, Fernando; Nualart, Francisco J

    2013-12-01

    Glioblastomas are lethal brain tumors that resist current cytostatic therapies. Vitamin C may antagonize the effects of reactive oxygen species (ROS) generating therapies; however, it is often used to reduce therapy-related side effects despite its effects on therapy or tumor growth. Because the mechanisms of vitamin C uptake in gliomas are currently unknown, we evaluated the expression of the sodium-vitamin C cotransporter (SVCT) and facilitative hexose transporter (GLUT) families in human glioma cells. In addition, as microglial cells can greatly infiltrate high-grade gliomas (constituting up to 45% of cells in glioblastomas), the effect of TC620 glioma cell interactions with microglial-like HL60 cells on vitamin C uptake (Bystander effect) was determined. Although glioma cells expressed high levels of the SVCT isoform-2 (SVCT2), low functional activity, intracellular localization and the expression of the dominant-negative isoform (dnSVCT2) were observed. The increased glucose metabolic activity of glioma cells was evident by the high 2-Deoxy-d-glucose and dehydroascorbic acid (DHA) uptake rates through the GLUT isoform-1 (GLUT1), the main DHA transporter in glioblastoma. Co-culture of glioma cells and activated microglial-like HL60 cells resulted in extracellular ascorbic acid oxidation and high DHA uptake by glioma cells. This Bystander effect may explain the high antioxidative potential observed in high-grade gliomas. This study strongly suggests that the Bystander effect, that is, glioma cell interaction with oxidant-producing microglia, could be an important mechanism for glioma vitamin C loading in the absence of functional sodium-vitamin C cotransporter 2 (SVCT2) expression. The high cellular vitamin C load in glioma cells results from a high uptake of extracellular dehydroascorbic acid (DHA) generated by neighboring microglia. This Bystander effect may explain the high antioxidative potential observed in high-grade gliomas, considering that high

  4. Early signaling by vascular endothelial growth factor and placental growth factor in human bone marrow-derived endothelial cells is mediated by superoxide.

    PubMed

    Dhar-Mascareno, Manya; Mascareno, Eduardo; Golde, David W

    2009-01-01

    We investigated the role of superoxide O(2)(-) during the initiation of vascular endothelial growth factor (VEGF)- and placental growth factor (PlGF)-mediated signal transduction in bone marrow-derived endothelial cells. BMhTERT cells were treated with VEGF or PlGF in the presence or absence of antioxidants. The signaling pathways downstream were analyzed by immunoprecipitation and Western blotting. Superoxide and reactive oxygen species (ROS) were measured using Superluminol or 2',7'-dichlorofluorescein fluorescence measurements. We show here that VEGF and PlGF generate extracellular and intracellular O(2)(-) that regulates their downstream signaling transduction pathways. Indeed, the extracellular O(2)(-) generated treatment of endothelial cells (using hypoxanthine/xanthine oxidase) was sufficient to initiate receptor phosphorylation of VEGF receptor 2. The PlGF treatment of endothelial cells increased the generation of intracellular ROS in an extracellular O(2)(-) dependent manner. Quenching of intracellular ROS by resveratrol inhibits PlGF- and VEGF-dependent induction of MAP kinase phosphorylation. Additionally, we found that the interaction of VEGF and PlGF with their specific receptors generates O(2)(-) in a cell-free system. Endothelial cells treated with VEGF stop proliferation in the presence of extracellular catalase, superoxide dismutase or peroxiredoxin IV. Our studies underscore the role of O(2)(-) as a critical regulator of VEGF and PlGF signal transduction initiation in endothelial cells. Copyright 2009 S. Karger AG, Basel.

  5. High Throughput Sequencing of Extracellular RNA from Human Plasma

    PubMed Central

    Danielson, Kirsty M.; Rubio, Renee; Abderazzaq, Fieda; Das, Saumya; Wang, Yaoyu E.

    2017-01-01

    The presence and relative stability of extracellular RNAs (exRNAs) in biofluids has led to an emerging recognition of their promise as ‘liquid biopsies’ for diseases. Most prior studies on discovery of exRNAs as disease-specific biomarkers have focused on microRNAs (miRNAs) using technologies such as qRT-PCR and microarrays. The recent application of next-generation sequencing to discovery of exRNA biomarkers has revealed the presence of potential novel miRNAs as well as other RNA species such as tRNAs, snoRNAs, piRNAs and lncRNAs in biofluids. At the same time, the use of RNA sequencing for biofluids poses unique challenges, including low amounts of input RNAs, the presence of exRNAs in different compartments with varying degrees of vulnerability to isolation techniques, and the high abundance of specific RNA species (thereby limiting the sensitivity of detection of less abundant species). Moreover, discovery in human diseases often relies on archival biospecimens of varying age and limiting amounts of samples. In this study, we have tested RNA isolation methods to optimize profiling exRNAs by RNA sequencing in individuals without any known diseases. Our findings are consistent with other recent studies that detect microRNAs and ribosomal RNAs as the major exRNA species in plasma. Similar to other recent studies, we found that the landscape of biofluid microRNA transcriptome is dominated by several abundant microRNAs that appear to comprise conserved extracellular miRNAs. There is reasonable correlation of sets of conserved miRNAs across biological replicates, and even across other data sets obtained at different investigative sites. Conversely, the detection of less abundant miRNAs is far more dependent on the exact methodology of RNA isolation and profiling. This study highlights the challenges in detecting and quantifying less abundant plasma miRNAs in health and disease using RNA sequencing platforms. PMID:28060806

  6. Extracellular vesicles in human follicular fluid do not promote coagulation.

    PubMed

    Franz, Cordula; Böing, Anita N; Montag, Markus; Strowitzki, Thomas; Markert, Udo R; Mastenbroek, Sebastiaan; Nieuwland, Rienk; Toth, Bettina

    2016-11-01

    Body fluids contain extracellular vesicles expressing tissue factor on their surface and serve as an additional trigger for coagulation. During the menstrual cycle ovarian tissue restoration is mandatory and it is unknown whether follicular fluid might provide procoagulant substances. Within an observational study, follicular fluid from women undergoing IVF/intracytoplasmic sperm injection (ICSI) was analysed by fluorescence-activated cell sorting (FACS), electron microscopy, resistive pulse sensing (RPS), nanoparticle-tracking analysis (NTA) and fibrin generation tests (FGT). The presence of extracellular vesicles, especially CD9-positive extracellular vesicles in follicular fluid, was proven. However, clotting tests revealed no procoagulant properties of the detected extracellular vesicles. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  7. Examination of the role of arginine-143 in the human copper and zinc superoxide dismutase by site-specific mutagenesis.

    PubMed

    Beyer, W F; Fridovich, I; Mullenbach, G T; Hallewell, R

    1987-08-15

    The active site arginine-143 of human Cu,Zn superoxide dismutase has been replaced by lysine or by isoleucine. The mutant proteins were expressed at high levels in yeast, purified, and the amino acid substitution explored through the use of group specific reagents. The specific activities of these enzymes, measured by the xanthine oxidase/cytochrome c method and by using dry weight determination to establish protein concentration, were: native enzyme, 6570 units/mg; Lys-substituted enzyme, 2840 units/mg, Ile-substituted enzyme, 708 units/mg. The active site arginine thus plays an important, but not an essential, role in the catalytic process.

  8. Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase.

    PubMed

    Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph; Marquardt, Iris; Donsante, Anthony; Yi, Ling; Hicks, Julia D; Steinbach, Peter J; Wilson, Callum; Elpeleg, Orly; Møller, Lisbeth Birk; Christodoulou, John; Kaler, Stephen G; Gärtner, Jutta

    2012-08-01

    Copper (Cu) is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular Cu metabolism, an elaborate system of chaperones and transporters has evolved, although no human Cu chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a Cu chaperone. © 2012 Wiley Periodicals, Inc.

  9. Seasonal superoxide overproduction and endothelial activation in guinea-pig heart; seasonal oxidative stress in rats and humans.

    PubMed

    Konior, Anna; Klemenska, Emilia; Brudek, Magdalena; Podolecka, Ewa; Czarnowska, Elżbieta; Beręsewicz, Andrzej

    2011-04-01

    Seasonality in endothelial dysfunction and oxidative stress was noted in humans and rats, suggesting it is a common phenomenon of a potential clinical relevance. We aimed at studying (i) seasonal variations in cardiac superoxide (O(2)(-)) production in rodents and in 8-isoprostane urinary excretion in humans, (ii) the mechanism of cardiac O(2)(-) overproduction occurring in late spring/summer months in rodents, (iii) whether this seasonal O(2)(-)-overproduction is associated with a pro-inflammatory endothelial activation, and (iv) how the summer-associated changes compare to those caused by diabetes, a classical cardiovascular risk factor. Langendorff-perfused guinea-pig and rat hearts generated ~100% more O(2)(-), and human subjects excreted 65% more 8-isoprostane in the summer vs. other seasons. Inhibitors of NADPH oxidase, xanthine oxidase, and NO synthase inhibited the seasonal O(2)(-)-overproduction. In the summer vs. other seasons, cardiac NADPH oxidase and xanthine oxidase activity, and protein expression were increased, the endothelial NO synthase and superoxide dismutases were downregulated, and, in guinea-pig hearts, adhesion molecules upregulation and the endothelial glycocalyx destruction associated these changes. In guinea-pig hearts, the summer and a streptozotocin-induced diabetes mediated similar changes, yet, more severe endothelial activation associated the diabetes. These findings suggest that the seasonal oxidative stress is a common phenomenon, associated, at least in guinea-pigs, with the endothelial activation. Nonetheless, its biological meaning (regulatory vs. deleterious) remains unclear. Upregulated NADPH oxidase and xanthine oxidase and uncoupled NO synthase are the sources of the seasonal O(2)(-)-overproduction.

  10. Titanium dioxide nanoparticles enhance production of superoxide anion and alter the antioxidant system in human osteoblast cells.

    PubMed

    Niska, Karolina; Pyszka, Katarzyna; Tukaj, Cecylia; Wozniak, Michal; Radomski, Marek Witold; Inkielewicz-Stepniak, Iwona

    2015-01-01

    Titanium dioxide (TiO2) nanoparticles (NPs) are manufactured worldwide for a variety of engineering and bioengineering applications. TiO2NPs are frequently used as a material for orthopedic implants. However, to the best of our knowledge, the biocompatibility of TiO2NPs and their effects on osteoblast cells, which are responsible for the growth and remodeling of the human skeleton, have not been thoroughly investigated. In the research reported here, we studied the effects of exposing hFOB 1.19 human osteoblast cells to TiO2NPs (5-15 nm) for 24 and 48 hours. Cell viability, alkaline phosphatase (ALP) activity, cellular uptake of NPs, cell morphology, superoxide anion (O2 (•-2)) generation, superoxide dismutase (SOD) activity and protein level, sirtuin 3 (SIR3) protein level, correlation between manganese (Mn) SOD and SIR, total antioxidant capacity, and malondialdehyde were measured following exposure of hFOB 1.19 cells to TiO2NPs. Exposure of hFOB 1.19 cells to TiO2NPs resulted in: (1) cellular uptake of NPs; (2) increased cytotoxicity and cell death in a time- and concentration-dependent manner; (3) ultrastructure changes; (4) decreased SOD and ALP activity; (5) decreased protein levels of SOD1, SOD2, and SIR3; (6) decreased total antioxidant capacity; (7) increased O2 (•-) generation; and (8) enhanced lipid peroxidation (malondialdehyde level). The linear relationship between the protein level of MnSOD and SIR3 and between O2 (•-) content and SIR3 protein level was observed. Importantly, the cytotoxic effects of TiO2NPs were attenuated by the pretreatment of hFOB 1.19 cells with SOD, indicating the significant role of O2 (•-) in the cell damage and death observed. Thus, decreased expression of SOD leading to increased oxidizing stress may underlie the nanotoxic effects of TiO2NPs on human osteoblasts.

  11. Immunometabolism of human autoimmune diseases: from metabolites to extracellular vesicles.

    PubMed

    de Candia, Paola; De Rosa, Veronica; Gigantino, Vincenzo; Botti, Gerardo; Ceriello, Antonio; Matarese, Giuseppe

    2017-06-26

    Immunometabolism focuses on the mechanisms regulating the impact of metabolism on lymphocyte activity and autoimmunity outbreak. The adipose tissue is long known to release adipokines, either pro- or anti-inflammatory factors bridging nutrition and immune function. More recently, adipocytes were discovered to also release extracellular vesicles (EVs) containing a plethora of biological molecules, including metabolites and microRNAs, which can regulate cell function/metabolism in distant tissues, suggesting that immune regulatory function by the adipose tissue may be far more complex than originally thought. Moreover, EVs were also identified as important mediators of immune cell-to-cell communication, adding a further microenvironmental mechanism of plasticity to fine-tune specific lymphocyte responses. This Review will first focus on the known mechanisms by which metabolism impacts immune function, presenting a systemic (nutrition and long-ranged adipokines) and a cellular point of view (metabolic pathway derangement in autoimmunity). It will then discuss the new discoveries concerning how EVs may act as nanometric vehicles integrating immune/metabolic responses at the level of the extracellular environment and affecting pathological processes. © 2017 Federation of European Biochemical Societies.

  12. Aortic ascorbic acid, trace elements, and superoxide dismutase activity in human aneurysmal and occlusive disease.

    PubMed

    Dubick, M A; Hunter, G C; Casey, S M; Keen, C L

    1987-02-01

    Altered trace elements and ascorbic acid metabolism have been implicated in the pathogenesis of atherosclerotic cardiovascular disease. However, their role in the disease process, or the effect of atherosclerosis on their tissue levels within plaque, is poorly understood. The present study analyzes the concentrations of Fe, Cu, Zn, and Mn, and ascorbic acid and superoxide dismutase (SOD) activity in tissue samples from 29 patients with abdominal aortic aneurysms (AAA) and 14 patients with atherosclerotic occlusive disease (AOD). It was observed that the Fe and Mn concentrations in AAA and AOD tissue were higher than the levels in nondiseased control aorta, whereas Cu and Zn levels in AAA and AOD tissue were similar to the levels in controls. The Zn:Cu ratio was significantly lower in the AAA tissue in comparison to both AOD and control tissue. In addition, AAA and AOD tissue had low ascorbic acid levels and low Cu,Zn-SOD activity with Cu,Zn-SOD:Mn-SOD ratios of 0.27 and 0.19, respectively, compared to a ratio of 3.20 in control aorta. These data indicate that aorta affected by aneurysms and occlusive disease have altered trace element and ascorbic acid concentrations, as well as low Cu,Zn-SOD activity. Although these observations do not directly support the hypothesis that AAA is associated with aortic Cu deficiency they do suggest a role for oxygen radicals or increased lipid peroxidation in occlusive and aneurysmal disease of the aorta.

  13. Aortic ascorbic acid, trace elements, and superoxide dismutase activity in human aneurysmal and occlusive disease

    SciTech Connect

    Dubick, M.A.; Hunter, G.C.; Casey, S.M.; Keen, C.L.

    1987-02-01

    Altered trace elements and ascorbic acid metabolism have been implicated in the pathogenesis of atherosclerotic cardiovascular disease. However, their role in the disease process, or the effect of atherosclerosis on their tissue levels within plaque, is poorly understood. The presence study analyzes the concentrations of Fe, Cu, Zn, and Mn, and ascorbic acid and superoxide dismutase (SOD) activity in tissue samples from 29 patients with abdominal aortic aneurysms (AAA) and 14 patients with atherosclerotic occlusive disease (AOD). It was observed that the Fe and Mn concentrations in AAA and AOD tissue were higher than the levels in nondiseased control aorta, whereas Cu and Zn levels in AAA and AOD tissue were similar to the levels in controls. The Zn:Cu ratio was significantly lower in the AAA tissue in comparison to both AOD and control tissue. In addition, AAA and AOD tissue had low ascorbic acid levels and low Cu, Zn-SOD activity with Cu,Zn-SOD:Mn-SOD ratios of 0.27 and 0.19, respectively, compared to a ratio of 3.20 in control aorta. These data indicate that aorta affected by aneurysms and occlusive disease have altered trace element and ascorbic acid concentrations, as well as low Cu,Zn-SOD activity. Although these observations do not directly support the hypothesis that AAA is associated with aortic Cu deficiency they do suggest a role for oxygen radicals or increased lipid peroxidation in occlusive and aneurysmal disease of the aorta.

  14. The impact of cationic solid lipid nanoparticles on human neutrophil activation and formation of neutrophil extracellular traps (NETs).

    PubMed

    Hwang, Tsong-Long; Aljuffali, Ibrahim A; Hung, Chi-Feng; Chen, Chun-Han; Fang, Jia-You

    2015-06-25

    Cationic solid lipid nanoparticles (cSLNs) are extensively employed as the nanocarriers for drug/gene targeting to tumors and the brain. Investigation into the possible immune response of cSLNs is still lacking. The aim of this study was to evaluate the impact of cSLNs upon the activation of human polymorphonuclear neutrophil cells (PMNs). The cytotoxicity, pro-inflammatory mediators, Ca(2+) mobilization, mitogen-activated protein kinases (MAPKs), and neutrophil extracellular traps (NETs) as the indicators of PMN stimulation were examined in this work. The cSLNs presented a diameter of 195 nm with a zeta potential of 44 mV. The cSLNs could interact with the cell membrane to produce a direct membrane lysis and the subsequent cytotoxicity according to lactate dehydrogenase (LDH) elevation. The interaction of cSLNs with the membrane also triggered a Ca(2+) influx, followed by the induction of oxidative stress and degranulation. The cationic nanoparticles elevated the levels of superoxide anion and elastase by 24- and 9-fold, respectively. The PMN activation by cSLNs promoted the phosphorylation of p38 and Jun-N-terminal kinases (JNK) but not extracellular signal-regulated kinases (ERK). The imaging of scanning electron microscopy (SEM) and immunofluorescence demonstrated the production of NETs by cSLNs. This phenomenon was not significant for the neutral SLNs (nSLNs), although histones in NETs also increased after treatment of nSLNs. Our results suggest an important role of cSLNs in governing the activation of human neutrophils.

  15. Pseudomonas aeruginosa binds to extracellular matrix deposited by human corneal epithelial cells.

    PubMed

    Esco, Miechia A; Hazlett, Linda D; Kurpakus-Wheater, Michelle

    2002-12-01

    To measure the effect of extracellular matrix substrate, pH, and O(2) on Pseudomonas aeruginosa binding. Extracellular matrix substrates were prepared from human corneal epithelial cells cultured in 2% or 20% O(2). P. aeruginosa strains ATCC 19660 or PAO1 (suspended in pH 7.0 or 7.5 buffer) were cultured on extracellular matrix substrates in 2% or 20% O(2). The mean number of adherent bacteria per counted per field +/- SEM (n = 15) was determined for combinations of bacteria, extracellular matrix substrate, pH, and O(2). Binding in the presence of antibodies directed against laminin-5 was also measured. Extracellular matrix substrates produced by cells cultured in 20% O(2), combined with an environment of pH 7.0, provided the least favorable conditions for binding of strain 19660. In contrast, extracellular matrix substrates produced by cells cultured in 2% O(2), combined with an environment of pH 7.0, provided the most favorable conditions for binding of strain 19660. Binding of PAO1, however, as a function of extracellular matrix substrate and pH, did not similarly compare with binding of strain 19660. Antibodies against laminin-5 chains served to increase the number of strain 19660 bacteria bound to extracellular matrix substrates compared with the control. The extracellular matrix secreted by hypoxic corneal epithelial cells is a substrate for binding of P. aeruginosa. Results in previous studies have shown that hypoxic extracellular matrix contains less laminin-5 protein than normoxic matrix. The antibody studies in this report suggest that the decrease in laminin-5 content in hypoxic matrix, relative to matrix secreted by normoxic corneal epithelium, may be responsible for increased bacterial adhesion.

  16. Extracellular N-Acetylaspartate in Human Traumatic Brain Injury.

    PubMed

    Shannon, Richard J; van der Heide, Susan; Carter, Eleanor L; Jalloh, Ibrahim; Menon, David K; Hutchinson, Peter J; Carpenter, Keri L H

    2016-02-15

    N-acetylaspartate (NAA) is an amino acid derivative primarily located in the neurons of the adult brain. The function of NAA is incompletely understood. Decrease in brain tissue NAA is presently considered symptomatic and a potential biomarker of acute and chronic neuropathological conditions. The aim of this study was to use microdialysis to investigate the behavior of extracellular NAA (eNAA) levels after traumatic brain injury (TBI). Sampling for this study was performed using cerebral microdialysis catheters (M Dialysis 71) perfused at 0.3 μL/min. Extracellular NAA was measured in microdialysates by high-performance liquid chromatography in 30 patients with severe TBI and for comparison, in radiographically "normal" areas of brain in six non-TBI neurosurgical patients. We established a detailed temporal eNAA profile in eight of the severe TBI patients. Microdialysate concentrations of glucose, lactate, pyruvate, glutamate, and glycerol were measured on an ISCUS clinical microdialysis analyzer. Here, we show that the temporal profile of microdialysate eNAA was characterized by highest levels in the earliest time-points post-injury, followed by a steady decline; beyond 70 h post-injury, average levels were 40% lower than those measured in non-TBI patients. There was a significant inverse correlation between concentrations of eNAA and pyruvate; eNAA showed significant positive correlations with glycerol and the lactate/pyruvate (L/P) ratio measured in microdialysates. The results of this on-going study suggest that changes in eNAA after TBI relate to the release of intracellular components, possibly due to neuronal death or injury, as well as to adverse brain energy metabolism.

  17. Altered protein levels in the isolated extracellular matrix of failing human hearts with dilated cardiomyopathy.

    PubMed

    DeAguero, Joshua L; McKown, Elizabeth N; Zhang, Liwen; Keirsey, Jeremy; Fischer, Edgar G; Samedi, Von G; Canan, Benjamin D; Kilic, Ahmet; Janssen, Paul M L; Delfín, Dawn A

    Dilated cardiomyopathy (DCM) is associated with extensive pathological cardiac remodeling and involves numerous changes in the protein expression profile of the extracellular matrix of the heart. We obtained seven human, end-stage, failing hearts with DCM (DCM-failing) and nine human, nonfailing donor hearts and compared their extracellular matrix protein profiles. We first showed that the DCM-failing hearts had indeed undergone extensive remodeling of the left ventricle myocardium relative to nonfailing hearts. We then isolated the extracellular matrix from a subset of these hearts and performed a proteomic analysis on the isolated matrices. We found that the levels of 26 structural proteins were altered in the DCM-failing isolated cardiac extracellular matrix compared to nonfailing isolated cardiac extracellular matrix. Overall, most of the extracellular matrix proteins showed reduced levels in the DCM-failing hearts, while all of the contractile proteins showed increased levels. There was a mixture of increased and decreased levels of cytoskeletal and nuclear transport proteins. Using immunoprobing, we verified that collagen IV (α2 and α6 isoforms), zyxin, and myomesin protein levels were reduced in the DCM-failing hearts. We expect that these data will add to the understanding of the pathology associated with heart failure with DCM.

  18. The potential for targeting extracellular LOX proteins in human malignancy.

    PubMed

    Mayorca-Guiliani, Alejandro; Erler, Janine T

    2013-11-25

    The extracellular matrix (ECM) is the physical scaffold where cells are organized into tissues and organs. The ECM may be modified during cancer to allow and promote proliferation, invasion, and metastasis. The family of lysyl oxidase (LOX) enzymes cross-links collagens and elastin and, therefore, is a central player in ECM deposition and maturation. Extensive research has revealed how the LOX proteins participate in every stage of cancer progression, and two family members, LOX and LOX-like 2, have been linked to metastasis, the final stage of cancer responsible for over 90% of cancer patient deaths. However, LOX biosynthesis results in by-product with antiproliferative properties in certain cancers, and LOX enzymes may have different effects depending on the molecular network in which they are active. Therefore, the design of therapies targeting the LOX family needs to be guided by the molecular makeup of the individual disease and will probably require other agents to act on both the LOX enzymes and their associated network.

  19. The potential for targeting extracellular LOX proteins in human malignancy

    PubMed Central

    Mayorca-Guiliani, Alejandro; Erler, Janine T

    2013-01-01

    The extracellular matrix (ECM) is the physical scaffold where cells are organized into tissues and organs. The ECM may be modified during cancer to allow and promote proliferation, invasion, and metastasis. The family of lysyl oxidase (LOX) enzymes cross-links collagens and elastin and, therefore, is a central player in ECM deposition and maturation. Extensive research has revealed how the LOX proteins participate in every stage of cancer progression, and two family members, LOX and LOX-like 2, have been linked to metastasis, the final stage of cancer responsible for over 90% of cancer patient deaths. However, LOX biosynthesis results in by-product with antiproliferative properties in certain cancers, and LOX enzymes may have different effects depending on the molecular network in which they are active. Therefore, the design of therapies targeting the LOX family needs to be guided by the molecular makeup of the individual disease and will probably require other agents to act on both the LOX enzymes and their associated network. PMID:24348049

  20. Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface

    SciTech Connect

    Borgstahl, G.E.O.; Hickey, M.J.; Johnson, M.J.

    1996-04-09

    Human manganese superoxide dismutase (MnSOD) is a homotetrameric enzyme which protects mitochondria against oxygen-mediated free radical damage. Within each subunit, both the N-terminal helical hairpin and C-terminal {alpha}/{beta} domains contribute ligands to the catalytic manganese site. Two identical four-helix bundles,symmetrically assembled form the N-terminal helical hairpins, form a novel tetrameric interface that stabilizes the active sites. The 2.5 {angstrom} crystallographic structure of the naturally occurring polymorphic variant Ile58Thr MnSOD reveals that the helical hairpin mutation Thr58 causes two packing defects in each of the two four-helix bundles of the tetrameric interface. Similar mutations, expected to cause packing defects in the Cu,ZnSOD dimer interface, are associated with the degenerative disease amyotrophic lateral sclerosis. Ile58Thr MnSOD is primarily dimeric in solution and is significantly less thermostable than the normal enzyme, with decreases of 15{degrees}C in the main melting temperature and 20{degrees}C in the heat-inactivation temperature. Consequently, this mutant MnSOD is compromised at normal body temperatures: thermal inactivation, predicted from the decrease in thermal stability, occurs with a theoretical half-life of only 3.2h at 37{degrees}C (1.4 h at 41 {degrees}C), compared with 3.1 years for native MnSOD. This prediction is supported by direct measurements: incubation at 41.7{degrees}C for 3 h has no effect on the activity of native MnSOD but completely inactivates mutant MnSOD. Rapid inactivation of Ile58Thr MnSOD at the elevated temperatures associated with fever and inflammation could provide an early advantage by killing infected cells, but also would increase superoxide-mediated oxidative damage and perhaps contribute to late-onset diseases. 63 refs., 7 figs., 2 tabs.

  1. Superoxide microsensor integrated into a Sensing Cell Culture Flask microsystem using direct oxidation for cell culture application.

    PubMed

    Flamm, H; Kieninger, J; Weltin, A; Urban, G A

    2015-03-15

    A new electrochemical sensor system for reliable and continuous detection of superoxide radical release from cell culture was developed utilizing direct oxidation of superoxide on polymer covered gold microelectrodes. Direct superoxide oxidation was demonstrated to provide robust measurement principle for sensitive and selective reactive oxygen species (ROS) quantification without the need for biocomponent supported conversion. Sensor performance was investigated by using artificial enzymatic superoxide production revealing a sensitivity of 2235AM(-1)m(-2). An electrode protection layer with molecular weight cut-off property from adsorbed linear branched polyethylenimine was successfully introduced for long term and selectivity improvement. Thin-film based sensor chip fabrication with implemented three-electrode setup and full integration into the technological platform Sensing Cell Culture Flask was described. Cell culturing directly on-chip and free radical release by phorbol-12-myristate-13-acetate (PMA) stimulation was demonstrated using T-47D human breast cancer carcinoma cell model. Transient extracellular superoxide production upon stimulation was successfully observed from amperometric monitoring. Signal inhibition from scavenging of extracellular superoxide by specific superoxide dismutase (SOD) showed the applicability for selective in vitro ROS determination. The results confirm the possibility of direct superoxide oxidation, with exclusion of the main interfering substances uric acid and hydrogen peroxide. This offers new insights into the development of reliable and robust ROS sensors.

  2. Potential Dengue Virus-Triggered Apoptotic Pathway in Human Neuroblastoma Cells: Arachidonic Acid, Superoxide Anion, and NF-κB Are Sequentially Involved

    PubMed Central

    Jan, Jia-Tsrong; Chen, Bor-Horng; Ma, Shiou-Hwa; Liu, Chiu-I; Tsai, Hui-Ping; Wu, Han-Chung; Jiang, Shian-Yuan; Yang, Kuen-Der; Shaio, Men-Fang

    2000-01-01

    Direct in vivo evidence for the susceptibility of human neuronal cells to dengue virus has not been reported. In this study, we demonstrated that type 2 dengue (DEN-2) virus infection induced extensive apoptosis in the human neuroblastoma cell line SK-N-SH. Phospholipase A2 (PLA2) was activated by DEN-2 infection, which led to the generation of arachidonic acid (AA). Inhibition of PLA2 activity by the PLA2 inhibitors, AACOCF3 and ONO-RS-082, diminished DEN-2 virus-induced apoptosis. In contrast, the cyclooxygenase inhibitors aspirin and indomethacin, thought to increase AA accumulation by blocking AA catabolism, enhanced apoptosis. Exogenous AA induced apoptosis in a dose-dependent manner. Superoxide anion, which is thought to be generated through the AA-activated NADPH oxidase, was increased after infection. Pretreatment with superoxide dismutase (SOD) protected cells against DEN-2 virus-induced apoptosis. Furthermore, generation of superoxide anion was blocked by AACOCF3. In addition, the transcription factors, NF-κB and c-Jun, were found to be activated after DEN-2 virus infection. However, pretreatment of cells with oligodeoxynucleotides containing NF-κB, but not c-Jun, binding sites (transcription factor decoy) strongly prevented dengue virus-induced apoptosis. The finding that AACOCF3 and SOD significantly block activation of NF-κB suggests that this activation is derived from the AA-superoxide anion pathway. Our results indicate that DEN-2 virus infection of human neuroblastoma cells triggers an apoptotic pathway through PLA2 activation to superoxide anion generation and subsequently to NF-κB activation. This apoptotic effect can be either directly derived from the action of AA and superoxide anion on mitochondria or indirectly derived from the products of apoptosis-related genes activated by NF-κB. PMID:10954569

  3. Extracellular Matrix Induced Gene Expression in Human Breast Cancer Cells

    PubMed Central

    Garamszegi, Nandor; Garamszegi, Susanna P.; Shehadeh, Lina A.; Scully, Sean P.

    2009-01-01

    Extracellular matrix (ECM) molecules modify gene expression through attachment-dependent (i.e., focal adhesion related) integrin receptor signalling. It was previously unknown whether the same molecules acting as soluble peptides could generate signal cascades without the associated mechanical anchoring, a condition that may be encountered during matrix remodelling, degradation and relevant to invasion and metastatic processes. In the current study the role of ECM ligand regulated gene expression through this attachment independent process was examined. It was observed that fibronectin, laminin, collagens type I and II induce Smad2 activation in MCF-10A and MCF-7 cells. This activation is not caused by TGFβ ligand contamination or autocrine TGF involvement and is 3–5 fold less robust than the TGFβ1 ligand. The resulting nuclear translocation of Smad4 in response to ECM ligand indicates downstream transcriptional responses occurring. Co-immunoprecipitation experiments determined that type II collagen and laminin act through interaction with integrin α2β1 receptor complex. The ECM ligand induced Smad activation (termed signalling crosstalk) resulted cell type and ligand specific transcriptional changes which are distinct from the TGFβ ligand induced responses. These findings demonstrate that cell-matrix communication is more complex than previously thought. Soluble ECM peptides drive transcriptional regulation through corresponding adhesion and non-attachment related processes. The resultant gene expressional patterns correlate with pathway activity and not by the extent of Smad activation. These results extend the complexity and the existing paradigms of ECM-cell communication to ECM ligand regulation without the necessity of mechanical coupling. PMID:19276183

  4. Priming of Human Neutrophils Is Necessary for Their Activation by Extracellular DNA.

    PubMed

    Prikhodko, A S; Vitushkina, M V; Zinovkina, L A; Popova, E N; Zinovkin, R A

    2016-06-01

    Extracellular plasma DNA is thought to act as a damage-associated molecular pattern causing activation of immune cells. However, purified preparations of mitochondrial and nuclear DNA were unable to induce neutrophil activation in vitro. Thus, we examined whether granulocyte-macrophage colony-stimulating factor (GM-CSF) acting as a neutrophil priming agent can promote the activation of neutrophils by different types of extracellular DNA. GM-CSF pretreatment greatly increased p38 MAPK phosphorylation and promoted CD11b/CD66b expression in human neutrophils treated with mitochondrial and, to a lesser extent, with nuclear DNA. Our experiments clearly indicate that GM-CSF-induced priming of human neutrophils is necessary for their subsequent activation by extracellular DNA.

  5. Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase

    PubMed Central

    Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph; Marquardt, Iris; Donsante, Anthony; Yi, Ling; Hicks, Julia D.; Steinbach, Peter J.; Wilson, Callum; Elpeleg, Orly; Møller, Lisbeth Birk; Christodoulou, John; Kaler, Stephen G.; Gärtner, Jutta

    2012-01-01

    Copper is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular copper metabolism, an elaborate system of chaperones and transporters has evolved, although no human copper chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited a homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the copper chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163 with tryptophan in domain II of CCS, which interacts directly with SOD1. Biochemical analyses of the patient’s fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal copper homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a copper chaperone. PMID:22508683

  6. Extracellular matrix remodelling in response to venous hypertension: proteomics of human varicose veins

    PubMed Central

    Barallobre-Barreiro, Javier; Oklu, Rahmi; Lynch, Marc; Fava, Marika; Baig, Ferheen; Yin, Xiaoke; Barwari, Temo; Potier, David N.; Albadawi, Hassan; Jahangiri, Marjan; Porter, Karen E.; Watkins, Michael T.; Misra, Sanjay; Stoughton, Julianne; Mayr, Manuel

    2016-01-01

    Aims Extracellular matrix remodelling has been implicated in a number of vascular conditions, including venous hypertension and varicose veins. However, to date, no systematic analysis of matrix remodelling in human veins has been performed. Methods and results To understand the consequences of venous hypertension, normal and varicose veins were evaluated using proteomics approaches targeting the extracellular matrix. Varicose saphenous veins removed during phlebectomy and normal saphenous veins obtained during coronary artery bypass surgery were collected for proteomics analysis. Extracellular matrix proteins were enriched from venous tissues. The proteomics analysis revealed the presence of >150 extracellular matrix proteins, of which 48 had not been previously detected in venous tissue. Extracellular matrix remodelling in varicose veins was characterized by a loss of aggrecan and several small leucine-rich proteoglycans and a compensatory increase in collagen I and laminins. Gene expression analysis of the same tissues suggested that the remodelling process associated with venous hypertension predominantly occurs at the protein rather than the transcript level. The loss of aggrecan in varicose veins was paralleled by a reduced expression of aggrecanases. Chymase and tryptase β1 were among the up-regulated proteases. The effect of these serine proteases on the venous extracellular matrix was further explored by incubating normal saphenous veins with recombinant enzymes. Proteomics analysis revealed extensive extracellular matrix degradation after digestion with tryptase β1. In comparison, chymase was less potent and degraded predominantly basement membrane-associated proteins. Conclusion The present proteomics study provides unprecedented insights into the expression and degradation of structural and regulatory components of the vascular extracellular matrix in varicosis. PMID:27068509

  7. Extracellular matrix remodelling in response to venous hypertension: proteomics of human varicose veins.

    PubMed

    Barallobre-Barreiro, Javier; Oklu, Rahmi; Lynch, Marc; Fava, Marika; Baig, Ferheen; Yin, Xiaoke; Barwari, Temo; Potier, David N; Albadawi, Hassan; Jahangiri, Marjan; Porter, Karen E; Watkins, Michael T; Misra, Sanjay; Stoughton, Julianne; Mayr, Manuel

    2016-06-01

    Extracellular matrix remodelling has been implicated in a number of vascular conditions, including venous hypertension and varicose veins. However, to date, no systematic analysis of matrix remodelling in human veins has been performed. To understand the consequences of venous hypertension, normal and varicose veins were evaluated using proteomics approaches targeting the extracellular matrix. Varicose saphenous veins removed during phlebectomy and normal saphenous veins obtained during coronary artery bypass surgery were collected for proteomics analysis. Extracellular matrix proteins were enriched from venous tissues. The proteomics analysis revealed the presence of >150 extracellular matrix proteins, of which 48 had not been previously detected in venous tissue. Extracellular matrix remodelling in varicose veins was characterized by a loss of aggrecan and several small leucine-rich proteoglycans and a compensatory increase in collagen I and laminins. Gene expression analysis of the same tissues suggested that the remodelling process associated with venous hypertension predominantly occurs at the protein rather than the transcript level. The loss of aggrecan in varicose veins was paralleled by a reduced expression of aggrecanases. Chymase and tryptase β1 were among the up-regulated proteases. The effect of these serine proteases on the venous extracellular matrix was further explored by incubating normal saphenous veins with recombinant enzymes. Proteomics analysis revealed extensive extracellular matrix degradation after digestion with tryptase β1. In comparison, chymase was less potent and degraded predominantly basement membrane-associated proteins. The present proteomics study provides unprecedented insights into the expression and degradation of structural and regulatory components of the vascular extracellular matrix in varicosis. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Cardiology.

  8. Yops of Yersinia enterocolitica Inhibit Receptor-Dependent Superoxide Anion Production by Human Granulocytes

    PubMed Central

    Visser, L. G.; Seijmonsbergen, E.; Nibbering, P. H.; van den Broek, P. J.; van Furth, R.

    1999-01-01

    The virulence plasmid-borne genes encoding Yersinia adhesin A (YadA) and several Yersinia secreted proteins (Yops) are involved in the inhibition of phagocytosis and killing of Yersinia enterocolitica by human granulocytes. One of these Yops, YopH, dephosphorylates multiple tyrosine-phosphorylated proteins in eukaryotic cells and is involved in the inhibition of phagocytosis of Y. enterocolitica by human granulocytes. We investigated whether antibody- and complement-opsonized plasmid-bearing (pYV+) Y. enterocolitica inhibits O2− production by human granulocytes in response to various stimuli and whether YopH is involved. Granulocytes were preincubated with mutant strains unable to express YadA or to secrete Yops or YopH. O2− production by granulocytes during stimulation was assessed by measuring the reduction of ferricytochrome c. PYV+ Y. enterocolitica inhibited O2− production by granulocytes incubated with opsonized Y. enterocolitica or N-formyl-Met-Leu-Phe (f-MLP). This inhibitory effect mediated by pYV did not affect receptor-independent O2− production by granulocytes in response to phorbol myristate acetate, indicating that NADPH activity remained unaffected after activation of protein kinase C. The inhibition of f-MLP-induced O2− production by granulocytes depends on the secretion of Yops and not on the expression of YadA. Insertional inactivation of the yopH gene abrogated the inhibition of phagocytosis of antibody- and complement-opsonized Y. enterocolitica by human granulocytes but not of the f-MLP-induced O2− production by granulocytes or tyrosine phosphorylation of granulocyte proteins. These findings suggest that the specific targets for YopH are not present in f-MLP receptor-linked signal transduction and that other Yop-mediated mechanisms are involved. PMID:10024567

  9. Yops of Yersinia enterocolitica inhibit receptor-dependent superoxide anion production by human granulocytes.

    PubMed

    Visser, L G; Seijmonsbergen, E; Nibbering, P H; van den Broek, P J; van Furth, R

    1999-03-01

    The virulence plasmid-borne genes encoding Yersinia adhesin A (YadA) and several Yersinia secreted proteins (Yops) are involved in the inhibition of phagocytosis and killing of Yersinia enterocolitica by human granulocytes. One of these Yops, YopH, dephosphorylates multiple tyrosine-phosphorylated proteins in eukaryotic cells and is involved in the inhibition of phagocytosis of Y. enterocolitica by human granulocytes. We investigated whether antibody- and complement-opsonized plasmid-bearing (pYV+) Y. enterocolitica inhibits O2- production by human granulocytes in response to various stimuli and whether YopH is involved. Granulocytes were preincubated with mutant strains unable to express YadA or to secrete Yops or YopH. O2- production by granulocytes during stimulation was assessed by measuring the reduction of ferricytochrome c. PYV+ Y. enterocolitica inhibited O2- production by granulocytes incubated with opsonized Y. enterocolitica or N-formyl-Met-Leu-Phe (f-MLP). This inhibitory effect mediated by pYV did not affect receptor-independent O2- production by granulocytes in response to phorbol myristate acetate, indicating that NADPH activity remained unaffected after activation of protein kinase C. The inhibition of f-MLP-induced O2- production by granulocytes depends on the secretion of Yops and not on the expression of YadA. Insertional inactivation of the yopH gene abrogated the inhibition of phagocytosis of antibody- and complement-opsonized Y. enterocolitica by human granulocytes but not of the f-MLP-induced O2- production by granulocytes or tyrosine phosphorylation of granulocyte proteins. These findings suggest that the specific targets for YopH are not present in f-MLP receptor-linked signal transduction and that other Yop-mediated mechanisms are involved.

  10. Extracellular depolymerization of hyaluronic acid in cultured human skin fibroblasts

    SciTech Connect

    Nakamura, T.; Takagaki, K.; Kubo, K.; Morikawa, A.; Tamura, S.; Endo, M. )

    1990-10-15

    The chain length of ({sup 3}H)hyaluronic acid synthesized by cultivating human skin fibroblasts in the presence of ({sup 3}H)glucosamine was investigated. ({sup 3}H)Hyaluronic acid obtained from the matrix fraction was excluded from a Sepharose CL-2B column irrespective of the incubation period, whereas that from the medium was depolymerized into a constant chain length (Mr = 40,000). The reducing and non-reducing terminals of the depolymerized hyaluronic acid were N-acetylglucosamine and glucuronic acid, respectively. Prolonged incubation produced no oligosaccharides as shown by examination of hyaluronidase digests, suggesting the presence of a novel endo-beta-N-acetylglucosaminidase in cultured human skin fibroblasts.

  11. Extracellular Matrix Molecular Remodeling in Human Liver Fibrosis Evolution

    PubMed Central

    Baiocchini, Andrea; Montaldo, Claudia; Conigliaro, Alice; Grimaldi, Alessio; Correani, Virginia; Mura, Francesco; Ciccosanti, Fabiola; Rotiroti, Nicolina; Brenna, Alessia; Montalbano, Marzia; D’Offizi, Gianpiero; Capobianchi, Maria Rosaria; Alessandro, Riccardo; Piacentini, Mauro; Schininà, Maria Eugenia; Maras, Bruno; Del Nonno, Franca; Tripodi, Marco; Mancone, Carmine

    2016-01-01

    Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis). Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies. PMID:26998606

  12. Plasma extracellular superoxide dismutase concentration, allelic variations in the SOD3 gene and risk of myocardial infarction and all-cause mortality in people with type 1 and type 2 diabetes.

    PubMed

    Mohammedi, Kamel; Bellili-Muñoz, Naïma; Marklund, Stefan L; Driss, Fathi; Le Nagard, Hervé; Patente, Thiago A; Fumeron, Frédéric; Roussel, Ronan; Hadjadj, Samy; Marre, Michel; Velho, Gilberto

    2015-01-15

    Oxidative stress is involved in development of diabetes complications. Extracellular superoxide dismutase (EC-SOD, SOD3) is a major extracellular antioxidant enzyme and is highly expressed in arterial walls. Advanced oxidation protein products (AOPP) and 8-iso-prostaglandin (isoprostane) are markers of oxidative stress. We investigated association of SOD3 gene variants, plasma concentrations of EC-SOD, AOPP and isoprostane with myocardial infarction and mortality in diabetic patients. We studied three cohorts designed to evaluate the vascular complications of diabetes: the GENEDIAB study (469 participants with type 1 diabetes at baseline; follow-up data for 259 participants), the GENESIS study (603 participants with type 1 diabetes at baseline; follow-up data for 525 participants) and the DIABHYCAR study (3137 participants with type 2 diabetes at baseline and follow-up). Duration of follow-up was 9, 5, and 5 years, respectively. Main outcome measures were incidence of myocardial infarction, and cardiovascular and total mortality during follow-up. Six single nucleotide polymorphisms in the SOD3 locus were genotyped in the three cohorts. Plasma concentrations of EC-SOD, AOPP, and isoprostane were measured in baseline samples of GENEDIAB participants. In GENEDIAB/GENESIS pooled cohorts, the minor T-allele of rs2284659 variant was inversely associated with the prevalence at baseline (Odds Ratio 0.48, 95% CI 0.29-0.78, p = 0.004) and the incidence during follow-up of myocardial infarction (Hazard Ratio 0.58, 95% CI 0.40-0.83, p = 0.003) and with cardiovascular (HR 0.33, 95% CI 0.08-0.74, p = 0.004) and all-cause mortality (HR 0.44, 95% CI 0.21-0.73, p = 0.0006). The protective allele was associated with higher plasma EC-SOD and lower plasma AOPP concentrations in GENEDIAB. It was also inversely associated with incidence of myocardial infarction (HR 0.75, 95% CI 0.59-0.94, p = 0.01) and all-cause mortality (HR 0.87, 95% CI 0.79-0.97, p = 0

  13. Extracellular stimulation with human "noisy" electromyographic patterns facilitates myotube activity.

    PubMed

    Sciancalepore, M; Coslovich, T; Lorenzon, P; Ziraldo, G; Taccola, G

    2015-10-01

    Electrical stimulation (ES) of skeletal muscle partially mimics the benefits of physical activity. However, the stimulation protocols applied clinically to date, often cause unpleasant symptoms and muscle fatigue. Here, we compared the efficiency of a "noisy" stimulus waveform derived from human electromyographic (EMG) muscle patterns, with stereotyped 45 and 1 Hz electrical stimulations applied to mouse myotubes in vitro. Human gastrocnemius medialis electromyograms recorded from volunteers during real locomotor activity were used as a template for a noisy stimulation, called EMGstim. The stimulus-induced electrical activity, intracellular Ca(2+) dynamics and mechanical twitches in the myotubes were assessed using whole-cell perforated patch-clamp, Ca(2+) imaging and optical visualization techniques. EMGstim was more efficient in inducing myotube cell firing, [Ca(2+)]i changes and contractions compared with more conventional electrical stimulation. Its stimulation strength was also much lower than the minimum required to induce contractions via stereotyped stimulation protocols. We conclude that muscle cells in vitro can be more efficiently depolarized using the "noisy" stochastic stimulation pattern, EMGstim, a finding that suggests a way to favor a higher level of electrical activity in a larger number of cells.

  14. Increased digitalis-like activity in human cerebrospinal fluid after expansion of the extracellular fluid volume

    SciTech Connect

    Halperin, J.A.; Martin, A.M.; Malave, S.

    1985-08-12

    The present study was designed to determine whether acute expansion of the extracellular fluid volume influenced the digitalis-like activity of human cerebrospinal fluid (CSF), previously described. Human CSF samples, drawn before and 30 minutes after the intravenous infusion of 1 liter of either saline or glucose solutions, were assayed for digitalis-like activity by inhibition of either the /sup 86/Rb/sup +/ uptake into human erythrocytes or by the activity of a purified Na/sup +/-K/sup +/ ATPase. The CSF inhibitory activity on both systems significantly increased after the infusion of sodium solutions but did not change after the infusion of glucose. These results indicate that the digitalis-like factor of human CSF might be involved in the regulation of the extracellular fluid volume and electrolyte content and thereby in some of the physiological responses to sodium loading. 31 references, 2 figures, 1 table.

  15. Human cecum content modulates production of extracellular proteins by food and probiotic bacteria.

    PubMed

    Sánchez, Borja; Ruiz, Lorena; Suárez, Adolfo; de Los Reyes-Gavilán, Clara G; Margolles, Abelardo

    2011-11-01

    Lactic acid bacteria (LAB) are responsible for different types of food fermentations that provide humans with many different classes of fermented products. During the 20th century, some LAB strains as well as several members of the genus Bifidobacterium started to be extensively used in human nutrition as probiotics because of their health-promoting effects. Nowadays, the subset of extracellular proteins is being investigated as potential mediators of the process known as bacteria-host molecular crosstalk. Inclusion of human cecum extracts in laboratory culture medium modified the production of extracellular proteins by food and probiotic microorganisms. By proteomic and genetic means, the specific overproduction of two proteins was revealed to occur at transcriptional level. This work sheds light on the potential molecular effectors that food bacteria could use for interacting with the human gut and revealed that they may be produced under very specific environmental conditions.

  16. In vitro effects of Ala16Val manganese superoxide dismutase gene polymorphism on human white blood cells exposed to methylmercury.

    PubMed

    Algarve, T D; Barbisan, F; Ribeiro, E E; Duarte, M M M F; Mânica-Cattani, M F; Mostardeiro, C P; Lenz, A F; da Cruz, I B M

    2013-10-29

    Environmental contamination by methylmercury (MeHg) is an enormous public health problem in world regions such as Amazonia. MeHg toxic effects seem to be influenced by environmental and genetic factors. However, few studies have evaluated the genetic influences of MeHg toxicity in humans. Therefore, the aim of this study was to evaluate the genetic influence of Ala16Val manganese superoxide dismutase gene polymorphism (Ala16Val-MnSOD) on the cytotoxic effects of in vitro human leukocytes exposed to MeHg. Subjects were selected from 100 individuals aged 26.4 ± 7.3 years genotyped to Ala16Val-MnSOD polymorphism (AA = 6, VV = 6, and AV = 12) to perform in vitro testing using white blood cells (WBCs). Reactive oxygen species production was measured using 2',7'-dichlorofluorescein diacetate fluorimetric assay, and cell viability was measured using MTT assay on WBC samples from the same subjects that were both exposed and not exposed to MeHg (2.5 µM for 6 h). The results showed that AA- and VV-WBCs exposed to MeHg did not display increased reactive oxygen species levels compared to those in cells that were not exposed. However, AV-leukocytes exposed to MeHg displayed increased ROS levels. Cellular viability comparison among genotypes exposed to MeHg showed that the viability of AA-WBCs was lower than that of VV-WBC, with mean values of 3.46 ± 0.13 and 3.08 ± 0.77 (standard error), respectively (P = 0.033), whereas heterozygous cells (AV) displayed intermediate values. This difference was likely due to the higher basal H2O2 production of AA-WBCs compared to that of other genotypes. These results suggest that the Ala16Val-MnSOD polymorphism has toxicogenetic effects in human cells exposed to MeHg.

  17. The induction of human superoxide dismutase and catalase in vivo: a fundamentally new approach to antioxidant therapy.

    PubMed

    Nelson, Sally K; Bose, Swapan K; Grunwald, Gary K; Myhill, Paul; McCord, Joe M

    2006-01-15

    A composition consisting of extracts of five widely studied medicinal plants (Protandim) was administered to healthy human subjects ranging in age from 20 to 78 years. Individual ingredients were selected on the basis of published findings of induction of superoxide dismutase (SOD) and/or catalase in rodents in vivo, combined with evidence of decreasing lipid peroxidation. Each ingredient was present at a dosage sufficiently low to avoid any accompanying unwanted pharmacological effects. Blood was analyzed before supplementation and after 30 and 120 days of supplementation (675 mg/day). Erythrocytes were assayed for SOD and catalase, and plasma was assayed for lipid peroxidation products as thiobarbituric acid-reacting substances (TBARS), as well as uric acid, C-reactive protein, and cholesterol (total, LDL, and HDL). Before supplementation, TBARS showed a strong age-dependent increase. After 30 days of supplementation, TBARS declined by an average of 40% (p = 0.0001) and the age-dependent increase was eliminated. By 120 days, erythrocyte SOD increased by 30% (p < 0.01) and catalase by 54% (p < 0.002). We conclude that modest induction of the catalytic antioxidants SOD and catalase may be a much more effective approach than supplementation with antioxidants (such as vitamins C and E) that can, at best, stoichiometrically scavenge a very small fraction of total oxidant production.

  18. 28-day repeated dose oral toxicity of human copper-zinc superoxide dismutase from recombinant Pichia pastori in rats.

    PubMed

    Zhu, Liang; Tian, Ying-Juan; Zhu, Si-Ming

    2012-04-01

    Human copper/zinc superoxide dismutase from recombinant Pichia pastori (RH-Cu/Zn-SOD) was orally administered, via gavage, to Sprague-Dawley rats at 500, 1,000, and 2,000 mg/kg body weight/day for 28 days. During the 28-day period, animals were examined for evidence of toxicity; there were no deaths, and in-life physical signs were normal. On day 29, the animals were exsanguinated, examined for gross pathology, and tissues were preserved for histopathology. Although statistical differences were noted in some hematology and clinical chemistry, they were of questionable biological significance. The results of the 28-day oral administration demonstrated a lack of toxicity of RH-Cu/Zn-SOD in rats. There were no treatment-related, toxicologically relevant changes in clinical signs, growth, food consumption, hematology, clinical chemistry, organ weights, or pathology. The no observed adverse effect level was greater than 2,000 mg/kg/day for RH-Cu/Zn-SOD in rats.

  19. In Vitro Effect of Sodium Fluoride on Malondialdehyde Concentration and on Superoxide Dismutase, Catalase, and Glutathione Peroxidase in Human Erythrocytes

    PubMed Central

    Gutiérrez-Salinas, José; García-Ortíz, Liliana; Morales González, José A.; Hernández-Rodríguez, Sergio; Ramírez-García, Sotero; Núñez-Ramos, Norma R.; Madrigal-Santillán, Eduardo

    2013-01-01

    The aim of this paper was to describe the in vitro effect of sodium fluoride (NaF) on the specific activity of the major erythrocyte antioxidant enzymes, as well as on the membrane malondialdehyde concentration, as indicators of oxidative stress. For this purpose, human erythrocytes were incubated with NaF (0, 7, 28, 56, and 100 μg/mL) or NaF (100 μg/mL) + vitamin E (1, 2.5, 5 and 10 μg/mL). The malondialdehyde (MDA) concentration on the surface of the erythrocytes was determined, as were the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GlPx). Our results demonstrated that erythrocytes incubated with increasing NaF concentrations had an increased MDA concentration, along with decreased activity of antioxidant enzymes. The presence of vitamin E partially reversed the toxic effects of NaF on erythrocytes. These findings suggest that NaF induces oxidative stress in erythrocytes in vitro, and this stress is partially reversed by the presence of vitamin E. PMID:24223512

  20. Superoxide anion radical (O2(-)) degrades methylmercury to inorganic mercury in human astrocytoma cell line (CCF-STTG1).

    PubMed

    Mailloux, Ryan J; Yumvihoze, Emmanuel; Chan, Hing Man

    2015-09-05

    Methylmercury (MeHg) is a global pollutant that is affecting the health of millions of people worldwide. However, the mechanism of MeHg toxicity still remains somewhat elusive and there is no treatment. It has been known for some time that MeHg can be progressively converted to inorganic mercury (iHg) in various tissues including the brain. Recent work has suggested that cleavage of the carbon-metal bond in MeHg in a biological environment is facilitated by reactive oxygen species (ROS). However, the oxyradical species that actually mediates this process has not been identified. Here, we provide evidence that superoxide anion radical (O2(-)) can convert MeHg to iHg. The calculated second-order rate constant for the degradation of 1μM MeHg by O2(-) generated by xanthine/xanthine oxidase was calculated to be 2×10(5)M(-1)s(-1). We were also able to show that this bioconversion can proceed in intact CCF-STTG1 human astrocytoma cells exposed to paraquat (PQ), a O2(-) generating viologen. Notably, exposure of cells to increasing amounts of PQ led to a dose dependent increase in both MeHg and iHg. Indeed, a 24h exposure to 500μM PQ induced a ∼13-fold and ∼18-fold increase in intracellular MeHg and iHg respectively. These effects were inhibited by superoxide dismutase mimetic MnTBAP. In addition, we also observed that a 24h exposure to a biologically relevant concentration of MeHg (1μM) did not induce cell death, oxidative stress, or even changes in cellular O2(-) and H2O2. However, co-exposure to PQ enhanced MeHg toxicity which was associated with a robust increase in cell death and oxidative stress. Collectively our results show that O2(-) can bioconvert MeHg to iHg in vitro and in intact cells exposed to conditions that simulate high intracellular O2(-) production. In addition, we show for the first time that O2(-) mediated degradation of MeHg to iHg enhances the toxicity of MeHg by facilitating an accumulation of both MeHg and iHg in the intracellular

  1. Molecular heterogeneity in chronic granulomatous disease: a human model of defective phagocyte superoxide production.

    PubMed

    Gabig, T G; Lefker, B A

    1985-01-01

    Chronic granulomatous disease (CGD) is a genetically transmitted disorder thought to result from defect(s) in the activation or turnover of the NADPH dependent O2- generating oxidase enzyme system of human neutrophils and monocytes. The normal oxidase may be a flavoprotein-cytochrome b559 complex; therefore, these components of the oxidase were quantitated in the neutrophils from patients and family members of two unrelated CGD kindreds. The male propositus from an X-linked recessive kindred had a neutrophil oxidase fraction with low FAD content (26 pmol/mg protein) and undetectable cytochrome b559 (less than 5 pmol/mg protein). The male propositus from an autosomal recessive kindred had a neutrophil oxidase fraction with low FAD content (34 pmol FAD/mg protein), but normal cytochrome b559 content (170 pmol cytochrome b559/mg protein). Both parents of this latter CGD patient had normal FAD and cytochrome b559 content in their neutrophil oxidase fraction. We conclude that the carrier state in certain X-linked recessive female carriers of CGD can be detected by partial deficiencies of both flavoprotein and cytochrome b559 components of the oxidase, whereas presumed heterozygous carriers of certain autosomal recessive CGD kindreds cannot be detected by this means.

  2. Functional relationship of the cytochrome b to the superoxide-generating oxidase of human neutrophils.

    PubMed

    Gabig, T G; Schervish, E W; Santinga, J T

    1982-04-25

    A subcellular particulate fraction containing the NADPH-dependent O2.--generating oxidase from stimulated human neutrophils was prepared. This fraction was depleted of certain enzyme markers of primary and secondary granules and was devoid of measurable myeloperoxidase, both enzymatically and spectrally. When prepared from neutrophils which had been previously stimulated with phorbal myristate acetate, this fraction contained cyanide-insensitive, pyridine nucleotide-dependent O2.--generating activity with a specific activity of 260 nmol min-1 mg-1. O2.--generating activity is completely ablated by p-chloromercuribenzoate exposure. Preparations from normal unstimulated neutrophils or stimulated neutrophils from a male patient with chronic granulomatous disease had negligible amounts of this O2.--generating enzymatic activity. The dominant chromophore in this preparation was a b-type cytochrome, the spectral and functional characteristics of which are further described herein. Pyridine nucleotide-dependent reduction of the intrinsic cytochrome b closely parallels O2.- generation in this preparation. Specifically, reduction occurs in preparations from phorbal myristate acetate-stimulated neutrophils and is absent in unstimulated or stimulated p-chloromercuribenzoate-inactivated preparations.

  3. High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro.

    PubMed

    Sil, Payel; Yoo, Dae-Goon; Floyd, Madison; Gingerich, Aaron; Rada, Balazs

    2016-06-18

    Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

  4. Effects of extracellular modulation through hypoxia on the glucose metabolism of human breast cancer stem cells

    NASA Astrophysics Data System (ADS)

    Yustisia, I.; Jusman, S. W. A.; Wanandi, S. I.

    2017-08-01

    Cancer stem cells have been reported to maintain stemness under certain extracellular changes. This study aimed to analyze the effect of extracellular O2 level modulation on the glucose metabolism of human CD24-/CD44+ breast cancer stem cells (BCSCs). The primary BCSCs (CD24-/CD44+ cells) were cultured under hypoxia (1% O2) for 0.5, 4, 6, 24 and 48 hours. After each incubation period, HIF1α, GLUT1 and CA9 expressions, as well as glucose metabolism status, including glucose consumption, lactate production, O2 consumption and extracellular pH (pHe) were analyzed using qRT-PCR, colorimetry, fluorometry, and enzymatic reactions, respectively. Hypoxia caused an increase in HIF1α mRNA expressions and protein levels and shifted the metabolic states to anaerobic glycolysis, as demonstrated by increased glucose consumption and lactate production, as well as decreased O2 consumption and pHe. Furthermore, we demonstrated that GLUT1 and CA9 mRNA expressions simultaneously increased, in line with HIF1α expression. In conclusion, modulation of the extracellular environment of human BCSCs through hypoxia shifedt the metabolic state of BCSCs to anaerobic glycolysis, which might be associated with GLUT1 and CA9 expressions regulated by HIFlα transcription factor.

  5. TCDD-induced mitochondrial superoxide production does not lead to mitochondrial degeneration or genomic instability in human SH-SY5Y neuroblastoma cells.

    PubMed

    Luukkonen, Jukka; Höytö, Anne; Viluksela, Matti; Juutilainen, Jukka; Naarala, Jonne

    2017-10-01

    Several genotoxic and non-genotoxic agents have been reported to cause delayed genetic damage in the progeny of the exposed cells. Such induced genomic instability (IGI) may be a driving force in carcinogenesis, and it is thus highly important to understand the cellular events accompanying it. The aim of this study was to investigate whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects mitochondrial integrity and can consequently induce genomic instability. Mitochondrial integrity was evaluated by measuring mitochondrial superoxide production, mitochondrial membrane potential, and mitochondrial activity. Micronucleus formation was used to assess immediate genetic damage and IGI. The assays were performed either immediately, 8 or 15d after the exposure. Mitochondrial superoxide production was increased by TCDD immediately after the exposure. No consistent effects on mitochondrial integrity were observed at later time points, although slightly decreased mitochondrial membrane potential at 8d and increased mitochondrial superoxide potential production at 15 after exposure were observed in the TCDD-exposed cells. TCDD did not cause immediate genetic damage, and significant IGI was not observed. In conclusion, the present results suggest that immediate TCDD-induced increase in mitochondrial superoxide level does not lead to persistent loss of mitochondrial integrity or IGI in human SH-SY5Y neuroblastoma cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Modulatory effect of interleukin-10 on the production of platelet-activating factor and superoxide anions by human leucocytes.

    PubMed Central

    Bussolati, B; Mariano, F; Montrucchio, G; Piccoli, G; Camussi, G

    1997-01-01

    We observed that human monocytes (MO) and polymorphonuclear neutrophils (PMN) stimulated by lipopolysaccharide (LPS) produce platelet-activating factor (PAF) in a pattern characterized by an early and a delayed peak of synthesis. The early peak of PAF synthesis was due to a direct stimulation of these cells through mCD14 receptor as it was inhibited by anti-CD14 monoclonal antibody. The delayed and sustained peak of PAF synthesis was dependent on protein synthesis and cytokine production as shown by the inhibitory effect of cycloheximide on both MO and PMN, and of anti-tumour necrosis factor-alpha (anti-TNF-alpha) and of anti-interleukin-8 (anti-IL-8) neutralizing antibodies on MO and PMN respectively. IL-10 completely prevented this second, cytokine-dependent peak of PAF synthesis. In contrast, IL-10 markedly enhanced the first peak of PAF synthesis both in MO and PMN. Moreover, IL-10 was shown to modulate the production of superoxide anions (O2-) on both MO and PMN. As suggested by previous studies, IL-10 inhibited the delayed production of O2-. In the present study, we observed that IL-10 directly stimulated an early production of O2-. In addition, IL-10 enhanced the synthesis of O2- by MO and PMN challenged with LPS. The IL-10-induced O2- production was dependent, at least in part, from its effect on PAF synthesis, as it was inhibited by the PAF receptor antagonist WEB 2170. These results suggest that IL-10 may upregulate the early synthesis of PAF and O2- triggered by direct LPS stimulation, whereas it may downregulate the delayed production of these mediators. PMID:9155653

  7. A Multinuclear Copper(I) Cluster Forms the Dimerization Interface in Copper-Loaded Human Copper Chaperone for Superoxide Dismutase

    SciTech Connect

    Stasser, J.P.; Siluvai, G.S.; Barry, A.N.; Blackburn, N.J.

    2009-06-04

    Copper binding and X-ray aborption spectroscopy studies are reported on untagged human CCS (hCCS; CCS = copper chaperone for superoxide dismutase) isolated using an intein self-cleaving vector and on single and double Cys to Ala mutants of the hCCS MTCQSC and CSC motifs of domains 1 (D1) and 3 (D3), respectively. The results on the wild-type protein confirmed earlier findings on the CCS-MBP (maltose binding protein) constructs, namely, that Cu(I) coordinates to the CXC motif, forming a cluster at the interface of two D3 polypeptides. In contrast to the single Cys to Ser mutations of the CCS-MBP protein (Stasser, J. P., Eisses, J. F., Barry, A. N., Kaplan, J. H., and Blackburn, N. J. (2005) Biochemistry 44, 3143-3152), single Cys to Ala mutations in D3 were sufficient to eliminate cluster formation and significantly reduce CCS activity. Analysis of the intensity of the Cu-Cu cluster interaction in C244A, C246A, and C244/246A variants suggested that the nuclearity of the cluster was greater than 2 and was most consistent with a Cu4S6 adamantane-type species. The relationship among cluster formation, oligomerization, and metal loading was evaluated. The results support a model in which Cu(I) binding converts the apo dimer with a D2-D2 interface to a new dimer connected by cluster formation at two D3 CSC motifs. The predominance of dimer over tetramer in the cluster-containing species strongly suggests that the D2 dimer interface remains open and available for sequestering an SOD1 monomer. This work implicates the copper cluster in the reactive form and adds detail to the cluster nuclearity and how copper loading affects the oligomerization states and reactivity of CCS for its partner SOD1.

  8. Effect of a Korean traditional formulation, Hwaotang, on superoxide generation in human neutrophils, platelet aggregation in human blood, and nitric oxide, prostaglandin E2 production and paw oedema induced by carrageenan in mice.

    PubMed

    Park, Won-Hwan; Park, Soo-Young; Kim, Hyung-Min; Kim, Cheorl-Ho

    2004-02-01

    Hwaotang, a traditional Korean medicinal formulation, is a dried decoctum of a mixture of 7 herbal medicines, consisting of Angelica gigantis Radix, Rehmanniae radix, Paeoniae radix, Ciniamomi cortex, Cnidii rhizoma, Persicae semen and Carthami flos. We have investigated that Hwaotang water extract (HOT) has various effects on stimulus-induced superoxide generation in human neutrophils. The effects of HOT on superoxide generation in human neutrophils were investigated. HOT significantly inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in a concentration-dependent manner, but not that induced by arachidonic acid (AA). On the other hand, HOT enhanced superoxide generation induced by phorbol 12-myristate 13-acetate (PMA) in a concentration-dependent manner. The superoxide generation induced by PMA with HOT was suppressed by staurosporine, an inhibitor of protein kinase C, but was not suppressed by genistein, an inhibitor of protein tyrosine kinase. Tyrosyl phosphorylation of a 58 kDa protein, which was increased by fMLP, was inhibited by HOT. HOT also inhibited the generation of a 47 kDa protein and platelet aggregation in human blood. The results suggest that protein tyrosine kinase participates in fMLP-mediated superoxide generation by HOT-treated human neutrophils. HOT inhibited neutrophil functions, including degranulation, superoxide generation, and leukotriene B4 production, without any effect on 5-lipoxygenase activity. HOT reduced nitric oxide (NO) and prostaglandin E2 production in mouse peritoneal macrophages stimulated with lipopolysaccharide, whereas no influence on the activity of iNOS, COX-2 or COX-1 was observed. HOT significantly reduced mouse paw oedema induced by carrageenan. Western blot analysis showed that HOT reduced the expression of iNOS and COX-2. The results indicate that HOT exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and prostaglandin E2 production, which

  9. The extracellular interactome of the human adenovirus family reveals diverse strategies for immunomodulation

    PubMed Central

    Martinez-Martin, Nadia; Ramani, Sree R.; Hackney, Jason A.; Tom, Irene; Wranik, Bernd J.; Chan, Michelle; Wu, Johnny; Paluch, Maciej T.; Takeda, Kentaro; Hass, Philip E.; Clark, Hilary; Gonzalez, Lino C.

    2016-01-01

    Viruses encode secreted and cell-surface expressed proteins essential to modulate host immune defenses and establish productive infections. However, to date there has been no systematic study of the extracellular interactome of any human virus. Here we utilize the E3 proteins, diverse and rapidly evolving transmembrane-containing proteins encoded by human adenoviruses, as a model system to survey the extracellular immunomodulatory landscape. From a large-scale protein interaction screen against a microarray of more than 1,500 human proteins, we find and validate 51 previously unidentified virus–host interactions. Our results uncover conserved strategies as well as substantial diversity and multifunctionality in host targeting within and between viral species. Prominent modulation of the leukocyte immunoglobulin-like and signalling lymphocyte activation molecule families and a number of inhibitory receptors were identified as hubs for viral perturbation, suggesting unrecognized immunoregulatory strategies. We describe a virus–host extracellular interaction map of unprecedented scale that provides new insights into viral immunomodulation. PMID:27145901

  10. Morphological Characterization of Organized Extracellular Matrix Deposition by Ascorbic Acid-Stimulated Human Corneal Fibroblasts

    PubMed Central

    Guo, Xiaoqing; Hutcheon, Audrey E. K.; Melotti, Suzanna A.; Zieske, James D.; Trinkaus-Randall, Vickery; Ruberti, Jeffrey W.

    2016-01-01

    Purpose To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures. Methods Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of Vitamin C. The cells were allowed to synthesize a fibrillar ECM for up to five weeks. Constructs were assessed via light (phase contrast and differential interference contrast) and transmission (standard and quick freeze/deep etch) microscopy. Results Electron micrographs revealed stratified constructs with multiple parallel layers of cells and an extracellular matrix comprising parallel arrays of small, polydisperse fibrils (27–51 nm) which often alternate in direction. Differential interference contrast images demonstrated oriented ECM fibril arrays parallel to the plane of the construct while quick-freeze deep etch micrographs showed the details of the matrix interaction with fibroblasts via arrays of membrane surface structures. Conclusions Human keratocytes, cultured in a stable Vitamin C derivative, are capable of assembling extracellular matrix which comprise parallel arrays of ECM fibrils. The resulting constructs, which are highly cellular, exhibit morphology similar to the developing mammalian stroma where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggest a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes which govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue-engineering a biomimetic stroma. PMID:17724187

  11. Superoxide and Peroxynitrite in Atherosclerosis

    NASA Astrophysics Data System (ADS)

    White, C. Roger; Brock, Tommy A.; Chang, Ling-Yi; Crapo, James; Briscoe, Page; Ku, David; Bradley, William A.; Gianturco, Sandra H.; Gore, Jeri; Freeman, Bruce A.; Tarpey, Margaret M.

    1994-02-01

    The role of reactive oxygen species in the vascular pathology associated with atherosclerosis was examined by testing the hypothesis that impaired vascular reactivity results from the reaction of nitric oxide (^.NO) with superoxide (O^-_2), yielding the oxidant peroxynitrite (ONOO^-). Contractility studies were performed on femoral arteries from rabbits fed a cholesterol-supplemented diet. Cholesterol feeding shifted the EC50 for acetylcholine (ACh)-induced relaxation and impaired the maximal response to ACh. We used pH-sensitive liposomes to deliver CuZn superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) to critical sites of ^.NO reaction with O^-_2. Intravenously injected liposomes (3000 units of SOD per ml) augmented ACh-induced relaxation in the cholesterol-fed group to a greater extent than in controls. Quantitative immunocytochemistry demonstrated enhanced distribution of SOD in both endothelial and vascular smooth muscle cells as well as in the extracellular matrix. SOD activity in vessel homogenates of liposome-treated rabbits was also increased. Incubation of β very low density lipoprotein with ONOO^- resulted in the rapid formation of conjugated dienes and thiobarbituric acid-reactive substances. Our results suggest that the reaction of O^-_2 with ^.NO is involved in the development of atherosclerotic disease by yielding a potent mediator of lipoprotein oxidation, as well as by limiting ^.NO stimulation of vascular smooth muscle guanylate cyclase activity.

  12. Differential responses of normal human melanocytes to intra- and extracellular dsRNA.

    PubMed

    Wang, Suiquan; Liu, Dongyin; Jin, Rong; Zhu, Yiping; Xu, Aie

    2015-06-01

    Viral factor has been implicated in the etiopathogenesis of vitiligo. To elucidate the effects of viral double-stranded RNA (dsRNA) on melanocytes and to explore the underlying mechanisms, primary cultured normal human melanocytes were treated with synthetic viral dsRNA analog poly(I:C). The results demonstrated that poly(I:C)-triggered apoptosis when transfected into melanocytes, while extracellular poly(I:C) did not have that effect. Intracellular poly(I:C)-induced melanocyte death was decreased by RIG-I or MDA5 siRNA, but not by TLR3 siRNA. Both intracellular and extracellular poly(I:C) induced the expression of IFNB, TNF, IL6, and IL8. However, extracellular poly(I:C) demonstrated a much weaker induction capacity of cytokine genes than intracellular poly(I:C). Further analysis revealed that phosphorylation of TBK1, IRF3, IRF7, and TAK1 was differentially induced by intra- or extracellular poly(I:C). NFκB inhibitor Bay 11-7082 decreased the induction of all the cytokines by poly(I:C), suggesting the ubiquitous role of NFκB in the process. Poly(I:C) treatment also induced the phosphorylation of p38 and JNK in melanocytes. Both JNK and p38 inhibitors showed suppression on the cytokine induction by intra- or extracellular poly(I:C). However, only the JNK inhibitor decreased the intracellular poly(I:C)-induced melanocyte death. Taken together, this study provides the possible mechanism of viral factor in the pathogenesis of vitiligo.

  13. Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

    PubMed Central

    Mello, Paola de Andrade; Filippi-Chiela, Eduardo Cremonese; Nascimento, Jéssica; Beckenkamp, Aline; Santana, Danielle Bertodo; Kipper, Franciele; Casali, Emerson André; Nejar Bruno, Alessandra; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Wink, Marcia Rosângela; Lenz, Guido; Buffon, Andréia

    2014-01-01

    In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells. PMID:25103241

  14. Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein

    PubMed Central

    Kamphorst, Jurre J.; Nofal, Michel; Commisso, Cosimo; Hackett, Sean R.; Lu, Wenyun; Grabocka, Elda; Vander Heiden, Matthew G.; Miller, George; Drebin, Jeffrey A.; Bar-Sagi, Dafna; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-01-01

    Glucose and amino acids are key nutrients supporting cell growth. Amino acids are imported as monomers, but an alternative route induced by oncogenic KRAS involves uptake of extracellular proteins via macropinocytosis and subsequent lysosomal degradation of these proteins as a source of amino acids. In this study, we examined the metabolism of pancreatic ductal adenocarcinoma (PDAC), a poorly vascularized lethal KRAS-driven malignancy. Metabolomic comparisons of human PDAC and benign adjacent tissue revealed that tumor tissue was low in glucose, upper glycolytic intermediates, creatine phosphate and the amino acids glutamine and serine, two major metabolic substrates. Surprisingly, PDAC accumulated essential amino acids. Such accumulation could arise from extracellular proteins being degraded through macropinocytosis in quantities necessary to meet glutamine requirements, which in turn produces excess of most other amino acids. Consistent with this hypothesis, active macropinocytosis is observed in primary human PDAC specimens. Moreover, in the presence of physiological albumin, we found that cultured murine PDAC cells grow indefinitely in media lacking single essential amino acids, and replicate once in the absence of free amino acids. Growth under these conditions was characterized by simultaneous glutamine depletion and essential amino acid accumulation. Overall, our findings argue that the scavenging of extracellular proteins is an important mode of nutrient uptake in PDAC. PMID:25644265

  15. Extracellular Recordings of Patterned Human Pluripotent Stem Cell-Derived Cardiomyocytes on Aligned Fibers

    PubMed Central

    Minami, Itsunari; Yu, Leqian; Nakajima, Minako; Qiao, Jing; Shimono, Ken; Nakatsuji, Norio; Kotera, Hitetoshi; Chen, Yong

    2016-01-01

    Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) hold high potential for use in drug assessment and myocardial regeneration. To create tissue-like constructs of CMs for extracellular monitoring, we placed aligned fibers (AFs) on the surface of a microelectrode array and then seeded hiPSC-CMs for subsequent monitoring for 14 days. As expected, the CMs organized into anisotropic and matured tissue and the extracellular recordings showed reduced premature beating higher signal amplitude and a higher probability of T-wave detection as compared to the culture without fibers. The CMs on the aligned fibers samples also exhibited anisotropic propagation of the field potential. These results therefore suggest that the hiPSC-CMs cultured on AFs can be used more reliably for cell based assays. PMID:27446217

  16. Mitochondrial manganese superoxide dismutase mRNA expression in human chorioamniotic membranes and its association with labor, inflammation, and infection.

    PubMed

    Than, Nandor Gabor; Romero, Roberto; Tarca, Adi L; Draghici, Sorin; Erez, Offer; Chaiworapongsa, Tinnakorn; Kim, Yeon Mee; Kim, Sun Kwon; Vaisbuch, Edi; Tromp, Gerard

    2009-11-01

    Human parturition is characterized by the activation of genes involved in acute inflammatory responses in the fetal membranes. Manganese superoxide dismutase (Mn SOD) is a mitochondrial enzyme that scavenges reactive oxygen species (ROS). Mn SOD is up-regulated in sites of inflammation and has an important role in the down-regulation of acute inflammatory processes. Therefore, the aim of this study was to determine the differences in Mn SOD mRNA expression in the fetal membranes in patients with term and preterm labor (PTL) as well as in acute chorioamnionitis. Fetal membranes were obtained from patients in the following groups: (1) term not in labor (n = 29); (2) term in labor (n = 29); (3) spontaneous PTL with intact mebranes (n = 16); (4) PTL with histological chorioamnionitis (n = 12); (5) preterm prelabor rupture of the membranes (PPROM; n = 17); and (6) PPROM with histological chorioamnionitis (n = 21). Mn SOD mRNA expression in the membranes was determined by quantitative real-time reverse transcription-polymerase chain reaction. (1) Mn SOD mRNA expression was higher in the fetal membranes of patients at term in labor than those not in labor (2.4-fold; p = 0.02); (2) the amount of Mn SOD mRNA in the fetal membranes was higher in PTL than in term labor or in PPROM (7.2-fold, p = 0.03; 3.2-fold, p = 0.03, respectively); (3) Mn SOD mRNA expression was higher when histological chorioamnionitis was present both among patients with PPROM (3.8-fold, p = 0.02) and with PTL (5.4-fold, p = 0.02) than in patients with these conditions without histological chorioamnionitis; (4) expression of Mn SOD mRNA was higher in PTL with chorioamnionitis than in PPROM with chorioamnionitis (4.3-fold, p = 0.03). The increase in Mn SOD mRNA expression by fetal membranes in term labor and in histological chorioamnionitis in PTL and PPROM suggests that the fetus deploys anti-oxidant mechanisms to constrain the inflammatory processes in the chorioamniotic membranes.

  17. UV-induced extracellular factor from human fibroblasts communicates the UV response to nonirradiated cells

    SciTech Connect

    Schorpp, M.; Mallick, U.; Rahmsdorf, H.J.; Herrlich, P.

    1984-07-01

    Ultraviolet light enhances the synthesis of at least eight abundant proteins in human fibroblasts within 2 hr. These proteins are identical with those induced by the tumor promoter TPA. The inducing signal is generated by DNA damage, as these proteins are induced by lower doses of UV in fibroblasts from patients with Cockayne's syndrome or Xeroderma pigmentosum. In the supernatant of UV-treated cells, a heat-labile ammonium sulfate precipitable factor of more than 10 kd (EPIF) was detected which, upon transfer to nonirradiated cells, mimicked UV in the UV-induced synthesis of gene products. The response to UV, TPA, or EPIF was inhibited by fluocinolone acetonide, but not by retinoic acid, protease inhibitors, or superoxide dismutase.

  18. Development of biomimetic nanocomposites as bone extracellular matrix for human osteoblastic cells.

    PubMed

    Bhowmick, Arundhati; Mitra, Tapas; Gnanamani, Arumugam; Das, Manas; Kundu, Patit Paban

    2016-05-05

    Here, we have developed biomimetic nanocomposites containing chitosan, poly(vinyl alcohol) and nano-hydroxyapatite-zinc oxide as bone extracellular matrix for human osteoblastic cells and characterized by Fourier transform infrared spectroscopy, powder X-ray diffraction. Scanning electron microscopy images revealed interconnected macroporous structures. Moreover, in this study, the problem related to fabricating a porous composite with good mechanical strength has been resolved by incorporating 5wt% of nano-hydroxyapatite-zinc oxide into chitosan-poly(vinyl alcohol) matrix; the present composite showed high tensile strength (20.25MPa) while maintaining appreciable porosity (65.25%). These values are similar to human cancellous bone. These nanocomposites also showed superior water uptake, antimicrobial and biodegradable properties than the previously reported results. Compatibility with human blood and pH was observed, indicating nontoxicity of these materials to the human body. Moreover, proliferation of osteoblastic MG-63 cells onto the nanocomposites was also observed without having any negative effect.

  19. The effects of caffeic, coumaric and ferulic acids on proliferation, superoxide production, adhesion and migration of human tumor cells in vitro.

    PubMed

    Nasr Bouzaiene, Nouha; Kilani Jaziri, Soumaya; Kovacic, Hervé; Chekir-Ghedira, Leila; Ghedira, Kamel; Luis, José

    2015-11-05

    Reactive oxygen species are well-known mediators of various biological responses. In this study, we examined the effect of three phenolic acids, caffeic, coumaric and ferulic acids, on superoxide anion production, adhesion and migration of human lung (A549) and colon adenocarcinoma (HT29-D4) cancer cell lines. Proliferation of both tumor cells was inhibited by phenolic acids. Caffeic, coumaric and ferulic acids also significantly inhibited superoxide production in A549 and HT29-D4 cells. Superoxide anion production decreased by 92% and 77% at the highest tested concentration (200 µM) of caffeic acid in A549 and HT29-D4 cell lines respectively. Furthermore, A549 and HT29-D4 cell adhesion was reduced by 77.9% and 79.8% respectively at the higher tested concentration of ferulic acid (200 µM). Migration assay performed towards A549 cell line, revealed that tested compounds reduced significantly cell migration. At the highest concentration tested (200 µM), the covered surface was 7.7%, 9.5% and 35% for caffeic, coumaric or ferulic acids, respectively. These results demonstrate that caffeic, coumaric and ferulic acids may participate as active ingredients in anticancer agents against lung and colon cancer development, at adhesion and migration steps of tumor progression.

  20. Bavachalcone-induced manganese superoxide dismutase expression through the AMP-activated protein kinase pathway in human endothelial cells.

    PubMed

    Dang, Yanqi; Ling, Shuang; Duan, Ju; Ma, Jing; Ni, Rongzhen; Xu, Jin-Wen

    2015-01-01

    Mitochondrial oxidative stress has been suggested as a major etiological factor in cardiovascular diseases. Manganese superoxide dismutase (MnSOD) is an essential antioxidant mitochondrial enzyme. Although polyphenols can induce MnSOD expression, their mechanism of action remains unclear. We examined the effect of bavachalcone, a bioactive compound isolated from Psoralea corylifolia, on MnSOD protein expression and explored whether this effect is mediated through the AMP-activated protein kinase (AMPK) signaling pathway. Our data showed that bavachalcone enhanced the luciferase activity of the MnSOD promoter and increased MnSOD mRNA and protein expressions. Moreover, bavachalcone suppressed the mitochondrial superoxide production in endothelial cells. Conversely, bavachalcone stimulated liver kinase B1 and AMPKα phosphorylation. mRNA interference by using short hairpin RNA (shRNA) of AMPK inhibited bavachalcone-induced MnSOD expression. A-769662, an AMPK activator, also stimulated AMPK activity and increased MnSOD expression. Furthermore, AMPK knockdown by shRNA-AMPK reversed the inhibitory effects of bavachalcone on mitochondrial superoxide production in endothelial cells. These findings indicate that bavachalcone can protect the endothelial function by increasing AMPK activity and MnSOD expression and reducing mitochondrial oxidative stress. .

  1. Host-Parasite Interaction: Parasite-Derived and -Induced Proteases That Degrade Human Extracellular Matrix

    PubMed Central

    Piña-Vázquez, Carolina; Reyes-López, Magda; Ortíz-Estrada, Guillermo; de la Garza, Mireya; Serrano-Luna, Jesús

    2012-01-01

    Parasitic protozoa are among the most important pathogens worldwide. Diseases such as malaria, leishmaniasis, amoebiasis, giardiasis, trichomoniasis, and trypanosomiasis affect millions of people. Humans are constantly threatened by infections caused by these pathogens. Parasites engage a plethora of surface and secreted molecules to attach to and enter mammalian cells. The secretion of lytic enzymes by parasites into host organs mediates critical interactions because of the invasion and destruction of interstitial tissues, enabling parasite migration to other sites within the hosts. Extracellular matrix is a complex, cross-linked structure that holds cells together in an organized assembly and that forms the basement membrane lining (basal lamina). The extracellular matrix represents a major barrier to parasites. Therefore, the evolution of mechanisms for connective-tissue degradation may be of great importance for parasite survival. Recent advances have been achieved in our understanding of the biochemistry and molecular biology of proteases from parasitic protozoa. The focus of this paper is to discuss the role of protozoan parasitic proteases in the degradation of host ECM proteins and the participation of these molecules as virulence factors. We divide the paper into two sections, extracellular and intracellular protozoa. PMID:22792442

  2. Extracellular protonation modulates cell-cell interaction mechanics and tissue invasion in human melanoma cells.

    PubMed

    Hofschröer, Verena; Koch, Kevin Alexander; Ludwig, Florian Timo; Friedl, Peter; Oberleithner, Hans; Stock, Christian; Schwab, Albrecht

    2017-02-13

    Detachment of cells from the primary tumour precedes metastatic progression by facilitating cell release into the tissue. Solid tumours exhibit altered pH homeostasis with extracellular acidification. In human melanoma, the Na(+)/H(+) exchanger NHE1 is an important modifier of the tumour nanoenvironment. Here we tested the modulation of cell-cell-adhesion by extracellular pH and NHE1. MV3 tumour spheroids embedded in a collagen matrix unravelled the efficacy of cell-cell contact loosening and 3D emigration into an environment mimicking physiological confinement. Adhesive interaction strength between individual MV3 cells was quantified using atomic force microscopy and validated by multicellular aggregation assays. Extracellular acidification from pHe7.4 to 6.4 decreases cell migration and invasion but increases single cell detachment from the spheroids. Acidification and NHE1 overexpression both reduce cell-cell adhesion strength, indicated by reduced maximum pulling forces and adhesion energies. Multicellular aggregation and spheroid formation are strongly impaired under acidification or NHE1 overexpression. We show a clear dependence of melanoma cell-cell adhesion on pHe and NHE1 as a modulator. These effects are opposite to cell-matrix interactions that are strengthened by protons extruded via NHE1. We conclude that these opposite effects of NHE1 act synergistically during the metastatic cascade.

  3. Extracellular protonation modulates cell-cell interaction mechanics and tissue invasion in human melanoma cells

    PubMed Central

    Hofschröer, Verena; Koch, Kevin Alexander; Ludwig, Florian Timo; Friedl, Peter; Oberleithner, Hans; Stock, Christian; Schwab, Albrecht

    2017-01-01

    Detachment of cells from the primary tumour precedes metastatic progression by facilitating cell release into the tissue. Solid tumours exhibit altered pH homeostasis with extracellular acidification. In human melanoma, the Na+/H+ exchanger NHE1 is an important modifier of the tumour nanoenvironment. Here we tested the modulation of cell-cell-adhesion by extracellular pH and NHE1. MV3 tumour spheroids embedded in a collagen matrix unravelled the efficacy of cell-cell contact loosening and 3D emigration into an environment mimicking physiological confinement. Adhesive interaction strength between individual MV3 cells was quantified using atomic force microscopy and validated by multicellular aggregation assays. Extracellular acidification from pHe7.4 to 6.4 decreases cell migration and invasion but increases single cell detachment from the spheroids. Acidification and NHE1 overexpression both reduce cell-cell adhesion strength, indicated by reduced maximum pulling forces and adhesion energies. Multicellular aggregation and spheroid formation are strongly impaired under acidification or NHE1 overexpression. We show a clear dependence of melanoma cell-cell adhesion on pHe and NHE1 as a modulator. These effects are opposite to cell-matrix interactions that are strengthened by protons extruded via NHE1. We conclude that these opposite effects of NHE1 act synergistically during the metastatic cascade. PMID:28205573

  4. Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency.

    PubMed Central

    Levy, D N; Refaeli, Y; Weiner, D B

    1995-01-01

    The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics. PMID:7815499

  5. Bioactive secondary metabolites of a marine Bacillus sp. inhibit superoxide generation and elastase release in human neutrophils by blocking formyl peptide receptor 1.

    PubMed

    Yang, Shun-Chin; Lin, Chwan-Fwu; Chang, Wen-Yi; Kuo, Jimmy; Huang, Yin-Ting; Chung, Pei-Jen; Hwang, Tsong-Long

    2013-06-03

    It is well known that overwhelming neutrophil activation is closely related to acute and chronic inflammatory injuries. Formyl peptide receptor 1 (FPR1) plays an important role in activation of neutrophils and may represent a potent therapeutic target in inflammatory diseases. In the present study, we demonstrated that IA-LBI07-1 (IA), an extract of bioactive secondary metabolites from a marine Bacillus sp., has anti-inflammatory effects in human neutrophils. IA significantly inhibited superoxide generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated neutrophils, but failed to suppress the cell responses activated by non-FPR1 agonists. IA did not alter superoxide production and elastase activity in cell-free systems. IA also attenuated the downstream signaling from FPR1, such as the Ca2+, MAP kinases and AKT pathways. In addition, IA inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analogue of FMLP, to FPR1 in human neutrophils and FPR1-transfected HEK293 cells. Taken together, these results show that the anti-inflammatory effects of IA in human neutrophils are through the inhibition of FPR1. Also, our data suggest that IA may have therapeutic potential to decrease tissue damage induced by human neutrophils.

  6. Neutrophils extracellular traps damage Naegleria fowleri trophozoites opsonized with human IgG.

    PubMed

    Contis-Montes de Oca, A; Carrasco-Yépez, M; Campos-Rodríguez, R; Pacheco-Yépez, J; Bonilla-Lemus, P; Pérez-López, J; Rojas-Hernández, S

    2016-08-01

    Naegleria fowleri infects humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N. fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. NETs are composed of nuclear DNA combined with histones and antibacterial proteins, and these structures are released from the cell to direct its antimicrobial attack. In this work, we evaluate the capacity of N. fowleri to induce the liberation of NETs by human PMN cells. Neutrophils were cocultured with unopsonized or IgG-opsonized N. fowleri trophozoites. DNA, histone, myeloperoxidase (MPO) and neutrophil elastase (NE) were stained, and the formation of NETs was evaluated by confocal microscopy and by quantifying the levels of extracellular DNA. Our results showed N. fowleri induce the liberation of NETs including release of MPO and NE by human PMN cells as exposure interaction time is increased, but N. fowleri trophozoites evaded killing. However, when trophozoites were opsonized, they were susceptible to the neutrophils activity. Therefore, our study suggests that antibody-mediated PMNs activation through NET formation may be crucial for antimicrobial responses against N. fowleri. © 2016 John Wiley & Sons Ltd.

  7. Influence of extracellular matrix on cytokine stimulated pro-labour gene expression in human uterine myocytes.

    PubMed

    Engineer, Neelam; Sooranna, Suren R; Liang, Zhiqing; Bennett, Phillip R; Johnson, Mark R

    2008-11-01

    Cellular function is modulated by the interaction with the extracellular matrix within the myometrium. We formed the hypothesis that the cytokine-stimulated pro-labour gene expression by human uterine smooth muscle cells would be increased by growing the cells on collagen-coated plates. Primary cultures of human uterine smooth muscle cells grown on uncoated plates and on plates coated with collagen were exposed to the inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta and interleukin-6) and assessed the messenger RNA expression of oxytocin receptor, interleukin-8, prostaglandin H synthase type-2 and prostaglandin F(2) alpha receptor. Basal pro-labour gene expression was unaffected by collagen coating and the response to the inflammatory cytokines was similar for oxytocin receptor and prostaglandin H synthase type-2, but appeared to be reduced for interleukin-8 and enhanced for FP. Collagen coating made no significant impact on basal integrin expression and interleukin-1beta induced phosphorylation of extracellular-regulated-kinase1/2 and RelA subunit of nuclear factor-kappa B (p65). We conclude that growing human uterine smooth muscle cells on collagen-coated plates may modulate the pro-labour gene response to the inflammatory cytokines.

  8. Preferential chemotaxis of activated human CD4+ T cells by extracellular cyclophilin A

    PubMed Central

    Damsker, Jesse M.; Bukrinsky, Michael I.; Constant, Stephanie L.

    2010-01-01

    The recruitment and trafficking of leukocytes are essential aspects of the inflammatory process. Although chemokines are thought to be the main regulators of cell trafficking, extracellular cyclophilins have been shown recently to have potent chemoattracting properties for human leukocytes. Cyclophilins are secreted by a variety of cell types and are detected at high levels in tissues with ongoing inflammation. CD147 has been identified as the main signaling receptor for cyclophilin A (CypA) on human leukocytes. It is interesting that the expression of CD147 is elevated on leukocytes from inflamed tissue, suggesting a correlation among the presence of extracellular cyclophilins, CD147 expression, and inflammatory responses. Thus, cyclophilin-CD147 interactions may contribute directly to the recruitment of leukocytes into inflamed tissues. In the current studies, we show that activated human T lymphocytes express elevated levels of CD147, compared with resting T cells and that these activated T cells migrate more readily to CypA than resting cells. Furthermore, we show that unlike resting CD4+ T cells, the cyclophilin-mediated migration of activated T cells does not require interaction with heparan sulfate receptors but instead, is dependent on CD147 interaction alone. Such findings suggest that cyclophilin-CD147 interactions will be most potent when leukocytes are in an activated state, for example, during inflammatory responses. Thus, targeting cyclophilin-CD147 interactions may provide a novel approach for alleviating tissue inflammation. PMID:17540735

  9. Biological Superoxide In Manganese Oxide Formation

    NASA Astrophysics Data System (ADS)

    Hansel, C.; Learman, D.; Zeiner, C.; Santelli, C. M.

    2011-12-01

    Manganese (Mn) oxides are among the strongest sorbents and oxidants within the environment, controlling the fate and transport of numerous elements and the degradation of recalcitrant carbon. Both bacteria and fungi mediate the oxidation of Mn(II) to Mn(III/IV) oxides but the genetic and biochemical mechanisms responsible remain poorly understood. Furthermore, the physiological basis for microbial Mn(II) oxidation remains an enigma. We have recently reported that a common marine bacterium (Roseobacter sp. AzwK-3b) oxidizes Mn(II) via reaction with extracellular superoxide (O2-) produced during exponential growth. Here we expand this superoxide-mediated Mn(II) oxidation pathway to fungi, introducing a surprising homology between prokaryotic and eukaryotic metal redox processes. For instance, Stibella aciculosa, a common soil Ascomycete filamentous fungus, precipitates Mn oxides at the base of asexual reproductive structures (synnemata) used to support conidia (Figure 1). This distribution is a consequence of localized production of superoxide (and it's dismutation product hydrogen peroxide, H2O2), leading to abiotic oxidation of Mn(II) by superoxide. Disruption of NADPH oxidase activity using the oxidoreductase inhibitor DPI leads to diminished cell differentiation and subsequent Mn(II) oxidation inhibition. Addition of Cu(II) (an effective superoxide scavenger) leads to a concentration dependent decrease in Mn oxide formation. We predict that due to the widespread production of extracellular superoxide within the fungal and likely bacterial kingdoms, biological superoxide may be an important contributor to the cycling of Mn, as well as other metals (e.g., Hg, Fe). Current and future explorations of the genes and proteins involved in superoxide production and Mn(II) oxidation will ideally lend insight into the physiological and biochemical basis for these processes.

  10. Differential expression of intracellular and extracellular CB(2) cannabinoid receptor protein by human peripheral blood leukocytes.

    PubMed

    Castaneda, Julie T; Harui, Airi; Kiertscher, Sylvia M; Roth, Jeffrey D; Roth, Michael D

    2013-03-01

    mRNA encoding for the CB(2) cannabinoid receptor is expressed by many subsets of human peripheral blood leukocytes (PBL), but little is known about the resulting protein expression and function. Employing clones from the A549 and 293T cell lines that were constructed to express both full-length human CB(2) and GFP, we developed a flow cytometry assay for characterizing CB(2) protein expression. A monoclonal antibody directed against human CB(2) selectively stained the surface of transduced but not parental cell lines. When cells were fixed and permeabilized, imaging flow cytometry identified large stores of intracellular protein. Total cellular staining for CB(2) corresponded closely with the level of GFP expression. When exposed to Δ(9)-tetrahydrocannabinol, CB(2)-expressing cells internalized cell surface CB(2) receptors in a time- and dose-dependent manner. Applying these approaches to human PBL, CB(2) protein was identified on the surface of human B cells but not on T cells or monocytes. In contrast, when PBL were fixed and permeabilized, intracellular CB(2) expression was readily detected in all three subsets by both conventional and imaging flow cytometry. Similar to the protein expression pattern observed in fixed and permeabilized PBL, purified B cells, T cells, and monocytes expressed relatively equal levels of CB(2) mRNA by quantitative real-time RT-PCR. Our findings confirm that human PBL express CB(2) protein but that its distribution is predominantly intracellular with only B cells expressing CB(2) protein at the extracellular membrane. The differential role of intracellular and extracellular CB(2) receptors in mediating ligand signaling and immune function remains to be determined.

  11. The human Krebs cycle 2-oxoglutarate dehydrogenase complex creates an additional source of superoxide/hydrogen peroxide from 2-oxoadipate as alternative substrate.

    PubMed

    Nemeria, Natalia S; Gerfen, Gary; Guevara, Elena; Nareddy, Pradeep Reddy; Szostak, Michal; Jordan, Frank

    2017-07-01

    Recently, we reported that the human 2-oxoglutarate dehydrogenase (hE1o) component of the 2-oxoglutarate dehydrogenase complex (OGDHc) could produce the reactive oxygen species superoxide and hydrogen peroxide (detected by chemical means) from its substrate 2-oxoglutarate (OG), most likely concurrently with one-electron oxidation by dioxygen of the thiamin diphosphate (ThDP)-derived enamine intermediate to a C2α-centered radical (detected by Electron Paramagnetic Resonance) [Nemeria et al., 2014 [17]; Ambrus et al. 2015 [18

  12. Extracellular nucleotides inhibit growth of human oesophageal cancer cells via P2Y2-receptors

    PubMed Central

    Maaser, K; Höpfner, M; Kap, H; Sutter, A P; Barthel, B; von Lampe, B; Zeitz, M; Scherübl, H

    2002-01-01

    Extracellular ATP is known to inhibit growth of various tumours by activating specific purinergic receptors (P2-receptors). Since the therapy of advanced oesophageal cancer is unsatisfying, new therapeutic approaches are mandatory. Here, we investigated the functional expression and potential antiproliferative effects of P2-purinergic receptors in human oesophageal cancer cells. Prolonged incubation of primary cell cultures of human oesophageal cancers as well as of the squamous oesophageal cancer cell line Kyse-140 with ATP or its stable analogue ATPγS dose-dependently inhibited cell proliferation. This was due to both an induction of apoptosis and cell cycle arrest. The expression of P2-receptors was examined by RT-PCR, immunocytochemistry, and [Ca2+]i-imaging. Application of various extracellular nucleotides dose-dependently increased [Ca2+]i. The rank order of potency was ATP=UTP>ATPγS>ADP=UDP. 2-methylthio-ATP and α,β-methylene-ATP had no effects on [Ca2+]i. Complete cross-desensitization between ATP and UTP was observed. Moreover, the phospholipase C inhibitor U73122 dose-dependently reduced the ATP triggered [Ca2+]i signal. The pharmacological features strongly suggest the functional expression of G-protein coupled P2Y2-receptors in oesophageal squamous cancer cells. P2Y2-receptors are involved in the antiproliferative actions of extracellular nucleotides. Thus, P2Y2-receptors are promising target proteins for innovative approaches in oesophageal cancer therapy. British Journal of Cancer (2002) 86, 636–644. DOI: 10.1038/sj/bjc/6600100 www.bjcancer.com © 2002 Cancer Research UK PMID:11870549

  13. Human hamstring tenocytes survive when seeded into a decellularized porcine Achilles tendon extracellular matrix.

    PubMed

    Lohan, Anke; Stoll, Christiane; Albrecht, Marit; Denner, Andreas; John, Thilo; Krüger, Kay; Ertel, Wolfgang; Schulze-Tanzil, Gundula

    2013-01-01

    Tendon ruptures and defects remain major orthopaedic challenges. Tendon healing is a time-consuming process, which results in scar tissue with an altered biomechanical competence. Using a xenogeneic tendon extracellular matrix (ECM) as a natural scaffold, which can be reseeded with autologous human tenocytes, might be a promising approach to reconstruct damaged tendons. For this purpose, the porcine Achilles (AS) tendons serving as a scaffold were histologically characterized in comparison to human cell donor tendons. AS tendons were decellularized and then reseeded with primary human hamstring tenocytes using cell centrifuging, rotating culture and cell injection techniques. Vitality testing, histology and glycosaminoglycan/DNA quantifications were performed to document the success of tendon reseeding. Porcine AS tendons were characterized by a higher cell and sulfated glycosaminoglycan content than human cell donor tendons. Complete decellularization could be achieved, but led to a wash out of sulfated glycosaminoglycans. Nevertheless, porcine tendon could be recellularized with vital human tenocytes. The recellularization led to a slight increase in cell number compared to the native tendon and some glycosaminoglycan recovery. This study indicates that porcine tendon can be de- and recellularized using adult human tenocytes. Future work should optimize cell distribution within the recellularized tendon ECM and consider tendon- and donor species-dependent differences.

  14. Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency

    PubMed Central

    Gonzalez, Rodolfo; Jennings, Lori L.; Knuth, Mark; Orth, Anthony P.; Klock, Heath E.; Ou, Weija; Feuerhelm, Julie; Hull, Mitchell V.; Koesema, Eric; Wang, Yuping; Zhang, Jia; Wu, Chunlei; Cho, Charles Y.; Su, Andrew I.; Batalov, Serge; Chen, Hong; Johnson, Kristen; Laffitte, Bryan; Nguyen, Deborah G.; Snyder, Evan Y.; Schultz, Peter G.; Harris, Jennifer L.; Lesley, Scott A.

    2010-01-01

    Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genome-wide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote long-term pluripotent growth of human embryonic stem cells without bFGF or TGFβ/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epithelium-derived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays. PMID:20133595

  15. Extracellular cholesterol-rich microdomains generated by human macrophages and their potential function in reverse cholesterol transport

    PubMed Central

    Ong, Daniel S.; Anzinger, Joshua J.; Leyva, Francisco J.; Rubin, Noa; Addadi, Lia; Kruth, Howard S.

    2010-01-01

    Previous studies have shown that cholesterol in atherosclerotic plaques is present in both intracellular and extracellular forms. In the current study, we investigated a mechanism for extracellular cholesterol accumulation and examined the capacity of this pool of cholesterol to be removed by cholesterol acceptors, a step in reverse cholesterol transport. Human monocyte-derived macrophages differentiated with macrophage-colony stimulating factor were incubated with acetylated LDL to allow cholesterol enrichment and processing. These macrophages were subsequently labeled with a monoclonal antibody that specifically detects ordered cholesterol arrays, revealing the presence of unesterified cholesterol-rich microdomains on the cell surfaces and in the extracellular matrix. Similar unesterified cholesterol-rich microdomains were present in human atherosclerotic plaques. Actin microfilaments functioned in microdomain deposition or maintenance, and Src family kinases regulated transfer of these microdomains from the cell surface onto the extracellular matrix. Mediators of reverse cholesterol transport, apolipoprotein A-I (apoA-I), and HDL were capable of removing these extracellular un-esterified cholesterol-rich microdomains. However, apoA-I removed the microdomains only when macrophages were present. ApoA-I removal of microdomains was blocked by glyburide and inhibitor of ATP-binding cassette transporter A1 (ABCA1) function. In summary, cultures of cholesterol-enriched human monocyte-derived macrophages generate extracellular unesterified cholesterol-rich microdomains, which can subsequently be removed by cholesterol acceptors and therefore potentially function in reverse cholesterol transport. PMID:20421591

  16. Human retinal pigment epithelial lysis of extracellular matrix: functional urokinase plasminogen activator receptor, collagenase, and elastase.

    PubMed Central

    Elner, Susan G

    2002-01-01

    PURPOSE: To show (1) human retinal pigment epithelial (HRPE) expression of functional urokinase plasminogen activator receptor (uPAR; CD87), (2) HRPE secretion of collagenase and elastase, (3) uPAR-dependent HRPE migration, and (4) uPAR expression in diseased human retinal tissue. METHODS: Immunohistochemistry for uPAR was performed on cultured HRPE cells and in sections of human retina. Double-immunofluorescent staining of live human RPE cells with anti-CR3 antibody (CD11b) was performed to demonstrate the physical proximity of this beta 2 integrin with uPAR and determine whether associations were dependent on RPE confluence and polarity. Extracellular proteolysis by HRPE uPAR was evaluated using fluorescent bodipy-BSA and assessed for specificity by plasminogen activator inhibitor-1 (PAI-1) inhibition. The effect of interleukin-1 beta (IL-1 beta) on uPAR expression was assessed. Collagenase and elastase secretion by unstimulated and IL-1-stimulated HRPE cells was measured by 3H-labelled collagen and elastin cleavage. HRPE-associated collagenase was also assessed by cleavage of fluorescent DQ-collagen and inhibited by phenanthroline. Using an extracellular matrix assay, the roles of uPAR and collagenase in HRPE migration were assessed. RESULTS: Immunoreactive uPAR was detected on cultured HRPE cells and increased by IL-1. On elongated, live HRPE cells, uPAR dissociated from CD11b (CR3) and translocated to anterior poles of migrating cells. Extracellular proteolysis was concentrated at sites of uPAR expression and specifically inhibited by PAI-1. Cultured HRPE cells secreted substantial, functional collagenase and elastase. IL-1 upregulated uPAR, collagenase, and elastase activities. Specific inhibition of uPAR, and to a lesser degree collagenase, reduced HRPE migration in matrix/gel assays. Immunoreactive uPAR was present along the HRPE basolateral membrane in retinal sections and in sections of diseased retinal tissue. CONCLUSIONS: HRPE cells express functional u

  17. Aggrecan-based extracellular matrix is an integral part of the human basal ganglia circuit.

    PubMed

    Brückner, G; Morawski, M; Arendt, T

    2008-01-24

    The extracellular matrix is known to be involved in neuronal communication and the regulation of plastic changes, and also considered to protect neurons and synapses against damage. The goal of this study was to investigate how major extracellular matrix components (aggrecan, link protein, hyaluronan) constitute the pathways of the nigral system in the human basal ganglia circuit affected by neurodegeneration in Parkinson's disease. Here we show that aggrecan- and link protein-related components form clear regional distribution patterns, whereas hyaluronan is widely distributed in gray and white matter. Two predominant phenotypes of the aggrecan-based matrix can be discriminated: (1) perineuronal nets (PNs) and (2) axonal coats (ACs) encapsulating preterminal fibers and synaptic boutons. Clearly contoured PNs are associated with GABAergic projection neurons in the external and internal division of the globus pallidus, the lateral and reticular part of the substantia nigra, as well as subpopulations of striatal and thalamic inhibitory interneurons. Dopaminergic nigral neurons are devoid of PNs but are contacted to a different extent by matrix-coated boutons forming subnucleus-specific patterns. A very dense network of ACs is characteristic especially of the posterior lateral cell groups of the compact substantia nigra (nigrosome 1). In the subthalamic nucleus and the lateral thalamic nuclei numerous AC-associated axons were attached to principal neurons devoid of PNs. We conclude from the region-specific patterns that the aggrecan-based extracellular matrix is adapted to the fast processing of sensorimotor activities which are the therapeutic target of surgery and deep brain stimulation in the treatment of advanced stages of Parkinson's disease.

  18. Extracellular Ca(2+)-dependent enhancement of cytocidal potency of zoledronic acid in human oral cancer cells.

    PubMed

    Inoue, Sayaka; Arai, Naoya; Tomihara, Kei; Takashina, Michinori; Hattori, Yuichi; Noguchi, Makoto

    2015-08-15

    Direct antitumor effects of bisphosphonates (BPs) have been demonstrated in various cancer cells in vitro. However, the effective concentrations of BPs are typically much higher than their clinically relevant concentrations. Oral cancers frequently invade jawbone and may lead to the release of Ca(2+) in primary lesions. We investigated the effects of the combined application of zoledronic acid (ZA) and Ca(2+) on proliferation and apoptosis of oral cancer cells. Human oral cancer cells, breast cancer cells, and colon cancer cells were treated with ZA at a wide range of concentrations in different Ca(2+) concentration environments. Under a standard Ca(2+) concentration (0.6mM), micromolar concentrations of ZA were required to inhibit oral cancer cell proliferation. Increasing extracellular Ca(2+) concentrations greatly enhanced the potency of the ZA cytocidal effect. The ability of Ca(2+) to enhance the cytocidal effects of ZA was negated by the Ca(2+)-selective chelator EGTA. In contrast, the cytocidal effect of ZA was less pronounced in breast and colon cancer cells regardless of whether extracellular Ca(2+) was elevated. In oral cancer cells incubated with 1.6mM Ca(2+), ZA up-regulated mitochondrial Bax expression and increased mitochondrial Ca(2+) uptake. This was associated with decreased mitochondrial membrane potential and increased release of cytochrome c. We suggest that ZA can specifically produce potent cytocidal activity in oral cancer cells in an extracellular Ca(2+)-dependent manner, implying that BPs may be useful for treatment of oral squamous cell carcinoma with jawbone invasion leading to the hypercalcemic state. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Changes in extracellular matrix composition regulate cyclooxygenase-2 expression in human mesangial cells.

    PubMed

    Alique, Matilde; Calleros, Laura; Luengo, Alicia; Griera, Mercedes; Iñiguez, Miguel Ángel; Punzón, Carmen; Fresno, Manuel; Rodríguez-Puyol, Manuel; Rodríguez-Puyol, Diego

    2011-04-01

    Glomerular diseases are characterized by a sustained synthesis and accumulation of abnormal extracellular matrix proteins, such as collagen type I. The extracellular matrix transmits information to cells through interactions with membrane components, which directly activate many intracellular signaling events. Moreover, accumulating evidence suggests that eicosanoids derived from cyclooxygenase (COX)-2 participate in a number of pathological processes in immune-mediated renal diseases, and it is known that protein kinase B (AKT) may act through different transcription factors in the regulation of the COX-2 promoter. The present results show that progressive accumulation of collagen I in the extracellular medium induces a significant increase of COX-2 expression in human mesangial cells, resulting in an enhancement in PGE(2) production. COX-2 overexpression is due to increased COX-2 mRNA levels. The study of the mechanism implicated in COX-2 upregulation by collagen I showed focal adhesion kinase (FAK) activation. Furthermore, we observed that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by collagen I and collagen I-induced COX-2 overexpression was abolished by PI3K and AKT inhibitors. Additionally, we showed that the cAMP response element (CRE) transcription factor is implicated. Finally, we studied COX-2 expression in an animal model, N(G)-nitro-l-arginine methyl ester hypertensive rats. In renal tissue and vascular walls, COX-2 and collagen type I content were upregulated. In summary, our results provide evidence that collagen type I increases COX-2 expression via the FAK/PI3K/AKT/cAMP response element binding protein signaling pathway.

  20. Air Revitalization Using Superoxides

    NASA Technical Reports Server (NTRS)

    Wydeven, Theodore; Wood, Peter C.; Spitze, L. A.

    1988-01-01

    Pellets made from powder mixtures of potassium superoxide, KO2, and calcium superoxide, Ca(O2)2, proven markedly superior to pellets of pure KO2 for adding O2 to and removing CO2 from atmospheric-pressure flow of humidified CO2 in He. Superoxides used extensively to supply O2 and scrub CO2 in variety of ambient-pressure life-support applications, including portable self-contained breathing apparatuses, spacecraft, and undersea submersible craft.

  1. Extracellular low pH affects circadian rhythm expression in human primary fibroblasts.

    PubMed

    Lee, Sang Kil; Achieng, Elsie; Maddox, Connie; Chen, Suephy C; Iuvone, P Michael; Fukuhara, Chiaki

    2011-12-16

    Circadian rhythm is a fundamental biological system involved in the regulation of various physiological functions. However, little is known about a nature or function of circadian clock in human primary cells. In the present study, we have applied in vitro real time circadian rhythm monitoring to study human clock properties using primary skin fibroblasts. Among factors that affect human physiology, slightly lower extracellular pH was chosen to test its effects on circadian rhythm expression. We established human primary fibroblast cultures obtained from three healthy subjects, stably delivered a circadian reporter gene Bmal1-luciferase, and recorded circadian rhythms in the culture medium at pH 7.2 and 6.7. At pH 7.2, robust and sustained circadian rhythms were observed with average period length 24.47 ± 0.03 h. Such rhythms were also found at pH 6.7; however, period length was significantly shortened to 22.60 ± 0.20, amplitude was increased, and damping rate was decreased. The effect of exposure to low pH on the period length was reversible. The shortened period was unlikely caused by factors affecting cell viability because cell morphology and MTT assay showed no significant difference between the two conditions. In summary, our results showed that the circadian rhythm expression is affected at pH 6.7 in human primary fibroblasts without affecting cell viability. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Full-thickness skin wound healing using human placenta-derived extracellular matrix containing bioactive molecules.

    PubMed

    Choi, Ji Suk; Kim, Jae Dong; Yoon, Hyun Soo; Cho, Yong Woo

    2013-02-01

    The human placenta, a complex organ, which facilitates exchange between the fetus and the mother, contains abundant extracellular matrix (ECM) components and well-preserved endogenous growth factors. In this study, we designed a new dermal substitute from human placentas for full-thickness wound healing. Highly porous, decellularized ECM sheets were fabricated from human placentas via homogenization, centrifugation, chemical and enzymatic treatments, molding, and freeze-drying. The physical structure and biological composition of human placenta-derived ECM sheets dramatically supported the regeneration of full-thickness wound in vivo. At the early stage, the ECM sheet efficiently absorbed wound exudates and tightly attached to the wound surface. Four weeks after implantation, the wound was completely closed, epidermic cells were well arranged and the bilayer structure of the epidermis and dermis was restored. Moreover, hair follicles and microvessels were newly formed in the ECM sheet-implanted wounds. Overall, the ECM sheet produced a dermal substitute with similar cellular organization to that of normal skin. These results suggest that human placenta-derived ECM sheets provide a microenvironment favorable to the growth and differentiation of cells, and positive modulate the healing of full-thickness wounds.

  3. The extracellular calcium-sensing receptor regulates human fetal lung development via CFTR.

    PubMed

    Brennan, Sarah C; Wilkinson, William J; Tseng, Hsiu-Er; Finney, Brenda; Monk, Bethan; Dibble, Holly; Quilliam, Samantha; Warburton, David; Galietta, Luis J; Kemp, Paul J; Riccardi, Daniela

    2016-02-25

    Optimal fetal lung growth requires anion-driven fluid secretion into the lumen of the developing organ. The fetus is hypercalcemic compared to the mother and here we show that in the developing human lung this hypercalcaemia acts on the extracellular calcium-sensing receptor, CaSR, to promote fluid-driven lung expansion through activation of the cystic fibrosis transmembrane conductance regulator, CFTR. Several chloride channels including TMEM16, bestrophin, CFTR, CLCN2 and CLCA1, are also expressed in the developing human fetal lung at gestational stages when CaSR expression is maximal. Measurements of Cl(-)-driven fluid secretion in organ explant cultures show that pharmacological CaSR activation by calcimimetics stimulates lung fluid secretion through CFTR, an effect which in humans, but not mice, was also mimicked by fetal hypercalcemic conditions, demonstrating that the physiological relevance of such a mechanism appears to be species-specific. Calcimimetics promote CFTR opening by activating adenylate cyclase and we show that Ca(2+)-stimulated type I adenylate cyclase is expressed in the developing human lung. Together, these observations suggest that physiological fetal hypercalcemia, acting on the CaSR, promotes human fetal lung development via cAMP-dependent opening of CFTR. Disturbances in this process would be expected to permanently impact lung structure and might predispose to certain postnatal respiratory diseases.

  4. Decellularized extracellular matrix derived from human adipose tissue as a potential scaffold for allograft tissue engineering.

    PubMed

    Choi, Ji Suk; Kim, Beob Soo; Kim, Jun Young; Kim, Jae Dong; Choi, Young Chan; Yang, Hyun-Jin; Park, Kinam; Lee, Hee Young; Cho, Yong Woo

    2011-06-01

    Decellularized tissues composed of extracellular matrix (ECM) have been clinically used to support the regeneration of various human tissues and organs. Most decellularized tissues so far have been derived from animals or cadavers. Therefore, despite the many advantages of decellularized tissue, there are concerns about the potential for immunogenicity and the possible presence of infectious agents. Herein, we present a biomaterial composed of ECM derived from human adipose tissue, the most prevalent, expendable, and safely harvested tissue in the human body. The ECM was extracted by successive physical, chemical, and enzymatic treatments of human adipose tissue isolated by liposuction. Cellular components including nucleic acids were effectively removed without significant disruption of the morphology or structure of the ECM. Major ECM components were quantified, including acid/pepsin-soluble collagen, sulfated glycosaminoglycan (GAG), and soluble elastin. In an in vivo experiment using mice, the decellularized ECM graft exhibited good compatibility to surrounding tissues. Overall results suggest that the decellularized ECM containing biological and chemical cues of native human ECM could be an ideal scaffold material not only for autologous but also for allograft tissue engineering.

  5. Propofol inhibits superoxide production, elastase release, and chemotaxis in formyl peptide-activated human neutrophils by blocking formyl peptide receptor 1.

    PubMed

    Yang, Shun-Chin; Chung, Pei-Jen; Ho, Chiu-Ming; Kuo, Chan-Yen; Hung, Min-Fa; Huang, Yin-Ting; Chang, Wen-Yi; Chang, Ya-Wen; Chan, Kwok-Hon; Hwang, Tsong-Long

    2013-06-15

    Neutrophils play a critical role in acute and chronic inflammatory processes, including myocardial ischemia/reperfusion injury, sepsis, and adult respiratory distress syndrome. Binding of formyl peptide receptor 1 (FPR1) by N-formyl peptides can activate neutrophils and may represent a new therapeutic target in either sterile or septic inflammation. Propofol, a widely used i.v. anesthetic, has been shown to modulate immunoinflammatory responses. However, the mechanism of propofol remains to be established. In this study, we showed that propofol significantly reduced superoxide generation, elastase release, and chemotaxis in human neutrophils activated by fMLF. Propofol did not alter superoxide generation or elastase release in a cell-free system. Neither inhibitors of γ-aminobutyric acid receptors nor an inhibitor of protein kinase A reversed the inhibitory effects of propofol. In addition, propofol showed less inhibitory effects in non-FPR1-induced cell responses. The signaling pathways downstream from FPR1, involving calcium, AKT, and ERK1/2, were also competitively inhibited by propofol. These results show that propofol selectively and competitively inhibits the FPR1-induced human neutrophil activation. Consistent with the hypothesis, propofol inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analog of fMLF, to FPR1 in human neutrophils, differentiated THP-1 cells, and FPR1-transfected human embryonic kidney-293 cells. To our knowledge, our results identify, for the first time, a novel anti-inflammatory mechanism of propofol by competitively blocking FPR1 in human neutrophils. Considering the importance of N-formyl peptides in inflammatory processes, our data indicate that propofol may have therapeutic potential to attenuate neutrophil-mediated inflammatory diseases by blocking FPR1.

  6. Inhibition of human CYP3A4, UGT1A6, and P-glycoprotein with halogenated xanthene food dyes and prevention by superoxide dismutase.

    PubMed

    Furumiya, Kenji; Mizutani, Takaharu

    2008-01-01

    Synthetic food dyes are xenobiotics, and, after ingestion, portions of these dyes may be absorbed and metabolized by phase I and II drug-metabolizing enzymes, and excreted by transporters of phase III enzymes. In the previous report, it was shown that inhibition of UDP-glucuronosyltrasnferase 1A6 occurred following ingestion of phloxine, erythrosine, and rose bengal present in 12 permitted synthetic food dyes. In this report, the influence of dyes was examined on CYP3A4, a major phase I drug-metabolizing enzyme, and P-glycoprotein, a major transporter by synthetic food dyes. Human cytochrome P-450 (CYP) 3A4 and P-glycoprotein were inhibited by xanthene food dyes. The IC(50) values of these dyes to inhibit CYP3A4 and P-glycoprotein were the same as the level of inhibition of UGT1A6 produced by three haloganated xanthene food dyes in the previous report, except acid red, which inhibited only CYP3A4. Data suggest that inhibition by dyes is not enzyme specific but may be in a membrane-specific or protein-specific manner, such as conformational changes in protein. In the previous study, it was suggested that inhibition by dyes depended upon light irradiation due to generation of (1)O2 from these dyes. In this study, the influence of superoxide dismutase and catalase on inhibition by dyes was examined. Superoxide dismutase but not catalase was effective in preventing the inhibition of UGT1A6 by the dyes. Data suggest that superoxide anions, originating from dyes via light irradiation, may attack drug-metabolizing enzymes. It is possible that red cosmetics containing phloxine, erythrosine, or rose bengal react with proteins in skin and may lead to skin damage.

  7. Human Adipose Tissue Derived Extracellular Matrix and Methylcellulose Hydrogels Augments and Regenerates the Paralyzed Vocal Fold

    PubMed Central

    Kim, Eun Na; Sung, Myung Whun; Kwon, Tack-Kyun; Cho, Yong Woo; Kwon, Seong Keun

    2016-01-01

    Vocal fold paralysis results from various etiologies and can induce voice changes, swallowing complications, and issues with aspiration. Vocal fold paralysis is typically managed using injection laryngoplasty with fat or synthetic polymers. Injection with autologous fat has shown excellent biocompatibility. However, it has several disadvantages such as unpredictable resorption rate, morbidities associated with liposuction procedure which has to be done in operating room under general anesthesia. Human adipose-derived extracellular matrix (ECM) grafts have been reported to form new adipose tissue and have greater biostability than autologous fat graft. Here, we present an injectable hydrogel that is constructed from adipose tissue derived soluble extracellular matrix (sECM) and methylcellulose (MC) for use in vocal fold augmentation. Human sECM derived from adipose tissue was extracted using two major steps—ECM was isolated from human adipose tissue and was subsequently solubilized. Injectable sECM/MC hydrogels were prepared by blending of sECM and MC. Sustained vocal fold augmentation and symmetric vocal fold vibration were accomplished by the sECM/MC hydrogel in paralyzed vocal fold which were confirmed by laryngoscope, histology and a high-speed imaging system. There were increased number of collagen fibers and fatty granules at the injection site without significant inflammation or fibrosis. Overall, these results indicate that the sECM/MC hydrogel can enhance vocal function in paralyzed vocal folds without early resorption and has potential as a promising material for injection laryngoplasty for stable vocal fold augmentation which can overcome the shortcomings of autologous fat such as unpredictable duration and morbidity associated with the fat harvest. PMID:27768757

  8. Expression and purification of the extracellular domain of human myelin protein zero.

    PubMed

    Bond, J P; Saavedra, R A; Kirschner, D A

    2001-12-01

    Myelin protein zero (P0), an adhesion protein of the immunoglobulin superfamily, is the major protein of peripheral nervous system myelin in higher vertebrates. Protein zero is required for the formation and maintenance of myelin structure in the internode, likely through homophilic interactions at both the extracellular and the intracellular domains. Mutations and deletions in the P0 gene correlate with hereditary peripheral neuropathies of varying severity. Comparisons between the human and rat isoforms, whose three-dimensional structure has been determined by X-ray crystallography, suggest that these disease-associated genetic alterations lead to structural changes in the protein that alter P0-P0 interactions and hence affect myelin functionality. Knowing the crystal structures of native and altered human P0 isoforms could help to elucidate the structural changes in myelin membrane packing that underlie the altered functionality. Alterations of P0 extracellular domain (P0-ED) are of additional interest as previous X-ray diffraction studies on myelin membrane packing suggest that P0-ED molecules can assume distinct adhesive arrangements. Here, we describe an improved method to express and purify human P0-ED (hP0-ED) suitable for crystallographic analysis. A fusion protein consisting of maltose binding protein fused to hP0-ED was secreted to the periplasm of Escherichia coli to allow an appropriate folding pathway. The fusion protein was extracted via osmotic shock and purified by affinity chromatography. Factor Xa was used to cleave the fusion protein, and a combination of affinity and ion-exchange chromatography was used to further purify hP0-ED. We document several significant improvements to previous protocols, including bacterial growth to approximately 15 OD using orbital shakers and the use of diafiltration, which result in yields of approximately 150 mg highly pure protein per liter of medium.

  9. Gene expression in human hippocampus from cocaine abusers identifies genes which regulate extracellular matrix remodeling.

    PubMed

    Mash, Deborah C; ffrench-Mullen, Jarlath; Adi, Nikhil; Qin, Yujing; Buck, Andrew; Pablo, John

    2007-11-14

    The chronic effects of cocaine abuse on brain structure and function are blamed for the inability of most addicts to remain abstinent. Part of the difficulty in preventing relapse is the persisting memory of the intense euphoria or cocaine "rush". Most abused drugs and alcohol induce neuroplastic changes in brain pathways subserving emotion and cognition. Such changes may account for the consolidation and structural reconfiguration of synaptic connections with exposure to cocaine. Adaptive hippocampal plasticity could be related to specific patterns of gene expression with chronic cocaine abuse. Here, we compare gene expression profiles in the human hippocampus from cocaine addicts and age-matched drug-free control subjects. Cocaine abusers had 151 gene transcripts upregulated, while 91 gene transcripts were downregulated. Topping the list of cocaine-regulated transcripts was RECK in the human hippocampus (FC = 2.0; p<0.05). RECK is a membrane-anchored MMP inhibitor that is implicated in the coordinated regulation of extracellular matrix integrity and angiogenesis. In keeping with elevated RECK expression, active MMP9 protein levels were decreased in the hippocampus from cocaine abusers. Pathway analysis identified other genes regulated by cocaine that code for proteins involved in the remodeling of the cytomatrix and synaptic connections and the inhibition of blood vessel proliferation (PCDH8, LAMB1, ITGB6, CTGF and EphB4). The observed microarray phenotype in the human hippocampus identified RECK and other region-specific genes that may promote long-lasting structural changes with repeated cocaine abuse. Extracellular matrix remodeling in the hippocampus may be a persisting effect of chronic abuse that contributes to the compulsive and relapsing nature of cocaine addiction.

  10. Combinatorial Extracellular Matrices for Human Embryonic Stem Cell Differentiation in 3D

    PubMed Central

    Yang, Fan; Cho, Seung-Woo; Son, Sun Mi; Hudson, Sarah P.; Bogatyrev, Said; Keung, Lily; Kohane, Daniel S.; Langer, Robert

    2010-01-01

    Embryonic stem cells (ESCs) are promising cell sources for tissue engineering and regenerative medicine. Scaffolds for ESC-based tissue regeneration should provide not only structural support, but also signals capable of supporting appropriate cell differentiation and tissue development. Extracellular matrix (ECM) is a key component of stem cell niche in vivo and can influence stem cell fate via mediating cell attachment and migration, presenting chemical and physical cues, as well as binding soluble factors. Here we investigated the effects of combinatorial extracellular matrix proteins on controlled human ESC (hESC) differentiation. Varying ECM compositions in 3D markedly affects cell behavior, and optimal compositions of ECM hydrogels are identified which facilitate specific-lineage differentiation of stem cells. To our knowledge, this is the first combinatorial analysis of ECM hydrogels for their effects on hESC differentiation in 3D. The 3D matrices described herein may provide a useful platform for studying the interactive ECM signaling in influencing stem cell differentiation. PMID:20614932

  11. Hydrostatic pressure stimulation of human mesenchymal stem cells seeded on collagen-based artificial extracellular matrices.

    PubMed

    Hess, Ricarda; Douglas, Timothy; Myers, Kenneth A; Rentsch, Barbe; Rentsch, Claudia; Worch, Hartmut; Shrive, Nigel G; Hart, David A; Scharnweber, Dieter

    2010-02-01

    Human mesenchymal stem cells (hMSCs) from bone marrow are considered a promising cell source for bone tissue engineering applications because of their ability to differentiate into cells of the osteoblastic lineage. Mechanical stimulation is able to promote osteogenic differentiation of hMSC; however, the use of hydrostatic pressure (HP) has not been well studied. Artificial extracellular matrices containing collagen and chondroitin sulfate (CS) have promoted the expression of an osteoblastic phenotype by hMSCs. However, there has been little research into the combined effects of biochemical stimulation by matrices and simultaneous mechanical stimulation. In this study, artificial extracellular matrices generated from collagen and/or CS were coated onto polycaprolactone-co-lactide substrates, seeded with hMSCs and subjected to cyclic HP at various time points during 21 days after cell seeding to investigate the effects of biochemical, mechanical, and combined biochemical and mechanical stimulations. Cell differentiation was assessed by analyzing the expression of alkaline phosphatase (ALP) at the protein- and mRNA levels, as well as for calcium accumulation. The timing of HP stimulation affected hMSC proliferation and expression of ALP activity. HP stimulation after 6 days was most effective at promoting ALP activity. CS-containing matrices promoted the osteogenic differentiation of hMSCs. A combination of both CS-containing matrices and cyclic HP yields optimal effects on osteogenic differentiation of hMSCs on scaffolds compared with individual responses.

  12. Trafficking mechanisms of extracellular matrix macromolecules: insights from vertebrate development and human diseases.

    PubMed

    Unlu, Gokhan; Levic, Daniel S; Melville, David B; Knapik, Ela W

    2014-02-01

    Cellular life depends on protein transport and membrane traffic. In multicellular organisms, membrane traffic is required for extracellular matrix deposition, cell adhesion, growth factor release, and receptor signaling, which are collectively required to integrate the development and physiology of tissues and organs. Understanding the regulatory mechanisms that govern cargo and membrane flow presents a prime challenge in cell biology. Extracellular matrix (ECM) secretion remains poorly understood, although given its essential roles in the regulation of cell migration, differentiation, and survival, ECM secretion mechanisms are likely to be tightly controlled. Recent studies in vertebrate model systems, from fishes to mammals and in human patients, have revealed complex and diverse loss-of-function phenotypes associated with mutations in components of the secretory machinery. A broad spectrum of diseases from skeletal and cardiovascular to neurological deficits have been linked to ECM trafficking. These discoveries have directly challenged the prevailing view of secretion as an essential but monolithic process. Here, we will discuss the latest findings on mechanisms of ECM trafficking in vertebrates. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Visualization of Neutrophil Extracellular Traps and Fibrin Meshwork in Human Fibrinopurulent Inflammatory Lesions: II. Ultrastructural Study

    PubMed Central

    Onouchi, Takanori; Shiogama, Kazuya; Matsui, Takahiro; Mizutani, Yasuyoshi; Sakurai, Kouhei; Inada, Ken-ichi; Tsutsumi, Yutaka

    2016-01-01

    Neutrophil extracellular traps (NETs) represent an extracellular, spider’s web-like structure resulting from cell death of neutrophils. NETs play an important role in innate immunity against microbial infection, but their roles in human pathological processes remain largely unknown. NETs and fibrin meshwork both showing fibrillar structures are observed at the site of fibrinopurulent inflammation, as described in our sister paper [Acta Histochem. Cytochem. 49; 109–116, 2016]. In the present study, immunoelectron microscopic study was performed for visualizing NETs and fibrin fibrils (thick fibrils in our tongue) in formalin-fixed, paraffin-embedded sections of autopsied lung tissue of legionnaire’s pneumonia. Lactoferrin and fibrinogen gamma chain were utilized as markers of NETs and fibrin, respectively. Analysis of immuno-scanning electron microscopy indicated that NETs constructed thin fibrils and granular materials were attached onto the NETs fibrils. The smooth-surfaced fibrin fibrils were much thicker than the NETs fibrils. Pre-embedding immunoelectron microscopy demonstrated that lactoferrin immunoreactivities were visible as dots on the fibrils, whereas fibrinogen gamma chain immunoreactivities were homogeneously observed throughout the fibrils. Usefulness of immunoelectron microscopic analysis of NETs and fibrin fibrils should be emphasized. PMID:27682015

  14. Effects of extracellular magnesium on the differentiation and function of human osteoclasts.

    PubMed

    Wu, Lili; Luthringer, Bérengère J C; Feyerabend, Frank; Schilling, Arndt F; Willumeit, Regine

    2014-06-01

    Magnesium-based implants have been shown to influence the surrounding bone structure. In an attempt to partially reveal the cellular mechanisms involved in the remodelling of magnesium-based implants, the influence of increased extracellular magnesium content on human osteoclasts was studied. Peripheral blood mononuclear cells were driven towards an osteoclastogenesis pathway via stimulation with receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor for 28 days. Concomitantly, the cultures were exposed to variable magnesium concentrations (from either magnesium chloride or magnesium extracts). Osteoclast proliferation and differentiation were evaluated based on cell metabolic activity, total protein content, tartrate-resistant acid phosphatase activity, cathepsin K and calcitonin receptor immunocytochemistry, and cellular ability to form resorption pits. While magnesium chloride first enhanced and then opposed cell proliferation and differentiation in a concentration-dependent manner (peaking between 10 and 15mM magnesium chloride), magnesium extracts (with lower magnesium contents) appeared to decrease cell metabolic activity (≈50% decrease at day 28) while increasing osteoclast activity at a lower concentration (twofold higher). Together, the results indicated that (i) variations in the in vitro extracellular magnesium concentration affect osteoclast metabolism and (ii) magnesium extracts should be used preferentially in vitro to more closely mimic the in vivo environment. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  15. Trafficking Mechanisms of Extracellular Matrix Macromolecules: Insights from Vertebrate Development and Human Diseases

    PubMed Central

    Unlu, Gokhan; Levic, Daniel S.; Melville, David B.; Knapik, Ela W.

    2014-01-01

    Cellular life depends on protein transport and membrane traffic. In multicellular organisms, membrane traffic is required for extracellular matrix deposition, cell adhesion, growth factor release, and receptor signaling, which are collectively required to integrate the development and physiology of tissues and organs. Understanding the regulatory mechanisms that govern cargo and membrane flow presents a prime challenge in cell biology. Extracellular matrix (ECM) secretion remains poorly understood, although given its essential roles in the regulation of cell migration, differentiation, and survival, ECM secretion mechanisms are likely to be tightly controlled. Recent studies in vertebrate model systems, from fishes to mammals and in human patients, have revealed complex and diverse loss-of-function phenotypes associated with mutations in components of the secretory machinery. A broad spectrum of diseases from skeletal and cardiovascular to neurological deficits have been linked to ECM trafficking. These discoveries have directly challenged the prevailing view of secretion as an essential but monolithic process. Here, we will discuss the latest findings on mechanisms of ECM trafficking in vertebrates. PMID:24333299

  16. Factor H Binds to Extracellular DNA Traps Released from Human Blood Monocytes in Response to Candida albicans

    PubMed Central

    Halder, Luke D.; Abdelfatah, Mahmoud A.; Jo, Emeraldo A. H.; Jacobsen, Ilse D.; Westermann, Martin; Beyersdorf, Niklas; Lorkowski, Stefan; Zipfel, Peter F.; Skerka, Christine

    2017-01-01

    Upon systemic infection with human pathogenic yeast Candida albicans (C. albicans), human monocytes and polymorph nuclear neutrophilic granulocytes are the first immune cells to respond and come into contact with C. albicans. Monocytes exert immediate candidacidal activity and inhibit germination, mediate phagocytosis, and kill fungal cells. Here, we show that human monocytes spontaneously respond to C. albicans cells via phagocytosis, decondensation of nuclear DNA, and release of this decondensed DNA in the form of extracellular traps (called monocytic extracellular traps: MoETs). Both subtypes of monocytes (CD14++CD16−/CD14+CD16+) formed MoETs within the first hours upon contact with C. albicans. MoETs were characterized by the presence of citrullinated histone, myeloperoxidase, lactoferrin, and elastase. MoETs were also formed in response to Staphylococcus aureus and Escherichia coli, indicating a general reaction of monocytes to infectious microbes. MoET induction differs from extracellular trap formation in macrophages as MoETs are not triggered by simvastatin, an inhibitor of cholesterol synthesis and inducer of extracellular traps in macrophages. Extracellular traps from both monocytes and neutrophils activate complement and C3b is deposited. However, factor H (FH) binds via C3b to the extracellular DNA, mediates cofactor activity, and inhibits the induction of the inflammatory cytokine interleukin-1 beta in monocytes. Altogether, the results show that human monocytes release extracellular DNA traps in response to C. albicans and that these traps finally bind FH via C3b to presumably support clearance without further inflammation. PMID:28133459

  17. Advances in the quantification of mitochondrial function in primary human immune cells through extracellular flux analysis.

    PubMed

    Nicholas, Dequina; Proctor, Elizabeth A; Raval, Forum M; Ip, Blanche C; Habib, Chloe; Ritou, Eleni; Grammatopoulos, Tom N; Steenkamp, Devin; Dooms, Hans; Apovian, Caroline M; Lauffenburger, Douglas A; Nikolajczyk, Barbara S

    2017-01-01

    Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96) analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes.

  18. Advances in the quantification of mitochondrial function in primary human immune cells through extracellular flux analysis

    PubMed Central

    Ip, Blanche C.; Habib, Chloe; Ritou, Eleni; Grammatopoulos, Tom N.; Steenkamp, Devin; Dooms, Hans; Apovian, Caroline M.; Lauffenburger, Douglas A.

    2017-01-01

    Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96) analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes. PMID:28178278

  19. Tumor Immunotherapy Using Gene-Modified Human Mesenchymal Stem Cells Loaded into Synthetic Extracellular Matrix Scaffolds

    PubMed Central

    Compte, Marta; Cuesta, Ángel M; Sánchez-Martín, David; Alonso-Camino, Vanesa; Vicario, José Luís; Sanz, Laura; Álvarez-Vallina, Luís

    2009-01-01

    Mesenchymal stem cells (MSCs) are appealing as gene therapy cell vehicles given their ease of expansion and transduction. However, MSCs exhibit immunomodulatory and proangiogenic properties that may pose a risk in their use in anticancer therapy. For this reason, we looked for a strategy to confine MSCs to a determined location, compatible with a clinical application. Human MSCs genetically modified to express luciferase (MSCluc), seeded in a synthetic extracellular matrix (sECM) scaffold (sentinel scaffold) and injected subcutaneously in immunodeficient mice, persisted for more than 40 days, as assessed by bioluminescence imaging in vivo. MSCs modified to express a bispecific α-carcinoembryonic antigen (αCEA)/αCD3 diabody (MSCdAb) and seeded in an sECM scaffold (therapeutic scaffolds) supported the release of functional diabody into the bloodstream at detectable levels for at least 6 weeks after implantation. Furthermore, when therapeutic scaffolds were implanted into CEA-positive human colon cancer xenograft-bearing mice and human T lymphocytes were subsequently transferred, circulating αCEA/αCD3 diabody activated T cells and promoted tumor cell lysis. Reduction of tumor growth in MSCdAb-treated mice was statistically significant compared with animals that only received MSCluc. In summary, we report here for the first time that human MSCs genetically engineered to secrete a bispecific diabody, seeded in an sECM scaffold and implanted in a location distant from the primary tumor, induce an effective antitumor response and tumor regression. PMID:19096041

  20. Role of TGFβ/Smad Signaling in Gremlin Induction of Human Trabecular Meshwork Extracellular Matrix Proteins

    PubMed Central

    Sethi, Anirudh; Jain, Ankur; Zode, Gulab S.; Wordinger, Robert J.

    2011-01-01

    Purpose. The bone morphogenic protein (BMP) antagonist gremlin is elevated in glaucomatous trabecular meshwork (TM) cells and tissues and elevates intraocular pressure (IOP). Gremlin also blocks BMP4 inhibition of transforming growth factor (TGF)-β2 induction of TM extracellular matrix (ECM) proteins. The purpose of this study was to determine whether Gremlin regulates ECM proteins in cultured human TM cells. Methods. Human TM cells were treated with recombinant gremlin to determine the effects on ECM gene and protein expression. Expression of the ECM genes FN, COL1, PAI1, and ELN was examined in cultured human TM cells by quantitative RT-PCR and Western immunoblot analysis. TM cells were pretreated with TGFBR inhibitors (LY364947, SB431542 or TGFBR1/TGFB2 siRNAs), inhibitors of the Smad signaling pathway (SIS3 or Smad2/3/4 siRNAs), or CTGF siRNA to identify the signaling pathway(s) involved in gremlin induction of ECM gene and protein expression. Results. All ECM genes analyzed (FN, COL1, PAI1, and ELN) were induced by gremlin. This gremlin induction of ECM genes and protein expression was blocked by inhibitors of TGFBR and the canonical Smad2/3/4 and CTGF signaling pathways. Conclusions. Gremlin employs canonical TGFβ2/Smad signaling to induce ECM genes and proteins in cultured human TM cells. Gremlin also induces both TGFβ2 and CTGF, which can act downstream to mediate some of these ECM changes in TM cells. PMID:21642622

  1. Scanning Electron Microscopic Examination of the Extracellular Matrix in the Decellularized Mouse and Human Cochlea.

    PubMed

    Santi, Peter A; Aldaya, Robair; Brown, Alec; Johnson, Shane; Stromback, Tyler; Cureoglu, Sebahattin; Rask-Andersen, Helge

    2016-06-01

    Decellularized tissues have been used to investigate the extracellular matrix (ECM) in a number of different tissues and species. Santi and Johnson JARO 14:3-15 (2013) first described the decellularized inner ear in the mouse, rat, and human using scanning thin-sheet laser imaging microscopy (sTSLIM). The purpose of the present investigation is to examine decellularized cochleas in the mouse and human at higher resolution using scanning electron microscopy (SEM). Fresh cochleas were harvested and decellularized using detergent extraction methods. Following decellularization, the ECM of the bone, basilar membrane, spiral limbus, and ligament remained, and all of the cells were removed from the cochlea. A number of similarities and differences in the ECM of the mouse and human were observed. A novel, spirally directed structure was present on the basilar membrane and is located at the border between Hensen and Boettcher cells. These septa-like structures formed a single row in the mouse and multiple rows in the human. The basal lamina of the stria vascularis capillaries was present and appeared thicker in the human compared with the mouse. In the mouse, numerous openings beneath the spiral prominence that previously housed the root processes of the external sulcus cells were observed but in the human there was only a single row of openings. These and other anatomical differences in the ECM between the mouse and human may reflect functional differences and/or be due to aging; however, decellularized cochleas provide a new way to examine the cochlear ECM and reveal new observations.

  2. Extracellular adenosine triphosphate affects the response of human macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Dubois-Colas, Nicolas; Petit-Jentreau, Laetitia; Barreiro, Luis B; Durand, Sylvère; Soubigou, Guillaume; Lecointe, Cécile; Klibi, Jihène; Rezaï, Keyvan; Lokiec, François; Coppée, Jean-Yves; Gicquel, Brigitte; Tailleux, Ludovic

    2014-09-01

    Granulomas are the hallmark of Mycobacterium tuberculosis infection. As the host fails to control the bacteria, the center of the granuloma exhibits necrosis resulting from the dying of infected macrophages. The release of the intracellular pool of nucleotides into the surrounding medium may modulate the response of newly infected macrophages, although this has never been investigated. Here, we show that extracellular adenosine triphosphate (ATP) indirectly modulates the expression of 272 genes in human macrophages infected with M. tuberculosis and that it induces their alternative activation. ATP is rapidly hydrolyzed by the ecto-ATPase CD39 into adenosine monophosphate (AMP), and it is AMP that regulates the macrophage response through the adenosine A2A receptor. Our findings reveal a previously unrecognized role for the purinergic pathway in the host response to M. tuberculosis. Dampening inflammation through signaling via the adenosine A2A receptor may limit tissue damage but may also favor bacterial immune escape.

  3. Extracellular production of an intact and biologically active human growth hormone by the Bacillus brevis system.

    PubMed

    Kajino, T; Saito, Y; Asami, O; Yamada, Y; Hirai, M; Udata, S

    1997-10-01

    The characteristic features of the Bacillus brevis system are very high productivity of heterologous proteins and very low extracellular protease activity. However, degradation of some heterologous proteins, especially mammalian proteins, can be observed and resulted in a lowering of protein productivity. By using a mutant expressing low levels of proteases and the addition of EDTA to the medium, intact human growth hormone (hGH) was successfully produced with the B. brevis system. Signal peptide modification with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a twelve-fold increase in hGH production. The hGH yield was further elevated to 240 mg L-1 by optimization of culture conditions. Thus, biologically active and mature hGH can be efficiently produced directly in the medium with the B. brevis system.

  4. Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange.

    PubMed

    Stock, Christian; Gassner, Birgit; Hauck, Christof R; Arnold, Hannelore; Mally, Sabine; Eble, Johannes A; Dieterich, Peter; Schwab, Albrecht

    2005-08-15

    Their glycolytic metabolism imposes an increased acid load upon tumour cells. The surplus protons are extruded by the Na+/H+ exchanger (NHE) which causes an extracellular acidification. It is not yet known by what mechanism extracellular pH (pHe) and NHE activity affect tumour cell migration and thus metastasis. We studied the impact of pHe and NHE activity on the motility of human melanoma (MV3) cells. Cells were seeded on/in collagen I matrices. Migration was monitored employing time lapse video microscopy and then quantified as the movement of the cell centre. Intracellular pH (pHi) was measured fluorometrically. Cell-matrix interactions were tested in cell adhesion assays and by the displacement of microbeads inside a collagen matrix. Migration depended on the integrin alpha2beta1. Cells reached their maximum motility at pHe approximately 7.0. They hardly migrated at pHe 6.6 or 7.5, when NHE was inhibited, or when NHE activity was stimulated by loading cells with propionic acid. These procedures also caused characteristic changes in cell morphology and pHi. The changes in pHi, however, did not account for the changes in morphology and migratory behaviour. Migration and morphology more likely correlate with the strength of cell-matrix interactions. Adhesion was the strongest at pHe 6.6. It weakened at basic pHe, upon NHE inhibition, or upon blockage of the integrin alpha2beta1. We propose that pHe and NHE activity affect migration of human melanoma cells by modulating cell-matrix interactions. Migration is hindered when the interaction is too strong (acidic pHe) or too weak (alkaline pHe or NHE inhibition).

  5. Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange

    PubMed Central

    Stock, Christian; Gassner, Birgit; Hauck, Christof R; Arnold, Hannelore; Mally, Sabine; Eble, Johannes A; Dieterich, Peter; Schwab, Albrecht

    2005-01-01

    Their glycolytic metabolism imposes an increased acid load upon tumour cells. The surplus protons are extruded by the Na+/H+ exchanger (NHE) which causes an extracellular acidification. It is not yet known by what mechanism extracellular pH (pHe) and NHE activity affect tumour cell migration and thus metastasis. We studied the impact of pHe and NHE activity on the motility of human melanoma (MV3) cells. Cells were seeded on/in collagen I matrices. Migration was monitored employing time lapse video microscopy and then quantified as the movement of the cell centre. Intracellular pH (pHi) was measured fluorometrically. Cell–matrix interactions were tested in cell adhesion assays and by the displacement of microbeads inside a collagen matrix. Migration depended on the integrin α2β1. Cells reached their maximum motility at pHe∼7.0. They hardly migrated at pHe 6.6 or 7.5, when NHE was inhibited, or when NHE activity was stimulated by loading cells with propionic acid. These procedures also caused characteristic changes in cell morphology and pHi. The changes in pHi, however, did not account for the changes in morphology and migratory behaviour. Migration and morphology more likely correlate with the strength of cell–matrix interactions. Adhesion was the strongest at pHe 6.6. It weakened at basic pHe, upon NHE inhibition, or upon blockage of the integrin α2β1. We propose that pHe and NHE activity affect migration of human melanoma cells by modulating cell–matrix interactions. Migration is hindered when the interaction is too strong (acidic pHe) or too weak (alkaline pHe or NHE inhibition). PMID:15946960

  6. Lefty Glycoproteins in Human Embryonic Stem Cells: Extracellular Delivery Route and Posttranslational Modification in Differentiation.

    PubMed

    Khalkhali-Ellis, Zhila; Galat, Vasiliy; Galat, Yekaterina; Gilgur, Alina; Seftor, Elisabeth A; Hendrix, Mary J C

    2016-09-19

    Lefty is a member of transforming growth factor-beta (TGF-β) superfamily and a potent antagonist of the TGF-β/Nodal/Activin signaling pathway. Lefty is critical in sustaining self-renewal/pluripotency status, and implicated in the differentiation of embryonic stem cells (ESCs). However, emerging studies depict Lefty as a multifaceted protein involved in myriad cellular events. Lefty proteins (human Lefty A and B) are secreted glycoproteins, but their mode of secretion and the significance of their "glycan" moiety remain mostly unexplored. By employing an in vitro system of human ESCs (hESCs), we observed that Lefty protein(s) are encased in exosomes for extracellular release. The exosomal- and cell-associated Lefty diverge in their proteolytic processing, and possess N-glycan structures of high mannose and complex nature. Differentiation of hESCs to mesenchymal cells (MSCs) or neuronal progenitor cells (NPCs) entails distinct changes in the Lefty A/Lefty B gene(s), and protein expression. Specifically, the proteolytic cleavage and N-glycan composition of the cell-associated and exosomal Lefty differ in the differentiated progenies. These modifications affected Lefty's inhibitory effect on Nodal signaling in aggressive melanoma cells. The microheterogeneity in the processing and glycosylation of Lefty protein(s) between hESCs, MSCs, and NPCs could present efficient means of diversifying the endogenous functions of Lefty. Whether Lefty's diverse functions in embryonic patterning, as well as its diffusion range in the extracellular environment, are similarly affected remains to be determined. Our studies underscore the potential relevance of Lefty-packaged exosomes for combating debilitating diseases such as cancer.

  7. Three-dimensional culture of human meniscal cells: Extracellular matrix and proteoglycan production

    PubMed Central

    Gruber, Helen E; Mauerhan, David; Chow, Yin; Ingram, Jane A; Norton, H James; Hanley, Edward N; Sun, Yubo

    2008-01-01

    Background The meniscus is a complex tissue whose cell biology has only recently begun to be explored. Published models rely upon initial culture in the presence of added growth factors. The aim of this study was to test a three-dimensional (3D) collagen sponge microenvironment (without added growth factors) for its ability to provide a microenvironment supportive for meniscal cell extracellular matrix (ECM) production, and to test the responsiveness of cells cultured in this manner to transforming growth factor-β (TGF-β). Methods Experimental studies were approved prospectively by the authors' Human Subjects Institutional Review Board. Human meniscal cells were isolated from surgical specimens, established in monolayer culture, seeded into a 3D scaffold, and cell morphology and extracellular matrix components (ECM) evaluated either under control condition or with addition of TGF-β. Outcome variables were evaluation of cultured cell morphology, quantitative measurement of total sulfated proteoglycan production, and immunohistochemical study of the ECM components chondroitin sulfate, keratan sulfate, and types I and II collagen. Result and Conclusion Meniscal cells attached well within the 3D microenvironment and expanded with culture time. The 3D microenvironment was permissive for production of chondroitin sulfate, types I and II collagen, and to a lesser degree keratan sulfate. This microenvironment was also permissive for growth factor responsiveness, as indicated by a significant increase in proteoglycan production when cells were exposed to TGF-β (2.48 μg/ml ± 1.00, mean ± S.D., vs control levels of 1.58 ± 0.79, p < 0.0001). Knowledge of how culture microenvironments influence meniscal cell ECM production is important; the collagen sponge culture methodology provides a useful in vitro tool for study of meniscal cell biology. PMID:18582376

  8. Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

    PubMed Central

    Cerqueira, Carla; Samperio Ventayol, Pilar; Vogeley, Christian

    2015-01-01

    ABSTRACT The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved. IMPORTANCE Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural

  9. Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells.

    PubMed

    Cerqueira, Carla; Samperio Ventayol, Pilar; Vogeley, Christian; Schelhaas, Mario

    2015-07-01

    The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved. Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural alterations that

  10. Effect of sulfur amino acids on stimulus-induced superoxide generation and translocation of p47phox and p67phox to cell membrane in human neutrophils and the scavenging of free radical.

    PubMed

    Kitaoka, Noriko; Liu, Gang; Masuoka, Noriyoshi; Yamashita, Koichi; Manabe, Masanobu; Kodama, Hiroyuki

    2005-03-01

    Various cystathionine metabolites are in the urine of the patients with cystathioninuria. Among these metabolites, cystathionine ketimine significantly enhanced N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in parallel with tyrosyl phosphorylation of 45 kDa protein in human neutrophils. We investigated the effect of various sulfur amino acids on fMLP-, phorbol-12-myristate-13-acetate (PMA)- and arachidonic acid (AA)-induced superoxide generation in human neutrophils. In addition, the effects of these sulfur amino acids on the membrane translocation of cytosolic compounds p47(phox) and p67(phox) and on the scavenging of superoxide anions were investigated. When the cells were preincubated with various sulfur amino acids, fMLP-induced superoxide generation was enhanced by D,L-homocysteine and D,L-homocysteine-thiolactone but was inhibited by other sulfur amino acids in a concentration-dependent manner. The AA-induced superoxide was enhanced by L-cysteine, N-acetyl-L-cysteine and D,L-homocysteine. The strength of enhancing effect was: L-cysteine>N-acetyl-L-cysteine>D,L-homocysteine. On the other hand, the superoxide generation was weakly inhibited by L-cystathionine. The superoxide generation induced by PMA was weakly inhibited by L-cysteine, N-acetyl-L-cysteine and L-cystathionine. Homocysteine and D,L-homocysteine-thiolactone had no effect. In addition, D,L-homocysteine also enhanced translocation to the cell membrane of cytosolic compounds p47(phox) and p67(phox). Conversely, L-cystathionine and N-acetyl-L-cysteine inhibited the translocation to membrane of p47(phox) and p67(phox) in a concentration-dependent manner. N-acetyl-L-cysteine and L-cysteine revealed scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The sulfur amino acids tested also indicated radical scavenging activity on superoxide anion generated by phenazine methoxysulfate (PMS)-NADH system. D,L-homocysteine and D

  11. Human papillomavirus 16 E6 and E7 oncoprotein expression alters microRNA expression in extracellular vesicles.

    PubMed

    Harden, Mallory E; Munger, Karl

    2017-08-01

    Extracellular vesicles released by cancer cells are mediators of intercellular communication that have been reported to contribute to carcinogenesis. Since they are readily detected in bodily fluids, they may also be used as cancer biomarkers. The E6/E7 oncoproteins drive human papillomavirus (HPV)-associated cancers, which account for approximately 5% of all human cancers worldwide. Here, we investigate how HPV16 E6/E7 oncogene expression in primary human epithelial cells alters miR expression in extracellular vesicles and compare these to changes in intracellular miR expression. Examining a panel of 68 cancer related miRs revealed that many miRs had similar expression patterns in cells and in extracellular vesicles, whereas some other miRs had different expression patterns and may be selectively packaged into extracellular vesicles. Interestingly, the set of miRs that may be selectively packaged in HPV16 E6/E7 extracellular vesicles is predicted to inhibit necrosis and apoptosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance*

    PubMed Central

    Soteriou, Despina; Iskender, Banu; Byron, Adam; Humphries, Jonathan D.; Borg-Bartolo, Simon; Haddock, Marie-Claire; Baxter, Melissa A.; Knight, David; Humphries, Martin J.; Kimber, Susan J.

    2013-01-01

    Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal. PMID:23658023

  13. Preparation of cardiac extracellular matrix scaffolds by decellularization of human myocardium.

    PubMed

    Oberwallner, Barbara; Brodarac, Andreja; Choi, Yeong-Hoon; Saric, Tomo; Anić, Petra; Morawietz, Lars; Stamm, Christof

    2014-09-01

    Extracellular matrix (ECM) derived by tissue decellularization has applications as a tissue engineering scaffold and for support of cellular regeneration. Myocardial ECM from animals has been produced by whole-organ perfusion or immersion processes, but methods for preparation of human myocardial ECM for therapy and research have not been compared in detail, yet. We analyzed the impact of decellularization processes on human myocardial ECM, and tested its ability to serve as a scaffold for cell seeding. Sodium dodecyl sulfate (SDS)-based decellularization, but not treatments based on Triton X-100, deoxycholate or hypo/hypertonic incubations, removed cells satisfactorily, and incubation with fetal bovine serum (FBS) eliminated residual DNA. ECM architecture was best preserved by a protocol consisting of 2 h lysis, 6 h SDS, and 3 h FBS, but age and pathology of the donor tissue are highly important for producing reproducible, high-quality scaffolds. We also studied ECM repopulation with mesenchymal stem cells (CB-MSC), cardiomyocytes derived from induced pluripotent stem cells (iPS-CM), and na€ıve neonatal mouse cardiomyocytes. Cells attached to the matrix and proliferated and displayed higher viability than in standard culture. We conclude that human cardiac ECM sheets may be suitable scaffold for cell-matrix interaction studies and as a biomaterial for tissue regeneration and engineering.

  14. Human Leukocytes Kill Brugia malayi Microfilariae Independently of DNA-Based Extracellular Trap Release

    PubMed Central

    McCoy, Ciaran J.; Reaves, Barbara J.; Giguère, Steeve; Coates, Ruby; Rada, Balázs

    2017-01-01

    Background Wuchereria bancrofti, Brugia malayi and Brugia timori infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Some parasite carriers are amicrofilaremic whilst others facilitate mosquito-based disease transmission through blood-circulating microfilariae (Mf). Recent findings, obtained largely from animal model systems, suggest that polymorphonuclear leukocytes (PMNs) contribute to parasitic nematode-directed type 2 immune responses. When exposed to certain pathogens PMNs release extracellular traps (NETs) in the form of chromatin loaded with various antimicrobial molecules and proteases. Principal findings In vitro, PMNs expel large amounts of NETs that capture but do not kill B. malayi Mf. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to human neutrophil elastase, myeloperoxidase and citrullinated histone H4. A fluorescent, extracellular DNA release assay was used to quantify and observe Mf induced NETosis over time. Blinded video analyses of PMN-to-worm attachment and worm survival during Mf-leukocyte co-culture demonstrated that DNase treatment eliminates PMN attachment in the absence of serum, autologous serum bolsters both PMN attachment and PMN plus peripheral blood mononuclear cell (PBMC) mediated Mf killing, and serum heat inactivation inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement inhibitor compstatin did not impede Mf killing and had little effect on PMN attachment. Both human PMNs and monocytes, but not lymphocytes, are able to kill B. malayi Mf in vitro and NETosis does not significantly contribute to this killing. Leukocytes derived from presumably parasite-naïve U.S. resident donors vary in their ability to kill Mf in vitro, which may reflect the pathological heterogeneity associated with filarial parasitic infections. Conclusions/Significance Human innate immune cells are able to

  15. Structure and Dynamics of Extracellular Loops in Human Aquaporin-1 from Solid-State NMR and Molecular Dynamics.

    PubMed

    Wang, Shenlin; Ing, Christopher; Emami, Sanaz; Jiang, Yunjiang; Liang, Hongjun; Pomès, Régis; Brown, Leonid S; Ladizhansky, Vladimir

    2016-09-22

    Multiple moderate-resolution crystal structures of human aquaporin-1 have provided a foundation for understanding the molecular mechanism of selective water translocation in human cells. To gain insight into the interfacial structure and dynamics of human aquaporin-1 in a lipid environment, we performed nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations. Using magic angle spinning solid-state NMR, we report a near complete resonance assignment of the human aquaporin-1. Chemical shift analysis of the secondary structure identified pronounced deviations from crystallographic structures in extracellular loops A and C, including the cis Y37-P38 bond in loop A, as well as ordering and immobilization of loop C. Site-specific H/D exchange measurements identify a number of protected nitrogen-bearing side chains and backbone amide groups, involved in stabilizing the loops. A combination of molecular dynamics simulations with NMR-derived restraints and filtering based on solvent accessibility allowed for the determination of a structural model of extracellular loops largely consistent with NMR results. The simulations reveal loop stabilizing interactions that alter the extracellular surface of human AQP1, with possible implications for water transport regulation through the channel. Modulation of water permeation may occur as a result of rearrangement of side chains from loop C in the extracellular vestibule of hAQP1, affecting the aromatic arginine selectivity filter.

  16. Crystal structure of the extracellular domain of human myelin protein zero

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Yedidi, Ravikiran S.; Brunzelle, Joseph S.; Kovari, Iulia A.; Sohi, Jasloveleen; Kamholz, John; Kovari, Ladislau C.

    2012-03-27

    Charcot-Marie-Tooth disease (CMT), a hereditary motor and sensory neuropathy, is the most common genetic neuropathy with an incidence of 1 in 2600. Several forms of CMT have been identified arising from different genomic abnormalities such as CMT1 including CMT1A, CMT1B, and CMTX. CMT1 with associated peripheral nervous system (PNS) demyelination, the most frequent diagnosis, demonstrates slowed nerve conduction velocities and segmental demyelination upon nerve biopsy. One of its subtypes, CMT1A, presents a 1.5-Mb duplication in the p11-p12 region of the human chromosome 17 which encodes peripheral myelin protein 22 (PMP22). CMT1B, a less common form, arises from the mutations in the myelin protein zero (MPZ) gene on chromosome 1, region q22-q23, which encodes the major structural component of the peripheral myelin. A rare type of CMT1 has been found recently and is caused by point mutations in early growth response gene 2 (EGR2), encoding a zinc finger transcription factor in Schwann cells. In addition, CMTX, an X-linked form of CMT, arises from a mutation in the connexin-32 gene. Myelin protein zero, associated with CMT1B, is a transmembrane protein of 219 amino acid residues. Human MPZ consists of three domains: 125 residues constitute the glycosylated immunoglobulin-like extracellular domain; 27 residues span the membrane; and 67 residues comprise the highly basic intracellular domain. MPZ makes up approximately 50% of the protein content of myelin, and is expressed predominantly in Schwann cells, the myelinating cell of the PNS. Myelin protein zero, a homophilic adhesion molecule, is a member of the immunoglobulin super-family and is essential for normal myelin structure and function. In addition, MPZ knockout mice displayed abnormal myelin that severely affects the myelination pathway, and overexpression of MPZ causes congenital hypomyelination of peripheral nerves. Myelin protein zero mutations account for {approx}5% of patients with CMT. To date, over 125

  17. The carbonate radical anion-induced covalent aggregation of human copper, zinc superoxide dismutase, and alpha-synuclein: intermediacy of tryptophan- and tyrosine-derived oxidation products.

    PubMed

    Zhang, Hao; Andrekopoulos, Christopher; Joseph, Joy; Crow, John; Kalyanaraman, B

    2004-06-01

    In this review, we describe the free radical mechanism of covalent aggregation of human copper, zinc superoxide dismutase (hSOD1). Bicarbonate anion (HCO3-) enhances the covalent aggregation of hSOD1 mediated by the SOD1 peroxidase-dependent formation of carbonate radical anion (CO3*-), a potent and selective oxidant. This species presumably diffuses out the active site of hSOD1 and reacts with tryptophan residue located on the surface of hSOD1. The oxidative degradation of tryptophan to kynurenine and N-formyl kynurenine results in the covalent crosslinking and aggregation of hSOD1. Implications of oxidant-mediated aggregation of hSOD1 in the increased cytotoxicity of motor neurons in amyotrophic lateral sclerosis are discussed.

  18. Wnt Interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells

    PubMed Central

    Rappa, Germana; Mercapide, Javier; Anzanello, Fabio; Le, Thuc T.; G, Mary; Johlfs, Ronald R. Fiscus; Wilsch-Bräuninger, Michaela; Corbeil, Denis; Lorico, Aurelio

    2013-01-01

    Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that three distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1–positive structures appeared in three sizes (small, ≤ 40 nm; intermediates ~40–80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1–containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma. PMID:23318676

  19. Human monocytes respond to extracellular cAMP through A2A and A2B adenosine receptors

    PubMed Central

    Sciaraffia, Ester; Riccomi, Antonella; Lindstedt, Ragnar; Gesa, Valentina; Cirelli, Elisa; Patrizio, Mario; De Magistris, Maria Teresa; Vendetti, Silvia

    2014-01-01

    In this study, we test the hypothesis that cAMP, acting as an extracellular mediator, affects the physiology and function of human myeloid cells. The cAMP is a second messenger recognized as a universal regulator of several cellular functions in different organisms. Many studies have shown that extracellular cAMP exerts regulatory functions, acting as first mediator in multiple tissues. However, the impact of extracellular cAMP on cells of the immune system has not been fully investigated. We found that human monocytes exposed to extracellular cAMP exhibit higher expression of CD14 and lower amount of MHC class I and class II molecules. When cAMP-treated monocytes are exposed to proinflammatory stimuli, they exhibit an increased production of IL-6 and IL-10 and a lower amount of TNF-α and IL-12 compared with control cells, resembling the features of the alternative-activated macrophages or M2 macrophages. In addition, we show that extracellular cAMP affects monocyte differentiation into DCs, promoting the induction of cells displaying an activated, macrophage-like phenotype with reduced capacity of polarized, naive CD4+ T cells into IFN-γ-producing lymphocytes compared with control cells. The effects of extracellular cAMP on monocytes are mediated by CD73 ecto-5′-nucleotidase and A2A and A2B adenosine receptors, as selective antagonists could reverse its effects. Of note, the expression of CD73 molecules has been found on the membrane of a small population of CD14+CD16+ monocytes. These findings suggest that an extracellular cAMP-adenosine pathway is active in cells of the immune systems. PMID:24652540

  20. Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans

    PubMed Central

    Ueki, Shigeharu; Melo, Rossana C. N.; Ghiran, Ionita; Spencer, Lisa A.; Dvorak, Ann M.; Weller, Peter F.

    2013-01-01

    Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane–enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets. PMID:23303825

  1. Passaged human chondrocytes accumulate extracellular matrix when induced by bovine chondrocytes.

    PubMed

    Ahmed, Nazish; Taylor, Drew W; Wunder, Jay; Nagy, Andras; Gross, Allan E; Kandel, Rita A

    2010-03-01

    A source of sufficient number of cells is a major limiting factor for cartilage tissue engineering. To circumvent this problem, we developed a co-culture method to induce redifferentiation in bovine articular chondrocytes, which had undergone dedifferentiation following serial passage in monolayer culture. In this study we determine whether human osteoarthritic (OA) and non-diseased passaged dedifferentiated chondrocytes will respond similarly. Human passaged chondrocytes were co-cultured for 4 weeks with primary bovine chondrocytes and their redifferentiation status was determined. Afterwards the cells were cultured either independently or in co-culture with cryopreserved passaged cells for functional analysis. The co-culture of passaged cells with primary chondrocytes resulted in reversion of their phenotype towards articular chondrocytes, as shown by increased gene expression of type II collagen and COMP, decreased type I collagen expression and extracellular matrix formation in vitro. Furthermore, this redifferentiation was stable, as those cells not only formed hyaline-like cartilage tissue when grown on their own but also they could induce redifferentiation of passaged chondrocytes in co-culture. These data suggest that it may be possible to use autologous chondrocytes obtained from osteoarthritic cartilage to form tissue suitable to use for cartilage repair. Copyright (c) 2009 John Wiley & Sons, Ltd.

  2. Wound healing effect of acellular artificial dermis containing extracellular matrix secreted by human skin fibroblasts.

    PubMed

    Seo, Young-Kwon; Song, Kye-Yong; Kim, Young-Jin; Park, Jung-Keug

    2007-07-01

    In this study, an acellular artificial dermis, composed of human collagen and glycosaminoglycan (GAG) secreted by cultured human fibroblasts on a bovine collagen sponge, was developed. Much of the newly secreted extracellular matrix (ECM) remained after the cell removal process. The main theme of this study focused on the matrix, rather than the viable cell components of the skin, as the major dermal deficit in the wound. Both the acellular artificial and bioartificial dermises, containing viable cells with ECM, were significantly less soluble than the collagen sponge, and the relative GAG content in the bioartificial and acellular artificial dermises was approximately 115-120% of the chondroitin-6-sulfate (CS) content found in the collagen sponge. In the group receiving the collagen sponge, the wound area gradually decreased to approximately 10% of its original area, while in the groups receiving the bioartificial and acellular artificial dermises, the wound area also gradually decreased to approximately 60 and 50%, respectively, of the original size over the 5 weeks after grafting. Both the bioartificial and acellular artificial dermises formed thicker, denser collagen fibers; more new blood vessel formation was observed in both cases. The basement membrane of the regenerated epidermal-dermal junction was thicker and more linear in the acellular artificial dermis graft than in the collagen sponge graft. In conclusion, the wound healing effects of acellular artificial dermis are no less than those of the bioartificial dermis, and much better than the collagen sponge graft with respect to wound contraction, angiogenesis, collagen formation, and basement membrane repair.

  3. In vitro immunotoxicity assessment of culture-derived extracellular vesicles in human monocytes

    PubMed Central

    Rosas, Lucia E.; Elgamal, Ola A.; Mo, Xiaokui; Phelps, Mitch A.; Schmittgen, Thomas D.; Papenfuss, Tracey L.

    2016-01-01

    The potential to engineer extracellular vesicles (EV) that target specific cells and deliver a therapeutic payload has propelled a growing interest in their development as promising therapeutics. These EV are often produced from cultured cells. Very little is known about the interaction of cell culture-derived EV with cells of the immune system and their potential immunomodulatory effects. The present study evaluated potential immunotoxic effects of HEK293T-derived EV on the human monocytic cell lines THP-1 and U937. Incubation of cells with different doses of EV for 16–24 h was followed by assessment of cytotoxicity and cell function by flow cytometry. Changes in cell functionality were evaluated by the capacity of cells to phagocytize fluorescent microspheres. In addition, the internalization of labeled EV in THP-1 and U937 cells was evaluated. Exposure to EV did not affect the viability of THP-1 or U937 cells. Although lower doses of the EV increased phagocytic capacity in both cell lines, phagocytic efficiency of individual cells was not affected by EV exposure at any of the doses evaluated. This study also demonstrated that THP-1 and U937 monocytic cells are highly permissive to EV entry in a dose-response manner. These results suggest that, although HEK293T-derived EV are efficiently internalized by human monocytic cells, they do not exert a cytotoxic effect or alter phagocytic efficiency on the cell lines evaluated. PMID:27075513

  4. Maintenance of human hepatocyte function in vitro by liver-derived extracellular matrix gels.

    PubMed

    Sellaro, Tiffany L; Ranade, Aarati; Faulk, Denver M; McCabe, George P; Dorko, Kenneth; Badylak, Stephen F; Strom, Stephen C

    2010-03-01

    Tissue engineering and regenerative medicine (TE&RM) approaches to treating liver disease have the potential to provide temporary support with biohybrid-liver-assist devices or long-term therapy by replacing the diseased liver with functional constructs. A rate-limiting step for TE&RM strategies has been the loss of hepatocyte-specific functions after hepatocytes are isolated from their highly specialized in vivo microenvironment and placed in in vitro culture systems. The identification of a biologic substrate that can maintain a functional hepatocyte differentiation profile during in vitro culture would advance potential TE&RM therapeutic strategies. The present study compared two different biologic substrates for their ability to support human hepatocyte function in vitro: porcine-liver-derived extracellular matrix (PLECM) or Matrigel. Because Matrigel has been shown to be the most useful matrix for static, traditional hepatocyte culture, we directly compared PLECM with Matrigel in each experiment. Albumin secretion, hepatic transport activity, and ammonia metabolism were used to determine hepatocyte function. Hepatocytes cultured between two layers of PLECM or Matrigel showed equally high levels of albumin expression and secretion, ammonia metabolism, and hepatic transporter expression and function. We conclude that like Matrigel, PLECM represents a favorable substrate for in vitro culture of human hepatocytes.

  5. Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation.

    PubMed

    Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

    2016-01-01

    Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

  6. Inhibitory effects of extracellular products from oral bacteria on human fibroblasts and stimulated lymphocytes.

    PubMed

    Higerd, T B; Vesole, D H; Goust, J M

    1978-08-01

    Extracellular products of 12 strains of Streptococcus mutans and 5 additional species of oral bacteria were analyzed for their ability to inhibit proliferation of fibroblastoid cells (HeLa and AV3) and blast transformation of human peripheral blood lymphocytes obtained from normal individuals. Products from S. mutans strains AHT and BHT, Streptococcus intermedius, and Actinomyces viscosus inhibited [3H]thymidine uptake by fibroblastoid cells and phytohemagglutinin-stimulated lymphocytes. Products from S. mutans E49, Streptococcus salivarius, and Actinomyces naeslundii inhibited blast transformation of human lymphocytes but did not significantly inhibit the growth of fibroblastoid cells. Preparations from S. intermedius gave the greatest inhibitory activity against both target cell types; initial characterization of this preparation suggested a single factor active in both assays, in that the heat lability and Sephadex G-200 elution profile were similar for the inhibitory activity seen with the two cell types. The molecular weight of the inhibitor, estimated by gel filtration on Sephadex G-200 and Ultragel AcA34, was approximately 160,000. The results strongly suggest that oral bacteria produce heat-labile substances that interfere with fibroblast proliferation and alter the lymphocytic immunological response.

  7. Effect of bovine pericardial extracellular matrix scaffold niche on seeded human mesenchymal stem cell function

    PubMed Central

    Liu, Zhi Zhao; Wong, Maelene L.; Griffiths, Leigh G.

    2016-01-01

    Numerous studies have focused on generation of unfixed bovine pericardium (BP) extracellular matrix (ECM) for clinical application. However, the extent to which maintenance of native ECM niche is capable of directing behavior of repopulating cells remains relatively unexplored. By exploiting the sidedness of BP scaffolds (i.e., serous or fibrous surface), this study aims to determine the effect of ECM niche preservation on cellular repopulation using different scaffold generation methods. BP underwent either sodium dodecyl sulfate (SDS) decellularization or stepwise, solubilization-based antigen removal using amidosulfobetaine-14 (ASB-14). SDS scaffolds were toxic to repopulating human mesenchymal stem cells (hMSC). Scanning electron microscopy revealed distinct surface ultrastructure of ASB-14 scaffolds based on native BP sidedness. Basement membrane structures on the serous side stimulated hMSC cell monolayer formation, whereas fibrous side facilitated cell penetration into scaffold. Additionally, serous side seeding significantly increased hMSC adhesion and proliferation rate compared to the fibrous side. Furthermore, scaffold ECM niche stimulated sidedness dependent differential hMSC human leukocyte antigen expression, angiogenic and inflammatory cytokine secretion. This work demonstrates that ECM scaffold preparation method and preservation of BP side-based niches critically affects in vitro cell growth patterns and behavior, which has implications for use of such ECM biomaterials in clinical practice. PMID:27845391

  8. Curcumin suppresses Streptococcus mutans adherence to human tooth surfaces and extracellular matrix proteins.

    PubMed

    Song, J; Choi, B; Jin, E-J; Yoon, Y; Choi, K-H

    2012-07-01

    Streptococcus mutans is the key causative agent of caries and infective endocarditis. The first step in biofilm development and the consequent initiation of further disease is bacterial adherence to host cell surfaces. Therefore, the aim of this study was to evaluate the inhibitory effect of curcumin on S. mutans adherence to extracellular matrices and tooth surfaces. The effect of curcumin on the ability of S. mutans to adhere to glass surfaces coated with collagen and fibronectin was tested in order to determine whether the decrease of the bacterial adhesion by curcumin is achieved by hindering the bacteria in adhering to collagen and/or fibronectin. Also, human teeth inoculated with S. mutans were treated with curcumin in vitro in order to assess the relevance of the anti-adhesive effect to oral conditions in vivo. The minimum inhibitory concentration (MIC) at which curcumin completely inhibited bacterial growth was 128 μg/mL. The addition of curcumin below the MIC diminished bacterial adherence onto both collagen- and fibronectin-coated glass surfaces and human tooth surfaces. It appears that the anti-adhesive effect of curcumin against S. mutans is mediated through collagen and fibronectin. These results support the widespread use of curcumin as a food-based antimicrobial agent.

  9. Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation

    PubMed Central

    Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

    2016-01-01

    Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil. PMID:27034964

  10. FIB-SEM tomography of human skin telocytes and their extracellular vesicles.

    PubMed

    Cretoiu, Dragos; Gherghiceanu, Mihaela; Hummel, Eric; Zimmermann, Hans; Simionescu, Olga; Popescu, Laurentiu M

    2015-04-01

    We have shown in 2012 the existence of telocytes (TCs) in human dermis. TCs were described by transmission electron microscopy (TEM) as interstitial cells located in non-epithelial spaces (stroma) of many organs (see www.telocytes.com). TCs have very long prolongations (tens to hundreds micrometers) named Telopodes (Tps). These Tps have a special conformation with dilated portions named podoms (containing mitochondria, endoplasmic reticulum and caveolae) and very thin segments (below resolving power of light microscopy), called podomers. To show the real 3D architecture of TC network, we used the most advanced available electron microscope technology: focused ion beam scanning electron microscopy (FIB-SEM) tomography. Generally, 3D reconstruction of dermal TCs by FIB-SEM tomography revealed the existence of Tps with various conformations: (i) long, flattened irregular veils (ribbon-like segments) with knobs, corresponding to podoms, and (ii) tubular structures (podomers) with uneven calibre because of irregular dilations (knobs) - the podoms. FIB-SEM tomography also showed numerous extracellular vesicles (diameter 438.6 ± 149.1 nm, n = 30) released by a human dermal TC. Our data might be useful for understanding the role(s) of TCs in intercellular signalling and communication, as well as for comprehension of pathologies like scleroderma, multiple sclerosis, psoriasis, etc. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  11. On-chip immunoelectrophoresis of extracellular vesicles released from human breast cancer cells.

    PubMed

    Akagi, Takanori; Kato, Kei; Kobayashi, Masashi; Kosaka, Nobuyoshi; Ochiya, Takahiro; Ichiki, Takanori

    2015-01-01

    Extracellular vesicles (EVs) including exosomes and microvesicles have attracted considerable attention in the fields of cell biology and medicine. For a better understanding of EVs and further exploration of their applications, the development of analytical methods for biological nanovesicles has been required. In particular, considering the heterogeneity of EVs, methods capable of measuring individual vesicles are desired. Here, we report that on-chip immunoelectrophoresis can provide a useful method for the differential protein expression profiling of individual EVs. Electrophoresis experiments were performed on EVs collected from the culture supernatant of MDA-MB-231 human breast cancer cells using a measurement platform comprising a microcapillary electrophoresis chip and a laser dark-field microimaging system. The zeta potential distribution of EVs that reacted with an anti-human CD63 (exosome and microvesicle marker) antibody showed a marked positive shift as compared with that for the normal immunoglobulin G (IgG) isotype control. Thus, on-chip immunoelectrophoresis could sensitively detect the over-expression of CD63 glycoproteins on EVs. Moreover, to explore the applicability of on-chip immunoelectrophoresis to cancer diagnosis, EVs collected from the blood of a mouse tumor model were analyzed by this method. By comparing the zeta potential distributions of EVs after their immunochemical reaction with normal IgG, and the anti-human CD63 and anti-human CD44 (cancer stem cell marker) antibodies, EVs of tumor origin circulating in blood were differentially detected in the real sample. The result indicates that the present method is potentially applicable to liquid biopsy, a promising approach to the low-invasive diagnosis of cancer.

  12. Effect of extracellular vesicles of human adipose tissue on insulin signaling in liver and muscle cells.

    PubMed

    Kranendonk, Mariëtte E G; Visseren, Frank L J; van Herwaarden, Joost A; Nolte-'t Hoen, Esther N M; de Jager, Wilco; Wauben, Marca H M; Kalkhoven, Eric

    2014-10-01

    Insulin resistance (IR) is a key mechanism in obesity-induced cardiovascular disease. To unravel mechanisms whereby human adipose tissue (AT) contributes to systemic IR, the effect of human AT-extracellular vesicles (EVs) on insulin signaling in liver and muscle cells was determined. EVs released from human subcutaneous (SAT) and omental AT (OAT)-explants ex vivo were used for stimulation of hepatocytes and myotubes in vitro. Subsequently, insulin-induced Akt phosphorylation and expression of gluconeogenic genes (G6P, PEPCK) was determined. AT-EV adipokine levels were measured by multiplex immunoassay, and AT-EVs were quantified by high-resolution flow cytometry. In hepatocytes, AT-EVs from the majority of patients inhibited insulin-induced Akt phosphorylation, while EVs from some patients stimulated insulin-induced Akt phosphorylation. In myotubes AT-EVs exerted an ambiguous effect on insulin signaling. Hepatic Akt phosphorylation related negatively to G6P-expression by both SAT-EVs (r = -0.60, P = 0.01) and OAT-EVs (r = -0.74, P = 0.001). MCP-1, IL-6, and MIF concentrations were higher in OAT-EVs compared to SAT-EVs and differently related to lower Akt phosphorylation in hepatocytes. Finally, the number of OAT-EVs correlated positively with liver enzymes indicative for liver dysfunction. Human AT-EVs can stimulate or inhibit insulin signaling in hepatocytes- possibly depending on their adipokine content- and may thereby contribute to systemic IR. Copyright © 2014 The Obesity Society.

  13. Adipose tissue transcriptomic signature highlights the pathological relevance of extracellular matrix in human obesity

    PubMed Central

    Henegar, Corneliu; Tordjman, Joan; Achard, Vincent; Lacasa, Danièle; Cremer, Isabelle; Guerre-Millo, Michèle; Poitou, Christine; Basdevant, Arnaud; Stich, Vladimir; Viguerie, Nathalie; Langin, Dominique; Bedossa, Pierre; Zucker, Jean-Daniel; Clement, Karine

    2008-01-01

    Background Investigations performed in mice and humans have acknowledged obesity as a low-grade inflammatory disease. Several molecular mechanisms have been convincingly shown to be involved in activating inflammatory processes and altering cell composition in white adipose tissue (WAT). However, the overall importance of these alterations, and their long-term impact on the metabolic functions of the WAT and on its morphology, remain unclear. Results Here, we analyzed the transcriptomic signature of the subcutaneous WAT in obese human subjects, in stable weight conditions and after weight loss following bariatric surgery. An original integrative functional genomics approach was applied to quantify relations between relevant structural and functional themes annotating differentially expressed genes in order to construct a comprehensive map of transcriptional interactions defining the obese WAT. These analyses highlighted a significant up-regulation of genes and biological themes related to extracellular matrix (ECM) constituents, including members of the integrin family, and suggested that these elements could play a major mediating role in a chain of interactions that connect local inflammatory phenomena to the alteration of WAT metabolic functions in obese subjects. Tissue and cellular investigations, driven by the analysis of transcriptional interactions, revealed an increased amount of interstitial fibrosis in obese WAT, associated with an infiltration of different types of inflammatory cells, and suggest that phenotypic alterations of human pre-adipocytes, induced by a pro-inflammatory environment, may lead to an excessive synthesis of ECM components. Conclusion This study opens new perspectives in understanding the biology of human WAT and its pathologic changes indicative of tissue deterioration associated with the development of obesity. PMID:18208606

  14. Extracellular Microvesicle Production by Human Eosinophils Activated by “Inflammatory” Stimuli

    PubMed Central

    Akuthota, Praveen; Carmo, Lívia A. S.; Bonjour, Kennedy; Murphy, Ryann O.; Silva, Thiago P.; Gamalier, Juliana P.; Capron, Kelsey L.; Tigges, John; Toxavidis, Vasilis; Camacho, Virginia; Ghiran, Ionita; Ueki, Shigeharu; Weller, Peter F.; Melo, Rossana C. N.

    2016-01-01

    A key function of human eosinophils is to secrete cytokines, chemokines and cationic proteins, trafficking, and releasing these mediators for roles in inflammation and other immune responses. Eosinophil activation leads to secretion of pre-synthesized granule-stored mediators through different mechanisms, but the ability of eosinophils to secrete extracellular vesicles (EVs), very small vesicles with preserved membrane topology, is still poorly understood. In the present work, we sought to identify and characterize EVs released from human eosinophils during different conditions: after a culturing period or after isolation and stimulation with inflammatory stimuli, which are known to induce eosinophil activation and secretion: CCL11 (eotaxin-1) and tumor necrosis factor alpha (TNF-α). EV production was investigated by nanoscale flow cytometry, conventional transmission electron microscopy (TEM) and pre-embedding immunonanogold EM. The tetraspanins CD63 and CD9 were used as EV biomarkers for both flow cytometry and ultrastructural immunolabeling. Nanoscale flow cytometry showed that human eosinophils produce EVs in culture and that a population of EVs expressed detectable CD9, while CD63 was not consistently detected. When eosinophils were stimulated immediately after isolation and analyzed by TEM, EVs were clearly identified as microvesicles (MVs) outwardly budding off the plasma membrane. Both CCL11 and TNF-α induced significant increases of MVs compared to unstimulated cells. TNF-α induced amplified release of MVs more than CCL11. Eosinophil MV diameters varied from 20 to 1000 nm. Immunonanogold EM revealed clear immunolabeling for CD63 and CD9 on eosinophil MVs, although not all MVs were labeled. Altogether, we identified, for the first time, that human eosinophils secrete MVs and that this production increases in response to inflammatory stimuli. This is important to understand the complex secretory activities of eosinophils underlying immune responses. The

  15. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix.

    PubMed

    Gronthos, S; Simmons, P J; Graves, S E; Robey, P G

    2001-02-01

    To date, the precise interactions between bone marrow stromal cells and the extracellular matrix that govern stromal cell development remain unclear. The integrin super-family of cell-surface adhesion molecules represents a major pathway used by virtually all cell types to interact with different extracellular matrix components. In this study, purified populations of stromal precursor cells were isolated from the STRO-1-positive fraction of normal human marrow, by fluoresence-activated cell sorting, and then assayed for their ability to initiate clonogenic growth in the presence of various integrin ligands. Bone marrow-derived stromal progenitors displayed differential growth to fibronectin, vitronectin, and laminin, over collagen types I and III, but showed a similar affinity for collagen type IV. The integrin heterodimers alpha1beta1, alpha2beta1, alpha5beta1, alpha6beta1, alpha(v)beta3, and alpha(v)beta5 were found to coexpress with the STRO-1 antigen on the cell surface of CFU-F, using dual-color analysis. Furthermore, only a proportion of stromal precursors expressed the integrin alpha4beta1, while no measurable levels of the integrin alpha3beta1 could be detected. Subsequent adhesion studies using functional blocking antibodies to different integrin alpha/beta heterodimers showed that stromal cell growth on collagen, laminin, and fibronectin was mediated by multiple beta1 integrins. In contrast, cloning efficiency in the presence of vitronectin was mediated in part by alpha(v)beta3. When human marrow stromal cells were cultured under osteoinductive conditions, their ability to form a mineralized matrix in vitro was significantly diminished in the presence of a functional blocking monoclonal antibody to the beta1 integrin subunit. The results of this study indicate that beta1 integrins appear to be the predominant adhesion receptor subfamily utilized by stromal precursor cells to adhere and proliferate utilizing matrix glycoproteins commonly found in the bone

  16. Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

    PubMed Central

    Blans, Kristine; Hansen, Maria S.; Sørensen, Laila V.; Hvam, Michael L.; Howard, Kenneth A.; Möller, Arne; Wiking, Lars; Larsen, Lotte B.; Rasmussen, Jan T.

    2017-01-01

    ABSTRACT Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk. PMID:28386391

  17. Extracellular Matrix Mediated Maturation of Human Pluripotent Stem Cell Derived Cardiac Monolayer Structure and Electrophysiological Function

    PubMed Central

    Herron, Todd J.; Rocha, Andre Monteiro Da; Campbell, Katherine; Ponce-Balbuena, Daniela; Willis, B. Cicero; Guerrero-Serna, Guadalupe; Liu, Qinghua; Klos, Matt; Musa, Hassan; Zarzoso, Manuel; Bizy, Alexandra; Furness, Jamie; Anumonwo, Justus; Mironov, Sergey; Jalife, José

    2016-01-01

    Background Human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electrical impulse propagation velocity and immature action potential profiles. Methods and Results Here we have identified an optimal extracellular matrix (ECM) for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal ECM combination have impulse propagation velocities ~2X faster than previously reported (43.6±7.0 cm·s−1 n=9) and have mature cardiomyocyte action potential profiles including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s, N=5 monolayers). In addition the optimal ECM promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1 and Connexin43) and myofilament markers (cTroponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase (FAK) activation prevented structural maturation. Conclusions Maturation of human stem cell derived cardiomyocyte monolayers is achieved in a one week period by plating cardiomyocytes on PDMS coverslips rather than on conventional 2D cell culture formats such as glass coverslips or plastic dishes. Activation of integrin signaling and FAK are essential for significant maturation of human cardiac monolayers. PMID:27069088

  18. Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis

    PubMed Central

    Bonne-Année, Sandra; Kerepesi, Laura A.; Hess, Jessica A.; Wesolowski, Jordan; Paumet, Fabienne; Lok, James B.; Nolan, Thomas J.; Abraham, David

    2014-01-01

    Neutrophils are multifaceted cells that are often the immune system’s first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system. PMID:24642003

  19. The origin, function, and diagnostic potential of RNA within extracellular vesicles present in human biological fluids

    PubMed Central

    Taylor, Douglas D.; Gercel-Taylor, Cicek

    2013-01-01

    We have previously demonstrated that tumor cells release membranous structures into their extracellular environment, which are termed exosomes, microvesicles or extracellular vesicles depending on specific characteristics, including size, composition and biogenesis pathway. These cell-derived vesicles can exhibit an array of proteins, lipids and nucleic acids derived from the originating tumor. This review focuses of the transcriptome (RNA) of these extracellular vesicles. Based on current data, these vesicular components play essential roles as conveyers of intercellular communication and mediators of many of the pathological conditions associated with cancer development, progression and therapeutic failures. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, signal pathway activation through growth factor/receptor transfer, chemoresistance, and genetic exchange. These tumor-derived extracellular vesicles not only to represent a central mediator of the tumor microenvironment, but their presence in the peripheral circulation may serve as a surrogate for tumor biopsies, enabling real-time diagnosis and disease monitoring. PMID:23908664

  20. The origin, function, and diagnostic potential of RNA within extracellular vesicles present in human biological fluids.

    PubMed

    Taylor, Douglas D; Gercel-Taylor, Cicek

    2013-01-01

    We have previously demonstrated that tumor cells release membranous structures into their extracellular environment, which are termed exosomes, microvesicles or extracellular vesicles depending on specific characteristics, including size, composition and biogenesis pathway. These cell-derived vesicles can exhibit an array of proteins, lipids and nucleic acids derived from the originating tumor. This review focuses of the transcriptome (RNA) of these extracellular vesicles. Based on current data, these vesicular components play essential roles as conveyers of intercellular communication and mediators of many of the pathological conditions associated with cancer development, progression and therapeutic failures. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodeling, signal pathway activation through growth factor/receptor transfer, chemoresistance, and genetic exchange. These tumor-derived extracellular vesicles not only to represent a central mediator of the tumor microenvironment, but their presence in the peripheral circulation may serve as a surrogate for tumor biopsies, enabling real-time diagnosis and disease monitoring.

  1. Extracellular matrix from human umbilical cord-derived mesenchymal stem cells as a scaffold for peripheral nerve regeneration

    PubMed Central

    Xiao, Bo; Rao, Feng; Guo, Zhi-yuan; Sun, Xun; Wang, Yi-guo; Liu, Shu-yun; Wang, Ai-yuan; Guo, Quan-yi; Meng, Hao-ye; Zhao, Qing; Peng, Jiang; Wang, Yu; Lu, Shi-bi

    2016-01-01

    The extracellular matrix, which includes collagens, laminin, or fibronectin, plays an important role in peripheral nerve regeneration. Recently, a Schwann cell-derived extracellular matrix with classical biomaterial was used to mimic the neural niche. However, extensive clinical use of Schwann cells remains limited because of the limited origin, loss of an autologous nerve, and extended in vitro culture times. In the present study, human umbilical cord-derived mesenchymal stem cells (hUCMSCs), which are easily accessible and more proliferative than Schwann cells, were used to prepare an extracellular matrix. We identified the morphology and function of hUCMSCs and investigated their effect on peripheral nerve regeneration. Compared with a non-coated dish tissue culture, the hUCMSC-derived extracellular matrix enhanced Schwann cell proliferation, upregulated gene and protein expression levels of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor in Schwann cells, and enhanced neurite outgrowth from dorsal root ganglion neurons. These findings suggest that the hUCMSC-derived extracellular matrix promotes peripheral nerve repair and can be used as a basis for the rational design of engineered neural niches. PMID:27630705

  2. Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations

    PubMed Central

    Iwai, Kazuya; Minamisawa, Tamiko; Suga, Kanako; Yajima, Yasutomo; Shiba, Kiyotaka

    2016-01-01

    Diagnostic methods that focus on the extracellular vesicles (EVs) present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA) and microRNA (miRNA), which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation. This article establishes a protocol for the isolation of EVs from human saliva using density gradient centrifugation. The fractionated salivary EVs were characterized by atomic force microscopy, western blot and reverse transcription polymerase chain reaction. The results indicate that salivary EVs have a smaller diameter (47.8±12.3 nm) and higher density (1.11 g/ml) than EVs isolated from conditioned cell media (74.0±23.5 nm and 1.06 g/ml, respectively). Additionally, to improve the throughput of density-based fractionation of EVs, the original protocol was further modified by using a fixed angle rotor instead of a swinging rotor. It was also confirmed that several miRNAs were expressed strongly in the EV-marker-expressing fractions. PMID:27193612

  3. Second harmonic generation microscopy analysis of extracellular matrix changes in human idiopathic pulmonary fibrosis

    PubMed Central

    Tilbury, Karissa; Hocker, James; Wen, Bruce L.; Sandbo, Nathan; Singh, Vikas; Campagnola, Paul J.

    2014-01-01

    Abstract. Patients with idiopathic fibrosis (IPF) have poor long-term survival as there are limited diagnostic/prognostic tools or successful therapies. Remodeling of the extracellular matrix (ECM) has been implicated in IPF progression; however, the structural consequences on the collagen architecture have not received considerable attention. Here, we demonstrate that second harmonic generation (SHG) and multiphoton fluorescence microscopy can quantitatively differentiate normal and IPF human tissues. For SHG analysis, we developed a classifier based on wavelet transforms, principle component analysis, and a K-nearest-neighbor algorithm to classify the specific alterations of the collagen structure observed in IPF tissues. The resulting ROC curves obtained by varying the numbers of principal components and nearest neighbors yielded accuracies of >95%. In contrast, simpler metrics based on SHG intensity and collagen coverage in the image provided little or no discrimination. We also characterized the change in the elastin/collagen balance by simultaneously measuring the elastin autofluorescence and SHG intensities and found that the IPF tissues were less elastic relative to collagen. This is consistent with known mechanical consequences of the disease. Understanding ECM remodeling in IPF via nonlinear optical microscopy may enhance our ability to differentiate patients with rapid and slow progression and, thus, provide better prognostic information. PMID:25134793

  4. Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice.

    PubMed

    Thomas, Grace M; Carbo, Carla; Curtis, Brian R; Martinod, Kimberly; Mazo, Irina B; Schatzberg, Daphne; Cifuni, Stephen M; Fuchs, Tobias A; von Andrian, Ulrich H; Hartwig, John H; Aster, Richard H; Wagner, Denisa D

    2012-06-28

    Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related death. The biologic processes contributing to TRALI are poorly understood. All blood products can cause TRALI, and no specific treatment is available. A "2-event model" has been proposed as the trigger. The first event may include surgery, trauma, or infection; the second involves the transfusion of antileukocyte antibodies or bioactive lipids within the blood product. Together, these events induce neutrophil activation in the lungs, causing endothelial damage and capillary leakage. Neutrophils, in response to pathogens or under stress, can release their chromatin coated with granule contents, thus forming neutrophil extracellular traps (NETs). Although protective against infection, these NETs are injurious to tissue. Here we show that NET biomarkers are present in TRALI patients' blood and that NETs are produced in vitro by primed human neutrophils when challenged with anti-HNA-3a antibodies previously implicated in TRALI. NETs are found in alveoli of mice experiencing antibody-mediated TRALI. DNase 1 inhalation prevents their alveolar accumulation and improves arterial oxygen saturation even when administered 90 minutes after TRALI onset. We suggest that NETs form in the lungs during TRALI, contribute to the disease process, and thus could be targeted to prevent or treat TRALI.

  5. Clinical Application of Human Urinary Extracellular Vesicles in Kidney and Urologic Diseases

    PubMed Central

    De Palma, Giuseppe; Sallustio, Fabio; Schena, Francesco Paolo

    2016-01-01

    Extracellular vesicles (EVs) have been isolated in different body fluids, including urine. The cargo of urinary EVs is composed of nucleic acids and proteins reflecting the physiological and possibly pathophysiological state of cells lining the nephron and the urinary tract. Urinary EVs have been confirmed to contain low amounts of various types of RNA that play a role in intercellular communication by transferring genetic information. This communication through EV RNAs includes both continuation of normal physiological processes and conditioning in disease mechanisms. Although proteins included in urinary EVs represent only 3% of the whole-urine proteome, urinary EVs can influence cells in the renal epithelia not only by delivering RNA cargo, but also by delivering a wide range of proteins. Since urine is a readily available biofluid, the discovery of EVs has opened a new field of biomarker research. The potential use of urinary EV RNAs and proteins as diagnostic biomarkers for various kidney and urologic diseases is currently being explored. Here, we review recent studies that deal in identifying biomarker candidates for human kidney and urologic diseases using urinary EVs and might help to understand the pathophysiology. PMID:27376269

  6. Biocompatibility of pure titanium modified by human endothelial cell-derived extracellular matrix

    NASA Astrophysics Data System (ADS)

    Xue, Xiaoqing; Wang, Jin; Zhu, Ying; Tu, Qiufen; Huang, Nan

    2010-04-01

    Extracellular matrix (ECM) used to modify biomaterial surface is a promising method for improving cardiovascular material hemocompatibility. In the present work, human umbilical vein endothelial cells (HUVECs) are cultured and native ECM is obtained on pure titanium surface. Fourier infrared spectrum (FTIR) test proves the existence of amide I and amide II band on the modified titanium surface. X-ray photoelectron spectroscopy (XPS) further confirms the chemical composition and binding types of the ECM proteins on the titanium substrate. The results of light microscopy and atomic force microscopy (AFM) exhibit the morphology of HUVEC derived ECM. There are higher water contact angles on the ECM modified samples. Furthermore, some ECM components, including fibronectin (FN), laminin (LN) and type IV collagen (IV-COL) are presented on ECM-covered titanium surface by immunofluorescence staining. The biological behavior of cultured HUVECs and adherent platelets on different samples are investigated by in vitro HUVECs culture and platelet adhesion. Cells exhibit better morphology and their proliferation ability greatly improve on the ECM-covered titanium. At the same time, the platelet adhesion and spreading are inhibited on ECM-covered titanium surface. These investigations demonstrate that ECM produced by HUVECs cannot only improve adhesion and proliferation ability of endothelial cell but also inhibit adhesion and activation of platelets. Thus, the approach described here may provide a basis for preparation of modified surface in cardiovascular implants application.

  7. Human Amniotic Membrane Mesenchymal Stem Cells inhibit Neutrophil Extracellular Traps through TSG-6.

    PubMed

    Magaña-Guerrero, Fátima Sofía; Domínguez-López, Alfredo; Martínez-Aboytes, Pamela; Buentello-Volante, Beatriz; Garfias, Yonathan

    2017-09-29

    The mesenchymal stem cells obtained from human amniotic membrane (hAMSC) possess immunosuppressive functions through soluble factors such as prostanoids and proteins; thus, they have been proposed to ameliorate inflammatory processes. On the other hand, activated neutrophils are cells of the first line of immune defense that are able to release extracellular traps (NETs). NETs are formed of DNA and granular components; however, the excessive release of NETs is associated with the development of autoimmune and chronic inflammatory diseases. In this study, we identified that conditioned medium (CM) from hAMSC was able to diminish NETs release, as well as the production of reactive oxygen species (ROS) and the mitochondrial membrane potential from LPS-stimulated mouse bone marrow-derived neutrophils (BMN). Interestingly, NETs inhibition, ROS levels decrease and mitochondrial membrane potential loss were reverted when LPS-stimulated murine derived BMN were exposed to the CM from hAMSC transfected with TSG-6-siRNA. Finally, rhTSG6 was able to significantly diminish NETs release in BMN. These data suggest an inhibition mechanism of NETs ROS-dependent in which TSG-6 participates. Consequently, we propose the hAMSC use as a therapeutic candidate in the treatment of inflammatory diseases in which NETs are involved.

  8. Extracellular matrix signatures of human mammary carcinoma identify novel metastasis promoters

    PubMed Central

    Naba, Alexandra; Clauser, Karl R; Lamar, John M; Carr, Steven A; Hynes, Richard O

    2014-01-01

    The extracellular matrix (ECM) is a major component of tumors and a significant contributor to cancer progression. In this study, we use proteomics to investigate the ECM of human mammary carcinoma xenografts and show that primary tumors of differing metastatic potential differ in ECM composition. Both tumor cells and stromal cells contribute to the tumor matrix and tumors of differing metastatic ability differ in both tumor- and stroma-derived ECM components. We define ECM signatures of poorly and highly metastatic mammary carcinomas and these signatures reveal up-regulation of signaling pathways including TGFβ and VEGF. We further demonstrate that several proteins characteristic of highly metastatic tumors (LTBP3, SNED1, EGLN1, and S100A2) play causal roles in metastasis, albeit at different steps. Finally we show that high expression of LTBP3 and SNED1 correlates with poor outcome for ER−/PR−breast cancer patients. This study thus identifies novel biomarkers that may serve as prognostic and diagnostic tools. DOI: http://dx.doi.org/10.7554/eLife.01308.001 PMID:24618895

  9. The transcriptional signature of human ovarian carcinoma macrophages is associated with extracellular matrix reorganization

    PubMed Central

    Adhikary, Till; Wortmann, Annika; Hoffmann, Nathalie; Bieringer, Tim; Nist, Andrea; Stiewe, Thorsten; Jansen, Julia M.; Wagner, Uwe; Müller-Brüsselbach, Sabine; Müller, Rolf

    2016-01-01

    Macrophages occur as resident cells of fetal origin or as infiltrating blood monocyte-derived cells. Despite the critical role of tumor-associated macrophages (TAMs) in tumor progression, the contribution of these developmentally and functionally distinct macrophage subsets and their alteration by the tumor microenvironment are poorly understood. We have addressed this question by comparing TAMs from human ovarian carcinoma ascites, resident peritoneal macrophages (pMPHs) and monocyte-derived macrophages (MDMs). Our study revealed striking a similarity between TAMs and pMPHs, which was considerably greater that the resemblance of TAMs and MDMs, including their transcriptomes, their inflammation-related activation state, the presence of receptors mediating immune functions and the expression of tumor-promoting mediators. Consistent with these results, TAMs phagocytized bacteria, presented peptide antigens and activated cytotoxic T cells within their pathophysiological environment. These observations support the notion that tumor-promoting properties of TAMs may reflect, at least to some extent, normal features of resident macrophages rather than functions induced by the tumor microenvironment. In spite of these surprising similarities between TAMs and pMPHs, bioinformatic analyses identified a TAM-selective signature of 30 genes that are upregulated relative to both pMPHs and MDMs. The majority of these genes is linked to extracellular matrix (ECM) remodeling, supporting a role for TAMs in cancer cell invasion and ovarian cancer progression. PMID:27659538

  10. Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1.

    PubMed

    Saitoh, Tatsuya; Komano, Jun; Saitoh, Yasunori; Misawa, Takuma; Takahama, Michihiro; Kozaki, Tatsuya; Uehata, Takuya; Iwasaki, Hidenori; Omori, Hiroko; Yamaoka, Shoji; Yamamoto, Naoki; Akira, Shizuo

    2012-07-19

    Neutrophils contribute to pathogen clearance by producing neutrophil extracellular traps (NETs), which are genomic DNA-based net-like structures that capture bacteria and fungi. Although NETs also express antiviral factors, such as myeloperoxidase and α-defensin, the involvement of NETs in antiviral responses remains unclear. We show that NETs capture human immunodeficiency virus (HIV)-1 and promote HIV-1 elimination through myeloperoxidase and α-defensin. Neutrophils detect HIV-1 by Toll-like receptors (TLRs) TLR7 and TLR8, which recognize viral nucleic acids. Engagement of TLR7 and TLR8 induces the generation of reactive oxygen species that trigger NET formation, leading to NET-dependent HIV-1 elimination. However, HIV-1 counteracts this response by inducing C-type lectin CD209-dependent production of interleukin (IL)-10 by dendritic cells to inhibit NET formation. IL-10 suppresses the reactive oxygen species-dependent generation of NETs induced upon TLR7 and TLR8 engagement, resulting in disrupted NET-dependent HIV-1 elimination. Therefore, NET formation is an antiviral response that is counteracted by HIV-1. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. A bioinformatics analysis of alternative exon usage in human genes coding for extracellular matrix proteins.

    PubMed

    Sakabe, Noboru Jo; Vibranovski, Maria Dulcetti; de Souza, Sandro José

    2004-12-30

    Alternative splicing increases protein diversity through the generation of different mRNA molecules from the same gene. Although alternative splicing seems to be a widespread phenomenon in the human transcriptome, it is possible that different subgroups of genes present different patterns, related to their biological roles. Analysis of a subgroup may enhance common features of its members that would otherwise disappear amidst a heterogeneous population. Extracellular matrix (ECM) proteins are a good set for such analyses since they are structurally and functionally related. This family of proteins is involved in a large variety of functions, probably achieved by the combinatorial use of protein domains through exon shuffling events. To determine if ECM genes have a different pattern of alternative splicing, we compared clusters of expressed sequences of ECM to all other genes regarding features related to the most frequent type of alternative splicing, alternative exon usage (AEU), such as: the number of alternative exon-intron structures per cluster, the number of AEU events per exon-intron structure, the number of exons per event, among others. Although we did not find many differences between the two sets, we observed a higher frequency of AEU events involving entire protein domains in the ECM set, a feature that could be associated with their multi-domain nature. As other subgroups or even the ECM set in different tissues could present distinct patterns of AEU, it may be premature to conclude that alternative splicing is homogeneous among groups of related genes.

  12. Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR).

    PubMed

    Gallart-Palau, Xavier; Serra, Aida; Wong, Andrew See Weng; Sandin, Sara; Lai, Mitchell K P; Chen, Christopher P; Kon, Oi Lian; Sze, Siu Kwan

    2015-09-30

    Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications.

  13. Quantitative proteomics of extracellular vesicles derived from human primary and metastatic colorectal cancer cells.

    PubMed

    Choi, Dong-Sic; Choi, Do-Young; Hong, Bok Sil; Jang, Su Chul; Kim, Dae-Kyum; Lee, Jaewook; Kim, Yoon-Keun; Kim, Kwang Pyo; Gho, Yong Song

    2012-01-01

    Cancer cells actively release extracellular vesicles (EVs), including exosomes and microvesicles, into surrounding tissues. These EVs play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteomic differences between primary and metastatic cancer cell-derived EVs remain unclear. Here, we conducted comparative proteomic analysis between EVs derived from human primary colorectal cancer cells (SW480) and their metastatic derivatives (SW620). Using label-free quantitation, we identified 803 and 787 proteins in SW480 EVs and SW620 EVs, respectively. Based on comparison between the estimated abundance of EV proteins, we identified 368 SW480 EV-enriched and 359 SW620 EV-enriched proteins. SW480 EV-enriched proteins played a role in cell adhesion, but SW620 EV-enriched proteins were associated with cancer progression and functioned as diagnostic indicators of metastatic cancer; they were overexpressed in metastatic colorectal cancer and played roles in multidrug resistance. As the first proteomic analysis comparing primary and metastatic cancer-derived EVs, this study increases our understanding of the pathological function of EVs in the metastatic process and provides useful biomarkers for cancer metastasis.

  14. Distribution volumes of macromolecules in human ovarian and endometrial cancers--effects of extracellular matrix structure.

    PubMed

    Haslene-Hox, Hanne; Oveland, Eystein; Woie, Kathrine; Salvesen, Helga B; Tenstad, Olav; Wiig, Helge

    2015-01-01

    Elements of the extracellular matrix (ECM), notably collagen and glucosaminoglycans, will restrict part of the space available for soluble macromolecules simply because the molecules cannot occupy the same space. This phenomenon may influence macromolecular drug uptake. To study the influence of steric and charge effects of the ECM on the distribution volumes of macromolecules in human healthy and malignant gynecologic tissues we used as probes 15 abundant plasma proteins quantified by high-resolution mass spectrometry. The available distribution volume (VA) of albumin was increased in ovarian carcinoma compared with healthy ovarian tissue. Furthermore, VA of plasma proteins between 40 and 190 kDa decreased with size for endometrial carcinoma and healthy ovarian tissue, but was independent of molecular weight for the ovarian carcinomas. An effect of charge on distribution volume was only found in healthy ovaries, which had lower hydration and high collagen content, indicating that a condensed interstitium increases the influence of negative charges. A number of earlier suggested biomarker candidates were detected in increased amounts in malignant tissue, e.g., stathmin and spindlin-1, showing that interstitial fluid, even when unfractionated, can be a valuable source for tissue-specific proteins. We demonstrate that the distribution of abundant plasma proteins in the interstitium can be elucidated by mass spectrometry methods and depends markedly on hydration and ECM structure. Our data can be used in modeling of drug uptake, and give indications on ECM components to be targeted to increase the uptake of macromolecular substances. Copyright © 2015 the American Physiological Society.

  15. Random sequential adsorption of human adenovirus 2 onto polyvinylidene fluoride surface influenced by extracellular polymeric substances.

    PubMed

    Lu, Ruiqing; Li, Qi; Nguyen, Thanh H

    2016-03-15

    Virus removal by membrane bioreactors depends on virus-membrane and virus-foulant interactions. The adsorption of human adenovirus 2 (HAdV-2) on polyvinylidene fluoride (PVDF) membrane and a major membrane foulant, extracellular polymeric substances (EPS), were measured in a quartz crystal microbalance. In 3-100mM CaCl2 solutions, irreversible adsorption of HAdV-2 was observed on both pristine and EPS-fouled PVDF surfaces. The HAdV-2 adsorption kinetics was successfully fitted with the random sequential adsorption (RSA) model. The applicability of the RSA model for HAdV-2 adsorption is confirmed by comparing the two fitting parameters, adsorption rate constant k(a) and area occupied by each adsorbed HAdV-2 particle a, with experimentally measured parameters. A linear correlation between the fitting parameter k(a) and the measured attachment efficiency was found, suggesting that the RSA model correctly describes the interaction forces dominating the HAdV-2 adsorption. By comparing the fitting parameter d(ads) with the hydrodynamic diameter of HAdV-2, we conclude that virus-virus and virus-surface interactions determine the area occupied by each adsorbed HAdV-2 particle, and thus influence the adsorption capacity. These results provide insights into virus retention and will benefit improving virus removal in membrane filtration.

  16. Superoxide production by a manganese-oxidizing bacterium facilitates iodide oxidation.

    PubMed

    Li, Hsiu-Ping; Daniel, Benjamin; Creeley, Danielle; Grandbois, Russell; Zhang, Saijin; Xu, Chen; Ho, Yi-Fang; Schwehr, Kathy A; Kaplan, Daniel I; Santschi, Peter H; Hansel, Colleen M; Yeager, Chris M

    2014-05-01

    The release of radioactive iodine (i.e., iodine-129 and iodine-131) from nuclear reprocessing facilities is a potential threat to human health. The fate and transport of iodine are determined primarily by its redox status, but processes that affect iodine oxidation states in the environment are poorly characterized. Given the difficulty in removing electrons from iodide (I(-)), naturally occurring iodide oxidation processes require strong oxidants, such as Mn oxides or microbial enzymes. In this study, we examine iodide oxidation by a marine bacterium, Roseobacter sp. AzwK-3b, which promotes Mn(II) oxidation by catalyzing the production of extracellular superoxide (O2(-)). In the absence of Mn(2+), Roseobacter sp. AzwK-3b cultures oxidized ∼90% of the provided iodide (10 μM) within 6 days, whereas in the presence of Mn(II), iodide oxidation occurred only after Mn(IV) formation ceased. Iodide oxidation was not observed during incubations in spent medium or with whole cells under anaerobic conditions or following heat treatment (boiling). Furthermore, iodide oxidation was significantly inhibited in the presence of superoxide dismutase and diphenylene iodonium (a general inhibitor of NADH oxidoreductases). In contrast, the addition of exogenous NADH enhanced iodide oxidation. Taken together, the results indicate that iodide oxidation was mediated primarily by extracellular superoxide generated by Roseobacter sp. AzwK-3b and not by the Mn oxides formed by this organism. Considering that extracellular superoxide formation is a widespread phenomenon among marine and terrestrial bacteria, this could represent an important pathway for iodide oxidation in some environments.

  17. Superoxide Production by a Manganese-Oxidizing Bacterium Facilitates Iodide Oxidation

    PubMed Central

    Li, Hsiu-Ping; Daniel, Benjamin; Creeley, Danielle; Grandbois, Russell; Zhang, Saijin; Xu, Chen; Ho, Yi-Fang; Schwehr, Kathy A.; Kaplan, Daniel I.; Santschi, Peter H.; Hansel, Colleen M.

    2014-01-01

    The release of radioactive iodine (i.e., iodine-129 and iodine-131) from nuclear reprocessing facilities is a potential threat to human health. The fate and transport of iodine are determined primarily by its redox status, but processes that affect iodine oxidation states in the environment are poorly characterized. Given the difficulty in removing electrons from iodide (I−), naturally occurring iodide oxidation processes require strong oxidants, such as Mn oxides or microbial enzymes. In this study, we examine iodide oxidation by a marine bacterium, Roseobacter sp. AzwK-3b, which promotes Mn(II) oxidation by catalyzing the production of extracellular superoxide (O2−). In the absence of Mn2+, Roseobacter sp. AzwK-3b cultures oxidized ∼90% of the provided iodide (10 μM) within 6 days, whereas in the presence of Mn(II), iodide oxidation occurred only after Mn(IV) formation ceased. Iodide oxidation was not observed during incubations in spent medium or with whole cells under anaerobic conditions or following heat treatment (boiling). Furthermore, iodide oxidation was significantly inhibited in the presence of superoxide dismutase and diphenylene iodonium (a general inhibitor of NADH oxidoreductases). In contrast, the addition of exogenous NADH enhanced iodide oxidation. Taken together, the results indicate that iodide oxidation was mediated primarily by extracellular superoxide generated by Roseobacter sp. AzwK-3b and not by the Mn oxides formed by this organism. Considering that extracellular superoxide formation is a widespread phenomenon among marine and terrestrial bacteria, this could represent an important pathway for iodide oxidation in some environments. PMID:24561582

  18. Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells

    SciTech Connect

    Rappa, Germana; Mercapide, Javier; Anzanello, Fabio; Le, Thuc T.; Johlfs, Mary G.; Fiscus, Ronald R.; Wilsch-Bräuninger, Michaela; Corbeil, Denis; Lorico, Aurelio

    2013-04-01

    Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that three distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1–positive structures appeared in three sizes (small, ≤40 nm; intermediates ∼40–80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1–containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma. - Highlights: ► First report of release of prominin-1–containing microvesicles from cancer cells. ► Pro-metastatic role of prominin-1–containing microvesicles in

  19. Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Ye, C.; Vassilev, P. M.; Sanders, J. L.; Brown, E. M.

    2001-01-01

    We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

  20. Immunohistochemical localization of extracellular matrix components in human diabetic glomerular lesions.

    PubMed Central

    Nerlich, A.; Schleicher, E.

    1991-01-01

    The immunohistochemical localization of the extracellular matrix was examined in 31 cases with different degrees of human diabetic nephropathy using antisera to human collagen types I, III, IV, V, fibronectin, laminin, and basement-membrane-associated heparan sulfate proteoglycan (HSPG). In normal glomeruli, HSPG was predominantly localized in the glomerular basement membrane and in the mesangium, and to minor extent in the basement membranes of tubules and Bowman's capsule. Collagen IV and laminin were distributed in glomerular basement membrane and mesangium in minor amounts. Interstitial collagens usually do not occur within glomeruli except for collagen V which has a light microscopic glomerular distribution similar to collagen IV. In diabetic diffuse glomerulosclerosis, the enlarged mesangial matrix showed an increased staining reaction for collagen IV, V, laminin, and fibronectin whereas the staining pattern of HSPG was markedly reduced. Early, small nodular lesions in diabetic glomeruli were similarly positive for most of the basement membrane components, whereas HSPG remained absent. With an increase in the diameter of the noduli, however, the staining reaction for all basement membrane components diminished, whereas interstitial collagens V and III, but not collagen I, were present in these noduli in substantial amounts. These initial studies provide evidence that the changes in the glomerular matrix in diabetic nephropathy may be divided into distinct and progressing stages of lesions. The reduced amount of HSPG even in slight, early lesions may represent the morphologic correlate to the impaired filter function of the glomerular basement membrane. Images Figure 3 Figure 5 Figure 1 Figure 2 Figure 4 PMID:1928305

  1. Effects of ranibizumab on the extracellular matrix production by human Tenon's fibroblast.

    PubMed

    Md Noh, Siti Munirah; Sheikh Abdul Kadir, Siti H; Bannur, Zakaria M; Froemming, Gabriele Anisah; Abdul Hamid Hasani, Narimah; Mohd Nawawi, Hapizah; Crowston, Jonathan G; Vasudevan, Sushil

    2014-10-01

    Anti-Vascular Endothelial Growth Factors (Anti-VEGF) agents have received recent interest as potential anti-fibrotic agents for their concurrent use with trabeculectomy. Preliminary cohort studies have revealed improved bleb morphology following trabeculectomy augmented with ranibizumab. The effects of this humanized monoclonal antibody on human Tenon's fibroblast (HTF), the key player of post trabeculectomy scar formation, are not fully understood. This study was conducted to understand the effects of ranibizumab on extracellular matrix production by HTF. The effect of ranibizumab on HTF proliferation and cell viability was determined using MTT assay (3-(4,5-dimethylthiazone-2-yl)-2,5-diphenyl tetrazolium). Ranibizumab at concentrations ranging from 0.01 to 0.5 mg/mL were administered for 24, 48 and 72 h in serum and serum free conditions. Supernatants and cell lysates from samples were assessed for collagen type 1 alpha 1 and fibronectin mRNA and protein level using quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). After 48-h, ranibizumab at 0.5 mg/mL, significantly induced cell death under serum-free culture conditions (p < 0.05). Ranibizumab caused significant reduction of collagen type 1 alpha 1 (COL1A1) mRNA, but not for fibronectin (FN). Meanwhile, COL1A1 and FN protein levels were found upregulated in treated monolayers compared to control monolayers. Ranibizumab at 0.5 mg/mL significantly reduced cell viability in cultured HTF. From this study, we found that single application of ranibizumab is inadequate to induce the anti-fibrotic effects on HTF, suggesting the importance of adjunctive therapy. Further studies are underway to understand mechanism of actions of ranibizumab on HTF. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors.

    PubMed

    Moritz, T; Patel, V P; Williams, D A

    1994-04-01

    Direct contact between hematopoietic cells and viral packaging cell lines or other sources of stroma has been shown to increase the efficiency of retroviral-mediated gene transfer into these target cells compared with infection with viral supernatant. We have investigated the role of defined bone marrow extracellular matrix molecules (ECM) in this phenomenon. Here we report that infection of cells adhering to the carboxy-terminal 30/35-kD fragment of the fibronectin molecule (30/35 FN), which contains the alternatively spliced CS-1 cell adhesion domain, significantly increases gene transfer into hematopoietic cells. Two retroviral vectors differing in recombinant viral titer were used. Gene transfer into committed progenitor cells and long-term culture-initiating cells, an in vitro assay for human stem cells, was significantly increased when the cells were infected while adherent to 30/35 FN-coated plates compared with cells infected on BSA-coated control plates or plates coated with other bone marrow ECM molecules. Although gene transfer into committed progenitor cells and to a lesser degree into long-term culture-initiating cells was increased on intact fibronectin as well, increased gene transfer efficiency into hematopoietic cells on 30/35 FN was dependent on CS-1 sequence since infection on a similar FN fragment lacking CS-1 (42 FN) was suboptimal. 30/35 FN has previously been shown by our laboratory and other investigators to mediate adhesion of primitive murine and human hematopoietic stem cells to the hematopoietic microenvironment. Additional studies showed that neither soluble 30/35 FN nor nonspecific binding of hematopoietic cells to poly-L-lysine-coated plates had any appreciable effect on the infection efficiency of these cells. Our findings indicate that hematopoietic stem cell adhesion to specific ECM molecules alters retroviral infection efficiency. These findings should aid in the design of gene transfer protocols using hematopoietic progenitor and

  3. Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Ye, C.; Vassilev, P. M.; Sanders, J. L.; Brown, E. M.

    2001-01-01

    We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

  4. Impact of acetaminophen consumption and resistance exercise on extracellular matrix gene expression in human skeletal muscle.

    PubMed

    Patel, Shivam H; D'Lugos, Andrew C; Eldon, Erica R; Curtis, Donald; Dickinson, Jared M; Carroll, Chad C

    2017-07-01

    Acetaminophen (APAP) given during chronic exercise reduces skeletal muscle collagen and cross-linking in rats. We propose that the effect of APAP on muscle extracellular matrix (ECM) may, in part, be mediated by dysregulation of the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). The purpose of this study was to evaluate the impact of APAP consumption during acute resistance exercise (RE) on several regulators of the ECM in human skeletal muscle. In a double-blinded, placebo-controlled, randomized crossover design, recreationally active men (n = 8, 25 ± 2 yr) performed two trials of knee extension. Placebo (PLA) or APAP (1,000 mg/6 h) was given for 24 h before and immediately following RE. Vastus lateralis biopsies were taken at baseline and 1 and 3 h post-RE. Quantitative RT-PCR was used to determine differences in mRNA expression. MMP-2, type I collagen, and type III collagen mRNA expression was not altered by exercise or APAP (P > 0.05). When compared with PLA, TIMP-1 expression was lower at 1 h post-RE during APAP conditions but greater than PLA at 3 h post-RE (P < 0.05). MMP-9 expression and protein levels were elevated at 3 h post-RE independent of treatment (P < 0.05). Lysyl oxidase expression was greater at 3 h post-RE during APAP consumption (P < 0.05) compared with PLA. MMP-2 and TIMP-1 protein was not altered by RE or APAP (P > 0.05). Phosphorylation of ERK1/2 and p38-MAPK increased (P < 0.05) with RE but was not influenced by APAP. Our findings do not support our hypothesis and suggest that short-term APAP consumption before RE has a small impact on the measured ECM molecules in human skeletal muscle following acute RE. Copyright © 2017 the American Physiological Society.

  5. Expression of functional NK1 receptors in human alveolar macrophages: superoxide anion production, cytokine release and involvement of NF-kappaB pathway.

    PubMed

    Bardelli, Claudio; Gunella, Gabriele; Varsaldi, Federica; Balbo, Pietro; Del Boca, Elisa; Bernardone, Ilaria Seren; Amoruso, Angela; Brunelleschi, Sandra

    2005-06-01

    1 Substance P (SP) is deeply involved in lung pathophysiology and plays a key role in the modulation of inflammatory-immune processes. We previously demonstrated that SP activates guinea-pig alveolar macrophages (AMs) and human monocytes, but a careful examination of its effects on human AMs is still scarce. 2 This study was undertaken to establish the role of SP in human AM isolated from healthy smokers and non-smokers, by evaluating the presence of tachykinin NK(1) receptors (NK-1R) and SP's ability to induce superoxide anion (O(2)(-)) production and cytokine release, as well as activation of the nuclear factor-kappaB (NF-kappaB) pathway. 3 By Western blot analysis and immunofluorescence, we demonstrate that authentic NK-1R are present on human AMs, a three-fold enhanced expression being observed in healthy smokers. These NK-1R are functional, as SP and NK(1) agonists dose-dependently induce O(2)(-) production and cytokine release. In AMs from healthy smokers, SP evokes an enhanced respiratory burst and a significantly increased release of tumor necrosis factor-alpha as compared to healthy non-smokers, but has inconsistent effects on IL-10 release. The NK(1) selective antagonist CP 96,345 ((2S,3S)-cis-2-diphenylmethyl-N[(2-methoxyphenyl)-methyl]-1-azabicyclo-octan-3-amine)) competitively antagonized SP-induced effects. 4 SP activates the transcription factor NF-kappaB, a three-fold increased nuclear translocation being observed in AMs from healthy smokers. This effect is receptor-mediated, as it is reproduced by the NK(1) selective agonist [Sar(9)Met(O(2))(11)]SP and reverted by CP 96,345. 5 These results clearly indicate that human AMs possess functional NK-1R on their surface, which are upregulated in healthy smokers, providing new insights on the mechanisms involved in tobacco smoke toxicity.

  6. Comparison of the crystal structures of the human manganese superoxide dismutase and the homologous Aspergillus fumigatus allergen at 2-A resolution.

    PubMed

    Flückiger, Sabine; Mittl, Peer R E; Scapozza, Leonardo; Fijten, Helmi; Folkers, Gerd; Grütter, Markus G; Blaser, Kurt; Crameri, Reto

    2002-02-01

    Manganese superoxide dismutase (MnSOD) of Aspergillus fumigatus, a fungus involved in many pulmonary complications, has been identified as IgE-binding protein. It has been shown also that MnSODs from other organisms, including human, are recognized by IgE Abs from individuals sensitized to A. fumigatus MnSOD. Comparison of the fungal and the human crystal structure should allow the identification of structural similarities responsible for IgE-mediated cross-reactivity. The three-dimensional structure of A. fumigatus MnSOD has been determined at 2-A resolution by x-ray diffraction analysis. Crystals belonged to space group P2(1)2(1)2(1) with unit cell dimensions of a = 65.88 A, b = 98.7 A, and c = 139.28 A. The structure was solved by molecular replacement using the structure of the human MnSOD as a search model. The final refined model included four chains of 199-200 amino acids, four manganese ions, and 745 water molecules, with a crystallographic R-factor of 19.4% and a free R-factor of 23.3%. Like MnSODs of other eukaryotic organisms, A. fumigatus MnSOD forms a homotetramer with the manganese ions coordinated by three histidines, one aspartic acid, and one water molecule. The fungal and the human MnSOD share high similarity on the level of both primary and tertiary structure. We identified conserved amino acids that are solvent exposed in the fungal and the human crystal structure and are therefore potentially involved in IgE-mediated cross-reactivity.

  7. Expression of extracellular calcium (Ca2+o)-sensing receptor in human peripheral blood monocytes

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Olozak, I.; Chattopadhyay, N.; Butters, R. R.; Kifor, O.; Scadden, D. T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor playing key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone turnover and may play a role in the "reversal" phase of skeletal remodeling that follows osteoclastic resorption and precedes osteoblastic bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for such mononuclear cells present locally within the bone marrow microenvironment. Indeed, previous studies by other investigators have shown that raising Ca2+o either in vivo or in vitro stimulated the release of interleukin-6 (IL-6) from human peripheral blood monocytes, suggesting that these cells express a Ca2+o-sensing mechanism. In these earlier studies, however, the use of reverse transcription-polymerase chain reaction (RT-PCR) failed to detect transcripts for the CaR previously cloned from parathyroid and kidney in peripheral blood monocytes. Since we recently found that non-specific esterase-positive, putative monocytes isolated from murine bone marrow express the CaR, we reevaluated the expression of this receptor in human peripheral blood monocytes. Immunocytochemistry, flow cytometry, and Western blot analysis, performed using a polyclonal antiserum specific for the CaR, detected CaR protein in human monocytes. In addition, the use of RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products, identified CaR transcripts in the cells. Therefore, taken together, our data show that human peripheral blood monocytes possess both CaR protein and mRNA very similar if not identical to those expressed in parathyroid and kidney that could mediate the previously described, direct effects of Ca2+o on these cells. Furthermore, since mononuclear cells isolated from bone marrow also express the CaR, the latter might play some role in

  8. Monocytes increase human cardiac myofibroblast-mediated extracellular matrix remodeling through TGF-β1.

    PubMed

    Mewhort, Holly E M; Lipon, Brodie D; Svystonyuk, Daniyil A; Teng, Guoqi; Guzzardi, David G; Silva, Claudia; Yong, V Wee; Fedak, Paul W M

    2016-03-15

    Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P < 0.01) and increased local ECM remodeling quantified by confocal microscopy. Under coculture conditions that allow indirect cellular interaction via paracrine factors but prevent direct cell-cell contact, monocytes had minimal effects on myofibroblast activity (17.9 ± 11.1% vs. 6.4 ± 7.0% increase, respectively; P < 0.01). When cells were cultured under direct contact conditions, multiplex analysis of the coculture media revealed an increase in the paracrine factors TGF-β1 and matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P < 0.001). TGF-β blockade abolished the monocyte-induced increase in cardiac myofibroblast activity. These data suggest that direct cell-cell interaction between monocytes and cardiac myofibroblasts stimulates TGF-β-mediated myofibroblast activity and increases remodeling of local matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-β1

  9. Expression of extracellular calcium (Ca2+o)-sensing receptor in human peripheral blood monocytes

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Olozak, I.; Chattopadhyay, N.; Butters, R. R.; Kifor, O.; Scadden, D. T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor playing key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone turnover and may play a role in the "reversal" phase of skeletal remodeling that follows osteoclastic resorption and precedes osteoblastic bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for such mononuclear cells present locally within the bone marrow microenvironment. Indeed, previous studies by other investigators have shown that raising Ca2+o either in vivo or in vitro stimulated the release of interleukin-6 (IL-6) from human peripheral blood monocytes, suggesting that these cells express a Ca2+o-sensing mechanism. In these earlier studies, however, the use of reverse transcription-polymerase chain reaction (RT-PCR) failed to detect transcripts for the CaR previously cloned from parathyroid and kidney in peripheral blood monocytes. Since we recently found that non-specific esterase-positive, putative monocytes isolated from murine bone marrow express the CaR, we reevaluated the expression of this receptor in human peripheral blood monocytes. Immunocytochemistry, flow cytometry, and Western blot analysis, performed using a polyclonal antiserum specific for the CaR, detected CaR protein in human monocytes. In addition, the use of RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products, identified CaR transcripts in the cells. Therefore, taken together, our data show that human peripheral blood monocytes possess both CaR protein and mRNA very similar if not identical to those expressed in parathyroid and kidney that could mediate the previously described, direct effects of Ca2+o on these cells. Furthermore, since mononuclear cells isolated from bone marrow also express the CaR, the latter might play some role in

  10. Biological Activities of Extracellular Vesicles and Their Cargos from Bovine and Human Milk in Humans and Implications for Infants.

    PubMed

    Zempleni, Janos; Aguilar-Lozano, Ana; Sadri, Mahrou; Sukreet, Sonal; Manca, Sonia; Wu, Di; Zhou, Fang; Mutai, Ezra

    2017-01-01

    Extracellular vesicles (EVs) in milk harbor a variety of compounds, including lipids, proteins, noncoding RNAs, and mRNAs. Among the various classes of EVs, exosomes are of particular interest, because cargo sorting in exosomes is a regulated, nonrandom process and exosomes play essential roles in cell-to-cell communication. Encapsulation in exosomes confers protection against enzymatic and nonenzymatic degradation of cargos and provides a pathway for cellular uptake of cargos by endocytosis of exosomes. Compelling evidence suggests that exosomes in bovine milk are transported by intestinal cells, vascular endothelial cells, and macrophages in human and rodent cell cultures, and bovine-milk exosomes are delivered to peripheral tissues in mice. Evidence also suggests that cargos in bovine-milk exosomes, in particular RNAs, are delivered to circulating immune cells in humans. Some microRNAs and mRNAs in bovine-milk exosomes may regulate the expression of human genes and be translated into protein, respectively. Some exosome cargos are quantitatively minor in the diet compared with endogenous synthesis. However, noncanonical pathways have been identified through which low concentrations of dietary microRNAs may alter gene expression, such as the accumulation of exosomes in the immune cell microenvironment and the binding of microRNAs to Toll-like receptors. Phenotypes observed in infant-feeding studies include higher Mental Developmental Index, Psychomotor Development Index, and Preschool Language Scale-3 scores in breastfed infants than in those fed various formulas. In mice, supplementation with plant-derived MIR-2911 improved the antiviral response compared with controls. Porcine-milk exosomes promote the proliferation of intestinal cells in mice. This article discusses the above-mentioned advances in research concerning milk exosomes and their cargos in human nutrition. Implications for infant nutrition are emphasized, where permitted, but data in infants are

  11. Regulation of extracellular matrix proteins and integrin cell substratum adhesion receptors on epithelium during cutaneous human wound healing in vivo.

    PubMed Central

    Juhasz, I.; Murphy, G. F.; Yan, H. C.; Herlyn, M.; Albelda, S. M.

    1993-01-01

    Although changes in extracellular matrix proteins during wound healing have been well documented, little is known about the regulation of corresponding extracellular matrix adhesion receptors (integrins). To study this process in a human in vivo model, full thickness human skin grafts were transplanted onto severe combined immunodeficient mice and deep excisional wounds involving both the epidermal and dermal layers were then made. The changes in the expression of cell matrix proteins and epithelial integrins over time were analyzed with specific antibodies using immunohistochemistry. Wounding was associated with alterations in extracellular matrix proteins, namely, loss of laminin and type IV collagen in the region of the wound and expression of tenascin and fibronectin. Changes were also noted in the integrins on the migrating keratinocytes. There was marked up-regulation of the alpha v subunit and de novo expression of the fibronectin receptor (alpha 5 beta 1) during the stage of active migration (days 1 to 3 after wounding). In the later stages of wound healing, after epithelial integrity had been established, redistribution of the alpha 2, alpha 3, alpha 6, and beta 4 collagen/laminin-binding integrin subunits to suprabasal epidermal layers was noted. Thus, during cutaneous wound healing, keratinocytes up-regulate fibronectin/fibrinogen-binding integrins and redistribute collagen/laminin-binding integrins. This study demonstrates that the human skin/severe combined immunodeficient chimera provides a useful model to study events during human wound repair. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7694470

  12. Preparation of a functional fluorescent human Fas ligand extracellular domain derivative using a three-dimensional structure guided site-specific fluorochrome conjugation.

    PubMed

    Muraki, Michiro

    2016-01-01

    Human Fas ligand extracellular domain has been investigated as an important target protein in the field of medical biotechnology. In a recent study, the author developed an effective method to produce biologically active human Fas ligand extracellular domain derivatives using site-specific chemical modifications. A human Fas ligand extracellular domain derivative containing a reactive cysteine residue within its N-terminal tag sequence, which locates not proximal to the binding interface between the ligand and the receptor in terms of the three-dimensional structure, was modified by Fluorescein-5-Maleimide without impairing the specific binding activity toward human Fas receptor extracellular domain. The purified protein sample free of low molecular-weight contaminants showed a characteristic fluorescence spectrum derived from the attached Fluorescein moieties, and formed a stable binding complex with human Fas receptor extracellular domain-human IgG1 Fc domain fusion protein in solution. The conjugation number of the fluorochrome was estimated to be 2.5 per a single human Fas ligand extracellular domain trimer from the ratio of the absorbance value at 280 nm to that at 495 nm. A functional fluorescent human Fas ligand extracellular domain derivative was prepared via a site-specific conjugation of fluorochrome, which was guided by the three-dimensional structure information on the ligand-receptor complex. Fluorescent derivatives created by this method may contribute to the development of an improved diagnosis system for the diseases related to Fas receptor.

  13. Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity.

    PubMed

    Torri, Anna; Carpi, Donatella; Bulgheroni, Elisabetta; Crosti, Maria-Cristina; Moro, Monica; Gruarin, Paola; Rossi, Riccardo L; Rossetti, Grazisa; Di Vizio, Dolores; Hoxha, Mirjam; Bollati, Valentina; Gagliani, Cristina; Tacchetti, Carlo; Paroni, Moira; Geginat, Jens; Corti, Laura; Venegoni, Luigia; Berti, Emilio; Pagani, Massimiliano; Matarese, Giuseppe; Abrignani, Sergio; de Candia, Paola

    2017-02-17

    Upon T cell receptor stimulation, CD4(+) T helper (Th) lymphocytes release extracellular vesicles (EVs) containing microRNAs. However, no data are available on whether human CD4(+) T cell subsets release EVs containing different pattern of microRNAs. The present work aimed at filling this gap by assessing the microRNA content in EVs released upon in vitro T cell receptor stimulation of Th1, Th17, and T regulatory (Treg) cells. Our results indicate that EVs released by Treg cells are significantly different compared with those released by the other subsets. In particular, miR-146a-5p, miR-150-5p, and miR-21-5p are enriched, whereas miR-106a-5p, miR-155-5p, and miR-19a-3p are depleted in Treg-derived EVs. The in vitro identified EV-associated microRNA signature was increased in serum of autoimmune patients with psoriasis and returned to healthy levels upon effective treatment with etanercept, a biological drug targeting the TNF pathway and suppressing inflammation. Moreover, Gene Set Enrichment Analysis showed an over-representation of genes relevant for T cell activation, such as CD40L, IRAK1, IRAK2, STAT1, and c-Myb in the list of validated targets of Treg-derived EV miRNAs. At functional level, Treg-derived (but not Th1/Th17-derived) EVs inhibited CD4(+) T cell proliferation and suppressed two relevant targets of miR-146a-5p: STAT1 and IRAK2. In conclusion, our work identified the miRNAs specifically released by different human CD4(+) T cell subsets and started to unveil the potential use of their quantity in human serum to mark the pathological elicitation of these cells in vivo and their biological effect in cell to cell communication during the adaptive immune response. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Identification of Extracellular Matrix Components and Biological Factors in Micronized Dehydrated Human Amnion/Chorion Membrane

    PubMed Central

    Lei, Jennifer; Priddy, Lauren B.; Lim, Jeremy J.; Massee, Michelle; Koob, Thomas J.

    2017-01-01

    Objective: The use of bioactive extracellular matrix (ECM) grafts such as amniotic membranes is an attractive treatment option for enhancing wound repair. In this study, the concentrations, activity, and distribution of matrix components, growth factors, proteases, and inhibitors were evaluated in PURION® Processed, micronized, dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Inc.). Approach: ECM components in dHACM tissue were assessed by using immunohistochemical staining, and growth factors, cytokines, proteases, and inhibitors were quantified by using single and multiplex ELISAs. The activities of proteases that were native to the tissue were determined via gelatin zymography and EnzChek® activity assay. Results: dHACM tissue contained the ECM components collagens I and IV, hyaluronic acid, heparin sulfate proteoglycans, fibronectin, and laminin. In addition, numerous growth factors, cytokines, chemokines, proteases, and protease inhibitors that are known to play a role in the wound-healing process were quantified in dHACM. Though matrix metalloproteinases (MMPs) were present in dHACM tissues, inhibitors of MMPs overwhelmingly outnumbered the MMP enzymes by an overall molar ratio of 28:1. Protease activity assays revealed that the MMPs in the tissue existed primarily either in their latent form or complexed with inhibitors. Innovation: This is the first study to characterize components that function in wound healing, including inhibitor and protease content and activity, in micronized dHACM. Conclusion: A variety of matrix components and growth factors, as well as proteases and their inhibitors, were identified in micronized dHACM, providing a better understanding of how micronized dHACM tissue can be used to effectively promote wound repair. PMID:28224047

  15. Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages

    PubMed Central

    Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

    2015-01-01

    HIV type 1 (HIV-1) infects CD4+ T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as “Trojan horses” carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages. PMID:26056317

  16. An extracellular matrix culture system for induced pluripotent stem cells derived from human dental pulp cells.

    PubMed

    Chen, Y; Zheng, Y-L; Qiu, D-B; Sun, Y-P; Kuang, S-J; Xu, Y; He, F; Gong, Y-H; Zhang, Z-G

    2015-11-01

    Induced pluripotent stem cells (iPSCs) have emerged as a promising tool for treating incurable diseases. The current challenges are to avoid potential xenopathogenic transmission and immune rejection potentially caused by exposure of iPSCs to animal-derived products. In addition, an efficient feeder cell-free culture condition will be required for minimizing batch-to-batch variation and facilitating scale-up. Therefore, establishing an efficient extracellular matrix (ECM) culture system is considered as a prerequisite for the future clinical application of iPSC-based cell therapies. In this study, we evaluated the feasibility of culturing iPSCs in ECM derived from human dental pulp cells (hDCP). iPSCs growing in Matrigel were transferred to ECM or Matrigel and cultured in mTeSRTM1 medium. The number of adherent cells in the ECM group was higher than that in the Matrigel group after incubation for 8, 12, and 24 h, indicating that the ECM could enhance cell adherence. The adhesion of cells to ECM not only depends on simple physical attachment with ECM, but also mediated by fibronectin in the ECM. The hDPC-iPSCs showed orderly growth in the ECM, suggesting that the ECM could promote the growth and proliferation of hDPC-iPSCs. We also observed that stem cells growed along to avoid contact inhibition. The iPSCs maintained undifferentiated state when cultured in ECM when the iPSCs and ECM are of the same cell origin. ECM and mTeSRTM1, can both be used as new culture medium for iPSCs that facilitates the clinical application of iPSC-based cell therapies in the future.

  17. IAPP modulates cellular autophagy, apoptosis, and extracellular matrix metabolism in human intervertebral disc cells

    PubMed Central

    Wu, Xinghuo; Song, Yu; Liu, Wei; Wang, Kun; Gao, Yong; Li, Shuai; Duan, Zhenfeng; Shao, Zengwu; Yang, Shuhua; Yang, Cao

    2017-01-01

    The pathogenic process of intervertebral disc degeneration (IDD) is characterized by imbalance in the extracellular matrix (ECM) metabolism. Nucleus pulposus (NP) cells have important roles in maintaining the proper structure and tissue homeostasis of disc ECM. These cells need adequate supply of glucose and oxygen. Islet amyloid polypeptide (IAPP) exerts its biological effects by regulating glucose metabolism. The purpose of this study was to investigate the expression of IAPP in degenerated IVD tissue, and IAPP modulation of ECM metabolism in human NP cells, especially the crosstalk mechanism between apoptosis and autophagy in these cells. We found that the expression of IAPP and Calcr-RAMP decreased considerably during IDD progression, along with the decrease in the expression of AG, BG, and Col2A1. Induction of IAPP in NP cells by transfection with pLV-IAPP enhanced the synthesis of aggrecan and Col2A1 and attenuated the expression of pro-inflammatory factors, tumor necrosis factor (TNF)-α, and interleukin (IL)-1. Upregulation of IAPP also affected the expression of the catabolic markers—matrix metalloproteinases (MMPs) 3, 9 and 13 and ADAMTS 4 and 5. Downregulation of IAPP by siRNA inhibited the expression of anabolic genes but increased the expression of catabolic genes and inflammatory factors. The expressions of autophagic and apoptotic markers in NP cells transfected with pLV-IAPP were upregulated, including BECLIN1, ATG5, ATG7, LC3 II/I and Bcl-2, while significantly increase in the expression of Bax and Caspase-3 in NP cells transfected with pLV-siIAPP. Mechanistically, PI3K/AKT-mTOR and p38/JNK MAPK signal pathways were involved. We propose that IAPP might play a pivotal role in the development of IDD, by regulating ECM metabolism and controlling the crosstalk between apoptosis and autophagy in NP, thus potentially offering a novel therapeutic approach to the treatment of IDD. PMID:28149534

  18. Handling and storage of human body fluids for analysis of extracellular vesicles.

    PubMed

    Yuana, Yuana; Böing, Anita N; Grootemaat, Anita E; van der Pol, Edwin; Hau, Chi M; Cizmar, Petr; Buhr, Egbert; Sturk, Auguste; Nieuwland, Rienk

    2015-01-01

    Because procedures of handling and storage of body fluids affect numbers and composition of extracellular vesicles (EVs), standardization is important to ensure reliable and comparable measurements of EVs in a clinical environment. We aimed to develop standard protocols for handling and storage of human body fluids for EV analysis. Conditions such as centrifugation, single freeze-thaw cycle, effect of time delay between blood collection and plasma preparation and storage were investigated. Plasma is the most commonly studied body fluid in EV research. We mainly focused on EVs originating from platelets and erythrocytes and investigated the behaviour of these 2 types of EVs independently as well as in plasma samples of healthy subjects. EVs in urine and saliva were also studied for comparison. All samples were analysed simultaneously before and after freeze-thawing by resistive pulse sensing, nanoparticle tracking analysis, conventional flow cytometry (FCM) and transmission (scanning) electron microscopy. Our main finding is that the effect of centrifugation markedly depends on the cellular origin of EVs. Whereas erythrocyte EVs remain present as single EVs after centrifugation, platelet EVs form aggregates, which affect their measured concentration in plasma. Single erythrocyte and platelet EVs are present mainly in the range of 100-200 nm, far below the lower limit of what can be measured by conventional FCM. Furthermore, the effects of single freeze-thaw cycle, time delay between blood collection and plasma preparation up to 1 hour and storage up to 1 year are insignificant (p>0.05) on the measured concentration and diameter of EVs from erythrocyte and platelet concentrates and EVs in plasma, urine and saliva. In conclusion, in standard protocols for EV studies, centrifugation to isolate EVs from collected body fluids should be avoided. Freezing and storage of collected body fluids, albeit their insignificant effects, should be performed identically for

  19. Identification of Extracellular Matrix Components and Biological Factors in Micronized Dehydrated Human Amnion/Chorion Membrane.

    PubMed

    Lei, Jennifer; Priddy, Lauren B; Lim, Jeremy J; Massee, Michelle; Koob, Thomas J

    2017-02-01

    Objective: The use of bioactive extracellular matrix (ECM) grafts such as amniotic membranes is an attractive treatment option for enhancing wound repair. In this study, the concentrations, activity, and distribution of matrix components, growth factors, proteases, and inhibitors were evaluated in PURION(®) Processed, micronized, dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Inc.). Approach: ECM components in dHACM tissue were assessed by using immunohistochemical staining, and growth factors, cytokines, proteases, and inhibitors were quantified by using single and multiplex ELISAs. The activities of proteases that were native to the tissue were determined via gelatin zymography and EnzChek(®) activity assay. Results: dHACM tissue contained the ECM components collagens I and IV, hyaluronic acid, heparin sulfate proteoglycans, fibronectin, and laminin. In addition, numerous growth factors, cytokines, chemokines, proteases, and protease inhibitors that are known to play a role in the wound-healing process were quantified in dHACM. Though matrix metalloproteinases (MMPs) were present in dHACM tissues, inhibitors of MMPs overwhelmingly outnumbered the MMP enzymes by an overall molar ratio of 28:1. Protease activity assays revealed that the MMPs in the tissue existed primarily either in their latent form or complexed with inhibitors. Innovation: This is the first study to characterize components that function in wound healing, including inhibitor and protease content and activity, in micronized dHACM. Conclusion: A variety of matrix components and growth factors, as well as proteases and their inhibitors, were identified in micronized dHACM, providing a better understanding of how micronized dHACM tissue can be used to effectively promote wound repair.

  20. Cadmium activates extracellular signal-regulated kinase 5 in HK-2 human renal proximal tubular cells

    SciTech Connect

    Kondo, Mio; Inamura, Hisako; Matsumura, Ken-ichi; Matsuoka, Masato

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Cadmium exposure induces ERK5 phosphorylation in HK-2 renal proximal tubular cells. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced ERK5 but not ERK1/2 phosphorylation. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced CREB and c-Fos phosphorylation. Black-Right-Pointing-Pointer ERK5 activation by cadmium exposure may play an anti-apoptotic role in HK-2 cells. -- Abstract: We examined the effects of cadmium chloride (CdCl{sub 2}) exposure on the phosphorylation and functionality of extracellular signal-regulated kinase 5 (ERK5), a recently identified member of the mitogen-activated protein kinase (MAPK) family, in HK-2 human renal proximal tubular cells. Following exposure to CdCl{sub 2}, ERK5 phosphorylation increased markedly, but the level of total ERK5 was unchanged. ERK5 phosphorylation following CdCl{sub 2} exposure was rapid and transient, similar to the time course of ERK1/2 phosphorylation. Treatment of HK-2 cells with the MAPK/ERK kinase 5 inhibitor, BIX02189, suppressed CdCl{sub 2}-induced ERK5 but not ERK1/2 phosphorylation. The CdCl{sub 2}-induced increase of phosphorylated cAMP response element-binding protein (CREB) and activating transcription factor-1 (ATF-1), as well as the accumulation of mobility-shifted c-Fos protein, were suppressed by BIX02189 treatment. Furthermore, BIX02189 treatment enhanced cleavage of poly(ADP-ribose) polymerase and increased the level of cytoplasmic nucleosomes in HK-2 cells exposed to CdCl{sub 2}. These findings suggest that ERK5 pathway activation by CdCl{sub 2} exposure might induce the phosphorylation of cell survival-transcription factors, such as CREB, ATF-1, and c-Fos, and may exert a partial anti-apoptotic role in HK-2 cells.

  1. Silicon-based quantum dots induce inflammation in human lung cells and disrupt extracellular matrix homeostasis.

    PubMed

    Stan, Miruna-Silvia; Sima, Cornelia; Cinteza, Ludmila Otilia; Dinischiotu, Anca

    2015-08-01

    Quantum dots (QDs) are nanocrystalline semiconductor materials that have been tested for biological applications such as cancer therapy, cellular imaging and drug delivery, despite the serious lack of information of their effects on mammalian cells. The present study aimed to evaluate the potential of Si/SiO2 QDs to induce an inflammatory response in MRC-5 human lung fibroblasts. Cells were exposed to different concentrations of Si/SiO2 QDs (25-200 μg·mL(-1)) for 24, 48, 72 and 96 h. The results obtained showed that uptake of QDs was dependent on biocorona formation and the stability of nanoparticles in various biological media (minimum essential medium without or with 10% fetal bovine serum). The cell membrane damage indicated by the increase in lactate dehydrogenase release after exposure to QDs was dose- and time-dependent. The level of lysosomes increased proportionally with the concentration of QDs, whereas an accumulation of autophagosomes was also observed. Cellular morphology was affected, as shown by the disruption of actin filaments. The enhanced release of nitric oxide and the increase in interleukin-6 and interleukin-8 protein expression suggested that nanoparticles triggered an inflammatory response in MRC-5 cells. QDs decreased the protein expression and enzymatic activity of matrix metalloproteinase (MMP)-2 and MMP-9 and also MMP-1 caseinase activity, whereas the protein levels of MMP-1 and tissue inhibitor of metalloproteinase-1 increased. The present study reveals for the first time that silicon-based QDs are able to generate inflammation in lung cells and cause an imbalance in extracellular matrix turnover through a differential regulation of MMPs and tissue inhibitor of metalloproteinase-1 protein expression.

  2. Effects of nicotine on proliferation and extracellular matrix production of human gingival fibroblasts in vitro.

    PubMed

    Tipton, D A; Dabbous, M K

    1995-12-01

    Normal function of gingival fibroblasts is essential for maintenance of the gingival extracellular matrix (ECM), but under inflammatory conditions in gingival tissue which may occur with tobacco use, they can also act in its destruction. The purpose of this study was to determine the effects of nicotine, a major component of tobacco, on gingival fibroblast proliferation, the production of fibronectin (FN), and the production and breakdown of type I collagen to elucidate its role in periodontal destruction associated with its use. A human gingival fibroblast strain derived from a healthy individual with non-inflamed gingiva was used in this study. Nicotine at concentrations > 0.075% caused cell death, and at 0.075% and 0.05% it caused transient vacuolization of the fibroblasts. At concentrations of 0.001% to 0.075%, nicotine significantly inhibited proliferation (P < or = 0.03), measured by the incorporation of [3H]-thymidine into DNA. The production of FN and type I collagen was significantly inhibited by nicotine at > or = 0.05% (P < or = 0.001), measured using specific ELISAs. On the other hand, nicotine at > or = 0.025% significantly increased collagenase activity (P < or = 0.008), using [3H]-gly and [14C]-pro-labeled type I collagen gels as substrate. The results show that, in vitro, nicotine inhibits the growth of gingival fibroblasts and their production of FN and collagen, while also promoting collagen breakdown. This suggests that nicotine itself may augment the destruction of the gingival ECM occurring during periodontal inflammation associated with smokeless tobacco use.

  3. High extracellular levels of potassium and trace metals in human brain abscess.

    PubMed

    Dahlberg, Daniel; Ivanovic, Jugoslav; Mariussen, Espen; Hassel, Bjørnar

    2015-03-01

    Brain abscesses frequently cause symptoms such as seizures, delirium, paresis and sensory deficits that could reflect brain edema, increased intracranial pressure, or tissue destruction. However, it is also possible that pus constituents could disturb neuronal function in the surrounding brain tissue. In pus from 16 human brain abscesses, extracellular potassium ([K(+)]o) was 10.6 ± 4.8 mmol/L (mean ± SD; maximum value 22.0 mmol/L). In cerebrospinal fluid (CSF), [K(+)]o was 2.7 ± 0.6 mmol/L (N = 14; difference from pus p < 0.001), which is similar to previous control values for [K(+)]o in CSF and brain parenchyma. Zinc and iron were >40-fold higher in pus than in CSF; calcium, copper, manganese, and chromium were also higher, whereas sodium and magnesium were similar. Pus from 10 extracerebral abscesses (empyemas) also had higher [K(+)]o, zinc, iron, calcium, copper, manganese, and chromium than did CSF. Brain abscess [K(+)]o was significantly higher than serum potassium (3.8 ± 0.5 mmol/L; p = 0.0001), indicating that the elevated abscess [K(+)]o originated from damaged cells (e.g. brain cells and leukocytes), not from serum. High [K(+)]o could depolarize neurons, high levels of zinc could inhibit glutamate and GABA receptors, and high levels of iron and copper could cause oxidative damage, all of which could contribute to neuronal dysfunction in brain abscess patients. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Acceleration of Apoptosis by Extracellular Basic pH in a 3D Human Skin Equivalent System.

    PubMed

    Park, Gunhyuk; Oh, Dal-Seok; Kim, Yong-Ung; Park, Moon Ki

    2017-01-01

    Previously, we have shown that extracellular basic pH plays a significant role in both the direct and indirect regulation of cellular processes in a wound; this in turn affects the wound-healing process. Several studies have demonstrated the importance of apoptosis modulation in the wound-healing process, especially in removing inflammatory cells and in inhibiting scar formation. However, the effects of extracellular basic pH on wound healing-related skin damage are yet to be examined. Therefore, we investigated the induction of accelerated apoptosis by extracellular basic pH in skin. Apoptosis-related protein levels were measured using an array kit, target protein expression levels were detected by immunostaining, lactate dehydrogenase was analyzed spectrophotometrically, and Annexin V levels were measured by fluorescence staining. Basic pH (8.40) strongly upregulated extrinsic apoptosis proteins (Fas, high temperature requirement A, and p21) and slightly upregulated intrinsic apoptosis proteins (cytochrome c, B-cell lymphoma 2 [Bcl-2], Bcl-2-associated death promoter, and Bcl-2-like protein 4) in a 3D human skin equivalent system. Moreover, basic pH (8.40) induced heat shock protein (HSP) 60 and 70. In addition, basic pH-exposed Fas- and HSP60-knockdown cells showed significantly decreased levels of apoptosis. Taken together, these results indicate that extracellular basic pH increases early-stage apoptosis through Fas/FasL via modulation of HSP60 and HSP70. © 2017 S. Karger AG, Basel.

  5. Endoplasmic reticulum stress modulates nicotine-induced extracellular matrix degradation in human periodontal ligament cells.

    PubMed

    Lee, S-I; Kang, K-L; Shin, S-I; Herr, Y; Lee, Y-M; Kim, E-C

    2012-06-01

    Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine-induced cytotoxicity and ECM degradation. Cytotoxicity and cell death were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT-PCR and western blot analysis. Treatment with nicotine reduced cell viability and increased the proportion of annexin V-negative, propidium iodide-positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2α and glucose-regulated protein-78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP-1, MMP-2, MMP-8 and MMP-9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine-induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small

  6. Cissus quadrangularis ethanol extract upregulates superoxide dismutase, glutathione peroxidase and endothelial nitric oxide synthase expression in hydrogen peroxide-injured human ECV304 cells.

    PubMed

    Sapsrithong, Tarat; Kaewprem, Weeraya; Tongumpai, Sarunya; Nusuetrong, Punnee; Meksuriyen, Duangdeun

    2012-09-28

    Cissus quadrangularis has been widely used in traditional medicine for the treatment of hemorrhoid. However, the detailed mechanism of antioxidant defense of C. quadrangularis in endothelial cells under oxidative stress remains unclear. The present study aims to elucidate the protective role of ethanol extract of C. quadrangularis (CQE) including its constituents, quercetin and resveratrol, on hydrogen peroxide (H(2)O(2))-injured human umbilical vein endothelial ECV304 cells. Viability, genotoxicity and protein expression of ECV304 cells were analyzed by MTT, alkaline comet and Western blot, respectively. Production of intracellular reactive oxygen species (ROS) was determined using dichlorofluorescein fluorescence dye. After exposing cells to CQE containing quercetin and resveratrol, DNA damage was not observed. CQE including quercetin and resveratrol significantly attenuated ROS in H(2)O(2)-injured ECV304 cells in a concentration-dependent manner. The protein expression of superoxide dismutase (Cu/Zn-SOD, Mn-SOD), glutathione peroxidase (GPx) and endothelial nitric oxide synthase (eNOS) increased in the cells treated with CQE, quercetin or resveratrol prior to H(2)O(2) exposure, as compared with control. The results provide a molecular mechanism of C. quadrangularis, which could be partially related to quercetin and resveratrol, in restoring ROS in endothelial cells through the upregulation of Cu/Zn-SOD, Mn-SOD, GPx and eNOS. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  7. PPAR{gamma} activation abolishes LDL-induced proliferation of human aortic smooth muscle cells via SOD-mediated down-regulation of superoxide

    SciTech Connect

    Heo, Kyung-Sun; Kim, Dong-Uk; Ryoo, Sungwoo; Nam, Miyoung; Baek, Seung Tae; Kim, Lila; Park, Song-Kyu; Myung, Chang-Seon; Hoe, Kwang-Lae . E-mail: kwanghoe@kribb.re.kr

    2007-08-10

    Native LDL would be a mitogenic and chemotactic stimulus of VSMC proliferation and differentiation in the atherosclerotic lesion where endothelial disruption occurred. In previous studies, our group investigated the molecular mechanisms by which LDL induces IL-8 production and by which PPAR{alpha} activation abolishes LDL effects in human aortic SMCs (hAoSMCs). Herein is the first report of PPAR{gamma} activation by troglitazone (TG) exerting its inhibitory effects on LDL-induced cell proliferation via generation not of H{sub 2}O{sub 2}, but of O2?-, and the subsequent activation of Erk1/2 in hAoSMCs. Moreover, in this study TG abolished the LDL-accelerated G{sub 1}-S progression to control levels via down-regulation of active cyclinD1/CDK4 and cyclinE/CDK2 complexes and up-regulation of p21{sup Cip1} expression. TG exerted its anti-proliferative effects through the up-regulation of basal superoxide dismutase (SOD) expression. This data suggests that the regulation of O2?- is located at the crossroads between LDL signaling and cell proliferation.

  8. Collaborative effects of Photobacterium CuZn superoxide dismutase (SODs) and human AP endonuclease in DNA repair and SOD-deficient Escherichia coli under oxidative stress.

    PubMed

    Kim, Young Gon

    2004-01-15

    The defenses against free radical damage include specialized repair enzymes that correct oxidative damage in DNA and detoxification systems such as superoxide dismutases (SODs). These defenses may be coordinated genetically as global responses. We hypothesized that the expression of SOD and DNA repair genes would inhibit DNA damage under oxidative stress. Therefore, protection of Escherichia coli mutants deficient in SOD and DNA repair genes (sod-, xth-, and nfo-) was demonstrated by transforming the mutant strain with a plasmid pYK9 that encoded Photobacterium leiognathi CuZnSOD and human AP endonuclease. The results show that survival rates were increased in sod+ xth- nfo+ cells compared with sod- xth- ape-, sod- xth- ape-, and sod+ xth- ape- cells under oxidative stress generated with 0.1 mM paraquat or 3 mM H2O2. The data suggest that, at the least, SOD and DNA repair enzymes may collaborate on protection and repair of damaged DNA. Additionally, both enzymes are required for protection against free radicals.

  9. The solution structure of a monomeric, reduced form of human copper,zinc superoxide dismutase bearing the same charge as the native protein.

    PubMed

    Banci, L; Bertini, I; Del Conte, R; Fadin, R; Mangani, S; Viezzoli, M S

    1999-12-01

    The solution structure of a mutated (Phe50Glu, Gly51Glu, Val148Lys, Ile151Lys), reduced, monomeric form of human copper,zinc superoxide dismutase (SOD; 153 amino acids) has been determined through 2237 meaningful nuclear Overhauser enhancements, out of 2492, and 43 dihedral angle constraints. A characteristic of this mutant is that of having the same overall charge as the dimeric protein, but an activity of only 20% with respect to wild-type SOD. This protein, at variance with a previously characterized monomeric form (Phe50Glu, Gly51Glu, Glu133Gln), does not contain mutations in the active site. Therefore, its characterization allows us to understand the structural changes independently induced by the monomerization and by the active site mutation. The family of 36 conformers, which have a target function with respect to the experimental constraints lower than 1.5 A2, has RMSD values with respect to the average structure of 0.94 +/- 0.14 A2 and 1.50 +/- 0.14 A2 for the backbone and the heavy atoms, respectively. The overall folding, which includes the classical eight-stranded Greek-key beta-barrel and a short alpha-helix, is very close to that of the previously characterized monomeric mutant E133QM2SOD and to that of wild-type SOD. The region involved in the subunit-subunit interactions in the dimeric protein is confirmed to be disordered in the monomeric species. It is also observed that a sizable rearrangement of the charged groups of the electrostatic loop and of Arg143 takes place in the monomeric species. The width of the active site channel, both at its entrance and at the bottleneck of the active site, is discussed in the light of the influence on the enzymatic activity and the latter with respect to the overall charge. It is also confirmed that the NH proton of His63 shields the Cu(I) from the bulk solvent, thus supporting the suggestion that superoxide may interact with the reduced metal ion in an outer-sphere fashion.

  10. [Effects of Gremlin on transdifferentiation and extracellular matrix synthesis in human lens epithelium cells].

    PubMed

    Pei, Cheng; Ma, Bo; Kang, Qianyan; Qin, Li; Cui, Lijun

    2015-08-01

    To explore the effects of Gremlin on transdifferentiation and extracellular matrix synthesis in cultured human lens epithelium cells (HLEC). This is an experimental research. HLEC were incubated with different concentrations of Gremlin (0, 100, 200 and 400 µg/L) for 24 h. The morphological changes of HLEC were observed by inverted microscope. Real-time PCR and Western-Blot were used to evaluate the expression of α-smooth muscle actin (α-SMA) (as a landmark protein of epithelial mesenchymal transition), fibronectin (Fn) and collagen type 1 (COL-1) (as major components of extracellular matrix) after stimulation with different time (0, 12, 24, 48, 72 h) by 200 µg/L Gremlin. The same parameters were observed in Gremlin. siRNA transfected HLEC which treated with 1.0 µg/L TGF-β2. α-SMA, Fn and COL-1 protein and mRNA expressions comparison with control group were analyzed using one-way and two-way ANOVA, while the difference between groups were compared using Turkey HSD and LSD-t test. The normal morphology of HLEC showed polygonal and anchorage-dependent. After the incubation of different concentrations of Gremlin for 24 h, morphological feature of HLEC were changed from monolayer and polygonal to multilayer and spindle fibroblast-like cells, and the intercellular space widened. The expression of α-SMA, Fn and COL-1 were increased with prolonging of Gremlin treatment time (α-SMA gene induction: 1.00 ± 0.00, 1.62 ± 0.57, 3.40 ± 0.83, 5.90 ± 0.49, 7.97 ± 0.91; F = 61.64, P < 0.05, q = 6.43, 13.13, 18.66, P < 0.05; Fn gene induction: 1.00 ± 0.00, 3.26 ± 0.23, 5.86 ± 0.90, 10.17 ± 2.16, 12.89 ± 1.63; F = 42.03, P < 0.05, q = 6.45, 12.18, 15.79, P < 0.05; COL-1 gene induction: 1.00 ± 0.00, 1.78 ± 0.88, 6.80 ± 0.44, 12.76 ± 2.46, 21.12 ± 3.66; F = 51.79, P < 0.05, q = 4.97, 10.08, 17.26, P < 0.05) (α-SMA protein expression: 0.13 ± 0.02, 0.26 ± 0.02, 0.29 ± 0.09, 0.47 ± 0.06, 0.68 ± 0.05; F = 45.14, q = 5.11, 10.67, 17.40, P < 0.05; Fn protein

  11. Extracellular nucleotide inhibits cell proliferation and negatively regulates Toll-like receptor 4 signalling in human progenitor endothelial cells.

    PubMed

    Xiao, Zhilin; Yang, Mei; Fang, Li; Lv, Qingshan; He, Qing; Deng, Minjie; Liu, Xueting; Chen, Xiaobin; Chen, Meifang; Xie, Xiumei; Hu, Jinyue

    2012-07-01

    Extracellular nucleotides mediate a wide range of physiological effects by interacting with plasma membrane P2 purinergic receptors. P2 receptors are expressed in certain kinds of stem cells, and function to induce cytokine expression and to modulate cell proliferation. We have analysed the expression and the function of P2 receptors in human umbilical cord blood-derived EPCs (endothelial progenitor cells). EPCs expressed P2X4,6,7 and P2Y2,4,11,13,14 receptors and extracellular ATP inhibited EPCs proliferation. As in a previous study, EPCs expressed functional TLR4 (Toll-like receptor 4) and activation of TLR4 by LPS (lipopolysaccharide) evoked a pro-inflammatory immune response. When human EPCs were stimulated with LPS and nucleotides, ATP or UTP inhibited the expression of pro-inflammatory cytokines including MCP-1 (monocyte chemoattractant protein-1), IFNα (interferon α), TNFα (tumour necrosis factor α) and adhesion molecule VCAM-1 (vascular cell adhesion molecule 1) induced by LPS. ATP and UTP also down-regulated the gene expression of TLR4, CD14 and MyD88 (myeloid differentiation factor 88), a TLR adaptor molecule, and protein expression of CD14 and MyD88. Moreover, the phosphorylation of NF-κB (nuclear factor κB) p65 induced by TLR4 activation was inhibited partly by ATP or UTP at concentrations of 1-5 μM. These results suggest that extracellular nucleotides negatively regulate EPCs proliferation and TLR4 signalling.

  12. Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta.

    PubMed

    Watson, Chris J; Phelan, Dermot; Collier, Patrick; Horgan, Stephen; Glezeva, Nadia; Cooke, Gordon; Xu, Maojia; Ledwidge, Mark; McDonald, Kenneth; Baugh, John A

    2014-06-01

    Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFβ1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFβ1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.

  13. Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes

    PubMed Central

    Leal Denis, M. Florencia; Alvarez, H. Ariel; Lauri, Natalia; Alvarez, Cora L.; Chara, Osvaldo; Schwarzbaum, Pablo J.

    2016-01-01

    Introduction The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. Methods and Treatments We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. Results In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40–50% and swelling by 40–60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. Analysis and Discussion Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop

  14. Regulation of Extracellular ATP in Human Erythrocytes Infected with Plasmodium falciparum

    PubMed Central

    Alvarez, Cora Lilia; Schachter, Julieta; de Sá Pinheiro, Ana Acacia; Silva, Leandro de Souza; Verstraeten, Sandra Viviana; Persechini, Pedro Muanis; Schwarzbaum, Pablo Julio

    2014-01-01

    In human erythrocytes (h-RBCs) various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics) depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages). A “3V” mixture containing isoproterenol (β-adrenergic agonist), forskolin (adenylate kinase activator) and papaverine (phosphodiesterase inhibitor) was used to induce cAMP-dependent ATP release. ATPe kinetics of r-RBCs (ring-infected RBCs), t-RBCs (trophozoite-infected RBCs) and s-RBCs (schizont-infected RBCs) showed [ATPe] to peak acutely to a maximum value followed by a slower time dependent decrease. In all intraerythrocytic stages, values of ΔATP1 (difference between [ATPe] measured 1 min post-stimulus and basal [ATPe]) increased nonlinearly with parasitemia (from 2 to 12.5%). Under 3V exposure, t-RBCs at parasitemia 94% (t94-RBCs) showed 3.8-fold higher ΔATP1 values than in h-RBCs, indicative of upregulated ATP release. Pre-exposure to either 100 µM carbenoxolone, 100 nM mefloquine or 100 µM NPPB reduced ΔATP1 to 83–87% for h-RBCs and 63–74% for t94-RBCs. EctoATPase activity, assayed at both low nM concentrations (300–900 nM) and 500 µM exogenous ATPe concentrations increased approx. 400-fold in t94-RBCs, as compared to h-RBCs, while intracellular ATP concentrations of t94-RBCs were 65% that of h-RBCs. In t94-RBCs, production of nitric oxide (NO) was approx. 7-fold higher than in h-RBCs, and was partially inhibited by L-NAME pre-treatment. In media with L-NAME, ΔATP1 values were 2.7-times higher in h-RBCs and 4.2-times higher in t94-RBCs, than without L-NAME. Results suggest that P. falciparum infection of h-RBCs strongly activates ATP release via Pannexin 1 in these cells. Several processes partially counteracted ATPe accumulation

  15. Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells

    SciTech Connect

    Ziegelstein, Roy C.; Xiong Yali; He Chaoxia; Hu Qinghua . E-mail: qinghuaa@jhmi.edu

    2006-03-31

    Extracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub o}) regulates the functions of many cell types through a G protein-coupled [Ca{sup 2+}]{sub o}-sensing receptor (CaR). Whether the receptor is functionally expressed in vascular endothelial cells is largely unknown. In cultured human aortic endothelial cells (HAEC), RT-PCR yielded the expected 555-bp product corresponding to the CaR, and CaR protein was demonstrated by fluorescence immunostaining and Western blot. RT-PCR also demonstrated the expression in HAEC of alternatively spliced variants of the CaR lacking exon 5. Although stimulation of fura 2-loaded HAEC by several CaR agonists (high [Ca{sup 2+}]{sub o}, neomycin, and gadolinium) failed to increase intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}), the CaR agonist spermine stimulated an increase in [Ca{sup 2+}]{sub i} that was diminished in buffer without Ca{sup 2+} and was abolished after depletion of an intracellular Ca{sup 2+} pool with thapsigargin or after blocking IP{sub 3}- and ryanodine receptor-mediated Ca{sup 2+} release with xestospongin C and with high concentration ryanodine, respectively. Spermine stimulated an increase in DAF-FM fluorescence in HAEC, consistent with NO production. Both the increase in [Ca{sup 2+}]{sub i} and in NO production were reduced or absent in HAEC transfected with siRNA specifically targeted to the CaR. HAEC express a functional CaR that responds to the endogenous polyamine spermine with an increase in [Ca{sup 2+}]{sub i}, primarily due to release of IP{sub 3}- and ryanodine-sensitive intracellular Ca{sup 2+} stores, leading to the production of NO. Expression of alternatively spliced variants of the CaR may result in the absence of a functional response to other known CaR agonists in HAEC.

  16. Extracellular matrix metabolism disorder induced by mechanical strain on human parametrial ligament fibroblasts.

    PubMed

    Min, Jie; Li, Bingshu; Liu, Cheng; Guo, Wenjun; Hong, Shasha; Tang, Jianming; Hong, Li

    2017-03-24

    Pelvic organ prolapse (POP) is a global health problem that may seriously impact the quality of life of the sufferer. The present study aimed to investigate the potential mechanisms underlying alterations in extracellular matrix (ECM) metabolism in the pathogenesis of POP, by investigating the expression of ECM components in human parametrial ligament fibroblasts (hPLFs) subject to various mechanical strain loads. Fibroblasts derived from parametrial ligaments were cultured from patients with POP and without malignant tumors, who underwent vaginal hysterectomy surgery. Fibroblasts at generations 3‑6 of exponential phase cells were selected, and a four‑point bending device was used for 0, 1,333 or 5,333 µ mechanical loading of cells at 0.5 Hz for 4 h. mRNA and protein expression levels of collagen type I α 1 chain (COL1A1), collagen type III α 1 chain (COL3A1), elastin, matrix metalloproteinase (MMP) ‑2 and ‑9, and transforming growth factor (TGF)‑β1 were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. Under increased mechanical strain (5,333 µ), mRNA and protein expression levels of COL1A1, COL3A1 elastin and TGF‑β1 decreased, particularly COL1A1; however, mRNA and protein expression levels of MMP‑2 and ‑9 were significantly increased, compared with the control group (0 µ strain). Following 1,333 µ mechanical strain, mRNA and protein expression levels of COL1A1, COL3A1 elastin and MMP‑2 increased, and MMP‑9 decreased, whereas no significant differences were observed in TGF‑β1 mRNA and protein expression levels. In conclusion, ECM alterations may be involved in pathogenesis of POP, with decreased synthesis and increased degradation of collagen and elastin. Furthermore, the TGF‑β1 signaling pathway may serve an important role in this process and thus may supply a new target and strategy for understanding the etiology and therapy of POP.

  17. Superoxide flashes in single mitochondria.

    PubMed

    Wang, Wang; Fang, Huaqiang; Groom, Linda; Cheng, Aiwu; Zhang, Wanrui; Liu, Jie; Wang, Xianhua; Li, Kaitao; Han, Peidong; Zheng, Ming; Yin, Jinhu; Wang, Weidong; Mattson, Mark P; Kao, Joseph P Y; Lakatta, Edward G; Sheu, Shey-Shing; Ouyang, Kunfu; Chen, Ju; Dirksen, Robert T; Cheng, Heping

    2008-07-25

    In quiescent cells, mitochondria are the primary source of reactive oxygen species (ROS), which are generated by leakiness of the electron transport chain (ETC). High levels of ROS can trigger cell death, whereas lower levels drive diverse and important cellular functions. We show here by employing a newly developed mitochondrial matrix-targeted superoxide indicator, that individual mitochondria undergo spontaneous bursts of superoxide generation, termed "superoxide flashes." Superoxide flashes occur randomly in space and time, exhibit all-or-none properties, and provide a vital source of superoxide production across many different cell types. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore stimulating superoxide production by the ETC. Furthermore, we observe a flurry of superoxide flash activity during reoxygenation of cardiomyocytes after hypoxia, which is inhibited by the cardioprotective compound adenosine. We propose that superoxide flashes could serve as a valuable biomarker for a wide variety of oxidative stress-related diseases.

  18. Adaptive regulation of human intestinal thiamine uptake by extracellular substrate level: a role for THTR-2 transcriptional regulation

    PubMed Central

    Nabokina, Svetlana M.; Subramanian, Veedamali S.; Valle, Judith E.

    2013-01-01

    The intestinal thiamine uptake process is adaptively regulated by the level of vitamin in the diet, but the molecular mechanism involved is not fully understood. Here we used the human intestinal epithelial Caco-2 cells exposed to different levels of extracellular thiamine to delineate the molecular mechanism involved. Our results showed that maintaining Caco-2 cells in a thiamine-deficient medium resulted in a specific and significant increase of [3H]thiamine uptake compared with cell exposure to a high level of thiamine (1 mM). This adaptive regulation was also associated with a higher level of mRNA expression of thiamine transporter-2 (THTR-2), but not thiamine transporter-1 (THTR-1), in the deficient condition and a higher level of promoter activity of gene encoding THTR-2 (SLC19A3). Using 5′-truncated promoter-luciferase constructs, we identified the thiamine level-responsive region in the SLC19A3 promoter to be between −77 and −29 (using transcriptional start site as +1). By means of mutational analysis, a key role for a stimulating protein-1 (SP1)/guanosine cytidine box in mediating the effect of extracellular thiamine level on SLC19A3 promoter was established. Furthermore, extracellular level of thiamine was found to affect SP1 protein expression and binding pattern to the thiamine level-responsive region of SLC19A3 promoter in Caco-2 cells as shown by Western blotting and electrophoretic mobility shift assay analysis, respectively. These studies demonstrate that the human intestinal thiamine uptake is adaptively regulated by the extracellular substrate level via transcriptional regulation of the THTR-2 system, and report that SP1 transcriptional factor is involved in this regulation. PMID:23989004

  19. Synthesis of calcium superoxide

    NASA Technical Reports Server (NTRS)

    Rewick, R. T.; Blucher, W. G.; Estacio, P. L.

    1972-01-01

    Efforts to prepare Ca(O2) sub 2 from reactions of calcium compounds with 100% O3 and with O(D-1) atoms generated by photolysis of O3 at 2537 A are described. Samples of Ca(OH) sub 2, CaO, CaO2, Ca metal, and mixtures containing suspected impurities to promote reaction have been treated with excess O3 under static and flow conditions in the presence and absence of UV irradiation. Studies with KO2 suggest that the superoxide anion is stable to radiation at 2537 A but reacts with oxygen atoms generated by the photolysis of O3 to form KO3. Calcium superoxide is expected to behave in an analogous.

  20. Superoxide Dismutase Assays

    DTIC Science & Technology

    1987-06-16

    rapidly catalyzed the dismutation of Superoxide free radical anions in the reaction : enzyme 0 * + 0’~ + 2H > O...its rate of dismutation both enzymically and spontaneously. By allowing the generator reaction to proceed for a specific time period before the addi...concentration is adjusted to give a reaction rate so that the change at 550 rm is 0.02 to 0.04 OD/min. Stock of Xanthine Oxidase BC 1.1.3.22 is at

  1. Cu/Zn superoxide dismutases in developing cotton fibers

    USDA-ARS?s Scientific Manuscript database

    Hydrogen peroxide (H2O2) and other reactive oxygen species (ROS) are important signaling molecules in diverse physiological processes. Previously, we discovered superoxide dismutase (SOD) activity in extracellular protein preparations from fiber-bearing cotton (Gossypium hirsutum L.) seeds. We sho...

  2. Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme*

    PubMed Central

    Coelho, Fernando R.; Iqbal, Asif; Linares, Edlaine; Silva, Daniel F.; Lima, Filipe S.; Cuccovia, Iolanda M.; Augusto, Ohara

    2014-01-01

    The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp32 residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp32 residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1WT and hSOD1G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp32 residue in the process. The results showed that Trp32 residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp32 residue (bovine SOD1 and hSOD1W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp32 residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1. PMID:25237191

  3. Recombinant human manganese superoxide dismutase (rMnSOD): a positive effect on the immunohematological state of mice irradiated with protons

    NASA Astrophysics Data System (ADS)

    Ambesi-Impiombato, Francesco Saverio; Belov, Oleg; Bulinina, Taisia; Ivanov, Alexander; Mancini, Aldo; Borrelli, Antonella; Krasavin, Eugene A.

    Protons represent the largest component of space radiation. In this regard screening of radioprotective drugs capable of increasing radioresistance of astronauts obligatory includes studying these compounds using proton radiation injury models. The recombinant human manganese superoxide dismutase (rMnSOD) had previously demonstrated its efficacy on an in vivo X-ray induced injury model, when multiple intraperitoneal treatments allowed the survival of mice irradiated with doses which were lethal for the control animals (Borrelli A et al. “A recombinant MnSOD is radioprotective for normal cells and radiosensitizing for tumor cells”. Free Radic Biol Med. 2009, 46, 110-6). Using the model of sublethal whole-body irradiation with protons available at Phasotron of Joint Institute for Nuclear Research (Dubna, Russia), we reconstruct the bone-marrow form of the acute radiation sickness to test the radioprotective effect of rMnSOD. Male (CBAxC57Bl6) F1 hybrid SPF mice weighting approximately 24 g were exposed to 171 MeV protons at the dose of 4 Gy. After irradiation, the sixfold daily subcutaneous treatment with rMnSOD has provided a statistically significant acceleration of the recovery of thymus and spleen mass and of the number of leukocytes in mice peripheral blood. In the control, untreated and irradiated mice, these positive effects were not observed even on day 7 after exposure. The number of karyocytes in bone marrow of irradiated mice has even exceeded its basal level in the control group 7 days after irradiation. The rMnSOD-treated group has thus demonstrated a significant hyper-restoration of this characteristic. In the presentation, several possibilities of using of rMnSOD in space medicine will be discussed, taking into account various biomedically relevant effects of this enzyme.

  4. The Human Pancreas as a Source of Protolerogenic Extracellular Matrix Scaffold for a New-generation Bioartificial Endocrine Pancreas.

    PubMed

    Peloso, Andrea; Urbani, Luca; Cravedi, Paolo; Katari, Ravi; Maghsoudlou, Panagiotis; Fallas, Mario Enrique Alvarez; Sordi, Valeria; Citro, Antonio; Purroy, Carolina; Niu, Guoguang; McQuilling, John P; Sittadjody, Sivanandane; Farney, Alan C; Iskandar, Samy S; Zambon, Joao P; Rogers, Jeffrey; Stratta, Robert J; Opara, Emmanuel C; Piemonti, Lorenzo; Furdui, Cristina M; Soker, Shay; De Coppi, Paolo; Orlando, Giuseppe

    2016-07-01

    Our study aims at producing acellular extracellular matrix scaffolds from the human pancreas (hpaECMs) as a first critical step toward the production of a new-generation, fully human-derived bioartificial endocrine pancreas. In this bioartificial endocrine pancreas, the hardware will be represented by hpaECMs, whereas the software will consist in the cellular compartment generated from patient's own cells. Extracellular matrix (ECM)-based scaffolds obtained through the decellularization of native organs have become the favored platform in the field of complex organ bioengineering. However, the paradigm is now switching from the porcine to the human model. To achieve our goal, human pancreata were decellularized with Triton-based solution and thoroughly characterized. Primary endpoints were complete cell and DNA clearance, preservation of ECM components, growth factors and stiffness, ability to induce angiogenesis, conservation of the framework of the innate vasculature, and immunogenicity. Secondary endpoint was hpaECMs' ability to sustain growth and function of human islet and human primary pancreatic endothelial cells. Results show that hpaECMs can be successfully and consistently produced from human pancreata and maintain their innate molecular and spatial framework and stiffness, and vital growth factors. Importantly, hpaECMs inhibit human naïve CD4 T-cell expansion in response to polyclonal stimuli by inducing their apoptosis and promoting their conversion into regulatory T cells. hpaECMs are cytocompatible and supportive of representative pancreatic cell types. We, therefore, conclude that hpaECMs has the potential to become an ideal platform for investigations aiming at the manufacturing of a regenerative medicine-inspired bioartificial endocrine pancreas.

  5. Comparative power spectral analysis of simultaneous electroencephalographic and magnetoencephalographic recordings in humans suggests non-resistive extracellular media

    PubMed Central

    Dehghani, Nima; Bédard, Claude; Cash, Sydney S.; Halgren, Eric

    2015-01-01

    The resistive or non-resistive nature of the extracellular space in the brain is still debated, and is an important issue for correctly modeling extracellular potentials. Here, we first show theoretically that if the medium is resistive, the frequency scaling should be the same for electroencephalogram (EEG) and magnetoencephalogram (MEG) signals at low frequencies (<10 Hz). To test this prediction, we analyzed the spectrum of simultaneous EEG and MEG measurements in four human subjects. The frequency scaling of EEG displays coherent variations across the brain, in general between 1/f and 1/f2. In a given region, although the variability of the frequency scaling exponent was higher for MEG compared to EEG, both signals consistently scale with a different exponent. In some cases, the scaling was similar, but only when the signal-to-noise ratio of the MEG was low. Several methods of noise correction for environmental and instrumental noise were tested, and they all increased the difference between EEG and MEG scaling. In conclusion, there is a significant difference in frequency scaling between EEG and MEG, which can be explained if the extracellular medium (including other layers such as dura matter and skull) is globally non-resistive. PMID:20556640

  6. Comparative power spectral analysis of simultaneous elecroencephalographic and magnetoencephalographic recordings in humans suggests non-resistive extracellular media

    PubMed Central

    Dehghani, Nima; Bédard, Claude; Cash, Sydney S.; Halgren, Eric

    2010-01-01

    The resistive or non-resistive nature of the extracellular space in the brain is still debated, and is an important issue for correctly modeling extracellular potentials. Here, we first show theoretically that if the medium is resistive, the frequency scaling should be the same for electroencephalogram (EEG) and magnetoencephalogram (MEG) signals at low frequencies (<10 Hz). To test this prediction, we analyzed the spectrum of simultaneous EEG and MEG measurements in four human subjects. The frequency scaling of EEG displays coherent variations across the brain, in general between 1/f and 1/f2, and tends to be smaller in parietal/temporal regions. In a given region, although the variability of the frequency scaling exponent was higher for MEG compared to EEG, both signals consistently scale with a different exponent. In some cases, the scaling was similar, but only when the signal-to-noise ratio of the MEG was low. Several methods of noise correction for environmental and instrumental noise were tested, and they all increased the difference between EEG and MEG scaling. In conclusion, there is a significant difference in frequency scaling between EEG and MEG, which can be explained if the extracellular medium (including other layers such as dura matter and skull) is globally non-resistive. Electronic supplementary material The online version of this article (doi:10.1007/s10827-010-0263-2) contains supplementary material, which is available to authorized users. PMID:20697790

  7. HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation.

    PubMed

    González-Ramos, Marta; Calleros, Laura; López-Ongil, Susana; Raoch, Viviana; Griera, Mercedes; Rodríguez-Puyol, Manuel; de Frutos, Sergio; Rodríguez-Puyol, Diego

    2013-02-01

    The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases.

  8. Histochemical evidence of osteoclastic degradation of extracellular matrix in osteolytic metastasis originating from human lung small carcinoma (SBC-5) cells.

    PubMed

    Li, Minqi; Amizuka, Norio; Takeuchi, Kiichi; Freitas, Paulo H L; Kawano, Yoshiro; Hoshino, Masaaki; Oda, Kimimitsu; Nozawa-Inoue, Kayoko; Maeda, Takeyasu

    2006-02-01

    The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular

  9. Extracellular calcium sensing receptor stimulation in human colonic epithelial cells induces intracellular calcium oscillations and proliferation inhibition.

    PubMed

    Rey, Osvaldo; Young, Steven H; Jacamo, Rodrigo; Moyer, Mary P; Rozengurt, Enrique

    2010-10-01

    The extracellular Ca(2+)-sensing receptor (CaR) is increasingly implicated in the regulation of multiple cellular functions in the gastrointestinal tract, including secretion, proliferation and differentiation of intestinal epithelial cells. However, the signaling mechanisms involved remain poorly defined. Here we examined signaling pathways activated by the CaR, including Ca(2+) oscillations, in individual human colon epithelial cells. Single cell imaging of colon-derived cells expressing the CaR, including SW-480, HT-29, and NCM-460 cells, shows that stimulation of this receptor by addition of aromatic amino acids or by an elevation of the extracellular Ca(2+) concentration promoted striking intracellular Ca(2+) oscillations. The intracellular calcium oscillations in response to extracellular Ca(2+) were of sinusoidal pattern and mediated by the phospholipase C/diacylglycerol/inositol 1,4,5-trisphosphate pathway as revealed by a biosensor that detects the accumulation of diacylglycerol in the plasma membrane. The intracellular calcium oscillations in response to aromatic amino acids were of transient type, that is, Ca(2+) spikes that returned to baseline levels, and required an intact actin cytoskeleton, a functional Rho, Filamin A and the ion channel TRPC1. Further analysis showed that re-expression and stimulation of the CaR in human epithelial cells derived from normal colon and from colorectal adenocarcinoma inhibits their proliferation. This inhibition was associated with the activation of the signaling pathway that mediates the generation of sinusoidal, but not transient, intracellular Ca(2+) oscillations. Thus, these results indicate that the CaR can function in two signaling modes in human colonic epithelial cells offering a potential link between gastrointestinal responses and food/nutrients uptake and metabolism.

  10. Chemical reactivity and biological effects of superoxide radicals

    NASA Astrophysics Data System (ADS)

    Chuaqui, C. A.; Petkau, A.

    The chemical reactivity of the superoxide radical is described in terms of its dismutation to oxygen, its basicity and ability to act as a nucleophile in aprotic media, and in its capacity to mediate one electron transfer reactions and undergo conversion to other species of active oxygen. The biological role of the superoxide radical is discussed in terms of its diffusion and reactivity within membranes, its potential to inactivate enzymes and elicit DNA damage in human granulocytes activated by phorbol esters. Mechanisms for reduction of oxygen to the superoxide radical by riboflavin, nitroaromatic compounds, nitrofurazone and oxazolinone are described. Finally, inactivation of bone marrow progenitor cells by superoxide radicals generated photochemically is discussed to emphasize the sensitizing effect of the medium, the sequential toxicity of superoxide and hydrogen peroxide, and the protective effects of superoxide dismutase and catalase while acting in their respective time frames.

  11. [Interaction of chagasic autoantibodies with the third extracellular domain of the human heart muscarinic receptor. Functional and pathological implications].

    PubMed

    Goin, J C; Pérez Leirós, C; Borda, E; Sterin-Borda, L

    1996-01-01

    Herein we demonstrate by ELISA and immunoblotting the presence in the sera of chagasic patients of circulating autoantibodies against the third extracellular domain of human muscarinic acetylcholine receptors by using a synthetic peptide corresponding to the sequence 169-192 of the receptor. Immunoaffinity purified antipeptide antibodies displayed cardiac muscarinic activity as decreased contractility and cAMP production and increased cGMP levels. These effects were specifically blocked by the synthetic peptide and by atropine. A strong association between the existence of circulating autoantibodies and the presence of dysautonomia was shown, making these autoantibodies an appropriate marker of heart autonomic dysfunction.

  12. Importance of Thickness in Human Cardiomyocyte Network for Effective Electrophysiological Stimulation Using On-Chip Extracellular Microelectrodes

    NASA Astrophysics Data System (ADS)

    Hamada, Tomoyo; Nomura, Fumimasa; Kaneko, Tomoyuki; Yasuda, Kenji

    2012-06-01

    We have developed a three-dimensionally controlled in vitro human cardiomyocyte network assay for the measurements of drug-induced conductivity changes and the appearance of fatal arrhythmia such as ventricular tachycardia/fibrillation for more precise in vitro predictive cardiotoxicity. To construct an artificial conductance propagation model of a human cardiomyocyte network, first, we examined the cell concentration dependence of the cell network heights and found the existence of a height limit of cell networks, which was double-layer height, whereas the cardiomyocytes were effectively and homogeneously cultivated within the microchamber maintaining their spatial distribution constant and their electrophysiological conductance and propagation were successfully recorded using a microelectrode array set on the bottom of the microchamber. The pacing ability of a cardiomyocyte's electrophysiological response has been evaluated using microelectrode extracellular stimulation, and the stimulation for pacing also successfully regulated the beating frequencies of two-layered cardiomyocyte networks, whereas monolayered cardiomyocyte networks were hardly stimulated by the external electrodes using the two-layered cardiomyocyte stimulation condition. The stability of the lined-up shape of human cardiomyocytes within the rectangularly arranged agarose microchambers was limited for a two-layered cardiomyocyte network because their stronger force generation shrunk those cells after peeling off the substrate. The results indicate the importance of fabrication technology of thickness control of cellular networks for effective extracellular stimulation and the potential concerning thick cardiomyocyte networks for long-term cultivation.

  13. Improved isolation and purification of functional human Fas receptor extracellular domain using baculovirus-silkworm expression system.

    PubMed

    Muraki, Michiro; Honda, Shinya

    2011-11-01

    To achieve an efficient isolation of human Fas receptor extracellular domain (hFasRECD), a fusion protein of hFasRECD with human IgG1 heavy chain Fc domain containing thrombin cleavage sequence at the junction site was overexpressed using baculovirus-silkworm larvae expression system. The hFasRECD part was separated from the fusion protein by the effective cleavage of the recognition site with bovine thrombin. Protein G column treatment of the reaction mixture and the subsequent cation-exchange chromatography provided purified hFasRECD with a final yield of 13.5mg from 25.0 ml silkworm hemolymph. The functional activity of the product was examined by size-exclusion chromatography analysis. The isolated hFasRECD less strongly interacted with human Fas ligand extracellular domain (hFasLECD) than the Fc domain-bridged counterpart, showing the contribution of antibody-like avidity in the latter case. The purified glycosylated hFasRECD presented several discrete bands in the disulphide-bridge non-reducing SDS-PAGE analysis, and virtually all of the components were considered to participate in the binding to hFasLECD. The attached glycans were susceptible to PNGase F digestion, but mostly resistant to Endo Hf digestion under denaturing conditions. One of the components exhibited a higher susceptibility to PNGase F digestion under non-denaturing conditions.

  14. Extracellular complexes of the hematopoietic human and mouse CSF-1 receptor are driven by common assembly principles.

    PubMed

    Elegheert, Jonathan; Desfosses, Ambroise; Shkumatov, Alexander V; Wu, Xiongwu; Bracke, Nathalie; Verstraete, Kenneth; Van Craenenbroeck, Kathleen; Brooks, Bernard R; Svergun, Dmitri I; Vergauwen, Bjorn; Gutsche, Irina; Savvides, Savvas N

    2011-12-07

    The hematopoietic colony stimulating factor-1 receptor (CSF-1R or FMS) is essential for the cellular repertoire of the mammalian immune system. Here, we report a structural and mechanistic consensus for the assembly of human and mouse CSF-1:CSF-1R complexes. The EM structure of the complete extracellular assembly of the human CSF-1:CSF-1R complex reveals how receptor dimerization by CSF-1 invokes a ternary complex featuring extensive homotypic receptor contacts and striking structural plasticity at the extremities of the complex. Studies by small-angle X-ray scattering of unliganded hCSF-1R point to large domain rearrangements upon CSF-1 binding, and provide structural evidence for the relevance of receptor predimerization at the cell surface. Comparative structural and binding studies aiming to dissect the assembly principles of human and mouse CSF-1R complexes, including a quantification of the CSF-1/CSF-1R species cross-reactivity, show that bivalent cytokine binding to receptor coupled to ensuing receptor-receptor interactions are common denominators in extracellular complex formation.

  15. 2-methoxyestradiol does not inhibit superoxide dismutase.

    PubMed

    Kachadourian, R; Liochev, S I; Cabelli, D E; Patel, M N; Fridovich, I; Day, B J

    2001-08-15

    It has been reported in the literature that the endogenous estrogen metabolite 2-methoxyestradiol (2-ME) inhibits both manganese and copper,zinc superoxide dismutases (Mn and Cu,Zn SODs) and that this mechanism is responsible for 2-ME's ability to kill cancer cells. In fact, as demonstrated using several SOD assays including pulse radiolysis, 2-ME does not inhibit SOD but rather interferes with the SOD assay originally used. Nevertheless, as confirmed by aconitase inactivation measurements and lactate dehydrogenase release in human leukemia HL-60 cells, 2-ME does increase superoxide production in these cells and is more toxic than its non-O-methylated precursor 2-hydroxyestradiol. Other mechanisms previously suggested in the literature may explain 2-ME's ability to increase intracellular superoxide levels in tumor cells. Copyright 2001 Academic Press.

  16. EPR Detection of Cellular and Mitochondrial Superoxide Using Cyclic Hydroxylamines

    PubMed Central

    Dikalov, Sergey I.; Kirilyuk, Igor A.; Voinov, Maxim; Grigor’ev, Igor A.

    2014-01-01

    Superoxide (O2•) has been implicated in the pathogenesis of many human diseases but detection of the O2• radicals in biological systems is limited due to inefficiency of O2• spin trapping and lack of site-specific information. In this work we studied production of extracellular, intracellular and mitochondrial O2• in neutrophils, cultured endothelial cells and isolated mitochondria using new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O2• producing stable nitroxides and allowed site-specific O2• detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O2•. Inhibition of EPR signal by SOD2 overexpression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O2• both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected increase in cytoplasm O2• stimulated by PMA but only CM-H and mitoTEMPO-H showed increase in rotenone-induced mitochondrial O2•. These data show that new set of hydroxylamine spin probes provide unique information about site-specific production of O2• radical in extracellular or intracellular compartments, cytoplasm or mitochondria. PMID:21128732

  17. EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines.

    PubMed

    Dikalov, Sergey I; Kirilyuk, Igor A; Voinov, Maxim; Grigor'ev, Igor A

    2011-04-01

    Superoxide (O₂ⁱ⁻) has been implicated in the pathogenesis of many human diseases, but detection of the O(2)(•-) radicals in biological systems is limited due to inefficiency of O₂ⁱ⁻ spin trapping and lack of site-specific information. This work studied production of extracellular, intracellular and mitochondrial O₂ⁱ⁻ in neutrophils, cultured endothelial cells and isolated mitochondria using a new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O₂ⁱ⁻, producing stable nitroxides and allowing site-specific cO₂ⁱ⁻ detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O₂ⁱ⁻. Inhibition of EPR signal by SOD2 over-expression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O₂ⁱ⁻ both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected an increase in cytoplasm O₂ⁱ⁻ stimulated by PMA, but only CM-H and mitoTEMPO-H showed an increase in rotenone-induced mitochondrial O₂ⁱ⁻. These data show that a new set of hydroxylamine spin probes provide unique information about site-specific production of the O₂ⁱ⁻ radical in extracellular or intracellular compartments, cytoplasm or mitochondria.

  18. Microbiota/host crosstalk biomarkers: regulatory response of human intestinal dendritic cells exposed to Lactobacillus extracellular encrypted peptide.

    PubMed

    Bernardo, David; Sánchez, Borja; Al-Hassi, Hafid O; Mann, Elizabeth R; Urdaci, María C; Knight, Stella C; Margolles, Abelardo

    2012-01-01

    The human gastrointestinal tract is exposed to a huge variety of microorganisms, either commensal or pathogenic; at this site, a balance between immunity and immune tolerance is required. Intestinal dendritic cells (DCs) control the mechanisms of immune response/tolerance in the gut. In this paper we have identified a peptide (STp) secreted by Lactobacillus plantarum, characterized by the abundance of serine and threonine residues within its sequence. STp is encoded in one of the main extracellular proteins produced by such species, which includes some probiotic strains, and lacks cleavage sites for the major intestinal proteases. When studied in vitro, STp expanded the ongoing production of regulatory IL-10 in human intestinal DCs from healthy controls. STp-primed DC induced an immunoregulatory cytokine profile and skin-homing profile on stimulated T-cells. Our data suggest that some of the molecular dialogue between intestinal bacteria and DCs may be mediated by immunomodulatory peptides, encoded in larger extracellular proteins, secreted by commensal bacteria. These peptides may be used for the development of nutraceutical products for patients with IBD. In addition, this kind of peptides seem to be absent in the gut of inflammatory bowel disease patients, suggesting a potential role as biomarker of gut homeostasis.

  19. Microbiota/Host Crosstalk Biomarkers: Regulatory Response of Human Intestinal Dendritic Cells Exposed to Lactobacillus Extracellular Encrypted Peptide

    PubMed Central

    Al-Hassi, Hafid O.; Mann, Elizabeth R.; Urdaci, María C.; Knight, Stella C.; Margolles, Abelardo

    2012-01-01

    The human gastrointestinal tract is exposed to a huge variety of microorganisms, either commensal or pathogenic; at this site, a balance between immunity and immune tolerance is required. Intestinal dendritic cells (DCs) control the mechanisms of immune response/tolerance in the gut. In this paper we have identified a peptide (STp) secreted by Lactobacillus plantarum, characterized by the abundance of serine and threonine residues within its sequence. STp is encoded in one of the main extracellular proteins produced by such species, which includes some probiotic strains, and lacks cleavage sites for the major intestinal proteases. When studied in vitro, STp expanded the ongoing production of regulatory IL-10 in human intestinal DCs from healthy controls. STp-primed DC induced an immunoregulatory cytokine profile and skin-homing profile on stimulated T-cells. Our data suggest that some of the molecular dialogue between intestinal bacteria and DCs may be mediated by immunomodulatory peptides, encoded in larger extracellular proteins, secreted by commensal bacteria. These peptides may be used for the development of nutraceutical products for patients with IBD. In addition, this kind of peptides seem to be absent in the gut of inflammatory bowel disease patients, suggesting a potential role as biomarker of gut homeostasis. PMID:22606249

  20. Oxidation of the tryptophan 32 residue of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity triggers the non-amyloid aggregation of the enzyme.

    PubMed

    Coelho, Fernando R; Iqbal, Asif; Linares, Edlaine; Silva, Daniel F; Lima, Filipe S; Cuccovia, Iolanda M; Augusto, Ohara

    2014-10-31

    The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1(WT) and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp(32) residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp(32) residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1(WT) and hSOD1(G93A) mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp(32) residue in the process. The results showed that Trp(32) residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp(32) residue (bovine SOD1 and hSOD1(W32F) mutant). The results support a role for the oxidation products of the hSOD1-Trp(32) residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Hydrogen peroxide overload increases adriamycin-induced apoptosis of SaOS(2)FM, a manganese superoxide dismutase-overexpressing human osteosarcoma cell line.

    PubMed

    Wang, Yadi; Kuroda, Masahiro; Gao, Xian-Shu; Asaumi, Jun-Ichi; Shibuya, Kohichi; Kawasaki, Shoji; Akaki, Shiro; St Clair, Daret; Hiraki, Yoshio; Kanazawa, Susumu

    2005-05-01

    We previously developed a new microscopic observation system that enables time-lapse quantitative analysis of apoptosis and necrosis. With this system we quantitatively analyzed adriamycin (ADR)-induced cell death using manganese superoxide dismutase (MnSOD)- and wild-type p53-gene transfectants on SaOS(2), a p53-deficient human osteosarcoma cell line. A highly MnSOD-overexpressing cell line, SaOS(2)FM(H), acquired ADR-tolerance compared to the parent cell line SaOS(2). The ADR-tolerance of SaOS(2)FM(H) diminished by L-buthionine-[S,R]-sulfoximine (BSO), which did not change ADR-sensitivity of SaOS(2), to the similar ADR-sensitivity of SaOS(2). A wild-type p53-expressing cell line, SaOS(2)wtp53, significantly increased in ADR-sensitivity compared to SaOS(2). This ADR-sensitivity of SaOS(2)wtp53 was enhanced by BSO. When isosorbide 5-mononitrate was combined with BSO, isosorbide 5-mononitrate increased ADR sensitivity of a moderately MnSOD-overexpressing cell line, SaOS(2)FM(L), decreased that of SaOS(2) FM(H), and did not change those of SaOS(2) and SaOS(2)wtp53 compared to BSO alone. Time-lapse microscopic observations during ADR treatment for 24 h indicated that the most cells of each cell line underwent apoptosis, and a few cells (less than 11%) died by necrosis. When cells were treated with iso-concentration of ADR, apoptosis of SaOS(2)FM(H) was less than that of SaOS(2). BSO, which did not change ADR-sensitivity of SaOS(2), increased appearance rate of ADR-induced apoptosis, but not necrosis of MnSOD-overexpressing cell lines. When iso-survival dose of ADR, which reduced surviving fraction to 0.01, was given for each cell line, no difference was observed in appearance of either apoptosis or necrosis between SaOS(2) and MnSOD-overexpressing cell lines. On the other hands, appearance of both apoptosis and the following secondary necrosis of SaOS(2) wtp53 was significantly accelerated compared to those of SaOS(2). These findings indicate that hydrogen peroxide

  2. Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

    SciTech Connect

    Matsuzaki, Shinichi; Ishizuka, Tamotsu; Yamada, Hidenori; Kamide, Yosuke; Hisada, Takeshi; Ichimonji, Isao; Aoki, Haruka; Yatomi, Masakiyo; Komachi, Mayumi; Tsurumaki, Hiroaki; Ono, Akihiro; Koga, Yasuhiko; Dobashi, Kunio; Mogi, Chihiro; Sato, Koichi; Tomura, Hideaki; Mori, Masatomo; Okajima, Fumikazu

    2011-10-07

    Highlights: {yields} The involvement of extracellular acidification in airway remodeling was investigated. {yields} Extracellular acidification alone induced CTGF production in human ASMCs. {yields} Extracellular acidification enhanced TGF-{beta}-induced CTGF production in human ASMCs. {yields} Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. {yields} OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-{beta}-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G{sub q/11} protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP{sub 3}) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G{sub q/11} protein and inositol-1,4,5-trisphosphate-induced Ca{sup 2+} mobilization in human ASMCs.

  3. Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis.

    PubMed

    Jokela, Tiina; Kärnä, Riikka; Rauhala, Leena; Bart, Genevieve; Pasonen-Seppänen, Sanna; Oikari, Sanna; Tammi, Markku I; Tammi, Raija H

    2017-03-24

    The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10-100 μm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.

  4. Glucosepane is a major protein cross-link of the senescent human extracellular matrix. Relationship with diabetes.

    PubMed

    Sell, David R; Biemel, Klaus M; Reihl, Oliver; Lederer, Markus O; Strauch, Christopher M; Monnier, Vincent M

    2005-04-01

    The extracellular matrix in most tissues is characterized by progressive age-related stiffening and loss of proteolytic digestibility that are accelerated in diabetes and can be duplicated by the nonenzymatic reaction of reducing sugars and extracellular matrix proteins. However, most cross-links of the Maillard reaction described so far are present in quantities too low to account for these changes. Here we have determined in human skin and glomerular basement membrane (GBM) collagen the levels of the recently discovered lysine-arginine cross-links derived from glucose, methylglyoxal, glyoxal, and 3-deoxyglucosone, i.e. glucosepane, MODIC, GODIC, and DOGDIC, respectively. Insoluble preparations of skin collagen (n = 110) and glomerular basement membrane (GBM, n = 28) were enzymatically digested, and levels were measured by isotope dilution technique using liquid chromatography/mass spectrometry. In skin, all cross-links increased with age (p < 0.0001) except DOGDIC (p = 0.34). In nondiabetic controls, levels at 90 years were 2000, 30, and 15 pmol/mg for glucosepane, MODIC, and GODIC, respectively. Diabetes, but not renal failure, increased glucosepane to 5000 pmol/mg (p < 0.0001), and for all others, increased it to <60 pmol/mg (p < 0.01). In GBMs, glucosepane reached up to 500 pmol/mg of collagen and was increased in diabetes (p < 0.0001) but not old age. In conclusion, glucosepane is the single major cross-link of the senescent extracellular matrix discovered so far, accounting for up to >120 mole% of triple helical collagen modification in diabetes. Its presence in high quantities may contribute to a number of structural and cell matrix dysfunctions observed in aging and diabetes.

  5. Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils

    PubMed Central

    Funchal, Giselle A.; Jaeger, Natália; Czepielewski, Rafael S.; Machado, Mileni S.; Muraro, Stéfanie P.; Stein, Renato T.; Bonorino, Cristina B. C.; Porto, Bárbara N.

    2015-01-01

    Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV) is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs) are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis. PMID:25856628

  6. Models of Superoxide Dismutases

    SciTech Connect

    Cabelli, Diane E.; Riley, Dennis; Rodriguez, Jorge A.; Valentine, Joan Selverstone; Zhu, Haining

    1998-05-20

    In this review we have focused much of our discussion on the mechanistic details of how the native enzymes function and how mechanistic developments/insights with synthetic small molecule complexes possessing SOD activity have influenced our understanding of the electron transfer processes involved with the natural enzymes. A few overriding themes have emerged. Clearly, the SOD enzymes operate at near diffusion controlled rates and to achieve such catalytic turnover activity, several important physical principles must be operative. Such fast electron transfer processes requires a role for protons; i.e., proton-coupled electron transfer (''H-atom transfer'') solves the dilemma of charge separation developing in the transition state for the electron transfer step. Additionally, outer-sphere electron transfer is likely a most important pathway for manganese and iron dismutases. This situation arises because the ligand exchange rates on these two ions in water never exceed {approx}10{sup +7} s{sup -1}; consequently, 10{sup +9} catalytic rates require more subtle mechanistic insights. In contrast, copper complexes can achieve diffusion controlled (>10{sup +9}) exchange rates in water; thus inner-sphere electron transfer processes are more likely to be operative in the Cu/Zn enzymes. Recent studies have continued to expand our understanding of the mechanism of action of this most important class of redox active enzymes, the superoxide dismutases, which have been critical in the successful adaptation of life on this planet to an oxygen-based metabolism. The design of SOD mimic drugs, synthetic models compounds that incorporate this superoxide dismutase catalytic activity and are capable of functioning in vivo, offers clear potential benefits in the control of diseases, ranging from the control of neurodegenerative conditions, such as Parkinson's or Alzheimer's disease, to cancer.

  7. Effects of extracellular nucleotides on single cells and populations of human osteoblasts: contribution of cell heterogeneity to relative potencies

    PubMed Central

    Jane Dixon, C; Bowler, Wayne B; Walsh, Catherine A; Gallagher, James A

    1997-01-01

    Human osteoblasts responded to the application of extracellular nucleotides, acting at P2-receptors, with increases in cytosolic free calcium concentration ([Ca2+]i).In populations of human osteoblasts, adenosine 5′-diphosphate (ADP) evoked a rise in [Ca2+]i with less than 40% of the amplitude of that induced by adenosine 5′-triphosphate (ATP).ATP and uridine 5′-triphosphate (UTP) were applied to single human osteoblasts and induced [Ca2+]i rises of comparable amplitude in every cell tested.However, from the results of single cell studies with ADP (and 2-methylthioATP (2-meSATP)) two groups of cells were delineated: one group responded to ADP (or 2-meSATP) with a rise in [Ca2+]i indistinguishable from that evoked by ATP; whereas the second group failed completely to respond to ADP (or 2-meSATP).Therefore heterogeneity of receptor expression exists within this population of human osteoblasts. The limited distribution of the ADP-responsive receptor underlies the small response to ADP, compared with ATP, recorded in populations of human osteoblasts. This heterogeneity may reflect differences in the differentiation status of individual cells. PMID:9138681

  8. Light and electron microscopic study of epithelial cells from the human oviduct and uterus subcultured on extracellular matrix gel.

    PubMed

    Eslaminejad, Mohamadreza Baghaban; Valojerdi, Mojtaba Rezazadeh; Ashtiani, Saeed Kazemi; Eftekhari-Yazdi, Poopak

    2007-06-01

    To investigate the structure of epithelial cells from the human oviduct and uterus on extracellular matrix (ECM) gel in the first passage. Human oviducts and endometrial tissues were obtained from patients undergoing total hysterectomy; the epithelial cells, having been isolated by enzyme digestion, were cultured on polystyrene plastic surfaces. The epithelial nature of the cells was confirmed by immunocytochemistry, and their morphology was examined by microscopy. Cells of an epithelial nature were then trypsinized and cultured on an ECM gel-coated filter insert for 5 days. The cells, in parallel with the tissues, were subsequently prepared for transmission electron microscopy. Plastic-cultured cells had no sign of differentiation and appeared as elongated spindle cells in sections. These cells looked columnar and highly polarized after being cultured on ECM gel surfaces. They were similar to epithelial cells from the corresponding tissue fragment. Cultured on ECM gel, the ciliated epithelial cells of human oviducts appeared ultrastructurally similar to glandular cells from the human uterus. Cilia did not form under culture conditions. It seems that human uterine and oviduct epithelial cells can acquire polarized morphology and differentiated states on ECM gel after having lost it on plastic surfaces and that ECM gel by itself is not enough to induce cilia formation in culture.

  9. Specific inhibition of T-cell adhesion to extracellular matrix and proinflammatory cytokine secretion by human recombinant galectin-1

    PubMed Central

    RABINOVICH, G A; ARIEL, A; HERSHKOVIZ, R; HIRABAYASHI, J; KASAI, K-I; LIDER, O

    1999-01-01

    The migration of immune cells through the extracellular matrix (ECM) towards inflammatory sites is co-ordinated by receptors recognizing ECM glycoproteins, chemokines and proinflammatory cytokines. In this context, galectins are secreted to the extracellular milieu, where they recognize poly-N-acetyllactosamine chains on major ECM glycoproteins, such as fibronectin and laminin. We investigated the possibility that galectin-1 could modulate the adhesion of human T cells to ECM and ECM components. T cells were purified from human blood, activated with interleukin-2 (IL-2), labelled, and incubated further with intact immobilized ECM and ECM glycoproteins in the presence of increasing concentrations of human recombinant galectin-1, or its more stable, related, C2-S molecule obtained by site-directed mutagenesis. The presence of galectin-1 was shown to inhibit T-cell adhesion to intact ECM, laminin and fibronectin, and to a lesser extent to collagen type IV, in a dose-dependent manner. This effect was specifically blocked by anti-galectin-1 antibody and was dependent on the lectin’s carbohydrate-binding properties. The inhibition of T-cell adhesion by galectin-1 correlates with the ability of this molecule to block the re-organization of the activated cell’s actin cytoskeleton. Furthermore, tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production was markedly reduced when IL-2-activated T cells were incubated with galectin-1 or its mutant. This effect was prevented by β-galactoside-related sugars. The present study reveals an alternative inhibitory mechanism for explaining the suppressive properties of the galectin-1 subfamily on inflammatory and autoimmune processes. PMID:10447720

  10. Extracellular ATP and P2Y Receptor Activation Induce a Proinflammatory Host Response in the Human Urinary Tract▿

    PubMed Central

    Säve, Susanne; Persson, Katarina

    2010-01-01

    Extracellular ATP can be released by many cell types under conditions of cellular stress and signals through activation of purinergic receptors. Bladder uroepithelial cells grown in vitro have previously been shown to release ATP in response to stretch. In the present study, we investigated ATP release from uroepithelial cells infected with bacteria and the effect of ATP on the host cell proinflammatory interleukin 8 (IL-8) response. The human kidney epithelial cell line A498 and the human uroepithelial cell line UROtsa were grown in culture and stimulated by the uropathogenic Escherichia coli (UPEC) IA2 strain or the stable ATP analogue ATP-γ-S. ATP and IL-8 levels were measured in cell culture medium with a luciferin-luciferase assay and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that UPEC infection of uroepithelial cells for 1 h significantly increased (P < 0.01) the extracellular ATP levels. ATP-γ-S (10 and 100 μM) stimulated release of IL-8 from UROtsa and A498 cells after 6 and 24 h. Experiments with different purinoceptor agonists suggested that P2Y receptors, and not P2X receptors, were responsible for the ATP-γ-S-induced IL-8 release. The potency profile further suggested involvement of P2Y1, P2Y2, and/or P2Y11 receptors, and reverse transcription-PCR (RT-PCR) studies confirmed that the cells expressed these receptors. The amount of IL-8 released increased 12-fold in UPEC-infected cells, and apyrase, an enzyme that degrades ATP, reduced this increase by approximately 50%. The present study suggests that enhanced ATP release and P2Y receptor activation during urinary tract infection may represent a novel, non-TLR4-mediated mechanism for production of proinflammatory IL-8 in human urinary tract epithelial cells. PMID:20515921

  11. Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene - methyltetrazine reactions.

    PubMed

    Muraki, Michiro; Hirota, Kiyonori

    2017-07-03

    Fas ligand plays a key role in the human immune system as a major cell death inducing protein. The extracellular domain of human Fas ligand (hFasLECD) triggers apoptosis of malignant cells, and therefore is expected to have substantial potentials in medical biotechnology. However, the current application of this protein to clinical medicine is hampered by a shortage of the benefits relative to the drawbacks including the side-effects in systemic administration. Effective procedures for the engineering of the protein by attaching useful additional functions are required to overcome the problem. A procedure for the site-specific chemical conjugation of hFasLECD with a fluorochrome and functional proteins was devised using an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene group and methyltetrazine group. The conjugations in the present study were attained by using much less molar excess amounts of the compounds to be attached as compared with the conventional chemical modification reactions using maleimide derivatives in the previous study. The isolated conjugates of hFasLECD with sulfo-Cy3, avidin and rabbit IgG Fab' domain presented the functional and the structural integrities of the attached molecules without impairing the specific binding activity toward human Fas receptor extracellular domain. The present study provided a new fundamental strategy for the production of the engineered hFasLECDs with additional beneficial functions, which will lead to the developments of the improved diagnostic systems and the effective treatment methods of serious diseases by using this protein as a component of novel molecular tools.

  12. Nature of extracellular signal that triggers RhoA/ROCK activation for the basal internal anal sphincter tone in humans

    PubMed Central

    Singh, Jagmohan; Kumar, Sumit; Phillips, Benjamin

    2015-01-01

    The extracellular signal that triggers activation of rho-associated kinase (RhoA/ROCK), the major molecular determinant of basal internal anal sphincter (IAS) smooth muscle tone, is not known. Using human IAS tissues, we identified the presence of the biosynthetic machineries for angiotensin II (ANG II), thromboxane A2 (TXA2), and prostaglandin F2α (PGF2α). These end products of the renin-angiotensin system (RAS) (ANG II) and arachidonic acid (TXA2 and PGF2α) pathways and their effects in human IAS vs. rectal smooth muscle (RSM) were studied. A multipronged approach utilizing immunocytochemistry, Western blot analyses, and force measurements was implemented. Additionally, in a systematic analysis of the effects of respective inhibitors along different steps of biosynthesis and those of antagonists, their end products were evaluated either individually or in combination. To further describe the molecular mechanism for the IAS tone via these pathways, we monitored RhoA/ROCK activation and its signal transduction cascade. Data showed characteristically higher expression of biosynthetic machineries of RAS and AA pathways in the IAS compared with the RSM. Additionally, specific inhibition of the arachidonic acid (AA) pathway caused ∼80% decrease in the IAS tone, whereas that of RAS lead to ∼20% decrease. Signal transduction studies revealed that the end products of both AA and RAS pathways cause increase in the IAS tone via activation of RhoA/ROCK. Both AA and RAS (via the release of their end products TXA2, PGF2α, and ANG II, respectively), provide extracellular signals which activate RhoA/ROCK for the maintenance of the basal tone in human IAS. PMID:25882611

  13. Human muscle protein synthesis is modulated by extracellular, not intramuscular amino acid availability: a dose-response study.

    PubMed

    Bohé, Julien; Low, Aili; Wolfe, Robert R; Rennie, Michael J

    2003-10-01

    To test the hypothesis that muscle protein synthesis (MPS) is regulated by the concentration of extracellular amino acids, we investigated the dose-response relationship between the rate of human MPS and the concentrations of blood and intramuscular amino acids. We increased blood mixed amino acid concentrations by up to 240 % above basal levels by infusion of mixed amino acids (Aminosyn 15, 44-261 mg kg-1 h-1) in 21 healthy subjects, (11 men 10 women, aged 29 +/- 2 years) and measured the rate of incorporation of D5-phenylalanine or D3-leucine into muscle protein and blood and intramuscular amino acid concentrations. The relationship between the fold increase in MPS and blood essential amino acid concentration ([EAA], mM) was hyperbolic and fitted the equation MPS = (2.68 x [EAA])/(1.51 + [EAA]) (P < 0.01). The pattern of stimulation of myofibrillar, sarcoplasmic and mitochondrial protein was similar. There was no clear relationship between the rate of MPS and the concentration of intramuscular EAAs; indeed, when MPS was increasing most rapidly, the concentration of intramuscular EAAs was below basal levels. We conclude that the rates of synthesis of all classes of muscle proteins are acutely regulated by the blood [EAA] over their normal diurnal range, but become saturated at high concentrations. We propose that the stimulation of protein synthesis depends on the sensing of the concentration of extracellular, rather than intramuscular EAAs.

  14. Human Muscle Protein Synthesis is Modulated by Extracellular, Not Intramuscular Amino Acid Availability: A Dose-Response Study

    PubMed Central

    Bohé, Julien; Low, Aili; Wolfe, Robert R; Rennie, Michael J

    2003-01-01

    To test the hypothesis that muscle protein synthesis (MPS) is regulated by the concentration of extracellular amino acids, we investigated the dose-response relationship between the rate of human MPS and the concentrations of blood and intramuscular amino acids. We increased blood mixed amino acid concentrations by up to 240 % above basal levels by infusion of mixed amino acids (Aminosyn 15, 44-261 mg kg−1 h−1) in 21 healthy subjects, (11 men 10 women, aged 29 ± 2 years) and measured the rate of incorporation of D5-phenylalanine or D3-leucine into muscle protein and blood and intramuscular amino acid concentrations. The relationship between the fold increase in MPS and blood essential amino acid concentration ([EAA], mM) was hyperbolic and fitted the equation MPS = (2.68 × [EAA])/(1.51 + [EAA]) (P < 0.01). The pattern of stimulation of myofibrillar, sarcoplasmic and mitochondrial protein was similar. There was no clear relationship between the rate of MPS and the concentration of intramuscular EAAs; indeed, when MPS was increasing most rapidly, the concentration of intramuscular EAAs was below basal levels. We conclude that the rates of synthesis of all classes of muscle proteins are acutely regulated by the blood [EAA] over their normal diurnal range, but become saturated at high concentrations. We propose that the stimulation of protein synthesis depends on the sensing of the concentration of extracellular, rather than intramuscular EAAs. PMID:12909668

  15. Extracellular matrix elasticity modulates TGF-β-induced p38 activation and myofibroblast transdifferentiation in human tenon fibroblasts.

    PubMed

    Meyer-ter-Vehn, Tobias; Han, Hong; Grehn, Franz; Schlunck, Günther

    2011-11-25

    Extracellular matrix and the cytokine TGF-β influence scar formation in an interdependent fashion. In this study, the impact of extracellular matrix elasticity on TGF-β-induced signal transduction and myofibroblast transdifferentiation was examined. Primary human tenon fibroblasts were seeded on collagen-coated glass coverslips (rigid environment) or collagen or polyacrylamide gels (elastic environment) of different compliance and stimulated with TGF-β. Myofibroblast transdifferentiation was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis for the marker gene α-smooth muscle actin (SMA), and SMA incorporation into stress fibers was determined by confocal immunofluorescence microscopy. CTGF transcription was assessed by RT-qPCR. Signaling pathways were examined by Western blot using phosphospecific antibodies and by immunofluorescence microscopy. TGF-β-dependent myofibroblast transdifferentiation was enhanced in a stiff environment. Increasing matrix elasticity attenuated TGF-β-induced myofibroblast transdifferentiation and the associated CTGF expression. TGF-β-induced p38 activation was reduced on elastic substrates. The results suggest that matrix elasticity influences TGF-β-dependent activation of p38 signaling and subsequent myofibroblast transdifferentiation. Biomechanical cues represent an important determinant of scarring processes. Therefore, cellular signals elicited by mechanotransduction deserve consideration in the design of novel antifibrotic strategies.

  16. Human resistin promotes neutrophil pro-inflammatory activation, neutrophil extracellular trap formation, and increases severity of acute lung injury

    PubMed Central

    Jiang, Shaoning; Park, Dae Won; Tadie, Jean-Marc; Gregoire, Murielle; Deshane, Jessy; Pittet, Jean Francois; Abraham, Edward; Zmijewski, Jaroslaw W.

    2014-01-01

    Although resistin was recently found to modulate insulin resistance in preclinical models of type II diabetes and obesity, recent studies have also suggested that resistin has proinflammatory properties. In these studies, we examined if the human specific variant of resistin affects neutrophil activation as well as the severity of LPS-induced acute lung injury (ALI). Because human and mouse resistin have distinct patterns of tissue distribution, experiments were performed using resistin humanized mice that exclusively express human resistin (hRTN+/−/−), but are deficient in mouse resistin. Enhanced production of TNF-α or MIP-2 was found in LPS-treated hRtn+/−/−, compared to control Rtn−/−/− neutrophils. Expression of human resistin inhibited the activation of AMP-activated protein kinase (AMPK), a major sensor and regulator of cellular bioenergetics that is also implicated in inhibiting inflammatory activity of neutrophils and macrophages. In addition to the ability of resistin to sensitize neutrophils to LPS stimulation, human resistin also enhanced neutrophil extracellular trap formation. In LPS-induced ALI, humanized resistin mice demonstrated enhanced production of pro-inflammatory cytokines, more severe pulmonary edema, increased NET formation, and elevated concentration of the alarmins HMGB1 and histone 3 in the lungs. Our results suggest that human resistin may play an important contributory role in enhancing TLR4 induced inflammatory responses, and may be a target for future therapies aimed at diminishing the severity of acute lung injury and other inflammatory situations where neutrophils play a major role. PMID:24719460

  17. Large-scale isolation and cytotoxicity of extracellular vesicles derived from activated human natural killer cells

    PubMed Central

    Jong, Ambrose Y.; Wu, Chun-Hua; Li, Jingbo; Sun, Jianping; Fabbri, Muller; Wayne, Alan S.; Seeger, Robert C.

    2017-01-01

    ABSTRACT Extracellular vesicles (EVs) have been the focus of great interest, as they appear to be involved in numerous important cellular processes. They deliver bioactive macromolecules such as proteins, lipids, and nucleic acids, allowing intercellular communication in multicellular organisms. EVs are secreted by all cell types, including immune cells such as natural killer cells (NK), and they may play important roles in the immune system. Currently, a large-scale procedure to obtain functional NK EVs is lacking, limiting their use clinically. In this report, we present a simple, robust, and cost-effective method to isolate a large quantity of NK EVs. After propagating and activating NK cells ex vivo and then incubating them in exosome-free medium for 48 h, EVs were isolated using a polymer precipitation method. The isolated vesicles contain the tetraspanin CD63, an EV marker, and associated proteins (fibronectin), but are devoid of cytochrome C, a cytoplasmic marker. Nanoparticle tracking analysis showed a size distribution between 100 and 200 nm while transmission electron microscopy imaging displayed vesicles with an oval shape and comparable sizes, fulfilling the definition of EV. Importantly, isolated EV fractions were cytotoxic against cancer cells. Furthermore, our results demonstrate for the first time that isolated activated NK (aNK) cell EVs contain the cytotoxic proteins perforin, granulysin, and granzymes A and B, incorporated from the aNK cells. Activation of caspase -3, -7 and -9 was detected in cancer cells incubated with aNK EVs, and caspase inhibitors blocked aNK EV-induced cytotoxicity, suggesting that aNK EVs activate caspase pathways in target cells. The ability to isolate functional aNK EVs on a large scale may lead to new clinical applications. Abbreviations: NK: natural killer cells; activated NK (aNK) cells; EVs: extracellular vesicles; ALL: acute lymphoblastic leukaemia; aAPC: artificial antigen-presenting cell; TEM: transmission

  18. Candida albicans SOD5 represents the prototype of an unprecedented class of Cu-only superoxide dismutases required for pathogen defense.

    PubMed

    Gleason, Julie E; Galaleldeen, Ahmad; Peterson, Ryan L; Taylor, Alexander B; Holloway, Stephen P; Waninger-Saroni, Jessica; Cormack, Brendan P; Cabelli, Diane E; Hart, P John; Culotta, Valeria Cizewski

    2014-04-22

    The human fungal pathogens Candida albicans and Histoplasma capsulatum have been reported to protect against the oxidative burst of host innate immune cells using a family of extracellular proteins with similarity to Cu/Zn superoxide dismutase 1 (SOD1). We report here that these molecules are widespread throughout fungi and deviate from canonical SOD1 at the primary, tertiary, and quaternary levels. The structure of C. albicans SOD5 reveals that although the β-barrel of Cu/Zn SODs is largely preserved, SOD5 is a monomeric copper protein that lacks a zinc-binding site and is missing the electrostatic loop element proposed to promote catalysis through superoxide guidance. Without an electrostatic loop, the copper site of SOD5 is not recessed and is readily accessible to bulk solvent. Despite these structural deviations, SOD5 has the capacity to disproportionate superoxide with kinetics that approach diffusion limits, similar to those of canonical SOD1. In cultures of C. albicans, SOD5 is secreted in a disulfide-oxidized form and apo-pools of secreted SOD5 can readily capture extracellular copper for rapid induction of enzyme activity. We suggest that the unusual attributes of SOD5-like fungal proteins, including the absence of zinc and an open active site that readily captures extracellular copper, make these SODs well suited to meet challenges in zinc and copper availability at the host-pathogen interface.

  19. Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium.

    PubMed Central

    Mason, S. J.; Paradiso, A. M.; Boucher, R. C.

    1991-01-01

    1 The role of extracellular nucleotides in regulation of ion transport activities (short circuit current, Isc) of human respiratory epithelia was studied. 2 Application of nucleotides to the apical or basolateral membrane of human nasal epithelium induced a concentration-dependent increase in Isc. 3 The rank order of potency of purine- or pyrimidine-induced changes in Isc of normal human nasal epithelium when applied to the apical membrane (UTP greater than or equal to ATP greater than ATP gamma S greater than 2MeSATP greater than ADP beta S much greater than beta gamma MeATP greater than or equal to alpha beta MeATP) or basolateral membrane (2MeSATP greater than UTP greater than ATP greater than ATP gamma S greater than alpha beta MeATP greater than beta gamma MeATP) is consistent with involvement of a P2 purinoceptor. A similar rank order of potencies was observed for nucleotide effects on intracellular calcium measured by Fura-2 fluorescence using microspectrofluorimetry. 4 Similar nucleotide potency in the regulation of ion transport and intracellular calcium in cystic fibrosis (CF) airway epithelium (UTP greater than or equal to ATP) was observed, suggesting purinoceptors might be used to stimulate ion transport processes that would promote hydration of airway secretions and facilitate their clearance from CF lungs. 5 These data provide evidence for the regulation of ion transport by P2 purinoceptors in normal and cystic fibrosis human airway epithelium. PMID:1718521

  20. Expression of extracellular matrix molecules typical of articular cartilage in the human scapholunate interosseous ligament

    PubMed Central

    Milz, S; Aktas, T; Putz, R; Benjamin, M

    2006-01-01

    The scapholunate interosseous ligament (SLIL) connects the scaphoid and lunate bones and plays a crucial role in carpal kinematics. Its rupture leads to carpal instability and impairment of radiocarpal joint function. As the ligament is one of the first structures affected in rheumatoid arthritis, we conducted an immunohistochemical study of cadaveric tissue to determine whether it contains known autoantigens for rheumatoid arthritis. We immunolabelled the ligament from one hand in 12 cadavers with monoclonal antibodies directed against a wide range of extracellular matrix (ECM) molecules associated with both fibrous and cartilaginous tissues. The labelling profile has also enabled us to comment on how the molecular composition of the ligament relates to its mechanical function. All regions of the ligament labelled for types I, III and VI collagens, chondroitin 4 and 6 sulphates, keratan sulphate, dermatan sulphate, versican, tenascin and cartilage oligomeric matrix protein (COMP). However, both entheses labelled strongly for type II collagen, aggrecan and link protein and were distinctly fibrocartilaginous. In some regions, the ligament attached to bone via a region of hyaline cartilage that was continuous with articular cartilage. Labelling for cartilage molecules in the midsubstance was most evident dorsally. We conclude that the SLIL has an ECM which is typical of other highly fibrocartilaginous ligaments that experience both tensile load and shear. The presence of aggrecan, link protein, COMP and type II collagen could explain why the ligament may be a target for autoantigenic destruction in some forms of rheumatoid arthritis. PMID:16761970

  1. Changes in vascular extracellular matrix composition during decidual spiral arteriole remodeling in early human pregnancy.

    PubMed

    Smith, Samantha D; Choudhury, Ruhul H; Matos, Patricia; Horn, James A; Lye, Stephen J; Dunk, Caroline E; Aplin, John D; Jones, Rebecca L; Harris, Lynda K

    2016-05-01

    Uterine spiral arteriole (SA) remodeling in early pregnancy involves a coordinated series of events including decidual immune cell recruitment, vascular cell disruption and loss, and colonization by placental-derived extravillous trophoblast (EVT). During this process, decidual SA are converted from narrow, muscular vessels into dilated channels lacking vasomotor control. We hypothesized that this extensive alteration in SA architecture must require significant reorganization and/or breakdown of the vascular extracellular matrix (ECM). First trimester decidua basalis (30 specimens) was immunostained to identify spiral arterioles undergoing trophoblast-independent and -dependent phases of remodeling. Serial sections were then immunostained for a panel of ECM markers, to examine changes in vascular ECM during the remodeling process. The initial stages of SA remodeling were characterized by loss of laminin, elastin, fibrillin, collagen types III, IV and VI from the basement membrane, vascular media and/or adventitia, and surrounding decidual stromal cells. Loss of ECM correlated with disruption and disorganization of vascular smooth muscle cells, and the majority of changes occurred prior to extensive colonization of the vessel wall by EVT. The final stages of SA remodeling, characterized by the arrival of EVT, were associated with the increased mural deposition of fibronectin and fibrinoid. This study provides the first detailed analysis of the spatial and temporal loss of ECM from the walls of remodeling decidual SA in early pregnancy.

  2. Predicting total body water and extracellular fluid volumes from bioelectrical measurements of the human body.

    PubMed

    Johnson, H L; Virk, S P; Mayclin, P; Barbieri, T

    1992-10-01

    Two biological impedance analyzers, a 50 kHz (RJL) and 20-100 kHz (BMA) instrument, and a total body electrical conductivity (TOBEC) instrument were used to estimate total body water (TBW), extracellular (ECF) and intracellular (ICF) fluid volumes by repeated measurements of 16 normal men (19-38 years old) to assess which, if any, would provide the best estimates. At 3-week intervals, TBW was determined by deuterium dilution, ECF by bromide dilution, ICF by difference (TBW-ECF) and lean body mass by density. Prediction equations were obtained by regression; predicted values for the body fluid volumes were calculated and the results were statistically evaluated. Both the TOBEC and the BMA provided rapid and reliable estimates for body fluid volumes with standard errors of the estimates of about 0.5-1.1 L for ECF, 1.0-1.8 L for TBW, and 1.0-1.3 L for ICF. Part of the error was attributable to standard tracer-dilution methods.

  3. Manufacturing of Human Extracellular Vesicle-Based Therapeutics for Clinical Use

    PubMed Central

    Gimona, Mario; Pachler, Karin; Laner-Plamberger, Sandra; Schallmoser, Katharina; Rohde, Eva

    2017-01-01

    Extracellular vesicles (EVs) derived from stem and progenitor cells may have therapeutic effects comparable to their parental cells and are considered promising agents for the treatment of a variety of diseases. To this end, strategies must be designed to successfully translate EV research and to develop safe and efficacious therapies, whilst taking into account the applicable regulations. Here, we discuss the requirements for manufacturing, safety, and efficacy testing of EVs along their path from the laboratory to the patient. Development of EV-therapeutics is influenced by the source cell types and the target diseases. In this article, we express our view based on our experience in manufacturing biological therapeutics for routine use or clinical testing, and focus on strategies for advancing mesenchymal stromal cell (MSC)-derived EV-based therapies. We also discuss the rationale for testing MSC-EVs in selected diseases with an unmet clinical need such as critical size bone defects, epidermolysis bullosa and spinal cord injury. While the scientific community, pharmaceutical companies and clinicians are at the point of entering into clinical trials for testing the therapeutic potential of various EV-based products, the identification of the mode of action underlying the suggested potency in each therapeutic approach remains a major challenge to the translational path. PMID:28587212

  4. Expression of extracellular matrix molecules typical of articular cartilage in the human scapholunate interosseous ligament.

    PubMed

    Milz, S; Aktas, T; Putz, R; Benjamin, M

    2006-06-01

    The scapholunate interosseous ligament (SLIL) connects the scaphoid and lunate bones and plays a crucial role in carpal kinematics. Its rupture leads to carpal instability and impairment of radiocarpal joint function. As the ligament is one of the first structures affected in rheumatoid arthritis, we conducted an immunohistochemical study of cadaveric tissue to determine whether it contains known autoantigens for rheumatoid arthritis. We immunolabelled the ligament from one hand in 12 cadavers with monoclonal antibodies directed against a wide range of extracellular matrix (ECM) molecules associated with both fibrous and cartilaginous tissues. The labelling profile has also enabled us to comment on how the molecular composition of the ligament relates to its mechanical function. All regions of the ligament labelled for types I, III and VI collagens, chondroitin 4 and 6 sulphates, keratan sulphate, dermatan sulphate, versican, tenascin and cartilage oligomeric matrix protein (COMP). However, both entheses labelled strongly for type II collagen, aggrecan and link protein and were distinctly fibrocartilaginous. In some regions, the ligament attached to bone via a region of hyaline cartilage that was continuous with articular cartilage. Labelling for cartilage molecules in the midsubstance was most evident dorsally. We conclude that the SLIL has an ECM which is typical of other highly fibrocartilaginous ligaments that experience both tensile load and shear. The presence of aggrecan, link protein, COMP and type II collagen could explain why the ligament may be a target for autoantigenic destruction in some forms of rheumatoid arthritis.

  5. Manufacturing of Human Extracellular Vesicle-Based Therapeutics for Clinical Use.

    PubMed

    Gimona, Mario; Pachler, Karin; Laner-Plamberger, Sandra; Schallmoser, Katharina; Rohde, Eva

    2017-06-03

    Extracellular vesicles (EVs) derived from stem and progenitor cells may have therapeutic effects comparable to their parental cells and are considered promising agents for the treatment of a variety of diseases. To this end, strategies must be designed to successfully translate EV research and to develop safe and efficacious therapies, whilst taking into account the applicable regulations. Here, we discuss the requirements for manufacturing, safety, and efficacy testing of EVs along their path from the laboratory to the patient. Development of EV-therapeutics is influenced by the source cell types and the target diseases. In this article, we express our view based on our experience in manufacturing biological therapeutics for routine use or clinical testing, and focus on strategies for advancing mesenchymal stromal cell (MSC)-derived EV-based therapies. We also discuss the rationale for testing MSC-EVs in selected diseases with an unmet clinical need such as critical size bone defects, epidermolysis bullosa and spinal cord injury. While the scientific community, pharmaceutical companies and clinicians are at the point of entering into clinical trials for testing the therapeutic potential of various EV-based products, the identification of the mode of action underlying the suggested potency in each therapeutic approach remains a major challenge to the translational path.

  6. Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

    SciTech Connect

    Manes, Nathan P.; Estep, Ryan D.; Mottaz, Heather M.; Moore, Ronald J.; Clauss, Therese RW; Monroe, Matthew E.; Du, Xiuxia; Adkins, Joshua N.; Wong, Scott; Smith, Richard D.

    2008-03-07

    Orthopoxviruses are the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, virulent (monkeypox virus) and benign (vaccinia virus) orthopoxviruses were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest™ surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by reversed-phase LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST® and X! Tandem resulted in the identification of hundreds of monkeypox, vaccinia, and copurified host proteins. The unfractionated samples were additionally analyzed by LC-MS on an LTQ-Orbitrap™, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially expressed orthopoxvirus genes are discussed.

  7. Loop Dynamics of the Extracellular Domain of Human Tissue Factor and Activation of Factor VIIa

    PubMed Central

    Minazzo, Agnese S.; Darlington, Reuben C.; Ross, J.B. Alexander

    2009-01-01

    Abstract In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and active-site-inhibited VIIa, residues 91 and 92 in the Pro79-Pro92 loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu84 resides in the Pro79-Pro92 loop, and Thr121 resides in the turn between the first and second antiparallel β-strands of the sTF subdomain that interacts with the Gla and EGF1 domains of VIIa; neither Glu84 nor Thr121 makes direct contact with VIIa. Probes bound to T121C report limited segmental flexibility in free sTF, which is lost after VIIa binding. Probes bound to E84C report substantial segmental flexibility in the Pro79-Pro92 loop in free sTF, which is greatly reduced after VIIa binding. Thus, VIIa binding reduces dynamic motions in sTF. In particular, the decrease in the Pro79-Pro92 loop motions indicates that loop entropy has a role in the thermodynamics of the protein-protein interactions involved in allosteric control of VIIa activation. PMID:19167313

  8. HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

    PubMed

    González, Mariela Natacha; de Mello, Wallace; Butler-Browne, Gillian S; Silva-Barbosa, Suse Dayse; Mouly, Vincent; Savino, Wilson; Riederer, Ingo

    2017-10-10

    The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving

  9. Production of extracellular matrix proteins by human pulp fibroblasts in contact with papacárie and carisolv.

    PubMed

    Bussadori, Sandra Kalil; Amancio, Olga Maria; Martins, Manoela Domingues; Guedes, Carolina Cardoso; Alfaya, Thays Almeida; Santos, Elaine Marcílio; França, Cristiane Miranda

    2014-01-01

    To evaluate the effect of two products for the chemomecanical removal of carious tissue (Papacárie and Carisolv) on human dental pulp fibroblasts and the synthesis of extracellular matrix proteins. Fibroblasts were divided into three groups: group 1 (control), group 2 (Papacárie) and group 3 (Carisolv). Collagen I, III , fibronectin and osteonectin were analysed by immunofluorescence and compared among the groups. The groups exhibited similar immunolabeling for vimentin, type I collagen and fibronectin, but were negative for type III collagen. Osteonectin staining was strongly positive in the cells treated with Papacárie and Carisolv and weakly positive in the control group. The findings of the present study showed that Papacárie and Carisolv are not cytotoxic to pulp fibroblast cells. Moreover, these products stimulate fibroblasts to produce osteonectin, likely leading to the formation of dentin matrix. These findings confirm the safe, beneficial use of both gels in minimally invasive techniques.

  10. A Lipid Mediator Hepoxilin A3 Is a Natural Inducer of Neutrophil Extracellular Traps in Human Neutrophils

    PubMed Central

    Douda, David N.; Grasemann, Hartmut; Pace-Asciak, Cecil

    2015-01-01

    Pulmonary exacerbations in cystic fibrosis airways are accompanied by inflammation, neutrophilia, and mucous thickening. Cystic fibrosis sputum contains a large amount of uncleared DNA contributed by neutrophil extracellular trap (NET) formation from neutrophils. The exact mechanisms of the induction of NETosis in cystic fibrosis airways remain unclear, especially in uninfected lungs of patients with early cystic fibrosis lung disease. Here we show that Hepoxilin A3, a proinflammatory eicosanoid, and the synthetic analog of Hepoxilin B3, PBT-3, directly induce NETosis in human neutrophils. Furthermore, we show that Hepoxilin A3-mediated NETosis is NADPH-oxidase-dependent at lower doses of Hepoxilin A3, while it is NADPH-oxidase-independent at higher doses. Together, these results demonstrate that Hepoxilin A3 is a previously unrecognized inducer of NETosis in cystic fibrosis lungs and may represent a new therapeutic target for treating cystic fibrosis and other inflammatory lung diseases. PMID:25784781

  11. Iron-chelating agent desferrioxamine stimulates formation of neutrophil extracellular traps (NETs) in human blood-derived neutrophils

    PubMed Central

    Völlger, Lena; Akong-Moore, Kathryn; Cox, Linda; Goldmann, Oliver; Wang, Yanming; Schäfer, Simon T.; Naim, Hassan Y.; Nizet, Victor; von Köckritz-Blickwede, Maren

    2016-01-01

    Neutrophil extracellular trap (NET) formation is a significant innate immune defense mechanism against microbial infection that complements other neutrophil functions including phagocytosis and degranulation of antimicrobial peptides. NETs are decondensed chromatin structures in which antimicrobial components (histones, antimicrobial peptides and proteases) are deployed and mediate immobilization of microbes. Here we describe an effect of iron chelation on the phenotype of NET formation. Iron-chelating agent desferrioxamine (DFO) showed a modest but significant induction of NETs by freshly isolated human neutrophils as visualized and quantified by immunocytochemistry against histone–DNA complexes. Further analyses revealed that NET induction by iron chelation required NADPH-dependent production of reactive oxygen species (ROS) as well as protease and peptidyl-arginine-deiminase 4 (PAD4) activities, three key mechanistic pathways previously linked to NET formation. Our results demonstrate that iron chelation by DFO contributes to the formation of NETs and suggest a target for pharmacological manipulation of NET activity. PMID:27129288

  12. Integrin-mediated Ras–Extracellular Regulated Kinase (ERK) Signaling Regulates Interferon γ Production in Human Natural Killer Cells

    PubMed Central

    Mainiero, Fabrizio; Gismondi, Angela; Soriani, Alessandra; Cippitelli, Marco; Palmieri, Gabriella; Jacobelli, Jordan; Piccoli, Mario; Frati, Luigi; Santoni, Angela

    1998-01-01

    Recent evidence indicates that integrin engagement results in the activation of biochemical signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that β1 integrin ligation on human natural killer (NK) cells results in the activation of Ras/mitogen-activated protein kinase pathways. Formation of Shc–growth factor receptor–bound protein 2 (Grb2) and Shc–proline-rich tyrosine kinase 2–Grb2 complexes are the receptor-proximal events accompanying the β1 integrin–mediated Ras activation. In addition, we demonstrate that ligation of β1 integrins results in the stimulation of interferon γ (IFN-γ) production, which is under the control of extracellular signal–regulated kinase 2 activation. Overall, our data indicate that β1 integrins, by delivering signals capable of triggering IFN-γ production, may function as NK-activating receptors. PMID:9763606

  13. Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin.

    PubMed

    Bae, Jung-Soo; Han, Mira; Shin, Hee Soon; Kim, Min-Kyoung; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

    2017-01-04

    Perilla frutescens (L.) Britt. (Lamiaceae) is a traditional herb that is consumed in East Asian countries as a traditional medicine. This traditional herb has been documented for centuries to treat various diseases such as depression, allergies, inflammation and asthma. However, the effect of Perilla frutescens on skin has not been characterized well. The present study aimed to investigate the effect of Perilla frutescens leaves extract (PLE) on ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin. Human dermal fibroblasts and Skh-1 hairless mice were irradiated with UV and treated with PLE. Protein and mRNA levels of various target molecules were analyzed by western blotting and quantitative RT-PCR, respectively. Histological changes of mouse skin were analyzed by H&E staining. To elucidate underlying mechanism of PLE, activator protein-1 (AP-1) DNA binding assay and the measurement of reactive oxygen species (ROS) were performed. PLE significantly inhibited basal and UV-induced MMP-1 and MMP-3 expression dose-dependently, and also decreased UV-induced phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinases. This inhibitory effects of PLE on MMP-1 and MMP-3 were mediated by reduction of ROS generation and AP-1 DNA binding activity induced by UV. Furthermore, PLE promoted type I procollagen production irrespective of UV irradiation. In the UV-irradiated animal model, PLE significantly reduced epidermal skin thickness and MMP-13 expression induced by UV. Our results demonstrate that PLE has the protective effect against UV-induced dermal matrix damage. Therefore, we suggest that PLE can be a potential agent for prevention of skin aging. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Comparison of four decontamination treatments on porcine renal decellularized extracellular matrix structure, composition, and support of human renal cortical tubular epithelium cells.

    PubMed

    Poornejad, Nafiseh; Nielsen, Jeffery J; Morris, Ryan J; Gassman, Jason R; Reynolds, Paul R; Roeder, Beverly L; Cook, Alonzo D

    2016-03-01

    Engineering whole organs from porcine decellularized extracellular matrix and human cells may lead to a plentiful source of implantable organs. Decontaminating the porcine decellularized extracellular matrix scaffolds is an essential step prior to introducing human cells. However, decontamination of whole porcine kidneys is a major challenge because the decontamination agent or irradiation needs to diffuse deep into the structure to eliminate all microbial contamination while minimizing damage to the structure and composition of the decellularized extracellular matrix. In this study, we compared four decontamination treatments that could be applicable to whole porcine kidneys: 70% ethanol, 0.2% peracetic acid in 1 M NaCl, 0.2% peracetic acid in 4% ethanol, and gamma (γ)-irradiation. Porcine kidneys were decellularized by perfusion of 0.5% (w/v) aqueous solution of sodium dodecyl sulfate and the four decontamination treatments were optimized using segments (n = 60) of renal tissue to ensure a consistent comparison. Although all four methods were successful in decontamination, γ-irradiation was very damaging to collagen fibers and glycosaminoglycans, leading to less proliferation of human renal cortical tubular epithelium cells within the porcine decellularized extracellular matrix. The effectiveness of the other three optimized solution treatments were then all confirmed using whole decellularized porcine kidneys (n = 3). An aqueous solution of 0.2% peracetic acid in 1 M NaCl was determined to be the best method for decontamination of porcine decellularized extracellular matrix.

  15. Growth Differentiation Factor-15–Induced Contractile Activity and Extracellular Matrix Production in Human Trabecular Meshwork Cells

    PubMed Central

    Muralidharan, Arumugam Ramachandran; Maddala, Rupalatha; Skiba, Nikolai P.; Rao, Ponugoti Vasantha

    2016-01-01

    Purpose To determine the role and regulation of growth differentiation factor-15 (GDF-15), a TGF-β–related cytokine in human trabecular meshwork (TM) cells in the context of aqueous humor (AH) outflow and IOP. Methods Regulation of expression by external cues, and the distribution and secretion of GDF-15 by human TM primary cell cultures, and the effects of recombinant (r) GDF-15 on TM cell contractile characteristics, actin cytoskeleton, cell adhesion, extracellular matrix (ECM), α-smooth muscle actin (αSMA), SMAD signaling, and gene expression were determined by immunoblot, immunofluorescence, mass spectrometry, cDNA microarray, and real-time quantitative PCR (RT-qPCR) analyses. Results Growth differentiation factor-15, a common constituent of ECM derived from the human TM cells, was confirmed to be distributed throughout the conventional aqueous humor outflow pathway of the human eye. Growth differentiation factor-15 protein levels were significantly increased in human TM cells in response to TGF-β2, dexamethasone, endothelin-1, lysophosphatidic acid, TNF-α, IL-1β treatment, and by cyclic mechanical stretch. Stimulation of human TM cells with rGDF-15 caused a significant increase in the formation of actin stress fibers and focal adhesions, myosin light chain phosphorylation, SMAD signaling, gene expression, and the levels of αSMA and ECM proteins. Conclusions The results of this study, including a robust induction of GDF-15 expression by several external factors known to elevate IOP, and rGDF-15–induced increase in contractility, cell adhesion, and the levels of ECM proteins and αSMA in TM cells, collectively suggest a potential role for GDF-15 in homeostasis and dysregulation of AH outflow and IOP in normal and glaucomatous eyes, respectively. PMID:27918822

  16. Regulation of superoxide dismutase synthesis in Candida albicans.

    PubMed

    Gunasekaran, U; Yang, R; Gunasekaran, M

    1998-01-01

    The synthesis of superoxide dismutase [SOD: EC 1.15.1.1] in response to various cultural conditions was examined in Candida albicans, an opportunistic yeast which causes candidiasis in immunosuppressed patients. SOD plays an important role in protecting cells from teh oxidative damage of superoxide radicals. Maximum SOD activity was found after 72 hrs of yeast growth. The optimum pH and temperature for the SOD activity were 7 and 40 degrees C, respectively. The major SOD activity was found in the cytosol fraction and the level of extracellular SOD was very low. The enzyme was stimulated to varying degrees by cholic acid, procaine and tocopherol. On the basis of inhibitor studies and other enzyme properties, the isolated enzyme from C. albicans is identified as a copper and zinc superoxide dismutase.

  17. Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix.

    PubMed

    Gründel, Anne; Jacobs, Enno; Dumke, Roger

    2016-12-01

    Mycoplasma pneumoniae is a major cause of community-acquired respiratory infections worldwide. Due to the strongly reduced genome, the number of virulence factors expressed by this cell wall-less pathogen is limited. To further understand the processes during host colonization, we investigated the interactions of the previously confirmed surface-located glycolytic enzymes of M. pneumoniae (pyruvate dehydrogenase A-C [PdhA-C], glyceraldehyde-3-phosphate dehydrogenase [GapA], lactate dehydrogenase [Ldh], phosphoglycerate mutase [Pgm], pyruvate kinase [Pyk] and transketolase [Tkt]) to the human extracellular matrix (ECM) proteins fibrinogen (Fn), fibronectin (Fc), lactoferrin (Lf), laminin (Ln) and vitronectin (Vc), respectively. Concentration-dependent interactions between Fn and Vc and all eight recombinant proteins derived from glycolytic enzymes, between Ln and PdhB-C, GapA, Ldh, Pgm, Pyk and Tkt, between Lf and PdhA-C, GapA and Pyk, and between Fc and PdhC and GapA were demonstrated. In most cases, these associations are significantly influenced by ionic forces and by polyclonal sera against recombinant proteins. In immunoblotting, the complex of human plasminogen, activator (tissue-type or urokinase plasminogen activator) and glycolytic enzyme was not able to degrade Fc, Lf and Ln, respectively. In contrast, degradation of Vc was confirmed in the presence of all eight enzymes tested. Our data suggest that the multifaceted associations of surface-localized glycolytic enzymes play a potential role in the adhesion and invasion processes during infection of human respiratory mucosa by M. pneumoniae.

  18. Extracellular-like matrices and leukaemia inhibitory factor for in vitro culture of human primordial follicles.

    PubMed

    Younis, Assiel J; Lerer-Serfaty, Galit; Stav, Dana; Sabbah, Bethsabee; Shochat, Tzippy; Kessler-Icekson, Gania; Zahalka, Muayad A; Shachar-Goldenberg, Michal; Ben-Haroush, Avi; Fisch, Benjamin; Abir, Ronit

    2017-02-01

    The possibility of maturing human primordial follicles in vitro would assist fertility restoration without the danger of reseeding malignancies. Leukaemia inhibitory factor (LIF) and certain culture matrices may promote human follicular growth. The present study compared human primordial follicular growth on novel culture matrices, namely human recombinant vitronectin (hrVit), small intestine submucosa (SIS), alginate scaffolds and human recombinant virgin collagen bioengineered in tobacco plant lines (CollPlant). The frozen-thawed ovarian samples that were used had been obtained from girls or young women undergoing fertility preservation. In the first part of the study, 20 samples were cultured for 6 days on hrVit or SIS with basic culture medium alone or supplemented with one of two concentrations of LIF (10ngmL-1 and 100ngmL-1), with and without LIF-neutralising antibody. In the second part of the study, 15 samples were cultured for 6 days on alginate scaffolds or CollPlant matrices with basic culture medium. Follicular development was assessed by follicular counts and classification, Ki67 immunohistochemistry and 17β-oestradiol and anti-Müllerian hormone measurements in spent media samples. Primordial follicular growth was not enhanced by LIF. Despite some significant differences among the four matrices, none appeared to have a clear advantage, apart from significantly more Ki67-stained follicles on alginate and CollPlant matrices. Further studies of other culture matrices and medium supplements are needed to obtain an optimal system.

  19. Effect of ion concentration changes in the limited extracellular spaces on sarcolemmal ion transport and Ca2+ turnover in a model of human ventricular cardiomyocyte.

    PubMed

    Hrabcová, Dana; Pásek, Michal; Šimurda, Jiří; Christé, Georges

    2013-12-13

    We have developed a computer model of human cardiac ventricular myocyte (CVM), including t-tubular and cleft spaces with the aim of evaluating the impact of accumulation-depletion of ions in restricted extracellular spaces on transmembrane ion transport and ionic homeostasis in human CVM. The model was based on available data from human CVMs. Under steady state, the effect of ion concentration changes in extracellular spaces on [Ca2+]i-transient was explored as a function of critical fractions of ion transporters in t-tubular membrane (not documented for human CVM). Depletion of Ca2+ and accumulation of K+ occurring in extracellular spaces slightly affected the transmembrane Ca2+ flux, but not the action potential duration (APD90). The [Ca2+]i-transient was reduced (by 2%-9%), depending on the stimulation frequency, the rate of ion exchange between t-tubules and clefts and fractions of ion-transfer proteins in the t-tubular membrane. Under non-steady state, the responses of the model to changes of stimulation frequency were analyzed. A sudden increase of frequency (1-2.5 Hz) caused a temporal decrease of [Ca2+] in both extracellular spaces, a reduction of [Ca2+]i-transient (by 15%) and APD90 (by 13 ms). The results reveal different effects of activity-related ion concentration changes in human cardiac t-tubules (steady-state effects) and intercellular clefts (transient effects) in the modulation of membrane ion transport and Ca2+ turnover.

  20. Manganese Superoxide Dismutase Protects against 6-Hydroxydopamine Injury in Mouse Brains*

    PubMed Central

    Callio, Jason; Oury, Tim D.; Chu, Charleen T.

    2007-01-01

    Dopaminergic neurons of the substantia nigra are susceptible to toxin-based insults. Intrastriatal injection of 6-hydroxydopamine results in selective toxicity to these neurons. A mechanistic role for reactive oxygen species is supported by observations that antioxidants confer protection from 6-hydroxydopamine. Although cell culture studies have suggested extracellular or nonmitochondrial mechanisms in 6-hydroxydopamine toxicity, the compartmentalization of oxidative injury mechanisms is incompletely defined in vivo. Transgenic mice overexpressing mitochondrial manganese superoxide dismutase or extracellular superoxide dismutase received unilateral intrastriatal injections of 6-hydroxydopamine. Mice that overexpress manganese superoxide dismutase showed significantly smaller striatal lesions than littermate controls. There were no differences in nonspecific striatal injury associated with contralateral vehicle injection. Manganese superoxide dismutase overexpression also protected against loss of neuronal cell bodies in the substantia nigra. In contrast, mice overexpressing extracellular superoxide dismutase showed no protection from 6-hydroxydopamine toxicity in either brain region. Protection of the nigrostriatal system by overexpression of manganese super-oxide dismutase supports a role for mitochondrially derived superoxide in 6-hydroxydopamine toxicity. Mitochondrial oxidative stress appears to be a common mechanism among diverse models of Parkinson disease, whether involving toxins, mutated genes, or cybrid cells containing patient mitochondria. Antioxidant therapies that target this subcellular compartment may prove promising. PMID:15755737

  1. BPA‐toxicity via superoxide anion overload and a deficit in β‐catenin signaling in human bone mesenchymal stem cells

    PubMed Central

    Oh, Seikwan; Kang, Hong‐Je; Kim, Jung‐Hwa; Yoon, Juno

    2016-01-01

    ABSTRACT Bisphenol A (BPA), used in the manufacture of products based on polycarbonate plastics and epoxy resins, is well known as an endocrine‐disrupting monomer. In the current study, BPA increased cytotoxicity in hBMSCs in a dose‐ and time‐dependent manner, concomitantly with increased lipid peroxidation. Increased cell death in BPA‐treated cells was markedly blocked by pretreatment with the superoxide dismutase mimetic MnTBAP and MnTMPyP, but not by catalase, glutathione, the glutathione peroxidase mimetic ebselen, the NOS inhibitor NAME, or the xanthine oxidase inhibitor allopurinol. Furthermore, the decline in nuclear β‐catenin and cyclin D1 levels in hBMSCs exposed to BPA was reversed by MnTBAP treatment. Finally, treatment of hBMSCs with the GSK3β inhibitor LiCl2 increased nuclear β‐catenin levels and significantly attenuated cytotoxicity compared with BPA treatment. Our current results in hBMSCs exposed to BPA suggest that BPA causes a disturbance in β‐catenin signaling via a superoxide anion overload. © 2016 The Authors Environmental Toxicology Published by Wiley Periodicals, Inc. Environ Toxicol 32: 344–352, 2017. PMID:26822619

  2. BPA-toxicity via superoxide anion overload and a deficit in β-catenin signaling in human bone mesenchymal stem cells.

    PubMed

    Leem, Yea-Hyun; Oh, Seikwan; Kang, Hong-Je; Kim, Jung-Hwa; Yoon, Juno; Chang, Jae-Suk

    2017-01-01

    Bisphenol A (BPA), used in the manufacture of products based on polycarbonate plastics and epoxy resins, is well known as an endocrine-disrupting monomer. In the current study, BPA increased cytotoxicity in hBMSCs in a dose- and time-dependent manner, concomitantly with increased lipid peroxidation. Increased cell death in BPA-treated cells was markedly blocked by pretreatment with the superoxide dismutase mimetic MnTBAP and MnTMPyP, but not by catalase, glutathione, the glutathione peroxidase mimetic ebselen, the NOS inhibitor NAME, or the xanthine oxidase inhibitor allopurinol. Furthermore, the decline in nuclear β-catenin and cyclin D1 levels in hBMSCs exposed to BPA was reversed by MnTBAP treatment. Finally, treatment of hBMSCs with the GSK3β inhibitor LiCl2 increased nuclear β-catenin levels and significantly attenuated cytotoxicity compared with BPA treatment. Our current results in hBMSCs exposed to BPA suggest that BPA causes a disturbance in β-catenin signaling via a superoxide anion overload. © 2016 The Authors Environmental Toxicology Published by Wiley Periodicals, Inc. Environ Toxicol 32: 344-352, 2017.

  3. In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps.

    PubMed

    Noubouossie, Denis F; Whelihan, Matthew F; Yu, Yuan-Bin; Sparkenbaugh, Erica; Pawlinski, Rafal; Monroe, Dougald M; Key, Nigel S

    2017-02-23

    NETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins. © 2017 by The American Society of Hematology.

  4. Impact of cyclic mechanical stimulation on the expression of extracellular matrix proteins in human primary rotator cuff fibroblasts.

    PubMed

    Lohberger, Birgit; Kaltenegger, Heike; Stuendl, Nicole; Rinner, Beate; Leithner, Andreas; Sadoghi, Patrick

    2016-12-01

    Mechanical stimulation plays an important role in the development and remodelling of tendons. The aim of the study was to evaluate the effects of mechanical stimulation on the expression of extracellular matrix proteins in human primary rotator cuff (RC) fibroblasts. RC fibroblasts were isolated from patients with degenerative RC tears and characterized using flow cytometry and immunohistochemistry. Cells were stimulated using the Flexcell FX5K™ Tension System. The stimulation regime was a uniaxial sinusoidal waveform with 10 % elongation and a frequency of 0.5 Hz, whereby each cycle consists of 10-s strain and 30-s relaxation. Data were normalized to mechanically unstimulated control groups for every experimental condition. RT-qPCR was performed to determine relative mRNA levels, and collagen production was measured by a colorimetric assay. The positive expression of CD91 and CD10, and negativity for CD45 and CD4 confirmed the fibroblast phenotype of RC primary cells. RT-qPCR revealed that 10 % continuous cyclic strain for 7 and 14 days induced a significant increase in the mRNA expression both on the matrix metalloproteinases MMP1, MMP3, MMP13, and MMP14 and on the extracellular matrix proteins decorin, tenascin-C, and scleraxis. Furthermore, mechanically stimulated groups produced significantly higher amounts of total collagen. These results may contribute to a better understanding of strain-induced tendon remodelling and will form the basis for the correct choice of applied force in rehabilitation after orthopaedic surgery. These findings underline the fact that early passive motion of the joint in order to induce remodelling of the tendon should be included within a rehabilitation protocol for rotator cuff repair.

  5. Effect of extracellular glucose and K+ on intracellular osmolytes and volume in a human kidney cell line.

    PubMed

    Hubert, Marcy D; Indyk, Elisabeth; Pena-Rasgado, Cecilia; Pierce, Sydney K; Rasgado-Flores, Hector; Garber, Sarah S

    2007-05-01

    The goal of this study was to assess the effect of extracellular glucose and K+ ((K)o) on the intracellular osmolyte content and cell volume maintenance and regulation in a human embryonic kidney cell line (tsA201a). Cell volume maintenance was studied by isotonic (313 +/- 5 mOsm) replacement of culture media by a glucose-free Ringer solution containing (in mM) 0, 3, 6, or 10 K+. Cell volume regulation was studied by exposing cells to hypotonic (250 +/- 5 mOsm) glucose-free Ringer solution containing the various (K)o. The results showed that: 1) intracellular osomlyte content (i.e. Na+, Cl-, Urea and free amino acids (FAA)) and cell volume increased when culture media was replaced with isotonic Ringer at all (K)o; 2) osmolyte content decreased with continuous exposure to isotonic Ringer at all (K)o but cell volume changes depended on (K)o. Volume recovery occurred at 6 and 10 mM K+; 3) exposure to hypotonic Ringer induced swelling at all (K)o followed by a reduction in measured intracellular osmolytes. Regulatory volume decrease occurred in 6 or 10 mM K+ but swelling continued in 0 or 3 mM K+; and 4) addition of ouabain produced swelling without recovery under iso- and hypotonic conditions. These results indicate that the removal of extracellular glucose produced a transient inhibition of the Na+/K+ ATPase resulting in a transient increase in the intracellular content of Urea, FAA and cell volume and (K)o regulated an as yet unidentified intracellular osmolyte.

  6. Decellularized extracellular matrix of human umbilical vein endothelial cells promotes endothelial differentiation of stem cells from exfoliated deciduous teeth.

    PubMed

    Gong, Ting; Heng, Boon Chin; Xu, Jianguang; Zhu, Shaoyue; Yuan, Changyong; Lo, Edward Chin Man; Zhang, Chengfei

    2017-04-01

    Dental stem cells can serve as a potential source of functional endothelial cells for tissue engineering applications, but the endothelial-lineage differentiation efficiency is rather low even with growth factors and mechanical stimuli, which greatly limits their clinical applications. This is partly due to the deficiency of standard two-dimensional (2-D) culture systems, which is unable to recapitulate the three-dimensional (3-D) in vivo milieu that is rich in extracellular matrix. Hence, we extracted decellularized extracellular matrix from human umbilical vein endothelial cells (HUVECs-DECM) to provide a bioactive substratum conducive to the endothelial differentiation of dental stem cells. Compared to cells plated on tissue culture polystyrene (TCP), stem cells from exfoliated deciduous teeth (SHED) cultured on the HUVECs-DECM demonstrated more regular arrangement and elongated morphology. HUVECs-DECM significantly enhanced the rapid adhesion and proliferation rates of SHED, as demonstrated by WST-8 assay and immunocytochemistry indicating higher expression levels of vinculin by newly adherent SHED on HUVECs-DECM versus TCP. In addition, there was twofold to fivefold higher mRNA expression levels of endothelial-specific markers CD31 and VEGFR-2 in SHED after seven days of culture on DECM versus TCP. Functional testing with in vitro matrigel angiogenesis assay identified more capillary-like structure formation with significantly higher tubule length in SHED induced by DECM versus TCP. Hence, the results of this study provide a better understanding of the unique characteristics of cell-specific ECM and demonstrated the potential use of HUVECs-DECM as a culture substratum conducive for stimulating the endothelial differentiation of SHED for therapeutic angiogenic applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1083-1093, 2017.

  7. Role of Age-Associated Alterations of the Dermal Extracellular Matrix Microenvironment in Human Skin Aging

    PubMed Central

    Quan, Taihao; Fisher, Gary J

    2015-01-01

    Human skin is largely composed of a collagen-rich connective tissue, which provides structural and functional support. The collagen-rich connective tissue is produced, organized, and maintained by dermal fibroblasts. During aging, dermal collagen fibrils undergo progressive loss and fragmentation, leading to thin and structurally weakened skin. Age-related alterations of collagen fibrils impairs skin structure and function and creates a tissue microenvironment that promotes age-related skin diseases, such as delayed wound healing and skin cancer development. This review describes cellular mechanisms that give rise to self-perpetuating, collagen fibril fragmentation that creates an age-associated dermal microenvironment (AADM), which contributes to decline of human skin function. PMID:25660807

  8. Microdialysis in the human brain: extracellular measurements in the thalamus of parkinsonian patients.

    PubMed

    Meyerson, B A; Linderoth, B; Karlsson, H; Ungerstedt, U

    1990-01-01

    Microdialysis in the human brain has been performed for the first time during thalamotomy intended to relieve tremor in patients with Parkinson's disease. The aim was to test the reliability of the microdialysis technique for biochemical characterization of a target area in the human brain during a routine operation. Microdialysis probes were introduced through the same trajectory as the lesioning electrode thus causing no additional damage to the brain. Dopamine, DOPAC, HVA, 5-HIAA, hypoxanthine, inosine, guanosine, adenosine, GABA, taurine, aspartate and glutamate were measured in the perfusate from the target region - the Vim nucleus. The results show initial high levels that reach baseline levels after 10-20 minutes. Surprisingly, consistent and reproducible levels were found, the only exception being one patient on 1-DOPA therapy who had elevated DA and metabolite levels.

  9. Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

    PubMed Central

    Coopman, P J; Thomas, D M; Gehlsen, K R; Mueller, S C

    1996-01-01

    The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3

  10. Superoxide dismutases in chronic gastritis.

    PubMed

    Švagelj, Dražen; Terzić, Velimir; Dovhanj, Jasna; Švagelj, Marija; Cvrković, Mirta; Švagelj, Ivan

    2016-04-01

    Human gastric diseases have shown significant changes in the activity and expression of superoxide dismutase (SOD) isoforms. The aim of this study was to detect Mn-SOD activity and expression in the tissue of gastric mucosa, primarily in chronic gastritis (immunohistochemical Helicobacter pylori-negative gastritis, without other pathohistological changes) and to evaluate their possible connection with pathohistological diagnosis. We examined 51 consecutive outpatients undergoing endoscopy for upper gastrointestinal symptoms. Patients were classified based on their histopathological examinations and divided into three groups: 51 patients (archive samples between 2004-2009) with chronic immunohistochemical Helicobacter pylori-negative gastritis (mononuclear cells infiltration were graded as absent, moderate, severe) divided into three groups. Severity of gastritis was graded according to the updated Sydney system. Gastric tissue samples were used to determine the expression of Mn-SOD with anti-Mn-SOD Ab immunohistochemically. The Mn-SOD expression was more frequently present in specimens with severe and moderate inflammation of gastric mucosa than in those with normal mucosa. In patients with normal histological finding, positive immunoreactivity of Mn-SOD was not found. Our results determine the changes in Mn-SOD expression occurring in the normal gastric mucosa that had undergone changes in the intensity of chronic inflammatory infiltrates in the lamina propria.

  11. Meniscus tissue engineering using a novel combination of electrospun scaffolds and human meniscus cells embedded within an extracellular matrix hydrogel.

    PubMed

    Baek, Jihye; Chen, Xian; Sovani, Sujata; Jin, Sungho; Grogan, Shawn P; D'Lima, Darryl D

    2015-04-01

    Meniscus injury and degeneration have been linked to the development of secondary osteoarthritis (OA). Therapies that successfully repair or replace the meniscus are, therefore, likely to prevent or delay OA progression. We investigated the novel approach of building layers of aligned polylactic acid (PLA) electrospun (ES) scaffolds with human meniscus cells embedded in extracellular matrix (ECM) hydrogel to lead to formation of neotissues that resemble meniscus-like tissue. PLA ES scaffolds with randomly oriented or aligned fibers were seeded with human meniscus cells derived from vascular or avascular regions. Cell viability, cell morphology, and gene expression profiles were monitored via confocal microscopy, scanning electron microscopy (SEM), and real-time polymerase chain reaction (PCR), respectively. Seeded scaffolds were used to produce multilayered constructs and were examined via histology and immunohistochemistry. Morphology and mechanical properties of PLA scaffolds (with and without cells) were influenced by fiber direction of the scaffolds. Both PLA scaffolds supported meniscus tissue formation with increased COL1A1, SOX9, and COMP, yet no difference in gene expression was found between random and aligned PLA scaffolds. Overall, ES materials, which possess mechanical strength of meniscus and can support neotissue formation, show potential for use in cell-based meniscus regeneration strategies.

  12. The generation of hybrid electrospun nanofiber layer with extracellular matrix derived from human pluripotent stem cells, for regenerative medicine applications.

    PubMed

    Shtrichman, Ronit; Zeevi-Levin, Naama; Zaid, Rinat; Barak, Efrat; Fishman, Bettina; Ziskind, Anna; Shulman, Rita; Novak, Atara; Avrahami, Ron; Livne, Erella; Lowenstein, Lior; Zussman, Eyal; Itskovitz-Eldor, Joseph

    2014-10-01

    Extracellular matrix (ECM) has been utilized as a biological scaffold for tissue engineering applications in a variety of body systems, due to its bioactivity and biocompatibility. In the current study we developed a modified protocol for the efficient and reproducible derivation of mesenchymal progenitor cells (MPCs) from human embryonic stem cells as well as human induced pluripotent stem cells (hiPSCs) originating from hair follicle keratinocytes (HFKTs). ECM was produced from these MPCs and characterized in comparison to adipose mesenchymal stem cell ECM, demonstrating robust ECM generation by the excised HFKT-iPSC-MPCs. Exploiting the advantages of electrospinning we generated two types of electrospun biodegradable nanofiber layers (NFLs), fabricated from polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA), which provide mechanical support for cell seeding and ECM generation. Elucidating the optimized decellularization treatment we were able to generate an available "off-the-shelf" implantable product (NFL-ECM). Using rat subcutaneous transplantation model we demonstrate that this stem-cell-derived construct is biocompatible and biodegradable and holds great potential for tissue regeneration applications.

  13. Electrical stimulation of neural stem cells mediated by humanized carbon nanotube composite made with extracellular matrix protein.

    PubMed

    Kam, Nadine Wong Shi; Jan, Edward; Kotov, Nicholas A

    2009-01-01

    One of the key challenges to engineering neural interfaces is to minimize their immune response toward implanted electrodes. One potential approach is to manufacture materials that bear greater structural resemblance to living tissues and by utilizing neural stem cells. The unique electrical and mechanical properties of carbon nanotubes make them excellent candidates for neural interfaces, but their adoption hinges on finding approaches for "humanizing" their composites. Here we demonstrated the fabrication of layer-by-layer assembled composites from single-walled carbon nanotubes (SWNTs) and laminin, which is an essential part of human extracellular matrix. Laminin-SWNT thin films were found to be conducive to neural stem cells (NSC) differentiation and suitable for their successful excitation. We observed extensive formation of functional neural network as indicated by the presence of synaptic connections. Calcium imaging of the NSCs revealed generation of action potentials upon the application of a lateral current through the SWNT substrate. These results indicate that the protein-SWNT composite can serve as materials foundation of neural electrodes with chemical structure better adapted with long-term integration with the neural tissue.

  14. Quantitative proteomics of extracellular vesicles released from human monocyte-derived macrophages upon β-glucan stimulation.

    PubMed

    Cypryk, Wojciech; Ohman, Tiina; Eskelinen, Eeva-Liisa; Matikainen, Sampsa; Nyman, Tuula A

    2014-05-02

    Fungal infections (mycoses) are common diseases of varying severity that cause problems, especially to immunologically compromised people. Fungi express a variety of pathogen-associated molecular patterns on their surface including β-glucans, which are important immunostimulatory components of fungal cell walls. During stimulatory conditions of infection and colonization, besides intensive intracellular response, human cells actively communicate on the intercellular level by secreting proteins and other biomolecules with several mechanisms. Vesicular secretion remains one of the most important paths for the proteins to exit the cell. Here, we have used high-throughput quantitative proteomics combined with bioinformatics to characterize and quantify vesicle-mediated protein release from β-glucan-stimulated human macrophages differentiated in vitro from primary blood monocytes. We show that β-glucan stimulation induces vesicle-mediated protein secretion. Proteomic study identified 540 distinct proteins from the vesicles, and the identified proteins show a proteomic signature characteristic for their cellular origin. Importantly, we identified several receptors, including cation-dependent mannose-6-phosphate receptor, macrophage scavenger receptor, and P2X7 receptor, that have not been identified from vesicles before. Proteomic data together with detailed pathway and network analysis showed that integrins and their cytoplasmic cargo proteins are highly abundant in extracellular vesicles released upon β-glucan stimulation. In conclusion, the present data provides a solid basis for further studies on the functional role of vesicular protein secretion upon fungal infection.

  15. Proteomic signatures of extracellular vesicles secreted by nonmineralizing and mineralizing human osteoblasts and stimulation of tumor cell growth.

    PubMed

    Morhayim, Jess; van de Peppel, Jeroen; Demmers, Jeroen A A; Kocer, Gulistan; Nigg, Alex L; van Driel, Marjolein; Chiba, Hideki; van Leeuwen, Johannes P

    2015-01-01

    Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. We focused on the proteomic characterization of nonmineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV-HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic, and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast-specific EV proteins. Label-free quantification demonstrated mineralization stage-dependent 5-fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV-regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival and growth. This was verified by in vitro proliferation assays where osteoblast EV uptake led to 2-fold increase in PC3 cell growth compared to cell-free culture medium-derived vesicle controls. Our findings elucidate the mineralization stage-specific protein content of osteoblast-secreted EVs, show a novel way by which osteoblasts communicate with prostate cancer, and open up innovative avenues for therapeutic intervention.

  16. Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix

    PubMed Central

    Pak, Jaewoo; Lee, Jung Hun; Park, Kwang Seung; Jeong, Byeong Chul; Lee, Sang Hee

    2016-01-01

    Abstract This clinical case series demonstrates that percutaneous injections of autologous adipose tissue-derived stem cells (ADSCs) and homogenized extracellular matrix (ECM) in the form of adipose stromal vascular fraction (SVF), along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, could regenerate cartilage-like tissue in human knee osteoarthritis (OA) patients. Autologous lipoaspirates were obtained from adipose tissue of the abdominal origin. Afterward, the lipoaspirates were minced to homogenize the ECM. These homogenized lipoaspirates were then mixed with collagenase and incubated. The resulting mixture of ADSCs and ECM in the form of SVF was injected, along with HA and PRP activated by calcium chloride, into knees of three Korean patients with OA. The same affected knees were reinjected weekly with additional PRP activated by calcium chloride for 3 weeks. Pretreatment and post-treatment magnetic resonance imaging (MRI) data, functional rating index, range of motion (ROM), and pain score data were then analyzed. All patients' MRI data showed cartilage-like tissue regeneration. Along with MRI evidence, the measured physical therapy outcomes in terms of ROM, subjective pain, and functional status were all improved. This study demonstrates that percutaneous injection of ADSCs with ECM contained in autologous adipose SVF, in conjunction with HA and PRP activated by calcium chloride, is a safe and potentially effective minimally invasive therapy for OA of human knees. PMID:27588219

  17. Shell Extracts from the Marine Bivalve Pecten maximus Regulate the Synthesis of Extracellular Matrix in Primary Cultured Human Skin Fibroblasts

    PubMed Central

    Latire, Thomas; Legendre, Florence; Bigot, Nicolas; Carduner, Ludovic; Kellouche, Sabrina; Bouyoucef, Mouloud; Carreiras, Franck; Marin, Frédéric; Lebel, Jean-Marc; Galéra, Philippe; Serpentini, Antoine

    2014-01-01

    Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the −112/−61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications. PMID:24949635

  18. Sub-toxic nicotine concentrations affect extracellular matrix and growth factor signaling gene expressions in human osteoblasts.

    PubMed

    Marinucci, Lorella; Bodo, Maria; Balloni, Stefania; Locci, Paola; Baroni, Tiziano

    2014-12-01

    Exposure to nicotine and other compounds contained in cigarette smoking affects human health. This study examined the effects of exposure to a single or multiple sub-toxic nicotine concentrations on human osteoblasts. Cell growth and expression of genes involved in bone differentiation, extracellular matrix (ECM) metabolism, and growth factor signaling pathways were investigated in nicotine-treated cells compared to untreated cells. Depending on osteoblast concentration and maturation stages, nicotine differently regulated cell growth. Real-time PCR showed regulated expressions of genes expressed by nicotine-treated osteoblasts compared to untreated cells. Among ECM genes, type I collagen was down-regulated and osteonectin was up-regulated in nicotine-treated osteoblasts; similarly, fibroblast growth factor-1 (FGF1) and fibroblast growth factor-2 (FGF2), two members of FGF signaling system, were discordantly modulated; genes involved in osteoblast maturation and differentiation such as alkaline phosphatase (ALP), runt-related transcription factor-2 (RUNX2), and bone sialoprotein (BSP) were over-expressed after drug treatment. Our results show a positive association between nicotine exposure and osteoblast phenotype and illustrate for the first time a mechanism whereby acute or chronic exposure to sub-toxic nicotine concentrations may affect bone formation through the impairment of growth factor signaling system and ECM metabolism.

  19. Extracellular O-Linked N-Acetylglucosamine Is Enriched in Stem Cells Derived from Human Umbilical Cord Blood

    PubMed Central

    Hirvonen, Tia; Ritamo, Ilja; Natunen, Suvi; Tuimala, Jarno; Laitinen, Saara; Anderson, Heidi; Nystedt, Johanna; Räbinä, Jarkko; Valmu, Leena

    2014-01-01

    Abstract Stem cells have a unique ability to self-renew and differentiate into diverse cell types. Currently, stem cells from various sources are being explored as a promising new treatment for a variety of human diseases. A diverse set of functional and phenotypical markers are used in the characterization of specific therapeutic stem cell populations. The glycans on the stem cell surface respond rapidly to alterations in cellular state and signaling and are therefore ideal for identifying even minor changes in cell populations. Many stem cell markers are based on cell surface glycan epitopes including the widely used markers SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81. We have now discovered by mRNA analysis that a novel glycosyltranferase, epidermal growth factor (EGF) domain-specific O-linked GlcNAc transferase (EOGT), is highly expressed in stem cells. EOGT is responsible for adding O-linked N-acetylglucosamine (O-GlcNAc) to folded EGF domains on extracellular proteins, such as those on the Notch receptors. We were able to show by immunological assays that human umbilical cord blood–derived mesenchymal stromal cells display O-GlcNAc, the product of EOGT, and that O-GlcNAc is further elongated with galactose to form O-linked N-acetyllactosamine. We suggest that these novel glycans are involved in the fine tuning of Notch receptor signaling pathways in stem cells. PMID:24804163

  20. Autoimmune disease-associated variants of extracellular endoplasmic reticulum aminopeptidase 1 induce altered innate immune responses by human immune cells

    PubMed Central

    Aldhamen, Yasser A.; Pepelyayeva, Yuliya; Rastall, David P.W.; Seregin, Sergey S.; Zervoudi, Efthalia; Koumantou, Despoina; Aylsworth, Charles F.; Quiroga, Dionisia; Godbehere, Sarah; Georgiadis, Dimitris; Stratikos, Efstratios; Amalfitano, Andrea

    2015-01-01

    ERAP1 gene polymorphisms have been linked to several autoimmune diseases; however, the molecular mechanisms underlying these associations are not well understood. Recently, we have demonstrated that ERAP1 regulates key aspects of the innate immune response. Moreover, previous studies show ERAP1 to be ER-localized and secreted during inflammation. Herein, we investigate the possible roles that ERAP1 polymorphic variants may have in modulating innate immune responses of human PBMCs using two experimental methods: extracellular exposure of hPBMCs to ERAP1 variants and adenovirus-based ERAP1 expression. We found that exposure of hPBMCs to ERAP1 variant proteins as well as ERAP1 overexpression by Ad vectors increased inflammatory cytokine and chemokine production, and enhanced immune cell activation. Investigating the molecular mechanisms behind these responses revealed that ERAP1 is able to activate innate immunity via multiple pathways, including the NLRP3 inflammasome. Importantly, these responses varied if autoimmune-disease-associated variants of ERAP1 were examined in the assay systems. Unexpectedly, blocking ERAP1 cellular internalization augmented IL-1β production. To our knowledge, this is the first report identifying ERAP1 as being involved in modulating innate responses of human immune cells, a finding that may explain why ERAP1 has been genetically associated with several autoimmune diseases. PMID:25591727

  1. Meniscus Tissue Engineering Using a Novel Combination of Electrospun Scaffolds and Human Meniscus Cells Embedded within an Extracellular Matrix Hydrogel

    PubMed Central

    Baek, Jihye; Chen, Xian; Sovani, Sujata; Jin, Sungho; Grogan, Shawn P; D’Lima, Darryl D

    2015-01-01

    Meniscus injury and degeneration have been linked to the development of secondary osteoarthritis (OA). Therapies that successfully repair or replace the meniscus are therefore likely to prevent or delay OA progression. We investigated the novel approach of building layers of aligned polylactic acid (PLA) electrospun (ES) scaffolds with human meniscus cells embedded in extracellular matrix (ECM) hydrogel to lead to formation of neotissues that resemble meniscus-like tissue. PLA ES scaffolds with randomly oriented or aligned fibers were seeded with human meniscus cells derived from vascular or avascular regions. Cell viability, cell morphology, and gene expression profiles were monitored via confocal microscopy, scanning electron microscopy (SEM), and real-time PCR, respectively. Seeded scaffolds were used to produce multilayered constructs and were examined via histology and immunohistochemistry. Morphology and mechanical properties of PLA scaffolds (with and without cells) were influenced by fiber direction of the scaffolds. Both PLA scaffolds supported meniscus tissue formation with increased COL1A1, SOX9, COMP, yet no difference in gene expression was found between random and aligned PLA scaffolds. Overall, ES materials, which possess mechanical strength of meniscus and can support neotissue formation, show potential for use in cell-based meniscus regeneration strategies. PMID:25640671

  2. Induction of extracellular matrix synthesis in normal human fibroblasts by anthraquinone isolated from Morinda citrifolia (Noni) fruit.

    PubMed

    Kim, Sung-Woo; Jo, Byoung-Kee; Jeong, Ji-Hean; Choi, Sun-Uk; Hwang, Yong-Il

    2005-01-01

    In previous studies we found that Morinda citrifolia (Noni) fruit extract up-regulated biosynthesis of type I collagen and glycosaminoglycans in primary cultures of normal human fibroblasts. The objective of this study was to identify the active ingredients in Noni fruit extract. An active single compound having a type I collagen-stimulating effect was isolated and identified as 1,4-dihydroxy-2-methoxy-7-methylanthraquinone by nuclear magnetic resonance, infrared, and mass analysis. It was revealed that anthraquinone showed significantly increased elaboration of procollagen type I C-terminal peptide and glycosaminoglycans and reduced expression of the collagenase matrix metalloproteinase-1 dose-dependently in human dermal fibroblasts. Furthermore, in a clinical trial, a nano-emulsion containing anthraquinone predominantly increased the dermal type I procollagen in nude mouse skin. These results suggest that anthraquinone derived from Noni extract is a good candidate for use as a new anti-wrinkle agent due to its strong induction of biosynthetic activity of extracellular matrix components.

  3. Thiamine and benfotiamine prevent apoptosis induced by high glucose-conditioned extracellular matrix in human retinal pericytes.

    PubMed

    Beltramo, Elena; Nizheradze, Konstantin; Berrone, Elena; Tarallo, Sonia; Porta, Massimo

    2009-10-01

    Early and selective loss of pericytes and thickening of the basement membrane are hallmarks of diabetic retinopathy. We reported red