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Sample records for biosynthesis induces matrix

  1. Dynamic compression of rabbit adipose-derived stem cells transfected with insulin-like growth factor 1 in chitosan/gelatin scaffolds induces chondrogenesis and matrix biosynthesis.

    PubMed

    Li, Jianjun; Zhao, Qun; Wang, Enbo; Zhang, Chuanhui; Wang, Guangbin; Yuan, Quan

    2012-05-01

    Articular cartilage is routinely subjected to mechanical forces and growth factors. Adipose-derived stem cells (ASCs) are multi-potent adult stem cells and capable of chondrogenesis. In the present study, we investigated the comparative and interactive effects of dynamic compression and insulin-like growth factor-I (IGF-I) on the chondrogenesis of rabbit ASCs in chitosan/gelatin scaffolds. Rabbit ASCs with or without a plasmid overexpressing of human IGF-1 were cultured in chitosan/gelatin scaffolds for 2 days, then subjected to cyclic compression with 5% strain and 1 Hz for 4 h per day for seven consecutive days. Dynamic compression induced chondrogenesis of rabbit ASCs by activating calcium signaling pathways and up-regulating the expression of Sox-9. Dynamic compression plus IGF-1 overexpression up-regulated expression of chondrocyte-specific extracellular matrix genes including type II collagen, Sox-9, and aggrecan with no effect on type X collagen expression. Furthermore, dynamic compression and IGF-1 expression promoted cellular proliferation and the deposition of proteoglycan and collagen. Intracellular calcium ion concentration and peak currents of Ca(2+) ion channels were consistent with chondrocytes. The tissue-engineered cartilage from this process had excellent mechanical properties. When applied together, the effects achieved by the two stimuli (dynamic compression and IGF-1) were greater than those achieved by either stimulus alone. Our results suggest that dynamic compression combined with IGF-1 overexpression might benefit articular cartilage tissue engineering in cartilage regeneration.

  2. Ethylene Biosynthesis-Inducing Xylanase 1

    PubMed Central

    Dean, Jeffrey F. D.; Anderson, J. D.

    1991-01-01

    The ethylene biosynthesis-inducing endoxylanase (EIX) from xylan-induced cultures of the fungus, Trichoderma viride, was purified to near homogeneity and compared with the EIX isolated from Cellulysin. Both enzymes migrate as 9.2 kilodalton proteins during gel filtration chromatography under nondenaturing conditions, but the mature polypeptide migrates as a 22 kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the 22 kilodalton polypeptide is enriched by Gly, Ser, Thr, Trp, and Tyr, but depleted in Ala, Glx, Leu, and Lys. Both proteins lack sulfur-containing amino acids. The protein is glycosylated, and inhibition of EIX synthesis by tunicamycin suggests that at least some of the sugar moieties are linked to asparagine residues. EIX appears to be synthesized initially as a 25 kilodalton precursor protein that is processed to 22 kilodalton during secretion. Images Figure 2 Figure 4 Figure 5 Figure 6 PMID:16667971

  3. Jasmonate-induced biosynthesis of andrographolide in Andrographis paniculata.

    PubMed

    Sharma, Shiv Narayan; Jha, Zenu; Sinha, Rakesh Kumar; Geda, Arvind Kumar

    2015-02-01

    Andrographolide is a prominent secondary metabolite found in Andrographis paniculata that exhibits enormous pharmacological effects. In spite of immense value, the normal biosynthesis of andrographolide results in low amount of the metabolite. To induce the biosynthesis of andrographolide, we attempted elicitor-induced activation of andrographolide biosynthesis in cell cultures of A. paniculata. This was carried out by using methyl jasmonate (MeJA) as an elicitor. Among the various concentrations of MeJA tested at different time periods, 5 µM MeJA yielded 5.25 times more andrographolide content after 24 h of treatment. The accumulation of andrographolide was correlated with the expression level of known regulatory genes (hmgs, hmgr, dxs, dxr, isph and ggps) of mevalonic acid (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. These results established the involvement of MeJA in andrographolide biosynthesis by inducing the transcription of its biosynthetic pathways genes. The coordination of isph, ggps and hmgs expression highly influenced the andrographolide biosynthesis.

  4. Tumors perturbing extracellular matrix biosynthesis. The case of von Recklinghausen's disease.

    PubMed

    Robert, L

    2014-04-01

    This is a short review of neurofibromatosis-1 or von Recklinghausen's disease, due to a loss of function mutation of the gene neurofibromin-1, which normally inhibits the Ras MAPK-pathways. Among its symptoms, the strong oversynthesis of several collagen types designates this disease as producing a deregulation of extracellular matrix biosynthesis involved in tumor formation. Up to about 40% of the skin tumors consist of collagens. A short summary of the clinical manifestations and pathological and genetic mechanisms are also described.

  5. Peroxidase Enzymes Regulate Collagen Biosynthesis and Matrix Mineralization by Cultured Human Osteoblasts.

    PubMed

    DeNichilo, Mark O; Shoubridge, Alexandra J; Panagopoulos, Vasilios; Liapis, Vasilios; Zysk, Aneta; Zinonos, Irene; Hay, Shelley; Atkins, Gerald J; Findlay, David M; Evdokiou, Andreas

    2016-03-01

    The early recruitment of inflammatory cells to sites of bone fracture and trauma is a critical determinant in successful fracture healing. Released by infiltrating inflammatory cells, myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, whose functional involvement in bone repair has mainly been studied in the context of providing a mechanism for oxidative defense against invading microorganisms. We report here novel findings that show peroxidase enzymes have the capacity to stimulate osteoblastic cells to secrete collagen I protein and generate a mineralized extracellular matrix in vitro. Mechanistic studies conducted using cultured osteoblasts show that peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl hydroxylase-dependent manner, which does not require ascorbic acid. Our studies demonstrate that osteoblasts rapidly bind and internalize both MPO and EPO, and the catalytic activity of these peroxidase enzymes is essential to support collagen I biosynthesis and subsequent release of collagen by osteoblasts. We show that EPO is capable of regulating osteogenic gene expression and matrix mineralization in culture, suggesting that peroxidase enzymes may play an important role not only in normal bone repair, but also in the progression of pathological states where infiltrating inflammatory cells are known to deposit peroxidases.

  6. Succinate is a preferential metabolic stimulus-coupling signal for glucose-induced proinsulin biosynthesis translation.

    PubMed

    Alarcon, Cristina; Wicksteed, Barton; Prentki, Marc; Corkey, Barbara E; Rhodes, Christopher J

    2002-08-01

    The secondary signals emanating from increased glucose metabolism, which lead to specific increases in proinsulin biosynthesis translation, remain elusive. It is known that signals for glucose-stimulated insulin secretion and proinsulin biosynthesis diverge downstream of glycolysis. Consequently, the mitochondrial products ATP, Krebs cycle intermediates, glutamate, and acetoacetate were investigated as candidate stimulus-coupling signals specific for glucose-induced proinsulin biosynthesis in rat islets. Decreasing ATP levels by oxidative phosphorylation inhibitors showed comparable effects on proinsulin biosynthesis and total protein synthesis. Although it is a cofactor, ATP is unlikely to be a metabolic stimulus-coupling signal specific for glucose-induced proinsulin biosynthesis. Neither glutamic acid methyl ester nor acetoacetic acid methyl ester showed a specific effect on glucose-stimulated proinsulin biosynthesis. Interestingly, among Krebs cycle intermediates, only succinic acid monomethyl ester specifically stimulated proinsulin biosynthesis. Malonic acid methyl ester, an inhibitor of succinate dehydrogenase, also specifically increased glucose-induced proinsulin biosynthesis without affecting islet ATP levels or insulin secretion. Glucose caused a 40% increase in islet intracellular succinate levels, but malonic acid methyl ester showed no further effect, probably due to efficient conversion of succinate to succinyl-CoA. In this regard, a GTP-dependent succinyl-CoA synthetase activity was found in cytosolic fractions of pancreatic islets. Thus, succinate and/or succinyl-CoA appear to be preferential metabolic stimulus-coupling factors for glucose-induced proinsulin biosynthesis translation.

  7. Proteolytic Pathways Induced by Herbicides That Inhibit Amino Acid Biosynthesis

    PubMed Central

    Zulet, Amaia; Gil-Monreal, Miriam; Villamor, Joji Grace; Zabalza, Ana; van der Hoorn, Renier A. L.; Royuela, Mercedes

    2013-01-01

    Background The herbicides glyphosate (Gly) and imazamox (Imx) inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolytic activities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling. Results Several common changes in proteolytic activity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides. Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment. Conclusion These results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolytic activity remains to be determined. PMID:24040092

  8. Sulfur deficiency–induced repressor proteins optimize glucosinolate biosynthesis in plants

    PubMed Central

    Aarabi, Fayezeh; Kusajima, Miyuki; Tohge, Takayuki; Konishi, Tomokazu; Gigolashvili, Tamara; Takamune, Makiko; Sasazaki, Yoko; Watanabe, Mutsumi; Nakashita, Hideo; Fernie, Alisdair R.; Saito, Kazuki; Takahashi, Hideki; Hubberten, Hans-Michael; Hoefgen, Rainer; Maruyama-Nakashita, Akiko

    2016-01-01

    Glucosinolates (GSLs) in the plant order of the Brassicales are sulfur-rich secondary metabolites that harbor antipathogenic and antiherbivory plant-protective functions and have medicinal properties, such as carcinopreventive and antibiotic activities. Plants repress GSL biosynthesis upon sulfur deficiency (−S); hence, field performance and medicinal quality are impaired by inadequate sulfate supply. The molecular mechanism that links –S to GSL biosynthesis has remained understudied. We report here the identification of the –S marker genes sulfur deficiency induced 1 (SDI1) and SDI2 acting as major repressors controlling GSL biosynthesis in Arabidopsis under –S condition. SDI1 and SDI2 expression negatively correlated with GSL biosynthesis in both transcript and metabolite levels. Principal components analysis of transcriptome data indicated that SDI1 regulates aliphatic GSL biosynthesis as part of –S response. SDI1 was localized to the nucleus and interacted with MYB28, a major transcription factor that promotes aliphatic GSL biosynthesis, in both yeast and plant cells. SDI1 inhibited the transcription of aliphatic GSL biosynthetic genes by maintaining the DNA binding composition in the form of an SDI1-MYB28 complex, leading to down-regulation of GSL biosynthesis and prioritization of sulfate usage for primary metabolites under sulfur-deprived conditions. PMID:27730214

  9. Alterations in the biosynthesis of extracellular matrix molecules in connective tissues by electric and magnetic fields

    SciTech Connect

    Ciombor, D.M.

    1992-01-01

    Pulsed electromagnetic fields (PEMFs) of certain configurations have been shown to be effective clinically in promoting the healing of fracture non-unions and are believed to enhance calcification of extracellular matrix. In vitro studies have suggested that PEMFs may also have the effect of modifying the extracellular matrix by promoting the synthesis of matrix molecules. This study examines the effect of one particular type of PEMF and a sinusoidal continuous wave upon the extracellular matrix and calcification of endochondral ossification in vivo. The pulsed magnetic field (SS-22) utilized in these studies is being used clinically for the treatment of fracture non-unions, a condition in which the bone is not restored to form or function. The sinusoidal continuous wave was designed to provide a 5 Gauss amplitude at a 15 Hz. rate. The synthesis of cartilage molecules is enhanced by this type of PEMF and since wave and subsequent endochondral calcification is stimulated. Histomorphometric studies indicate that the maturation of bone trabeculae is also promoted by this type of PEMF stimulation. These results indicate that a specific PEMF or continuous waveform can change the composition of cartilage extracellular matrix in vivo and raises the possibility that the effects on other processes of endochondral ossification (e.g., fracture healing and growth plates) may occur through a similar mechanism.

  10. Salt-stress induced modulation of chlorophyll biosynthesis during de-etiolation of rice seedlings.

    PubMed

    Turan, Satpal; Tripathy, Baishnab C

    2015-03-01

    Chlorophyll biosynthesis in plants is subjected to modulation by various environmental factors. To understand the modulation of the chlorophyll (Chl) biosynthesis during greening process by salt, 100-200 mM NaCl was applied to the roots of etiolated rice seedlings 12 h prior to the transfer to light. Application of 200 mM NaCl to rice seedlings that were grown in light for further 72 h resulted in reduced dry matter production (-58%) and Chl accumulation (-66%). Ionic imbalance due to salinity stress resulted in additional downregulation (41-45%) of seedling dry weight, Chl and carotenoid contents over and above that of similar osmotic stress induced by polyethylene glycol. Downregulation of Chl biosynthesis may be attributed to decreased activities of Chl biosynthetic pathway enzymes, i.e. 5-aminolevulinic acid (ALA) dehydratase (EC-2.4.1.24), porphobilinogen deaminase (EC-4.3.1.8), coproporphyrinogen III oxidase (EC-1.3.3.3), protoporphyrinogen IX oxidase (EC-1.3.3.4), Mg-protoporphyrin IX chelatase (EC-6.6.1.1) and protochlorophyllide oxidoreductase (EC-1.3.33.1). Reduced enzymatic activities were due to downregulation of their protein abundance and/or gene expression in salt-stressed seedlings. The extent of downregulation of ALA biosynthesis nearly matched with that of protochlorophyllide and Chl to prevent the accumulation of highly photosensitive photodynamic tetrapyrroles that generates singlet oxygen under stress conditions. Although, ALA synthesis decreased, the gene/protein expression of glutamyl-tRNA reductase (EC-1.2.1.70) increased suggesting it may play a role in acclimation to salt stress. The similar downregulation of both early and late Chl biosynthesis intermediates in salt-stressed seedlings suggests a regulatory network of genes involved in tetrapyrrole biosynthesis.

  11. Mycolic acid-containing bacteria induce natural-product biosynthesis in Streptomyces species.

    PubMed

    Onaka, Hiroyasu; Mori, Yukiko; Igarashi, Yasuhiro; Furumai, Tamotsu

    2011-01-01

    Natural products produced by microorganisms are important starting compounds for drug discovery. Secondary metabolites, including antibiotics, have been isolated from different Streptomyces species. The production of these metabolites depends on the culture conditions. Therefore, the development of a new culture method can facilitate the discovery of new natural products. Here, we show that mycolic acid-containing bacteria can influence the biosynthesis of cryptic natural products in Streptomyces species. The production of red pigment by Streptomyces lividans TK23 was induced by coculture with Tsukamurella pulmonis TP-B0596, which is a mycolic acid-containing bacterium. Only living cells induced this pigment production, which was not mediated by any substances. T. pulmonis could induce natural-product synthesis in other Streptomyces strains too: it altered natural-product biosynthesis in 88.4% of the Streptomyces strains isolated from soil. The other mycolic acid-containing bacteria, Rhodococcus erythropolis and Corynebacterium glutamicum, altered biosynthesis in 87.5 and 90.2% of the Streptomyces strains, respectively. The coculture broth of T. pulmonis and Streptomyces endus S-522 contained a novel antibiotic, which we named alchivemycin A. We concluded that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the direct interaction between the mycolic acid-containing bacteria and Streptomyces. We used these results to develop a new coculture method, called the combined-culture method, which facilitates the screening of natural products.

  12. Ethanol-induced impairment in the biosynthesis of N-linked glycosylation.

    PubMed

    Welti, Michael; Hülsmeier, Andreas J

    2014-04-01

    Deficiency in N-linked protein glycosylation is a long-known characteristic of alcoholic liver disease and congenital disorders of glycosylation. Previous investigations of ethanol-induced glycosylation deficiency demonstrated perturbations in the early steps of substrate synthesis and in the final steps of capping N-linked glycans in the Golgi. The significance of the biosynthesis of N-glycan precursors in the endoplasmic reticulum, however, has not yet been addressed in alcoholic liver disease. Ethanol-metabolizing hepatoma cells were treated with increasing concentrations of ethanol. Transcript analysis of genes involved in the biosynthesis of N-glycans, activity assays of related enzymes, dolichol-phosphate quantification, and analysis of dolichol-linked oligosaccharides were performed. Upon treatment of cells with ethanol, we found a decrease in the final N-glycan precursor Dol-PP-GlcNAc(2) Man(9) Glc(3) and in C95- and C100-dolichol-phosphate levels. Transcript analysis of genes involved in N-glycosylation showed a 17% decrease in expression levels of DPM1, a subunit of the dolichol-phosphate-mannose synthase, and an 8% increase in RPN2, a subunit of the oligosaccharyl transferase. Ethanol treatment decreases the biosynthesis of dolichol-phosphate. Consequently, the formation of N-glycan precursors is affected, resulting in an aberrant precursor assembly. Messenger RNA levels of genes involved in N-glycan biosynthesis are slightly affected by ethanol treatment, indicating that the assembly of N-glycan precursors is not regulated at the transcriptional level. This study confirms that ethanol impairs N-linked glycosylation by affecting dolichol biosynthesis leading to impaired dolichol-linked oligosaccharide assembly. Together our data help to explain the underglycosylation phenotype observed in alcoholic liver disease and congenital disorders of glycosylation.

  13. A Synthetic Chalcone as a Potent Inducer of Glutathione Biosynthesis

    PubMed Central

    Kachadourian, Remy; Day, Brian J.; Pugazhenti, Subbiah; Franklin, Christopher C.; Genoux-Bastide, Estelle; Mahaffey, Gregory; Gauthier, Charlotte; Di Pietro, Attilio; Boumendjel, Ahcène

    2014-01-01

    Chalcones continue to attract considerable interest due to their anti-inflammatory and antiangiogenic properties. We recently reported the ability of 2′,5′-dihydroxychalcone (2′,5′-DHC) to induce both breast cancer resistance protein-mediated export of glutathione (GSH) and c-Jun N-terminal kinase-mediated increased intracellular GSH levels. Herein, we report a structure–activity relationship study of a series of 30 synthetic chalcone derivatives with hydroxyl, methoxyl, and halogen (F and Cl) substituents and their ability to increase intracellular GSH levels. This effect was drastically improved with one or two electrowithdrawing groups on phenyl ring B and up to three methoxyl and/or hydroxyl groups on phenyl ring A. The optimal structure, 2-chloro-4′,6′-dimethoxy-2′-hydroxychalcone, induced both a potent NF-E2-related factor 2-mediated transcriptional response and an increased formation of glutamate cysteine ligase holoenzyme, as shown using a human breast cancer cell line stably expressing a luciferase reporter gene driven by antioxidant response elements. PMID:22239485

  14. Prediction of thermal cycling induced matrix cracking

    NASA Technical Reports Server (NTRS)

    Mcmanus, Hugh L.

    1992-01-01

    Thermal fatigue has been observed to cause matrix cracking in laminated composite materials. A method is presented to predict transverse matrix cracks in composite laminates subjected to cyclic thermal load. Shear lag stress approximations and a simple energy-based fracture criteria are used to predict crack densities as a function of temperature. Prediction of crack densities as a function of thermal cycling is accomplished by assuming that fatigue degrades the material's inherent resistance to cracking. The method is implemented as a computer program. A simple experiment provides data on progressive cracking of a laminate with decreasing temperature. Existing data on thermal fatigue is also used. Correlations of the analytical predictions to the data are very good. A parametric study using the analytical method is presented which provides insight into material behavior under cyclical thermal loads.

  15. Binary stress induces an increase in indole alkaloid biosynthesis in Catharanthus roseus

    PubMed Central

    Zhu, Wei; Yang, Bingxian; Komatsu, Setsuko; Lu, Xiaoping; Li, Ximin; Tian, Jingkui

    2015-01-01

    Catharanthus roseus is an important medicinal plant, which produces a variety of indole alkaloids of significant pharmaceutical relevance. In the present study, we aimed to investigate the potential stress-induced increase of indole alkaloid biosynthesis in C. roseus using proteomic technique. The contents of the detectable alkaloids ajmalicine, vindoline, catharanthine, and strictosidine in C. roseus were significantly increased under binary stress. Proteomic analysis revealed that the abundance of proteins related to tricarboxylic acid cycle and cell wall was largely increased; while, that of proteins related to tetrapyrrole synthesis and photosynthesis was decreased. Of note, 10-hydroxygeraniol oxidoreductase, which is involved in the biosynthesis of indole alkaloid was two-fold more abundant in treated group compared to the control. In addition, mRNA expression levels of genes involved in the indole alkaloid biosynthetic pathway indicated an up-regulation in their transcription in C. roseus under UV-B irradiation. These results suggest that binary stress might negatively affect the process of photosynthesis in C. roseus. In addition, the induction of alkaloid biosynthesis appears to be responsive to binary stress. PMID:26284098

  16. Phenytoin potentiates interleukin-1-induced prostaglandin biosynthesis in human gingival fibroblasts.

    PubMed Central

    Modéer, T.; Brunius, G.; Iinuma, M.; Lerner, U. H.

    1992-01-01

    1. The effect of phenytoin (PHT) on prostaglandin E2 (PGE2) biosynthesis in human gingival fibroblasts stimulated by interleukin-1 (IL-1 alpha, IL-1 beta) or by tumour necrosis factor alpha (TNF alpha) was studied. 2. IL-1 alpha (1.5-6.0 ng ml-1) and IL-1 beta (30-300 pg ml-1), dose-dependently, stimulated PGE2 formation, in 24 h cultures, with IL-beta being the most potent agonist. 3. PHT (2.5-20 micrograms ml-1) did not induce PGE2 formation itself but potentiated IL-1 alpha- and IL-1 beta-induced PGE2 formation in the gingival fibroblasts in a manner dependent on the concentrations of both IL-1 and PHT. 4. IL-1 beta (0.1-1.0 ng ml-1) induced release of [3H]-arachidonic acid ([3H]-AA) from prelabelled fibroblasts that was potentiated by PHT (20 micrograms ml-1). 5. TNF-alpha (greater than or equal to 0.01 micrograms ml-1) significantly stimulated the biosynthesis of PGE2 by a process that was potentiated by PHT. 6. Addition of exogenous arachidonic acid (AA) (greater than or equal to 1 microM) caused an increase of PGE2 formation in the fibroblasts that was not potentiated by PHT (20 micrograms ml-1). 7. The results indicate that treatment with PHT results in upregulation of prostaglandin biosynthesis in gingival fibroblasts challenged with IL-1 or TNF alpha, at least partly due to enhanced level of phospholipase A2 activity. PMID:1504741

  17. Involvement of polyamine biosynthesis in somatic embryogenesis of Valencia sweet orange (Citrus sinensis) induced by glycerol.

    PubMed

    Wu, Xiao-Ba; Wang, Jing; Liu, Ji-Hong; Deng, Xiu-Xin

    2009-01-01

    Culture of Citrus sinensis embryogenic callus on the embryo-inducing medium (EIM) containing glycerol gave rise to a large number of embryos, whereas very few embryos were observed on the callus growth medium (CGM). In the current paper, attempts were made to investigate whether polyamine biosynthesis was involved in glycerol-mediated somatic embryogenesis. Quantification of free polyamines by high-performance liquid chromatography showed that the cultures on EIM had less putrescine than those on CGM. However, increase in spermidine and spermine was detected in cultures on EIM during the first 20d of culture, coincident with abundant somatic embryogenesis. The globular embryos contained more polyamines than embryos at other stages. Semi-quantitative reverse transcriptase-polymerase chain reaction assay showed that expression levels of all of the five key genes involved in polyamine biosynthesis, with the exception of S-adenosylmethionine decarboxylase, were induced in cultures on EIM, and that their transcriptional levels were increased with maturation of the embryos. Addition of alpha-difluoromethylornithine, a polyamine biosynthesis inhibitor, to EIM resulted in remarkable inhibition of somatic embryogenesis, concurrent with notable reduction of endogenous putrescine and spermidine, particularly at higher concentrations. Exogenous application of 1mM putrescine to EIM together with 5mM alpha-difluoromethylornithine led to dramatic enhancement of endogenous polyamines, which successfully restored somatic embryogenesis. All of these, collectively, demonstrated that free polyamines, at least spermidine and spermine herein, were involved in glycerol-mediated promotion of somatic embryogenesis, which will open a new avenue for establishing a sophisticated system for somatic embryogenesis based on the modulation of endogenous polyamines.

  18. Ultrastructural alterations induced by two ergosterol biosynthesis inhibitors, ketoconazole and terbinafine, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi.

    PubMed Central

    Lazardi, K; Urbina, J A; de Souza, W

    1990-01-01

    We report the ultrastructural alterations induced during the proliferative stages of Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease, by two ergosterol biosynthesis inhibitors, ketoconazole and terbinafine, which had previously been shown to be potent growth inhibitors whose effects are potentiated when used in combination (J. A. Urbina, K. Lazardi, T. Aguirre, M. M. Piras, and R. Piras, Antimicrob. Agents Chemother. 32:1237-1242, 1988). Epimastigotes treated with a low concentration of ketoconazole (1 microM), which blocks ergosterol biosynthesis at the level of C-14 demethylation of lanosterol and induces cell lysis coincident with total ergosterol depletion, showed gross alterations of the kinetoplast-mitochondrion complex, which swelled and lost the organization of its inner membrane and the electron-dense bodies of its matrix. Thus, coincident with the beginning of cell lysis, the kinetoplast-mitochondrion complex occupied greater than 80% of the cell volume, while other subcellular structures such as the nucleus and subpellicular microtubules were not affected. Terbinafine, which blocks ergosterol synthesis in these cells at the level of squalene synthetase and thus leads to almost immediate arrest of growth at concentrations greater than 1 microM, produced proliferation of glycosomelike bodies, binucleated cells (arrest at cytokinesis), and eventually massive vacuolization. When the drugs were combined, the predominant effect was mitochondrial swelling, which was more drastic and took place earlier than that observed in cells treated with ketoconazole alone. In amastigotes proliferating in Vero cells, ketoconazole at the concentration required to eradicate the parasites (10 nM) produced mitochondrial swelling, the appearance of autophagic vacuoles containing partially degraded subcellular material, and finally a general breakdown of the subcellular structures. Terbinafine at 3 microM induced more limited ultrastructural damage to

  19. Crucial role of androgen receptor in vascular H2S biosynthesis induced by testosterone

    PubMed Central

    Brancaleone, V; Vellecco, V; Matassa, D S; d'Emmanuele di Villa Bianca, R; Sorrentino, R; Ianaro, A; Bucci, M; Esposito, F; Cirino, G

    2015-01-01

    Background and Purpose Hydrogen sulphide (H2S) is a gaseous mediator strongly involved in cardiovascular homeostasis, where it provokes vasodilatation. Having previously shown that H2S contributes to testosterone-induced vasorelaxation, here we aim to uncover the mechanisms underlying this effect. Experimental Approach H2S biosynthesis was evaluated in rat isolated aortic rings following androgen receptor (NR3C4) stimulation. Co-immunoprecipitation and surface plasmon resonance analysis were performed to investigate mechanisms involved in NR3C4 activation. Key Results Pretreatment with NR3C4 antagonist nilutamide prevented testosterone-induced increase in H2S and reduced its vasodilator effect. Androgen agonist mesterolone also increased H2S and induced vasodilatation; effects attenuated by the selective cystathionine-γ lyase (CSE) inhibitor propargylglycine. The NR3C4-multicomplex-derived heat shock protein 90 (hsp90) was also involved in this effect; its specific inhibitor geldanamycin strongly reduced testosterone-induced H2S production. Neither progesterone nor 17-β-oestradiol induced H2S release. Furthermore, we demonstrated that CSE, the main vascular H2S-synthesizing enzyme, is physically associated with the NR3C4/hsp90 complex and the generation of such a ternary system represents a key event leading to CSE activation. Finally, H2S levels in human blood collected from male healthy volunteers were higher than those in female samples. Conclusions and Implications We demonstrated that selective activation of the NR3C4 is essential for H2S biosynthesis within vascular tissue, and this event is based on the formation of a ternary complex between cystathionine-γ lyase, NR3C4and hsp90. This novel molecular mechanism operating in the vasculature, corroborated by higher H2S levels in males, suggests that the L-cysteine/CSE/H2S pathway may be preferentially activated in males leading to gender-specific H2S biosynthesis. Linked Articles This article is part of a

  20. Emodin Decreases Hepatic Hypoxia-Inducible Factor-1[Formula: see text] by Inhibiting its Biosynthesis.

    PubMed

    Ma, Feifei; Hu, Lijuan; Yu, Ming; Wang, Feng

    2016-01-01

    Hypoxia-inducible factor-1 (HIF-1) is an [Formula: see text] dimeric transcription factor. Because HIF-1[Formula: see text] is instable with oxygen, HIF-1 is scarce in normal mammalian cells. However, HIF-1[Formula: see text] is expressed in pathological conditions such as cancer and obesity. Inhibiting HIF-1[Formula: see text] may be of therapeutic value for these pathologies. Here, we investigated whether emodin, derived from the herb of Rheum palmatum L, which is also known as Chinese rhubarb, and is native to China, regulates HIF-1[Formula: see text] expression. Male C57BL/6 mice without or with diet-induced obesity were treated with emodin for two weeks, while control mice were treated with vehicle. HIF-1[Formula: see text] expression was determined by Western blot. We found that emodin inhibited obesity-induced HIF-1[Formula: see text] expression in liver and skeletal muscle but did not regulate HIF-1[Formula: see text] expression in the kidneys or in intra-abdominal fat. In vitro, emodin inhibited HIF-1[Formula: see text] expression in human HepG2 hepatic cells and Y1 adrenocortical cells. Further, we investigated the mechanisms of HIF-1[Formula: see text] expression in emodin-treated HepG2 cells. First, we found that HIF-1[Formula: see text] had normal stability in the presence of emodin. Thus, emodin did not decrease HIF-1[Formula: see text] by stimulating its degradation. Importantly, emodin decreased the activity of the signaling pathways that led to HIF-1[Formula: see text] biosynthesis. Interestingly, emodin increased HIF-1[Formula: see text] mRNA in HepG2 cells. This may be a result of feedback in response to the emodin-induced decrease in the protein of HIF-1[Formula: see text]. In conclusion, emodin decreases hepatic HIF-1[Formula: see text] by inhibiting its biosynthesis.

  1. Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex.

    PubMed

    Bartholdson, S Josefin; Brown, Alan R; Mewburn, Ben R; Clarke, David J; Fry, Stephen C; Campopiano, Dominic J; Govan, John R W

    2008-08-01

    The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET12 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.

  2. High Temperature Induces Expression of Tobacco Transcription Factor NtMYC2a to Regulate Nicotine and JA Biosynthesis

    PubMed Central

    Yang, Liming; Li, Junying; Ji, Jianhui; Li, Ping; Yu, Liangliang; Abd_Allah, Elsayed F.; Luo, Yuming; Hu, Liwei; Hu, Xiangyang

    2016-01-01

    Environmental stress elevates the level of jasmonic acid (JA) and activates the biosynthesis of nicotine and related pyridine alkaloids in tobacco (Nicotiana tabacum L.) by up-regulating the expression of putrescine N-methyltransferase 1 (NtPMT1), which encodes a putrescine N-methyl transferase that catalyzes nicotine formation. The JA signal suppressor JASMONATE ZIM DOMAIN 1 (NtJAZ1) and its target protein, NtMYC2a, also regulate nicotine biosynthesis; however, how these proteins interact to regulate abiotic-induced nicotine biosynthesis is poorly understood. In this study, we found that high-temperature (HT) treatment activated transcription of NtMYC2a, which subsequently stimulated the transcription of genes associated with JA biosynthesis, including Lipoxygenase (LOX), Allene oxide synthase (AOS), Allene oxide cyclase (AOC), and 12-oxophytodienodate reductase (OPR). Overexpression of NtMYC2a increased nicotine biosynthesis by enhancing its binding to the promoter of NtPMT1. Overexpression of either NtJAZ1 or proteasome-resistant NtJAZ1ΔC suppressed nicotine production under normal conditions, but overexpression only of the former resulted in low levels of nicotine under HT treatment. These data suggest that HT induces NtMYC2a accumulation through increased transcription to activate nicotine synthesis; meanwhile, HT-induced NtMYC2a can activate JA synthesis to promote additional NtMYC2a activity by degrading NtJAZ1 at the post-transcriptional level. PMID:27833561

  3. Starch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-Induced Stomatal Closing1[OPEN

    PubMed Central

    Stephan, Aaron B.; Schroeder, Julian I.

    2016-01-01

    Starch metabolism is involved in stomatal movement regulation. However, it remains unknown whether starch-deficient mutants affect CO2-induced stomatal closing and whether starch biosynthesis in guard cells and/or mesophyll cells is rate limiting for high CO2-induced stomatal closing. Stomatal responses to [CO2] shifts and CO2 assimilation rates were compared in Arabidopsis (Arabidopsis thaliana) mutants that were either starch deficient in all plant tissues (ADP-Glc-pyrophosphorylase [ADGase]) or retain starch accumulation in guard cells but are starch deficient in mesophyll cells (plastidial phosphoglucose isomerase [pPGI]). ADGase mutants exhibited impaired CO2-induced stomatal closure, but pPGI mutants did not, showing that starch biosynthesis in guard cells but not mesophyll functions in CO2-induced stomatal closing. Nevertheless, starch-deficient ADGase mutant alleles exhibited partial CO2 responses, pointing toward a starch biosynthesis-independent component of the response that is likely mediated by anion channels. Furthermore, whole-leaf CO2 assimilation rates of both ADGase and pPGI mutants were lower upon shifts to high [CO2], but only ADGase mutants caused impairments in CO2-induced stomatal closing. These genetic analyses determine the roles of starch biosynthesis for high CO2-induced stomatal closing. PMID:27208296

  4. Salicylic acid induces mitochondrial injury by inhibiting ferrochelatase heme biosynthesis activity.

    PubMed

    Gupta, Vipul; Liu, Shujie; Ando, Hideki; Ishii, Ryohei; Tateno, Shumpei; Kaneko, Yuki; Yugami, Masato; Sakamoto, Satoshi; Yamaguchi, Yuki; Nureki, Osamu; Handa, Hiroshi

    2013-12-01

    Salicylic acid is a classic nonsteroidal anti-inflammatory drug. Although salicylic acid also induces mitochondrial injury, the mechanism of its antimitochondrial activity is not well understood. In this study, by using a one-step affinity purification scheme with salicylic acid-immobilized beads, ferrochelatase (FECH), a homodimeric enzyme involved in heme biosynthesis in mitochondria, was identified as a new molecular target of salicylic acid. Moreover, the cocrystal structure of the FECH-salicylic acid complex was determined. Structural and biochemical studies showed that salicylic acid binds to the dimer interface of FECH in two possible orientations and inhibits its enzymatic activity. Mutational analysis confirmed that Trp301 and Leu311, hydrophobic amino acid residues located at the dimer interface, are directly involved in salicylic acid binding. On a gel filtration column, salicylic acid caused a shift in the elution profile of FECH, indicating that its conformational change is induced by salicylic acid binding. In cultured human cells, salicylic acid treatment or FECH knockdown inhibited heme synthesis, whereas salicylic acid did not exert its inhibitory effect in FECH knockdown cells. Concordantly, salicylic acid treatment or FECH knockdown inhibited heme synthesis in zebrafish embryos. Strikingly, the salicylic acid-induced effect in zebrafish was partially rescued by FECH overexpression. Taken together, these findings illustrate that FECH is responsible for salicylic acid-induced inhibition of heme synthesis, which may contribute to its antimitochondrial and anti-inflammatory function. This study establishes a novel aspect of the complex pharmacological effects of salicylic acid.

  5. Disruption of sphingolipid biosynthesis in hepatocyte nodules: selective proliferative stimulus induced by fumonisin B1.

    PubMed

    van der Westhuizen, Liana; Gelderblom, Wentzel C A; Shephard, Gordon S; Swanevelder, Sonja

    2004-07-15

    In order to investigate the role of sphingolipid disruption in the cancer promoting potential of fumonisin B(1) (FB(1)) in the development of hepatocyte nodules, male Fischer 344 rats were subjected to cancer initiation (FB(1) containing diet or diethylnitrosamine (DEN) by i.p. injection) and promotion (2-acetylaminofluorene with partial hepatectomy, 2-AAF/PH) treatments followed by a secondary FB(1) dietary regimen. Sphinganine (Sa) and sphingosine (So) levels were measured by high performance liquid chromatography in control, surrounding and nodular liver tissues of the rats. The disruption of sphingolipid biosynthesis by the secondary FB(1) treatment in the control rats was significantly (P < 0.05) enhanced by the 2-AAF/PH cancer promotion treatment. The nodular and surrounding Sa levels returned to baseline following FB(1) initiation and 2-AAF/PH promotion. When comparing the groups subjected to the secondary FB(1) treatment, the initiation effected by FB(1) was less (P < 0.01) sensitive to the accumulation of Sa in the nodular and surrounding tissues than DEN initiation and the 2-AAF/PH control treatment. In contrast, the So level of FB(1) initiation was marginally increased in the nodules compared to the surrounding liver after 2-AAF/PH promotion and significantly (P < 0.05) higher with the secondary FB(1) treatment. Although, the FB(1)-induced hepatocyte nodules were not resistant to the disruption of sphingolipid biosynthesis, the nodular So levels were increased and might provide a selective growth stimulus possibly induced by bio-active sphingoid intermediates such as sphingosine 1-phosphate (S1P).

  6. Kinetics and localization of wound-induced DNA biosynthesis in potato tuber.

    PubMed

    Lulai, Edward C; Neubauer, Jonathan D; Suttle, Jeffrey C

    2014-11-01

    Tuber wounding induces a cascade of biological responses that are involved in processes required to heal and protect surviving plant tissues. Little is known about the coordination of these processes, including essential wound-induced DNA synthesis, yet they play critical roles in maintaining marketability of the harvested crop and tubers cut for seed. A sensitive "Click-iT EdU Assay" employing incorporation of the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), in conjunction with 4',6-diamindino-2-phenylindole (DAPI) counter labeling, was employed to objectively identify and determine the time course and spatial distribution of tuber nuclei that were wound-induced to enter S-phase of the cell cycle. Both labeling procedures are rapid and sensitive in situ. Following wounding, EdU incorporation (indicating DNA synthesis) was not detectable until after 12h, rapidly reached a maximum at about 18h and then declined to near zero at 48h. About 28% of the nuclei were EdU labeled at 18h reflecting the proportion of cells in S-phase of the cell cycle. During the ∼30h in which induced cells were progressing through S-phase, de novo DNA synthesis extended 7-8 cell layers below the wound surface. Cessation of nuclear DNA synthesis occurred about 4 d prior to completion of wound closing layer formation. Initiation of wound periderm development followed at 7 d, i.e. about 5 d after cessation of nuclear DNA biosynthesis; at this time the phellogen developed and meristematic activity was detected via the production of new phellem cells. Collectively, these results provide new insight into the coordination of wound-induced nucleic acid synthesis with associated tuber wound-healing processes.

  7. Does restraining nitric oxide biosynthesis rescue from toxins-induced parkinsonism and sporadic Parkinson's disease?

    PubMed

    Gupta, Satya Prakash; Yadav, Sharawan; Singhal, Naveen Kumar; Tiwari, Manindra Nath; Mishra, Sarad Kumar; Singh, Mahendra Pratap

    2014-02-01

    Nitric oxide (NO) is an important inorganic molecule of the biological system owing to diverse physiological implications. NO is synthesised from a semi-essential amino acid L-arginine. NO biosynthesis is catalysed by a family of enzymes referred to as nitric oxide synthases (NOSs). NO is accused in many acute and chronic illnesses, which include central nervous system disorders, inflammatory diseases, reproductive impairments, cancer and cardiovascular anomalies. Owing to very unstable nature, NO gets converted into nitrite, peroxynitrite and other reactive nitrogen species that could lead to nitrosative stress in the nigrostriatal system. Nitrosative stress is widely implicated in Parkinson's disease (PD), and its beneficial and harmful effects are demonstrated in in vitro, rodent and primate models of toxins-induced parkinsonism and in the blood, cerebrospinal fluid and nigrostriatal tissues of sporadic PD patients. The current article updates the roles of NO and NOSs in sporadic PD and toxins-induced parkinsonism in rodents along with the scrutiny of how inhibitors of NOSs could open a new line of approach to moderately rescue from PD pathogenesis based on the existing literature. The article also provides a perspective concerning the lack of ample admiration to such an approach and how to minimise the underlying lacunae.

  8. Expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinases during mouse embryonic development.

    PubMed

    Chen, Li; Nakai, Masaaki; Belton, Robert J; Nowak, Romana A

    2007-02-01

    Mouse embryo implantation is a highly invasive and controlled process that involves remodeling and degradation of the extracellular matrix of the uterus. Matrix metalloproteinases (MMPs) are the main proteinases facilitating this process. Extracellular matrix metalloproteinase inducer (EMMPRIN) can stimulate the production of MMPs and is required for successful implantation in the mouse. The aims of the present study were to examine the expression profiles of mRNA and proteins for EMMPRIN and MMPs in the developing mouse embryo in vitro, and to study whether EMMPRIN protein induces the production of MMPs by mouse blastocysts. EMMPRIN mRNA, detected by RT-PCR, was present at all stages of embryo development from the one-cell to the blastocyst outgrowth. EMMPRIN protein, observed by confocal microscopy, was present on the cell surface at the same stages of development as was the mRNA. Of seven MMPs studied, murine collagenase-like A (Mcol-A), murine collagenase-like B (Mcol-B) and gelatinase A (MMP-2) mRNAs were detected only in blastocyst outgrowths by RT-PCR. Gelatinase B (MMP-9) mRNA was detected both in expanded blastocysts and blastocyst outgrowths. MMP-2 and -9 proteins were detected in the cytoplasm of outgrowing trophoblast cells. Collagenase-2 (MMP-8), collagenase-3 (MMP-13), or stromelysin-1 (MMP-3) mRNAs were not present at any stage of pre- or peri-implantation mouse embryo development. Quantitative RT-PCR analyses showed that recombinant EMMPRIN protein did not stimulate MMP-2 or -9 expression by mouse blastocyst outgrowths. These data suggest that EMMPRIN may regulate physiological functions other than MMP production by mouse embryos during implantation.

  9. Extracellular matrix in cutaneous ageing: the effects of 0.1% copper-zinc malonate-containing cream on elastin biosynthesis.

    PubMed

    Mahoney, My G; Brennan, Donna; Starcher, Barry; Faryniarz, Joseph; Ramirez, Jose; Parr, Lisa; Uitto, Jouni

    2009-03-01

    Cutaneous ageing, as visualized at the exposed areas of skin, reflects dramatic alterations in the structure and function of the extracellular matrix of connective tissues. Among them, the elastic fibre network, which is responsible for the physiological elasticity and resilience of normal skin, undergoes degradative changes leading to loss of functional elastic fibres. A potential strategy to counteract these degenerative changes entails topical application of a compound that may lead to regeneration of the elastic fibre network. In this study, we have evaluated the effects of a bi-metal, 0.1% copper-zinc malonate-containing cream that has been shown to efface wrinkles in clinical trials. An effect on elastin biosynthesis and elastic tissue accumulation in skin biopsies was observed in 21 female patients with photoaged facial skin, as measured at baseline and at 6 weeks of treatment. Histopathological evaluation revealed evidence of elastic fibre regeneration, including those extending perpendicularly towards the dermo-epidermal junction within the papillary dermis. Elastin biosynthesis, measured by semi-quantitative immunofluorescence with an antibody recognizing only the newly synthesized, uncrosslinked tropoelastin molecules, suggested statistically significant enhancement of elastin biosynthesis by the bi-metal compound when applied twice daily. Accumulation of elastic fibres was confirmed by assay of desmosine, an elastin-specific crosslink compound. These results suggest that the bi-metal, 0.1% copper-zinc malonate-containing cream has the propensity to increase elastin synthesis in human skin in vivo, and that regeneration of elastic fibres may contribute to wrinkle effacement in female patients with photoaged facial skin.

  10. Repression by H-NS of genes required for the biosynthesis of the Vibrio cholerae biofilm matrix is modulated by the second messenger cyclic diguanylic acid

    PubMed Central

    Ayala, Julio C.; Wang, Hongxia; Silva, Anisia J.; Benitez, Jorge A.

    2015-01-01

    Summary Expression of Vibrio cholerae genes required for the biosynthesis of exopolysacchide (vps) and protein (rbm) components of the biofilm matrix is enhanced by cyclic diguanylate (c-di-GMP). In a previous study, we reported that the H-NS protein represses the transcription of vpsA, vpsL and vpsT. Here we demonstrate that the regulator VpsT can disrupt repressive H-NS nucleoprotein complexes at the vpsA and vpsL promoters in the presence of c-di-GMP while H-NS could disrupt the VpsT-promoter complexes in the absence of c-di-GMP. ChiP-Seq showed a remarkable trend for H-NS to cluster at loci involved in biofilm development such as the rbmABCDEF genes. We show that the antagonistic relationship between VpsT and H-NS regulates the expression of the rbmABCDEF cluster. Epistasis analysis demonstrated that VpsT functions as an antirepressor at the rbmA/F, vpsU and vpsA/L promoters. Deletion of vpsT increased H-NS occupancy at these promoters while increasing the c-di-GMP pool had the opposite effect and included the vpsT promoter. The negative effect of c-di-GMP on H-NS occupancy at the vpsT promoter required the regulator VpsR. These results demonstrate that c-di-GMP activates the transcription of genes required for the biosynthesis of the biofilm matrix by triggering a coordinated VpsR- and VpsT-dependent H-NS antirepression cascade. PMID:25982817

  11. MyD88 regulates physical inactivity-induced skeletal muscle inflammation, ceramide biosynthesis signaling, and glucose intolerance.

    PubMed

    Kwon, Oh Sung; Tanner, Ruth E; Barrows, Katherine M; Runtsch, Marah; Symons, J David; Jalili, Thunder; Bikman, Benjamin T; McClain, Donald A; O'Connell, Ryan M; Drummond, Micah J

    2015-07-01

    Physical inactivity in older adults is a risk factor for developing glucose intolerance and impaired skeletal muscle function. Elevated inflammation and ceramide biosynthesis have been implicated in metabolic disruption and are linked to Toll-like receptor (TLR)/myeloid differentiation primary response 88 (MyD88) signaling. We hypothesize that a physical inactivity stimulus, capable of inducing glucose intolerance, would increase skeletal muscle inflammation and ceramide biosynthesis signaling and that this response would be regulated by the TLR/MyD88 pathway. Therefore, we subjected wild-type (WT) and MyD88(-/-) mice to hindlimb unloading (HU) for 14 days or an ambulatory control period. We observed impaired glucose uptake, muscle insulin signaling (p-Akt), and increased markers of NF-κB signaling (p-IκBα), inflammation (p-JNK, IL-6), TLR4, and the rate-limiting enzyme of ceramide biosynthesis, SPT2, with HU WT (P < 0.05), but not in HU MyD88(-/-) mice. Concurrently, we found that 5 days of bed rest in older adults resulted in whole body glucose dysregulation, impaired skeletal muscle insulin signaling, and upregulation of muscle IL-6 and SPT2 (P < 0.05). Post-bed rest TLR4 abundance was tightly correlated with impaired postprandial insulin and glucose levels. In conclusion, MyD88 signaling is necessary for the increased inflammation, ceramide biosynthesis signaling, and compromised metabolic function that accompanies physical inactivity.

  12. MyD88 regulates physical inactivity-induced skeletal muscle inflammation, ceramide biosynthesis signaling, and glucose intolerance

    PubMed Central

    Kwon, Oh Sung; Tanner, Ruth E.; Barrows, Katherine M.; Runtsch, Marah; Symons, J. David; Jalili, Thunder; Bikman, Benjamin T.; McClain, Donald A.; O'Connell, Ryan M.

    2015-01-01

    Physical inactivity in older adults is a risk factor for developing glucose intolerance and impaired skeletal muscle function. Elevated inflammation and ceramide biosynthesis have been implicated in metabolic disruption and are linked to Toll-like receptor (TLR)/myeloid differentiation primary response 88 (MyD88) signaling. We hypothesize that a physical inactivity stimulus, capable of inducing glucose intolerance, would increase skeletal muscle inflammation and ceramide biosynthesis signaling and that this response would be regulated by the TLR/MyD88 pathway. Therefore, we subjected wild-type (WT) and MyD88−/− mice to hindlimb unloading (HU) for 14 days or an ambulatory control period. We observed impaired glucose uptake, muscle insulin signaling (p-Akt), and increased markers of NF-κB signaling (p-IκBα), inflammation (p-JNK, IL-6), TLR4, and the rate-limiting enzyme of ceramide biosynthesis, SPT2, with HU WT (P < 0.05), but not in HU MyD88−/− mice. Concurrently, we found that 5 days of bed rest in older adults resulted in whole body glucose dysregulation, impaired skeletal muscle insulin signaling, and upregulation of muscle IL-6 and SPT2 (P < 0.05). Post-bed rest TLR4 abundance was tightly correlated with impaired postprandial insulin and glucose levels. In conclusion, MyD88 signaling is necessary for the increased inflammation, ceramide biosynthesis signaling, and compromised metabolic function that accompanies physical inactivity. PMID:25968578

  13. Examination of the Abscission-Associated Transcriptomes for Soybean, Tomato, and Arabidopsis Highlights the Conserved Biosynthesis of an Extensible Extracellular Matrix and Boundary Layer

    PubMed Central

    Kim, Joonyup; Sundaresan, Srivignesh; Philosoph-Hadas, Sonia; Yang, Ronghui; Meir, Shimon; Tucker, Mark L.

    2015-01-01

    Abscission zone (AZ) development and the progression of abscission (detachment of plant organs) have been roughly separated into four stages: first, AZ differentiation; second, competence to respond to abscission signals; third, activation of abscission; and fourth, formation of a protective layer and post-abscission trans-differentiation. Stage three, activation of abscission, is when changes in the cell wall and extracellular matrix occur to support successful organ separation. Most abscission research has focused on gene expression for enzymes that disassemble the cell wall within the AZ and changes in phytohormones and other signaling events that regulate their expression. Here, transcriptome data for soybean, tomato and Arabidopsis were examined and compared with a focus not only on genes associated with disassembly of the cell wall but also on gene expression linked to the biosynthesis of a new extracellular matrix. AZ-specific up-regulation of genes associated with cell wall disassembly including cellulases (beta-1,4-endoglucanases, CELs), polygalacturonases (PGs), and expansins (EXPs) were much as expected; however, curiously, changes in expression of xyloglucan endotransglucosylase/hydrolases (XTHs) were not AZ-specific in soybean. Unexpectedly, we identified an early increase in the expression of genes underlying the synthesis of a waxy-like cuticle. Based on the expression data, we propose that the early up-regulation of an abundance of small pathogenesis-related (PR) genes is more closely linked to structural changes in the extracellular matrix of separating cells than an enzymatic role in pathogen resistance. Furthermore, these observations led us to propose that, in addition to cell wall loosening enzymes, abscission requires (or is enhanced by) biosynthesis and secretion of small proteins (15–25 kDa) and waxes that form an extensible extracellular matrix and boundary layer on the surface of separating cells. The synthesis of the boundary layer

  14. Wounding of potato tubers induces increases in ABA biosynthesis and catabolism and alters expression of ABA metabolic genes.

    PubMed

    Suttle, Jeffrey C; Lulai, Edward C; Huckle, Linda L; Neubauer, Jonathan D

    2013-04-15

    The effects of physical wounding on ABA biosynthesis and catabolism and expression of genes encoding key ABA metabolic enzymes were determined in potato tubers. An increase in ABA and ABA metabolite content was observed 48h after wounding and remained elevated through 96h. Wounding induced dramatic increases in the expression of the ABA metabolic genes encoding zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), and ABA-8'-hydroxylase. Although the patterns of wound-induced expression of individual genes varied, increased gene expression was observed within 3h of wounding and remained elevated through 96h. An apparent correlation between expression of the gene encoding ZEP and the increase in ABA content suggested that the wound-induced increase in ABA biosynthesis was regulated by both substrate availability and increased NCED activity. Suppression of wound-induced jasmonic acid accumulation by rinsing the wounded tissue with water did not inhibit the subsequent increase in ABA content. Exogenous ethylene completely suppressed the wound-induced increase in ABA content and dramatically reduced wound-induced up-regulation of ABA metabolic genes. This study is the first to identify the molecular bases for increased ABA accumulation following physical trauma in potato tubers and highlights the complex physiological interactions between various wound-induced hormones.

  15. Surface modification of ceramic matrix composites induced by laser treatment

    NASA Astrophysics Data System (ADS)

    Costil, S.; Lukat, S.; Langlade, C.; Coddet, C.

    2008-12-01

    Ceramics or ceramic composites present many advantages (hardness, chemical resistance, low density, etc.) which induce some more and more important applications particularly from the industrial point of view. The evolution of technology can also be beneficial to enlarge their global application areas. This is particularly the aim of this work which consists in applying a laser beam on the ceramic in order to clean its surface. A Nd:YAG laser has been used to study the basic mechanism roughening the surface of silicon carbide composite (ceramic matrix composite (CMC)). Investigations on different surfaces (two chemical compositions) show a strong influence of the nature of the material on the development of a characteristic conic structure. Microscopic studies (SEM) and elementary analyses (EDS and RMS) demonstrated the formation of a regular cone-like structure with a kinetic and a chemical modification specific to each material.

  16. Prediction of thermal cycling induced cracking in polmer matrix composites

    NASA Technical Reports Server (NTRS)

    Mcmanus, Hugh L.

    1994-01-01

    The work done in the period August 1993 through February 1994 on the 'Prediction of Thermal Cycling Induced Cracking In Polymer Matrix Composites' program is summarized. Most of the work performed in this period, as well as the previous one, is described in detail in the attached Master's thesis, 'Analysis of Thermally Induced Damage in Composite Space Structures,' by Cecelia Hyun Seon Park. Work on a small thermal cycling and aging chamber was concluded in this period. The chamber was extensively tested and calibrated. Temperatures can be controlled very precisely, and are very uniform in the test chamber. Based on results obtained in the previous period of this program, further experimental progressive cracking studies were carried out. The laminates tested were selected to clarify the differences between the behaviors of thick and thin ply layers, and to explore other variables such as stacking sequence and scaling effects. Most specimens tested were made available from existing stock at Langley Research Center. One laminate type had to be constructed from available prepreg material at Langley Research Center. Specimens from this laminate were cut and prepared at MIT. Thermal conditioning was carried out at Langley Research Center, and at the newly constructed MIT facility. Specimens were examined by edge inspection and by crack configuration studies, in which specimens were sanded down in order to examine the distribution of cracks within the specimens. A method for predicting matrix cracking due to decreasing temperatures and/or thermal cycling in all plies of an arbitrary laminate was implemented as a computer code. The code also predicts changes in properties due to the cracking. Extensive correlations between test results and code predictions were carried out. The computer code was documented and is ready for distribution.

  17. Enhanced salt-induced antioxidative responses involve a contribution of polyamine biosynthesis in grapevine plants.

    PubMed

    Ikbal, Fatima Ezzohra; Hernández, José Antonio; Barba-Espín, Gregorio; Koussa, Tayeb; Aziz, Aziz; Faize, Mohamed; Diaz-Vivancos, Pedro

    2014-06-15

    The possible involvement of polyamines in the salt stress adaptation was investigated in grapevine (Vitis vinifera L.) plantlets focusing on photosynthesis and oxidative metabolism. Salt stress resulted in the deterioration of plant growth and photosynthesis, and treatment of plantlets with methylglyoxal-bis(guanylhydrazone) (MGBG), a S-adenosylmethionine decarboxylase (SAMDC) inhibitor, enhanced the salt stress effect. A decrease in PSII quantum yield (Fv/Fm), effective PSII quantum yield (Y(II)) and coefficient of photochemical quenching (qP) as well as increases in non-photochemical quenching (NPQ) and its coefficient (qN) was observed by these treatments. Salt and/or MGBG treatments also triggered an increase in lipid peroxidation and reactive oxygen species (ROS) accumulation as well as an increase of superoxide dismutase (SOD) and peroxidase (POX) activities, but not ascorbate peroxidase (APX) activity. Salt stress also resulted in an accumulation of oxidized ascorbate (DHA) and a decrease in reduced glutathione. MGBG alone or in combination with salt stress increased monodehydroascorbate reductase (MDHAR), SOD and POX activities and surprisingly no accumulation of DHA was noticed following treatment with MGBG. These salt-induced responses correlated with the maintaining of high level of free and conjugated spermidine and spermine, whereas a reduction of agmatine and putrescine levels was observed, which seemed to be amplified by the MGBG treatment. These results suggest that maintaining polyamine biosynthesis through the enhanced SAMDC activity in grapevine leaf tissues under salt stress conditions could contribute to the enhanced ROS scavenging activity and a protection of photosynthetic apparatus from oxidative damages.

  18. Abscisic acid induces biosynthesis of bisbibenzyls and tolerance to UV-C in the liverwort Marchantia polymorpha.

    PubMed

    Kageyama, Akito; Ishizaki, Kimitsune; Kohchi, Takayuki; Matsuura, Hideyuki; Takahashi, Kosaku

    2015-09-01

    Environmental stresses are effective triggers for the biosynthesis of various secondary metabolites in plants, and phytohormones such as jasmonic acid and abscisic acid are known to mediate such responses in flowering plants. However, the detailed mechanism underlying the regulation of secondary metabolism in bryophytes remains unclear. In this study, the induction mechanism of secondary metabolites in the model liverwort Marchantia polymorpha was investigated. Abscisic acid (ABA) and ultraviolet irradiation (UV-C) were found to induce the biosynthesis of isoriccardin C, marchantin C, and riccardin F, which are categorized as bisbibenzyls, characteristic metabolites of liverworts. UV-C led to the significant accumulation of ABA. Overexpression of MpABI1, which encodes protein phosphatase 2C (PP2C) as a negative regulator of ABA signaling, suppressed accumulation of bisbibenzyls in response to ABA and UV-C irradiation and conferred susceptibility to UV-C irradiation. These data show that ABA plays a significant role in the induction of bisbibenzyl biosynthesis, which might confer tolerance against UV-C irradiation in M. polymorpha.

  19. Viral serine palmitoyltransferase induces metabolic switch in sphingolipid biosynthesis and is required for infection of a marine alga

    PubMed Central

    Ziv, Carmit; Malitsky, Sergey; Ben-Dor, Shifra; Wei, Yu; Zheng, Shuning; Aharoni, Asaph; Vardi, Assaf

    2016-01-01

    Marine viruses are the most abundant biological entities in the oceans shaping community structure and nutrient cycling. The interaction between the bloom-forming alga Emiliania huxleyi and its specific large dsDNA virus (EhV) is a major factor determining the fate of carbon in the ocean, thus serving as a key host-pathogen model system. The EhV genome encodes for a set of genes involved in the de novo sphingolipid biosynthesis, not reported in any viral genome to date. We combined detailed lipidomic and biochemical analyses to characterize the functional role of this virus-encoded pathway during lytic viral infection. We identified a major metabolic shift, mediated by differential substrate specificity of virus-encoded serine palmitoyltransferase, a key enzyme of sphingolipid biosynthesis. Consequently, unique viral glycosphingolipids, composed of unusual hydroxylated C17 sphingoid bases (t17:0) were highly enriched in the infected cells, and their synthesis was found to be essential for viral assembly. These findings uncover the biochemical bases of the virus-induced metabolic rewiring of the host sphingolipid biosynthesis during the chemical “arms race” in the ocean. PMID:26984500

  20. Prediction of thermal cycling induced cracking in polymer matrix composites

    NASA Technical Reports Server (NTRS)

    Mcmanus, Hugh L.

    1993-01-01

    This report summarizes the work done in the period February 1993 through July 1993 on the 'Prediction of Thermal Cycling Induced Cracking In Polymer Matrix Composites' program. An oral presentation of this work was given to Langley personnel in September of 1993. This document was prepared for archival purposes. Progress studies have been performed on the effects of spatial variations in material strength. Qualitative agreement was found with observed patterns of crack distribution. These results were presented to NASA Langley personnel in November 1992. The analytical methodology developed by Prof. McManus in the summer of 1992 (under an ASEE fellowship) has been generalized. A method for predicting matrix cracking due to decreasing temperatures and/or thermal cycling in all plies of an arbitrary laminate has been implemented as a computer code. The code also predicts changes in properties due to the cracking. Experimental progressive cracking studies on a variety of laminates were carried out at Langley Research Center. Results were correlated to predictions using the new methods. Results were initially mixed. This motivated an exploration of the configuration of cracks within laminates. A crack configuration study was carried out by cutting and/or sanding specimens in order to examine the distribution of cracks within the specimens. These investigations were supplemented by dye-penetrant enhanced X-ray photographs. The behavior of thin plies was found to be different from the behavior of thicker plies (or ply groups) on which existing theories are based. Significant edge effects were also noted, which caused the traditional metric of microcracking (count of cracks on a polished edge) to be very inaccurate in some cases. With edge and configuration taken into account, rough agreement with predictions was achieved. All results to date were reviewed with NASA Langley personnel in September 1993.

  1. Prostacyclin biosynthesis and reduced 5-HT uptake after complement-induced endothelial injury in the dog isolated lung.

    PubMed Central

    Bult, H.; Heiremans, J. J.; Herman, A. G.; Malcorps, C. M.; Peeters, F. A.

    1988-01-01

    1. Pulmonary prostacyclin (PGI2) biosynthesis was evaluated in relation to endothelial integrity before and after complement activation in isolated plasma-perfused lung lobes of the dog. 2. The plasma was activated with zymosan (ZAP, n = 4), yeast cells (YAP, n = 4) or yeast with 3 microM indomethacin (Indo + YAP, n = 3). Immunoreactive 6-oxo-prostaglandin F1 alpha (i-6-oxo-PGF1 alpha) and thromboxane B2 (iTXB2) were measured to monitor PGI2 and TXA2 biosynthesis. 3. The kinetic parameters Km and Vmax of 5-hydroxytryptamine (5-HT) uptake were calculated on the basis of multiple indicator diffusion data to evaluate endothelial integrity. 4. YAP and ZAP induced a biphasic increase of the arterial perfusion pressure. The immediate pressure peak was partly mediated by TXA2 and the TXB2 was subsequently cleared by the lung. 5. The apparent Vmax of 5-HT uptake remained constant throughout the experiment. Thus, complement activation did not affect the number of endothelial 5-HT carrier sites available to the perfusate. 6. The apparent Km of 5-HT uptake was enhanced in 9 lungs exposed to activated plasma complement for 20 min. This decreased affinity for 5-HT probably reflects endothelial injury. It was transient as the apparent Km had returned to the baseline value after 60 min. 7. PGI2 clearance and biosynthesis were virtually absent in the control period. PGI2 formation increased drastically after infusion of ZAP or YAP and was proportional to the endothelial injury expressed as elevated Km or pulmonary oedema. Thus, PGI2 biosynthesis might be a marker of severe endothelial distress. PMID:3291998

  2. Overexpression of an ABA biosynthesis gene using a stress inducible promoter enhances drought resistance in petunia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants respond to drought stress by closing their stomata and reducing transpirational water loss. The plant hormone abscisic acid (ABA) regulates growth and stomatal closure particularly when the plant is under environmental stresses. One of the key enzymes in the ABA biosynthesis of higher plants ...

  3. Investigation of Dynamics Biosynthesis of Phytoalexins induced in Malva Sylvestris by the Verticilium Dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biosynthesis of phytoalexins is an active mechanism utilized by plants to protect against pathogens. Phytoalexins from wild plant species may be more potent than those produced in cultivated plants. Two terpenoid substances from the pathogen-infected plant Malva sylvestris L. were isolated using t...

  4. Oxidative stress induces the biosynthesis of citrinin by Penicillium verrucosum at the expense of ochratoxin.

    PubMed

    Schmidt-Heydt, Markus; Stoll, Dominic; Schütz, Peter; Geisen, Rolf

    2015-01-02

    Penicillium verrucosum is a fungus that can produce ochratoxin A and citrinin, two structurally related nephrotoxic mycotoxins. P. verrucosum usually occurs on wheat but can occasionally also be found in NaCl rich habitats such as salted cheeses or olives, indicating that this fungus can adapt to different environments. The ratio of ochratoxin A to citrinin produced by P. verrucosum is shifted to one of either mycotoxin at the expense of the other dependent on the environmental conditions. High NaCl concentrations shift secondary metabolite biosynthesis towards ochratoxin A production. P. verrucosum copes with NaCl stress by increased ochratoxin A biosynthesis, ensuring chloride homeostasis. Ochratoxin A carries chlorine in its molecule and can excrete chlorine from the cell. It was further shown that the regulation of ochratoxin A by high NaCl conditions is mediated by the HOG MAP kinase signal transduction pathway. Here it is shown that high oxidative stress conditions, evoked for example by increasing concentrations of Cu(2+) cations in the growth medium, shift secondary metabolite biosynthesis of P. verrucosum from ochratoxin A to citrinin. The production of citrinin normalizes the oxidative status of the fungal cell under oxidative stress conditions leading to an adaptation to these environmental conditions and protects against increased oxidative stress caused by increased Cu(2+) concentrations. Moreover citrinin also protects against light of short wavelength, which may also increase the oxidative status of the environment. The biosynthesis of citrinin is apparently regulated by a cAMP/PKA signaling pathway, because increasing amounts of external cAMP reduce citrinin biosynthesis in a concentration dependent manner. These conditions lead to the cross-regulation of the ochratoxin A/citrinin secondary metabolite pair and support the adaptation of P. verrucosum to different environments.

  5. Exogenous GA₃ Application Enhances Xylem Development and Induces the Expression of Secondary Wall Biosynthesis Related Genes in Betula platyphylla.

    PubMed

    Guo, Huiyan; Wang, Yucheng; Liu, Huizi; Hu, Ping; Jia, Yuanyuan; Zhang, Chunrui; Wang, Yanmin; Gu, Shan; Yang, Chuanping; Wang, Chao

    2015-09-23

    Gibberellin (GA) is a key signal molecule inducing differentiation of tracheary elements, fibers, and xylogenesis. However the molecular mechanisms underlying the effect of GA on xylem elongation and secondary wall development in tree species remain to be determined. In this study, Betula platyphylla (birch) seeds were treated with 300 ppm GA₃ and/or 300 ppm paclobutrazol (PAC), seed germination was recorded, and transverse sections of hypocotyls were stained with toluidine blue; the two-month-old seedlings were treated with 50 μM GA₃ and/or 50 μM PAC, transverse sections of seedling stems were stained using phloroglucinol-HCl, and secondary wall biosynthesis related genes expression was analyzed by real-time quantitative PCR. Results indicated that germination percentage, energy and time of seeds, hypocotyl height and seedling fresh weight were enhanced by GA₃, and reduced by PAC; the xylem development was wider in GA₃-treated plants than in the control; the expression of NAC and MYB transcription factors, CESA, PAL, and GA oxidase was up-regulated during GA₃ treatment, suggesting their role in GA₃-induced xylem development in the birch. Our results suggest that GA₃ induces the expression of secondary wall biosynthesis related genes to trigger xylogenesis in the birch plants.

  6. Regulation of ascorbate biosynthesis in green algae has evolved to enable rapid stress-induced response via the VTC2 gene encoding GDP-l-galactose phosphorylase.

    PubMed

    Vidal-Meireles, André; Neupert, Juliane; Zsigmond, Laura; Rosado-Souza, Laise; Kovács, László; Nagy, Valéria; Galambos, Anikó; Fernie, Alisdair R; Bock, Ralph; Tóth, Szilvia Z

    2017-04-01

    Ascorbate (vitamin C) plays essential roles in stress resistance, development, signaling, hormone biosynthesis and regulation of gene expression; however, little is known about its biosynthesis in algae. In order to provide experimental proof for the operation of the Smirnoff-Wheeler pathway described for higher plants and to gain more information on the regulation of ascorbate biosynthesis in Chlamydomonas reinhardtii, we targeted the VTC2 gene encoding GDP-l-galactose phosphorylase using artificial microRNAs. Ascorbate concentrations in VTC2 amiRNA lines were reduced to 10% showing that GDP-l-galactose phosphorylase plays a pivotal role in ascorbate biosynthesis. The VTC2 amiRNA lines also grow more slowly, have lower chlorophyll content, and are more susceptible to stress than the control strains. We also demonstrate that: expression of the VTC2 gene is rapidly induced by H2 O2 and (1) O2 resulting in a manifold increase in ascorbate content; in contrast to plants, there is no circadian regulation of ascorbate biosynthesis; photosynthesis is not required per se for ascorbate biosynthesis; and Chlamydomonas VTC2 lacks negative feedback regulation by ascorbate in the physiological concentration range. Our work demonstrates that ascorbate biosynthesis is also highly regulated in Chlamydomonas albeit via mechanisms distinct from those previously described in land plants.

  7. Extracellular matrix and sex-inducing pheromone in Volvox.

    PubMed

    Hallmann, Armin

    2003-01-01

    During evolution of multicellularity it was imperative to create a complex, multifunctional extracellular matrix (ECM) out of the simple cell wall of a unicellular ancestor. The green alga Volvox represents one of the simplest multicellular organisms, but even so, it already has a highly developed ECM. This ECM is mainly composed of an assortment of glycoproteins, many of which are hydroxyproline rich and extensively sulfated. Several ECM proteins are cross-linked and might have only structural functions. However, the ECM does not represent a static but rather a dynamic and multifunctional interface between a cell and its neighboring cells or its environment. It not only provides protection and structural support for the shape of each cell and the organism as a whole, but also plays a broad range of biological roles in growth, development, reproduction, and responses to environmental stress or wounding. The variety of functions of the ECM requires many glycoproteins to do the work. To attain a high flexibility and adaptability, almost all ECM glycoproteins from Volvox consist of modules, defined as functional subunits that form modular mosaic proteins with an outstanding combinatorial potential. The ECM's functions are not only extensive but also change under developmental control or by environmental incidents. The changing scope of duties necessitates a permanent ECM turnover and remodeling. In Volvox carteri one particularly challenging trigger of such ECM modifications is a sex-inducing pheromone, which is one of the most potent biological effector molecules known: the glycoprotein pheromone is fully effective for inducing sexual development in males and females at concentrations as low as 10(-16) M. The earliest detectable response to the pheromone is the synthesis of ECM glycoproteins.

  8. Transgenic analysis reveals LeACS-1 as a positive regulator of ethylene-induced shikonin biosynthesis in Lithospermum erythrorhizon hairy roots.

    PubMed

    Fang, Rongjun; Wu, Fengyao; Zou, Ailan; Zhu, Yu; Zhao, Hua; Zhao, Hu; Liao, Yonghui; Tang, Ren-Jie; Yang, Tongyi; Pang, Yanjun; Wang, Xiaoming; Yang, Rongwu; Qi, Jinliang; Lu, Guihua; Yang, Yonghua

    2016-03-01

    The phytohormone ethylene (ET) is a crucial signaling molecule that induces the biosynthesis of shikonin and its derivatives in Lithospermum erythrorhizon shoot cultures. However, the molecular mechanism and the positive regulators involved in this physiological process are largely unknown. In this study, the function of LeACS-1, a key gene encoding the 1-aminocyclopropane-1-carboxylic acid synthase for ET biosynthesis in L. erythrorhizon hairy roots, was characterized by using overexpression and RNA interference (RNAi) strategies. The results showed that overexpression of LeACS-1 significantly increased endogenous ET concentration and shikonin production, consistent with the up-regulated genes involved in ET biosynthesis and transduction, as well as the genes related to shikonin biosynthesis. Conversely, RNAi of LeACS-1 effectively decreased endogenous ET concentration and shikonin production and down-regulated the expression level of above genes. Correlation analysis showed a significant positive linear relationship between ET concentration and shikonin production. All these results suggest that LeACS-1 acts as a positive regulator of ethylene-induced shikonin biosynthesis in L. erythrorhizon hairy roots. Our work not only gives new insights into the understanding of the relationship between ET and shikonin biosynthesis, but also provides an efficient genetic engineering target gene for secondary metabolite production in non-model plant L. erythrorhizon.

  9. Involvement of lipids in dimethoate-induced inhibition of testosterone biosynthesis in rat interstitial cells.

    PubMed

    Astiz, Mariana; Hurtado de Catalfo, Graciela E; de Alaniz, María J T; Marra, Carlos Alberto

    2009-08-01

    The mechanism involved in the inhibition of testosterone (Te) biosynthesis after a sub-chronic exposure to low doses of dimethoate (D) was studied in rat interstitial cells (IC). Expression of COX-2 in IC isolated from D-treated rats increased by 44% over C data, while transcription of StAR decreased by approx. 50% and the expression of this protein was diminished by approximately 40%. PGE(2) and PGF(2alpha) were increased by 61 and 78%, respectively. Te concentration decreased by 49% in IC homogenates. Concomitantly, plasma concentration of LH and FSH both increased. Araquidonate (ARA) and C(22) fatty acyl chains in phospholipids from IC mitochondrial fraction decreased by approx. 30% after D treatment. Protein carbonyls, lipoperoxides and nitrite content increased while alpha-tocopherol and the antioxidant capacity of the soluble cellular fraction decreased significantly. Stimulation with h-CG 10 nM overnight failed to overcome the inhibition caused by D on both Te biosynthesis and 3beta- and 17beta-hydroxysteroid dehydrogenases. Decreased Te biosynthesis may be attributed to (1) inhibition of StAR protein activity due to the stimulation of COX-2 and the overproduction of PGF(2alpha), (2) decreased stimulatory effect of ARA on StAR with a subsequent reduction in the availability of CHO for the androgenic pathway, and/or (3) indirect inhibition of steroidogenic enzymes by a lower transcriptional rate caused by elevated PGF(2alpha). Rofecoxib administration prevents the deleterious effect(s) exerted by D.

  10. Decellularized zebrafish cardiac extracellular matrix induces mammalian heart regeneration

    PubMed Central

    Chen, William C. W.; Wang, Zhouguang; Missinato, Maria Azzurra; Park, Dae Woo; Long, Daniel Ward; Liu, Heng-Jui; Zeng, Xuemei; Yates, Nathan A.; Kim, Kang; Wang, Yadong

    2016-01-01

    Heart attack is a global health problem that leads to significant morbidity, mortality, and health care burden. Adult human hearts have very limited regenerative capability after injury. However, evolutionarily primitive species generally have higher regenerative capacity than mammals. The extracellular matrix (ECM) may contribute to this difference. Mammalian cardiac ECM may not be optimally inductive for cardiac regeneration because of the fibrotic, instead of regenerative, responses in injured adult mammalian hearts. Given the high regenerative capacity of adult zebrafish hearts, we hypothesize that decellularized zebrafish cardiac ECM (zECM) made from normal or healing hearts can induce mammalian heart regeneration. Using zebrafish and mice as representative species of lower vertebrates and mammals, we show that a single administration of zECM, particularly the healing variety, enables cardiac functional recovery and regeneration of adult mouse heart tissues after acute myocardial infarction. zECM-treated groups exhibit proliferation of the remaining cardiomyocytes and multiple cardiac precursor cell populations and reactivation of ErbB2 expression in cardiomyocytes. Furthermore, zECM exhibits pro-proliferative and chemotactic effects on human cardiac precursor cell populations in vitro. These contribute to the structural preservation and correlate with significantly higher cardiac contractile function, notably less left ventricular dilatation, and substantially more elastic myocardium in zECM-treated hearts than control animals treated with saline or decellularized adult mouse cardiac ECM. Inhibition of ErbB2 activity abrogates beneficial effects of zECM administration, indicating the possible involvement of ErbB2 signaling in zECM-mediated regeneration. This study departs from conventional focuses on mammalian ECM and introduces a new approach for cardiac tissue regeneration. PMID:28138518

  11. CPU86017-RS attenuate hypoxia-induced testicular dysfunction in mice by normalizing androgen biosynthesis genes and pro-inflammatory cytokines

    PubMed Central

    Zhang, Guo-lin; Yu, Feng; Dai, De-zai; Cheng, Yu-si; Zhang, Can; Dai, Yin

    2012-01-01

    Aim: Downregulation of androgen biosynthesis genes StAR (steroidogenic acute regulatory) and 3β-HSD (3β-hydroxysteroid dehydrogenase) contributes to low testosterone levels in hypoxic mice and is possibly related to increased expression of pro-inflammatory cytokines in the testis. The aim of this study is to investigate the effects of CPU86017-RS that block Ca2+ influx on hypoxia-induced testis insult in mice. Methods: Male ICR mice were divided into 5 groups: control group, hypoxia group, hypoxia group treated with nifedipine (10 mg/kg), hypoxia groups treated with CPU86017-RS (60 or 80 mg/kg). Hypoxia was induced by placing the mice in a chamber under 10%±0.5% O2 for 28 d (8 h per day). The mice were orally administered with drug in the last 14 d. At the end of experiment the testes of the mice were harvested. The mRNA and protein levels of StAR, 3β-HSD, connexin 43 (Cx43), matrix metalloprotease 9 (MMP9), endothelin receptor A (ETAR) and leptin receptor (OBRb) were analyzed using RT-PCR and Western blotting, respectively. The malondialdehyde (MDA), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and acid phosphatase (ACP) levels were measured using biochemical kits. Serum testosterone concentration was measured with radioimmunoassay. Results: Hypoxia significantly increased the MDA level, and decreased the LDH, ACP and SDH activities in testes. Meanwhile, hypoxia induced significant downregulation of StAR and 3β-HSD in testes responsible for reduced testosterone biosynthesis. It decreased the expression of Cx43, and increased the expression of MMP9, ETAR and OBRb, leading to abnormal testis function and structure. These changes were effectively diminished by CPU86017-RS (80 mg/kg) or nifedipine (10 mg/kg). Conclusion: Low plasma testosterone level caused by hypoxia was due to downregulation of StAR and 3β-HSD genes, in association with an increased expression of pro-inflammatory cytokines. These changes can be alleviated by CPU86017-RS or

  12. Rhubarb Antagonizes Matrix Metalloproteinase-9-induced Vascular Endothelial Permeability

    PubMed Central

    Cui, Yun-Liang; Zhang, Sheng; Tian, Zhao-Tao; Lin, Zhao-Fen; Chen, De-Chang

    2016-01-01

    Background: Intact endothelial structure and function are critical for maintaining microcirculatory homeostasis. Dysfunction of the latter is an underlying cause of various organ pathologies. In a previous study, we showed that rhubarb, a traditional Chinese medicine, protected intestinal mucosal microvascular endothelial cells in rats with metastasizing septicemia. In this study, we investigated the effects and mechanisms of rhubarb on matrix metalloproteinase-9 (MMP9)-induced vascular endothelial (VE) permeability. Methods: Rhubarb monomers were extracted and purified by a series of chromatography approaches. The identity of these monomers was analyzed by hydrogen-1 nuclear magnetic resonance (NMR), carbon-13 NMR, and distortionless enhancement by polarization transfer magnetic resonance spectroscopy. We established a human umbilical vein endothelial cell (HUVEC) monolayer on a Transwell insert. We measured the HUVEC permeability, proliferation, and the secretion of VE-cadherin into culture medium using fluorescein isothiocyanate-dextran assay, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, and enzyme-linked immunosorbent assay, respectively, in response to treatment with MMP9 and/or rhubarb monomers. Results: A total of 21 rhubarb monomers were extracted and identified. MMP9 significantly increased the permeability of the HUVEC monolayer, which was significantly reduced by five individual rhubarb monomer (emodin, 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid, 1-O-caffeoyl-2-(4-hydroxyl-O-cinnamoyl)-β-D-glucose, daucosterol linoleate, and rhein) or a combination of all five monomers (1 μmol/L for each monomer). Mechanistically, the five-monomer mixture at 1 μmol/L promoted HUVEC proliferation. In addition, MMP9 stimulated the secretion of VE-cadherin into the culture medium, which was significantly inhibited by the five-monomer mixture. Conclusions: The rhubarb mixture of emodin, 3,8-dihydroxy-1-methyl-anthraquinone-2

  13. Photo-induced biosynthesis of silver nanoparticles from aqueous extract of Dunaliella salina and their anticancer potential.

    PubMed

    Singh, Ankit Kumar; Tiwari, Ratnakar; Kumar, Vijay; Singh, Prabhakar; Riyazat Khadim, S K; Tiwari, Anupam; Srivastava, Vikas; Hasan, S H; Asthana, R K

    2017-01-01

    The synthesis of silver nanoparticles (AgNPs) via green route, using biological entities is an area of interest, because one of the potential applications in the nanomedicine. In the present study, we have developed photo-induced, ecofriendly, low cost method for biosynthesis of the stable silver nanoparticles using aqueous extract of Dunaliella salina (AED) which act as both reducing as well as stabilizing agent. Biosynthesis of the AgNPs was optimized as: sunlight exposure (30min), AED (5% (v/v)) and AgNO3 (4mM). Biosynthesis of AgNPs was monitored by using UV-Vis spectroscopy which exhibited sharp SPR band at 430nm after 30min of bright sunlight exposure. SEM and TEM analyses confirmed the presence of spherical AgNPs with average size of 15.26nm. Crystalline nature of AgNPs was confirmed by SAED and XRD analyses where Braggs reflection pattern at (111), (200), (220) and (311) corresponded to face centered cubic crystal lattice of metallic silver. FTIR analysis revealed the involvement of various functional groups present in AED. AFM analysis confirmed the average surface roughness of synthesized AgNPs as 8.48nm. AgNPs were also screened for anticancer potential using assay of calcein AM/PI, Annexin/PI and cancer biomarkers against cancer cell line (MCF-7), while normal cell line (MCF-10A) were kept as control. Interestingly, anticancer potential was comparable to the known anticancer drug (Cisplatin), and was not detrimental to the normal cell line. Therefore, such green synthesized AgNPs may be explored as anticancer agent.

  14. TAA1-regulated local auxin biosynthesis in the root-apex transition zone mediates the aluminum-induced inhibition of root growth in Arabidopsis.

    PubMed

    Yang, Zhong-Bao; Geng, Xiaoyu; He, Chunmei; Zhang, Feng; Wang, Rong; Horst, Walter J; Ding, Zhaojun

    2014-07-01

    The transition zone (TZ) of the root apex is the perception site of Al toxicity. Here, we show that exposure of Arabidopsis thaliana roots to Al induces a localized enhancement of auxin signaling in the root-apex TZ that is dependent on TAA1, which encodes a Trp aminotransferase and regulates auxin biosynthesis. TAA1 is specifically upregulated in the root-apex TZ in response to Al treatment, thus mediating local auxin biosynthesis and inhibition of root growth. The TAA1-regulated local auxin biosynthesis in the root-apex TZ in response to Al stress is dependent on ethylene, as revealed by manipulating ethylene homeostasis via the precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid, the inhibitor of ethylene biosynthesis aminoethoxyvinylglycine, or mutant analysis. In response to Al stress, ethylene signaling locally upregulates TAA1 expression and thus auxin responses in the TZ and results in auxin-regulated root growth inhibition through a number of auxin response factors (ARFs). In particular, ARF10 and ARF16 are important in the regulation of cell wall modification-related genes. Our study suggests a mechanism underlying how environmental cues affect root growth plasticity through influencing local auxin biosynthesis and signaling.

  15. Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica napus.

    PubMed

    Rodríguez-Sanz, Héctor; Solís, María-Teresa; López, María-Fernanda; Gómez-Cadenas, Aurelio; Risueño, María C; Testillano, Pilar S

    2015-07-01

    Isolated microspores are reprogrammed in vitro by stress, becoming totipotent cells and producing embryos and plants via a process known as microspore embryogenesis. Despite the abundance of data on auxin involvement in plant development and embryogenesis, no data are available regarding the dynamics of auxin concentration, cellular localization and the expression of biosynthesis genes during microspore embryogenesis. This work involved the analysis of auxin concentration and cellular accumulation; expression of TAA1 and NIT2 encoding enzymes of two auxin biosynthetic pathways; expression of the PIN1-like efflux carrier; and the effects of inhibition of auxin transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-chlorophenoxy) isobutyric acid (PCIB) during Brassica napus microspore embryogenesis. The results indicated de novo auxin synthesis after stress-induced microspore reprogramming and embryogenesis initiation, accompanying the first cell divisions. The progressive increase of auxin concentration during progression of embryogenesis correlated with the expression patterns of TAA1 and NIT2 genes of auxin biosynthetic pathways. Auxin was evenly distributed in early embryos, whereas in heart/torpedo embryos auxin was accumulated in apical and basal embryo regions. Auxin efflux carrier PIN1-like gene expression was induced in early multicellular embryos and increased at the globular/torpedo embryo stages. Inhibition of polar auxin transport (PAT) and action, by NPA and PCIB, impaired embryo development, indicating that PAT and auxin action are required for microspore embryo progression. NPA also modified auxin embryo accumulation patterns. These findings indicate that endogenous auxin biosynthesis, action and polar transport are required in stress-induced microspore reprogramming, embryogenesis initiation and progression.

  16. Stress-induced neutral lipid biosynthesis in microalgae - Molecular, cellular and physiological insights.

    PubMed

    Zienkiewicz, Krzysztof; Du, Zhi-Yan; Ma, Wei; Vollheyde, Katharina; Benning, Christoph

    2016-09-01

    Photosynthetic microalgae have promise as biofuel feedstock. Under certain conditions, they produce substantial amounts of neutral lipids, mainly in the form of triacylglycerols (TAGs), which can be converted to fuels. Much of our current knowledge on the genetic and molecular basis of algal neutral lipid metabolism derives mainly from studies of plants, i.e. seed tissues, and to a lesser extent from direct studies of algal lipid metabolism. Thus, the knowledge of TAG synthesis and the cellular trafficking of TAG precursors in algal cells is to a large extent based on genome predictions, and most aspects of TAG metabolism have yet to be experimentally verified. The biofuel prospects of microalgae have raised the interest in mechanistic studies of algal TAG biosynthesis in recent years and resulted in an increasing number of publications on lipid metabolism in microalgae. In this review we summarize the current findings on genetic, molecular and physiological studies of TAG accumulation in microalgae. Special emphasis is on the functional analysis of key genes involved in TAG synthesis, molecular mechanisms of regulation of TAG biosynthesis, as well as on possible mechanisms of lipid droplet formation in microalgal cells. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

  17. Effects of freezing-induced cell-fluid-matrix interactions on the cells and extracellular matrix of engineered tissues.

    PubMed

    Teo, Ka Yaw; DeHoyos, Tenok O; Dutton, J Craig; Grinnell, Frederick; Han, Bumsoo

    2011-08-01

    The two most significant challenges for successful cryopreservation of engineered tissues (ETs) are preserving tissue functionality and controlling highly tissue-type dependent preservation outcomes. In order to address these challenges, freezing-induced cell-fluid-matrix interactions should be understood, which determine the post-thaw cell viability and extracellular matrix (ECM) microstructure. However, the current understanding of this tissue-level biophysical interaction is still limited. In this study, freezing-induced cell-fluid-matrix interactions and their impact on the cells and ECM microstructure of ETs were investigated using dermal equivalents as a model ET. The dermal equivalents were constructed by seeding human dermal fibroblasts in type I collagen matrices with varying cell seeding density and collagen concentration. While these dermal equivalents underwent an identical freeze/thaw condition, their spatiotemporal deformation during freezing, post-thaw ECM microstructure, and cellular level cryoresponse were characterized. The results showed that the extent and characteristics of freezing-induced deformation were significantly different among the experimental groups, and the ETs with denser ECM microstructure experienced a larger deformation. The magnitude of the deformation was well correlated to the post-thaw ECM structure, suggesting that the freezing-induced deformation is a good indicator of post-thaw ECM structure. A significant difference in the extent of cellular injury was also noted among the experimental groups, and it depended on the extent of freezing-induced deformation of the ETs and the initial cytoskeleton organization. These results suggest that the cells have been subjected to mechanical insult due to the freezing-induced deformation as well as thermal insult. These findings provide insight on tissue-type dependent cryopreservation outcomes, and can help to design and modify cryopreservation protocols for new types of tissues from

  18. Cannabinoids attenuate norepinephrine-induced melatonin biosynthesis in the rat pineal gland by reducing arylalkylamine N-acetyltransferase activity without involvement of cannabinoid receptors.

    PubMed

    Koch, Marco; Dehghani, Faramarz; Habazettl, Iris; Schomerus, Christof; Korf, Horst-Werner

    2006-07-01

    Cannabinoids modulate neuronal and neuroendocrine circuits by binding to cannabinoid receptors acting upon cAMP/Ca(2+)-mediated intracellular signaling cascades. The rat pineal represents an established model to investigate intracellular signaling processes because a well defined input, the neurotransmitter norepinephrine, is transformed via cAMP/Ca(2+)-dependent mechanisms into an easily detectable output signal, the biosynthesis of melatonin. Here we investigated the impact of cannabinoids on norepinephrine-regulated melatonin biosynthesis in the rat pineal. We demonstrated that treatment of cultured rat pineals with 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), cannabidiol or cannabinol significantly reduced norepinephrine-induced arylalkylamine N-acetyltransferase (AANAT) activity and melatonin biosynthesis. These effects were not mimicked by the cannabinoid receptor agonist WIN55,212-2 and were not blocked by cannabinoid 1 and 2 receptor antagonists. The cannabinoids used did not affect norepinephrine-induced increases in cAMP/Ca(2+) levels. Notably, cannabinoids were found to directly inhibit AANAT activity in lysates of the pineal gland. This effect was specific in so far as cannabinoids did not influence the activity of hydroxyindole-O-methyltransferase (HIOMT), the last enzyme in melatonin biosynthesis. Taken together, our data strongly suggest that cannabinoids inhibit AANAT activity and attenuate melatonin biosynthesis through intracellular actions without involvement of classical cannabinoid receptor-dependent signaling cascades.

  19. Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice.

    PubMed

    Holland, William L; Bikman, Benjamin T; Wang, Li-Ping; Yuguang, Guan; Sargent, Katherine M; Bulchand, Sarada; Knotts, Trina A; Shui, Guanghou; Clegg, Deborah J; Wenk, Markus R; Pagliassotti, Michael J; Scherer, Philipp E; Summers, Scott A

    2011-05-01

    Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid-induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKβ. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes.

  20. Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid–induced ceramide biosynthesis in mice

    PubMed Central

    Holland, William L.; Bikman, Benjamin T.; Wang, Li-Ping; Yuguang, Guan; Sargent, Katherine M.; Bulchand, Sarada; Knotts, Trina A.; Shui, Guanghou; Clegg, Deborah J.; Wenk, Markus R.; Pagliassotti, Michael J.; Scherer, Philipp E.; Summers, Scott A.

    2011-01-01

    Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid–induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKβ. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes. PMID:21490391

  1. Overexpression of an ABA biosynthesis gene using a stress-inducible promoter enhances drought resistance in petunia.

    PubMed

    Estrada-Melo, Alejandro C; Chao; Reid, Michael S; Jiang, Cai-Zhong

    2015-01-01

    The response of plants to drought stress includes reduced transpiration as stomates close in response to increased abscisic acid (ABA) concentrations. Constitutive overexpression of 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in ABA biosynthesis, increases drought resistance, but causes negative pleiotropic effects on plant growth and development. We overexpressed the tomato NCED (LeNCED1) in petunia plants under the control of a stress-inducible promoter, rd29A. Under water stress, the transgenic plants had increased transcripts of NCED mRNA, elevated leaf ABA concentrations, increased concentrations of proline, and a significant increase in drought resistance. The transgenic plants also displayed the expected decreases in stomatal conductance, transpiration, and photosynthesis. After 14 days without water, the control plants were dead, but the transgenic plants, though wilted, recovered fully when re-watered. Well-watered transgenic plants grew like non-transformed control plants and there was no effect of the transgene on seed dormancy.

  2. Overexpression of an ABA biosynthesis gene using a stress-inducible promoter enhances drought resistance in petunia

    PubMed Central

    Estrada-Melo, Alejandro C; Chao; Reid, Michael S; Jiang, Cai-Zhong

    2015-01-01

    The response of plants to drought stress includes reduced transpiration as stomates close in response to increased abscisic acid (ABA) concentrations. Constitutive overexpression of 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in ABA biosynthesis, increases drought resistance, but causes negative pleiotropic effects on plant growth and development. We overexpressed the tomato NCED (LeNCED1) in petunia plants under the control of a stress-inducible promoter, rd29A. Under water stress, the transgenic plants had increased transcripts of NCED mRNA, elevated leaf ABA concentrations, increased concentrations of proline, and a significant increase in drought resistance. The transgenic plants also displayed the expected decreases in stomatal conductance, transpiration, and photosynthesis. After 14 days without water, the control plants were dead, but the transgenic plants, though wilted, recovered fully when re-watered. Well-watered transgenic plants grew like non-transformed control plants and there was no effect of the transgene on seed dormancy. PMID:26504568

  3. Transcription Factor Amr1 Induces Melanin Biosynthesis and Suppresses Virulence in Alternaria brassicicola

    SciTech Connect

    Cho, Yangrae; Srivastava, Akhil; Ohm, Robin A.; Lawrence, Christopher B.; Wang, Koon-Hui; Grigoriev, Igor V.; Marahatta, Sharadchandra P.

    2012-05-01

    Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen. Several A. brassicicola genes have been characterized as affecting pathogenesis of Brassica species. To study regulatory mechanisms of pathogenesis, we mined 421 genes in silico encoding putative transcription factors in a machine-annotated, draft genome sequence of A. brassicicola. In this study, targeted gene disruption mutants for 117 of the transcription factor genes were produced and screened. Three of these genes were associated with pathogenesis. Disruption mutants of one gene (AbPacC) were nonpathogenic and another gene (AbVf8) caused lesions less than half the diameter of wild-type lesions. Unexpectedly, mutants of the third gene, Amr1, caused lesions with a two-fold larger diameter than the wild type and complementation mutants. Amr1 is a homolog of Cmr1, a transcription factor that regulates melanin biosynthesis in several fungi. We created gene deletion mutants of ?amr1 and characterized their phenotypes. The ?amr1 mutants used pectin as a carbon source more efficiently than the wild type, were melanin-deficient, and more sensitive to UV light and glucanase digestion. The AMR1 protein was localized in the nuclei of hyphae and in highly melanized conidia during the late stage of plant pathogenesis. RNA-seq analysis revealed that three genes in the melanin biosynthesis pathway, along with the deleted Amr1 gene, were expressed at low levels in the mutants. In contrast, many hydrolytic enzyme-coding genes were expressed at higher levels in the mutants than in the wild type during pathogenesis. The results of this study suggested that a gene important for survival in nature negatively affected virulence, probably by a less efficient use of plant cell-wall materials. We speculate that the functions of the Amr1 gene are important to the success of A. brassicicola as a competitive saprophyte and plant parasite.

  4. Transcription Factor Amr1 Induces Melanin Biosynthesis and Suppresses Virulence in Alternaria brassicicola

    PubMed Central

    Cho, Yangrae; Srivastava, Akhil; Ohm, Robin A.; Lawrence, Christopher B.; Wang, Koon-Hui; Grigoriev, Igor V.; Marahatta, Sharadchandra P.

    2012-01-01

    Alternaria brassicicola is a successful saprophyte and necrotrophic plant pathogen. Several A. brassicicola genes have been characterized as affecting pathogenesis of Brassica species. To study regulatory mechanisms of pathogenesis, we mined 421 genes in silico encoding putative transcription factors in a machine-annotated, draft genome sequence of A. brassicicola. In this study, targeted gene disruption mutants for 117 of the transcription factor genes were produced and screened. Three of these genes were associated with pathogenesis. Disruption mutants of one gene (AbPacC) were nonpathogenic and another gene (AbVf8) caused lesions less than half the diameter of wild-type lesions. Unexpectedly, mutants of the third gene, Amr1, caused lesions with a two-fold larger diameter than the wild type and complementation mutants. Amr1 is a homolog of Cmr1, a transcription factor that regulates melanin biosynthesis in several fungi. We created gene deletion mutants of Δamr1 and characterized their phenotypes. The Δamr1 mutants used pectin as a carbon source more efficiently than the wild type, were melanin-deficient, and more sensitive to UV light and glucanase digestion. The AMR1 protein was localized in the nuclei of hyphae and in highly melanized conidia during the late stage of plant pathogenesis. RNA-seq analysis revealed that three genes in the melanin biosynthesis pathway, along with the deleted Amr1 gene, were expressed at low levels in the mutants. In contrast, many hydrolytic enzyme-coding genes were expressed at higher levels in the mutants than in the wild type during pathogenesis. The results of this study suggested that a gene important for survival in nature negatively affected virulence, probably by a less efficient use of plant cell-wall materials. We speculate that the functions of the Amr1 gene are important to the success of A. brassicicola as a competitive saprophyte and plant parasite. PMID:23133370

  5. Modulatory effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells.

    PubMed

    Zhang, Min; Lee, Hak Ju; Park, Keun Hong; Park, Hyun Jin; Choi, Hyun Sook; Lim, Sung Cil; Lee, Myung Koo

    2012-06-01

    The effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Sesamin at concentration ranges of 20-75 μM exhibited a significant increase in intracellular dopamine levels at 24 h: 50 μM sesamin increased dopamine levels to 133% and tyrosine hydroxylase (TH) activity to 128.2% of control levels. Sesamin at 20-100 μM rapidly increased the intracellular levels of cyclic AMP (cAMP) to 158.3%-270.3% of control levels at 30 min. At 50 μM, sesamin combined with L-DOPA (50, 100 and 200 μM) further increased the intracellular dopamine levels for 24 h compared to L-DOPA alone. In the absence or presence of L-DOPA (100 and 200 μM), sesamin (50 μM) increased the phosphorylation of TH, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), as well as the mRNA levels of TH and CREB for 24 h, an effect which was reduced by L-DOPA (100 and 200 μM). In addition, 50 μM sesamin exhibited a protective effect against L-DOPA (100 and 200 μM)-induced cytotoxicity via the inhibition of reactive oxygen species (ROS) production and superoxide dismutase reduction, induction of extracellular signal-regulated kinase (ERK)1/2 and BadSer112 phosphorylation and Bcl-2 expression, and inhibition of cleaved-caspase-3 formation. These results suggested that sesamin enhanced dopamine biosynthesis and L-DOPA-induced increase in dopamine levels by inducing TH activity and TH gene expression, which was mediated by cAMP-PKA-CREB systems. Sesamin also protected against L-DOPA (100-200 μM)-induced cytotoxicity through the suppression of ROS activity via the modulation of ERK1/2, BadSer112, Bcl-2, and caspase-3 pathways in PC12 cells. Therefore, sesamin might serve as an adjuvant phytonutrient for neurodegenerative diseases.

  6. Characterization of a midgut-specific chitin synthase gene (LmCHS2) responsible for biosynthesis of chitin of peritrophic matrix in Locusta migratoria.

    PubMed

    Liu, Xiaojian; Zhang, Huanhuan; Li, Sheng; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2012-12-01

    Chitin, an essential component of peritrophic matrix (PM), is produced by a series of biochemical reactions. Chitin synthase plays a crucial role in chitin polymerization in chitin biosynthetic pathway. In this study, we identified and characterized a full-length cDNA of chitin synthase 2 gene (LmCHS2) from Locusta migratoria. The cDNA contains an open reading frame of 4569 nucleotides that encode 1523 amino acid residues, and 76- and 373-nucleotides for 5'- and 3'-noncoding regions, respectively. Analysis of LmCHS2 transcript in different tissues of the locust by using real-time quantitative PCR indicated that LmCHS2 was exclusively expressed in midgut and gastric caeca (a part of the midgut). The highest expression was found in the anterior midgut with a decline of the transcript level from the anterior to posterior regions. During growth and development of locusts, there was only a slight expression in eggs, but the expression gradually increased from nymphs to adults. In situ hybridization further revealed that LmCHS2 transcript mainly presented in the apical regions of brush border forming columnar cells of gastric caeca. LmCHS2 dsRNA was injected to fifth-instar nymphs to further explore biological functions of LmCHS2. Significantly down-regulated transcript of LmCHS2 resulted in a cessation of feeding and a high mortality of the insect. However, no visible abnormal morphological change of locusts was observed until insects molted to adults. After dissection, we found that the average length of midguts from the LmCHS2 dsRNA-injected locusts was shorter than that of the control insects that were injected with dsGFP. Furthermore, microsection of midguts showed that the PM of the LmCHS2 dsRNA-injected nymphs was amorphous and thin as compared with the controls. Our results demonstrate that LmCHS2 is responsible for the biosynthesis of chitin associated with PM and plays an essential role in locust growth and development.

  7. Effect of inhibitors of polyamine biosynthesis on gibberellin-induced internode growth in light-grown dwarf peas

    NASA Technical Reports Server (NTRS)

    Kaur-Sawhney, R.; Dai, Y. R.; Galston, A. W.

    1986-01-01

    When gibberellic acid (GA3) is sprayed on 9-day-old light-brown dwarf Progress pea (Pisum sativum) seedlings, arginine decarboxylase (ADC; EC 4.1.1.9) activity increases within 3 h and peaks at about 9 h after GA3 application. This is followed by a second lower peak at about 30 h; both peaks were higher than the corresponding peaks in the controls. In contrast, no appreciable effect of GA3 on internode length was observed until about 12 h, after which time a dramatic increase in growth rate occurred and persisted for about 12 h. Specific (DL-alpha-difluoromethylarginine) and non-specific (D-arginine and L-canavanine) inhibitors of ADC strongly inhibited ADC activity and to a lesser extent internode growth. The inhibition was reversed only slightly by the addition of polyamines. Actinomycin D and cycloheximide inhibited the rise in ADC activity induced by GA3. The half-life of the enzyme was increased by GA3 treatment. The results suggest that part of the GA3-induced increase in internode growth may result from enhanced polyamine biosynthesis through the ADC pathway. Furthermore, the GA3 induced increase in ADC activity probably requires de novo synthesis of both RNA and protein.

  8. Hispidulin induces mitochondrial apoptosis in acute myeloid leukemia cells by targeting extracellular matrix metalloproteinase inducer

    PubMed Central

    Gao, Hui; Liu, Yongji; Li, Kan; Wu, Tianhui; Peng, Jianjun; Jing, Fanbo

    2016-01-01

    Acute myeloid leukemia (AML) represents a heterogeneous group of hematological neoplasms with marked heterogeneity in response to both standard therapy and survival. Hispidulin, a flavonoid compound that is anactive ingredient in the traditional Chinese medicinal herb Salvia plebeia R. Br, has recently been reported to have anantitumor effect against solid tumors in vitro and in vivo. The aim of the present study was to investigate the effects of hispidulin on the human leukemia cell line in vitro and the underlying mechanisms of its actions on these cells. Our results showed that hispidulin inhibits AML cell proliferation in a dose- and time-dependent manner, and induces cell apoptosis throughan intrinsic mitochondrial pathway. Our results also revealed that hispidulin treatment significantly inhibits extracellular matrix metalloproteinase inducer (EMMPRIN) expression in both tested AML cell lines in a dose-dependent manner, and that the overexpression of EMMPRIN protein markedly attenuates hispidulin-induced cell apoptosis. Furthermore, our results strongly indicated that the modulating effect of hispidulin on EMMPRIN is correlated with its inhibitory effect on both the Akt and STAT3 signaling pathways. PMID:27158398

  9. Hardening with salicylic acid induces concentration-dependent changes in abscisic acid biosynthesis of tomato under salt stress.

    PubMed

    Horváth, Edit; Csiszár, Jolán; Gallé, Ágnes; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2015-07-01

    The role of salicylic acid (SA) in the control of abscisic acid (ABA) biosynthesis is controversial although both plant growth regulators may accumulate in tissues under abiotic and biotic stress conditions. Hardening of tomato plants to salinity stress with 10(-4)M SA ("high SA") resulted in an up-regulation of ABA biosynthesis genes, zeaxanthin epoxidase (SlZEP1), 9-cis-epoxycarotenoid dioxygenase (SlNCED1) and aldehyde oxidases (SlAO1 and SlAO2) in the roots and led to ABA accumulation both in root and leaf tissues. In plants pre-treated with lower concentration of SA (10(-7)M, "low SA"), the up-regulation of SlNCED1 in the roots promoted ABA accumulation in the root tissues but the hormone concentration remained at control level in the leaves. Salt stress induced by 100mM NaCl reduced the transcript abundance of ABA biosynthetic genes and inhibited SlAO activity in plants hardened with "high SA", but the tissues maintained root ABA level over the untreated control. The combined effect of "high SA" and ABA under salt stress led to partially recovered photosynthetic activity, reduced ethylene production in root apices, and restored root growth, which is one of the main features of salt tolerance. Unlike "high SA", hardening with "low SA" had no influence on ethylene production, and led to reduced elongation of roots in plants exposed to 100mM NaCl. The up-regulation of carotenoid cleavage dioxygenases SlCCD1A and SlCCD1B by SA, which produce apocarotenoids, may open new pathways in SA sensing and signalling processes.

  10. Arabidopsis FHY3 and FAR1 Regulate Light-Induced myo-Inositol Biosynthesis and Oxidative Stress Responses by Transcriptional Activation of MIPS1.

    PubMed

    Ma, Lin; Tian, Tian; Lin, Rongcheng; Deng, Xing-Wang; Wang, Haiyang; Li, Gang

    2016-04-04

    myo-Inositol-1-phosphate synthase (MIPS) catalyzes the limiting step of inositol biosynthesis and has crucial roles in plant growth and development. In response to stress, the transcription of MIPS1 is induced and the biosynthesis of inositol or inositol derivatives is promoted by unknown mechanisms. Here, we found that the light signaling protein FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1) regulate light-induced inositol biosynthesis and oxidative stress responses by activating the transcription of MIPS1. Disruption of FHY3 and FAR1 caused light-induced cell death after dark-light transition, precocious leaf senescence, and increased sensitivity to oxidative stress. Reduction of salicylic acid (SA) accumulation by overexpression of SALICYLIC ACID 3-HYDROXYLASE largely suppressed the cell death phenotype of fhy3 far1 mutant plants, suggesting that FHY3- and FAR1-mediated cell death is dependent on SA. Furthermore, comparative analysis of chromatin immunoprecipitation sequencing and microarray results revealed that FHY3 and FAR1 directly target both MIPS1 and MIPS2. The fhy3 far1 mutant plants showed severely decreased MIPS1/2 transcript levels and reduced inositol levels. Conversely, constitutive expression of MIPS1 partially rescued the inositol contents, caused reduced transcript levels of SA-biosynthesis genes, and prevented oxidative stress in fhy3 far1. Taken together, our results indicate that the light signaling proteins FHY3 and FAR1 directly bind the promoter of MIPS1 to activate its expression and thereby promote inositol biosynthesis to prevent light-induced oxidative stress and SA-dependent cell death.

  11. Fluoride-induced changes in haem biosynthesis pathway, neurological variables and tissue histopathology of rats.

    PubMed

    Chouhan, Swapnila; Lomash, Vinay; Flora, S J S

    2010-01-01

    This study intended to determine the effects of various concentrations of fluoride (1, 10, 50 and 100 ppm) in drinking water for a period of 12 weeks on changes in haem biosynthesis pathway, oxidative stress and neurological variables supported by histopathological observations and fluoride in rats. The data indicates significant alterations in the parameters related to haeme synthesis pathway like inhibition of blood delta-aminolevulinic acid dehydratase, delta-aminolevulinic acid synthetase, oxidative stress like depletion of glutathione (GSH) and increase in oxidized glutathione (GSSG) and thiobarbituric acid reactive substances. These changes were accompanied by depletion in GSH:GSSG ratio, whole brain biogenic amine levels and a dose-dependent increase in fluoride concentration. Interestingly and most significantly, these changes were more pronounced at lower concentrations of fluoride compared with higher fluoride dose. Biochemical changes were supported by the histological observations, which also revealed that at high concentrations of fluoride, toxic effects and damages to organs were more pronounced. These changes support our earlier findings regarding the role of decreased ionic mobility of fluoride ion at higher concentrations, leading to less pronounced toxicity.

  12. Inhibition of de novo ceramide biosynthesis by FTY720 protects rat retina from light-induced degeneration[S

    PubMed Central

    Chen, Hui; Tran, Julie-Thu A.; Eckerd, Annette; Huynh, Tuan-Phat; Elliott, Michael H.; Brush, Richard S.; Mandal, Nawajes A.

    2013-01-01

    Light-induced retinal degeneration (LIRD) in albino rats causes apoptotic photoreceptor cell death. Ceramide is a second messenger for apoptosis. We tested whether increases in ceramide mediate photoreceptor apoptosis in LIRD and if inhibition of ceramide synthesis protects the retina. Sprague-Dawley rats were exposed to 2,700 lux white light for 6 h, and the retinal levels of ceramide and its intermediary metabolites were measured by GC-MS or electrospray ionization tandem mass spectrometry. Enzymes of the de novo biosynthetic and sphingomyelinase pathways of ceramide generation were assayed, and gene expression was measured. The dosage and temporal effect of the ceramide synthase inhibitor FTY720 on the LIRD retina were measured by histological and functional analyses. Retinal ceramide levels increased coincident with the increase of dihydroceramide at various time points after light stress. Light stress in retina induces ceramide generation predominantly through the de novo pathway, which was prevented by systemic administration of FTY720 (10 mg/kg) leading to the protection of retinal structure and function. The neuroprotection of FTY720 was independent of its immunosuppressive action. We conclude that ceramide increase by de novo biosynthesis mediates photoreceptor apoptosis in the LIRD model and that inhibition of ceramide production protects the retina against light stress. PMID:23468130

  13. Disruption of plant carotenoid biosynthesis through virus-induced gene silencing affects oviposition behaviour of the butterfly Pieris rapae.

    PubMed

    Zheng, Si-Jun; Snoeren, Tjeerd A L; Hogewoning, Sander W; van Loon, Joop J A; Dicke, Marcel

    2010-05-01

    Optical plant characteristics are important cues to plant-feeding insects. In this article, we demonstrate for the first time that silencing the phytoene desaturase (PDS) gene, encoding a key enzyme in plant carotenoid biosynthesis, affects insect oviposition site selection behaviour. Virus-induced gene silencing employing tobacco rattle virus was used to knock down endogenous PDS expression in three plant species (Arabidopsis thaliana, Brassica nigra and Nicotiana benthamiana) by its heterologous gene sequence from Brassica oleracea. We investigated the consequences of the silencing of PDS on oviposition behaviour by Pieris rapae butterflies on Arabidopsis and Brassica plants; first landing of the butterflies on Arabidopsis plants (to eliminate an effect of contact cues); first landing on Arabidopsis plants enclosed in containers (to eliminate an effect of volatiles); and caterpillar growth on Arabidopsis plants. Our results show unambiguously that P. rapae has an innate ability to visually discriminate between green and variegated green-whitish plants. Caterpillar growth was significantly lower on PDS-silenced than on empty vector control plants. This study presents the first analysis of PDS function in the interaction with an herbivorous insect. We conclude that virus-induced gene silencing is a powerful tool for investigating insect-plant interactions in model and nonmodel plants.

  14. Salinity-induced regulation of the myo-inositol biosynthesis pathway in tilapia gill epithelium.

    PubMed

    Sacchi, Romina; Li, Johnathon; Villarreal, Fernando; Gardell, Alison M; Kültz, Dietmar

    2013-12-15

    The myo-inositol biosynthesis (MIB) pathway converts glucose-6-phosphate to the compatible osmolyte myo-inositol that protects cells from osmotic stress. Using proteomics, the enzymes that constitute the MIB pathway, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1), are identified in tilapia (Oreochromis mossambicus) gill epithelium. Targeted, quantitative, label-free proteomics reveals that they are both upregulated during salinity stress. Upregulation is stronger when fish are exposed to severe (34 ppt acute and 90 ppt gradual) relative to moderate (70 ppt gradual) salinity stress. IMPA1 always responds more strongly than MIPS, suggesting that MIPS is more stable during salinity stress. MIPS is N-terminally acetylated and the corresponding peptide increases proportionally to MIPS protein, while non-acetylated N-terminal peptide is not detectable, indicating that MIPS acetylation is constitutive and may serve to stabilize the protein. Hyperosmotic induction of MIPS and IMPA1 is confirmed using western blot and real-time qPCR and is much higher at the mRNA than at the protein level. Two distinct MIPS mRNA variants are expressed in the gill, but one is more strongly regulated by salinity than the other. A single MIPS gene is encoded in the tilapia genome whereas the zebrafish genome lacks MIPS entirely. The genome of euryhaline tilapia contains four IMPA genes, two of which are expressed, but only one is salinity regulated in gill epithelium. The genome of stenohaline zebrafish contains a single IMPA gene. We conclude that the MIB pathway represents a major salinity stress coping mechanism that is regulated at multiple levels in euryhaline fish but absent in stenohaline zebrafish.

  15. Process-induced extracellular matrix alterations affect the mechanisms of soft tissue repair and regeneration

    PubMed Central

    Xu, Hui; Sandor, Maryellen; Lombardi, Jared

    2013-01-01

    Extracellular matrices derived from animal tissues for human tissue repairs are processed by various methods of physical, chemical, or enzymatic decellularization, viral inactivation, and terminal sterilization. The mechanisms of action in tissue repair vary among bioscaffolds and are suggested to be associated with process-induced extracellular matrix modifications. We compared three non-cross-linked, commercially available extracellular matrix scaffolds (Strattice, Veritas, and XenMatrix), and correlated extracellular matrix alterations to in vivo biological responses upon implantation in non-human primates. Structural evaluation showed significant differences in retaining native tissue extracellular matrix histology and ultrastructural features among bioscaffolds. Tissue processing may cause both the condensation of collagen fibers and fragmentation or separation of collagen bundles. Calorimetric analysis showed significant differences in the stability of bioscaffolds. The intrinsic denaturation temperature was measured to be 51°C, 38°C, and 44°C for Strattice, Veritas, and XenMatrix, respectively, demonstrating more extracellular matrix modifications in the Veritas and XenMatrix scaffolds. Consequently, the susceptibility to collagenase degradation was increased in Veritas and XenMatrix when compared to their respective source tissues. Using a non-human primate model, three bioscaffolds were found to elicit different biological responses, have distinct mechanisms of action, and yield various outcomes of tissue repair. Strattice permitted cell repopulation and was remodeled over 6 months. Veritas was unstable at body temperature, resulting in rapid absorption with moderate inflammation. XenMatrix caused severe inflammation and sustained immune reactions. This study demonstrates that extracellular matrix alterations significantly affect biological responses in soft tissue repair and regeneration. The data offer useful insights into the rational design of

  16. Angiogenic response induced by acellular femoral matrix in vivo

    PubMed Central

    Conconi, Maria Teresa; Nico, Beatrice; Rebuffat, Piera; Crivellato, Enrico; Parnigotto, Pier Paolo; Nussdorfer, Gastone G; Ribatti, Domenico

    2005-01-01

    We investigated the angiogenic response induced by acellular femoral matrices implanted in vivo on to the chick embryo chorioallantoic membrane (CAM), a useful model for such investigation. The results showed that acellular matrices were able to induce a strong angiogenic response, comparable with that of fibroblast growth factor-2 (FGF-2), a well-known angiogenic cytokine. The angiogenic response was further increased when exogenous FGF-2 or transforming growth factor beta-1 (TGF-β1) was added to the matrices and inhibited by the addition of anti-FGF-2 or anti-TGF-β1 antibodies. The response may be considered to be dependent on a direct angiogenic effect exerted by the matrices, and also in part by the presence of FGF-2 and TGF-β1 in the acellular matrices. PMID:16011546

  17. A Stress-Inducible Resveratrol O-Methyltransferase Involved in the Biosynthesis of Pterostilbene in Grapevine1

    PubMed Central

    Schmidlin, Laure; Poutaraud, Anne; Claudel, Patricia; Mestre, Pere; Prado, Emilce; Santos-Rosa, Maria; Wiedemann-Merdinoglu, Sabine; Karst, Francis; Merdinoglu, Didier; Hugueney, Philippe

    2008-01-01

    Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl3 treatment. PMID:18799660

  18. Prolidase-dependent mechanism of (Z)-8,9-epoxyheptadeca-1,11,14-triene-induced inhibition of collagen biosynthesis in cultured human skin fibroblasts.

    PubMed

    Szoka, Lukasz; Karna, Ewa; Nazaruk, Jolanta; Palka, Jerzy A

    2016-01-01

    The effects of polyolefinic compound from roots of Cirsium palustre, (Z)-8,9-epoxyheptadeca-1,11,14-triene (EHT) on collagen biosynthesis, prolidase activity, expression of insulin-like growth factor receptor (IGF-IR), β1 integrin, MAP kinases (pERK1/2), the transcription factors such as nuclear factor kappa B (NF-κB) and hypoxia-inducible factor-1α (HIF-1α) were evaluated in human dermal fibroblasts treated with micromolar concentrations (40-200 μM) for 24 h. It was found that EHT-dependent inhibition of collagen biosynthesis was accompanied by parallel inhibition in prolidase activity. Since IGF-I is the most potent regulator of both processes and prolidase is regulated by β1 integrin signalling, the effect of EHT on IGF-IR and β1 integrin receptor expressions were evaluated. Exposure of the cells to EHT contributed to distinct increase in IGF-IR and slight increase in β1 integrin receptor expressions. It was accompanied by decrease in expression of pERK1/2, HIF-1α and NF-κB. EHT-dependent inhibition of collagen biosynthesis results from inhibition of prolidase activity, the enzyme involved in collagen biosynthesis.

  19. Bacterial lipopolysaccharides induce in vitro degradation of cartilage matrix through chondrocyte activation.

    PubMed Central

    Jasin, H E

    1983-01-01

    The present studies demonstrate that bacterial lipopolysaccharides (LPS) induce cartilage matrix degradation in live explants in organ culture. Quintuplicate bovine nasal fibrocartilage explants cultured for 8 d with three different purified LPS preparations derived from Escherichia coli and Salmonella typhosa at concentrations ranging from 1.0 to 25.0 micrograms/ml resulted in matrix proteoglycan depletion of 33.3 +/- 5.8 to 92.5 +/- 2.0% (medium control depletion 17.7 +/- 0.7 to 32.4 +/- 1.4%). Matrix degradation depended on the presence of live chondrocytes because frozen-thawed explants incubated with LPS failed to show any proteoglycan release. Moreover, the addition of Polymyxin B (25 micrograms/ml) to live explants incubated with LPS abolished matrix release, whereas Polymyxin B had no effect on the matrix-degrading activity provided by blood mononuclear cell factors. A highly purified Lipid A preparation induced matrix degradation at a concentration of 0.01 micrograms/ml. Cartilage matrix collagen and proteoglycan depletion also occurred with porcine articular cartilage explants (collagen release: 18.3 +/- 3.5%, medium control: 2.1 +/- 0.5%; proteoglycan release: 79.0 +/- 5.9%, medium control: 28.8 +/- 4.8%). Histochemical analysis of the cultured explants confirmed the results described above. Gel chromatography of the proteoglycans released in culture indicated that LPS induced significant degradation of the high molecular weight chondroitin sulfate-containing aggregates. These findings suggest that bacterial products may induce cartilage damage by direct stimulation of chondrocytes. This pathogenic mechanism may play a role in joint damage in septic arthritis and in arthropathies resulting from the presence of bacterial products derived from the gastrointestinal tract. Images PMID:6358260

  20. Role of cells in freezing-induced cell-fluid-matrix interactions within engineered tissues.

    PubMed

    Seawright, Angela; Ozcelikkale, Altug; Dutton, Craig; Han, Bumsoo

    2013-09-01

    During cryopreservation, ice forms in the extracellular space resulting in freezing-induced deformation of the tissue, which can be detrimental to the extracellular matrix (ECM) microstructure. Meanwhile, cells dehydrate through an osmotically driven process as the intracellular water is transported to the extracellular space, increasing the volume of fluid for freezing. Therefore, this study examines the effects of cellular presence on tissue deformation and investigates the significance of intracellular water transport and cell-ECM interactions in freezing-induced cell-fluid-matrix interactions. Freezing-induced deformation characteristics were examined through cell image deformetry (CID) measurements of collagenous engineered tissues embedded with different concentrations of MCF7 breast cancer cells versus microspheres as their osmotically inactive counterparts. Additionally, the development of a biophysical model relates the freezing-induced expansion of the tissue due to the cellular water transport and the extracellular freezing thermodynamics for further verification. The magnitude of the freezing-induced dilatation was found to be not affected by the cellular water transport for the cell concentrations considered; however, the deformation patterns for different cell concentrations were different suggesting that cell-matrix interactions may have an effect. It was, therefore, determined that intracellular water transport during freezing was insignificant at the current experimental cell concentrations; however, it may be significant at concentrations similar to native tissue. Finally, the cell-matrix interactions provided mechanical support on the ECM to minimize the expansion regions in the tissues during freezing.

  1. Auxin Biosynthesis

    PubMed Central

    Zhao, Yunde

    2014-01-01

    lndole-3-acetic acid (IAA), the most important natural auxin in plants, is mainly synthesized from the amino acid tryptophan (Trp). Recent genetic and biochemical studies in Arabidopsis have unambiguously established the first complete Trp-dependent auxin biosynthesis pathway. The first chemical step of auxin biosynthesis is the removal of the amino group from Trp by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) family of transaminases to generate indole-3-pyruvate (IPA). IPA then undergoes oxidative decarboxylation catalyzed by the YUCCA (YUC) family of flavin monooxygenases to produce IAA. This two-step auxin biosynthesis pathway is highly conserved throughout the plant kingdom and is essential for almost all of the major developmental processes. The successful elucidation of a complete auxin biosynthesis pathway provides the necessary tools for effectively modulating auxin concentrations in plants with temporal and spatial precision. The progress in auxin biosynthesis also lays a foundation for understanding polar auxin transport and for dissecting auxin signaling mechanisms during plant development. PMID:24955076

  2. Studies of matrix vesicle-induced mineralization in a gelatin gel

    NASA Technical Reports Server (NTRS)

    Boskey, A. L.; Boyan, B. D.; Doty, S. B.; Feliciano, A.; Greer, K.; Weiland, D.; Swain, L. D.; Schwartz, Z.

    1992-01-01

    Matrix vesicles isolated from fourth-passage cultures of chondrocytes were tested for their ability to induce hydroxyapatite formation in a gelatin gel in order to gain insight into the function of matrix vesicles in in situ mineralization. These matrix vesicles did not appear to be hydroxyapatite nucleators per se since the extent of mineral accumulation in the gel diffusion system was not altered by the presence of matrix vesicles alone, and in the vesicle containing gels, mineral crystals were formed whether associated with vesicles or not. In gels with these matrix vesicles and beta-glycerophosphate, despite the presence of alkaline phosphatase activity, there was no increase in mineral deposition. This suggested that in the gel system these culture-derived vesicles did not increase local phosphate concentrations. However, when known inhibitors of mineral crystal formation and growth (proteoglycan aggregates [4 mg/ml], or ATP [1 mM], or both proteoglycan and ATP) were included in the gel, more mineral was deposited in gels with the vesicles than in comparable gels without vesicles, indicating that enzymes within these vesicles were functioning to remove the inhibition. These data support the suggestion that one function of the extracellular matrix vesicles is to transport enzymes for matrix modification.

  3. Extracellular Matrix Remodeling During the Progression of Volume Overload-Induced Heart Failure

    PubMed Central

    Hutchinson, Kirk R.; Stewart, James A.; Lucchesi, Pamela A.

    2009-01-01

    Volume overload-induced heart failure results in progressive left ventricular remodeling characterized by chamber dilation, eccentric cardiac myocyte hypertrophy and changes in extracellular matrix (ECM) remodeling changes. The ECM matrix scaffold is an important determinant of the structural integrity of the myocardium and actively participates in force transmission across the LV wall. In response to this hemodynamic overload, the ECM undergoes a distinct pattern of remodeling that differs from pressure overload. Once thought to be a static entity, the ECM is now regarded to be a highly adaptive structure that is dynamically regulated by mechanical stress, neurohormonal activation, inflammation and oxidative stress, that result in alterations in collagen and other matrix components and a net change in matrix metalloproteinase (MMP) expression and activation. These changes dictate overall ECM turnover during volume overload hear failure progression. This review will discuss the cellular and molecular mechanisms that dictate the temporal patterns of ECM remodeling during heart disease progression. PMID:19524591

  4. Skin resistance to oxidative stress induced by resveratrol: from Nrf2 activation to GSH biosynthesis.

    PubMed

    Soeur, J; Eilstein, J; Léreaux, G; Jones, C; Marrot, L

    2015-01-01

    Skin is particularly exposed to oxidative stress, either from environmental insults such as sunlight or pollution or as a consequence of specific impairments in antioxidant status resulting from pathologies or aging. Traditionally, antioxidant products are exogenously provided to neutralize pro-oxidant species. However, another approach based on stimulation of endogenous antioxidant defense pathways is more original. Resveratrol (RSV) was reported to display such a behavior in various tissues, but data about the mechanisms of action in skin are scarce. We show here that, in primary culture of normal human keratinocytes (NHKs) or in full-thickness reconstructed human skin, RSV activated the Nrf2 pathway at nontoxic doses, from 20 µM up to 100µM. Among the Nrf2 downstream genes, glutamylcysteinyl ligase and glutathione peroxidase-2 were induced at the mRNA and protein levels. In parallel, a significant increase in glutathione content, assessed by LC/MS analysis, was observed in both models. Nrf2 gene silencing experiments performed in NHKs confirmed that Nrf2 was involved in RSV-induced modulation of cellular antioxidant status, in part by increasing cellular glutathione content. Finally, improvement of endogenous defenses induced in RSV-pretreated reconstructed skin ensured protection against the toxic oxidative effects of cumene hydroperoxide (CHP). In fact after RSV pretreatment, in response to CHP stress, glutathione content did not decrease as in unprotected samples. Cellular alterations at the dermal-epidermal junction were clearly prevented. Together, these complementary experiments demonstrated the beneficial effects of RSV on skin, beyond its direct antioxidant properties, by upregulation of a cutaneous endogenous antioxidant pathway.

  5. Quercetin induces HepG2 cell apoptosis by inhibiting fatty acid biosynthesis

    PubMed Central

    ZHAO, PENG; MAO, JUN-MIN; ZHANG, SHU-YUN; ZHOU, ZE-QUAN; TAN, YANG; ZHANG, YU

    2014-01-01

    Quercetin can inhibit the growth of cancer cells with the ability to act as a ‘chemopreventer’. Its cancer-preventive effect has been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis, as well as its antioxidant functions. Quercetin can also reduce adipogenesis. Previous studies have shown that quercetin has potent inhibitory effects on animal fatty acid synthase (FASN). In the present study, activity of quercetin was evaluated in human liver cancer HepG2 cells. Intracellular FASN activity was calculated by measuring the absorption of NADPH via a spectrophotometer. MTT assay was used to test the cell viability, immunoblot analysis was performed to detect FASN expression levels and the apoptotic effect was detected by Hoechst 33258 staining. In the present study, it was found that quercetin could induce apoptosis in human liver cancer HepG2 cells with overexpression of FASN. This apoptosis was accompanied by the reduction of intracellular FASN activity and could be rescued by 25 or 50 μM exogenous palmitic acids, the final product of FASN-catalyzed synthesis. These results suggested that the apoptosis induced by quercetin was via the inhibition of FASN. These findings suggested that quercetin may be useful for preventing human liver cancer. PMID:25009654

  6. Tumor necrosis factor alpha-induced angiogenesis depends on in situ platelet-activating factor biosynthesis

    PubMed Central

    1994-01-01

    Tumor necrosis factor (TNF) alpha, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Therefore, it was suggested that the angiogenic properties of this agent might be consequent to the production of secondary mediators. Since TNF-alpha stimulates the synthesis of platelet-activating factor (PAF) by monocytes and endothelial cells, we investigated the possible involvement of PAF in the angiogenic effect of TNF-alpha. Angiogenesis was studied in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model the angiogenesis induced by TNF-alpha was shown to be inhibited by WEB 2170, a specific PAF receptor antagonist. Moreover, in mice injected with TNF-alpha, PAF was detected within the Matrigel, 6 and 24 h after TNF-alpha injection. The synthesis of PAF within the Matrigel was concomitant with the early migration of endothelial cells and infiltration of monocytes. No infiltration of lymphocytes or polymorphonuclear leukocytes was observed. Synthetic PAF as well as PAF extracted and purified from mice challenged with TNF-alpha induced a rapid angiogenic response, inhibited by WEB 2170. These results suggest that the angiogenic effect of TNF-alpha is, at least in part, mediated by PAF synthesized from monocytes and/or endothelial cells infiltrating the Matrigel plug. PMID:7516414

  7. Quercetin induces HepG2 cell apoptosis by inhibiting fatty acid biosynthesis.

    PubMed

    Zhao, Peng; Mao, Jun-Min; Zhang, Shu-Yun; Zhou, Ze-Quan; Tan, Yang; Zhang, Yu

    2014-08-01

    Quercetin can inhibit the growth of cancer cells with the ability to act as a 'chemopreventer'. Its cancer-preventive effect has been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis, as well as its antioxidant functions. Quercetin can also reduce adipogenesis. Previous studies have shown that quercetin has potent inhibitory effects on animal fatty acid synthase (FASN). In the present study, activity of quercetin was evaluated in human liver cancer HepG2 cells. Intracellular FASN activity was calculated by measuring the absorption of NADPH via a spectrophotometer. MTT assay was used to test the cell viability, immunoblot analysis was performed to detect FASN expression levels and the apoptotic effect was detected by Hoechst 33258 staining. In the present study, it was found that quercetin could induce apoptosis in human liver cancer HepG2 cells with overexpression of FASN. This apoptosis was accompanied by the reduction of intracellular FASN activity and could be rescued by 25 or 50 μM exogenous palmitic acids, the final product of FASN-catalyzed synthesis. These results suggested that the apoptosis induced by quercetin was via the inhibition of FASN. These findings suggested that quercetin may be useful for preventing human liver cancer.

  8. The regulatory mechanism of fungal elicitor-induced secondary metabolite biosynthesis in medical plants.

    PubMed

    Zhai, Xin; Jia, Min; Chen, Ling; Zheng, Cheng-Jian; Rahman, Khalid; Han, Ting; Qin, Lu-Ping

    2017-03-01

    A wide range of external stress stimuli trigger plant cells to undergo complex network of reactions that ultimately lead to the synthesis and accumulation of secondary metabolites. Accumulation of such metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. Throughout evolution, endophytic fungi, an important constituent in the environment of medicinal plants, have known to form long-term stable and mutually beneficial symbiosis with medicinal plants. The endophytic fungal elicitor can rapidly and specifically induce the expression of specific genes in medicinal plants which can result in the activation of a series of specific secondary metabolic pathways resulting in the significant accumulation of active ingredients. Here we summarize the progress made on the mechanisms of fungal elicitor including elicitor signal recognition, signal transduction, gene expression and activation of the key enzymes and its application. This review provides guidance on studies which may be conducted to promote the efficient synthesis and accumulation of active ingredients by the endogenous fungal elicitor in medicinal plant cells, and provides new ideas and methods of studying the regulation of secondary metabolism in medicinal plants.

  9. Impact of Gas Adsorption Induced Coal Matrix Damage on the Evolution of Coal Permeability

    NASA Astrophysics Data System (ADS)

    Zhu, W. C.; Wei, C. H.; Liu, J.; Xu, T.; Elsworth, D.

    2013-11-01

    It has been widely reported that coal permeability can change from reduction to enhancement due to gas adsorption even under the constant effective stress condition, which is apparently inconsistent with the classic theoretical solutions. This study addresses this inconsistency through explicit simulations of the dynamic interactions between coal matrix swelling/shrinking induced damage and fracture aperture alteration, and translations of these interactions to permeability evolution under the constant effective stress condition. We develop a coupled coal-gas interaction model that incorporates the material heterogeneity and damage evolution of coal, which allows us to couple the progressive development of damage zone with gas adsorption processes within the coal matrix. For the case of constant effective stress, coal permeability changes from reduction to enhancement while the damage zone within the coal matrix develops from the fracture wall to further inside the matrix. As the peak Langmuir strain is approached, the decrease of permeability halts and permeability increases with pressure. The transition of permeability reduction to permeability enhancement during gas adsorption, which may be closely related to the damage zone development in coal matrix, is controlled by coal heterogeneity, external boundary condition, and adsorption-induced swelling.

  10. Rice chalky ring formation caused by temporal reduction in starch biosynthesis during osmotic adjustment under foehn-induced dry wind.

    PubMed

    Wada, Hiroshi; Masumoto-Kubo, Chisato; Gholipour, Yousef; Nonami, Hiroshi; Tanaka, Fukuyo; Erra-Balsells, Rosa; Tsutsumi, Koichi; Hiraoka, Kenzo; Morita, Satoshi

    2014-01-01

    Foehn-like extreme hot and dry wind conditions (34°C, >2.5 kPa vapor pressure deficit, and 7 m s(-1)) strongly affect grain quality in rice (Oryza sativa L.). This is a current concern because of the increasing frequency and intensity of combined heat and water-deficit stress under climate change. Foehn-induced dry wind conditions during the grain-filling stage increase ring-shaped chalkiness as a result of spatiotemporal reduction in starch accumulation in the endosperm, but kernel growth is sometimes maintained by osmotic adjustment. Here, we assess the effects of dry wind on chalky ring formation in environmentally controlled growth chambers. Our results showed that hot and dry wind conditions that lasted for >24 h dramatically increased chalky ring formation. Hot and dry wind conditions temporarily reduced panicle water potential to -0.65 MPa; however, kernel growth was maintained by osmotic adjustment at control levels with increased transport of assimilate to the growing kernels. Dynamic tracer analysis with a nano-electrospray-ionization Orbitrap mass spectrometer and quantitative polymerase chain reaction analysis revealed that starch degradation was negligible in the short-term treatment. Overall expression of starch synthesis-related genes was found to be down-regulated at moderately low water potential. Because the events observed at low water potential preceded the packing of starch granules in cells, we concluded that reduced rates of starch biosynthesis play a central role in the events of cellular metabolism that are altered at osmotic adjustment, which leads to chalky ring formation under short-term hot and dry wind conditions.

  11. Rice Chalky Ring Formation Caused by Temporal Reduction in Starch Biosynthesis during Osmotic Adjustment under Foehn-Induced Dry Wind

    PubMed Central

    Wada, Hiroshi; Masumoto-Kubo, Chisato; Gholipour, Yousef; Nonami, Hiroshi; Tanaka, Fukuyo; Erra-Balsells, Rosa; Tsutsumi, Koichi; Hiraoka, Kenzo; Morita, Satoshi

    2014-01-01

    Foehn-like extreme hot and dry wind conditions (34°C, >2.5 kPa vapor pressure deficit, and 7 m s−1) strongly affect grain quality in rice (Oryza sativa L.). This is a current concern because of the increasing frequency and intensity of combined heat and water-deficit stress under climate change. Foehn-induced dry wind conditions during the grain-filling stage increase ring-shaped chalkiness as a result of spatiotemporal reduction in starch accumulation in the endosperm, but kernel growth is sometimes maintained by osmotic adjustment. Here, we assess the effects of dry wind on chalky ring formation in environmentally controlled growth chambers. Our results showed that hot and dry wind conditions that lasted for >24 h dramatically increased chalky ring formation. Hot and dry wind conditions temporarily reduced panicle water potential to –0.65 MPa; however, kernel growth was maintained by osmotic adjustment at control levels with increased transport of assimilate to the growing kernels. Dynamic tracer analysis with a nano-electrospray-ionization Orbitrap mass spectrometer and quantitative polymerase chain reaction analysis revealed that starch degradation was negligible in the short-term treatment. Overall expression of starch synthesis-related genes was found to be down-regulated at moderately low water potential. Because the events observed at low water potential preceded the packing of starch granules in cells, we concluded that reduced rates of starch biosynthesis play a central role in the events of cellular metabolism that are altered at osmotic adjustment, which leads to chalky ring formation under short-term hot and dry wind conditions. PMID:25330305

  12. SlCCD7 controls strigolactone biosynthesis, shoot branching and mycorrhiza-induced apocarotenoid formation in tomato.

    PubMed

    Vogel, Jonathan T; Walter, Michael H; Giavalisco, Patrick; Lytovchenko, Anna; Kohlen, Wouter; Charnikhova, Tatsiana; Simkin, Andrew J; Goulet, Charles; Strack, Dieter; Bouwmeester, Harro J; Fernie, Alisdair R; Klee, Harry J

    2010-01-01

    The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch-inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C(13) cyclohexenone and C(14) mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source-sink interactions and production of arbuscular mycorrhiza-induced apocarotenoids.

  13. Damage initiation, progression and failure of polymer matrix composites due to manufacturing induced defects

    NASA Astrophysics Data System (ADS)

    Chowdhury, Khairul Alam

    In polymer matrix composites (PMCs) manufacturing processes can induce defects, e.g., voids, fiber misalignment, irregular fiber distribution in the cross-section and broken fibers. The effects of such defects can be beneficial or deleterious depending on whether they cause failure suppression or enhancement by localized deformation processes e.g., crazing, shear yielding and fiber-matrix debonding. In this study, a computational approach is formulated and implemented to develop solutions for general boundary-value problems for PMC microstructures that accounts for micromechanics-based constitutive relations including fine scale mechanisms of material failure. The defects considered are voids, and the microstructure is explicitly represented by a distribution of fibers and voids embedded in a polymer matrix. Fiber is modeled as a linearly elastic material while the polymer matrix is modeled as an elastic-viscoplastic material. Two distinct models for the matrix behavior are implemented: (i) Drucker-Prager type Bodner model that accounts for rate and pressure-sensitivity, and (ii) improved macromolecular constitutive model that also accounts for temperature dependence, small-strain softening and large-strain hardening. Damage is simulated by the Gearing-Anand craze model as a reference model and by a new micromechanical craze model, developed to account for craze initiation, growth and breakdown. Critical dilatational energy density criterion is utilized to predict fiber-matrix debonding through cavitation induced matrix cracking. An extensive parametric study is conducted in which the roles of void shape, size and distribution relative to fiber in determining damage initiation and evolution are investigated under imposed temperature and strain rate conditions. Results show there are significant effects of voids on microstructural damage as well as on the overall deformational and failure response of composites.

  14. Modifications in stromal extracellular matrix of aged corneas can be induced by ultraviolet A irradiation

    PubMed Central

    Gendron, Sébastien P; Rochette, Patrick J

    2015-01-01

    With age, structural and functional changes can be observed in human cornea. Some studies have shown a loss of corneal transparency and an increase in turbidity associated with aging. These changes are caused by modifications in the composition and arrangement of extracellular matrix in the corneal stroma. In human skin, it is well documented that exposure to solar radiation, and mainly to the UVA wavelengths, leads to phenotypes of photoaging characterized by alteration in extracellular matrix of the dermis. Although the cornea is also exposed to solar radiation, the extracellular matrix modifications observed in aging corneas have been mainly attributed to chronological aging and not to solar exposure. To ascertain the real implication of UVA exposure in extracellular matrix changes observed with age in human cornea, we have developed a model of photoaging by chronically exposing corneal stroma keratocytes with a precise UVA irradiation protocol. Using this model, we have analyzed UVA-induced transcriptomic and proteomic changes in corneal stroma. Our results show that cumulative UVA exposure causes changes in extracellular matrix that are found in corneal stromas of aged individuals, suggesting that solar exposure catalyzes corneal aging. Indeed, we observe a downregulation of collagen and proteoglycan gene expression and a reduction in proteoglycan production and secretion in response to cumulative UVA exposure. This study provides the first evidence that chronic ocular exposure to sunlight affects extracellular matrix composition and thus plays a role in corneal changes observed with age. PMID:25728164

  15. Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples.

    PubMed

    Takos, Adam M; Jaffé, Felix W; Jacob, Steele R; Bogs, Jochen; Robinson, Simon P; Walker, Amanda R

    2006-11-01

    Anthocyanins are secondary metabolites found in higher plants that contribute to the colors of flowers and fruits. In apples (Malus domestica Borkh.), several steps of the anthocyanin pathway are coordinately regulated, suggesting control by common transcription factors. A gene encoding an R2R3 MYB transcription factor was isolated from apple (cv Cripps' Pink) and designated MdMYB1. Analysis of the deduced amino acid sequence suggests that this gene encodes an ortholog of anthocyanin regulators in other plants. The expression of MdMYB1 in both Arabidopsis (Arabidopsis thaliana) plants and cultured grape cells induced the ectopic synthesis of anthocyanin. In the grape (Vitis vinifera) cells MdMYB1 stimulated transcription from the promoters of two apple genes encoding anthocyanin biosynthetic enzymes. In ripening apple fruit the transcription of MdMYB1 was correlated with anthocyanin synthesis in red skin sectors of fruit. When dark-grown fruit were exposed to sunlight, MdMYB1 transcript levels increased over several days, correlating with anthocyanin synthesis in the skin. MdMYB1 gene transcripts were more abundant in red skin apple cultivars compared to non-red skin cultivars. Several polymorphisms were identified in the promoter of MdMYB1. A derived cleaved amplified polymorphic sequence marker designed to one of these polymorphisms segregated with the inheritance of skin color in progeny from a cross of an unnamed red skin selection (a sibling of Cripps' Pink) and the non-red skin cultivar Golden Delicious. We conclude that MdMYB1 coordinately regulates genes in the anthocyanin pathway and the expression level of this regulator is the genetic basis for apple skin color.

  16. Regulation of Extracellular Matrix Remodeling Proteins by Osteoblasts in Titanium Nanoparticle-Induced Aseptic Loosening Model.

    PubMed

    Xie, Jing; Hou, Yanhua; Fu, Na; Cai, Xiaoxiao; Li, Guo; Peng, Qiang; Lin, Yunfeng

    2015-10-01

    Titanium (Ti)-wear particles, formed at the bone-implant interface, are responsible for aseptic loosening, which is a main cause of total joint replacement failure. There have been many studies on Ti particle-induced function changes in mono-cultured osteoblasts and synovial cells. However, little is known on extracellular matrix remodeling displayed by osteoblasts when in coexistence with Synovial cells. To further mimic the bone-implant interface environment, we firstly established a nanoscaled-Ti particle-induced aseptic loosening system by co-culturing osteoblasts and Synovial cells. We then explored the impact of the Synovial cells on Ti particle-engulfed osteoblasts in the mimicked flamed niche. The matrix metalloproteinases and lysyl oxidases expression levels, two protein families which are critical in osseointegration, were examined under induction by tumor necrosis factor-alpha. It was found that the co-culture between the osteoblasts and Synovial cells markedly increased the migration and proliferation of the osteoblasts, even in the Ti-particle engulfed osteoblasts. Importantly, the Ti-particle engulfed osteoblasts, induced by TNF-alpha after the co-culture, enhanced the release of the matrix metalloproteinases and reduced the expressions of lysyl oxidases. The regulation of extracellular matrix remodeling at the protein level was further assessed by investigations on gene expression of the matrix metalloproteinases and lysyl oxidases, which also suggested that the regulation started at the genetic level. Our research work has therefore revealed the critical role of multi cell-type interactions in the extracellular matrix remodeling within the peri-prosthetic tissues, which provides new insights on aseptic loosening and brings new clues about incomplete osseointegration between the implantation materials and their surrounding bones.

  17. Inducible De Novo Biosynthesis of Isoflavonoids in Soybean Leaves by Spodoptera litura Derived Elicitors: Tracer Techniques Aided by High Resolution LCMS.

    PubMed

    Nakata, Ryu; Kimura, Yuki; Aoki, Kenta; Yoshinaga, Naoko; Teraishi, Masayoshi; Okumoto, Yutaka; Huffaker, Alisa; Schmelz, Eric A; Mori, Naoki

    2016-12-01

    Isoflavonoids are a characteristic family of natural products in legumes known to mediate a range of plant-biotic interactions. For example, in soybean (Glycine max: Fabaceae) multiple isoflavones are induced and accumulate in leaves following attack by Spodoptera litura (Lepidoptera: Noctuidae) larvae. To quantitatively examine patterns of activated de novo biosynthesis, soybean (Var. Enrei) leaves were treated with a combination of plant defense elicitors present in S. litura gut content extracts and L-α-[(13)C9, (15)N]phenylalanine as a traceable isoflavonoid precursor. Combined treatments promoted significant increases in (13)C-labeled isoflavone aglycones (daidzein, formononetin, and genistein), (13)C-labeled isoflavone 7-O-glucosides (daidzin, ononin, and genistin), and (13)C-labeled isoflavone 7-O-(6″-O-malonyl-β-glucosides) (malonyldaidzin, malonylononin, and malonylgenistin). In contrast levels of (13)C-labeled flavones and flavonol (4',7-dihydroxyflavone, kaempferol, and apigenin) were not significantly altered. Curiously, application of fatty acid-amino acid conjugate (FAC) elicitors present in S. litura gut contents, namely N-linolenoyl-L-glutamine and N-linoleoyl-L-glutamine, both promoted the induced accumulation of isoflavone 7-O-glucosides and isoflavone 7-O-(6″-O-malonyl-β-glucosides), but not isoflavone aglycones in the leaves. These results demonstrate that at least two separate reactions are involved in elicitor-induced soybean leaf responses to the S. litura gut contents: one is the de novo biosynthesis of isoflavone conjugates induced by FACs, and the other is the hydrolysis of the isoflavone conjugates to yield isoflavone aglycones. Gut content extracts alone displayed no hydrolytic activity. The quantitative analysis of isoflavone de novo biosynthesis, with respect to both aglycones and conjugates, affords a useful bioassay system for the discovery of additional plant defense elicitor(s) in S. litura gut contents that specifically

  18. Graphene nanoplatelets induced heterogeneous bimodal structural magnesium matrix composites with enhanced mechanical properties

    PubMed Central

    Xiang, Shulin; Wang, Xiaojun; Gupta, Manoj; Wu, Kun; Hu, Xiaoshi; Zheng, Mingyi

    2016-01-01

    In this work, graphene nanoplatelets (GNPs) reinforced magnesium (Mg) matrix composites were synthesised using the multi-step dispersion route. Well-dispersed but inhomogeneously distributed GNPs were obtained in the matrix. Compared with the monolithic alloy, the nanocomposites exhibited dramatically enhanced Young’s modulus, yield strength and ultimate tensile strength and relatively high plasticity, which mainly attributed to the significant heterogeneous laminated microstructure induced by the addition of GNPs. With increasing of the concentration of GNPs, mechanical properties of the composites were gradually improved. Especially, the strengthening efficiency of all the composites exceeded 100%, which was significantly higher than that of carbon nanotubes reinforced Mg matrix composites. The grain refinement and load transfer provided by the two-dimensional and wrinkled surface structure of GNPs were the dominated strengthening mechanisms of the composites. This investigation develops a new method for incorporating GNPs in metals for fabricating high-performance composites. PMID:27941839

  19. Graphene nanoplatelets induced heterogeneous bimodal structural magnesium matrix composites with enhanced mechanical properties

    NASA Astrophysics Data System (ADS)

    Xiang, Shulin; Wang, Xiaojun; Gupta, Manoj; Wu, Kun; Hu, Xiaoshi; Zheng, Mingyi

    2016-12-01

    In this work, graphene nanoplatelets (GNPs) reinforced magnesium (Mg) matrix composites were synthesised using the multi-step dispersion route. Well-dispersed but inhomogeneously distributed GNPs were obtained in the matrix. Compared with the monolithic alloy, the nanocomposites exhibited dramatically enhanced Young’s modulus, yield strength and ultimate tensile strength and relatively high plasticity, which mainly attributed to the significant heterogeneous laminated microstructure induced by the addition of GNPs. With increasing of the concentration of GNPs, mechanical properties of the composites were gradually improved. Especially, the strengthening efficiency of all the composites exceeded 100%, which was significantly higher than that of carbon nanotubes reinforced Mg matrix composites. The grain refinement and load transfer provided by the two-dimensional and wrinkled surface structure of GNPs were the dominated strengthening mechanisms of the composites. This investigation develops a new method for incorporating GNPs in metals for fabricating high-performance composites.

  20. Graphene nanoplatelets induced heterogeneous bimodal structural magnesium matrix composites with enhanced mechanical properties.

    PubMed

    Xiang, Shulin; Wang, Xiaojun; Gupta, Manoj; Wu, Kun; Hu, Xiaoshi; Zheng, Mingyi

    2016-12-12

    In this work, graphene nanoplatelets (GNPs) reinforced magnesium (Mg) matrix composites were synthesised using the multi-step dispersion route. Well-dispersed but inhomogeneously distributed GNPs were obtained in the matrix. Compared with the monolithic alloy, the nanocomposites exhibited dramatically enhanced Young's modulus, yield strength and ultimate tensile strength and relatively high plasticity, which mainly attributed to the significant heterogeneous laminated microstructure induced by the addition of GNPs. With increasing of the concentration of GNPs, mechanical properties of the composites were gradually improved. Especially, the strengthening efficiency of all the composites exceeded 100%, which was significantly higher than that of carbon nanotubes reinforced Mg matrix composites. The grain refinement and load transfer provided by the two-dimensional and wrinkled surface structure of GNPs were the dominated strengthening mechanisms of the composites. This investigation develops a new method for incorporating GNPs in metals for fabricating high-performance composites.

  1. Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

    PubMed Central

    Gil, Carmen; Solano, Cristina; Burgui, Saioa; Latasa, Cristina; García, Begoña; Toledo-Arana, Alejandro

    2014-01-01

    The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections. PMID:24343648

  2. Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots.

    PubMed

    Armendáriz, O; Gil-Monreal, M; Zulet, A; Zabalza, A; Royuela, M

    2016-05-01

    The objective of this work was to ascertain whether there is a general pattern of carbon allocation and utilisation in plants following herbicide supply, independent of the site of application: sprayed on leaves or supplied to nutrient solution. The herbicides studied were the amino acid biosynthesis-inhibiting herbicides (ABIH): glyphosate, an inhibitor of aromatic amino acid biosynthesis, and imazamox, an inhibitor of branched-chain amino acid biosynthesis. All treated plants showed impaired carbon metabolism; carbohydrate accumulation was detected in both leaves and roots of the treated plants. The accumulation in roots was due to lack of use of available sugars as growth was arrested, which elicited soluble carbohydrate accumulation in the leaves due to a decrease in sink strength. Under aerobic conditions, ethanol fermentative metabolism was enhanced in roots of the treated plants. This fermentative response was not related to a change in total respiration rates or cytochrome respiratory capacity, but an increase in alternative oxidase capacity was detected. Pyruvate accumulation was detected after most of the herbicide treatments. These results demonstrate that both ABIH induce the less-efficient, ATP-producing pathways, namely fermentation and alternative respiration, by increasing the key metabolite, pyruvate. The plant response was similar not only for the two ABIH but also after foliar or residual application.

  3. Notch modulates VEGF action in endothelial cells by inducing Matrix Metalloprotease activity

    PubMed Central

    2011-01-01

    Background In the vasculature, Notch signaling functions as a downstream effecter of Vascular Endothelial Growth Factor (VEGF) signaling. VEGF regulates sprouting angiogenesis in part by inducing and activating matrix metalloproteases (MMPs). This study sought to determine if VEGF regulation of MMPs was mediated via Notch signaling and to determine how Notch regulation of MMPs influenced endothelial cell morphogenesis. Methods and Results We assessed the relationship between VEGF and Notch signaling in cultured human umbilical vein endothelial cells. Overexpression of VEGF-induced Notch4 and the Notch ligand, Dll4, activated Notch signaling, and altered endothelial cell morphology in a fashion similar to that induced by Notch activation. Expression of a secreted Notch antagonist (Notch1 decoy) suppressed VEGF-mediated activation of endothelial Notch signaling and endothelial morphogenesis. We demonstrate that Notch mediates VEGF-induced matrix metalloprotease activity via induction of MMP9 and MT1-MMP expression and activation of MMP2. Introduction of a MMP inhibitor blocked Notch-mediated endothelial morphogenesis. In mice, analysis of VEGF-induced dermal angiogenesis demonstrated that the Notch1 decoy reduced perivascular MMP9 expression. Conclusions Taken together, our data demonstrate that Notch signaling can act downstream of VEGF signaling to regulate endothelial cell morphogenesis via induction and activation of specific MMPs. In a murine model of VEGF-induced dermal angiogenesis, Notch inhibition led to reduced MMP9 expression. PMID:21349159

  4. α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

    SciTech Connect

    Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-02-01

    We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blot and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated.

  5. Mechanisms of fluid-flow-induced matrix production in bone tissue engineering.

    PubMed

    Morris, H L; Reed, C I; Haycock, J W; Reilly, G C

    2010-12-01

    Matrix production by tissue-engineered bone is enhanced when the growing tissue is subjected to mechanical forces and/or fluid flow in bioreactor culture. Cells deposit collagen and mineral, depending upon the mechanical loading that they receive. However, the molecular mechanisms of flow-induced signal transduction in bone are poorly understood. The hyaluronan (HA) glycocalyx has been proposed as a potential mediator of mechanical forces in bone. Using a parallel-plate flow chamber the effects of removal of HA on flow-induced collagen production and NF-kappaB activation in MLO-A5 osteoid osteocytes were investigated. Short periods of fluid flow significantly increased collagen production and induced translocation of the NF-kappaB subunit p65 to the cell's nuclei in 65 per cent of the cell population. Enzymatic removal of the HA coat and antibody blocking of CD44 (a transmembrane protein that binds to HA) eliminated the fluid-flow-induced increase in collagen production but had no effect on the translocation of p65. HA and CD44 appear to play roles in transducing the flow signals that modulate collagen production over long-term culture but not in the short-term flow-induced activation of NF-kappaB, implying that multiple signalling events are initiated from the commencement of flow. Understanding the mechanotransduction events that enable fluid flow to stimulate bone matrix production will allow the optimization of bioreactor design and flow profiles for bone tissue engineering.

  6. Polyphosphate induces matrix metalloproteinase-3-mediated proliferation of odontoblast-like cells derived from induced pluripotent stem cells

    SciTech Connect

    Ozeki, Nobuaki; Hase, Naoko; Yamaguchi, Hideyuki; Hiyama, Taiki; Kawai, Rie; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-05-01

    Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously reported that cytokine-induced matrix metalloproteinase (MMP)-3 accelerates the proliferation of purified odontoblast-like cells. In this study, MMP-3 small interfering RNA (siRNA) was transfected into odontoblast-like cells derived from induced pluripotent stem cells to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation and differentiation into odontoblast-like cells. Treatment with Poly(P) led to an increase in both cell proliferation and additional odontoblastic differentiation. Poly(P)-treated cells showed a small but significant increase in dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) mRNA expression, which are markers of mature odontoblasts. The cells also acquired additional odontoblast-specific properties including adoption of an odontoblastic phenotype typified by high alkaline phosphatase (ALP) activity and a calcification capacity. In addition, Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA-mediated disruption of the expression of these effectors potently suppressed the expression of odontoblastic biomarkers ALP, DSPP, and DMP-1, and blocked calcification. Interestingly, upon siRNA-mediated silencing of MMP-3, we noted a potent and significant decrease in cell proliferation. Using specific siRNAs, we revealed that a unique signaling cascade, Poly(P)→MMP-3→DSPP and/or DMP-1, was intimately involved in the proliferation of odontoblast-like cells. - Highlights: • Polyphosphate increases proliferation of iPS cell-derived odontoblast-like cells. • Polyphosphate-induced MMP-3 results in an increase of cell proliferation. • Induced cell proliferation involves MMP-3, DSPP, and/or DMP-1 sequentially. • Induced MMP-3 also results in an increase of odontoblastic

  7. Passive Strain-Induced Matrix Synthesis and Organization in Shape-Specific, Cartilaginous Neotissues

    PubMed Central

    MacBarb, Regina F.; Paschos, Nikolaos K.; Abeug, Reedge; Makris, Eleftherios A.; Hu, Jerry C.

    2014-01-01

    Tissue-engineered musculoskeletal soft tissues typically lack the appropriate mechanical robustness of their native counterparts, hindering their clinical applicability. With structure and function being intimately linked, efforts to capture the anatomical shape and matrix organization of native tissues are imperative to engineer functionally robust and anisotropic tissues capable of withstanding the biomechanically complex in vivo joint environment. The present study sought to tailor the use of passive axial compressive loading to drive matrix synthesis and reorganization within self-assembled, shape-specific fibrocartilaginous constructs, with the goal of developing functionally anisotropic neotissues. Specifically, shape-specific fibrocartilaginous neotissues were subjected to 0, 0.01, 0.05, or 0.1 N axial loads early during tissue culture. Results found the 0.1-N load to significantly increase both collagen and glycosaminoglycan synthesis by 27% and 67%, respectively, and to concurrently reorganize the matrix by promoting greater matrix alignment, compaction, and collagen crosslinking compared with all other loading levels. These structural enhancements translated into improved functional properties, with the 0.1-N load significantly increasing both the relaxation modulus and Young's modulus by 96% and 255%, respectively, over controls. Finite element analysis further revealed the 0.1-N uniaxial load to induce multiaxial tensile and compressive strain gradients within the shape-specific neotissues, with maxima of 10.1%, 18.3%, and −21.8% in the XX-, YY-, and ZZ-directions, respectively. This indicates that strains created in different directions in response to a single axis load drove the observed anisotropic functional properties. Together, results of this study suggest that strain thresholds exist within each axis to promote matrix synthesis, alignment, and compaction within the shape-specific neotissues. Tailoring of passive axial loading, thus, presents

  8. Directional conductivity in SWNT-collagen-fibrin composite biomaterials through strain-induced matrix alignment.

    PubMed

    Voge, Christopher M; Kariolis, Mihalis; MacDonald, Rebecca A; Stegemann, Jan P

    2008-07-01

    Composite biomaterials incorporating fibroblast cells, collagen Type I, fibrin, and 2 wt % carboxylated SWNT were created, and their properties were compared with similar control constructs without SWNT. Alignment of the matrix was stimulated by application of 8% cyclic strain for three 12-h periods over three days. All constructs underwent cell-mediated gel compaction to 15-20% of their initial volume, which was not affected by SWNT loading. Mechanical strain increased the rate of compaction, and strained constructs were significantly more compacted than unstrained controls by day 3. Cell viability and morphology were similar in both control and SWNT-loaded constructs, but unstrained samples exhibited a more stellate appearance with more numerous cellular projections. Application of mechanical strain caused clear alignment of both the cells and matrix in the direction of the applied strain. Bioimpedance measurements showed that SWNT loading increased the electrical conductivity of composite constructs, and that mechanically-induced alignment of the matrix/SWNT caused a further increase in conductivity. These results demonstrate that SWNT can be used to augment the electrical properties of 3D protein hydrogels, and that anisotropy in the matrix further enhances these properties. Such electrically conductive biopolymers may have a variety of applications in tissue engineering and biosensor development.

  9. Three-dimensional collagen matrix induces a mechanosensitive invasive epithelial phenotype

    PubMed Central

    Carey, Shawn P.; Martin, Karen E.; Reinhart-King, Cynthia A.

    2017-01-01

    A critical step in breast cancer progression is local tissue invasion, during which cells pass from the epithelial compartment to the stromal compartment. We recently showed that malignant leader cells can promote the invasion of otherwise non-invasive epithelial follower cells, but the effects of this induced-invasion phenomenon on follower cell phenotype remain unclear. Notably, this process can expose epithelial cells to the stromal extracellular matrix (ECM), which is distinct from the ECM within the normal epithelial microenvironment. Here, we used a 3D epithelial morphogenesis model in which cells were cultured in biochemically and mechanically defined matrices to examine matrix-mediated gene expression and the associated phenotypic response. We found that 3D collagen matrix promoted expression of mesenchymal genes including MT1-MMP, which was required for collagen-stimulated invasive behavior. Epithelial invasion required matrix anchorage as well as signaling through Src, PI3K, and Rac1, and increasingly stiff collagen promoted dispersive epithelial cell invasion. These results suggest that leader cell-facilitated access to the stromal ECM may trigger an invasive phenotype in follower epithelial cells that could enable them to actively participate in local tissue invasion. PMID:28186196

  10. Sesamin inhibits lipopolysaccharide-induced inflammation and extracellular matrix catabolism in rat intervertebral disc.

    PubMed

    Li, Kang; Li, Yan; Xu, Bo; Mao, Lu; Zhao, Jie

    2016-09-01

    Intervertebral disc (IVD) degeneration contributes to most spinal degenerative diseases, while treatment inhibiting IVD degeneration is still in the experimental stage. Sesamin, a bioactive component extracted from sesame, has been reported to exert chondroprotective and anti-inflammatory effects. Here, we analyzed the anti-inflammatory and anti-catabolic effects of sesamin on rat IVD in vitro and ex vivo. Results show that sesamin significantly inhibits the lipopolysaccharide (LPS)-induced expression of catabolic enzymes (MMP-1, MMP-3, MMP-13, ADAMTS-4, ADAMTS-5) and inflammation factors (IL-1β, TNF-α, iNOS, NO, COX-2, PGE2) in a dose-dependent manner in vitro. It is also proven that migration of macrophages induced by LPS can be inhibited by treatment with sesamin. Organ culture experiments demonstrate that sesamin protects the IVD from LPS-induced depletion of the extracellular matrix ex vivo. Moreover, sesamin suppresses LPS-induced activation of the mitogen-activated protein kinase (MAPK) pathway through inhibiting phosphorylation of JNK, the common downstream signaling pathway of LPS and IL-1β, which may be the potential mechanism of the effects of sesamin. In light of our results, sesamin protects the IVD from inflammation and extracellular matrix catabolism, presenting positive prospects in the treatment of IVD degenerative diseases.

  11. Hydrogen peroxide, nitric oxide and UV RESISTANCE LOCUS8 interact to mediate UV-B-induced anthocyanin biosynthesis in radish sprouts

    PubMed Central

    Wu, Qi; Su, Nana; Zhang, Xiaoyan; Liu, Yuanyuan; Cui, Jin; Liang, Yongchao

    2016-01-01

    The cross talk among hydrogen peroxide (H2O2), nitric oxide (NO) and UV RESISTANCE LOCUS8 (UVR8) in UV-B-induced anthocyanin accumulation in the hypocotyls of radish sprouts was investigated. The results showed that UV-B irradiation significantly increased the anthocyanin accumulation and the expression of UVR8, and a similar trend appeared in radish sprouts subjected to cadmium, chilling and salt stresses regardless of light source. However, these responses disappeared under dark exposure. These results suggest that abiotic stress-induced anthocyanin accumulation and UVR8 expression were light-dependent. Moreover, abiotic stresses all enhanced the production of H2O2 and exogenous H2O2 addition significantly increased the anthocyanin concentration and UVR8 transcription, while these increases were severely inhibited by addition of dimethylthiourea (DMTU, a chemical trap for H2O2). It seems to suggest that H2O2 played an important role in the anthocyanin biosynthesis. Furthermore, addition of 0.5 mM sodium nitroprusside (SNP, a NO-releasing compound) substantially induced the anthocyanin accumulation, and H2O2-induced anthocyanin accumulation and UVR8 expression were significantly suppressed by co-treatment with 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO, a NO scavenger), which was parallel with the expression of anthocyanin biosynthesis-related transcription factors and structural genes. All these results demonstrate that both H2O2 and NO are involved in UV-B-induced anthocyanin accumulation, and there is a crosstalk between them as well as a classical UVR8 pathway. PMID:27404993

  12. Hydrogen peroxide, nitric oxide and UV RESISTANCE LOCUS8 interact to mediate UV-B-induced anthocyanin biosynthesis in radish sprouts.

    PubMed

    Wu, Qi; Su, Nana; Zhang, Xiaoyan; Liu, Yuanyuan; Cui, Jin; Liang, Yongchao

    2016-07-12

    The cross talk among hydrogen peroxide (H2O2), nitric oxide (NO) and UV RESISTANCE LOCUS8 (UVR8) in UV-B-induced anthocyanin accumulation in the hypocotyls of radish sprouts was investigated. The results showed that UV-B irradiation significantly increased the anthocyanin accumulation and the expression of UVR8, and a similar trend appeared in radish sprouts subjected to cadmium, chilling and salt stresses regardless of light source. However, these responses disappeared under dark exposure. These results suggest that abiotic stress-induced anthocyanin accumulation and UVR8 expression were light-dependent. Moreover, abiotic stresses all enhanced the production of H2O2 and exogenous H2O2 addition significantly increased the anthocyanin concentration and UVR8 transcription, while these increases were severely inhibited by addition of dimethylthiourea (DMTU, a chemical trap for H2O2). It seems to suggest that H2O2 played an important role in the anthocyanin biosynthesis. Furthermore, addition of 0.5 mM sodium nitroprusside (SNP, a NO-releasing compound) substantially induced the anthocyanin accumulation, and H2O2-induced anthocyanin accumulation and UVR8 expression were significantly suppressed by co-treatment with 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO, a NO scavenger), which was parallel with the expression of anthocyanin biosynthesis-related transcription factors and structural genes. All these results demonstrate that both H2O2 and NO are involved in UV-B-induced anthocyanin accumulation, and there is a crosstalk between them as well as a classical UVR8 pathway.

  13. R-Matrix Codes for Charged-particle Induced Reactionsin the Resolved Resonance Region

    SciTech Connect

    Leeb, Helmut; Dimitriou, Paraskevi; Thompson, Ian J.

    2017-01-01

    A Consultant’s Meeting was held at the IAEA Headquarters, from 5 to 7 December 2016, to discuss the status of R-matrix codes currently used in calculations of charged-particle induced reaction cross sections at low energies. The meeting was a follow-up to the R-matrix Codes meeting held in December 2015, and served the purpose of monitoring progress in: the development of a translation code to enable exchange of input/output parameters between the various codes in different formats, fitting procedures and treatment of uncertainties, the evaluation methodology, and finally dissemination. The details of the presentations and technical discussions, as well as additional actions that were proposed to achieve all the goals of the meeting are summarized in this report.

  14. Internal damping due to dislocation movements induced by thermal expansion mismatch between matrix and particles in metal matrix composites. [Al/SiC

    SciTech Connect

    Girand, C.; Lormand, G.; Fougeres, R.; Vincent, A. )

    1993-05-01

    In metal matrix composites (MMCs), the mechanical 1 of the reinforcement-matrix interface is an important parameter because it governs the load transfer from matrix to particles, from which the mechanical properties of these materials are derived. Therefore, it would be useful to set out an experimental method able to characterize the interface and the adjacent matrix behaviors. Thus, a study has been undertaken by means of internal damping (I.D.) measurements, which are well known to be very sensitive for studying irreversible displacements at the atomic scale. More especially, this investigation is based on the fact that, during cooling of MMC's, stress concentrations originating from differences in coefficients of thermal expansion (C.T.E.) of matrix and particles should induce dislocation movements in the matrix surrounding the reinforcement; that is, local microplastic strains occur. Therefore, during I.D. measurements vs temperature these movements should contribute to MMCs I.D. in a process similar to those involved around first order phase transitions in solids. The aim of this paper is to present, in the case of Al/SiC particulate composites, new developments of this approach that has previously led to promising results in the case of Al-Si alloys.

  15. Cordycepin inhibits LPS-induced inflammatory and matrix degradation in the intervertebral disc

    PubMed Central

    Mao, Lu; Han, Xiuguo; Zhang, Kai; Zhao, Changqing

    2016-01-01

    Cordycepin is a component of the extract obtained from Cordyceps militaris and has many biological activities, including anti-cancer, anti-metastatic and anti-inflammatory effects. Intervertebral disc degeneration (IDD) is a degenerative disease that is closely related to the inflammation of nucleus pulposus (NP) cells. The effect of cordycepin on NP cells in relation to inflammation and degeneration has not yet been studied. In our study, we used a rat NP cell culture and an intervertebral disc (IVD) organ culture model to examine the inhibitory effects of cordycepin on lipopolysaccharide (LPS)-induced gene expression and the production of matrix degradation enzymes (MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5) and oxidative stress-associated factors (nitric oxide and PGE2). We found a protective effect of cordycepin on NP cells and IVDs against LPS-induced matrix degradation and macrophage infiltration. In addition, western blot and luciferase assay results demonstrated that pretreatment with cordycepin significantly suppressed the LPS-induced activation of the NF-κB pathway. Taken together, the results of our research suggest that cordycepin could exert anti-inflammatory and anti-degenerative effects on NP cells and IVDs by inhibiting the activation of the NF-κB pathway. Therefore, cordycepin may be a potential treatment for IDD in the future. PMID:27190710

  16. Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

    SciTech Connect

    Palmieri, D.; Valli, M.; Viglio, S.; Ferrari, N.; Ledda, B.; Volta, C.; Manduca, P.

    2010-03-10

    Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

  17. Loss of Matrix Metalloproteinase-13 Attenuates Murine Radiation-Induced Pulmonary Fibrosis

    SciTech Connect

    Flechsig, Paul; Hartenstein, Bettina; Teurich, Sybille; Dadrich, Monika; Hauser, Kai; Abdollahi, Amir; Groene, Hermann-Josef; Angel, Peter; Huber, Peter E.

    2010-06-01

    Purpose: Pulmonary fibrosis is a disorder of the lungs with limited treatment options. Matrix metalloproteinases (MMPs) constitute a family of proteases that degrade extracellular matrix with roles in fibrosis. Here we studied the role of MMP13 in a radiation-induced lung fibrosis model using a MMP13 knockout mouse. Methods and Materials: We investigated the role of MMP13 in lung fibrosis by investigating the effects of MMP13 deficiency in C57Bl/6 mice after 20-Gy thoracic irradiation (6-MV Linac). The morphologic results in histology were correlated with qualitative and quantitative results of volume computed tomography (VCT), magnetic resonance imaging (MRI), and clinical outcome. Results: We found that MMP13 deficient mice developed less pulmonary fibrosis than their wildtype counterparts, showed attenuated acute pulmonary inflammation (days after irradiation), and a reduction of inflammation during the later fibrogenic phase (5-6 months after irradiation). The reduced fibrosis in MMP13 deficient mice was evident in histology with reduced thickening of alveolar septi and reduced remodeling of the lung architecture in good correlation with reduced features of lung fibrosis in qualitative and quantitative VCT and MRI studies. The partial resistance of MMP13-deficient mice to fibrosis was associated with a tendency towards a prolonged mouse survival. Conclusions: Our data indicate that MMP13 has a role in the development of radiation-induced pulmonary fibrosis. Further, our findings suggest that MMP13 constitutes a potential drug target to attenuate radiation-induced lung fibrosis.

  18. Current-induced forces in mesoscopic systems: A scattering-matrix approach.

    PubMed

    Bode, Niels; Kusminskiy, Silvia Viola; Egger, Reinhold; von Oppen, Felix

    2012-01-01

    Nanoelectromechanical systems are characterized by an intimate connection between electronic and mechanical degrees of freedom. Due to the nanoscopic scale, current flowing through the system noticeably impacts upons the vibrational dynamics of the device, complementing the effect of the vibrational modes on the electronic dynamics. We employ the scattering-matrix approach to quantum transport in order to develop a unified theory of nanoelectromechanical systems out of equilibrium. For a slow mechanical mode the current can be obtained from the Landauer-Büttiker formula in the strictly adiabatic limit. The leading correction to the adiabatic limit reduces to Brouwer's formula for the current of a quantum pump in the absence of a bias voltage. The principal results of the present paper are the scattering-matrix expressions for the current-induced forces acting on the mechanical degrees of freedom. These forces control the Langevin dynamics of the mechanical modes. Specifically, we derive expressions for the (typically nonconservative) mean force, for the (possibly negative) damping force, an effective "Lorentz" force that exists even for time-reversal-invariant systems, and the fluctuating Langevin force originating from Nyquist and shot noise of the current flow. We apply our general formalism to several simple models that illustrate the peculiar nature of the current-induced forces. Specifically, we find that in out-of-equilibrium situations the current-induced forces can destabilize the mechanical vibrations and cause limit-cycle dynamics.

  19. Sulforaphane, a cancer chemopreventive agent, induces pathways associated with membrane biosynthesis in response to tissue damage by aflatoxin B{sub 1}

    SciTech Connect

    Techapiesancharoenkij, Nirachara; Fiala, Jeannette L.A.; Navasumrit, Panida; Croy, Robert G.; Wogan, Gerald N.; Groopman, John D.; Ruchirawat, Mathuros; Essigmann, John M.

    2015-01-01

    Aflatoxin B{sub 1} (AFB{sub 1}) is one of the major risk factors for liver cancer globally. A recent study showed that sulforaphane (SF), a potent inducer of phase II enzymes that occurs naturally in widely consumed vegetables, effectively induces hepatic glutathione S-transferases (GSTs) and reduces levels of hepatic AFB{sub 1}-DNA adducts in AFB{sub 1}-exposed Sprague Dawley rats. The present study characterized the effects of SF pre-treatment on global gene expression in the livers of similarly treated male rats. Combined treatment with AFB{sub 1} and SF caused reprogramming of a network of genes involved in signal transduction and transcription. Changes in gene regulation were observable 4 h after AFB{sub 1} administration in SF-pretreated animals and may reflect regeneration of cells in the wake of AFB{sub 1}-induced hepatotoxicity. At 24 h after AFB{sub 1} administration, significant induction of genes that play roles in cellular lipid metabolism and acetyl-CoA biosynthesis was detected in SF-pretreated AFB{sub 1}-dosed rats. Induction of this group of genes may indicate a metabolic shift toward glycolysis and fatty acid synthesis to generate and maintain pools of intermediate molecules required for tissue repair, cell growth and compensatory hepatic cell proliferation. Collectively, gene expression data from this study provide insights into molecular mechanisms underlying the protective effects of SF against AFB{sub 1} hepatotoxicity and hepatocarcinogenicity, in addition to the chemopreventive activity of this compound as a GST inducer. - Highlights: • This study revealed sulforaphane (SF)-deregulated gene sets in aflatoxin B{sub 1} (AFB{sub 1})-treated rat livers. • SF redirects biochemical networks toward lipid biosynthesis in AFB{sub 1}-dosed rats. • SF enhanced gene sets that would be expected to favor cell repair and regeneration.

  20. Manganese-induced regulations in growth, yield formation, quality characters, rice aroma and enzyme involved in 2-acetyl-1-pyrroline biosynthesis in fragrant rice.

    PubMed

    Li, Meijuan; Ashraf, Umair; Tian, Hua; Mo, Zhaowen; Pan, Shenggang; Anjum, Shakeel Ahmad; Duan, Meiyang; Tang, Xiangru

    2016-06-01

    Micro-nutrient application is essential for normal plant growth while a little is known about manganese (Mn)-induced regulations in morpho-physiological attributes, aroma formation and enzyme involved in 2-acetyl-1-pyrroline (2-AP) biosynthesis in aromatic rice. Present study aimed to examine the influence of four levels of Mn i.e., Mn1 (100 mg MnSO4 pot(-1)), Mn2 (150 mg MnSO4 pot(-1)), Mn3 (200 mg MnSO4 pot(-1)), and Mn4 (250 mg MnSO4 pot(-1)) on the growth, yield formation, quality characters, rice aroma and enzyme involved in 2-acetyl-1-pyrroline biosynthesis in two fragrant rice cultivars i.e., Meixiangzhan and Nongxiang 18. Pots without Mn application were served as control (Ck). Each pot contained 15 kg of soil. Effects on agronomic characters, quality attributes, 2-AP contents and enzymes involved in 2-AP biosynthesis have been studied in early and late season rice. Results depicted that Mn improved rice growth, yield and related characters, and some quality attributes significantly. It further up-regulated proline, pyrroline-5-carboxylic acid (P5C) (precursors of 2-AP), soluble proteins and activities of proline dehydrogenase (ProDH), Δ(1) pyrroline-5-carboxylic acid synthetase (P5CS) ornithine aminotransferase (OAT) that led to enhanced 2-AP production in rice grains. Moreover, higher Mn levels resulted in increased grain Mn contents in both rice cultivars. Along with growth and yield improvement, Mn application significantly improved rice aromatic contents. Overall, Nongxiang 18 accumulated more 2-AP contents than Meixiangzhan in both seasons under Mn application. This study further explored the importance of Mn in rice aroma formation and signifies that micro-nutrients can play significant roles in rice aroma synthesis; however, intensive studies at molecular levels are still needed to understand the exact mechanisms of Mn to improve rice aroma formation.

  1. Polyamine-induced modulation of genes involved in ethylene biosynthesis and signalling pathways and nitric oxide production during olive mature fruit abscission

    PubMed Central

    Parra-Lobato, Maria C.; Gomez-Jimenez, Maria C.

    2011-01-01

    After fruit ripening, many fruit-tree species undergo massive natural fruit abscission. Olive (Olea europaea L.) is a stone-fruit with cultivars such as Picual (PIC) and Arbequina (ARB) which differ in mature fruit abscission potential. Ethylene (ET) is associated with abscission, but its role during mature fruit abscission remains largely uncharacterized. The present study investigates the possible roles of ET and polyamine (PA) during mature fruit abscission by modulating genes involved in the ET signalling and biosynthesis pathways in the abscission zone (AZ) of both cultivars. Five ET-related genes (OeACS2, OeACO2, OeCTR1, OeERS1, and OeEIL2) were isolated in the AZ and adjacent cells (AZ–AC), and their expression in various olive organs and during mature fruit abscission, in relation to interactions between ET and PA and the expression induction of these genes, was determined. OeACS2, OeACO2, and OeEIL2 were found to be the only genes that were up-regulated in association with mature fruit abscission. Using the inhibition of ET and PA biosynthesis, it is demonstrated that OeACS2 and OeEIL2 expression are under the negative control of PA while ET induces their expression in AZ–AC. Furthermore, mature fruit abscission depressed nitric oxide (NO) production present mainly in the epidermal cells and xylem of the AZ. Also, NO production was differentially responsive to ET, PA, and different inhibitors. Taken together, the results indicate that PA-dependent ET signalling and biosynthesis pathways participate, at least partially, during mature fruit abscission, and that endogenous NO and 1-aminocyclopropane-1-carboxylic acid maintain an inverse correlation, suggesting an antagonistic action of NO and ET in abscission signalling. PMID:21633085

  2. Multiscale alterations in bone matrix quality increased fragility in steroid induced osteoporosis

    PubMed Central

    Karunaratne, A.; Xi, L.; Bentley, L.; Sykes, D.; Boyde, A.; Esapa, C.T.; Terrill, N.J.; Brown, S.D.M.; Cox, R.D.; Thakker, R.V.; Gupta, H.S.

    2016-01-01

    A serious adverse clinical effect of glucocorticoid steroid treatment is secondary osteoporosis, enhancing fracture risk in bone. This rapid increase in bone fracture risk is largely independent of bone loss (quantity), and must therefore arise from degradation of the quality of the bone matrix at the micro- and nanoscale. However, we lack an understanding of both the specific alterations in bone quality n steroid-induced osteoporosis as well as the mechanistic effects of these changes. Here we demonstrate alterations in the nanostructural parameters of the mineralized fibrillar collagen matrix, which affect bone quality, and develop a model linking these to increased fracture risk in glucocorticoid induced osteoporosis. Using a mouse model with an N-ethyl-N-nitrosourea (ENU)-induced corticotrophin releasing hormone promoter mutation (Crh− 120/+) that developed hypercorticosteronaemia and osteoporosis, we utilized in situ mechanical testing with small angle X-ray diffraction, synchrotron micro-computed tomography and quantitative backscattered electron imaging to link altered nano- and microscale deformation mechanisms in the bone matrix to abnormal macroscopic mechanics. We measure the deformation of the mineralized collagen fibrils, and the nano-mechanical parameters including effective fibril modulus and fibril to tissue strain ratio. A significant reduction (51%) of fibril modulus was found in Crh− 120/+ mice. We also find a much larger fibril strain/tissue strain ratio in Crh− 120/+ mice (~ 1.5) compared to the wild-type mice (~ 0.5), indicative of a lowered mechanical competence at the nanoscale. Synchrotron microCT show a disruption of intracortical architecture, possibly linked to osteocytic osteolysis. These findings provide a clear quantitative demonstration of how bone quality changes increase macroscopic fragility in secondary osteoporosis. PMID:26657825

  3. Laser induced ultrasonic phased array using full matrix capture data acquisition and total focusing method.

    PubMed

    Stratoudaki, Theodosia; Clark, Matt; Wilcox, Paul D

    2016-09-19

    Laser ultrasonics is a technique where lasers are employed to generate and detect ultrasound. A data collection method (full matrix capture) and a post processing imaging algorithm, the total focusing method, both developed for ultrasonic arrays, are modified and used in order to enhance the capabilities of laser ultrasonics for nondestructive testing by improving defect detectability and increasing spatial resolution. In this way, a laser induced ultrasonic phased array is synthesized. A model is developed and compared with experimental results from aluminum samples with side drilled holes and slots at depths of 5 - 20 mm from the surface.

  4. Strain-Induced Localized States Within the Matrix Continuum of Self-Assembled Quantum Dots

    SciTech Connect

    Popescu, V.; Bester, G.; Zunger, A.

    2009-07-01

    Quantum dot-based infrared detectors often involve transitions from confined states of the dot to states above the minimum of the conduction band continuum of the matrix. We discuss the existence of two types of resonant states within this continuum in self-assembled dots: (i) virtual bound states, which characterize square wells even without strain and (ii) strain-induced localized states. The latter emerge due to the appearance of 'potential wings' near the dot, related to the curvature of the dots. While states (i) do couple to the continuum, states (ii) are sheltered by the wings, giving rise to sharp absorption peaks.

  5. Numerical study of deformation-induced fluid flows in periodic osteonal matrix under harmonic axial loading

    NASA Astrophysics Data System (ADS)

    Nguyen, Vu-Hieu; Lemaire, Thibault; Naili, Salah

    2009-05-01

    Living bone is a tissue that is constantly renewed. It has been demonstrated that bone fluid flow and induced shear effects on the bone cells are important players in triggering and signaling bone formation and remodelling. This Note presents a model studying interstitial fluid flow in cortical bone under axial harmonic loads. These living tissues are considered as saturated anisotropic poroelastic material characterized by three-dimensional periodic groups of osteons. Using a frequency-domain analysis, the fluid shear stress variations are studied for various loading conditions and geometrical or physical bone matrix parameters. To cite this article: V.-H. Nguyen et al., C. R. Mecanique 337 (2009).

  6. Increased potassium conductance of brain mitochondria induces resistance to permeability transition by enhancing matrix volume.

    PubMed

    Hansson, Magnus J; Morota, Saori; Teilum, Maria; Mattiasson, Gustav; Uchino, Hiroyuki; Elmér, Eskil

    2010-01-01

    Modulation of K(+) conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K(+) channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca(2+) and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K(+) or H(+) conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoK(ATP) channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K(+) conductance did not result in augmented DeltapH. The beneficial effect of valinomycin on CRC was not mediated by H(2)O(2)-induced protein kinase Cepsilon activation. Rather, increased K(+) conductance reduced H(2)O(2) generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.

  7. Echinomycin biosynthesis.

    PubMed

    Sato, Michio; Nakazawa, Takehito; Tsunematsu, Yuta; Hotta, Kinya; Watanabe, Kenji

    2013-08-01

    Echinomycin is an antitumor antibiotic secondary metabolite isolated from streptomycetes, whose core structure is biosynthesized by nonribosomal peptide synthetase (NRPS). The echinomycin biosynthetic pathway was successfully reconstituted in Escherichia coli. NRPS often contains a thioesterase domain at its C terminus for cyclorelease of the elongating peptide chain. Those thioesterase domains were shown to exhibit significant substrate tolerance. More recently, an oxidoreductase Ecm17, which forms the disulfide bridge in triostin A, was characterized. Surprisingly, an unrelated disulfide-forming enzyme GliT for gliotoxin biosynthesis was also able to catalyze the same reaction, providing another example of broad substrate specificity in secondary metabolite biosynthetic enzymes. Those promiscuous catalysts can be a valuable tool in generating diversity in natural products analogs we can produce heterologously.

  8. Biosynthesis of Gold Nanoparticles and Identification of Capping Agent Using Gas Chromatography-Mass Spectrometry and Matrix Assisted Laser Desorption Ionization-Mass Spectrometry.

    PubMed

    Karthick, V; Kumar, V Ganesh; Dhas, T Stalin; Govindaraju, K; Sinha, Sweta; Singaravelu, G

    2015-06-01

    In the present study, gold nanoparticles (AuNPs) were synthesized using leaf extract of Syzygium jambolanum and capping agent has been explored. The synthesized AuNPs have been characterized using UV-visible spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FTIR), high resolution transmission electron microscopy (HRTEM) and atomic force microscopic (AFM) analysis. The AuNPs show intense surface plasmon resonance (SPR) band at 528 nm and were found to be spherical and hexagonal in shape with particle size ranging from 20-30 nm. Transmission electron microscopy and atomic force microscopy were used to analyze the surface morphology of synthesized AuNPs. The capping ligand has been evaluated using matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) and gas chromatography-mass spectrometry (GC-MS) analysis.

  9. The effect of haem biosynthesis inhibitors and inducers on intestinal iron absorption and liver haem biosynthetic enzyme activities

    SciTech Connect

    Laftah, A.H.; Simpson, R.J. Peters, T.J.; Raja, K.B.

    2008-06-15

    The relation between haem biosynthesis and intestinal iron absorption is not well understood, we therefore investigated the effect of compounds that alter haem metabolism on duodenal iron absorption. CD1 mice were treated with either an inhibitor (succinyl acetone (SA)) or stimulator (2-allyl-2-isopropylacetamide (AIA)) of haem biosynthesis. 5-Aminolaevulinic acid (ALA) dehydratase and urinary ALA and porphobilinogen (PBG) levels, were determined. Intestinal iron absorption was assayed with in vivo and in vitro techniques. Liver hepcidin (Hamp1) and duodenal iron transporter mRNA levels were measured using RT-PCR. AIA caused increased hepatic ALA synthase (1.6-fold) and ALA dehydratase (1.4-fold, both p < 0.005) activities and increased urinary ALA and PBG excretion (2.1- and 1.4-fold, p < 0.005, p < 0.05, respectively). In vivo intestinal iron absorption was reduced to 49% of control (p < 0.005). Mice treated with SA showed decreased urinary ALA and PBG levels (75 and 55% control, both p < 0.005) and reductions in both ALA synthase and ALA dehydratase activities (77 and 56% control, p < 0.05, p < 0.005, respectively) in the liver. Liver and duodenal haem and cytochrome oxidase levels were not significantly decreased. Iron absorption was enhanced (1.26-fold, p < 0.05) and hepatic Hamp1 mRNA was reduced (53% of control, p < 0.05). In vitro duodenal iron uptake after mice were injected with SA also demonstrated an increase in Fe(III) reduction and uptake (1.27- and 1.41-fold, p < 0.01 respectively). Simultaneous injections of SA and ALA blocked the enhancing effect on iron absorption seen with SA alone. We conclude that alterations in haem biosynthesis can influence iron absorption and in particular, the intermediate ALA seems to be an inhibitor of iron absorption.

  10. Photo-induced biosynthesis of silver nanoparticles using aqueous extract of Erigeron bonariensis and its catalytic activity against Acridine Orange.

    PubMed

    Kumar, Vijay; Singh, Devendra K; Mohan, Sweta; Hasan, Syed Hadi

    2016-02-01

    The green synthesis of silver nanoparticles (AgNPs) has reduced the pollution load in the environment to a greater extent by avoiding the use of hazardous chemicals. In the present work we have developed an ecofriendly and zero cost approach for the green synthesis of more stable and spherical AgNPs using aqueous extract of Erigeron bonariensis (AEE) which act as both reducing and stabilizing agent. The reaction of AEE and AgNO3 was carried out in direct sunlight for the instant biosynthesis of AgNPs within minutes. The biosynthesis was monitored by UV-vis spectroscopy which exhibited a sharp SPR band at 442 nm and 435 nm after 5 and 35 min of sunlight exposure. The optimum conditions for biosynthesis of AgNPs were found to be 2.5mM AgNO3 concentration, 1.5% (v/v) of AEE inoculum dose and 35 min of sunlight exposure. Presence of spherical AgNPs with average size 13 nm was confirmed by SEM and TEM analysis. The XRD and SAED analysis confirmed the crystalline nature of the AgNPs where the Bragg's diffraction pattern at (111), (200), (220) and (311) corresponded to face centered cubic crystal lattice of metallic silver. The average roughness of the synthesized AgNPs was 3.21 nm which was confirmed by AFM analysis. FTIR analysis was recorded between 4000 and 400 cm(-1) which confirmed the involvement of various functional groups in the synthesis of AgNPs. The AgNPs thus obtained showed catalytic activity towards degradation of Acridine Orange (AO) without involvement of any hazardous reducing agent. The concentration dependent catalytic activity of the synthesized AgNPs was also monitored using 1, 2 and 3 mL of silver colloids and was found that the degradation of AO followed pseudo first-order kinetics.

  11. Platelet-rich fibrin matrix improves wound angiogenesis via inducing endothelial cell proliferation.

    PubMed

    Roy, Sashwati; Driggs, Jason; Elgharably, Haytham; Biswas, Sabyasachi; Findley, Muna; Khanna, Savita; Gnyawali, Urmila; Bergdall, Valerie K; Sen, Chandan K

    2011-11-01

    The economic, social, and public health burden of chronic ulcers and other compromised wounds is enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis, and tissue sealing. In this study, we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely platelet-rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study, for the first time, shows the kinetics of the viability of platelet-embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The vascular endothelial growth factor released from PRFM was primarily responsible for endothelial mitogenic response via extracellular signal-regulated protein kinase activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers.

  12. Lib, transcriptionally induced in senile plaque-associated astrocytes, promotes glial migration through extracellular matrix.

    PubMed

    Satoh, Kazuki; Hata, Mitsumi; Shimizu, Tomoko; Yokota, Hiroshi; Akatsu, Hiroyasu; Yamamoto, Takayuki; Kosaka, Kenji; Yamada, Tatsuo

    2005-09-23

    In an effort to identify astrocyte-derived molecules that may be intimately associated with progression of Alzheimer's disease (AD), Lib, a type I transmembrane protein belonging to leucine-rich repeat superfamily, has been identified as a distinctly inducible gene, responsive to beta-amyloid as well as pro-inflammatory cytokines in astrocytes. To evaluate the roles of Lib in AD, we investigated Lib expression in AD brain. In non-AD brain, Lib mRNA has been detected in neurons but not in quiescent astrocytes. On the contrary, in AD brain, Lib mRNA is expressed in activated astrocytes associated with senile plaques, but not expressed in neurons around lesions. Lib-expressing glioma cells displayed promotion of migration ability through reconstituted extracellular matrix and recombinant Lib protein bound to constituents of extracellular matrix. These observations suggest that Lib may contribute to regulation of cell-matrix adhesion interactions with respect to astrocyte recruitment around senile plaques in AD brain.

  13. Dynamic metabolic profiling together with transcription analysis reveals salinity-induced starch-to-lipid biosynthesis in alga Chlamydomonas sp. JSC4.

    PubMed

    Ho, Shih-Hsin; Nakanishi, Akihito; Kato, Yuichi; Yamasaki, Hiroaki; Chang, Jo-Shu; Misawa, Naomi; Hirose, Yuu; Minagawa, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

    2017-04-04

    Biodiesel production using microalgae would play a pivotal role in satisfying future global energy demands. Understanding of lipid metabolism in microalgae is important to isolate oleaginous strain capable of overproducing lipids. It has been reported that reducing starch biosynthesis can enhance lipid accumulation. However, the metabolic mechanism controlling carbon partitioning from starch to lipids in microalgae remains unclear, thus complicating the genetic engineering of algal strains. We here used "dynamic" metabolic profiling and essential transcription analysis of the oleaginous green alga Chlamydomonas sp. JSC4 for the first time to demonstrate the switching mechanisms from starch to lipid synthesis using salinity as a regulator, and identified the metabolic rate-limiting step for enhancing lipid accumulation (e.g., pyruvate-to-acetyl-CoA). These results, showing salinity-induced starch-to-lipid biosynthesis, will help increase our understanding of dynamic carbon partitioning in oleaginous microalgae. Moreover, we successfully determined the changes of several key lipid-synthesis-related genes (e.g., acetyl-CoA carboxylase, pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthetase and pyruvate ferredoxin oxidoreductase) and starch-degradation related genes (e.g., starch phosphorylases), which could provide a breakthrough in the marine microalgal production of biodiesel.

  14. Mildronate, an inhibitor of carnitine biosynthesis, induces an increase in gamma-butyrobetaine contents and cardioprotection in isolated rat heart infarction.

    PubMed

    Liepinsh, Edgars; Vilskersts, Reinis; Loca, Dagnija; Kirjanova, Olga; Pugovichs, Osvalds; Kalvinsh, Ivars; Dambrova, Maija

    2006-12-01

    The inhibition of gamma-butyrobetaine (GBB) hydroxylase, a key enzyme in the biosynthesis of carnitine, contributes to lay ground for the cardioprotective mechanism of action of mildronate. By inhibiting the biosynthesis of carnitine, mildronate is supposed to induce the accumulation of GBB, a substrate of GBB hydroxylase. This study describes the changes in content of carnitine and GBB in rat plasma and heart tissues during long-term (28 days) treatment of mildronate [i.p. (intraperitoneal) 100 mg/kg/daily]. Obtained data show that in concert with a decrease in carnitine concentration, the administration of mildronate caused a significant increase in GBB concentration. We detected about a 5-fold increase in GBB contents in the plasma and brain and a 7-fold increase in the heart. In addition, we tested the cardioprotective effect of mildronate in isolated rat heart infarction model after 3, 7, and 14 days of administration. We found a statistically significant decrease in necrotic area of infarcted rat hearts after 14 days of treatment with mildronate. The cardioprotective effect of mildronate correlated with an increase in GBB contents. In conclusion, our study, for the first time, provides experimental evidence that the long-term administration of mildronate not only decreases free carnitine concentration, but also causes a significant increase in GBB concentration, which correlates with the cardioprotection of mildronate.

  15. Dynamic metabolic profiling together with transcription analysis reveals salinity-induced starch-to-lipid biosynthesis in alga Chlamydomonas sp. JSC4

    PubMed Central

    Ho, Shih-Hsin; Nakanishi, Akihito; Kato, Yuichi; Yamasaki, Hiroaki; Chang, Jo-Shu; Misawa, Naomi; Hirose, Yuu; Minagawa, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

    2017-01-01

    Biodiesel production using microalgae would play a pivotal role in satisfying future global energy demands. Understanding of lipid metabolism in microalgae is important to isolate oleaginous strain capable of overproducing lipids. It has been reported that reducing starch biosynthesis can enhance lipid accumulation. However, the metabolic mechanism controlling carbon partitioning from starch to lipids in microalgae remains unclear, thus complicating the genetic engineering of algal strains. We here used “dynamic” metabolic profiling and essential transcription analysis of the oleaginous green alga Chlamydomonas sp. JSC4 for the first time to demonstrate the switching mechanisms from starch to lipid synthesis using salinity as a regulator, and identified the metabolic rate-limiting step for enhancing lipid accumulation (e.g., pyruvate-to-acetyl-CoA). These results, showing salinity-induced starch-to-lipid biosynthesis, will help increase our understanding of dynamic carbon partitioning in oleaginous microalgae. Moreover, we successfully determined the changes of several key lipid-synthesis-related genes (e.g., acetyl-CoA carboxylase, pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthetase and pyruvate ferredoxin oxidoreductase) and starch-degradation related genes (e.g., starch phosphorylases), which could provide a breakthrough in the marine microalgal production of biodiesel. PMID:28374798

  16. HpDTC1, a Stress-Inducible Bifunctional Diterpene Cyclase Involved in Momilactone Biosynthesis, Functions in Chemical Defence in the Moss Hypnum plumaeforme

    PubMed Central

    Okada, Kazunori; Kawaide, Hiroshi; Miyamoto, Koji; Miyazaki, Sho; Kainuma, Ryosuke; Kimura, Honoka; Fujiwara, Kaoru; Natsume, Masahiro; Nojiri, Hideaki; Nakajima, Masatoshi; Yamane, Hisakazu; Hatano, Yuki; Nozaki, Hiroshi; Hayashi, Ken-ichiro

    2016-01-01

    Momilactones, which are diterpenoid phytoalexins with antimicrobial and allelopathic functions, have been found only in rice and the moss Hypnum plumaeforme. Although these two evolutionarily distinct plant species are thought to produce momilactones as a chemical defence, the momilactone biosynthetic pathway in H. plumaeforme has been unclear. Here, we identified a gene encoding syn-pimara-7,15-diene synthase (HpDTC1) responsible for the first step of momilactone biosynthesis in the moss. HpDTC1 is a bifunctional diterpene cyclase that catalyses a two-step cyclization reaction of geranylgeranyl diphosphate to syn-pimara-7,15-diene. HpDTC1 transcription was up-regulated in response to abiotic and biotic stress treatments. HpDTC1 promoter-GUS analysis in transgenic Physcomitrella patens showed similar transcriptional responses as H. plumaeforme to the stresses, suggesting that a common response system to stress exists in mosses. Jasmonic acid (JA), a potent signalling molecule for inducing plant defences, could not activate HpDTC1 expression. In contrast, 12-oxo-phytodienoic acid, an oxylipin precursor of JA in vascular plants, enhanced HpDTC1 expression and momilactone accumulation, implying that as-yet-unknown oxylipins could regulate momilactone biosynthesis in H. plumaeforme. These results demonstrate the existence of an evolutionarily conserved chemical defence system utilizing momilactones and suggest the molecular basis of the regulation for inductive production of momilactones in H. plumaeforme. PMID:27137939

  17. Diet-Induced Obesity and Reduced Skin Cancer Susceptibility in Matrix Metalloproteinase 19-Deficient Mice

    PubMed Central

    Pendás, Alberto M.; Folgueras, Alicia R.; Llano, Elena; Caterina, John; Frerard, Françoise; Rodríguez, Francisco; Astudillo, Aurora; Noël, Agnès; Birkedal-Hansen, Henning; López-Otín, Carlos

    2004-01-01

    Matrix metalloproteinase 19 (MMP-19) is a member of the MMP family of endopeptidases that, in contrast to most MMPs, is widely expressed in human tissues under normal quiescent conditions. MMP-19 has been found to be associated with ovulation and angiogenic processes and is deregulated in diverse pathological conditions such as rheumatoid arthritis and cancer. To gain further insights into the in vivo functions of this protease, we have generated mutant mice deficient in Mmp19. These mice are viable and fertile and do not display any obvious abnormalities. However, Mmp19-null mice develop a diet-induced obesity due to adipocyte hypertrophy and exhibit decreased susceptibility to skin tumors induced by chemical carcinogens. Based on these results, we suggest that this enzyme plays an in vivo role in some of the tissue remodeling events associated with adipogenesis, as well as in pathological processes such as tumor progression. PMID:15169894

  18. TGF-β in dentin matrix extract induces osteoclastogenesis in vitro.

    PubMed

    Sriarj, Wannakorn; Aoki, Kazuhiro; Ohya, Keiichi; Takahashi, Mariko; Takagi, Yuzo; Shimokawa, Hitoyata

    2015-01-01

    Previously, we have demonstrated that the extracellular matrix from dentin affects osteoclastic activity in co-culture between osteoclast and osteoblast-rich fraction from mouse marrow cells. In the present study, we aimed to investigate the mechanisms of dentin matrix extract-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs). Dentin proteins were extracted from bovine incisor root dentin using 0.6 M HCl. BMMs were cultured in α-MEM containing macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-B ligand in the presence or absence of dentin matrix extract. Tartrate-resistant acid phosphatase (TRAP)-positive cell number, total TRAP activity, and the mRNA levels of osteoclast-related genes, assayed by real-time RT-PCR, were determined as markers of osteoclastogenesis. A neutralizing antibody against transforming growth factor-β1 (TGF-β1), SB431542, a TGF-β receptor inhibitor, and ELISA were used to determine the role of TGF-β1. We observed increases in TRAP-positive cell number, TRAP activity, and the mRNA levels of osteoclast-related genes of BMMs cultured with dentin extract. The use of a neutralizing antibody against TGF-β1 or SB431542 inhibited the inductive effect of dentin extract, suggesting TGF-β1 involvement. The addition of exogenous TGF-β1, but not bone morphogenic protein-2, also increased osteoclastogenesis, corresponding to the ELISA determination of TGF-β1 in the dentin extract. In conclusion, our results indicate that proteins from dentin matrix have an inductive effect in osteoclastogenesis, which is mediated, in part, by TGF-β1.

  19. The sesquiterpene biosynthesis and vessel-occlusion formation in stems of Aquilaria sinensis (Lour.) Gilg trees induced by wounding treatments without variation of microbial communities.

    PubMed

    Zhang, Zheng; Wei, Jianhe; Han, Xiaomin; Liang, Liang; Yang, Yun; Meng, Hui; Xu, Yanhong; Gao, Zhihui

    2014-12-18

    As widely recognized, agarwood formation in Aquilaria trees is induced by external wounding. Because agarwood usually harbors specific microbes, the function of microbes in agarwood formation has been debated for almost a century. In this study, two wounding methods, the burning-chisel-drilling method (BCD) and the whole-tree agarwood-inducing method (Agar-Wit), were used under the non-contamination of environmental microorganisms. After pyrosequencing the small rRNA subunits of the wounds induced by the BCD and Agar-Wit, no substantial variation was observed either in fungal and bacterial enrichment and diversity or in the relative abundances of taxa. By contrast, significant variations in fungal and bacterial communities were detected following the partial tree pruning (PTP)-wounding. The wound-induced sesquiterpene biosynthesis and vessel-occlusion formation, however, were found to be similar in all types of wounded trunks. We thus infer that wounding in the absence of variations in microbial communities may induce agarwood formation. This result does not support the long-standing notion that agarwood formation depends on microbes.

  20. The Sesquiterpene Biosynthesis and Vessel-Occlusion Formation in Stems of Aquilaria sinensis (Lour.) Gilg Trees Induced by Wounding Treatments without Variation of Microbial Communities

    PubMed Central

    Zhang, Zheng; Wei, Jianhe; Han, Xiaomin; Liang, Liang; Yang, Yun; Meng, Hui; Xu, Yanhong; Gao, Zhihui

    2014-01-01

    As widely recognized, agarwood formation in Aquilaria trees is induced by external wounding. Because agarwood usually harbors specific microbes, the function of microbes in agarwood formation has been debated for almost a century. In this study, two wounding methods, the burning-chisel-drilling method (BCD) and the whole-tree agarwood-inducing method (Agar-Wit), were used under the non-contamination of environmental microorganisms. After pyrosequencing the small rRNA subunits of the wounds induced by the BCD and Agar-Wit, no substantial variation was observed either in fungal and bacterial enrichment and diversity or in the relative abundances of taxa. By contrast, significant variations in fungal and bacterial communities were detected following the partial tree pruning (PTP)-wounding. The wound-induced sesquiterpene biosynthesis and vessel-occlusion formation, however, were found to be similar in all types of wounded trunks. We thus infer that wounding in the absence of variations in microbial communities may induce agarwood formation. This result does not support the long-standing notion that agarwood formation depends on microbes. PMID:25530613

  1. Ozone-induced kiwifruit ripening delay is mediated by ethylene biosynthesis inhibition and cell wall dismantling regulation.

    PubMed

    Minas, Ioannis S; Vicente, Ariel R; Dhanapal, Arun Prabhu; Manganaris, George A; Goulas, Vlasios; Vasilakakis, Miltiadis; Crisosto, Carlos H; Molassiotis, Athanassios

    2014-12-01

    Ozone treatments are used to preserve quality during cold storage of commercially important fruits due to its ethylene oxidizing capacity and its antimicrobial attributes. To address whether or not ozone also modulates ripening by directly affecting fruit physiology, kiwifruit (Actinidia deliciosa cv. 'Hayward') were stored in very low ethylene atmosphere at 0°C (95% RH) in air (control) or in the presence of ozone (0.3μLL(-1)) for 2 or 4 months and subsequently ripened at 20°C (90% RH) for up to 8d. Ozone-treated kiwifruit showed a significant delay of ripening during maintenance at 20°C, accompanied by a marked decrease in ethylene biosynthesis due to inhibited AdACS1 and AdACO1 expression and reduced ACC synthase (ACS) and ACC oxidase (ACO) enzyme activity. Furthermore, ozone-treated fruit exhibited a marked reduction in flesh softening and cell wall disassembly. This effect was associated with reduced cell wall swelling and pectin and neutral sugar solubilization and was correlated with the inhibition of cell wall degrading enzymes activity, such as polygalacturonase (PG) and endo-1,4-β-glucanase/1,4-β-glucosidase (EGase/glu). Conclusively, the present study indicated that ozone may exert major residual effects in fruit ripening physiology and suggested that ethylene biosynthesis and cell walls turnover are specifically targeted by ozone.

  2. HIV-1-infected macrophages induce astrogliosis by SDF-1{alpha} and matrix metalloproteinases

    SciTech Connect

    Okamoto, Mika; Wang, Xin; Baba, Masanori . E-mail: baba@m.kufm.kagoshima-u.ac.jp

    2005-11-04

    Brain macrophages/microglia and astrocytes are known to be involved in the pathogenesis of HIV-1-associated dementia (HAD). To clarify their interaction and contribution to the pathogenesis, HIV-1-infected or uninfected macrophages were used as a model of brain macrophages/microglia, and their effects on human astrocytes in vitro were examined. The culture supernatants of HIV-1-infected or uninfected macrophages induced significant astrocyte proliferation, which was annihilated with a neutralizing antibody to stromal cell-derived factor (SDF)-1{alpha} or a matrix metalloproteinase (MMP) inhibitor. In these astrocytes, CXCR4, MMP, and tissue inhibitors of matrix metalloproteinase mRNA expression and SDF-1{alpha} production were significantly up-regulated. The supernatants of infected macrophages were always more effective than those of uninfected cells. Moreover, the enhanced production of SDF-1{alpha} was suppressed by the MMP inhibitor. These results indicate that the activated and HIV-1-infected macrophages can indirectly induce astrocyte proliferation through up-regulating SDF-1{alpha} and MMP production, which implies a mechanism of astrogliosis in HAD.

  3. Protease induced plasticity: matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1

    PubMed Central

    Allen, Megan; Ghosh, Suhasini; Ahern, Gerard P.; Villapol, Sonia; Maguire-Zeiss, Kathleen A.; Conant, Katherine

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of secreted endopeptidases expressed by neurons and glia. Regulated MMP activity contributes to physiological synaptic plasticity, while dysregulated activity can stimulate injury. Disentangling the role individual MMPs play in synaptic plasticity is difficult due to overlapping structure and function as well as cell-type specific expression. Here, we develop a novel system to investigate the selective overexpression of a single MMP driven by GFAP expressing cells in vivo. We show that MMP-1 induces cellular and behavioral phenotypes consistent with enhanced signaling through the G-protein coupled protease activated receptor 1 (PAR1). Application of exogenous MMP-1, in vitro, stimulates PAR1 dependent increases in intracellular Ca2+ concentration and dendritic arborization. Overexpression of MMP-1, in vivo, increases dendritic complexity and induces biochemical and behavioral endpoints consistent with increased GPCR signaling. These data are exciting because we demonstrate that an astrocyte-derived protease can influence neuronal plasticity through an extracellular matrix independent mechanism. PMID:27762280

  4. Ultrasonic Assessment of Impact-Induced Damage and Microcracking in Polymer Matrix Composites

    NASA Technical Reports Server (NTRS)

    Liaw, Benjamin; Villars, Esther; Delmont, Frantz; Bowles, Kenneth J. (Technical Monitor)

    2001-01-01

    The main objective of this NASA FAR project is to conduct ultrasonic assessment of impact-induced damage and microcracking in polymer matrix composites at various temperatures. It is believed that the proposed study of impact damage assessment on polymer matrix composites will benefit several NASA missions and current interests, such as ballistic impact testing of composite fan containment and high strain rate deformation modeling of polymer matrix composites. Impact-induced damage mechanisms in GLARE and ARALL fiber-metal laminates subject to instrumented drop-weight impacts at various temperatures were studied. GLARE and ARALL are hybrid composites made of alternating layers of aluminum and glass (for GLARE) and aramid- (for ARALL) fiber-reinforced epoxy. Damage in pure aluminum panels impacted by foreign objects was mainly characterized by large plastic deformation surrounding a deep penetration dent. On the other hand, plastic deformation in fiber-metal laminates was often not as severe although the penetration dent was still produced. The more stiff fiber-reinforced epoxy layers provided better bending rigidity; thus, enhancing impact damage tolerance. Severe cracking, however, occurred due to the use of these more brittle fiber-reinforced epoxy layers. Fracture patterns, e.g., crack length and delamination size, were greatly affected by the lay-up configuration rather than by the number of layers, which implies that thickness effect was not significant for the panels tested in this study. Immersion ultrasound techniques were then used to assess damages generated by instrumented drop-weight impacts onto these fiber-metal laminate panels as well as 6061-T6 aluminum/cast acrylic sandwich plates adhered by epoxy. Depending on several parameters, such as impact velocity, mass, temperature, laminate configuration, sandwich construction, etc., various types of impact damage were observed, including plastic deformation, radiating cracks emanating from the impact site

  5. Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

    PubMed Central

    Smith, Michael L; Gourdon, Delphine; Little, William C; Kubow, Kristopher E; Eguiluz, R. Andresen; Luna-Morris, Sheila; Vogel, Viola

    2007-01-01

    Whether mechanically unfolded fibronectin (Fn) is present within native extracellular matrix fibrils is controversial. Fn extensibility under the influence of cell traction forces has been proposed to originate either from the force-induced lengthening of an initially compact, folded quaternary structure as is found in solution (quaternary structure model, where the dimeric arms of Fn cross each other), or from the force-induced unfolding of type III modules (unfolding model). Clarification of this issue is central to our understanding of the structural arrangement of Fn within fibrils, the mechanism of fibrillogenesis, and whether cryptic sites, which are exposed by partial protein unfolding, can be exposed by cell-derived force. In order to differentiate between these two models, two fluorescence resonance energy transfer schemes to label plasma Fn were applied, with sensitivity to either compact-to-extended conformation (arm separation) without loss of secondary structure or compact-to-unfolded conformation. Fluorescence resonance energy transfer studies revealed that a significant fraction of fibrillar Fn within a three-dimensional human fibroblast matrix is partially unfolded. Complete relaxation of Fn fibrils led to a refolding of Fn. The compactly folded quaternary structure with crossed Fn arms, however, was never detected within extracellular matrix fibrils. We conclude that the resting state of Fn fibrils does not contain Fn molecules with crossed-over arms, and that the several-fold extensibility of Fn fibrils involves the unfolding of type III modules. This could imply that Fn might play a significant role in mechanotransduction processes. PMID:17914904

  6. Products of dentin matrix protein-1 degradation by interleukin-1β-induced matrix metalloproteinase-3 promote proliferation of odontoblastic cells.

    PubMed

    Hase, Naoko; Ozeki, Nobuaki; Hiyama, Taiki; Yamaguchi, Hideyuki; Kawai, Rie; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-08-01

    We have previously reported that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in mouse embryonic stem cell (ESC)-derived odontoblast-like cells, suggesting that MMP-3 plays a potentially unique physiological role in regeneration by odontoblast-like cells. MMPs are able to process virtually any component of the extracellular matrix, including collagen, laminin and bioactive molecules. Because odontoblasts produce dentin matrix protein-1 (DMP-1), we examined whether the degraded products of DMP-1 by MMP-3 contribute to enhanced proliferation in odontoblast-like cells. IL-1β increased mRNA and protein levels of odontoblastic marker proteins, including DMP-1, but not osteoblastic marker proteins, such as osteocalcin and osteopontin. The recombinant active form of MMP-3 could degrade DMP-1 protein but not osteocalcin and osteopontin in vitro. The exogenous degraded products of DMP-1 by MMP-3 resulted in increased proliferation of odontoblast-like cells in a dose-dependent manner. Treatment with a polyclonal antibody against DMP-1 suppressed IL-1β-induced cell proliferation to a basal level, but identical treatment had no effect on the IL-1β-induced increase in MMP-3 expression and activity. Treatment with siRNA against MMP-3 potently suppressed the IL-1β-induced increase in DMP-1 expression and suppressed cell proliferation (p < 0.05). Similarly, treatment with siRNAs against Wnt5a and Wnt5b suppressed the IL-1β-induced increase in DMP-1 expression and suppressed cell proliferation (p < 0.05). Rat KN-3 cells, representative of authentic odontoblasts, showed similar responses to the odontoblast-like cells. Taken together, our current study demonstrates the sequential involvement of Wnt5, MMP-3, DMP-1 expression, and DMP-1 degradation products by MMP-3, in effecting IL-1β-induced proliferation of ESC-derived odontoblast-like cells.

  7. Misfit-induced changes of lattice parameters in two-phase systems: coherent/incoherent precipitates in a matrix

    PubMed Central

    Akhlaghi, Maryam; Steiner, Tobias; Meka, Sai Ramudu; Mittemeijer, Eric Jan

    2016-01-01

    Elastic accommodation of precipitation-induced or thermally induced misfit leads to lattice-parameter changes in crystalline multi-phase systems. Formulae for calculation of such misfit-induced lattice-parameter changes are presented for the aggregate (matrix + second-phase particles) and for the individual matrix and second phase, recognizing the occurrence of either coherent or incoherent diffraction by the matrix and second-phase particles. An overview and an (re)interpretation on the above basis is presented of published lattice-parameter data, obtained by X-ray diffraction analyses of aggregates of matrix plus second-phase particles. Examples for three types of systems consisting of a matrix with misfitting second-phase particles are dealt with, which differ in the origin of the misfit (precipitation or thermally induced) and in the type of diffraction (coherent or incoherent diffraction of matrix plus second-phase particles). The experimental data are shown to be in good to very good agreement with predictions according to the current treatment. PMID:26937236

  8. Prevotella intermedia induces matrix metalloproteinase-9 expression in human periodontal ligament cells.

    PubMed

    Guan, Su-Min; Shu, Lei; Fu, Shan-Min; Liu, Bin; Xu, Xiu-Li; Wu, Jun-Zheng

    2008-06-01

    Matrix metalloproteinases (MMPs) play pivotal roles in inflammatory diseases including chronic periodontitis. The effects of Prevotella intermedia, a major periodontal pathogen, on MMP-9 production in primary human periodontal ligament (hPDL) cells were examined in the present study. MMP-9 mRNA expression was measured by semiquantitative reverse transcriptase PCR and its protein secretion was assayed by gelatin zymography. Prevotella intermedia ATCC 25611 supernatant time and dose-dependently induced MMP-9 expression. In contrast, Porphyromanas gingivalis ATCC 33277 supernatants, Escherichia coli lipopolysacchride and IL-1beta exhibited no stimulatory effects on MMP-9 production in hPDL cells. Mitogen-activated protein kinases [MAPK, including extracellular signal-related kinases (ERK), c-jun N-terminal kinases (JNK) and p38] inhibitors exerted no effect on the P. intermedia-induced MMP-9 production, indicating that P. intermedia induced MMP-9 production through an MAPK-independent pathway. Our results demonstrated that P. intermedia may contribute to periodontal tissue destruction during chronic periodontitis by inducing MMP-9 production in hPDL cells.

  9. Biosynthesis of silver nanoparticles from Premna serratifolia L. leaf and its anticancer activity in CCl4-induced hepato-cancerous Swiss albino mice

    NASA Astrophysics Data System (ADS)

    Arockia John Paul, J.; Karunai Selvi, B.; Karmegam, N.

    2015-11-01

    In this study, we report the biosynthesis of silver nanoparticles using the ethanolic leaf powder extract of Premna serratifolia L. and its anticancer activity in carbon tetra chloride (CCl4)-induced liver cancer in Swiss albino mice (Balb/c). The synthesized silver nanoparticles were characterized by SEM, FTIR and XRD analyses. The Debye-Scherrer equation was used to calculate particle size and the average size of silver nanoparticles synthesized from P. serratifolia leaf extract was 22.97 nm. The typical pattern revealed that the sample contained cubic structure of silver nanoparticles. FTIR analysis confirmed that the bioreduction of silver ions to silver nanoparticles is due to reduction by capping material of the plant extract. The silver nanoparticles of P. serratifolia leaf extract were effective in treating liver cancer in Swiss albino mice when compared with P. serratifolia leaf extract with isoleucine.

  10. Betaine prevents homocysteine-induced memory impairment via matrix metalloproteinase-9 in the frontal cortex.

    PubMed

    Kunisawa, K; Nakashima, N; Nagao, M; Nomura, T; Kinoshita, S; Hiramatsu, M

    2015-10-01

    Betaine plays important roles that include acting as a methyl donor and converting homocysteine (Hcy) to methionine. Elevated plasma Hcy levels are known as hyperhomocysteinemia (HHcy) and contribute to impairments of learning and memory. Although it is commonly known that betaine plays an important role in Hcy metabolism, the effects of betaine on Hcy-induced memory impairment have not been investigated. Previously, we demonstrated the beneficial effects of betaine on acute stress and lipopolysaccharide-induced memory impairment. In the present study, we investigated whether betaine ameliorates Hcy-induced memory impairment and the underlying mechanisms of this putative effect. Mice were treated with Hcy (0.162mg/kg, s.c.) twice a day for nine days, and betaine (25mg/kg, s.c.) was administered 30min before the Hcy injections. The memory functions were evaluated using a spontaneous alternation performance test (Y-maze) at seven days and a step-down type passive avoidance test (SD) at nine and ten days after Hcy injection. We found that betaine suppressed the memory impairment induced by repeated Hcy injections. However, the blood concentrations of Hcy were significantly increased in the Hcy-treated mice immediately after the passive avoidance test, and betaine did not prevent this increase. Furthermore, Hcy induces redox stress in part by activating matrix metalloproteinase-9 (MMP-9), which leads to BBB dysfunction. Therefore, we tested whether betaine affected MMP-9 activity. Interestingly, treatment with betaine significantly inhibited Hcy-induced MMP-9 activity in the frontal cortex but not in the hippocampus after acute Hcy injection. These results suggest that the changes in MMP-9 activity after betaine treatment might have been partially responsible for the amelioration of the memory deficits and that MMP-9 might be a candidate therapeutic target for HHcy.

  11. A putative functional MYB transcription factor induced by low temperature regulates anthocyanin biosynthesis in purple kale (Brassica Oleracea var. acephala f. tricolor).

    PubMed

    Zhang, Bin; Hu, Zongli; Zhang, Yanjie; Li, Yali; Zhou, Shuang; Chen, Guoping

    2012-02-01

    The purple kale (Brassica Oleracea var. acephala f. tricolor) is a mutation in kales, giving the mutant phenotype of brilliant purple color in the interior. Total anthocyanin analysis showed that the amount of anthocyanins in the purple kale was up to 1.73 mg g(-1) while no anthocyanin was detected in the white kale. To elucidate the molecular mechanism of the anthocyanin biosynthesis in the purple kale, we analyzed the expression of structural genes and some transcription factors associated with anthocyanin biosynthesis in the purple cultivar "Red Dove" and the white cultivar "White Dove". The result showed that nearly all the anthocyanin biosynthetic genes showed higher expression levels in the purple cultivar than in the white cultivar, especially for DFR and ANS, they were barely detected in the white cultivar. Interestingly, the fact that a R2R3 MYB transcription factor named BoPAP1 was extremely up-regulated in the purple kale and induced by low temperature attracted our attention. Further sequence analysis showed that BoPAP1 shared high similarity with AtPAP1 and BoMYB1. In addition, the anthocyanin accumulation in the purple kale is strongly induced by the low temperature stress. The total anthocyanin contents in the purple kale under low temperature were about 50-fold higher than the plants grown in the greenhouse. The expression of anthocyanin biosynthetic genes C4H, F3H, DFR, ANS and UFGT were all enhanced under the low temperature. These evidences strongly suggest that BoPAP1 may play an important role in activating the anthocyanin structural genes for the abundant anthocyanin accumulation in the purple kale.

  12. UV-B and UV-C pre-treatments induce physiological changes and artemisinin biosynthesis in Artemisia annua L. - an antimalarial plant.

    PubMed

    Rai, Rashmi; Meena, Ram Prasad; Smita, Shachi Shuchi; Shukla, Aparna; Rai, Sanjay Kumar; Pandey-Rai, Shashi

    2011-12-02

    Present study was undertaken to investigate if short-term UV-B (4.2 kJ m(-2) day(-1)) and UV-C (5.7 kJ m(-2) day(-1)), pre-treatments can induce artemisinin biosynthesis in Artemisia annua. Twenty-one day old Artemisia seedlings were subjected to short-term (14 days) UV pre-treatment in an environmentally controlled growth chamber and then transplanted to the field under natural conditions. Treatment of A. annua with artificial UV-B and UV-C radiation not only altered the growth responses, biomass, pigment content and antioxidant enzyme activity but enhanced the secondary metabolites (artemisinin and flavonoid) content at all developmental stages as compared to non-irradiated plants. The extent of oxidative damage was measured in terms of the activities of enzymes such as catalase, superoxide dismutase and ascorbate peroxidase. Reinforcement in the antioxidative defense system seems to be a positive response of plants in ameliorating the negative effects of UV-B and UV-C radiations. While the carotenoid content was elevated, the chlorophyll content decreased under UV-B and UV-C pre-treatments. The reverse transcription PCR analysis of the genes associated in artemisinin/isoprenoid biosynthesis like 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), cytochrome P450 oxidoreductase (CPR) and amorpha-4,11-diene synthase (ADS) genes at different growth stages revealed UV induced significant over-expression of the above protein genes. UV-B and UV-C pre-treatments, led to an increase in the concentrations of artemisinin at full bloom stage by 10.5% and 15.7% than that of the control respectively. Thus, the result of our study suggests that short term UV-B pre-treatment of seedlings in greenhouse prior to transplantation into the field enhances artemisinin production with lesser yield related damages as compared to UV-C radiation in A. annua.

  13. Effect of Ultraviolet A-induced Crosslinking on Dentin Collagen Matrix

    PubMed Central

    Seseogullari-Dirihan, Roda; Tjäderhane, Leo; Pashley, David H; Tezvergil-Mutluay, Arzu

    2016-01-01

    Objectives The aim of this study was to evaluate the effect of using UVA-induced crosslinking with or without riboflavin as photosensitizers on degradation of dentin matrix by dentin proteases. Methods Demineralized dentin specimens (0.4×3×6mm, n=10/group) were subjected to: (RP1), 0.1% riboflavin-5 phosphate/UVA for 1 min; (RP5), 0.1% riboflavin-5 phosphate/UVA for 5 min; (R1), 0.1% riboflavin/UVA for 1 min; (R5), 0.1% riboflavin-UVA for 5 min; (UV1), UVA for 1 min; (UV5), UVA for 5 min. Specimens were incubated in 1 mL zinc and calcium containing media for 1 day and 1 week. An untreated group served as control (CM). After incubation, the loss of dry mass of samples was measured and aliquots of media were analyzed for the release of C-terminal fragment telopeptide (ICTP vs CTX) of collagen to evaluate for cathepsin K (CA-K) and total matrix metalloproteinase (MMP)-mediated degradation. Data were analyzed using repeated measures ANOVA at α=0.05. Results Although UVA radiation alone reduced dentin degradation, UVA-activated riboflavin or riboflavin-5 phosphate inhibited MMP and CA-K activities more than UVA alone. The effects of crosslinking were more pronounced in 7-day samples; only with CA-K were the effects of crosslinking with or without photosensitizer significantly different from controls in 1-day samples. Significance The use of bioactive forms (RP) or longer treatment time did not result with better effect. The use of UVA crosslinking reduces dentin matrix degradation, especially with photosensitizers. PMID:26314255

  14. Cardioprotective effects of lysyl oxidase inhibition against volume overload-induced extracellular matrix remodeling.

    PubMed

    El Hajj, Elia C; El Hajj, Milad C; Ninh, Van K; Gardner, Jason D

    2016-03-01

    A hallmark of heart failure (HF) is adverse extracellular matrix (ECM) remodeling, which is regulated by the collagen cross-linking enzyme, lysyl oxidase (LOX). In this study, we evaluate the efficacy of LOX inhibition to prevent adverse left ventricular (LV) remodeling and dysfunction using an experimental model of HF. Sprague-Dawley rats were subjected to surgically induced volume overload (VO) by creation of aortocaval fistula (ACF). A LOX inhibitor, beta-aminopropionitrile (BAPN; 100 mg/kg/day), was administered to rats with ACF or sham surgery at eight weeks postsurgery. Echocardiography was used to assess progressive alterations in cardiac ventricular structure and function. Left ventricular (LV) catheterization was used to assess alterations in contractility, stiffness, LV pressure and volume, and other indices of cardiac function. The LV ECM alterations were assessed by: (a) histological staining of collagen, (b) protein expression of collagen types I and III, (c) hydroxyproline assay, and (d) cross-linking assay. LOX inhibition attenuated VO-induced increases in cardiac stress, and attenuated increases in interstitial myocardial collagen, total collagen, and protein levels of collagens I and III. Both echocardiography and catheterization measurements indicated improved cardiac function post-VO in BAPN treated rats vs. untreated. Inhibition of LOX attenuated VO-induced decreases in LV stiffness and cardiac function. Overall, our data indicate that LOX inhibition was cardioprotective in the volume overloaded heart.

  15. HMGB1 Induces Secretion of Matrix Vesicles by Macrophages to Enhance Ectopic Mineralization

    PubMed Central

    Chen, Qiang; Bei, Jun-Jie; Liu, Chuan; Feng, Shi-Bin; Zhao, Wei-Bo; Zhou, Zhou; Yu, Zheng-Ping; Du, Xiao-Jun; Hu, Hou-Yuan

    2016-01-01

    Numerous clinical conditions have been linked to ectopic mineralization (EM). This process of pathological biomineralization is complex and not fully elucidated, but thought to be started within matrix vesicles (MVs). We hypothesized that high mobility group box 1 (HMGB1), a cytokine associated with biomineralizing process under physiological and pathological conditions, induces EM via promoting MVs secretion from macrophages. In this study, we found that HMGB1 significantly promoted secretion of MVs from macrophages and subsequently led to mineral deposition in elevated Ca/Pi medium in vitro. Transmission electron microscopy of calcifying MVs showed formation of hydroxyapatite crystals in the vesicle interior. Subcutaneous injection into mice with MVs derived from HMGB1-treated cells showed a greater potential to initiate regional mineralization. Mechanistic experiments revealed that HMGB1 activated neutral sphingomyelinase2 (nSMase2) that involved the receptor for advanced glycation end products (RAGE) and p38 MAPK (upstream of nSMase2). Inhibition of nSMase2 with GW4869 or p38 MAPK with SB-239063 prevented MVs secretion and mineral deposition. Collectively, HMGB1 induces MVs secretion from macrophages at least in part, via the RAGE/p38 MAPK/nSMase2 signaling pathway. Our findings thus reveal a novel mechanism by which HMGB1 induces ectopic mineralization. PMID:27243975

  16. Full matrix capture and the total focusing imaging algorithm using laser induced ultrasonic phased arrays

    NASA Astrophysics Data System (ADS)

    Stratoudaki, Theodosia; Clark, Matt; Wilcox, Paul D.

    2017-02-01

    Laser ultrasonics is a technique where lasers are used for the generation and detection of ultrasound instead of conventional piezoelectric transducers. The technique is broadband, non-contact, and couplant free, suitable for large stand-off distances, inspection of components of complex geometries and hazardous environments. In this paper, array imaging is presented by obtaining the full matrix of all possible laser generation, laser detection combinations in the array (Full Matrix Capture), at the nondestructive, thermoelastic regime. An advanced imaging technique developed for conventional ultrasonic transducers, the Total Focusing Method (TFM), is adapted for laser ultrasonics and then applied to the captured data, focusing at each point of the reconstruction area. In this way, the beamforming and steering of the ultrasound is done during the post processing. A 1-D laser induced ultrasonic phased array is synthesized with significantly improved spatial resolution and defect detectability. In this study, shear waves are used for the imaging, since they are more efficiently produced than longitudinal waves in the nondestructive, thermoelastic regime. Experimental results are presented from nondestructive, laser ultrasonic inspection of aluminum samples with side drilled holes and slots at depths varying between 5 and 20mm from the surface.

  17. Heat- and light-induced transformations of Yb trapping sites in an Ar matrix

    SciTech Connect

    Tao, L.-G.; Lambo, R. Zhou, X.-G.; Hu, S.-M.; Kleshchina, N. N.; Bezrukov, D. S.; Buchachenko, A. A.

    2015-11-07

    The low-lying electronic states of Yb isolated in a solid Ar matrix grown at 4.2 K are characterized through absorption and emission spectroscopy. Yb atoms are found to occupy three distinct thermally stable trapping sites labeled “red,” “blue,” and “violet” according to the relative positions of the absorption features they produce. Classical simulations of the site structure and relative stability broadly reproduced the experimentally observed matrix-induced frequency shifts and thus identified the red, blue, and violet sites as due to respective single substitutional (SS), tetravacancy (TV), and hexavacancy (HV) occupation. Prolonged excitation of the {sup 1}S → {sup 1}P transition was found to transfer the Yb population from HV sites into TV and SS sites. The process showed reversibility in that annealing to 24 K predominantly transferred the TV population back into HV sites. Population kinetics were used to deduce the effective rate parameters for the site transformation processes. Experimental observations indicate that the blue and violet sites lie close in energy, whereas the red one is much less stable. Classical simulations identify the blue site as the most stable one.

  18. Heat- and light-induced transformations of Yb trapping sites in an Ar matrix.

    PubMed

    Tao, L-G; Kleshchina, N N; Lambo, R; Buchachenko, A A; Zhou, X-G; Bezrukov, D S; Hu, S-M

    2015-11-07

    The low-lying electronic states of Yb isolated in a solid Ar matrix grown at 4.2 K are characterized through absorption and emission spectroscopy. Yb atoms are found to occupy three distinct thermally stable trapping sites labeled "red," "blue," and "violet" according to the relative positions of the absorption features they produce. Classical simulations of the site structure and relative stability broadly reproduced the experimentally observed matrix-induced frequency shifts and thus identified the red, blue, and violet sites as due to respective single substitutional (ss), tetravacancy (Tv), and hexavacancy (Hv) occupation. Prolonged excitation of the (1)S → (1)P transition was found to transfer the Yb population from hv sites into Tv and ss sites. The process showed reversibility in that annealing to 24 K predominantly transferred the Tv population back into Hv sites. Population kinetics were used to deduce the effective rate parameters for the site transformation processes. Experimental observations indicate that the blue and violet sites lie close in energy, whereas the red one is much less stable. Classical simulations identify the blue site as the most stable one.

  19. Laser-induced thermal characterization of nano Ag metal dispersed ceramic alumina matrix

    NASA Astrophysics Data System (ADS)

    George, Sajan D.; Anapara, Aji A.; Warrier, K. G. K.; Radhakrishnan, P.; Vallabhan, C. P. G.; Nampoori, V. P. N.

    2003-04-01

    In this paper, we report the measurements of thermal diffusivity of nano Ag metal dispersed ceramic alumina matrix sintered at different temperatures using laser induced non-destructive photoacoustic technique. Measurements of thermal diffusivity also have been carried out on specimens with various concentration of nano metal. Analysis of the data is done on the basis of one-dimensional model of Rosencwaig and Gersho. The present measurements on the thermal diffusivity of nano metal dispersed ceramic alumina shows that porosity has a great influence on the heat transport and the thermal diffusivity value. The present analysis also shows that the inclusion of nano metal into ceramic matrix increases its interconnectivity and hence the thermal diffusivity value. The present study on the samples sintered at different temperature shows that the porosity of the ceramics varies considerably with the change in sintering temperature. The results are interpreted in terms of phonon assisted heat transfer mechanism and the exclusion of pores with the increase in sintering temperature.

  20. Determination of the magnetic field induced circular birefringence using the Mueller matrix of FBGs

    NASA Astrophysics Data System (ADS)

    Descamps, Frédéric; Bette, Sébastien; Kinet, Damien; Caucheteur, Christophe

    2016-04-01

    Different methods have already been developed to measure the magnetic field with fiber Bragg gratings (FBGs). They are based on the use of a magnetic fluid or magnetostrictive materials. In addition to these methods, a direct measurement of the magnetic field is also possible by determining the circular birefringence created by the magnetic field inside the fiber. In standard optical fiber, this circular birefringence is of the same order as the intrinsic fiber birefringence or even below. The polarization properties of FBGs are therefore used to perform such measurement since they allow to determine weak birefringence with higher accuracy than standard read-out techniques. However, the obtained accuracy is usually low due to the influence of the intrinsic fiber birefringence. To mitigate this issue, we study in this work the use of the diattenuation vector. This parameter is obtained from the Mueller matrix and we show that it evolves in response to a magnetic field. In practice, we analyze its response by both simulation and experiment. In our simulations, we solve numerically the coupled mode equations of the FBG. For the experiments, the Mueller matrix is measured by an optical vector analyzer for the gratings connected in transmission. We apply an increasing magnetic field on different Bragg gratings photo-written in SMF28 fibers. The rotation of the diattenuation vector is then used to retrieve the magnetic field induced circular birefringence. A linear increase of the reconstructed circular birefringence is reported for increasing magnetic field values in the range 0-1T.

  1. Laser induced fluorescence spectra of fluorophenol cations in a Ne matrix

    USGS Publications Warehouse

    Bondybey, V.E.; English, J.H.; Miller, T.A.; Shiley, R.H.

    1983-01-01

    Laser induced fluorescence and/or absorption spectra of the cations of 2,3,5,6‐tetrafluorophenol, 2,3,5,6‐tetrafluorothiophenol, and 3,5‐difluorophenol have been obtained in a Ne matrix. The spectra of C6HF4OH+ are much better resolved than in the gas phase. The gas phase congestion is likely caused by the near degeneracy of the and electronic states whose separation is now measured at 207 cm−1. The spectrum of C6H3F2OH+ represents a deperturbed example of the Jahn–Teller distorted sym‐C6F3H3+ ion. C6H3F2SH+ shows only a broad featureless absorption.

  2. miR-221/222 induces pancreatic cancer progression through the regulation of matrix metalloproteinases.

    PubMed

    Xu, Qinhong; Li, Pei; Chen, Xin; Zong, Liang; Jiang, Zhengdong; Nan, Ligang; Lei, Jianjun; Duan, Wanxing; Zhang, Dong; Li, Xuqi; Sha, Huanchen; Wu, Zheng; Ma, Qingyong; Wang, Zheng

    2015-06-10

    MicroRNAs are involved in the initiation and progression of pancreatic cancer. In this study, we showed that miR-221/222 is overexpressed in pancreatic cancer. MiR-221/222 overexpression significantly promoted pancreatic cancer cell proliferation and invasion while inhibiting apoptosis. The expression of the matrix metalloproteinases (MMPs) MMP-2 and MMP-9 was increased in miR-221/222 mimic-transfected pancreatic cancer cells. Validation experiments identified TIMP-2 as a direct target of miR-221/222. These data indicate that overexpressed miR-221/222 may play an oncogenic role in pancreatic cancer by inducing the expression of MMP-2 and MMP-9, thus leading to cancer cell invasion.

  3. In response to partial plant shading, the lack of phytochrome A does not directly induce leaf senescence but alters the fine-tuning of chlorophyll biosynthesis.

    PubMed

    Brouwer, Bastiaan; Gardeström, Per; Keech, Olivier

    2014-07-01

    Phytochrome is thought to control the induction of leaf senescence directly, however, the signalling and molecular mechanisms remain unclear. In the present study, an ecophysiological approach was used to establish a functional connection between phytochrome signalling and the physiological processes underlying the induction of leaf senescence in response to shade. With shade it is important to distinguish between complete and partial shading, during which either the whole or only a part of the plant is shaded, respectively. It is first shown here that, while PHYB is required to maintain chlorophyll content in a completely shaded plant, only PHYA is involved in maintaining the leaf chlorophyll content in response to partial plant shading. Second, it is shown that leaf yellowing associated with strong partial shading in phyA-mutant plants actually correlates to a decreased biosynthesis of chlorophyll rather than to an increase of its degradation. Third, it is shown that the physiological impact of this decreased biosynthesis of chlorophyll in strongly shaded phyA-mutant leaves is accompanied by a decreased capacity to adjust the Light Compensation Point. However, the increased leaf yellowing in phyA-mutant plants is not accompanied by an increase of senescence-specific molecular markers, which argues against a direct role of PHYA in inducing leaf senescence in response to partial shade. In conclusion, it is proposed that PHYA, but not PHYB, is essential for fine-tuning the chlorophyll biosynthetic pathway in response to partial shading. In turn, this mechanism allows the shaded leaf to adjust its photosynthetic machinery to very low irradiances, thus maintaining a positive carbon balance and repressing the induction of leaf senescence, which can occur under prolonged periods of shade.

  4. Intramedullary Pressure and Matrix Strain Induced by Oscillatory Skeletal Muscle Stimulation and its Potential in Adaptation

    PubMed Central

    Qin, Yi-Xian; Lam, Hoyan

    2010-01-01

    Intramedullary pressure (ImP) and low-level bone strain induced by oscillatory muscle stimulation (MS) has the potential to mitigate bone loss induced by disuse osteopenia, i.e., hindlimb suspension (HLS). To test this hypothesis, we evaluated a) MS induced ImP and bone strain as function of stimulation frequency, and b) the adaptive responses to functional disuse, and disuse plus 1Hz and 20Hz stimulation in vivo. Femoral ImP and bone strain generated by MS were measured in the frequencies of 1Hz-100Hz in four rats. Forty retired breeder rats were used for the in vivo HLS study. The quadriceps muscle was stimulated at frequencies of 1 Hz and 20 Hz, 10min/d for 4 weeks. The metaphyseal trabecular bone quantity and microstructure at the distal femur were evaluated using μCT, while bone formation indices were analyzed using histomorphometric techniques. Oscillatory MS generated a maximum ImP of 45±9 mmHg at 20 Hz and produced a maximum matrix strain of 128±19 με at 10 Hz. Our analyses from the in vivo study showed that MS at 20 Hz was able to attenuate trabecular bone loss and partially maintain the microstructure induced by HLS. Conversely, there was no evidence of an adaptive effect of stimulation at 1 Hz on disused skeleton. The results suggested that oscillatory MS regulates fluid dynamics and mechanical strain in bone, which serves as a critical mediator of adaptation. These results clearly demonstrated the ability of MS in attenuating bone loss from the disuse osteopenia and could hold potential in mitigating skeletal degradation imposed by conditions of disuse, which may serve as a biomechanical intervention in clinic application. PMID:19081096

  5. Mifepristone treatment results in differential regulation of glycerolipid biosynthesis in baby hamster kidney cells expressing a mifepristone-inducible ABCA1.

    PubMed

    Hauff, Kristin D; Mitchell, Ryan W; Xu, Fred Y; Dembinski, Thomas; Mymin, David; Zha, Xiaohui; Choy, Patrick C; Hatch, Grant M

    2011-09-01

    ATP binding cassette A1 (ABCA1) transports cholesterol, phospholipids and lipophilic molecules to and across cellular membranes. We examined if ABCA1 expression altered cellular de novo glycerolipid biosynthesis in growing Baby hamster kidney (BHK) cells. Mock BHK cells or cells expressing a mifepristone-inducible ABCA1 (ABCA1) were incubated plus or minus mifepristone and then with [(3)H]serine or [(3)H]inositol or [(3)H]ethanolamine or [methyl-(3)H]choline or [(3)H]glycerol or [(14)C]oleate and radioactivity incorporated into glycerolipids determined. Mifepristone did not affect [1,3-(3)H]glycerol or [(14)C]oleate or [(3)H]ethanolamine or [methyl-(3)H]choline uptake in BHK cells. In contrast, [(3)H]glycerol and [(14)C]oleate incorporated into phosphatidylserine (PtdSer) were elevated 2.4-fold (p < 0.05) and 54% (p < 0.05), respectively, upon ABCA1 induction confirming increased PtdSer biosynthesis from these precursors. However, mifepristone inhibited [(3)H]serine uptake and incorporation into PtdSer indicating that PtdSer synthesis from serine in BHK cells is dependent on serine uptake. Mifepristone stimulated [(3)H]inositol uptake in mock and ABCA1 cells but not its incorporation into phosphatidylinositol indicating that its synthesis from inositol is independent of inositol uptake in BHK cells. [(3)H]glycerol and [(14)C]oleate incorporated into triacylglycerol were reduced and into diacylglycerol elevated only in mifepristone-induced ABCA1 expressing cells due to a decrease in diacylglycerol acyltransferase-1 (DGAT-1) activity. The presence of trichostatin A, a class I and II histone deacetylase inhibitor, reversed the ABCA1-mediated reduction in DGAT-1 activity but did not affect DGAT-1 mRNA expression. Thus, mifepristone has diverse effects on de novo glycerolipid synthesis. We suggest that caution should be exercised when using mifepristone-inducible systems for studies of glycerolipid metabolism in cells expressing glucocorticoid responsive receptors.

  6. LIM mineralization protein-1 suppresses TNF-α induced intervertebral disc degeneration by maintaining nucleus pulposus extracellular matrix production and inhibiting matrix metalloproteinases expression.

    PubMed

    Liu, Hui; Pan, Hehai; Yang, Hao; Wang, Jianru; Zhang, Kuibo; Li, Xiang; Wang, Hua; Ding, Wenbin; Li, Bingxue; Zheng, Zhaomin

    2015-03-01

    Imbalanced metabolism of Nucleus pulposus (NP) extracellular matrix (ECM) is closely correlated to Intervertebral Disc Degenerative Disease. LIM mineralization protein-1 (LMP-1) has been proven to induce sulfated glycosaminoglycan (sGAG) production in NP and have an anti-inflammatory effect in pre-osteoclast. However, whether it has any effect on the NP ECM production and degradation under inflammatory stimulation has not been studied. In the current study, a TNF-α induced cell model was established in vitro. Lentivirus encoding LMP-1 (LV-LMP-1) and short heparin LMP-1 (LV-shLMP-1) were constructed to overexpress and knockdown LMP-1 expression in NP cells. LMP-1 mRNA level was regulated in a dose-dependent manner after transfection. LV-LMP-1 increased whereas LV-shLMP-1 decreased collagen II, aggrecan, versican expression, and sGAG production. LV-LMP-1 abolished while LV-shLMP-1 aggravated TNF-α mediated down-regulation of the above matrix genes via ERK1/2 activation. Moreover, LV-LMP-1 abrogated TNF-α induced MMP-3 and MMP-13 expression via inhibiting p65 translocation and MMP-3 and MMP-13 promoter activity. These results indicated that LMP-1 had an ECM production maintenance effect under inflammatory stimulation. This effect was via up-regulation of matrix genes expression at least partially through ERK1/2 activation, and down-regulation of MMPs expression through NF-κB inhibition.

  7. Release of Matrix Metalloproteinase-8 During Physiological Trafficking and Induced Mobilization of Human Hematopoietic Stem Cells

    PubMed Central

    Steinl, Carolin; Essl, Mike; Schreiber, Thomas D.; Geiger, Konstanze; Prokop, Lea; Stevanović, Stefan; Pötz, Oliver; Abele, Harald; Wessels, Johannes T.; Aicher, Wilhelm K.

    2013-01-01

    Previous studies indicate that the release of proteases, including the gelatinase matrix metalloproteinase (MMP)-9, from mature granulocytes plays a crucial role in cytokine-induced hematopoietic stem and progenitor cell (HSPC) mobilization. However, studies with MMP-9-deficient mice revealed that HSPC mobilization was normal in these animals, suggesting that additional proteases must be active at clinically relevant cytokine concentrations. In the present study, we provide evidence that the collagenase MMP-8 is involved in stem cell mobilization. A rapid release of MMP-8 from isolated neutrophil granulocytes can be observed during an in vitro culture. During granulocyte colony-stimulating factor-induced HSPC mobilization, highly elevated serum concentrations of MMP-8 were observed on days 4 to 6 of the mobilization regimen, concomitantly with elevated MMP-9 serum levels and higher numbers of circulating CD34+ cells. Elevated serum concentrations of both proteases were also found in umbilical cord blood serum. In functional assays, adhesion of HSPC to osteoblasts as an essential component of the endosteal stem cell niche is negatively influenced by MMP-8. The chemokine CXCL12, which is critically involved in stem cell trafficking, can be proteolytically processed by MMP-8 treatment. This degradation has a strong inhibitory influence on HSPC migration. Taken together, our data strongly suggest that MMP-8 can be directly involved in hematopoietic stem cell mobilization and trafficking. PMID:23259856

  8. Creep and stress relaxation induced by interface diffusion in metal matrix composites

    NASA Astrophysics Data System (ADS)

    Li, Yinfeng; Li, Zhonghua

    2013-03-01

    An analytical solution is developed to predict the creep rate induced by interface diffusion in unidirectional fiber-reinforced and particle reinforced composites. The driving force for the interface diffusion is the normal stress acting on the interface, which is obtained from rigorous Eshelby inclusion theory. The closed-form solution is an explicit function of the applied stress, volume fraction and radius of the fiber, as well as the modulus ratio between the fiber and the matrix. It is interesting that the solution is formally similar to that of Coble creep in polycrystalline materials. For the application of the present solution in the realistic composites, the scale effect is taken into account by finite element analysis based on a unit cell. Based on the solution, a closed-form solution is also given as a description of stress relaxation induced by interfacial diffusion under constant strain. In addition, the analytical solution for the interface stress presented in this study gives some insight into the relationship between the interface diffusion and interface slip. This work was supported by the financial support from the Nature Science Foundation of China (No. 10932007), the National Basic Research Program of China (No. 2010CB631003/5), and the Doctoral Program of Higher Education of China (No. 20100073110006).

  9. Role of Matrix Metalloproteinases 2 in Spinal Cord Injury-Induced Neuropathic Pain

    PubMed Central

    Miranpuri, Gurwattan S.; Schomberg, Dominic T.; Alrfaei, Bahauddeen; King, Kevin C.; Rynearson, Bryan; Wesley, Vishwas S.; Khan, Nayab; Obiakor, Kristen; Wesley, Umadevi V.; Resnick, Daniel K.

    2016-01-01

    Neuropathic pain (NP) affects approximately 4 million people in the United States with spinal cord injury (SCI) being a common cause. Matrix metalloproteinases (MMPs) play an integral role in mediating inflammatory responses, cellular signaling, cell migration, extracellular matrix degradation and tissue remodeling and repair. As such, they are major components in the pathogenesis of secondary injury within the central nervous system. Other gene regulatory pathways, specifically MAPK/extracellular signaling-regulated kinase (ERK) and Wnt/β-catenin, are also believed to participate in secondary injury likely intersect. The study aims to examine the MMP-2 signaling pathway associated with ERK and Wnt/β-catenin activity during contusion SCI (cSCI)-induced NP in a rat model. This is an experimental study investigating the implication of MMP-2 in SCI-induced NP and its association with the cellular and molecular changes in the interactions between extracellular signaling kinase and β-catenin. Adult Sprague-Dawley rats received cSCI injury by NYU impactor by dropping 10 g weight from a height of 12.5 mm. Locomotor functional recovery of injured rats was measured on post cSCI day 1, and weekly thereafter for 6 weeks using Basso, Beattie and Bresnahan scores. Thermal hyperalgesia (TH) testing was performed on days 21, 28, 35 and 42 post cSCI. The expression and/or activity of MMP-2, β-catenin and ERK were studied following harvest of spinal cord tissues between 3 and 6 weeks post cSCI. All experiments were funded by the department of Neurological Surgery at the University of Wisconsin, School of Medicine and Public Health having no conflict of interest. MMP-2 and β-catenin expression were elevated and gradually increased from days 21 to 42 compared to sham-operated rats and injured rats that did not exhibit TH. The expression of phosphorylated ERK (phospho-ERK) increased on day 21 but returned to baseline levels on day 42 whereas total ERK levels remained relatively

  10. Differential role of mesangial cells and podocytes in TGF-beta-induced mesangial matrix synthesis in chronic glomerular disease.

    PubMed

    Lee, Hyun Soon; Song, Chi Young

    2009-07-01

    Glomerulosclerosis is characterized by mesangial matrix accumulation that is mediated primarily by activation of transforming growth factor-beta (TGF-beta). Unlike podocytes, mesangial cells secrete TGF-beta in response to common in vitro fibrogenic stimuli. However, mesangial immunostaining for active TGF-beta1 in chronic glomerular disease is almost negligible, despite increased mesangial TGF-beta1 mRNA expression, while podocytes covering the sclerotic glomerular segments exhibit increased TGF-beta1 protein expression. The mechanisms whereby TGF-beta is activated in the diseased glomeruli and how the activated TGF-beta leads to mesangial matrix overproduction are not clear. We provide evidence that TGF-beta secreted as latent complexes by mesangial cells is stored in the mesangial matrix, from which soluble forms of latent TGF-beta are released and localized to the podocyte surface in chronic glomerular disease. Podocyte-derived reactive oxygen species, plasmin and thrombospondin-1, particularly renin-angiotensin-aldosterone system-induced oxidative stress, seem to be involved in TGF-beta activation in podocytes. We also provide evidence that the TGF-beta-induced secretion of connective tissue growth factor and vascular endothelial growth factor by podocytes acts as a paracrine regulatory mechanism on mesangial cells, which may cause mesangial matrix accumulation culminating in the development of glomerulosclerosis. Collectively, these data bring new insights into our understanding of the roles of the mesangial cells and podocytes in the TGF-beta-induced mesangial matrix synthesis in chronic glomerular disease.

  11. MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples.

    PubMed

    An, Xiu-Hong; Tian, Yi; Chen, Ke-Qin; Liu, Xiao-Juan; Liu, Dan-Dan; Xie, Xing-Bin; Cheng, Cun-Gang; Cong, Pei-Hua; Hao, Yu-Jin

    2015-04-01

    Anthocyanin and proanthocyanidin (PA) are important secondary metabolites and beneficial to human health. Their biosynthesis is induced by jasmonate (JA) treatment and regulated by MYB transcription factors (TFs). However, which and how MYB TFs regulate this process is largely unknown in apple. In this study, MdMYB9 and MdMYB11 which were induced by methyl jasmonate (MeJA) were functionally characterized. Overexpression of MdMYB9 or MdMYB11 promoted not only anthocyanin but also PA accumulation in apple calluses, and the accumulation was further enhanced by MeJA. Subsequently, yeast two-hybrid, pull-down and bimolecular fluorescence complementation assays showed that both MYB proteins interact with MdbHLH3. Moreover, Jasmonate ZIM-domain (MdJAZ) proteins interact with MdbHLH3. Furthermore, chromatin immunoprecipitation-quantitative PCR and yeast one-hybrid assays demonstrated that both MdMYB9 and MdMYB11 bind to the promoters of ANS, ANR and LAR, whereas MdbHLH3 is recruited to the promoters of MdMYB9 and MdMYB11 and regulates their transcription. In addition, transient expression assays indicated that overexpression of MdJAZ2 inhibits the recruitment of MdbHLH3 to the promoters of MdMYB9 and MdMYB11. Our findings provide new insight into the mechanism of how MeJA regulates anthocyanin and PA accumulation in apple.

  12. Root cap-dependent gravitropic U-turn of maize root requires light-induced auxin biosynthesis via the YUC pathway in the root apex

    PubMed Central

    Suzuki, Hiromi; Yokawa, Ken; Nakano, Sayuri; Yoshida, Yuriko; Fabrissin, Isabelle; Okamoto, Takashi; Baluška, František; Koshiba, Tomokazu

    2016-01-01

    Gravitropism refers to the growth or movement of plants that is influenced by gravity. Roots exhibit positive gravitropism, and the root cap is thought to be the gravity-sensing site. In some plants, the root cap requires light irradiation for positive gravitropic responses. However, the mechanisms regulating this phenomenon are unknown. We herein report that maize roots exposed to white light continuously for ≥1–2h show increased indole-3-acetic acid (IAA) levels in the root tips, especially in the transition zone (1–3mm from the tip). Treatment with IAA biosynthesis inhibitors yucasin and l-kynurenine prevented any increases in IAA content and root curvature under light conditions. Analyses of the incorporation of a stable isotope label from tryptophan into IAA revealed that some of the IAA in roots was synthesized in the root apex. Furthermore, Zmvt2 and Zmyuc gene transcripts were detected in the root apex. One of the Zmyuc genes (ZM2G141383) was up-regulated by light irradiation in the 0–1mm tip region. Our findings suggest that IAA accumulation in the transition zone is due to light-induced activation of Zmyuc gene expression in the 0–1mm root apex region. Light-induced changes in IAA levels and distributions mediate the maize root gravitropic U-turn. PMID:27307546

  13. Calcification preceding new bone formation induced by demineralized bone matrix gelatin.

    PubMed

    Yamashita, K; Takagi, T

    1992-03-01

    Demineralized bone matrix gelatin (BMG) was implanted into the skeletal muscle of Sprague-Dawley (S.D.) rats, and histological changes were examined 3, 5, 7, 10 and 15 days later. Before bone formation, a specific calcification process was found in most of the BMG from day 5 and 7 after implantation. The heterotopic calcified sites were not always consistent with the sites of the alkaline phosphatase activity. It was considered that this calcification progresses without any cellular components, and we distinguished this type of calcification as "acellular mineral deposition" from the calcification which occurs in new bone formation. This "acellular mineral deposition" was first observed as small spherical calcified deposits in the BMG on day 7 after implantation; these deposits then gradually grew and fused with each other. Some multinucleated cells appeared near the site of calcification on day 7 after implantation, but osteoblasts or osteoblast-like cells were scarcely observed around the calcified deposits in BMG until day 7. Vascularization was often observed near the "acellular mineral deposition" and the new bone formation. Fourier transform infrared spectroscopy showed that the calcified deposits in BMG were composed of hydroxyapatite, carbonateapatite and other calcium phosphate components, and that the first two components became prominent with time. It is believed that the "acellular mineral deposition" is due to the deposition of calcium and phosphate into the BMG by a process of heterogenic nucleation that does not involve osteoblasts or matrix vesicles. Bone formation induced by the BMG occurred after the "acellular mineral deposition." The experimental calcification shown in this paper seems a useful model for the study of biocalcification.

  14. 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.

  15. Osteoclast precursor interaction with bone matrix induces osteoclast formation directly by an interleukin-1-mediated autocrine mechanism.

    PubMed

    Yao, Zhenqiang; Xing, Lianping; Qin, Chunlin; Schwarz, Edward M; Boyce, Brendan F

    2008-04-11

    Interleukin-1 (IL-1) and tumor necrosis factor (TNF) mediate bone resorption in a variety of diseases affecting bone. Like TNF, IL-1 is secreted by osteoclast precursors (OCPs), but unlike TNF, it does not induce osteoclast formation directly from OCPs in vitro. TNF induces IL-1 expression and activates c-Fos, a transcription factor required in OCPs for osteoclast formation. Here, we examined whether IL-1 can induce osteoclast formation directly from OCPs overexpressing c-Fos and whether interaction with bone matrix affects OCP cytokine expression. We infected OCPs with c-Fos or green fluorescent protein retrovirus, cultured them with macrophage colony-stimulating factor and IL-1 on bone slices or plastic dishes, and assessed osteoclast and resorption pit formation and expression of IL-1 by OCPs. We used a Transwell assay to determine whether OCPs secrete IL-1 when they interact with bone matrix. IL-1 induced osteoclast formation directly from c-Fos-expressing OCPs on plastic. c-Fos-expressing OCPs formed osteoclasts spontaneously on bone slices without addition of cytokines. OCPs on bone secreted IL-1, which induced osteoclast formation from c-Fos-expressing OCPs in the lower Transwell dishes. The bone matrix proteins dentin sialoprotein and osteopontin, but not transforming growth factor-beta, stimulated OCP expression of IL-1 and induced c-Fos-expressing OCP differentiation into osteoclasts. Osteoclasts eroding inflamed joints have higher c-Fos expression compared with osteoclasts inside bone. We conclude that OCPs expressing c-Fos may induce their differentiation directly into osteoclasts by an autocrine mechanism in which they produce IL-1 through interaction with bone matrix. TNF could induce c-Fos expression in OCPs at sites of inflammation in bone to promote this autocrine mechanism and thus amplify bone loss.

  16. On the thermally-induced residual stresses in thick fiber-thermoplastic matrix (PEEK) cross-ply laminated plates

    NASA Technical Reports Server (NTRS)

    Hu, Shoufeng; Nairn, John A.

    1992-01-01

    An analytical method for calculating thermally-induced residual stresses in laminated plates is applied to cross-ply PEEK laminates. We considered three cooling procedures: slow cooling (uniform temperature distribution); convective and radiative cooling; and rapid cooling by quenching (constant surface temperature). Some of the calculated stresses are of sufficient magnitude to effect failure properties such as matrix microcracking.

  17. Molybdenum metallization, and step-induced defects in active-matrix liquid crystal displays

    NASA Astrophysics Data System (ADS)

    Quli, Farhat Abbas

    The objective of this work was to identify the causes of the step-induced preferential etching which occurs during the patterning of molybdenum (Mo) interconnects for the source/drain line metallization of the thin-film transistor array of active matrix liquid crystal displays. The morphology of Mo films deposited on steps under various conditions using sputtering and electron beam physical vapor deposition (EB-PVD) was studied to identify the causes of preferential etching, and determine processing conditions leading to defect-free interconnects. It was found that columnar growth domains grown from the steps were misaligned with the columnar growth from the substrate, causing the creation of high-angle grain boundaries where the columns impinged upon one another. This boundary region is believed to be responsible for the preferential etching in the Mo films. Columnar growth of silicon dioxide, an amorphous film, did not result in preferential etching due to the lack of grain boundaries. Neon (Ne) sputtering of Mo did not lead to amorphous films due to higher film bombardment during Ne sputtering, as reported by other researchers. Instead, the low rate of deposition during Ne sputtering was found to cause the incorporation of high amounts of impurities that lead to amorphization of the growing film. At extremely low rates and high impurity concentrations, it was found that the film grew in a fcc rather than bcc structure. Mo deposited under high levels of argon ion bombardment was found to have an isotropic rather than columnar morphology which led to interconnects that were free from preferential etching. The maximum ratio of depositing atoms to bombarding ions necessary to modify the microstructure was found to be 1.5 for Mo deposited by EB-PVD at 400°C substrate temperature and --600 volts substrate bias. The processing window for bias sputtering conditions leading to an isotropic morphology was also identified. Thermally induced grain growth in metal films was

  18. Ultrasonic Assessment of Impact-Induced Damage and Microcracking in Polymer Matrix Composites

    NASA Technical Reports Server (NTRS)

    Gyekanyesi, John (Technical Monitor); Liaw, Benjamin; Villars, Esther; Delmont, Frantz

    2003-01-01

    The main objective of this NASA Faculty Awards for Research (FAR) project is to conduct ultrasonic assessment of impact-induced damage and microcracking in fiber-metal laminated (FML) composites at various temperatures. It is believed that the proposed study of impact damage assessment on FML composites will benefit several NASA's missions and current interests, such as ballistic impact testing of composite fan containment and high strain rate deformation modeling of polymer matrix composites. Impact-induced damage mechanisms in GLARE and ARALL fiber-metal laminates subject to instrumented drop-weight impacts at various temperatures were studied. GLARE and ARALL are hybrid composites made of alternating layers of aluminum and glass- (for GLARE) and aramid- (for ARALL) fiber reinforced epoxy. Damage in pure aluminum panels impacted by foreign objects was mainly characterized by large plastic deformation surrounding a deep penetration dent. On the other hand, plastic deformation in fiber-metal laminates was often not as severe although the penetration dent was still produced. The more stiff fiber-reinforced epoxy layers provided better bending rigidity; thus, enhancing impact damage tolerance. Severe cracking, however, occurred due to the use of these more brittle fiber-reinforced epoxy layers. Fracture patterns, e.g., crack length and delamination size, were greatly affected by the lay-up configuration rather than by the number of layers, which implies that thickness effect was not significant for the panels tested in this study. Immersion ultrasound techniques were then used to assess damages generated by instrumented drop-weight impacts onto these fiber-metal laminate panels as well as 2024-T3 aluminum/cast acrylic sandwich plates adhered by epoxy. Depending on several parameters, such as impact velocity, mass, temperature, laminate configuration, sandwich construction, etc., various types of impact damage were observed, including plastic deformation, radiating

  19. The HIV matrix protein p17 induces hepatic lipid accumulation via modulation of nuclear receptor transcriptoma

    PubMed Central

    Renga, Barbara; Francisci, Daniela; Carino, Adriana; Marchianò, Silvia; Cipriani, Sabrina; Chiara Monti, Maria; Del Sordo, Rachele; Schiaroli, Elisabetta; Distrutti, Eleonora; Baldelli, Franco; Fiorucci, Stefano

    2015-01-01

    Liver disease is the second most common cause of mortality in HIV-infected persons. Exactly how HIV infection per se affects liver disease progression is unknown. Here we have investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in HepG2 cells upon stimulation with the HIV matrix protein p17. This viral protein regulated mRNA expression of some NRs among which LXRα and its transcriptional co-activator MED1 were highly induced at mRNA level. Dissection of p17 downstream intracellular pathway demonstrated that p17 mediated activation of Jak/STAT signaling is responsible for the promoter dependent activation of LXR. The treatment of both HepG2 as well as primary hepatocytes with HIV p17 results in the transcriptional activation of LXR target genes (SREBP1c and FAS) and lipid accumulation. These effects are lost in HepG2 cells pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide as well as in HepG2 cells pre-incubated with the natural LXR antagonist gymnestrogenin. These results suggest that HIV p17 affects NRs and their related signal transduction thus contributing to the progression of liver disease in HIV infected patients. PMID:26469385

  20. The HIV matrix protein p17 induces hepatic lipid accumulation via modulation of nuclear receptor transcriptoma.

    PubMed

    Renga, Barbara; Francisci, Daniela; Carino, Adriana; Marchianò, Silvia; Cipriani, Sabrina; Chiara Monti, Maria; Del Sordo, Rachele; Schiaroli, Elisabetta; Distrutti, Eleonora; Baldelli, Franco; Fiorucci, Stefano

    2015-10-15

    Liver disease is the second most common cause of mortality in HIV-infected persons. Exactly how HIV infection per se affects liver disease progression is unknown. Here we have investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in HepG2 cells upon stimulation with the HIV matrix protein p17. This viral protein regulated mRNA expression of some NRs among which LXRα and its transcriptional co-activator MED1 were highly induced at mRNA level. Dissection of p17 downstream intracellular pathway demonstrated that p17 mediated activation of Jak/STAT signaling is responsible for the promoter dependent activation of LXR. The treatment of both HepG2 as well as primary hepatocytes with HIV p17 results in the transcriptional activation of LXR target genes (SREBP1c and FAS) and lipid accumulation. These effects are lost in HepG2 cells pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide as well as in HepG2 cells pre-incubated with the natural LXR antagonist gymnestrogenin. These results suggest that HIV p17 affects NRs and their related signal transduction thus contributing to the progression of liver disease in HIV infected patients.

  1. High throughput screening of cytokines, chemokines and matrix metalloproteinases in wound fluid induced by mammary surgery

    PubMed Central

    Gao, Ningning; Zhang, Hao; Jiang, Yanlin; Liu, Caigang; Wang, Shouyu; Zhao, Zuowei

    2015-01-01

    Objective To clarify the composition of wound fluid (WF) and investigate the impact of WF on breast cancer cell lines. Methods The proliferation and migration of WF-treated breast cancer cells MDA-MB-231 and MCF-7 were assessed with colony formation test, MTT cell proliferation test and scratch wound test. The quantitative profiles of WF were analyzed using Bio-Plex Pro kits. Results The proliferation and migration of WF-treated breast cancer cells were significantly higher than that of untreated cells. Fifteen cytokines, 29 chemokines and 9 matrix metalloproteinases (MMPs) were assessed in WF. The concentrations of these factors were influenced by post-surgery days, neoadjuvant chemotherapy (NAC), TNM stage, pathological type and molecular subtype. The WF harvested from patients underwent NAC showed significant higher profiles of interleukin-1β (IL-1β), IL-4, IL-6, IL-17F, IL-21, IL-23, IL-25, IL-31, Interferon γ (IFNγ), CD40 ligand (CD40L), tumor necrosis factor α (TNFα), CXCL1, CXCL2, CXCL5, CCL3, CCL7 and CCL20. Conclusions Surgery-induced WF promotes the proliferation and migration of breast cancer cells. The composition of WF is influenced by various clinical features and provides potential therapeutic targets to control local recurrence and tumor progression. PMID:26313265

  2. Synthesis and Characterization of a Model Extracellular Matrix that Induces Partial Regeneration of Adult Mammalian Skin

    NASA Astrophysics Data System (ADS)

    Yannas, I. V.; Lee, E.; Orgill, D. P.; Skrabut, E. M.; Murphy, G. F.

    1989-02-01

    Regeneration of the dermis does not occur spontaneously in the adult mammal. The epidermis is regenerated spontaneously provided there is a dermal substrate over which it can migrate. Certain highly porous, crosslinked collagen--glycosaminoglycan copolymers have induced partial morphogenesis of skin when seeded with dermal and epidermal cells and then grafted on standard, full-thickness skin wounds in the adult guinea pig. A mature epidermis and a nearly physiological dermis, which lacked hair follicles but was demonstrably different from scar, were regenerated over areas as large as 16 cm2. These chemical analogs of extracellular matrices were morphogenetically active provided that the average pore diameter ranged between 20 and 125 μ m, the resistance to degradation by collagenase exceeded a critical limit, and the density of autologous dermal and epidermal cells inoculated therein was >5 × 104 cells per cm2 of wound area. Unseeded copolymers with physical structures that were within these limits delayed the onset of wound contraction by about 10 days but did not eventually prevent it. Seeded copolymers not only delayed contraction but eventually arrested and reversed it while new skin was being regenerated. The data identify a model extracellular matrix that acts as if it were an insoluble growth factor with narrowly specified physicochemical structure, functioning as a transient basal lamina during morphogenesis of skin.

  3. Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin.

    PubMed Central

    Yannas, I V; Lee, E; Orgill, D P; Skrabut, E M; Murphy, G F

    1989-01-01

    Regeneration of the dermis does not occur spontaneously in the adult mammal. The epidermis is regenerated spontaneously provided there is a dermal substrate over which it can migrate. Certain highly porous, crosslinked collagen-glycosaminoglycan copolymers have induced partial morphogenesis of skin when seeded with dermal and epidermal cells and then grafted on standard, full-thickness skin wounds in the adult guinea pig. A mature epidermis and a nearly physiological dermis, which lacked hair follicles but was demonstrably different from scar, were regenerated over areas as large as 16 cm2. These chemical analogs of extracellular matrices were morphogenetically active provided that the average pore diameter ranged between 20 and 125 microns, the resistance to degradation by collagenase exceeded a critical limit, and the density of autologous dermal and epidermal cells inoculated therein was greater than 5 x 10(4) cells per cm2 of wound area. Unseeded copolymers with physical structures that were within these limits delayed the onset of wound contraction by about 10 days but did not eventually prevent it. Seeded copolymers not only delayed contraction but eventually arrested and reversed it while new skin was being regenerated. The data identify a model extracellular matrix that acts as if it were an insoluble growth factor with narrowly specified physiochemical structure, functioning as a transient basal lamina during morphogenesis of skin. Images PMID:2915988

  4. Enhanced cognitive flexibility in reversal learning induced by removal of the extracellular matrix in auditory cortex.

    PubMed

    Happel, Max F K; Niekisch, Hartmut; Castiblanco Rivera, Laura L; Ohl, Frank W; Deliano, Matthias; Frischknecht, Renato

    2014-02-18

    During brain maturation, the occurrence of the extracellular matrix (ECM) terminates juvenile plasticity by mediating structural stability. Interestingly, enzymatic removal of the ECM restores juvenile forms of plasticity, as for instance demonstrated by topographical reconnectivity in sensory pathways. However, to which degree the mature ECM is a compromise between stability and flexibility in the adult brain impacting synaptic plasticity as a fundamental basis for learning, lifelong memory formation, and higher cognitive functions is largely unknown. In this study, we removed the ECM in the auditory cortex of adult Mongolian gerbils during specific phases of cortex-dependent auditory relearning, which was induced by the contingency reversal of a frequency-modulated tone discrimination, a task requiring high behavioral flexibility. We found that ECM removal promoted a significant increase in relearning performance, without erasing already established-that is, learned-capacities when continuing discrimination training. The cognitive flexibility required for reversal learning of previously acquired behavioral habits, commonly understood to mainly rely on frontostriatal circuits, was enhanced by promoting synaptic plasticity via ECM removal within the sensory cortex. Our findings further suggest experimental modulation of the cortical ECM as a tool to open short-term windows of enhanced activity-dependent reorganization allowing for guided neuroplasticity.

  5. The Ebola virus matrix protein VP40 selectively induces vesiculation from phosphatidylserine-enriched membranes.

    PubMed

    Soni, Smita P; Stahelin, Robert V

    2014-11-28

    Ebola virus is from the Filoviridae family of viruses and is one of the most virulent pathogens known with ∼ 60% clinical fatality. The Ebola virus negative sense RNA genome encodes seven proteins including viral matrix protein 40 (VP40), which is the most abundant protein found in the virions. Within infected cells VP40 localizes at the inner leaflet of the plasma membrane (PM), binds lipids, and regulates formation of new virus particles. Expression of VP40 in mammalian cells is sufficient to form virus-like particles that are nearly indistinguishable from the authentic virions. However, how VP40 interacts with the PM and forms virus-like particles is for the most part unknown. To investigate VP40 lipid specificity in a model of viral egress we employed giant unilamellar vesicles with different lipid compositions. The results demonstrate VP40 selectively induces vesiculation from membranes containing phosphatidylserine (PS) at concentrations of PS that are representative of the PM inner leaflet content. The formation of intraluminal vesicles was not significantly detected in the presence of other important PM lipids including cholesterol and polyvalent phosphoinositides, further demonstrating PS selectivity. Taken together, these studies suggest that PM phosphatidylserine may be an important component of Ebola virus budding and that VP40 may be able to mediate PM scission.

  6. Density matrix reconstruction of three-level atoms via Rydberg electromagnetically induced transparency

    NASA Astrophysics Data System (ADS)

    Gavryusev, V.; Signoles, A.; Ferreira-Cao, M.; Zürn, G.; Hofmann, C. S.; Günter, G.; Schempp, H.; Robert-de-Saint-Vincent, M.; Whitlock, S.; Weidemüller, M.

    2016-08-01

    We present combined measurements of the spatially resolved optical spectrum and the total excited-atom number in an ultracold gas of three-level atoms under electromagnetically induced transparency conditions involving high-lying Rydberg states. The observed optical transmission of a weak probe laser at the center of the coupling region exhibits a double peaked spectrum as a function of detuning, while the Rydberg atom number shows a comparatively narrow single resonance. By imaging the transmitted light onto a charge-coupled-device camera, we record hundreds of spectra in parallel, which are used to map out the spatial profile of Rabi frequencies of the coupling laser. Using all the information available we can reconstruct the full one-body density matrix of the three-level system, which provides the optical susceptibility and the Rydberg density as a function of spatial position. These results help elucidate the connection between three-level interference phenomena, including the interplay of matter and light degrees of freedom and will facilitate new studies of many-body effects in optically driven Rydberg gases.

  7. Involvement of lipid peroxidation and organic peroxides in UVA-induced matrix metalloproteinase-1 expression.

    PubMed

    Polte, Tobias; Tyrrell, Rex M

    2004-06-15

    Ultraviolet A (UVA) irradiation causes human skin aging and skin cancer at least partially through the activation of matrix metalloproteinases (MMPs). MMP-1, the interstitial collagenase, is responsible for the degradation of collagen and is involved in tumor progression in human skin. The present study uses human skin fibroblast cells (FEK4) to investigate the involvement of lipid peroxidation and the role of peroxides as possible mediators in MMP-1 activation by UVA. Preincubation with the antioxidants butylated hydroxytoluene and Trolox reduced UVA-dependent MMP-1 upregulation, suggesting that peroxidation of membrane lipids is involved. Blocking the iron-driven generation of lipid peroxides and hydroxyl radicals by different iron chelators led to a decrease in UVA-induced MMP-1 mRNA accumulation. Moreover, modulation of glutathione peroxidase activity by use of the specific inhibitor mercaptosuccinate (MS) or by the depletion of glutathione (using buthionine-S, R-sulfoximine, BSO), enhanced the UVA-dependent MMP-1 response. Finally, UVA irradiation generated a significant increase in intracellular peroxide levels which is augmented by pretreatment of the cells with BSO or MS. Our results demonstrate that lipid peroxidation and the production of peroxides are important events in the signalling pathway of MMP-1 activation by UVA.

  8. Deciphering chondrocyte behaviour in matrix-induced autologous chondrocyte implantation to undergo accurate cartilage repair with hyaline matrix.

    PubMed

    Demoor, M; Maneix, L; Ollitrault, D; Legendre, F; Duval, E; Claus, S; Mallein-Gerin, F; Moslemi, S; Boumediene, K; Galera, P

    2012-06-01

    Since the emergence in the 1990s of the autologous chondrocytes transplantation (ACT) in the treatment of cartilage defects, the technique, corresponding initially to implantation of chondrocytes, previously isolated and amplified in vitro, under a periosteal membrane, has greatly evolved. Indeed, the first generations of ACT showed their limits, with in particular the dedifferentiation of chondrocytes during the monolayer culture, inducing the synthesis of fibroblastic collagens, notably type I collagen to the detriment of type II collagen. Beyond the clinical aspect with its encouraging results, new biological substitutes must be tested to obtain a hyaline neocartilage. Therefore, the use of differentiated chondrocytes phenotypically stabilized is essential for the success of ACT at medium and long-term. That is why researchers try now to develop more reliable culture techniques, using among others, new types of biomaterials and molecules known for their chondrogenic activity, giving rise to the 4th generation of ACT. Other sources of cells, being able to follow chondrogenesis program, are also studied. The success of the cartilage regenerative medicine is based on the phenotypic status of the chondrocyte and on one of its essential component of the cartilage, type II collagen, the expression of which should be supported without induction of type I collagen. The knowledge accumulated by the scientific community and the experience of the clinicians will certainly allow to relief this technological challenge, which influence besides, the validation of such biological substitutes by the sanitary authorities.

  9. The Tomato Hoffman's Anthocyaninless Gene Encodes a bHLH Transcription Factor Involved in Anthocyanin Biosynthesis That Is Developmentally Regulated and Induced by Low Temperatures.

    PubMed

    Qiu, Zhengkun; Wang, Xiaoxuan; Gao, Jianchang; Guo, Yanmei; Huang, Zejun; Du, Yongchen

    2016-01-01

    Anthocyanin pigments play many roles in plants, including providing protection against biotic and abiotic stresses. Many of the genes that mediate anthocyanin accumulation have been identified through studies of flowers and fruits; however, the mechanisms of genes involved in anthocyanin regulation in seedlings under low-temperature stimulus are less well understood. Genetic characterization of a tomato inbred line, FMTT271, which showed no anthocyanin pigmentation, revealed a mutation in a bHLH transcription factor (TF) gene, which corresponds to the ah (Hoffman's anthocyaninless) locus, and so the gene in FMTT271 at that locus was named ah. Overexpression of the wild type allele of AH in FMTT271 resulted in greater anthocyanin accumulation and increased expression of several genes in the anthocyanin biosynthetic pathway. The expression of AH and anthocyanin accumulation in seedlings was shown to be developmentally regulated and induced by low-temperature stress. Additionally, transcriptome analyses of hypocotyls and leaves from the near-isogenic lines seedlings revealed that AH not only influences the expression of anthocyanin biosynthetic genes, but also genes associated with responses to abiotic stress. Furthermore, the ah mutation was shown to cause accumulation of reactive oxidative species and the constitutive activation of defense responses under cold conditions. These results suggest that AH regulates anthocyanin biosynthesis, thereby playing a protective role, and that this function is particularly important in young seedlings that are particularly vulnerable to abiotic stresses.

  10. Methyl jasmonate induces ATP biosynthesis deficiency and accumulation of proteins related to secondary metabolism in Catharanthus roseus (L.) G. hairy roots.

    PubMed

    Ruiz-May, Eliel; De-la-Peña, Clelia; Galaz-Ávalos, Rosa M; Lei, Zhentian; Watson, Bonnie S; Sumner, Lloyd W; Loyola-Vargas, Víctor M

    2011-08-01

    Jasmonates are specific signal molecules in plants that are involved in a diverse set of physiological and developmental processes. However, methyl jasmonate (MeJA) has been shown to have a negative effect on root growth and, so far, the biochemical mechanism for this is unknown. Using Catharanthus roseus hairy roots, we were able to observe the effect of MeJA on growth inhibition, cell disorganization and cell death of the root cap. Hairy roots treated with MeJA induced the perturbation of mitochondrial membrane integrity and a diminution in ATP biosynthesis. Furthermore, several proteins were identified that were involved in energy and secondary metabolism; the changes in accumulation of these proteins were observed with 100 μM MeJA. In conclusion, our results suggest that a switch of the metabolic fate of hairy roots in response to MeJA could cause an increase in the accumulation of secondary metabolites. This is likely to have important consequences in the production of specific alkaloids important for the pharmaceutical industry.

  11. Molecular cloning and expression of Hedychium coronarium farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral and wounding/herbivory induced leaf volatile sesquiterpenoids.

    PubMed

    Lan, Jian-bin; Yu, Rang-cai; Yu, Yun-yi; Fan, Yan-ping

    2013-04-15

    Farnesyl pyrophosphate synthase (FPPS EC 2.5.1.10) catalyzes the production of farnesyl pyrophosphate (FPP), which is a key precursor for many sesquiterpenoids such as floral scent and defense volatiles against herbivore attack. Here we report a new full-length cDNA encoding farnesyl diphosphate synthase from Hedychium coronarium. The open reading frame for full-length HcFPPS encodes a protein of 356 amino acids, which is 1068 nucleotides long with calculated molecular mass of 40.7 kDa. Phylogenetic tree analysis indicates that HcFPPS belongs to the plant FPPS super-family and has strong relationship with FPPS from Musa acuminata. Expression of the HcFPPS gene in Escherichia coli yielded FPPS activity. Tissue-specific and developmental analyses of the HcFPPS mRNA and corresponding volatile sesquiterpenoid levels in H. coronarium flowers revealed that the HcFPPS might play a regulatory role in floral volatile sesquiterpenoid biosynthesis. The emission of the FPP-derived volatile terpenoid correlates with strong expression of HcFPPS induced by mechanical wounding and Udaspes folus-damage in leaves, which suggests that HcFPPS may have an important ecological function in H. coronarium vegetative organ.

  12. MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple.

    PubMed

    Li, Yuan-Yuan; Mao, Ke; Zhao, Cheng; Zhao, Xian-Yan; Zhang, Hua-Lei; Shu, Huai-Rui; Hao, Yu-Jin

    2012-10-01

    MdMYB1 is a crucial regulator of light-induced anthocyanin biosynthesis and fruit coloration in apple (Malus domestica). In this study, it was found that MdMYB1 protein accumulated in the light but degraded via a ubiquitin-dependent pathway in the dark. Subsequently, the MdCOP1-1 and MdCOP1-2 genes were isolated from apple fruit peel and were functionally characterized in the Arabidopsis (Arabidopsis thaliana) cop1-4 mutant. Yeast (Saccharomyces cerevisiae) two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays showed that MdMYB1 interacts with the MdCOP1 proteins. Furthermore, in vitro and in vivo experiments indicated that MdCOP1s are necessary for the ubiquitination and degradation of MdMYB1 protein in the dark and are therefore involved in the light-controlled stability of the MdMYB1 protein. Finally, a viral vector-based transformation approach demonstrated that MdCOP1s negatively regulate the peel coloration of apple fruits by modulating the degradation of the MdMYB1 protein. Our findings provide new insight into the mechanism by which light controls anthocyanin accumulation and red fruit coloration in apple and even other plant species.

  13. The Tomato Hoffman’s Anthocyaninless Gene Encodes a bHLH Transcription Factor Involved in Anthocyanin Biosynthesis That Is Developmentally Regulated and Induced by Low Temperatures

    PubMed Central

    Gao, Jianchang; Guo, Yanmei; Huang, Zejun; Du, Yongchen

    2016-01-01

    Anthocyanin pigments play many roles in plants, including providing protection against biotic and abiotic stresses. Many of the genes that mediate anthocyanin accumulation have been identified through studies of flowers and fruits; however, the mechanisms of genes involved in anthocyanin regulation in seedlings under low-temperature stimulus are less well understood. Genetic characterization of a tomato inbred line, FMTT271, which showed no anthocyanin pigmentation, revealed a mutation in a bHLH transcription factor (TF) gene, which corresponds to the ah (Hoffman's anthocyaninless) locus, and so the gene in FMTT271 at that locus was named ah. Overexpression of the wild type allele of AH in FMTT271 resulted in greater anthocyanin accumulation and increased expression of several genes in the anthocyanin biosynthetic pathway. The expression of AH and anthocyanin accumulation in seedlings was shown to be developmentally regulated and induced by low-temperature stress. Additionally, transcriptome analyses of hypocotyls and leaves from the near-isogenic lines seedlings revealed that AH not only influences the expression of anthocyanin biosynthetic genes, but also genes associated with responses to abiotic stress. Furthermore, the ah mutation was shown to cause accumulation of reactive oxidative species and the constitutive activation of defense responses under cold conditions. These results suggest that AH regulates anthocyanin biosynthesis, thereby playing a protective role, and that this function is particularly important in young seedlings that are particularly vulnerable to abiotic stresses. PMID:26943362

  14. VcBBX, VcMYB21, and VcR2R3MYB Transcription Factors Are Involved in UV-B-Induced Anthocyanin Biosynthesis in the Peel of Harvested Blueberry Fruit.

    PubMed

    Nguyen, Chau T T; Lim, Sooyeon; Lee, Jeong Gu; Lee, Eun Jin

    2017-03-15

    This study was carried out to better understand the mechanism responsible for increasing the anthocyanins in blueberries after UV-B radiation at 6.0 kJ m(-2) for 20 min. UV-B induced upregulation of genes involved in anthocyanin biosynthesis in blueberry fruit compared to a nontreated control. Phenylalanine ammonia lyase, chalcone synthase, and flavanone 3'-hydroxylase, which are enzymes that function upstream of anthocyanin biosynthesis, were significantly expressed by UV-B. Expression levels of VcBBX, VcMYB21, and VcR2R3MYB transcription factors (TFs) were upregulated by UV-B in the same manner as the anthocyanin biosynthesis genes. The significant increase in the expression of TFs occurred immediately after UV-B treatment and was then maximized within 3 h. In accordance with these changes, individual anthocyanin contents in the fruits treated with UV-B significantly increased within 6 h and were 2-3-fold higher than the control. Our results indicated that UV-B radiation stimulates an increase in anthocyanin biosynthesis, which could be upregulated by the TFs studied.

  15. Collagenase-3 (matrix metalloproteinase-13) expression is induced in oral mucosal epithelium during chronic inflammation.

    PubMed Central

    Uitto, V. J.; Airola, K.; Vaalamo, M.; Johansson, N.; Putnins, E. E.; Firth, J. D.; Salonen, J.; López-Otín, C.; Saarialho-Kere, U.; Kähäri, V. M.

    1998-01-01

    Increased proliferation of mucosal epithelium during inflammation is associated with degradation of subepithelial connective tissue matrix and local invasion of the epithelial cells. Here we have studied, whether collagenase-3 (MMP-13), a collagenolytic matrix metalloproteinase with an exceptionally wide substrate specificity, is expressed in the epithelium of chronically inflamed mucosa. Examination of human gingival tissue sections from subjects with chronic adult periodontitis with in situ hybridization revealed marked expression of MMP-13 in basal cells of some epithelial rete ridges expanding into connective tissue. Immunohistochemical staining demonstrated that these cells also expressed strongly laminin-5, suggesting that they are actively migrating cells. A strong signal for MMP-13 mRNA was occasionally also noted in the suprabasal epithelial cells facing the gingival pocket, whereas no collagenase-1 (MMP-1) mRNA was detected in any areas of the epithelium. MMP-13 expression was also detected in fibroblast-like cells associated with collagen fibers of the inflamed subepithelial connective tissue. In organ culture of human oral mucosa, MMP-13 mRNA expression was observed in epithelial cells growing into connective tissue of the specimens. Regulation of MMP-13 expression was examined in cultured normal nonkeratinizing epithelial cells isolated from porcine periodontal ligament. In these cells, MMP-13 expression at the mRNA and protein level was potently enhanced (up to sixfold) by tumor necrosis factor-alpha, transforming growth factor-beta(1), and transforming growth factor-alpha and by keratinocyte growth factor in the presence of heparin. In addition, plating periodontal ligament epithelial cells on type I collagen stimulated MMP-13 expression (sevenfold) as compared with cells grown on tissue culture plastic. The results of this study show, that expression of MMP-13 is specifically induced in undifferentiated epithelial cells during chronic inflammation

  16. Conformers, infrared spectrum and UV-induced photochemistry of matrix-isolated furfuryl alcohol.

    PubMed

    Araujo-Andrade, C; Gómez-Zavaglia, A; Reva, I D; Fausto, R

    2012-03-08

    The infrared spectra of furfuryl alcohol (2-furanmethanol, FFA) were investigated for FFA monomers isolated in low-temperature argon matrices. The structural interpretation of the obtained experimental spectra was assisted by analysis of the molecule's conformational landscape. According to the DFT(B3LYP)/6-311++G(d,p) calculations, five different minimum energy structures were found on the potential energy surface of the molecule. They can be defined by the orientation of the OCCO and CCOH dihedral angles: GG', GG, TG, TT, GT (G = +gauche, G' = -gauche, T = trans) and have a symmetry equivalent configuration: GG' = G'G, GG = G'G', TG = TG', GT = G'T. When zero-point energies are taken into account, only three (GG', GG, and TT) out of the five unique minima correspond to stable structures. The most stable conformer GG' (OCCO, 72.7°; CCOH, -59.3°), which in gas phase at room temperature accounts for ∼65% of the total population, was the only form isolated in the argon matrices at 14 K. The other two relevant forms convert into conformer GG' during matrix deposition. The low temperature glassy and crystalline states of FFA were also obtained and their infrared spectra assigned, suggesting the sole existence of the GG' conformer also in these phases. The photochemical behavior of FFA induced in situ, by tunable UV-laser, was also studied. The longest wavelength resulting in photochemical changes in the structure of the irradiated sample was found to be λ = 229 nm. Such UV irradiation of the matrix-isolated FFA led to production of formaldehyde and different isomeric C(4)H(4)O species. Cycloprop-2-ene-1-carbaldehyde and buta-2,3-dienal (two conformers) are the main initial C(4)H(4)O photoproducts formed upon short-time excitation at λ = 229 nm. But-3-ynal (two conformers) was the principal photoproduct resulting from prolonged excitation at λ= 229 nm, being consumed upon irradiation at shorter wavelengths (λ < 227.5 nm). Vinyl ketene is produced from FFA in the

  17. Silencing an N-Acyltransferase-Like Involved in Lignin Biosynthesis in Nicotiana attenuata Dramatically Alters Herbivory-Induced Phenolamide Metabolism

    PubMed Central

    Onkokesung, Nawaporn; Galis, Ivan; Baldwin, Ian T.

    2013-01-01

    In a transcriptomic screen of Manduca sexta-induced N-acyltransferases in leaves of Nicotiana attenuata, we identified an N-acyltransferase gene sharing a high similarity with the tobacco lignin-biosynthetic hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) gene whose expression is controlled by MYB8, a transcription factor that regulates the production of phenylpropanoid polyamine conjugates (phenolamides, PAs). To evaluate the involvement of this HCT-like gene in lignin production as well as the resulting crosstalk with PA metabolism during insect herbivory, we transiently silenced (by VIGs) the expression of this gene and performed non-targeted (UHPLC-ESI/TOF-MS) metabolomics analyses. In agreement with a conserved function of N. attenuata HCT-like in lignin biogenesis, HCT-silenced plants developed weak, soft stems with greatly reduced lignin contents. Metabolic profiling demonstrated large shifts (up to 12% deregulation in total extracted ions in insect-attacked leaves) due to a large diversion of activated coumaric acid units into the production of developmentally and herbivory-induced coumaroyl-containing PAs (N′,N′′-dicoumaroylspermidine, N′,N′′-coumaroylputrescine, etc) and to minor increases in the most abundant free phenolics (chlorogenic and cryptochlorogenic acids), all without altering the production of well characterized herbivory-responsive caffeoyl- and feruloyl-based putrescine and spermidine PAs. These data are consistent with a strong metabolic tension, exacerbated during herbivory, over the allocation of coumaroyl-CoA units among lignin and unusual coumaroyl-containing PAs, and rule out a role for HCT-LIKE in tuning the herbivory-induced accumulation of other PAs. Additionally, these results are consistent with a role for lignification as an induced anti-herbivore defense. PMID:23704878

  18. Biosynthesis and Defensive Function of Nδ-Acetylornithine, a Jasmonate-Induced Arabidopsis Metabolite[C][W

    PubMed Central

    Adio, Adewale M.; Casteel, Clare L.; De Vos, Martin; Kim, Jae Hak; Joshi, Vijay; Li, Baohua; Juéry, Caroline; Daron, Josquin; Kliebenstein, Daniel J.; Jander, Georg

    2011-01-01

    Since research on plant interactions with herbivores and pathogens is often constrained by the analysis of already known compounds, there is a need to identify new defense-related plant metabolites. The uncommon nonprotein amino acid Nδ-acetylornithine was discovered in a targeted search for Arabidopsis thaliana metabolites that are strongly induced by the phytohormone methyl jasmonate (MeJA). Stable isotope labeling experiments show that, after MeJA elicitation, Arg, Pro, and Glu are converted to Orn, which is acetylated by NATA1 to produce Nδ-acetylornithine. MeJA-induced Nδ-acetylornithine accumulation occurs in all tested Arabidopsis accessions, other Arabidopsis species, Capsella rubella, and Boechera stricta, but not in less closely related Brassicaceae. Both insect feeding and Pseudomonas syringae infection increase NATA1 expression and Nδ-acetylornithine accumulation. NATA1 transient expression in Nicotiana tabacum and the addition of Nδ-acetylornithine to an artificial diet both decrease Myzus persicae (green peach aphid) reproduction, suggesting a direct toxic or deterrent effect. However, since broad metabolic changes that are induced by MeJA in wild-type Arabidopsis are attenuated in a nata1 mutant strain, there may also be indirect effects on herbivores and pathogens. In the case of P. syringae, growth on a nata1 mutant is reduced compared with wild-type Arabidopsis, but growth in vitro is unaffected by Nδ-acetylornithine addition. PMID:21917546

  19. Biosynthesis of the general form of monooxygenase induced by xenobiotics of the methylcholanthrene type in the rat liver

    SciTech Connect

    Tsyrlov, I.B.; Chasovnikova, O.B.; Grishanova, A.Yu.; Lyakhovich, V.V.

    1986-10-10

    When five structurally different inducers of the MC type (polycyclic aromatic and heterocyclic hydrocarbons, chloro-derivatives of biphenyl and dibenzo-p-dioxin) are introduced into Wistar rats, de novo synthesis of two P-448 hemoproteins (M/sub r/ 56,000 and 53,000), differing in their functional and immunological parameters, occurs. A comparison of the catalytic and immunochemical properties of five cytochromes P-448 (M/sub r/ 56,000), the data of electrophoretic, proteolytic, and inhibitor analyses did not reveal any differences in the preparations compared, with the exception of TCDD-microsomes, where a low content of this form of cytochrome P-448 and high molecular activity of it with respect to 3,4-benzpyrene and 7-ethoxyresorufin were demonstrated. The results obtained do not support the hypothesis of the possibility of induced synthesis of such a variety of individual forms of monoxygenases as would correspond to the number of variants of the chemical structure of the xenobiotic inducers.

  20. Cerato-platanin induces resistance in Arabidopsis leaves through stomatal perception, overexpression of salicylic acid- and ethylene-signalling genes and camalexin biosynthesis.

    PubMed

    Baccelli, Ivan; Lombardi, Lara; Luti, Simone; Bernardi, Rodolfo; Picciarelli, Piero; Scala, Aniello; Pazzagli, Luigia

    2014-01-01

    Microbe-associated molecular patterns (MAMPs) lead to the activation of the first line of plant defence. Few fungal molecules are universally qualified as MAMPs, and proteins belonging to the cerato-platanin protein (CPP) family seem to possess these features. Cerato-platanin (CP) is the name-giving protein of the CPP family and is produced by Ceratocystis platani, the causal agent of the canker stain disease of plane trees (Platanus spp.). On plane tree leaves, the biological activity of CP has been widely studied. Once applied on the leaf surface, CP acts as an elicitor of defence responses. The molecular mechanism by which CP elicits leaves is still unknown, and the protective effect of CP against virulent pathogens has not been clearly demonstrated. In the present study, we tried to address these questions in the model plant Arabidopsis thaliana. Our results suggest that stomata rapidly sense CP since they responded to the treatment with ROS signalling and stomatal closure, and that CP triggers salicylic acid (SA)- and ethylene (ET)-signalling pathways, but not the jasmonic acid (JA)-signalling pathway, as revealed by the expression pattern of 20 marker genes. Among these, EDS1, PAD4, NPR1, GRX480, WRKY70, ACS6, ERF1a/b, COI1, MYC2, PDF1.2a and the pathogenesis-related (PR) genes 1-5. CP rapidly induced MAPK phosphorylation and induced the biosynthesis of camalexin within 12 hours following treatment. The induction of localised resistance was shown by a reduced susceptibility of the leaves to the infection with Botrytis cinerea and Pseudomonas syringae pv. tomato. These results contribute to elucidate the key steps of the signalling process underlying the resistance induction in plants by CP and point out the central role played by the stomata in this process.

  1. Functional link between TNF biosynthesis and CaM-dependent activation of inducible nitric oxide synthase in RAW 264.7 macrophages

    SciTech Connect

    Weber, Thomas J; Smallwood, Heather S; Kathmann, Loel E; Markillie, Lye MENG; Squier, Thomas C; Thrall, Brian D

    2006-01-18

    Inflammatory responses stimulated by bacterial endotoxin (lipopolysaccharide, LPS) involve calcium-mediated signaling, yet the cellular sensors that determine cell fate in response to LPS remain poorly understood. We report that exposure of RAW 264.7 macrophage-like cells to LPS induces a rapid increase in calmodulin (CaM) abundance, which is associated with the modulation of the inflammatory response. Increases in CaM abundance precedes nuclear localization of key transcription factors (i.e., NFκB p65 subunit, phospho-c-Jun, and Sp1) and subsequent increases in the pro-inflammatory cytokine tumor necrosis factor α (TNF) and inducible nitric oxide synthase (iNOS). Cellular apoptosis following LPS challenge is blocked following inhibition of iNOS activity, whether accomplished using the pharmacological inhibitor 1400W, through gene silencing of TNFα, or by increasing the level of cellular CaM by stable transfection. Increasing CaM expression also results in reductions in the cellular release of TNFα and iNOS, and activation of their transcriptional regulators, indicating the level of available CaM plays a key role in determining the expression of the pro-inflammatory and pro-apoptotic cascade during cellular activation by LPS. These results indicate a previously unrecognized central role for CaM in maintaining cellular homeostasis in response to LPS, such that under resting conditions cellular concentrations of CaM are sufficient to inhibit the biosynthesis of proinflammatory mediators associated with macrophage activation. Although CaM and iNOS protein levels are coordinately increased as part of the oxidative burst, limiting cellular concentrations of CaM due to association with iNOS (and other high-affinity binders) commit the cell to an unchecked inflammatory cascade leading to apoptosis.

  2. Epithelial expression of extracellular matrix metalloproteinase inducer/CD147 and matrix metalloproteinase-2 in neoplasms and precursor lesions derived from cutaneous squamous cells: An immunohistochemical study.

    PubMed

    Ayva, Sebnem Kupana; Karabulut, Ayse Anil; Akatli, Ayşe Nur; Atasoy, Pinar; Bozdogan, Onder

    2013-10-01

    Extracellular matrix metalloproteinase inducer (CD147) is a transmembrane glycoprotein involved in the regulation of matrix metalloproteinases (MMPs). The study investigated CD147 and MMP-2 expression in epidermis of cutaneous squamous lesions. CD147 and MMP-2 expressions were evaluated immunohistochemically in 44 specimens: 18 actinic keratoses (AK), 6 squamous cell carcinomas in situ (SCCIS), 13 squamous cell carcinomas (SCC; peritumoral and invasive portions assessed), and 7 normal skins. Patterns of expression were assessed, with MMP-2 in nuclei (MMP-2n) and cytoplasm (MMP-2c) evaluated separately. The expression of each marker was quantified using a calculated immunohistochemical/histologic score (H-score). Correlations were analyzed for the marker H-scores in each study group. Associations between H-scores and histopathologic parameters were also evaluated. CD147 H-score was the highest in SCC (invasive islands), followed by AK, SCCIS, and control specimens, respectively. MMP-2n and MMP-2c H-scores were the highest in AK, followed by SCCIS, SCC, and control specimens, respectively. MMP-2c and MMP-2n H-scores were significantly higher in peritumoral epidermis than in invasive islands of SCC. MMP-2c and CD147 H-scores were positively correlated in the peritumoral SCCs. CD147 H-score was positively correlated with tumor differentiation in SCC. The findings suggest that overexpression of CD147 plays a role in the development of SCC.

  3. Expression and activity of matrix metalloproteinases in the uterus of bitches after spontaneous and induced abortion.

    PubMed

    Kanca, H; Walter, I; Miller, I; Schäfer-Somi, S; Izgur, H; Aslan, S

    2011-04-01

    Aim of this study was to determine the intrauterine activity of matrix metalloproteinases (MMP)-2 and -9 after cessation of the local effect of progesterone. For this purpose, pregnancy was terminated in 10 bitches at mid-gestation with the progesterone receptor antagonist aglepristone (10 mg/kg body weight, sc, Alizine®; Virbac, France) at two subsequent days (group IRA = induced resorption/abortion). The IRA group was divided into two subgroups (Group I, n = 5, days 25-35 of pregnancy; group II, n = 5, days 36-45). Five further bitches were introduced with beginning abortion (group SRA = spontaneous resorption/abortion). Seven healthy bitches between day 25 and 45 of gestation served as controls. After ovariohysterectomy at the end of abortion and between days 25 and 45 of gestation, respectively, the distribution and activity of collagenases were investigated by immunohistochemistry and gelatin zymography. At placental sites, MMP-2 activity in the endometrium was significantly lower in IRA groups than in the SRA group (33.7 ± 11.8% and 39.3 ± 5.4% vs 52.2 ± 10.2%, p < 0.05); however, MMP-2 expression was lowest in the control group (control: 21.4 ± 6.3%; p < 0.01) and similarly in the myometrium (controls: 13.1 ± 2.5%; p < 0.05). MMP-9 activity was also lower in the endometrium and myometrium of the control group in comparison to SRA and IRA groups (11.8 ± 3.2%; p < 0.01 and 28.4 ± 32.8%; p < 0.05). At interplacental sites, the amount of active collagenases in the myometrium was significantly lower in the control group. It is concluded that the blockade of the biological progesterone effect was associated with an increase in activity of both collagenases.

  4. Cleavage of galectin-3 by matrix metalloproteases induces angiogenesis in breast cancer

    PubMed Central

    Nangia-Makker, Pratima; Wang, Yi; Raz, Tirza; Tait, Larry; Balan, Vitaly; Hogan, Victor; Raz, Avraham

    2012-01-01

    Galectin-3 cleavage is related to progression of human breast and prostate cancer and is partly responsible for tumor growth, angiogenesis and apoptosis resistance in mouse models. A functional polymorphism in galectin-3 gene, determining its susceptibility to cleavage by matrix metalloproteinases (MMPs)-2/-9 is related to racial disparity in breast cancer incidence in Asian and Caucasian women. The purpose of our study is to evaluate (i) if cleavage of galectin-3 could be related to angiogenesis during the progression of human breast cancer, (ii) the role of cleaved galectin-3 in induction of angiogenesis and (iii) determination of the galectin-3 domain responsible for induction of angiogenic response. Galectin-3 null breast cancer cells BT-459 were transfected with either cleavable full-length galectin-3 or its fragmented peptides. Chemotaxis, chemoinvasion, heterotypic aggregation, epithelial-endothelial cell interactions and angiogenesis were compared to noncleavable galectin-3. BT-549-H64 cells harboring cleavable galectin-3 exhibited increased chemotaxis, invasion and interactions with endothelial cells resulting in angiogenesis and 3D morphogenesis compared to BT-549-P64 cells harboring noncleavable galectin-3. BT-549-H64 cells induced increased migration and phosphorylation of focal adhesion kinase in migrating endothelial cells. Endothelial cells cocultured with BT-549 cells transfected with galectin-3 peptides indicate that amino acids 1–62 and 33–250 stimulate migration and morphogenesis of endothelial cells. Immunohistochemical analysis of blood vessel density and galectin-3 cleavage in a breast cancer progression tissue array support the in vitro findings. We conclude that the cleavage of the N terminus of galectin-3 followed by its release in the tumor microenvironment in part leads to breast cancer angiogenesis and progression. PMID:20162566

  5. Modulation of CD147-induced matrix metalloproteinase activity: role of CD147 N-glycosylation.

    PubMed

    Huang, Wan; Luo, Wen-Juan; Zhu, Ping; Tang, Juan; Yu, Xiao-Ling; Cui, Hong-Yong; Wang, Bin; Zhang, Yang; Jiang, Jian-Li; Chen, Zhi-Nan

    2013-01-15

    Degradation of the basement membrane by MMPs (matrix metalloproteinases) is one of the most critical steps in tumour progression. CD147 is a tumour-associated antigen that plays a key regulatory role for MMP activities. In the present study, mass spectrum analysis demonstrated that the purified native CD147 from human lung cancer tissue was N-glycosylated and contained a series of high-mannose and complex-type N-linked glycan structures. Moreover, native glycosylated CD147 existed exclusively as oligomers in solution and directly stimulated MMP production more efficiently than non-glycosylated prokaryotic CD147. The glycosylation site mutation results indicated that, among three N-glycan attachment sites, the N152Q mutants were retained in the endoplasmic reticulum and unfolded protein response signalling was activated. This improper intracellular accumulation impaired its MMP-inducing activity. Increased β1,6-branching of N-glycans as a result of overexpression of GnT-V (N-acetylglucosaminyltransferase V) plays an important role in tumour metastasis. In the present study, we identified CD147 as a target protein of GnT-V and found that overexpression of GnT-V resulted in an elevated level of CD147 at the plasma membrane and in cell-conditioned medium, thereby increasing the induction of MMPs. The present study reveals the important role of N-glycosylation of CD147 in its biological function and implied that targeting aberrant β1,6-branching of N-glycans on CD147 would be valuable for the development of novel therapeutic modalities against carcinoma.

  6. Endometrial inflammation and abnormal expression of extracellular matrix proteins induced by Mycoplasma bovis in dairy cows.

    PubMed

    Guo, Mengyao; Wang, Guoqing; Lv, Tingting; Song, Xiaojing; Wang, Tiancheng; Xie, Guanghong; Cao, Yongguo; Zhang, Naisheng; Cao, Rongfeng

    2014-03-15

    Mycoplasma bovis infection can cause endometrial inflammation leading to infertility and involuntary culling in dairy cows. Because extracellular matrix (ECM) proteins affect the adherence of mycoplasma to eukaryotic cell surface, they may play a role in the pathogenesis of the bacteria. The objective of the present study was to evaluate the endometrial inflammatory response and ECM protein expression induced by M bovis. Endometrial concentrations of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and mRNA and protein expression of collagen IV (CL-IV), fibronectin (FN), and laminin (LN) were evaluated 10, 20, and 30 days after M bovis intrauterine infusion in breed cows 18 days postpartum. The presence of the bacteria in the uterus was detected by nested polymerase chain reaction and denaturing gradient gel electrophoresis. Endometrial TNF-α, IL-1β, and IL-6 concentrations in the treatment group were greater (P < 0.05) than in the positive and negative control groups 20 and 30 days after infusion. Endometrial CL-IV, FN, and LN mRNA and protein expression increased (P < 0.01) 20 days after infusion in all groups. However, the increase was more pronounced in the treatment group and reactive expressions were greater (P < 0.05) than in the positive and negative control groups 10, 20, and 30 days after infusion. In conclusion, M bovis triggered endometrial inflammatory response and increased CL-IV, FN, and LN mRNA and protein expression. The abnormal expression of ECM these proteins may promote the pathogenic effects of M bovis that lead to endometrial tissue damage and infertility.

  7. Extracellular matrix metalloproteinase inducer enhances host resistance against pseudomonas aeruginosa infection through MAPK signaling pathway

    PubMed Central

    Li, Yongwei; Chen, Lu; Wang, Chunxia; Chen, Jianshe; Zhang, Xiaoqian; Hu, Yue; Niu, Xiaobin; Pei, Dongxu; He, Zhiqiang; Bi, Yongyi

    2016-01-01

    This study aims to explore the role of extra-cellular matrix metalloproteinase inducer (EMMPRIN) in the drug resistance of the pseudomonas aeruginosa (PA). The BALB/c mice were transfected with PA, then the mice were infected with the siRNA of EMMPRIN to silence the EMMPRIN gene. The EMMPRIN mRNA and protein were detected by using RT-PCR and western blot, respectively. In order to examine the function of EMMPRIN in drug resistance of PA, the BALB/c and C57BL/6 mice were treated with EMMPRIN siRNA. The cytokines, EMMPRIN and MMP9 were examined by the RP-PCR and ELISA, respectively, undergoing the silence of EMMPRIN siRNA. Moreover, the western blot assay was also used to test the phosphorylated MAPK in the murine macrophages after silenced by the EMMPRIN siRNA. The EMMPRIN was activated, with lipopolysaccharide stimulation and treated with the MAPK inhibitor, to evaluate whether the MAPK participates in the EMMPRIN-triggered drug resistance. The results indicated that the EMMPRIN expression was elevated in the infected BALB/c at 3 or 5 days post-infection. Silence of EMMPRIN Enhanced the Production of pro-inflammatory cytokines in PA keratitis. Silence of EMMPRIN significantly up-regulated Th1-type cytokines IFN-γ, IL-12, and IL-18, but down-regulated Th2-type cytokines IL-4, IL-5, and IL-10. MMP9 was increased in the cells with rEMMPRIN treatment. EMMPRIN inhibits pro-inflammatory cytokine production via a MAPK signaling pathway. In conclusion, EMMPRIN promotes host resistance against pseudomonas aeruginosa infection via MAPK signaling pathway. PMID:28078032

  8. Disruption of LH-induced testosterone biosynthesis in testicular Leydig cells by triclosan: probable mechanism of action.

    PubMed

    Kumar, Vikas; Balomajumder, Chandrajeet; Roy, Partha

    2008-09-04

    Triclosan (TCS) is an antimicrobial chemical widely used in different commercial preparations. The present study demonstrated the mechanism of action of TCS-induced anti-androgenicity in rat Leydig cells. Treatment of purified cells with increasing concentrations of TCS (0.001, 0.01, 0.1, 1 and 10 microM) resulted in a significantly decreased activity of adenylyl cyclase enzyme which was followed by a decreased synthesis of cAMP. This decreased cAMP level resulted in the disruption of entire steroidogenic cascade causing a depressed synthesis of testosterone. However, TCS-induced decrease in the production of testosterone returned to normalcy when cells were treated with forskolin (an adenylyl cyclase activator). Transcription followed by translational of four prominent steroidogenic enzyme/proteins, cytochrome P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and steroidogenic acute regulatory (StAR) protein, also decreased in a dose-dependent manner in TCS-treated Leydig cells as determined by RT-PCR, enzyme assay and Western blot. These results suggested that the disruption of the activity of adenylyl cyclase enzyme by TCS in turn leads to the disruption of intermediate steroidogenic cascade causing a depressed testosterone production. The study further confirmed the anti-androgenic activity of TCS in Leydig cells with highest effective concentration at 1 microM.

  9. Copper stress induces biosynthesis of octadecanoid and eicosanoid oxygenated derivatives in the brown algal kelp Laminaria digitata.

    PubMed

    Ritter, Andrés; Goulitquer, Sophie; Salaün, Jean-Pierre; Tonon, Thierry; Correa, Juan A; Potin, Philippe

    2008-01-01

    To better understand the toxicity and the orchestration of antioxidant defenses of marine brown algae in response to copper-induced stress, lipid peroxidation processes were investigated in the brown alga Laminaria digitata. The expression of genes involved in cell protection and anti-oxidant responses were monitored by semi-quantitative reverse transcriptase polymerase chain reaction and the lipid peroxidation products were further characterized by profiling oxylipin signatures using high-pressure liquid chromatography-mass spectrometry. Exposure to copper excess triggers lipoperoxide accumulation and upregulates the expression of stress related genes. It also increases the release of free polyunsaturated fatty acids, leading to an oxidative cascade through at least two distinct mechanisms. Incubations in presence of inhibitors of lipoxygenases and cycloxygenases showed that in addition to the reactive oxygen species-mediated processes, copper stress induces the synthesis of oxylipins through enzymatic mechanisms. Among complex oxylipins, cyclopentenones from C18 and C20 fatty acids such as 12-oxo-PDA and prostaglandins were detected for the first time in brown algae, as well as unique compounds such as the 18-hydroxy-17-oxo-eicosatetraenoic acid. These results suggest that lipid peroxidation participates in the toxic effects of copper and that lipid peroxidation derivatives may regulate protective mechanisms by employing plant-like octadecanoid signals but also eicosanoid oxylipins which are absent in vascular plants.

  10. Differential expression of extracellular matrix remodeling genes in a murine model of bleomycin-induced pulmonary fibrosis.

    PubMed Central

    Swiderski, R. E.; Dencoff, J. E.; Floerchinger, C. S.; Shapiro, S. D.; Hunninghake, G. W.

    1998-01-01

    Exposure to the chemotherapeutic drug bleomycin leads to pulmonary fibrosis in humans and has been widely used in animal models of the disease. Using C57BL/6 bleomycin-sensitive mice, pulmonary fibrosis was induced by multiple intraperitoneal injections of the drug. An increase in the relative amounts of steady-state alpha1(I) procollagen, alpha1(III) procollagen, and fibronectin mRNA as well as histopathological evidence of fibrosis was observed. The effect of bleomycin on the expression of the enzymes responsible for extracellular matrix degradation, the matrix metalloproteinases (MMPs), and their inhibitors (TIMPs), was selective and showed temporal differences during the development of fibrosis. Of the MMPs tested, bleomycin treatment resulted in the up-regulation of gelatinase A and macrophage metalloelastase gene expression in whole-lung homogenates, whereas gelatinase B, stromelysin-1, and interstitial collagenase gene expression was not significantly changed. Timp2 and Timp3, the murine homologues of the respective TIMP genes, were constitutively expressed, whereas Timp1 was markedly up-regulated during fibrosis. The strong correlation between enhanced extracellular matrix gene expression, differential MMP and TIMP gene expression, and histopathological evidence of fibrosis suggest that dysregulated matrix remodeling is likely to contribute to the pathology of bleomycin-induced pulmonary fibrosis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9502424

  11. Biosynthesis of terephthalic acid, isophthalic acid and their derivatives from the corresponding dinitriles by tetrachloroterephthalonitrile-induced Rhodococcus sp.

    PubMed

    He, Yu-Cai; Wu, Ya-Dong; Pan, Xue-He; Ma, Cui-Luan

    2014-02-01

    The nitrilase from Rhodococcus sp. CCZU10-1 catalyses the hydrolysis of dinitriles to acids without the formation of amides and cyanocarboxylic acids. It was induced by benzonitrile and its analogues (tetrachloroterephthalonitrile > ε-caprolactam > benzonitrile > phenylacetonitrile), and had activity towards aromatic nitriles (terephthalonitrile > tetrachloroterephthalonitrile > isophthalonitrile > tetrachloroisophthalonitrile > tetrafluoroterephthalonitrile > benzonitrile). After the optimization, the highest nitrilase induction [311 U/(g DCW)] was achieved with tetrachloroterephthalonitrile (1 mM) in the medium after 24 h at 30 °C after optimum enzyme activity was at pH 6.8 and at 30 °C. Efficient biocatalyst recycling was achieved by cell immobilization in calcium alginate, with a product-to-biocatalyst ratios of 776 g terephthalic acid/g DCW and 630 g isophthalic acid/g DCW.

  12. Experimental characterization of thermo-oxidation induced shrinkage and damage onset in polymer matrix composites at high temperature

    NASA Astrophysics Data System (ADS)

    Vu, D. Q.; Gigliotti, M.; Lafarie, M. C.; Grandidier, J. C.

    2010-06-01

    This paper focuses on the experimental characterization of thermo-oxidation in carbon fibre reinforced polymers (CFRP) exposed to “high” temperatures (up to 150°C) and “high” oxygen pressures (up to 5 bars). Thermo-oxidation induces matrix shrinkage and damage in CFRP. In this study these are both investigated at room temperature by means of confocal interferometric microscopy (CIM) and scanning electron microscopy (SEM).

  13. Tyramine biosynthesis is transcriptionally induced at low pH and improves the fitness of Enterococcus faecalis in acidic environments.

    PubMed

    Perez, Marta; Calles-Enríquez, Marina; Nes, Ingolf; Martin, Maria Cruz; Fernandez, Maria; Ladero, Victor; Alvarez, Miguel A

    2015-04-01

    Enterococcus faecalis is a commensal bacterium of the human gut that requires the ability to pass through the stomach and therefore cope with low pH. E. faecalis has also been identified as one of the major tyramine producers in fermented food products, where they also encounter acidic environments. In the present work, we have constructed a non-tyramine-producing mutant to study the role of the tyramine biosynthetic pathway, which converts tyrosine to tyramine via amino acid decarboxylation. Wild-type strain showed higher survival in a system that mimics gastrointestinal stress, indicating that the tyramine biosynthetic pathway has a role in acid resistance. Transcriptional analyses of the E. faecalis V583 tyrosine decarboxylase cluster showed that an acidic pH, together with substrate availability, induces its expression and therefore the production of tyramine. The protective role of the tyramine pathway under acidic conditions appears to be exerted through the maintenance of the cytosolic pH. Tyramine production should be considered important in the adaptability of E. faecalis to acidic environments, such as fermented dairy foods, and to survive passage through the human gastrointestinal tract.

  14. Influence of total parenteral nutrition on tumor growth and polyamine biosynthesis of fibrosarcoma-bearing rats after induced cachexia.

    PubMed

    Grossie, V B; Ota, D M; Ajani, J A; Chang, T H; Patenia, D; Nishioka, K

    1988-01-01

    The effect of a protein-free diet (PF) or a restricted intake of chow (RI) and subsequent host repletion with total parenteral nutrition (PF-TPN, RI-TPN) on tumor growth and polyamine metabolism of fibrosarcoma-bearing rats was examined. Host weight was significantly reduced by PF and RI. Tumor growth was reduced in malnourished rats with the PF regimen resulting in the greatest decrease. Rats receiving TPN after 14 days of the RI or PF regimens had higher host weight and plasma albumin levels than malnourished rats. Tumor growth during TPN was evaluated as the percent increase and compared with that of the respective malnourished rats. The percent increase for RI-TPN rats was significantly greater although a trend toward an increase was also evident for PF-TPN rats. Tumor ornithine decarboxylase (ODC) activity and putrescine levels were increased for PF rats and decreased for RI rats while tumor ODC activity was consistently increased by TPN. Tumor growth, ODC activity, and putrescine levels were simultaneously increased only for those rats fed the RI regimen prior to TPN. These results show a disparity in tumor ODC activity, putrescine levels, and tumor growth in malnourished rats. The results of this study suggest that the nutritional origin of cachexia influences the response of the tumor to TPN and emphasizes the importance of considering the methods to induce malnutrition in designing therapuetic regimens.

  15. Methyl jasmonate induces traumatic resin ducts, terpenoid resin biosynthesis, and terpenoid accumulation in developing xylem of Norway spruce stems.

    PubMed

    Martin, Diane; Tholl, Dorothea; Gershenzon, Jonathan; Bohlmann, Jörg

    2002-07-01

    Norway spruce (Picea abies L. Karst) produces an oleoresin characterized by a diverse array of terpenoids, monoterpenoids, sesquiterpenoids, and diterpene resin acids that can protect conifers against potential herbivores and pathogens. Oleoresin accumulates constitutively in resin ducts in the cortex and phloem (bark) of Norway spruce stems. De novo formation of traumatic resin ducts (TDs) is observed in the developing secondary xylem (wood) after insect attack, fungal elicitation, and mechanical wounding. Here, we characterize the methyl jasmonate-induced formation of TDs in Norway spruce by microscopy, chemical analyses of resin composition, and assays of terpenoid biosynthetic enzymes. The response involves tissue-specific differentiation of TDs, terpenoid accumulation, and induction of enzyme activities of both prenyltransferases and terpene synthases in the developing xylem, a tissue that constitutively lacks axial resin ducts in spruce. The induction of a complex defense response in Norway spruce by methyl jasmonate application provides new avenues to evaluate the role of resin defenses for protection of conifers against destructive pests such as white pine weevils (Pissodes strobi), bark beetles (Coleoptera, Scolytidae), and insect-associated tree pathogens.

  16. The Methoxyflavonoid Isosakuranetin Suppresses UV-B-Induced Matrix Metalloproteinase-1 Expression and Collagen Degradation Relevant for Skin Photoaging

    PubMed Central

    Jung, Hana; Lee, Eunjoo H.; Lee, Tae Hoon; Cho, Man-Ho

    2016-01-01

    Solar ultraviolet (UV) radiation is a main extrinsic factor for skin aging. Chronic exposure of the skin to UV radiation causes the induction of matrix metalloproteinases (MMPs), such as MMP-1, and consequently results in alterations of the extracellular matrix (ECM) and skin photoaging. Flavonoids are considered as potent anti-photoaging agents due to their UV-absorbing and antioxidant properties and inhibitory activity against UV-mediated MMP induction. To identify anti-photoaging agents, in the present study we examined the preventative effect of methoxyflavonoids, such as sakuranetin, isosakuranetin, homoeriodictyol, genkwanin, chrysoeriol and syringetin, on UV-B-induced skin photo-damage. Of the examined methoxyflavonoids, pretreatment with isosakuranetin strongly suppressed the UV-B-mediated induction of MMP-1 in human keratinocytes in a concentration-dependent manner. Isosakuranetin inhibited UV-B-induced phosphorylation of mitogen-activated protein kinase (MAPK) signaling components, ERK1/2, JNK1/2 and p38 proteins. This result suggests that the ERK1/2 kinase pathways likely contribute to the inhibitory effects of isosakuranetin on UV-induced MMP-1 production in human keratinocytes. Isosakuranetin also prevented UV-B-induced degradation of type-1 collagen in human dermal fibroblast cells. Taken together, our findings suggest that isosakuranetin has the potential for development as a protective agent for skin photoaging through the inhibition of UV-induced MMP-1 production and collagen degradation. PMID:27598131

  17. The Methoxyflavonoid Isosakuranetin Suppresses UV-B-Induced Matrix Metalloproteinase-1 Expression and Collagen Degradation Relevant for Skin Photoaging.

    PubMed

    Jung, Hana; Lee, Eunjoo H; Lee, Tae Hoon; Cho, Man-Ho

    2016-09-01

    Solar ultraviolet (UV) radiation is a main extrinsic factor for skin aging. Chronic exposure of the skin to UV radiation causes the induction of matrix metalloproteinases (MMPs), such as MMP-1, and consequently results in alterations of the extracellular matrix (ECM) and skin photoaging. Flavonoids are considered as potent anti-photoaging agents due to their UV-absorbing and antioxidant properties and inhibitory activity against UV-mediated MMP induction. To identify anti-photoaging agents, in the present study we examined the preventative effect of methoxyflavonoids, such as sakuranetin, isosakuranetin, homoeriodictyol, genkwanin, chrysoeriol and syringetin, on UV-B-induced skin photo-damage. Of the examined methoxyflavonoids, pretreatment with isosakuranetin strongly suppressed the UV-B-mediated induction of MMP-1 in human keratinocytes in a concentration-dependent manner. Isosakuranetin inhibited UV-B-induced phosphorylation of mitogen-activated protein kinase (MAPK) signaling components, ERK1/2, JNK1/2 and p38 proteins. This result suggests that the ERK1/2 kinase pathways likely contribute to the inhibitory effects of isosakuranetin on UV-induced MMP-1 production in human keratinocytes. Isosakuranetin also prevented UV-B-induced degradation of type-1 collagen in human dermal fibroblast cells. Taken together, our findings suggest that isosakuranetin has the potential for development as a protective agent for skin photoaging through the inhibition of UV-induced MMP-1 production and collagen degradation.

  18. Glutamine-induced protection of isolated rat heart from ischemia/reperfusion injury is mediated via the hexosamine biosynthesis pathway and increased protein O-GlcNAc levels

    PubMed Central

    Liu, Jia; Marchase, Richard B.; Chatham, John C.

    2007-01-01

    It has been shown that glutamine protects the heart from ischemia/reperfusion (I/R) injury; however, the mechanisms underlying this protection have not been identfied. Glutamine: fructose-6-phosphate amidotransferase (GFAT) regulates the entry of glucose into the hexosamine biosynthesis pathway (HBP) and activation of this pathway has been shown to be cardioprotective. Glutamine is required for metabolism of glucose via GFAT; therefore, the goal of this study was to determine whether glutamine cardioprotection could be attributed to increased flux through the HBP and elevated levels of O-linked N-acetyl-glucosamine (O-GlcNAc) on proteins. Hearts from male rats were isolated and perfused with Krebs-Henseliet buffer containing 5mM glucose, and global, no-flow ischemia was induced for 20 minutes followed by 60 minutes of reperfusion. 30 minute pre-treatment with 2.5 mM glutamine significantly improved functional recovery (RPP: 15.6±5.7% Vs. 59.4±6.1%; p<0.05) and decreased cardiac Troponin I release (25.4±3.0 Vs. 4.7±1.9; p<0.05) during reperfusion. This protection was associated with a significant increase in the levels of protein O-GlcNAc and ATP. Pre-treatment with 80 μM azaserine, an inhibitor of GFAT, completely reversed the protection seen with glutamine and prevented the increase in protein O-GlcNAc. O-GlcNAc transferase (OGT) catalyzes the formation of O-GlcNAc, and inhibition of OGT with 5mM alloxan also reversed the protection associated with glutamine. These data support the hypothesis that in the ex vivo perfused heart glutamine cardioprotection is due, at least in part, to enhanced flux through the HBP and increased protein O-GlcNAc levels. PMID:17069847

  19. Glucose Induces Sensitivity to Oxygen Deprivation and Modulates Insulin/IGF-1 Signaling and Lipid Biosynthesis in Caenorhabditis elegans

    PubMed Central

    Garcia, Anastacia M.; Ladage, Mary L.; Dumesnil, Dennis R.; Zaman, Khadiza; Shulaev, Vladimir; Azad, Rajeev K.; Padilla, Pamela A.

    2015-01-01

    Diet is a central environmental factor that contributes to the phenotype and physiology of individuals. At the root of many human health issues is the excess of calorie intake relative to calorie expenditure. For example, the increasing amount of dietary sugars in the human diet is contributing to the rise of obesity and type 2 diabetes. Individuals with obesity and type 2 diabetes have compromised oxygen delivery, and thus it is of interest to investigate the impact a high-sugar diet has on oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, we determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide synthesis, modulates anoxia survival. Additionally, a glucose-supplemented diet alters lipid localization and initiates a positive chemotaxis response. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified show homology to human genes that are differentially regulated in response to obesity or type 2 diabetes, suggesting that there may be conserved gene expression responses between C. elegans fed a glucose-supplemented diet and a diabetic and/or obesity state observed in humans. These findings support the utility of the C. elegans model for understanding the molecular mechanisms regulating dietary-induced metabolic diseases. PMID:25762526

  20. Senescence-Induced Serotonin Biosynthesis and Its Role in Delaying Senescence in Rice Leaves1[C][W][OA

    PubMed Central

    Kang, Kiyoon; Kim, Young-Soon; Park, Sangkyu; Back, Kyoungwhan

    2009-01-01

    Serotonin, which is well known as a pineal hormone in mammals, plays a key role in conditions such as mood, eating disorders, and alcoholism. In plants, although serotonin has been suggested to be involved in several physiological roles, including flowering, morphogenesis, and adaptation to environmental changes, its regulation and functional roles are as yet not characterized at the molecular level. In this study, we found that serotonin is greatly accumulated in rice (Oryza sativa) leaves undergoing senescence induced by either nutrient deprivation or detachment, and its synthesis is closely coupled with transcriptional and enzymatic induction of the tryptophan biosynthetic genes as well as tryptophan decarboxylase (TDC). Transgenic rice plants that overexpressed TDC accumulated higher levels of serotonin than the wild type and showed delayed senescence of rice leaves. However, transgenic rice plants, in which expression of TDC was suppressed through an RNA interference (RNAi) system, produced less serotonin and senesced faster than the wild type, suggesting that serotonin is involved in attenuating leaf senescence. The senescence-retarding activity of serotonin is associated with its high antioxidant activity compared to either tryptophan or chlorogenic acid. Results of TDC overexpression and TDC RNAi plants suggest that TDC plays a rate-limiting role for serotonin accumulation, but the synthesis of serotonin depends on an absolute amount of tryptophan accumulation by the coordinate induction of the tryptophan biosynthetic genes. In addition, immunolocalization analysis revealed that serotonin was abundant in the vascular parenchyma cells, including companion cells and xylem-parenchyma cells, suggestive of its involvement in maintaining the cellular integrity of these cells for facilitating efficient nutrient recycling from senescing leaves to sink tissues during senescence. PMID:19439571

  1. Prostanoid-induced expression of matrix metalloproteinase-1 messenger ribonucleic acid in rat osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Clohisy, J. C.; Connolly, T. J.; Bergman, K. D.; Quinn, C. O.; Partridge, N. C.

    1994-01-01

    Individual prostanoids have distinct potencies in activating intracellular signaling pathways and regulating gene expression in osteoblastic cells. The E-series prostaglandins (PGs) are known to stimulate matrix metalloproteinase-1 (MMP-1) synthesis and secretion in certain rodent and human osteoblastic cells, yet the intracellular events involved remain unclear. To further characterize this response and its signal transduction pathway(s), we examined prostanoid-induced expression of the MMP-1 gene in the rat osteoblastic osteosarcoma cell line UMR 106-01. Northern blot analysis demonstrated that prostaglandin E2 (PGE2) and PGE1 were very potent stimulators (40-fold) of MMP-1 transcript abundance, PGF2 alpha and prostacyclin were weak stimulators (4-fold), and thromboxane-B2 had no effect. The marked increase in MMP-1 transcript abundance after PGE2 treatment was first detected at 2 h, became maximal at 4 h, and persisted beyond 24 h. This response was dose dependent and elicited maximal and half-maximal effects with concentrations of 10(-6) and 0.6 x 10(-7) M, respectively. Cycloheximide, a protein synthesis inhibitor, completely blocked this effect of PGE2, suggesting that the expression of other genes is required. Nuclear run-on experiments demonstrated that PGE2 rapidly activates MMP-1 gene transcription, with a maximal increase at 2-4 h. The second messenger analog, 8-bromo-cAMP, mimicked the effects of PGE2 by stimulating a dose-dependent increase in MMP-1 messenger RNA (mRNA) levels, with a maximal effect quantitatively similar to that observed with PGE2. Thus, in UMR 106-01 cells, different prostanoids have distinct potencies in stimulating MMP-1 mRNA abundance. Our data suggest that PGE2 stimulation of MMP-1 synthesis is due to activation of MMP-1 gene transcription and a subsequent marked increase in MMP-1 mRNA abundance. This effect is dependent on de novo protein synthesis and is mimicked by protein kinase-A activation.

  2. Nitric Oxide Induces Cardiac Protection by Preventing Extracellular Matrix Degradation through the Complex Caveolin-3/EMMPRIN in Cardiac Myocytes.

    PubMed

    Cuadrado, Irene; Castejon, Borja; Martin, Ana M; Saura, Marta; Reventun-Torralba, Paula; Zamorano, Jose Luis; Zaragoza, Carlos

    2016-01-01

    Inhibition of Extracellular Matrix degradation by nitric oxide (NO) induces cardiac protection against coronary ischemia/reperfusion (IR). Glycosylation of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) stimulates enzymatic activation of matrix metalloproteinases (MMPs) in the heart, although the mechanisms leading to EMMPRIN glycosylation are poorly understood. We sought to determine if NO may induce cardiac protection by preventing glycosylation of EMMPRIN in a mouse model of IR. Here we found that Caveolin-3 binds to low glycosylated EMMPRIN (LG-EMMPRIN) in cardiac cells and in the hearts of healthy mice, whereas IR disrupted the complex in nitric oxide synthase 2 (NOS2) knockout (KO) mice. By contrast, the binding was partially restored when mice were fed with an NO donor (DEA-NO) in the drinking water, showing a significant reduction on infarct size (NOS2KO: 34.6±5 vs NOS2KO+DEA-NO: 20.7±9), in expression of matrix metalloproteinases, and cardiac performance was improved (left ventricular ejection fraction (LVEF). NOS2KO: 31±4 vs NOS2KO+DEA-NO: 46±6). The role of Caveolin-3/EMMPRIN in NO-mediated cardiac protection was further assayed in Caveolin-3 KO mice, showing no significant improvement on infarct size (Caveolin-3 KO: 34.8±3 vs Caveolin-3 KO+DEA-NO:33.7±5), or in the expression of MMPs, suggesting that stabilization of the complex Caveolin-3/LG-EMMPRIN may play a significant role in the cardioprotective effect of NO against IR.

  3. Nitric Oxide Induces Cardiac Protection by Preventing Extracellular Matrix Degradation through the Complex Caveolin-3/EMMPRIN in Cardiac Myocytes

    PubMed Central

    Cuadrado, Irene; Castejon, Borja; Martin, Ana M.; Saura, Marta; Reventun-Torralba, Paula; Zamorano, Jose Luis

    2016-01-01

    Inhibition of Extracellular Matrix degradation by nitric oxide (NO) induces cardiac protection against coronary ischemia/reperfusion (IR). Glycosylation of Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) stimulates enzymatic activation of matrix metalloproteinases (MMPs) in the heart, although the mechanisms leading to EMMPRIN glycosylation are poorly understood. We sought to determine if NO may induce cardiac protection by preventing glycosylation of EMMPRIN in a mouse model of IR. Here we found that Caveolin-3 binds to low glycosylated EMMPRIN (LG-EMMPRIN) in cardiac cells and in the hearts of healthy mice, whereas IR disrupted the complex in nitric oxide synthase 2 (NOS2) knockout (KO) mice. By contrast, the binding was partially restored when mice were fed with an NO donor (DEA-NO) in the drinking water, showing a significant reduction on infarct size (NOS2KO: 34.6±5 vs NOS2KO+DEA-NO: 20.7±9), in expression of matrix metalloproteinases, and cardiac performance was improved (left ventricular ejection fraction (LVEF). NOS2KO: 31±4 vs NOS2KO+DEA-NO: 46±6). The role of Caveolin-3/EMMPRIN in NO-mediated cardiac protection was further assayed in Caveolin-3 KO mice, showing no significant improvement on infarct size (Caveolin-3 KO: 34.8±3 vs Caveolin-3 KO+DEA-NO:33.7±5), or in the expression of MMPs, suggesting that stabilization of the complex Caveolin-3/LG-EMMPRIN may play a significant role in the cardioprotective effect of NO against IR. PMID:27649573

  4. Analysis of delamination in cross ply laminates initiating from impact induced matrix cracking

    NASA Technical Reports Server (NTRS)

    Salpekar, S. A.

    1991-01-01

    Several two dimensional finite element analyses of (0 sub 2/90 sub 8/0 sub 2) glass/epoxy and graphite-epoxy composite laminates were performed to study some of the characteristics of damage development due to an impact load. A cross section through the thickness of the laminate with fixed ends, and carrying a transverse load in the center was analyzed. Inclined matrix cracks such as those produced by low velocity impact were modeled in the 90 deg ply group. The introduction of the matrix cracks caused large interlaminar tension and shear stresses in the vicinity of both crack tips in the 0/90 and 90/0 interfaces. The large interlaminar stresses at the ends of the matrix cracks indicate that matrix cracking may give rise to delamination. The ratio of mode I to total strain energy release rate at the beginning of delamination calculated at the two matrix crack tips was 60 and 28 pct., respectively, in the glass/epoxy laminate. The corresponding ratio was 97 and 77 pct. in the graphite-epoxy laminate. Thus, a significant mode I component of strain energy release rate may be present at the delamination initiation due to an impact load.

  5. Multiphoton microscopy of engineered dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast contraction

    NASA Astrophysics Data System (ADS)

    Pena, Ana-Maria; Fagot, Dominique; Olive, Christian; Michelet, Jean-François; Galey, Jean-Baptiste; Leroy, Frédéric; Beaurepaire, Emmanuel; Martin, Jean-Louis; Colonna, Anne; Schanne-Klein, Marie-Claire

    2010-09-01

    Dermal fibroblasts are responsible for the generation of mechanical forces within their surrounding extracellular matrix and can be potentially targeted by anti-aging ingredients. Investigation of the modulation of fibroblast contraction by these ingredients requires the implementation of three-dimensional in situ imaging methodologies. We use multiphoton microscopy to visualize unstained engineered dermal tissue by combining second-harmonic generation that reveals specifically fibrillar collagen and two-photon excited fluorescence from endogenous cellular chromophores. We study the fibroblast-induced reorganization of the collagen matrix and quantitatively evaluate the effect of Y-27632, a RhoA-kinase inhibitor, on dermal substitute contraction. We observe that collagen fibrils rearrange around fibroblasts with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA-kinase inhibitor. Moreover, we show that the inhibitory effects are reversible. Our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the extracellular matrix induced by fibroblast contraction or other processes.

  6. Multiphoton microscopy of engineered dermal substitutes: assessment of 3D collagen matrix remodeling induced by fibroblasts contraction

    NASA Astrophysics Data System (ADS)

    Pena, A.-M.; Olive, C.; Michelet, J.-F.; Galey, J.-B.; Fagot, D.; Leroy, F.; Martin, J.-L.; Colonna, A.; Schanne-Klein, M.-C.

    2010-02-01

    One of the main functions of dermal fibroblasts is the generation of mechanical forces within their surrounding extracellular matrix. Investigating molecules that could modulate fibroblast contraction and act as potent anti aging ingredients requires the development of three-dimensional in situ imaging methodologies for dermal substitute analysis. Here we use multiphoton microscopy in order to investigate the fibroblast-induced collagen matrix reorganization in engineered dermal tissue and to evaluate the effect of Y27632, a RhoA kinase inhibitor on dermal substitutes contraction. We observe that collagen fibrils rearrange around fibroblast with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA kinase inhibitor. Moreover, when the culture medium containing the inhibitor was replaced with a control medium, the dermal substitutes presented the same 3D reorganization as the control samples, which indicates that the inhibitory effects are reversible. In conclusion, our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the matrix induced by fibroblast contraction.

  7. Wild-type amyloid beta 1-40 peptide induces vascular smooth muscle cell death independently from matrix metalloprotease activity.

    PubMed

    Blaise, Régis; Mateo, Véronique; Rouxel, Clotilde; Zaccarini, François; Glorian, Martine; Béréziat, Gilbert; Golubkov, Vladislav S; Limon, Isabelle

    2012-06-01

    Cerebral amyloid angiopathy (CAA) is an important cause of intracerebral hemorrhages in the elderly, characterized by amyloid-β (Aβ) peptide accumulating in central nervous system blood vessels. Within the vessel walls, Aβ-peptide deposits [composed mainly of wild-type (WT) Aβ(1-40) peptide in sporadic forms] induce impaired adhesion of vascular smooth muscle cells (VSMCs) to the extracellular matrix (ECM) associated with their degeneration. This process often results in a loss of blood vessel wall integrity and ultimately translates into cerebral ischemia and microhemorrhages, both clinical features of CAA. In this study, we decipher the molecular mechanism of matrix metalloprotease (MMP)-2 activation in WT-Aβ(1-40) -treated VSMC and provide evidence that MMP activity, although playing a critical role in cell detachment disrupting ECM components, is not involved in the WT-Aβ(1-40) -induced degeneration of VSMCs. Indeed, whereas this peptide clearly induced VSMC apoptosis, neither preventing MMP-2 activity nor hampering the expression of membrane type1-MMP, or preventing tissue inhibitors of MMPs-2 (TIMP-2) recruitment (two proteins evidenced here as involved in MMP-2 activation), reduced the number of dead cells. Even the use of broad-range MMP inhibitors (GM6001 and Batimastat) did not affect WT-Aβ(1-40) -induced cell apoptosis. Our results, in contrast to those obtained using the Aβ(1-40) Dutch variant suggesting a link between MMP-2 activity, VSMC mortality and degradation of specific matrix components, indicate that the ontogenesis of the Dutch familial and sporadic forms of CAAs is different. ECM degradation and VSMC degeneration would be tightly connected in the Dutch familial form while being two independent processes in sporadic forms of CAA.

  8. Biosynthesis: Imaging cell-wall biosynthesis live

    NASA Astrophysics Data System (ADS)

    Bugg, Timothy D. H.

    2013-01-01

    The biosynthesis of peptidoglycan is an important step in bacterial cell division and cell-wall maturation. Now it has been shown that fluorescent D-amino acids can be used to label the peptidoglycan cell wall of living bacteria, providing a new tool to study this important process.

  9. Minocycline attenuates experimental colitis in mice by blocking expression of inducible nitric oxide synthase and matrix metalloproteinases

    SciTech Connect

    Huang, T.-Y.; Chu, H.-C.; Lin, Y.-L.; Lin, C.-K.; Hsieh, T.-Y.; Chang, W.-K.; Chao, Y.-C.; Liao, C.-L.

    2009-05-15

    In addition to its antimicrobial activity, minocycline exerts anti-inflammatory effects in several disease models. However, whether minocycline affects the pathogenesis of inflammatory bowel disease has not been determined. We investigated the effects of minocycline on experimental colitis and its underlying mechanisms. Acute and chronic colitis were induced in mice by treatment with dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS), and the effect of minocycline on colonic injury was assessed clinically and histologically. Prophylactic and therapeutic treatment of mice with minocycline significantly diminished mortality rate and attenuated the severity of DSS-induced acute colitis. Mechanistically, minocycline administration suppressed inducible nitric oxide synthase (iNOS) expression and nitrotyrosine production, inhibited proinflammatory cytokine expression, repressed the elevated mRNA expression of matrix metalloproteinases (MMPs) 2, 3, 9, and 13, diminished the apoptotic index in colonic tissues, and inhibited nitric oxide production in the serum of mice with DSS-induced acute colitis. In DSS-induced chronic colitis, minocycline treatment also reduced body weight loss, improved colonic histology, and blocked expression of iNOS, proinflammatory cytokines, and MMPs from colonic tissues. Similarly, minocycline could ameliorate the severity of TNBS-induced acute colitis in mice by decreasing mortality rate and inhibiting proinflammatory cytokine expression in colonic tissues. These results demonstrate that minocycline protects mice against DSS- and TNBS-induced colitis, probably via inhibition of iNOS and MMP expression in intestinal tissues. Therefore, minocycline is a potential remedy for human inflammatory bowel diseases.

  10. Involvement of matrix metalloproteinases in human immunodeficiency virus type 1-induced replication by clinical Mycobacterium avium isolates.

    PubMed

    Dezzutti, C S; Swords, W E; Guenthner, P C; Sasso, D R; Wahl, L M; Drummond, A H; Newman, G W; King, C H; Quinn, F D; Lal, R B

    1999-10-01

    The role of Mycobacterium avium isolates in modulating human immunodeficiency virus type 1 (HIV-1) replication was examined by use of an in vitro, resting T cell system. Two human clinical isolates (serotypes 1 and 4) but not an environmental M. avium isolate (serotype 2) enhanced HIV-1 replication. The M. avium-induced HIV-1 replication was not associated with cell activation or differential cytokine production or utilization. Addition of matrix metalloproteinase (MMP) inhibitors and their in vivo regulators, tissue inhibitors of metalloproteinases-1 and -2, abrogated M. avium-induced HIV-1 replication 80%-95%. The MMP inhibitors did not have any effect on the HIV-1 protease activity, suggesting that they may affect cellular processes. Furthermore, MMP-9 protein was differentially expressed after infection with clinical M. avium isolates and paralleled HIV-1 p24 production. Collectively, these data suggest that M. avium-induced HIV-1 replication is mediated, in part, through the induction of MMP-9.

  11. Altered Liver Proteoglycan/Glycosaminoglycan Structure as a Manifestation of Extracellular Matrix Remodeling upon BCG-induced Granulomatosis in Mice.

    PubMed

    Kim, L B; Shkurupy, V A; Putyatina, A N

    2017-01-01

    Experimental BCG-induced granulomatosis in mice was used to study changes in the dynamics of individual liver proteoglycan components reflecting phasic extracellular matrix remodeling, determined by the host-parasite interaction and associated with granuloma development. In the early BCG-granulomatosis period, the increase in individual proteoglycan components promotes granuloma formation, providing conditions for mycobacteria adhesion to host cells, migration of phagocytic cells from circulation, and cell-cell interaction leading to granuloma development and fibrosis. Later, reduced reserve capacity of the extracellular matrix, development of interstitial fibrosis and granuloma fibrosis can lead to trophic shortage for cells within the granulomas, migration of macrophages out of them, and development of spontaneous necrosis and apoptosis typical of tuberculosis.

  12. Direct measurement of excited-state dipole matrix elements using electromagnetically induced transparency in the hyperfine Paschen-Back regime

    NASA Astrophysics Data System (ADS)

    Whiting, Daniel J.; Keaveney, James; Adams, Charles S.; Hughes, Ifan G.

    2016-04-01

    Applying large magnetic fields to gain access to the hyperfine Paschen-Back regime can isolate three-level systems in a hot alkali metal vapors, thereby simplifying usually complex atom-light interactions. We use this method to make the first direct measurement of the |<5 P ||e r ||5 D >| matrix element in 87Rb. An analytic model with only three levels accurately models the experimental electromagnetically induced transparency spectra and extracted Rabi frequencies are used to determine the dipole matrix element. We measure |<5 P3 /2||e r ||5 D5 /2>| =(2.290 ±0 .002stat±0 .04syst) e a0 , which is in excellent agreement with the theoretical calculations of Safronova, Williams, and Clark [Phys. Rev. A 69, 022509 (2004), 10.1103/PhysRevA.69.022509].

  13. Phytochrome-mediated Carotenoids Biosynthesis in Ripening Tomatoes.

    PubMed

    Thomas, R L; Jen, J J

    1975-09-01

    Red light induced and far red light inhibited carotenoid biosynthesis in ripening tomatoes (Lycopersicon esculentum Mill.) when compared to controls kept in the dark. Red illumination following far red illumination reversed the inhibitory action of far red light on carotenoid biosynthesis, suggesting a phytochrome-mediated process. Quantitation of individual carotenoids favored the hypothesis of two separate carotenoid biosynthetic pathways in tomatoes.

  14. TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

    SciTech Connect

    De Abrew, K. Nadira; Thomas-Virnig, Christina L.; Rasmussen, Cathy A.; Bolterstein, Elyse A.; Schlosser, Sandy J.; Allen-Hoffmann, B. Lynn

    2014-05-01

    The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin. - Highlights: • TCDD causes hyperkeratosis and basement membrane changes in a model of human skin. • TCDD induces MMP-10 expression in organotypic cultures

  15. Extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinases (MMPs) as regulators of tumor-host interaction in a spontaneous metastasis model in rats.

    PubMed

    Donadio, Ana Carolina; Remedi, María Mónica; Susperreguy, Sebastián; Frede, Silvia; Gilardoni, Mónica Beatriz; Tang, Yi; Pellizas, Claudia Gabriela; Yan, Li

    2008-12-01

    EMMPRIN has a role in invasion and metastasis through the induction of MMPs and the consequent modulation of cell-substrate and cell-cell adhesion processes. The present study evaluates the expression of EMMPRIN protein and MMP-2/9 activity in tumor and parenchymal cells in a spontaneous metastasis model in rats. Moreover, we explore the regulation of EMMPRIN and MMP-9 by tumor-epithelial cell interactions in vitro. By zymography, we observed an increased proMMP-9 expression in both metastasized liver and spleen samples from tumor bearing rats. Immunohistochemical studies showed EMMPRIN-positive tumor cells in tumor biopsies as well as in spleen and liver samples from tumor bearing rats. Interestingly, a significant increase in EMMPRIN expression in hepatic cells was also detected. The regulation of EMMPRIN expression in tumor and liver cells in response to tumor-host interaction was investigated in vitro through a tumor cell line culture on extracellular matrix (ECM) molecules or in co-culture with normal rat liver cells (BRL3A cells). No significant changes in EMMPRIN expression were detected in tumor cells cultured on ECM molecules. On the other hand, EMMPRIN protein and MMP-9 mRNA expression were induced in BRL3A cells. The increase in EMMPRIN expression in BRL3A cells was inhibited by an anti-EMMPRIN antibody. These results reinforce the main role of EMMPRIN mediating tumor-host interactions that may evolve new opportunities for therapeutic interventions.

  16. Analysis of delamination in cross-ply laminates initiating from impact induced matrix cracking

    NASA Technical Reports Server (NTRS)

    Salpekar, S. A.

    1993-01-01

    Two-dimensional finite element analyses of (02/90(8)/02) glass/epoxy and graphite/epoxy composite laminates were performed to investigate some of the characteristics of damage development due to an impact load. A cross section through the thickness of the laminate with fixed ends, and carrying a transverse load in the center, was analyzed. Inclined matrix cracks, such as those produced by a low-velocity impact, were modeled in the 90 deg ply group. The introduction of the matrix cracks caused large interlaminar tensile and shear stresses in the vicinity of both crack tips in the 0/90 and 90/0 interfaces, indicating that matrix cracking may give rise to delamination. The ratio of Mode I to total strain energy release rate, G(I)/G(total), at the beginning of delamination, calculated at the two (top and bottom) matrix crack tips was 60 and 28 percent, respectively, in the glass/epoxy laminate. The corresponding ratio was 97 and 77 percent in the graphite/epoxy laminate. Thus, a significant Mode I component of strain energy release rate may be present at the delamination initiation due to an impact load. The value of strain energy release rate at either crack tip increased due to an increase in the delamination length at the other crack tip and may give rise to an unstable delamination growth under constant load.

  17. Protein Biosynthesis in Mitochondria

    PubMed Central

    Kuzmenko, A. V.; Levitskii, S. A.; Vinogradova, E. N.; Atkinson, G. C.; Hauryliuk, V.; Zenkin, N.; Kamenski, P. A.

    2013-01-01

    Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis. PMID:24228873

  18. Protein biosynthesis in mitochondria.

    PubMed

    Kuzmenko, A V; Levitskii, S A; Vinogradova, E N; Atkinson, G C; Hauryliuk, V; Zenkin, N; Kamenski, P A

    2013-08-01

    Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis.

  19. Eicosapentaenoic acid inhibits TNF-{alpha}-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

    SciTech Connect

    Kim, Hyeon Ho; Lee, Youngae; Eun, Hee Chul Chung, Jin Ho

    2008-04-04

    Eicosapentaenoic acid (EPA) is an omega-3 ({omega}-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-{kappa}B activation induced by tumor necrosis factor (TNF)-{alpha} or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-{alpha}-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-{alpha} induced MMP-9 expression by NF-{kappa}B-dependent pathway. Pretreatment of EPA inhibited TNF-{alpha}-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect I{kappa}B-{alpha} phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-{kappa}B. EPA inhibited TNF-{alpha}-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKK{alpha}-dependent event. Taken together, we demonstrate that EPA inhibits TNF-{alpha}-induced MMP-9 expression through inhibition of p38 and Akt activation.

  20. BrMYB4, a suppressor of genes for phenylpropanoid and anthocyanin biosynthesis, is down-regulated by UV-B but not by pigment-inducing sunlight in turnip cv. Tsuda.

    PubMed

    Zhang, Lili; Wang, Yu; Sun, Mei; Wang, Jing; Kawabata, Saneyuki; Li, Yuhua

    2014-12-01

    The regulation of light-dependent anthocyanin biosynthesis in Brassica rapa subsp. rapa cv. Tsuda turnip was investigated using an ethyl methanesulfonate (EMS)-induced mutant R30 with light-independent pigmentation. TILLING (targeting induced local lesions in genomes) and subsequent analysis showed that a stop codon was inserted in the R2R3-MYB transcription factor gene BrMYB4 and that the encoded protein (BrMYB4mu) had lost its C-terminal region. In R30, anthocyanin accumulated in the below-ground portion of the storage root of 2-month-old plants. In 4-day-old seedlings and 2-month-old plants, expression of BrMYB4 was similar between R30 and the wild type (WT), but the expression of the cinnamate 4-hydroxylase gene (BrC4H) was markedly enhanced in R30 in the dark. In turnip seedlings, BrMYB4 expression was suppressed by UV-B irradiation in the WT, but this negative regulation was absent in R30. Concomitantly, BrC4H was repressed by UV-B irradiation in the WT, but stayed at high levels in R30. A gel-shift assay revealed that BrMYB4 could directly bind to the promoter region of BrC4H, but BrMYB4mu could not. The BrMYB4-enhanced green fluorescent protein (eGFP) protein could enter the nucleus in the presence of BrSAD2 (an importin β-like protein) nuclear transporter, but BrMYB4mu-eGFP could not. These results showed that BrMYB4 functions as a negative transcriptional regulator of BrC4H and mediates UV-B-dependent phenylpropanoid biosynthesis, while BrMYB4mu has lost this function. In the storage roots, the expression of anthocyanin biosynthesis genes was enhanced in R30 in the dark and in sunlight in both the WT and R30. However, in the WT, anthocyanin-inducing sunlight did not suppress BrMYB4 expression. Therefore, sunlight-induced anthocyanin biosynthesis does not seem to be regulated by BrMYB4.

  1. Osteoblast responses one hour after load-induced fluid flow in a three-dimensional porous matrix.

    PubMed

    Tanaka, Shigeo M; Sun, Hui B; Roeder, Ryan K; Burr, David B; Turner, Charles H; Yokota, Hiroki

    2005-04-01

    When bone is loaded, substrate strain is generated by the external force and this strain induces fluid flow that creates fluid shear stress on bone cells. Our current understanding of load-driven gene regulation of osteoblasts is based primarily on in vitro studies on planer two-dimensional tissue culture substrates. However, differences between a flat layer of cells and cells in 3-dimensional (3D) ECM are being recognized for signal transduction. Proliferation and differentiation of osteoblasts are affected by substrate geometry. Here we developed a novel 3D culture system that would mimic physiologically relevant substrate strain as well as strain-induced fluid flow in a 3D porous collagen matrix. The system allowed us to evaluate the responses of osteoblasts in a 3D stress-strain environment similar to a mechanical field to which bone is exposed. Using MC3T3-E1 osteoblasts grown in the 3D collagen matrix with and without hydroxyapatite deposition, we tested the role of strain and the strain-induced fluid flow in the expression of the load-responsive genes such as c-fos, egr1, cox2, osteopontin, and mmp1B involved in transcriptional regulation, osteogenesis, and rearrangement of ECM. Strain-induced fluid flow was visualized with a microspheres approximately 3 microm in diameter in real time, and three viscoelastic parameters were determined. The results obtained by semi-quantitative PCR, immunoblot assay, enzymatic activity assays for collagenase and gelatinase, and mechanical characterization of collagen matrices supported the dominant role of strain-induced fluid flow in expression of the selected genes one hour after the mechanical treatment.

  2. Class I to III histone deacetylases differentially regulate inflammation-induced matrix metalloproteinase 9 expression in primary amnion cells.

    PubMed

    Poljak, Marin; Lim, Ratana; Barker, Gillian; Lappas, Martha

    2014-06-01

    Matrix metalloproteinase (MMP) 9 plays an important role in the degradation of the extracellular matrix in fetal membranes, and pathological activation of MMP-9 can lead to preterm birth. In nongestational tissues, modulation of histone deacetylases (HDACs) regulates MMP-9 expression. The aim of this study was to determine whether class I to III HDACs regulate MMP-9 expression and activity in primary amnion cells. Class I and II HDAC regulation of MMP-9 was assessed using the general class I and II HDAC inhibitors (HDACi) trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), the class I HDACi MS-275, and the class II HDACi MC1568. Class III HDAC regulation of MMP-9 was assessed using the SIRT1 activators resveratrol and SRT1720 as well as SIRT1 small interfering RNA (siRNA). Primary amnion epithelial cells were incubated with 1 ng/mL interleukin (IL) 1β in the absence or presence of 0.3 μmol/L TSA, 5 μmol/L SAHA, 2.5 μmol/L MS-275, 2.5 μmol/L MC1568, 50 μmol/L resveratrol, or 10 μmol/L SRT1720 for 20 hours. We found that the class I and II HDACi TSA and SAHA and the class II HDACi MC1568 significantly decreased IL-β-induced MMP-9 gene and pro-MMP-9 expression in primary amnion cells. There was, however, no effect of the class I HDACi MS-275 on IL-β-induced MMP-9 expression. On the other hand, inhibition of class III HDAC SIRT1 using siRNA significantly augmented IL-1β-induced MMP-9, and SIRT1 activation using resveratrol and SRT1720 inhibited IL-1β-induced MMP-9 expression. In summary, class I to III HDACs differentially regulate inflammation-induced MMP-9 expression in primary amnion cells.

  3. Matrix isolation infrared spectroscopic study of 4-Pyridinecarboxaldehyde and of its UV-induced photochemistry

    NASA Astrophysics Data System (ADS)

    Cluyts, Liesel; Sharma, Archna; Kuş, Nihal; Schoone, Kristien; Fausto, Rui

    2017-01-01

    The structure, infrared spectrum, barrier to internal rotation, and photochemistry of 4-pyridinecarboxaldehyde (4PCA) were studied by low-temperature (10 K) matrix isolation infrared spectroscopy and quantum chemical calculations undertaken at both Moller-Plesset to second order (MP2) and density functional theory (DFT/B3LYP) levels of approximation. The molecule has a planar structure (Cs point group), with MP2/6-311 ++G(d,p) predicted internal rotation barrier of 26.6 kJ mol- 1, which is slightly smaller than that of benzaldehyde ( 30 kJ mol- 1), thus indicating a less important electron charge delocalization from the aromatic ring to the aldehyde moiety in 4PCA than in benzaldehyde. A complete assignment of the infrared spectrum of 4PCA isolated in an argon matrix has been done for the whole 4000-400 cm- 1 spectral range, improving over previously reported data. Both the geometric parameters and vibrational frequencies of the aldehyde group reveal the relevance in this molecule of the electronic charge back-donation effect from the oxygen trans lone electron pair to the aldehyde Csbnd H anti-bonding orbital. Upon in situ UV irradiation of the matrix-isolated compound, prompt decarbonylation was observed, leading to formation of pyridine.

  4. Delmopinol-induced matrix removal facilitates photodynamic therapy and chlorhexidine methods for disinfecting mixed oral biofilms

    NASA Astrophysics Data System (ADS)

    Rogers, Stephen Christopher

    It is often observed that the slimy matrixes of various bacterial-formed biofilms can limit their disinfection. This investigation demonstrated that disinfection effectiveness by either photodynamic therapy (PDT) or chlorhexidine irrigation is significantly improved by collapse of that matrix using the non-bactericidal reagent delmopinol as part of the treatment sequence. Cyclic shear-producing conditions were used to grow 4-day, whole salivary and growth media biofilms on glow-discharge-treated polystyrene (N=46) and mini-germanium internal reflection prisms to serve in a periodontal crypt model of disinfection by either methylene-blue-mediated PDT or by chlorhexidine irrigation. Assays for bacterial viability, with and without treatments, were performed by alamarBlueRTM fluorescent methods, statistically applied (ANOVA, Tukey's HSD). Multiple Attenuated Internal Reflection Infrared (MAIR-IR) assays confirmed selective removal of the predominantly polysaccharide matrix materials by the delmopinol treatment, but not by equivalent water or chlorhexidine methods. Confocal-IR microscopy showed that the delmopinol reagent, alone, caused about one-third of each wet biofilm to be removed, while bacterial re-growth was confirmed by alamarBlueRTM assay. Chlorhexidine and PDT suppression of bacterial activity without regrowth was significantly improved with the added delmopinol treatment, and is likely to provide similarly beneficial results in the effective disinfection of diverse biofilms in many settings.

  5. Protein kinase D2 induces invasion of pancreatic cancer cells by regulating matrix metalloproteinases

    PubMed Central

    Wille, Christoph; Köhler, Conny; Armacki, Milena; Jamali, Arsia; Gössele, Ulrike; Pfizenmaier, Klaus; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Pancreatic cancer cell invasion, metastasis, and angiogenesis are major challenges for the development of novel therapeutic strategies. Protein kinase D (PKD) isoforms are involved in controlling tumor cell motility, angiogenesis, and metastasis. In particular PKD2 expression is up-regulated in pancreatic cancer, whereas PKD1 expression is lowered. We report that both kinases control pancreatic cancer cell invasive properties in an isoform-specific manner. PKD2 enhances invasion in three-dimensional extracellular matrix (3D-ECM) cultures by stimulating expression and secretion of matrix metalloproteinases 7 and 9 (MMP7/9), by which MMP7 is likely to act upstream of MMP9. Knockdown of MMP7/9 blocks PKD2-mediated invasion in 3D-ECM assays and in vivo using tumors growing on chorioallantois membranes. Furthermore, MMP9 enhances PKD2-mediated tumor angiogenesis by releasing extracellular matrix–bound vascular endothelial growth factor A, increasing its bioavailability and angiogenesis. Of interest, specific knockdown of PKD1 in PKD2-expressing pancreatic cancer cells further enhanced the invasive properties in 3D-ECM systems by generating a high-motility phenotype. Loss of PKD1 thus may be beneficial for tumor cells to enhance their matrix-invading abilities. In conclusion, we define for the first time PKD1 and 2 isoform–selective effects on pancreatic cancer cell invasion and angiogenesis, in vitro and in vivo, addressing PKD isoform specificity as a major factor for future therapeutic strategies. PMID:24336522

  6. PPARα and PPARγ attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities

    PubMed Central

    Huang, Wen; Eum, Sung Yong; András, Ibolya E; Hennig, Bernhard; Toborek, Michal

    2009-01-01

    The blood-brain barrier (BBB) plays an important role in HIV trafficking into the brain and the development of the central nervous system complications in HIV infection. Tight junctions are the main structural and functional elements that regulate the BBB integrity. Exposure of human brain microvascular endothelial cells (hCMEC/D3 cell line) to HIV-infected monocytes resulted in decreased expression of tight junction proteins, such as junctional adhesion molecule-A (JAM)-A, occludin, and zonula occludens (ZO)-1. Control experiments involved exposure to uninfected monocytes. Alterations of tight junction protein expression were associated with increased endothelial permeability and elevated transendothelial migration of HIV-infected monocytes across an in vitro model of the BBB. Notably, overexpression of the peroxisome proliferator-activated receptor (PPAR)α or PPARγ attenuated HIV-mediated dysregulation of tight junction proteins. With the use of exogenous PPARγ agonists and silencing of PPARα or PPARγ, these protective effects were connected to down-regulation of matrix metalloproteinase (MMP) and proteasome activities. Indeed, the HIV-induced decrease in the expression of JAM-A and occludin was restored by inhibition of MMP activity. Moreover, both MMP and proteasome inhibitors attenuated HIV-mediated altered expression of ZO-1. The present data indicate that down-regulation of MMP and proteasome activities constitutes a novel mechanism of PPAR-induced protections against HIV-induced disruption of brain endothelial cells.—Huang, W., Eum, S. Y., András, I. E., Hennig, B., Toborek, M. PPARα and PPARγ attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities. PMID:19141539

  7. Infrared Spectrum and UV-Induced Photochemistry of Matrix-Isolated 5-Hydroxyquinoline.

    PubMed

    Kuş, Nihal; Sagdinc, Seda; Fausto, Rui

    2015-06-18

    The structure, infrared spectrum, and photochemistry of 5-hydroxyquinoline (5HQ) were studied by matrix isolation infrared spectroscopy, complemented by theoretical calculations performed at the DFT(B3LYP)/6-311++G(d,p) level of approximation. According to the calculations, the trans conformer of 5HQ (with the OH group pointing to the opposite direction of the pyridine ring of the molecule) is more stable than the cis form (by ∼8.8 kJ mol(-1)). The main factors determining the relative stability of the two conformers were rationalized through natural bond orbital (NBO) and charge density analyses. The compound was trapped in solid nitrogen at 10 K, and its infrared spectra registered and interpreted, showing the sole presence in the matrix of the more stable trans conformer. Broadband in situ UV irradiations (λ ≥ 288 nm and λ ≥ 235 nm) allowed for the observation of different chemical transformations, which started by excitation to the S1 state of 5HQ, followed by homolytic cleavage of the O-H bond, and subsequent reattachment of the H atom to the 5HQ radical to form quinolin-5(6H)-one and quinolin-5(8H)-one. The first of these two quinolinones was found to convert to open-ring isomeric ketenes, especially when irradiation was performed at higher energy, whereas the second is rather stable under the used experimental conditions. As a whole, the observed photochemistry of matrix-isolated 5HQ closely matches those previously reported for phenol and thiophenol. A detailed mechanistic interpretation for the observed photochemical processes is here proposed, which received support from time-dependent DFT calculations.

  8. Density Induced Phase Transitions in the Schwinger Model: A Study with Matrix Product States

    NASA Astrophysics Data System (ADS)

    Bañuls, Mari Carmen; Cichy, Krzysztof; Cirac, J. Ignacio; Jansen, Karl; Kühn, Stefan

    2017-02-01

    We numerically study the zero temperature phase structure of the multiflavor Schwinger model at nonzero chemical potential. Using matrix product states, we reproduce analytical results for the phase structure for two flavors in the massless case and extend the computation to the massive case, where no analytical predictions are available. Our calculations allow us to locate phase transitions in the mass-chemical potential plane with great precision and provide a concrete example of tensor networks overcoming the sign problem in a lattice gauge theory calculation.

  9. UV radiation-induced biosynthesis, stability and antioxidant activity of mycosporine-like amino acids (MAAs) in a unicellular cyanobacterium Gloeocapsa sp. CU2556.

    PubMed

    Rastogi, Rajesh P; Incharoensakdi, Aran

    2014-01-05

    The biosynthesis of natural sunscreening compounds as influenced by ultraviolet radiation, their stability and antioxidant activity were studied in the cyanobacterium Gloeocapsa sp. CU-2556. An analysis by high-performance liquid chromatography (HPLC) with photodiode-array (PDA) detection revealed the biosynthesis of two MAAs, shinorine (UVλmax 333nm) and an unknown MAA designated as M-307 (UVλmax 307nm) with retention times of 5.9 and 6.4min, respectively. Induction of the synthesis of MAAs was studied under 395 (PAR), 320 (PAR+UV-A) and 295 (PAR+UV-A+UV-B) nm cut-off filters. MAAs induction was significantly increased with an increase in exposure time up to 72h in the samples covered with 295nm cut-off filters. Contrary to shinorine, the biosynthesis of M-307 was more dominant in this unicellular cyanobacterium. Both MAAs were highly stable to some physico-chemical stressors such as UV radiation, heat and a strong oxidizing agent. The MAA M-307 was more stable under strong oxidative stress than shinorine. Moreover, UV-C radiation drastically decreased the stability of both MAAs. The MAAs (shinorine+M-307) also exhibited efficient antioxidant activity which was dose-dependent. The results indicate that MAAs may perform a vital role in survival and sustainability of Gloeocapsa sp. CU-2556 in harsh environmental conditions by its ability to absorb/screen short wavelength UV radiation and antioxidant function.

  10. Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis in cultured human fibroblasts

    PubMed Central

    Donejko, Magdalena; Przylipiak, Andrzej; Rysiak, Edyta; Miltyk, Wojciech; Galicka, Elżbieta; Przylipiak, Jerzy; Zaręba, Ilona; Surazynski, Arkadiusz

    2015-01-01

    Introduction The aim of the study was to evaluate the effect of ethanol on collagen biosynthesis in cultured human skin fibroblasts, and the role of hyaluronic acid (HA) in this process. Regarding the mechanism of ethanol action on human skin fibroblasts we investigated: expression of β1 integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), nuclear factor kappa B (NF-κB) transcription factor, cytotoxicity assay and apoptosis, metalloproteinase activity, as well as the influence of HA on these processes. Materials and methods Collagen biosynthesis, activity of prolidase, DNA biosynthesis, and cytotoxicity were measured in confluent human skin fibroblast cultures that have been treated with 25, 50, and 100 mM ethanol and with ethanol and 500 µg/mL HA. Western blot analysis and zymography were performed to evaluate expression of collagen type I, β1 integrin receptor, IGF-IR, NF-κB protein, phospho-Akt protein, kinase MAPK, caspase 9 activity, and matrix metalloproteinases (MMP-9 and MMP-2). Results Ethanol in a dose-dependent manner lead to the impairment of collagen biosynthesis in fibroblast cultures through decreasing prolidase activity and expression of β1 integrin and IGF-IR. This was accompanied by an increased cytotoxicity, apoptosis and lowered expression of the signaling pathway proteins induced by β1 integrin and IGF-IR, that is, MAPK (ERK1/2) kinases. The lowered amount of synthesized collagen and prolidase activity disturbance may also be due to the activation of NF-κB transcription factor, which inhibits collagen gene expression. It suggests that the decrease in fibroblast collagen production may be caused by the disturbance in its biosynthesis but not degradation. The application of HA has a protective effect on disturbances caused by the examined substances. It seems that regulatory mechanism of ethanol-induced collagen aberration take

  11. Acetylcholine induces neurite outgrowth and modulates matrix metalloproteinase 2 and 9.

    PubMed

    Anelli, Tonino; Mannello, Ferdinando; Salani, Monica; Tonti, Gaetana A; Poiana, Giancarlo; Biagioni, Stefano

    2007-10-19

    The matrix metalloproteinases (MMPs), responsible for the degradation of extracellular matrix (ECM) proteins, may regulate brain cellular functions. Choline acetyltransferase (ChAT) transfected murine neuroblastoma cell line N18TG2, that synthesize acetylcholine and show enhancement of several neurospecific markers (i.e., sinapsin I, voltage gated Na(+) channels, high affinity choline uptake) and fiber outgrowth, were studied for the MMP regulation during neuronal differentiation. Zymography of N18TG2 culture medium revealed no gelatinolytic activity, whereas after carbachol treatment of cells both MMP-9 and activated MMP-2 forms were detected. ChAT-transfected clone culture medium contains three MMP forms at 230, 92, and 66kDa. Carbachol treatment increased MMP-2 and MMP-9 gene expression in N18TG2 cells and higher levels for both genes were also observed in ChAT transfected cells. The data are consistent with the hypothesis that acetylcholine brings about the activation of an autocrine loop modulating MMP expression.

  12. 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

  13. Transitional Remodeling of the Hepatic Extracellular Matrix in Alcohol-Induced Liver Injury

    PubMed Central

    Poole, Lauren G.

    2016-01-01

    Alcohol consumption is a common custom worldwide, and the toxic effects of alcohol on several target organs are well understood. The liver is the primary site of alcohol metabolism and is therefore the major target of alcohol toxicity. Alcoholic liver disease is a spectrum of disease states, ranging from simple steatosis (fat accumulation), to inflammation, and eventually to fibrosis and cirrhosis if untreated. The fibrotic stage of ALD is primarily characterized by robust accumulation of extracellular matrix (ECM) proteins (collagens) which ultimately impairs the function of the organ. The role of the ECM in early stages of ALD is poorly understood, but recent research has demonstrated that a number of changes in the hepatic ECM in prefibrotic ALD not only are present, but may also contribute to disease progression. The purpose of this review is to summarize the established and proposed changes to the hepatic extracellular matrix (ECM) that may contribute to earlier stages of ALD development and to discuss potential mechanisms by which these changes may mediate the progression of the disease. PMID:27843941

  14. Effects of astragalosides from Radix Astragali on high glucose-induced proliferation and extracellular matrix accumulation in glomerular mesangial cells

    PubMed Central

    CHEN, XIAO; WANG, DONG-DONG; WEI, TONG; HE, SU-MEI; ZHANG, GUAN-YING; WEI, QUN-LI

    2016-01-01

    Diabetic nephropathy (DN) exhibits a deteriorating course that may lead to end-stage renal failure. Astragalosides have been clinically tested for the treatment of DN, but the mechanism is unclear at present. In this study, the effects of astragalosides were investigated on high glucose-induced proliferation and expression of transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), type IV collagen (colIV) and fibronectin (FN) in glomerular mesangial cells (MCs). Cell proliferation was determined by 5-bromo-2′-deoxyuridine assay, and the expression of TGF-β1, CTGF, colIV and FN mRNA and proteins in MCs was detected by reverse transcription-polymerase chain reaction and ELISA assay, respectively. The results showed that high glucose clearly induced the proliferation of MCs and increased the expression of TGF-β1, CTGF, colIV and FN. Treatment with 50, 100, 200 µg/ml astragalosides inhibited cell proliferation and the expression of TGF-β1, CTGF, colIV and FN induced by high glucose. Thus, it is concluded that astragalosides inhibit the increased cell proliferation and expression of major extracellular matrix proteins that are induced by high glucose, indicating their value for the prophylaxis and therapy of DN. PMID:27313676

  15. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

    SciTech Connect

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2008-06-26

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

  16. Sub-inhibitory tigecycline concentrations induce extracellular matrix binding protein Embp dependent Staphylococcus epidermidis biofilm formation and immune evasion.

    PubMed

    Weiser, Julian; Henke, Hanae A; Hector, Nina; Both, Anna; Christner, Martin; Büttner, Henning; Kaplan, Jeffery B; Rohde, Holger

    2016-09-01

    Biofilm-associated Staphylococcus epidermidis implant infections are notoriously reluctant to antibiotic treatment. Here we studied the effect of sub-inhibitory concentrations of penicillin, oxacillin, vancomycin, daptomycin, linezolid and tigecycline on S. epidermidis 1585 biofilm formation, expression of extracellular matrix binding protein (Embp) and potential implications for S. epidermidis - macrophage interactions. Penicillin, vancomycin, daptomycin, and linezolid had no biofilm augmenting effect at any of the concentrations tested. In contrast, at sub-inhibitory concentrations tigecycline and oxacillin exhibited significant biofilm inducing activity. In S. epidermidis 1585, SarA is a negative regulator of giant 1 MDa Embp, and down regulation of sarA induces Embp-dependent assembly of a multi-layered biofilm architecture. Dot blot immune assays, confocal laser scanning microscopy, and qPCR showed that under biofilm inducing conditions, tigecycline augmented embp expression compared to the control grown without antibiotics. Conversely, expression of regulator sarA was suppressed, suggesting that tigecycline exerts its effects on embp expression through SarA. Tigecycline failed to induce biofilm formation in embp transposon mutant 1585-M135, proving that under these conditions Embp up-regulation is necessary for biofilm accumulation. As a functional consequence, tigecycline induced biofilm formation significantly impaired the up-take of S. epidermidis by mouse macrophage-like cell line J774A.1. Our data provide novel evidence for the molecular basis of antibiotic induced biofilm formation, a phenotype associated with inherently increased antimicrobial tolerance. While this could explain failure of antimicrobial therapies, persistence of S. epidermidis infections in the presence of sub-inhibitory antimicrobials is additionally propelled by biofilm-related impairment of macrophage-mediated pathogen eradication.

  17. Ulex europaeus I lectin induces activation of matrix-metalloproteinase-2 in endothelial cells.

    PubMed

    Gomez, D E; Yoshiji, H; Kim, J C; Thorgeirsson, U P

    1995-11-02

    In this report, we show that the lectin Ulex europaeus agglutinin I (UEA I), which binds to alpha-linked fucose residues on the surface of endothelial cells, mediates activation of the 72-kDa matrix metalloproteinase-2 (MMP-2). A dose-dependent increase in the active 62-kDa form of MMP-2 was observed in conditioned medium from monkey aortic endothelial cells (MAEC) following incubation with concentrations of UEA I ranging from 2 to 100 micrograms/ml. The increase in the 62-kDa MMP-2 gelatinolytic activity was not reflected by a rise in MMP-2 gene expression. The UEA I-mediated activation of MMP-2 was blocked by L-fucose, which competes with UEA I for binding to alpha-fucose. These findings may suggest that a similar in vivo mechanism exists, whereby adhesive interactions between tumor cell lectins and endothelial cells can mediate MMP-2 activation.

  18. Aflatoxin biosynthesis: current frontiers.

    PubMed

    Roze, Ludmila V; Hong, Sung-Yong; Linz, John E

    2013-01-01

    Aflatoxins are among the principal mycotoxins that contaminate economically important food and feed crops. Aflatoxin B1 is the most potent naturally occurring carcinogen known and is also an immunosuppressant. Occurrence of aflatoxins in crops has vast economic and human health impacts worldwide. Thus, the study of aflatoxin biosynthesis has become a focal point in attempts to reduce human exposure to aflatoxins. This review highlights recent advances in the field of aflatoxin biosynthesis and explores the functional connection between aflatoxin biosynthesis, endomembrane trafficking, and response to oxidative stress. Dissection of the regulatory mechanisms involves a complete comprehension of the aflatoxin biosynthetic process and the dynamic network of transcription factors that orchestrates coordinated expression of the target genes. Despite advancements in the field, development of a safe and effective multifaceted approach to solve the aflatoxin food contamination problem is still required.

  19. Estrogen deficiency promotes cigarette smoke-induced changes in the extracellular matrix in the lungs of aging female mice.

    PubMed

    Glassberg, Marilyn K; Catanuto, Paola; Shahzeidi, Shahriar; Aliniazee, Muddassir; Lilo, Sarit; Rubio, Gustavo A; Elliot, Sharon J

    2016-12-01

    Female smokers have a faster decline in lung function with increasing age and overall develop a greater loss of lung function than male smokers. This raises the question of whether estrogen status in women affects susceptibility to cigarette smoke (CS)-induced lung disease. Mouse models suggest that female mice are more susceptible than males to CS-induced lung disease. Moreover, young CS-exposed female mice develop emphysema earlier than male mice. The purpose of this study was to characterize the relationship of estrogen status on the pattern and severity of CS-induced lung disease. In this study, 15-month-old female C57BL/6J mice were ovariectomized and administered either placebo (pla) or 17β-estradiol (E2, 0.025 mg) 2 weeks after ovariectomy. They were further divided into those that were exposed to CS and no-smoke controls (NSC). Mice were exposed to CS in stainless steel inhalation chambers 3 hours a day, 5 days a week for 6 months, and sacrificed after 24 weeks of CS exposure. Blood and urine were collected at sacrifice to measure estrogen and cotinine levels, a metabolite of nicotine. Uterine weight was recorded as an indicator of estrogen status. Results showed that CS in the absence of E2 induced a decrease in hydroxyproline content, macrophage number, and respiratory chain complex-1 protein. CS without E2 also resulted in an increase in matrix metalloproteinase-2 activity and apoptosis and a change in the ratio of estrogen receptor subtype. These findings were abrogated with administration of E2, suggesting that estrogen deficiency increases susceptibility to CS-induced lung disease.

  20. Protective effects of acetaminophen on ibuprofen-induced gastric mucosal damage in rats with associated suppression of matrix metalloproteinase.

    PubMed

    Fukushima, Eriko; Monoi, Noriyuki; Mikoshiba, Shigeo; Hirayama, Yutaka; Serizawa, Tetsushi; Adachi, Kiyo; Koide, Misao; Ohdera, Motoyasu; Murakoshi, Michiaki; Kato, Hisanori

    2014-04-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to cause gastric mucosal damage as a side effect. Acetaminophen, widely used as an analgesic and antipyretic drug, has gastroprotective effects against gastric lesions induced by absolute ethanol and certain NSAIDs. However, the mechanisms that underlie the gastroprotective effects of acetaminophen have not yet been clarified. In the present study, we examined the role and protective mechanism of acetaminophen on ibuprofen-induced gastric damage in rats. Ibuprofen and acetaminophen were administered orally, and the gastric mucosa was macroscopically examined 4 hours later. Acetaminophen decreased ibuprofen-induced gastric damage in a dose-dependent manner. To investigate the mechanisms involved, transcriptome analyses of the ibuprofen-damaged gastric mucosa were performed in the presence and absence of acetaminophen. Ingenuity pathway analysis (IPA) software revealed that acetaminophen suppressed the pathways related to cellular assembly and inflammation, whereas they were highly activated by ibuprofen. On the basis of gene classifications from the IPA Knowledge Base, we identified the following five genes that were related to gastric damage and showed significant changes in gene expression: interleukin-1β (IL-1β), chemokine (C-C motif) ligand 2 (CCL2), matrix metalloproteinase-10 (MMP-10), MMP-13, and FBJ osteosarcoma oncogene (FOS). Expression of these salient genes was confirmed using real-time polymerase chain reaction. The expression of MMP-13 was the most reactive to the treatments, showing strong induction by ibuprofen and suppression by acetaminophen. Moreover, MMP-13 inhibitors decreased ibuprofen-induced gastric damage. In conclusion, these results suggest that acetaminophen decreases ibuprofen-induced gastric mucosal damage and that the suppression of MMP-13 may play an important role in the gastroprotective effects of acetaminophen.

  1. Peroxynitrite-induced nitration of tyrosine hydroxylase: identification of tyrosines 423, 428, and 432 as sites of modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tyrosine-scanning mutagenesis.

    PubMed

    Kuhn, Donald M; Sadidi, Mahdieh; Liu, Xiuli; Kreipke, Christian; Geddes, Timothy; Borges, Chad; Watson, J Throck

    2002-04-19

    Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30-60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7-30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr(423), Tyr(428), and Tyr(432) as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine

  2. The extracellular matrix of plants: Molecular, cellular and developmental biology

    SciTech Connect

    1996-12-31

    A symposium entitled ``The Extracellular Matrix of Plants: Molecular, Cellular and Developmental Biology was held in Tamarron, Colorado, March 15--21, 1996. The following topics were explored in addresses by 43 speakers: structure and biochemistry of cell walls; biochemistry, molecular biology and biosynthesis of lignin; secretory pathway and synthesis of glycoproteins; biosynthesis of matrix polysaccharides, callose and cellulose; role of the extracellular matrix in plant growth and development; plant cell walls in symbiosis and pathogenesis.

  3. Melatonin attenuates angiotensin II-induced abdominal aortic aneurysm through the down-regulation of matrix metalloproteinases.

    PubMed

    Kong, Jing; Zhang, Ya; Liu, Shanshan; Li, Hongxuan; Liu, Shangming; Wang, Jingjing; Qin, Xiaoteng; Jiang, Xiuxin; Yang, Jianmin; Zhang, Cheng; Zhang, Wencheng

    2017-02-04

    Abdominal aortic aneurysm (AAA) affects more than 5% of the population in developed countries and the pharmacotherapies for AAA are limited. Here, we explored whether melatonin regulates the development of AAA. In smooth muscle cells, melatonin treatment decreases angiotensin II-induced matrix metalloproteinase 2 (MMP2) and MMP9 expression. Human antigen R (HuR) could bind with the adenylateuridylate-rich elements of MMP2 and MMP9 mRNAs 3' untranslated region, resulting in the increased stability of MMP2 and MMP9 mRNAs. HuR is required for angiotensin II-induced MMP2 and MMP9 expression. Moreover, melatonin suppresses angiotensin II-induced HuR expression through inhibiting NF-κB signaling, leading to decreased MMP2 and MMP9 levels. Finally, melatonin attenuates the development of AAA in ApoE-/- mice infused with angiotensin II in vivo. These data support a role of HuR in the development of AAA and possible therapeutic roles for melatonin and/or HuR inhibition in AAA.

  4. N-acetyl-cysteine attenuates remifentanil-induced postoperative hyperalgesia via inhibiting matrix metalloproteinase-9 in dorsal root ganglia.

    PubMed

    Liu, Yue; Ni, Yuan; Zhang, Wei; Sun, Yu-E; Ma, Zhengliang; Gu, Xiaoping

    2017-02-09

    Treatment of remifentanil-induced postoperative hyperalgesia (RIH) remains a clinical challenge because the mechanisms are not fully understood. Matrix metalloproteinase-9 (MMP-9) is a key component in neuroinflammation because of its facilitation of pro-inflammatory cytokine maturation. Therefore, inhibition of MMP-9 may represent a novel therapeutic approach to the treatment of RIH. Sprague-Dawley rats were randomly divided into three groups: Control, Incision and Remifentanil. A right plantar surgical incision was performed in Group Incision, and intraoperative remifentanil (0.04 mg/kg, 0.4 ml) was infused subcutaneously for 30 min in Group Remifentanil. The results indicated that intraoperative remifentanil induced an up-regulation and activation of MMP-9 in DRGs but not spinal cords. MMP-9 was expressed primarily in DRG neurons co-expressing mu opioid receptors (MOR), and elicited interleukin-1β (IL-1β) cleavage in DRG neurons and satellite glial cells (SGCs). Intraperitoneal injection of N-acetyl-cysteine (NAC), a broadly used safe drug, significantly attenuated RIH via suppressing the activation of MMP-9 in DRGs. NAC inhibited the cleavage of IL-1β in DRGs, which is a critical substrate of MMP-9, and markedly suppressed glial activation and neuron excitability in spinal dorsal horn induced by remifentanil. These results demonstrated that NAC can effectively alleviate RIH via powerfully inhibiting MMP-9 activation in DRGs.

  5. Glucose enhances indolic glucosinolate biosynthesis without reducing primary sulfur assimilation

    PubMed Central

    Miao, Huiying; Cai, Congxi; Wei, Jia; Huang, Jirong; Chang, Jiaqi; Qian, Hongmei; Zhang, Xin; Zhao, Yanting; Sun, Bo; Wang, Bingliang; Wang, Qiaomei

    2016-01-01

    The effect of glucose as a signaling molecule on induction of aliphatic glucosinolate biosynthesis was reported in our former study. Here, we further investigated the regulatory mechanism of indolic glucosinolate biosynthesis by glucose in Arabidopsis. Glucose exerted a positive influence on indolic glucosinolate biosynthesis, which was demonstrated by induced accumulation of indolic glucosinolates and enhanced expression of related genes upon glucose treatment. Genetic analysis revealed that MYB34 and MYB51 were crucial in maintaining the basal indolic glucosinolate accumulation, with MYB34 being pivotal in response to glucose signaling. The increased accumulation of indolic glucosinolates and mRNA levels of MYB34, MYB51, and MYB122 caused by glucose were inhibited in the gin2-1 mutant, suggesting an important role of HXK1 in glucose-mediated induction of indolic glucosinolate biosynthesis. In contrast to what was known on the function of ABI5 in glucose-mediated aliphatic glucosinolate biosynthesis, ABI5 was not required for glucose-induced indolic glucosinolate accumulation. In addition, our results also indicated that glucose-induced glucosinolate accumulation was due to enhanced sulfur assimilation instead of directed sulfur partitioning into glucosinolate biosynthesis. Thus, our data provide new insights into molecular mechanisms underlying glucose-regulated glucosinolate biosynthesis. PMID:27549907

  6. NF-κB Has a Direct Role in Inhibiting Bmp- and Wnt-Induced Matrix Protein Expression.

    PubMed

    Tarapore, Rohinton S; Lim, Jason; Tian, Chen; Pacios, Sandra; Xiao, Wenmei; Reid, Daniel; Guan, Hancheng; Mattos, Marcelo; Yu, Bo; Wang, Cun-Yu; Graves, Dana T

    2016-01-01

    The host response to pathogens through nuclear factor κB (NF-κB) is an essential defense mechanism for eukaryotic organisms. NF-κB-mediated host responses inhibit bone and other connective tissue synthesis and are thought to affect the transcription of matrix proteins through multiple indirect pathways. We demonstrate that inhibiting NF-κB in osteoblasts increases osteocalcin expression in vivo in mice with periodontal disease. Mutating NF-κB binding sites on osteocalcin (OC) or bone sialoprotein (Bsp) promoters rescues the negative impact of NF-κB on their transcription and that NF-κB can inhibit Wnt- and Bmp-induced OC and Bsp transcription, even when protein synthesis is inhibited, indicating a direct effect of NF-κB. This inhibition depends on p65-p50 NF-κB heterodimer formation and deacetylation by HDAC1 but is not affected by the noncanonical NF-κB pathway. Moreover, NF-κB reduces Runx2 and β-catenin binding to OC/Bsp promoters independently of their nuclear localization. Thus, inflammatory signals stimulate the direct interaction of NF-κB with response elements to inhibit binding of β-catenin and Runx2 binding to nearby consensus sites and reduce expression of matrix proteins. This direct mechanism provides a new explanation for the rapid decrease in new bone formation after inflammation-related NF-κB activation.

  7. The Matrix protein M1 from influenza C virus induces tubular membrane invaginations in an in vitro cell membrane model

    PubMed Central

    Saletti, David; Radzimanowski, Jens; Effantin, Gregory; Midtvedt, Daniel; Mangenot, Stéphanie; Weissenhorn, Winfried; Bassereau, Patricia; Bally, Marta

    2017-01-01

    Matrix proteins from enveloped viruses play an important role in budding and stabilizing virus particles. In order to assess the role of the matrix protein M1 from influenza C virus (M1-C) in plasma membrane deformation, we have combined structural and in vitro reconstitution experiments with model membranes. We present the crystal structure of the N-terminal domain of M1-C and show by Small Angle X-Ray Scattering analysis that full-length M1-C folds into an elongated structure that associates laterally into ring-like or filamentous polymers. Using negatively charged giant unilamellar vesicles (GUVs), we demonstrate that M1-C full-length binds to and induces inward budding of membrane tubules with diameters that resemble the diameter of viruses. Membrane tubule formation requires the C-terminal domain of M1-C, corroborating its essential role for M1-C polymerization. Our results indicate that M1-C assembly on membranes constitutes the driving force for budding and suggest that M1-C plays a key role in facilitating viral egress. PMID:28120862

  8. Double pulse laser induced breakdown spectroscopy: Experimental study of lead emission intensity dependence on the wavelengths and sample matrix

    NASA Astrophysics Data System (ADS)

    Piscitelli S, V.; Martínez L., M. A.; Fernández C., A. J.; González, J. J.; Mao, X. L.; Russo, R. E.

    2009-02-01

    Lead (Pb) emission intensity (atomic line 405.78 nm) dependence on the sample matrix (metal alloy) was studied by means of collinear double pulse (DP)-laser induced breakdown spectroscopy (LIBS). The measurement of the emission intensity produced by three different wavelength combinations (i.e. I:532 nm-II:1064 nm, I:532 nm-II:532 nm, and I:532 nm-II:355 nm) from three series of standard reference materials showed that the lead atomic line 405.78 nm emission intensity was dependent on the sample matrix for all the combination of wavelengths, however reduced dependency was found for the wavelength combination I:532 nm-II:355 nm. Two series of standard reference materials from the National Institute of Standards and Technology (NIST) and one series from the British Chemical Standards (BCS) were used for these experiments. Calibration curves for lead ablated from NIST 626-630 ("Zn 95Al 4Cu 1") provided higher sensitivity (slope) than those calibration curves produced from NIST 1737-1741 ("Zn 99.5Al 0.5") and with the series BCS 551-556 ("Cu 87Sn 11"). Similar trends between lead emission intensity (calibration curve sensitivities) and reported variations in plasma temperatures caused by the differing ionization potentials of the major and minor elements in these samples were established.

  9. 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.

  10. Pherophorins: a family of extracellular matrix glycoproteins from Volvox structurally related to the sex-inducing pheromone.

    PubMed

    Godl, K; Hallmann, A; Rappel, A; Sumper, M

    1995-01-01

    Pherophorins are extracellular matrix (ECM) glycoproteins from Volvox that share homology with the sex-inducing pheromone. A novel pherophorin (pherophorin III) was characterized both with respect to expression pattern and proteolytic processing in vivo. Furthermore, it was shown that the pherophorins represent a protein family of ECM glycoproteins exhibiting a modular composition: their N-terminally located domain is a homolog of a domain found in the ECM glycoprotein SSG 185. Together with SSG 185, pherophorin I is a main component of the cellular zone within the ECM. The Volvox genome contains a tandem arrangement of genes encoding pherophorin II-related polypeptides. Inhibition of proteolytic processing of pherophorin II and III in vivo appears to result in the suppression of sexual induction.

  11. The altered glucose metabolism in tumor and a tumor acidic microenvironment associated with extracellular matrix metalloproteinase inducer and monocarboxylate transporters

    PubMed Central

    Li, Xiaofeng; Yu, Xiaozhou; Dai, Dong; Song, Xiuyu; Xu, Wengui

    2016-01-01

    Extracellular matrix metalloproteinase inducer, also knowns as cluster of differentiation 147 (CD147) or basigin, is a widely distributed cell surface glycoprotein that is involved in numerous physiological and pathological functions, especially in tumor invasion and metastasis. Monocarboxylate transporters (MCTs) catalyze the proton-linked transport of monocarboxylates such as L-lactate across the plasma membrane to preserve the intracellular pH and maintain cell homeostasis. As a chaperone to some MCT isoforms, CD147 overexpression significantly contributes to the metabolic transformation of tumor. This overexpression is characterized by accelerated aerobic glycolysis and lactate efflux, and it eventually provides the tumor cells with a metabolic advantage and an invasive phenotype in the acidic tumor microenvironment. This review highlights the roles of CD147 and MCTs in tumor cell metabolism and the associated molecular mechanisms. The regulation of CD147 and MCTs may prove to be with a therapeutic potential for tumors through the metabolic modification of the tumor microenvironment. PMID:27009812

  12. Matrix protein CCN1 is critical for prostate carcinoma cell proliferation and TRAIL-induced apoptosis.

    PubMed

    Franzen, Carrie A; Chen, Chih-Chiun; Todorović, Viktor; Juric, Vladislava; Monzon, Ricardo I; Lau, Lester F

    2009-07-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays an important role in immune surveillance and preferentially induces apoptosis in cancer cells over normal cells, suggesting its potential in cancer therapy. However, the molecular basis for its selective killing of cancer cells is not well understood. Recent studies have identified the CCN family of integrin-binding matricellular proteins as important regulators of cell behavior, including cell adhesion, proliferation, migration, differentiation, and survival. We show here that CCN1 (CYR61) supports the adhesion of prostatic carcinoma cells as an adhesion substrate through integrins and heparan sulfate proteoglycans. Knockdown of CCN1 expression in PC-3 and DU-145 androgen-independent prostate cancer cells strongly inhibited their proliferation without causing apoptosis, indicating that CCN1 promotes their growth. However, CCN1 also significantly enhances TRAIL-induced apoptosis through interaction with integrins alphavbeta3 and alpha6beta4 and the cell-surface heparan sulfate proteoglycan syndecan-4, acting through a protein kinase Calpha-dependent mechanism without requiring de novo protein synthesis. Knockdown of CCN1 expression in PC-3, DU-145, and LNCaP cells severely blunted their sensitivity to TRAIL, an effect that was reversed by exogenously added CCN1 protein. These findings reveal a functional dichotomy for CCN1 in prostate carcinoma cells, because it contributes to both cell proliferation and TRAIL-induced cell death and suggest that CCN1 expression status may be an important parameter in assessing the efficacy of TRAIL-dependent cancer therapy.

  13. Quantum cascade laser modulation for correction of matrix-induced background changes in aqueous samples

    NASA Astrophysics Data System (ADS)

    Schaden, S.; Domínguez-Vidal, A.; Lendl, B.

    2007-01-01

    A robust method for the determination of carbon dioxide in sugar containing solutions using a single distributed feedback quantum cascade laser (QCL) has been developed. By switching the driving current between two selected values, 1.6 and 1 A, emission wavelengths at 2341.4 and 2341.6 cm-1 could be achieved. The method is based on absorbance measurements in transmission and the calculation of the absorbance differences between both wavenumbers. This allows the elimination of indirect matrix interference produced on carbon dioxide measurements with increasing sugar concentrations. A flow injection setup was employed to produce carbon dioxide standards from a series of bicarbonate solutions (0-3 g/l) by adjusting the pH with a sodium hydroxide/citric acid buffer solution to pH 3.13. Different concentrations (0-90 g/l) of sugar were also mixed on line with the analyte to study their influence on carbon dioxide measurement. As the difference in the two evaluated wavelengths is small compared to the absorption peak of CO2, the analytical readout of the QCL modulation can be seen as a proportional parameter to the first derivative of FTIR spectra in this spectral region.

  14. Onion extract and quercetin induce matrix metalloproteinase-1 in vitro and in vivo.

    PubMed

    Cho, Jae-We; Cho, Sun-Young; Lee, Seong-Ryong; Lee, Kyu-Suk

    2010-03-01

    A scar is usually developed by an imbalance of collagen synthesis and degradation. It is believed that the flavonoids (quercetin and kaempferol) in onion extract play a role in reducing scar formation through inhibition of fibroblast activities. Even though several commercial products are composed of onion extract, the precise molecular mechanisms of onion extract in reduction of scar formation in skin are still largely unknown. In this study we investigated the effect both of onion extract and quercetin on the proliferation of fibroblasts, expression of type I collagen and matrix metalloproteinase-1 (MMP-1). Our data show that proliferation rates of fibroblasts were decreased in a dose-dependent manner of the onion extract and quercetin. The expression of type I collagen was not markedly changed by the onion extract and quercetin. Interestingly, the expression of MMP-1 was markedly increased by both onion extract and quercetin in vitro and in vivo. Thus, our data indicate that onion extract and quercetin play a role in the anti-scar effect in skin through up-regulation of MMP-1 expression, implying this agent is a promising material for reducing scar formation.

  15. Fibulin1C peptide induces cell attachment and extracellular matrix deposition in lung fibroblasts

    PubMed Central

    Ge, Qi; Chen, Ling; Jaffar, Jade; Argraves, William Scott; Twal, Waleed O.; Hansbro, Phil; Black, Judith L.; Burgess, Janette K.; Oliver, Brian

    2015-01-01

    Fibulin-1 is an extracellular matrix (ECM) protein, levels of which are elevated in serum and lung tissue from patients with idiopathic pulmonary fibrosis compared to healthy volunteers. Inhibition of fibulin-1C, one of four fibulin-1 isoforms, reduced proliferation and wound healing in human airway smooth muscle (ASM) cells. This study identified the bioactive region/s of fibulin-1C which promotes fibrosis. Seven fibulin-1C peptides were synthesized and used to pre-coat tissue culture plates before lung derived ASM cells and fibroblasts from patients with pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD) or neither disease (Control) were plated. Peptide effects on in vitro measures of fibrosis: cell attachment, proliferation and viability, and ECM deposition, were examined. Among these peptides, peptide 1C1 (FBLN1C1) enhanced ASM cell and fibroblast attachment. FBLN1C1 increased mitochondrial activity and proliferation in fibroblasts. In addition, FBLN1C1 stimulated fibulin1 deposition in PF and COPD fibroblasts, and augmented fibronectin and perlecan deposition in all three groups. Peptides FBLN1C2 to FBLN1C7 had no activity. The active fibulin-1C peptide identified in this study describes a useful tool for future studies. Ongoing investigation of the role of fibulin-1 may reveal the mechanisms underlying the pathphysiology of chronic lung diseases. PMID:25834989

  16. Castor oil polymer induces bone formation with high matrix metalloproteinase-2 expression.

    PubMed

    Saran, Wallace Rocha; Chierice, Gilberto Orivaldo; da Silva, Raquel Assed Bezerra; de Queiroz, Alexandra Mussolino; Paula-Silva, Francisco Wanderley Garcia; da Silva, Léa Assed Bezerra

    2014-02-01

    The aim of this study was to evaluate the modulation of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) expression in newly formed bone tissue at the interface between implants derived from castor oil (Ricinus communis) polymer and the tibia medullary canal. Forty-four rabbits were assigned to either Group 1 (n = 12; control) or Group 2 (n = 30), which had the tibial medullary canals reamed bilaterally and filled with polymer. CT scans showed no space between the material surface and the bone at the implant/bone marrow interface, and the density of the tissues at this interface was similar to the density measured of other regions of the bone. At 90 days postimplantation, the interface with the polymer presented a thick layer of newly formed bone tissue rich in osteocytes. This tissue exhibited ongoing maturation at 120 and 150 days postimplantation. Overall, bone remodeling process was accompanied by positive modulation of MMP-2 and low MMP-9 expression. Differently, in control group, the internal surface close to the medullary canal was lined by osteoblasts, followed by a bone tissue zone with few lacunae filled with osteocytes. Maturation of the tissue of the medullary internal surface occurred in the inner region, with the bone being nonlamellar.

  17. Confinement of massless Dirac fermions in the graphene matrix induced by the B/N heteroatoms.

    PubMed

    Yu, Shansheng; Zheng, Weitao; Ao, Zhimin; Li, Sean

    2015-02-28

    In this work, the systems are constructed with the defect lines of B-B or N-N dimers embedded in a graphene matrix using density functional theory. It is found that the Dirac-cone dispersions appear at the Fermi level in the bands introduced by the B or N heteroatom, linear B-B or N-N dimers, demonstrating that the carrier mobility is ∼10(6) m s(-1) which is comparable with that of the pristine graphene. Most importantly, such dimer lines act as the quasi-1-D conducting nanowires whose charge carriers are confined around the linear defects in these dimers while the charge carriers in pristine graphene are dispersed two-dimensionally. Such systems suggest that heteroatoms in graphene can indeed contribute to the Dirac cone. In addition, the type of carriers (p-type or n-type) can be manipulated using the B or N heteroatoms, respectively. This will greatly enrich the electronic properties of Dirac semimetals.

  18. Pamidronate Down-regulates Tumor Necrosis Factor-alpha Induced Matrix Metalloproteinases Expression in Human Intervertebral Disc Cells

    PubMed Central

    Kang, Young-Mi; Hong, Seong-Hwan; Yang, Jae-Ho; Oh, Jin-Cheol; Park, Jin-Oh; Lee, Byung Ho; Lee, Sang-Yoon; Kim, Hak-Sun; Lee, Hwan-Mo

    2016-01-01

    Background N-containing bisphosphonates (BPs), such as pamidronate and risedronate, can inhibit osteoclastic function and reduce osteoclast number by inducing apoptotic cell death in osteoclasts. The aim of this study is to demonstrate the effect of pamidronate, second generation nitrogen-containing BPs and to elucidate matrix metallo-proteinases (MMPs) mRNA expression under serum starvation and/or tumor necrosis factor alpha (TNF-α) stimulation on metabolism of intervertebral disc (IVD) cells in vitro. Methods Firstly, to test the effect of pamidronate on IVD cells in vitro, various concentrations (10-12, 10-10, 10-8, and 10-6 M) of pamidronate were administered to IVD cells. Then DNA and proteoglycan synthesis were measured and messenger RNA (mRNA) expressions of type I collagen, type II collagen, and aggrecan were analyzed. Secondly, to elucidate the expression of MMPs mRNA in human IVD cells under the lower serum status, IVD cells were cultivated in full serum or 1% serum. Thirdly, to elucidate the expression of MMPs mRNA in IVD cells under the stimulation of 1% serum and TNF-α (10 ng/mL) In this study, IVD cells were cultivated in three dimensional alginate bead. Results Under the lower serum culture, IVD cells in alginate beads showed upregulation of MMP 2, 3, 9, 13 mRNA. The cells in lower serum and TNF-α also demonstrated upregulation of MMP-2, 3, 9, and 13 mRNA. The cells with various doses of pamidronate and lower serum and TNF-α were reveled partial down-regulation of MMPs. Conclusions Pamidronate, N-containing second generation BPs, was safe in metabolism of IVD in vitro maintaining chondrogenic phenotype and matrix synthesis, and down-regulated TNF-α induced MMPs expression. PMID:27622181

  19. Fenofibrate Attenuated Glucose-Induced Mesangial Cells Proliferation and Extracellular Matrix Synthesis via PI3K/AKT and ERK1/2

    PubMed Central

    Zhu, Fengming; Ma, Zufu; Liao, Wenhui; He, Yong; He, JinSeng; Li, Wei; Yang, Juan; Lu, Qian; Xu, Gang; Yao, Ying

    2013-01-01

    Excess mesangial extracellular matrix (ECM) and mesangial cell proliferation is the major pathologic feature of diabetic nephropathy (DN). Fenofibrate, a PPARα agonist, has been shown to attenuate extracellular matrix formation in diabetic nephropathy. However, the mechanisms underlying this effect remain to be elucidated. In this study, the effect of fenofibrate on high-glucose induced cell proliferation and extracellular matrix exertion and its mechanisms were investigated in cultured rat mesangial cells by the methylthiazoletetrazolium (MTT) assay, flow cytometry and western blot. The results showed that treatment of mesangial cells (MCs) with fenofibrate repressed high-glucose induced up-regulation of extracellular matrix Collagen-IV, and inhibited entry of cell cycle into the S phase. This G1 arrest and ECM inhibition was caused by the reduction of phosphorylation and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT. On the contrary, PPARα siRNA accelerated high glucose-induced cell cycle progression by ERK1/2 and AKT activation. Taken together, fenofibrate ameliorated glucose-induced mesangial cell proliferation and matrix production via its inhibition of PI3K/AKT and ERK1/2 signaling pathways. Such mechanisms may contribute to the favorable effects of treatment using fenofibrate in diabetic nephropathy. PMID:24130796

  20. Biosynthesis of Polyisoprenoids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The invention is a process for synthesis of a polymer with the same chemical structure as Natural Rubber (NR) obtained from Hevea brasiliensis and other plant species. The research collaborators recently proposed that NR biosynthesis proceeds via a carbocationic polymerization. Based on this theory...

  1. Beta-1,3-glucooligosaccharide induced activation of four enzymes responsible for N-p-coumaroyloctopamine biosynthesis in potato (Solanum tuberosum cv.) tuber tissue.

    PubMed

    Matsuda, F; Miyagawa, H; Ueno, T

    2000-01-01

    Potato tuber disks, when treated with laminarin, a beta-1,3-glucooligosaccharide from Laminaria digitata, accumulate a hydroxycinnamoyl amide compound, N-p-coumaroyloctopamine (p-CO). The biosynthesis of p-CO was investigated by feeding experiments, in order to show that the precursors of N-p-coumaroyl and octopamine moieties of p-CO are L-phenylalanine and L-tyrosine, respectively. The treatment of potato tuber tissue with laminarin resulted in elevated activities of four enzymes which are putatively involved in p-CO biosynthesis: phenylalanine ammonia lyase (PAL; EC 4.3.1.5), 4-hydroxycinnamic acid:CoA ligase (4CL; EC 6.2.1.12), hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25). Among these, the response of TyrDC was specific to laminarin treatment, thus indicating that the regulation of TyrDC activity is critical for the accumulation of p-CO in potato tuber tissue.

  2. Keratinocyte growth factor induces matrix metalloproteinase-9 expression and correlates with venous invasion in pancreatic cancer

    PubMed Central

    CHO, KAZUMITSU; MATSUDA, YOKO; UEDA, JUNJI; UCHIDA, EIJI; NAITO, ZENYA; ISHIWATA, TOSHIYUKI

    2012-01-01

    Keratinocyte growth factor (KGF), also known as fibroblast growth factor-7, and KGF receptor (KGFR) play important roles in the growth of epithelial cells and are overexpressed in a variety of malignant epithelial tumors, including pancreatic ductal adenocarcinoma (PDAC). We previously reported that co-expression of KGF and KGFR in PDAC is associated with venous invasion, enhanced vascular endothelial growth factor A expression and poor prognosis. Matrix metalloproteinase-9 (MMP-9) is known to participate in the degradation of type IV collagen, which is a primary component of extracellular matrices in the vascular basement membrane. In the present study, we examined the expression and roles of KGF, KGFR and MMP-9 in human PDAC cell lines and tissues. Quantitative real-time polymerase chain reaction analysis demonstrated the expression of MMP-9 mRNA in all eight PDAC cell lines. KGF, KGFR and MMP-9 were, respectively, expressed in 27 (43%), 23 (37%) and 35 (56%) of 63 patients. Each expression of KGF, KGFR or MMP-9 correlated positively with venous invasion. Furthermore, expression of KGF or MMP-9 correlated positively with liver metastasis. KGF-positive cases exhibited shorter survival than KGF-negative cases, while KGFR and MMP-9 expression were unrelated to prognosis. Administration of recombinant human KGF increased MMP-9 expression in PDAC cells, while transient transfection with short hairpin RNAs targeting KGF transcripts reduced MMP-9 expression in PDAC cells. Moreover, recombinant human KGF significantly enhanced migration and invasion of PDAC cells. These findings suggest that KGF and KGFR promote venous invasion via MMP-9 in PDAC, and closely correlate with liver metastasis. The KGF/KGFR pathway may be a critical therapeutic target for PDAC metastasis. PMID:22159401

  3. NF kappa B and Matrix Metalloproteinase induced Receptor Cleavage in the Spontaneously Hypertensive Rat

    PubMed Central

    Wu, Kwan-I Sharon; Schmid-Schönbein, Geert W.

    2011-01-01

    Recent evidence suggests that inflammation in the spontaneously hypertensive rat (SHR) is associated with an uncontrolled matrix metalloproteinase (MMP) activity. We hypothesize that the transcription factor nuclear factor kappa B (NF–κB) is overexpressed in the SHR, enhancing its MMP activity and enzymatic cleavage of the beta-2 adrenergic receptor (β2AR), thereby diminishing catecholamine-mediated arteriolar vasodilation. NF-κB expression level and translocation were compared between Wistar Kyoto rat (WKY) and SHR kidney, heart and brain. The animals were treated with a NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), for ten weeks and correlations between NF-κB and MMP activity were determined. Immunohistochemistry showed that NF-κB expression is increased in untreated SHR kidney (~ 14%) and brain hypothalamus (~ 22%) compared to that in WKY (p <0.05), but not in myocardium and cerebral cortex. After PDTC treatment, the SHR systolic blood pressure was reduced close to WKY levels. NF-κB expression level in treated-SHR was also decreased in kidney and hypothalamus compared to non-treated animals (p <0.05). Furthermore, MMP-2 and -9 activities in SHR plasma were significantly reduced (~41%) by PDTC treatment. Additionally, zymographic analyses and in situ zymography showed decreased MMP-2 activity in kidney homogenates and decreased MMP-1,-9 activities in brain. The level of the β2AR extracellular, but not intracellular, domain density was found reduced in kidney showing a receptor cleavage process that can be blocked by PDTC treatment. These results suggest NF-κB is an important transcription factor in the SHR and may be involved in the enhanced MMP activity and consequently receptor cleavage. PMID:21220710

  4. Matrix metalloproteinase 9-induced increase in intestinal epithelial tight junction permeability contributes to the severity of experimental DSS colitis

    PubMed Central

    Nighot, Prashant; Al-Sadi, Rana; Guo, Shuhong; Watterson, D. Martin; Ma, Thomas

    2015-01-01

    Recent studies have implicated a pathogenic role for matrix metalloproteinases 9 (MMP-9) in inflammatory bowel disease. Although loss of epithelial barrier function has been shown to be a key pathogenic factor for the development of intestinal inflammation, the role of MMP-9 in intestinal barrier function remains unclear. The aim of this study was to investigate the role of MMP-9 in intestinal barrier function and intestinal inflammation. Wild-type (WT) and MMP-9−/− mice were subjected to experimental dextran sodium sulfate (DSS) colitis by administration of 3% DSS in drinking water for 7 days. The mouse colonic permeability was measured in vivo by recycling perfusion of the entire colon using fluorescently labeled dextran. The DSS-induced increase in the colonic permeability was accompanied by an increase in intestinal epithelial cell MMP-9 expression in WT mice. The DSS-induced increase in intestinal permeability and the severity of DSS colitis was found to be attenuated in MMP-9−/− mice. The colonic protein expression of myosin light chain kinase (MLCK) and phospho-MLC was found to be significantly increased after DSS administration in WT mice but not in MMP-9−/− mice. The DSS-induced increase in colonic permeability and colonic inflammation was attenuated in MLCK−/− mice and MLCK inhibitor ML-7-treated WT mice. The DSS-induced increase in colonic surface epithelial cell MLCK mRNA was abolished in MMP-9−/− mice. Lastly, increased MMP-9 protein expression was detected within the colonic surface epithelial cells in ulcerative colitis cases. These data suggest a role of MMP-9 in modulation of colonic epithelial permeability and inflammation via MLCK. PMID:26514773

  5. Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3

    SciTech Connect

    Ozeki, Nobuaki; Hase, Naoko; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-02-01

    A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells. - Highlights: • Pro-inflammatory cytokines induce MMP-3 activity in human odontoblast-like cells. • Increased MMP-3 activity can promote cell proliferation in odontoblasts. • Specific loss of MMP-3 increases apoptosis in odontoblasts. • MMP-3 has potential as a promising new target for pupal repair and regeneration.

  6. Laser induced breakdown spectroscopy for heavy metal detection in a sand matrix

    NASA Astrophysics Data System (ADS)

    Michel, Anna P. M.; Sonnichsen, Frederick

    2016-11-01

    Sediments in many locations, including harbors and coastal areas, can become contaminated and polluted, for example, from anthropogenic inputs, shipping, human activities, and poor waste management. Sampling followed by laboratory analysis has been the traditional methodology for such analysis. In order to develop rapid methodologies for field analysis of sediment samples, especially for metals analyses, we look to Laser Induced Breakdown Spectroscopy as an option. Here through laboratory experiments, we demonstrate that dry sand samples can be rapidly analyzed for the detection of the heavy metals chromium, zinc, lead, and copper. We also demonstrate that cadmium and nickel are detectable in sand matrices at high concentrations.

  7. Light-induced director-controlled microassembly of dye molecules from a liquid crystal matrix

    NASA Astrophysics Data System (ADS)

    Voloschenko, D.; Lavrentovich, O. D.

    1999-11-01

    We report on a light-induced phenomenon in dye-doped liquid crystals (LCs) with the distinctive features of molecular transport and assembly at micron scales. Under single-beam laser irradiation, the dye molecules phase separate from the LC host and assemble onto the cell substrate. Although the intensity of incident light is uniform within the irradiated area, the density of the adsorbed dye is modulated in accord with the director modulation of the LC. The dye molecules form a surface imprint that portrays orientational distortions of the LC host.

  8. Gray level co-occurrence matrix algorithm as pattern recognition biosensor for oxidopamine-induced changes in lymphocyte chromatin architecture.

    PubMed

    Pantic, Igor; Dimitrijevic, Draga; Nesic, Dejan; Petrovic, Danica

    2016-10-07

    We demonstrate that a proapoptotic chemical agent, oxidopamine, induces dose dependent changes in chromatin textural patterns which can be quantified using the Gray level co-occurrence matrix (GLCM) method. Peripheral blood (heparin-pretreated) samples were treated with oxidopamine (6-OHDA, 6-hydroxydopamine) to achieve effective concentrations of 100, 200 and 300µM. The samples were smeared on microscope slides and fixated in methanol. The smears were stained using a modification of Feulgen method for DNA visualization. For each stained smear, a sample of 30 lymphocyte chromatin structures were visualized and analyzed. This way, textural parameters for a total of 120 nuclei micrographs were calculated. For each chromatin structure, five different GLCM features were calculated: angular second moment, GLCM entropy, inverse difference moment, GLCM correlation, and GLCM variance. Oxidopamine induced the rise of the values of GLCM entropy and variance, and the reduction of angular second moment, correlation, and inverse difference moment. The trends for GLCM parameter changes were found to be highly significant (p<0.001). These results indicate that GLCM mathematical algorithm might be successfully used in detection and evaluation of discrete early apoptotic structural changes in Feulgen-stained chromatin of peripheral blood lymphocytes that are not detectable using conventional microscopy/cell biology techniques.

  9. Phytolacca americana inhibits the high glucose-induced mesangial proliferation via suppressing extracellular matrix accumulation and TGF-beta production.

    PubMed

    Jeong, Seung Il; Kim, Kang Ju; Choo, Yong Kug; Keum, Kyung Soo; Choi, Bong Kyu; Jung, Kyu Yong

    2004-02-01

    This study describes a potential of Phytolaccaceae (Phytolacca americana var.) as an inhibitor of high glucose-stimulated production of extracellular matrix (ECM) proteins and TGF-beta in cultured glomerular mesangial cells (GMCs). Raising the ambient glucose concentration for 24 hrs caused a dose-dependent increase in [3H]thymidine incorporation of GMCs, and the maximal response was achieved at 20 mM. Phytolaccaceae extracts (2.5-20 microg/ml) inhibited the high glucose-induced [3H]thymidine incorporation in a dose-dependent manner, and the concentrations tested here did not affect to the cell viability. Exposure of the GMCs to 20 mM glucose caused both ECM (collagen and fibronectin) accumulation and TGF-beta secretion, and these changes were significantly diminished by treatment of GMCs with Phytolaccaceae (10 microg/ml). Taken together, these results indicate that Phytolaccaceae inhibits the high glucose-induced GMCs proliferation partially through suppressing accumulation of ECM components and TGF-beta production, suggesting that Phytolaccaceae may be a promising agent for treating the development and progression of diabetic glomerulopathy.

  10. Minocycline Attenuates Neonatal Germinal-Matrix-Hemorrhage-Induced Neuroinflammation and Brain Edema by Activating Cannabinoid Receptor 2.

    PubMed

    Tang, Jun; Chen, Qianwei; Guo, Jing; Yang, Liming; Tao, Yihao; Li, Lin; Miao, Hongping; Feng, Hua; Chen, Zhi; Zhu, Gang

    2016-04-01

    Germinal matrix hemorrhage (GMH) is the most common neurological disease of premature newborns leading to detrimental neurological sequelae. Minocycline has been reported to play a key role in neurological inflammatory diseases by controlling some mechanisms that involve cannabinoid receptor 2 (CB2R). The current study investigated whether minocycline reduces neuroinflammation and protects the brain from injury in a rat model of collagenase-induced GMH by regulating CB2R activity. To test this hypothesis, the effects of minocycline and a CB2R antagonist (AM630) were evaluated in male rat pups that were post-natal day 7 (P7) after GMH. We found that minocycline can lead to increased CB2R mRNA expression and protein expression in microglia. Minocycline significantly reduced GMH-induced brain edema, microglial activation, and lateral ventricular volume. Additionally, minocycline enhanced cortical thickness after injury. All of these neuroprotective effects of minocycline were prevented by AM630. A cannabinoid CB2 agonist (JWH133) was used to strengthen the hypothesis, which showed the identical neuroprotective effects of minocycline. Our study demonstrates, for the first time, that minocycline attenuates neuroinflammation and brain injury in a rat model of GMH, and activation of CBR2 was partially involved in these processes.

  11. Photo-induced anisotropy in ZnO/PVA nanocomposites prepared by modified electrochemical method in PMA matrix

    NASA Astrophysics Data System (ADS)

    Yanchuk, O. M.; Ebothé, J.; El-Naggar, A. M.; Albassam, A.; Tsurkova, L. V.; Marchuk, O. V.; Lakshminarayana, G.; Tkaczyk, S.; Kityk, I. V.; Fedorchuk, A. O.; Vykhryst, O. M.; Urubkov, I. V.

    2017-02-01

    Substantial photo-induced optical anisotropy was discovered in ZnO/PVA nanocomposites under the influence of external bicolor laser illumination. Zinc oxide nanoparticles were synthesized by electrolysis of a sodium chloride aqueous medium including poly-methacrylic acid (PMA) in a cell system having a soluble zinc anode. The structural analysis of the ZnO powder samples has been carried out by X-Ray Diffractometry (XRD) and Scanning Electron Microscopy (SEM). The polyvinyl alcohol (PVA) embedded ZnO films obtained from the powder samples possess larger grain sizes than those in powder form. The films were prepared from the same polymer matrix but elaborated with two different PVA contents which are respectively 15% and 30%. The photoinduced anisotropy was identified by using two bicolor Er: glass laser beams incident at different angles. Substantial influence of the technological processes on the embedded nanoparticle sizes and related birefringence was explored. The process of laser induced anisotropy shows an occurrence of birefringence saturation.

  12. The ginsenoside derivative 20(S)-protopanaxadiol inhibits solar ultraviolet light-induced matrix metalloproteinase-1 expression.

    PubMed

    Han, Eungmin; Lim, Tae-Gyu; Kim, Jong-Eun; Yang, Hee; Oh, Deok-Kun; Yoon Park, Jung Han; Yoon Park, Dalius; Kim, Hee Jung; Rhee, Young Kyoung; Lee, Ki Won

    2017-04-05

    Ginsenosides are major pharmacologically active compounds present in ginseng (Panax ginseng). Among the ginsenosides, 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (GPPD) and ginsenoside Rb1 (Rb1) have previously been reported to exhibit anti-wrinkle effects. In this study, 20(S)-protopanaxadiol (20(S)-PPD), an aglycone derivative of the Rb1 metabolite was investigated for its anti-wrinkle benefit and compared to GPPD and Rb1. The anti-wrinkle effect of 20(S)-PPD during solar UV light was investigated using a human skin equivalent model and human keratinocytes. 20(S)-PPD attenuated solar UV-induced matrix metalloproteinase (MMP)-1 expression to a greater extent than GPPD and Rb1. 20(S)-PPD treatment modulated MMP-1 mRNA expression and the transcriptional activity of activator protein (AP)-1, a major transcription factor of MMP-1. Two upstream signaling pathways for AP-1, the MEK1/2-ERK1/2-p90(RSK) and MEK3/6-p38 pathways, were also suppressed. Taken together, these findings highlight the potential of 20(S)-PPD for further development as a preventative agent for sunlight-induced skin wrinkle. This article is protected by copyright. All rights reserved.

  13. Toxicity induced enhanced extracellular matrix production in osteoblastic cells cultured on single-walled carbon nanotube networks

    NASA Astrophysics Data System (ADS)

    Tutak, Wojtek; Park, Ki Ho; Vasilov, Anatoly; Starovoytov, Valentin; Fanchini, Giovanni; Cai, Shi-Qing; Partridge, Nicola C.; Sesti, Federico; Chhowalla, Manish

    2009-06-01

    A central effort in biomedical research concerns the development of materials for sustaining and controlling cell growth. Carbon nanotube based substrates have been shown to support the growth of different kinds of cells (Hu et al 2004 Nano Lett. 4 507-11 Kalbacova et al 2006 Phys. Status Solidi b 13 243; Zanello et al 2006 Nano Lett. 6 562-7) however the underlying molecular mechanisms remain poorly defined. To address the fundamental question of mechanisms by which nanotubes promote bone mitosis and histogenesis, primary calvariae osteoblastic cells were grown on single-walled carbon nanotube thin film (SWNT) substrates. Using a combination of biochemical and optical techniques we demonstrate here that SWNT networks promote cell development through two distinct steps. Initially, SWNTs are absorbed in a process that resembles endocytosis, inducing acute toxicity. Nanotube-mediated cell destruction, however, induces a release of endogenous factors that act to boost the activity of the surviving cells by stimulating the synthesis of extracellular matrix.

  14. Optical absorption, induced bleaching, and photoluminescence of CdSe nanoplatelets grown in cadmium octanoate matrix.

    PubMed

    Lyashchova, Alina; Dmytruk, Andriy; Dmitruk, Igor; Klimusheva, Gertruda; Mirnaya, Tetyana; Asaula, Vitaliy

    2014-02-20

    CdSe nanoparticles (NPs) are chemically synthesized in thermotropic ionic liquid crystalline (LC) phase of cadmium octanoate that was used as a nanoreactor. The nanocomposite samples are obtained by the rapid cooling of the LC phase to room temperature. Observed doublet structure in absorption spectra of the nanocomposites is characteristic for the two-dimensional CdSe nanoplatelets (NPLs). The thicknesses of the CdSe NPLs are 1.6, 1.9 and 2.3 nm as determined from the absorption spectra, and correspond to 4, 5 and 6 CdSe monolayers, respectively. Induced simultaneous bleaching of the doublet components observed under femtosecond laser excitation, as well as photoluminescence spectra and their kinetics are found compatible with the model of excitons with heavy- and light-hole valence bands confined in nanoplatelets.

  15. Optical absorption, induced bleaching, and photoluminescence of CdSe nanoplatelets grown in cadmium octanoate matrix

    NASA Astrophysics Data System (ADS)

    Lyashchova, Alina; Dmytruk, Andriy; Dmitruk, Igor; Klimusheva, Gertruda; Mirnaya, Tetyana; Asaula, Vitaliy

    2014-02-01

    CdSe nanoparticles (NPs) are chemically synthesized in thermotropic ionic liquid crystalline (LC) phase of cadmium octanoate that was used as a nanoreactor. The nanocomposite samples are obtained by the rapid cooling of the LC phase to room temperature. Observed doublet structure in absorption spectra of the nanocomposites is characteristic for the two-dimensional CdSe nanoplatelets (NPLs). The thicknesses of the CdSe NPLs are 1.6, 1.9 and 2.3 nm as determined from the absorption spectra, and correspond to 4, 5 and 6 CdSe monolayers, respectively. Induced simultaneous bleaching of the doublet components observed under femtosecond laser excitation, as well as photoluminescence spectra and their kinetics are found compatible with the model of excitons with heavy- and light-hole valence bands confined in nanoplatelets.

  16. Radiation-induced transformations of methanol molecules in low-temperature solids: a matrix isolation study.

    PubMed

    Saenko, Elizaveta V; Feldman, Vladimir I

    2016-11-30

    The effect of X-ray irradiation on methanol molecules (CH3OH, CD3OH, and (13)CH3OH) isolated in solid noble gas matrices (Ne, Ar, Kr, and Xe) was studied by FTIR spectroscopy at 6 K. CH2OH˙, H2CO, HCO˙ and CO were found to be the main degradation products. Somewhat unexpectedly, the production of CO is quite prominent, even at low doses, and it strongly predominates in low-polarizable matrices (especially, in neon). This result is explained by inefficient quenching of excess energy in the H2CO molecules initially generated from methanol. Relatively small amounts of CH4, CH3˙ and CO2 were also observed directly after irradiation. The latter species presumably originates from methanol dimers or methanol-water complexes. The mechanisms of radiolysis and annealing-induced reactions are discussed and possible implications for the astrochemically relevant ices are considered.

  17. Transcriptional regulation of cuticle biosynthesis.

    PubMed

    Borisjuk, Nikolai; Hrmova, Maria; Lopato, Sergiy

    2014-01-01

    Plant cuticle is the hydrophobic protection layer that covers aerial plant organs and plays a pivotal role during plant development and interactions of plants with the environment. The mechanical structure and chemical composition of cuticle lipids and other secondary metabolites vary considerably between plant species, and in response to environmental stimuli and stresses. As the cuticle plays an important role in responses of plants to major abiotic stresses such as drought and high salinity, close attention has been paid to molecular processes underlying the stress-induced biosynthesis of cuticle components. This review addresses the genetic networks responsible for cuticle formation and in particular highlights the role of transcription factors that regulate cuticle formation in response to abiotic stresses.

  18. UV-A light induces anthocyanin biosynthesis in a manner distinct from synergistic blue + UV-B light and UV-A/blue light responses in different parts of the hypocotyls in turnip seedlings.

    PubMed

    Wang, Yu; Zhou, Bo; Sun, Mei; Li, Yuhua; Kawabata, Saneyuki

    2012-08-01

    The effects of irradiating blue, UV-A, UV-B and a combination of the lights on anthocyanin accumulation at different hypocotyl positions were investigated in seedlings of the purple top turnip 'Tsuda'. The location of anthocyanin accumulation varied depending on different light spectra. Stronger accumulation of anthocyanin was induced (i) at the upper hypocotyl positions by blue light; (ii) mainly at the upper position, but also at the middle position by UV-B light; and (iii) at the upper to lower position by UV-A light. There were synergistic effects between blue and UV-B, while such effects were not observed for the other light combinations. Among the six chalcone synthase (CHS) genes identified in the 'Tsuda' turnip, BrCHS1, 4 and 5 exhibited light-dependent expression patterns, while the other three showed no apparent light responses. The expression of BrCHS1, 4 and 5 was increased particularly by UV-A and blue + UV-B irradiation at the middle to lower hypocotyl positions, in accordance with anthocyanin accumulation patterns. The highest induction of gene expression was observed for BrCHS4 upon blue + UV-B co-irradiation. In contrast, CHS expression was induced only slightly at higher hypocotyl positions by blue light. The R2R3-type MYB transcription factor genes PAP1, MYB4, MYB12 and MYB111 exhibited differential expression patterns at different hypocotyl positions; these patterns were unique for different light spectra. These unique anthocyanin accumulation patterns and gene expression profiles depending on hypocotyl positions and light sources demonstrate that there is a distinct UV-A response, blue + UV-B synergistic response and blue/UV-A light response for anthocyanin biosynthesis in turnip. UV-A light-dependent anthocyanin biosynthesis appeared to be regulated in a manner that is distinct from that mediated by cryptochromes and UV-B photoreceptors.

  19. Modulation of corneal and stromal matrix metalloproteinase by the mannose-induced Acanthamoeba cytolytic protein

    PubMed Central

    Alizadeh, Hassan; Li, Haochuan; Neelam, Sudha; Niederkorn, Jerry Y.

    2008-01-01

    The involvement of the mannose-induced Acanthamoeba cytopathic protein (MIP-133) in tissue injury and activation of metalloproteinase of corneal and stromal cells was examined in vitro. Activation of MMP-1, MMP-2, MMP-3, and MMP-9 induced by MIP-133 on human corneal epithelial and stromal cell cultures was examined by reverse transcriptase polymerase chain reaction (RT-PCR), and ELISA. MMP-1, MMP-2, MMP-3, and MMP-9 mRNA were expressed in both cultured human corneal epithelial and stromal cells. When the epithelial cells were exposed to MIP-133 protein, the mRNA expression for MMP-1 and MMP-9 was unchanged. However, the transcript for MMP-2 and MMP-3 was decreased by two fold. By contrast, the expression of MMP-2 and MMP-3 was significantly up-regulated (2–4 fold) in the corneal stromal cells 1, 4, and 8 hours after MIP-133 stimulation. At the protein level, there was no significant difference in the level of MMPs between the corneal epithelial cells before and after stimulation with MIP-133. By contrast, the levels of MMP-2 and MMP-3 were significantly higher in the corneal stromal cells stimulated with MIP-133. The supernatants from corneal stromal cells stimulated with MIP-133 were incubated with PMSF and MIP-133 antibody and the level of MMP-2 was measured by ELISA. Activation of MMP-2 by MIP-133 was inhibited in the supernatants pretreated with the serine protease inhibitor, PMSF, and anti-MIP-133. Supernatants pretreated with the cysteine protease inhibitor E6 or control antibody produced the same amount of MMP-2 as the untreated supernatants. To verify the possible of homology between MMPs and A. castellanii proteases, the mRNA from A. castellanii was prepared and analyzed for the expression of MMP genes by PT-PCR. The results showed that A. castellanii did not express mRNA for MMP-1, MMP-2, MMP-3, or MMP-9. Thus, A. castellanii mRNA does not cross react with human MMPs. Furthermore, ELISA was used to determine the cross reactivity of MMP antibodies with

  20. Mechanical Strain Induced Expression of Matrix Metalloproteinase-9 via Stretch-Activated Channels in Rat Abdominal Aortic Dissection

    PubMed Central

    Qiu, ZhiHuang; Chen, LiangWan; Cao, Hua; Chen, Qiang; Peng, Hua

    2017-01-01

    Background The aim of the study was to investigate the expression of matrix metalloproteinase-9 (MMP-9) in rat abdominal aortic dissection (AD) induced by mechanical strain, so as to offer a better understanding of the possible mechanisms of AD. Material/Methods Experimental AD in rats was achieved by the injection of porcine pancreatic elastase. At days 0, 1, 3, 5, 7, 14, 21, and 30 after the establishment of AD model, serum MMP-9 levels were measured by enzyme-linked immunosorbent assay (ELISA). Four groups of vascular rings were stretched in vitro with a mechanical strength of 0 g, 1 g, 3 g, or 5 g for 30 min. Another four groups were pretreated with GdCl3, streptomycin, SN50, and SN50M, followed by stretching with 3 g for 30 min. The messenger RNA and the protein of MMP-9 were analyzed by real-time RT-PCR and Western blotting, and NF-κB p65 was detected by ELISA. Results After the establishment of rat abdominal AD model, the serum MMP-9 levels of AD groups increased significantly. The results showed increased expression of MMP-9 in rat AD vessels stretched with mechanical strength of 1 g, 3 g, and 5 g, but this effect was mostly blocked by Gd Cl3 and streptomycin. The NF-κB activity in aortic rings was activated by stretching with a mechanical strength of 3 g and was blocked by SN50, but not by SN50M. Conclusions The expression of MMP-9 in serum was increased significantly after rat abdominal AD formation. Mechanical strain induced MMP-9 expression in AD vessels, which was mediated through the activation of the stretch-activated channel-induced NF-κB pathway. PMID:28286334

  1. 2-Chloroethanol Induced Upregulation of Matrix Metalloproteinase-2 in Primary Cultured Rat Astrocytes Via MAPK Signal Pathways

    PubMed Central

    Sun, Qi; Liao, Yingjun; Wang, Tong; Tang, Hongge; Wang, Gaoyang; Zhao, Fenghong; Jin, Yaping

    2017-01-01

    This study was to explore the mechanisms underlying 1,2-dichloroethane (1,2-DCE) induced brain edema by focusing on alteration of matrix metalloproteinase-2 (MMP-2) in rat astrocytes induced by 2-chloroethanol (2-CE), an intermediate metabolite of 1,2-DCE in vivo. Protein and mRNA levels of MMP-2, and the phosphorylated protein levels of p38 MAPK (p-p38), extracellular signal regulated protein kinase (p-ERK1/2) and c-Jun N-terminal kinase (p-JNK1/2) in astrocytes were examined by immunostaining, western blot or real-time RT-PCR analysis. Findings from this study disclosed that protein levels of MMP-2 were upregulated by 2-CE in astrocytes. Meanwhile, protein levels of p-p38, p-ERK1/2 and p-JNK1/2 were also increased apparently in the cells treated with 2-CE. Moreover, pretreatment of astrocytes with SB202190 (inhibitor of p38 MAPK), U0126 (inhibitor of ERK1/2) or SP600125 (inhibitor of JNK1/2) could suppress the upregulated expression of p-p38, p-ERK1/2, and p-JNK1/2. In response to suppressed protein levels of p-p38 and p-JNK1/2, the protein levels of MMP-2 also decreased significantly, indicating that activation of MAPK signal pathways were involved in the mechanisms underlying 2-CE-induced upregulation of MMP-2 expression. PMID:28101000

  2. Effect of uric acid on gentamicin-induced nephrotoxicity in rats - role of matrix metalloproteinases 2 and 9.

    PubMed

    Romero, Freddy; Pérez, Mariela; Chávez, Maribel; Parra, Gustavo; Durante, Paula

    2009-12-01

    In this work, we aimed to study the effect of uric acid on gentamicin-induced nephrotoxicity. Male Sprague-Dawley rats were assigned to one of six groups (six rats each) which received intraperitoneal injections for 9 days: (S) saline; (UA) Uric acid alone; (G) Gentamicin alone; (G + UA) Gentamicin + uric acid; (G rec) Gentamicin recovery and (G + UA rec) Gentamicin + uric acid recovery. In (G rec) and (G + UA rec), rats recovered for 7 days after the last injection. Urine and blood samples were taken on day 0 and at the end of every stage. Kidneys were harvested for histological scoring, determination of renal malondialdehyde (MDA), zymography and western blots for matrix metalloprotease (MMP)-2 and MMP-9. Uric acid alone did not provoke changes in biochemical and histological parameters when compared to controls. Gentamicin alone increased significantly plasma creatinine and blood urea nitrogen and caused a moderate histological damage. When combined with uric acid, these conditions worsened. MMP-9 activity and expression was decreased in rats from group G + UA as compared with rats from group G, while activity of MMP-2 was similarly increased in both groups when compared to controls. The increase in renal MDA induced by gentamicin was not altered when it was combined with uric acid. During the recovery stage, all biochemical parameters returned to normal levels, though a trend for delay of tubular damage recovery was observed in group G + UA rec when compared with group G rec. The results indicate that uric acid worsens gentamicin-induced nephrotoxicity. The mechanism is likely to implicate down-regulation of MMP-9.

  3. The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates

    PubMed Central

    Knoblauch, Jan; Peters, Winfried S.; Knoblauch, Michael

    2016-01-01

    Background and Aims In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system. Methods The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy. Key Results Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates. Conclusions The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks. PMID:26929203

  4. [The role of fetus decalcified bone matrix (FDBM) in inducing pure titanium-bone implant integration].

    PubMed

    Zou, L; Zhang, D; Wang, W

    1998-05-01

    Because of its high biological compatibility, titanium has been a good biomaterial. The implanted artificial bone made from titanium can contact with the vital and mature osseous tissue directly within 3-6 months, the so-called osteointergration. In order to promote the process of osteointergration, FDBM of rabbit was prepared and was combined with pure titanium so as to speed up osteointergration. The study focused on bone density, bone intergration rate, new bone growth rate around the pure titanium, and the Ca2+ and PO(4)3- density of titanium-bone interface. A control group of pure titanium inplant without FDBM was set up. The results showed FDBM had no antigenicity. It could induce and speed up the new bone formation at titanium-bone interface. The titanium-bone intergration time was within 2 months. It was suggested that there were more bone morphogenesis protein (BMP) or other bone induction and bone formation factors in brephobone than that in child and adult bone. As a kind of bone induction material, FDBM was easy prepared, cheap in price, easy to storage, no antigenicity and obvious bone-inductive function.

  5. Acute morphine induces matrix metalloproteinase-9 up-regulation in primary sensory neurons to mask opioid-induced analgesia in mice

    PubMed Central

    2012-01-01

    Background Despite decades of intense research efforts, actions of acute opioids are not fully understood. Increasing evidence suggests that in addition to well-documented antinociceptive effects opioids also produce paradoxical hyperalgesic and excitatory effects on neurons. However, most studies focus on the pronociceptive actions of chronic opioid exposure. Matrix metalloproteinase 9 (MMP-9) plays an important role in neuroinflammation and neuropathic pain development. We examined MMP-9 expression and localization in dorsal root ganglia (DRGs) after acute morphine treatment and, furthermore, the role of MMP-9 in modulating acute morphine-induced analgesia and hyperalgesia in mice. Results Subcutaneous morphine induced a marked up-regulation of MMP-9 protein in DRGs but not spinal cords. Morphine also increased MMP-9 activity and mRNA expression in DRGs. MMP-9 up-regulation peaked at 2 h but returned to the baseline after 24 h. In DRG tissue sections, MMP-9 is expressed in small and medium-sized neurons that co-express mu opioid receptors (MOR). In DRG cultures, MOR agonists morphine, DAMGO, and remifentanil each increased MMP-9 expression in neurons, whereas the opioid receptor antagonist naloxone and the MOR-selective antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) suppressed morphine-induced MMP-9 expression. Notably, subcutaneous morphine-induced analgesia was enhanced and prolonged in Mmp9 knockout mice and also potentiated in wild-type mice receiving intrathecal injection of MMP-9 inhibitors. Consistently, intrathecal injection of specific siRNA targeting MMP-9 reduced MMP-9 expression in DRGs and enhanced and prolonged morphine analgesia. Subcutaneous morphine also produced heat hyperalgesia at 24 h, but this opioid-induced hyperalgesia was not enhanced after MMP-9 deletion or inhibition. Conclusions Transient MMP-9 up-regulation in DRG neurons can mask opioid analgesia, without modulating opioid-induced hyperalgesia. Distinct molecular

  6. Hydrogen isotope detection in metal matrix using double-pulse laser-induced breakdown-spectroscopy

    NASA Astrophysics Data System (ADS)

    Fantoni, Roberta; Almaviva, Salvatore; Caneve, Luisa; Colao, Francesco; Maddaluno, Giorgio; Gasior, Pawel; Kubkowska, Monika

    2017-03-01

    The amount of hydrogen isotopes retained in plasma facing components (PFCs) and the determination of their surface layer composition are among the most critical issues for the next generation fusion device, ITER, under construction in Cadarache (France). Laser Induced Breakdown Spectroscopy (LIBS) is currently under evaluation as a technique suitable for quantitative, in situ, non-invasive measurements of these quantities. In order to detect traces of contaminant in metallic samples and improve its limit of detection (LOD), the Double Pulse LIBS (DP-LIBS) variant can be used instead of the standard Single Pulse LIBS (SP-LIBS), as it has been proven by several authors that DP-LIBS can considerably raise the analytical performances of the technique. In this work Mo samples coated with a 1.5-1.8 μm thick W-Al mixed layer, contaminated with co-deposited deuterium (D) were measured by SP- and DP-LIBS under vacuum (p 5 × 10- 5 mbar), with an experimental set-up simulating conditions that can be found in a real fusion device between plasma discharges. A partial Calibration Free procedure (pCF) was applied to the LIBS data in order to retrieve the relative concentration of W and Al in the mixed layer. The amount of deuterium was then inferred by using tungsten as internal standard, accounting for the intensity ratio between the Dα line and nearby W I lines. The results are in satisfactory agreement with those obtained from preliminary Ion Beam Analysis measurements performed immediately after the specimen's realization.

  7. BIOSYNTHESIS OF YEAST CAROTENOIDS

    PubMed Central

    Simpson, Kenneth L.; Nakayama, T. O. M.; Chichester, C. O.

    1964-01-01

    Simpson, Kenneth L. (University of California, Davis), T. O. M. Nakayama, and C. O. Chichester. Biosynthesis of yeast carotenoids. J. Bacteriol. 88:1688–1694. 1964.—The biosynthesis of carotenoids was followed in Rhodotorula glutinis and in a new strain, 62-506. The treatment of the growing cultures by methylheptenone, or ionone, vapors permitted observations of the intermediates in the biosynthetic pathway. On the basis of concentration changes and accumulation in blocked pathways, the sequence of carotenoid formation is postulated as phytoene, phytofluene, ζ-carotene, neurosporene, β-zeacarotene, γ-carotene, torulin, a C40 aldehyde, and torularhodin. Torulin and torularhodin were established as the main carotenoids of 62-506. PMID:14240958

  8. Histidine biosynthesis in plants.

    PubMed

    Stepansky, A; Leustek, T

    2006-03-01

    The study of histidine metabolism has never been at the forefront of interest in plant systems despite the significant role that the analysis of this pathway has played in development of the field of molecular genetics in microbes. With the advent of methods to analyze plant gene function by complementation of microbial auxotrophic mutants and the complete analysis of plant genome sequences, strides have been made in deciphering the histidine pathway in plants. The studies point to a complex evolutionary origin of genes for histidine biosynthesis. Gene regulation studies have indicated novel regulatory networks involving histidine. In addition, physiological studies have indicated novel functions for histidine in plants as chelators and transporters of metal ions. Recent investigations have revealed intriguing connections of histidine in plant reproduction. The exciting new information suggests that the study of plant histidine biosynthesis has finally begun to flower.

  9. Relaxin induces an extracellular matrix-degrading phenotype in human lung fibroblasts in vitro and inhibits lung fibrosis in a murine model in vivo.

    PubMed Central

    Unemori, E N; Pickford, L B; Salles, A L; Piercy, C E; Grove, B H; Erikson, M E; Amento, E P

    1996-01-01

    Pulmonary fibrosis is the common end stage of a number of pneumopathies. In this study, we examined the ability of the human cytokine, relaxin, to block extracellular matrix deposition by human lung fibroblasts in vitro, and to inhibit lung fibrosis in a bleomycin-induced murine model. In vitro, relaxin (1-100 ng/ml) inhibited the transforming growth factor-beta-mediated over-expression of interstitial collagen types I and III by human lung fibroblasts by up to 45% in a dose-dependent manner. Relaxin did not affect basal levels of collagen expression in the absence of TGF-beta-induced stimulation. Relaxin also blocked transforming growth factor-beta-induced upregulation of fibronectin by 80% at the highest relaxin dose tested (100 ng/ml). The expression of matrix metalloproteinase-1, or procollagenase, was stimulated in a biphasic, dose-dependent manner by relaxin. In vivo, relaxin, at a steady state circulating concentration of approximately 50 ng/ml, inhibited bleomycin-mediated alveolar thickening compared with the vehicle only control group (P < 0.05). Relaxin also restored bleomycin-induced collagen accumulation, as measured by lung hydroxyproline content, to normal levels (P < 0.05). In summary, relaxin induced a matrix degradative phenotype in human lung fibroblasts in vitro and inhibited bleomycin-induced fibrosis in a murine model in vivo. These data indicate that relaxin may be efficacious in the treatment of pathologies characterized by lung fibrosis. PMID:8981919

  10. Biosynthesis of Hemes.

    PubMed

    Beale, Samuel I

    2007-04-01

    This review is concerned specifically with the structures and biosynthesis of hemes in E. coli and serovar Typhimurium. However, inasmuch as all tetrapyrroles share a common biosynthetic pathway, much of the material covered here is applicable to tetrapyrrole biosynthesis in other organisms. Conversely, much of the available information about tetrapyrrole biosynthesis has been gained from studies of other organisms, such as plants, algae, cyanobacteria, and anoxygenic phototrophs, which synthesize large quantities of these compounds. This information is applicable to E. coli and serovar Typhimurium. Hemes play important roles as enzyme prosthetic groups in mineral nutrition, redox metabolism, and gas-and redox-modulated signal transduction. The biosynthetic steps from the earliest universal precursor, 5-aminolevulinic acid (ALA), to protoporphyrin IX-based hemes constitute the major, common portion of the pathway, and other steps leading to specific groups of products can be considered branches off the main axis. Porphobilinogen (PBG) synthase (PBGS; also known as ALA dehydratase) catalyzes the asymmetric condensation of two ALA molecules to form PBG, with the release of two molecules of H2O. Protoporphyrinogen IX oxidase (PPX) catalyzes the removal of six electrons from the tetrapyrrole macrocycle to form protoporphyrin IX in the last biosynthetic step that is common to hemes and chlorophylls. Several lines of evidence converge to support a regulatory model in which the cellular level of available or free protoheme controls the rate of heme synthesis at the level of the first step unique to heme synthesis, the formation of GSA by the action of GTR.

  11. Basic fibroblast growth factor as a selective inducer of matrix Gla protein gene expression in proliferative chondrocytes.

    PubMed Central

    Stheneur, Chantal; Dumontier, Marie-France; Guedes, Claudie; Fulchignoni-Lataud, Marie-Claude; Tahiri, Khadija; Karsenty, Gerard; Corvol, Marie Thérèse

    2003-01-01

    Matrix Gla protein (MGP) is a member of the vitamin K-dependent gamma carboxylase protein family expressed in cartilage. Insulin-like growth factor I (IGF1) stimulates chondrocyte differentiation, whereas basic fibroblast growth factor (FGF2) acts in an opposite manner. We explored the differential expression and regulation by IGF1 and FGF2 of the MGP gene during chondrocyte differentiation. We used a primary culture system of rabbit epiphyseal chondrocytes to show that MGP mRNA is mainly expressed during serum-induced proliferation. Much lower MGP mRNA content is observed in post-mitotic chondrocytes, which newly express alpha 1X procollagen mRNA, a marker of late-differentiated cells. From studies of a series of growth factors, it was shown that IGF1 decreased chondrocyte MGP transcripts, whereas FGF2 had the opposite effect. FGF2 stimulated chondrocyte MGP production in a dose- and time-dependent manner at the mRNA and protein levels. FGF2 acted in a dose- and time-dependent manner, reaching a maximum at 10 ng/ml at 20 h. The protein synthesis inhibitor cycloheximide did not modify FGF2 action, in agreement with a direct effect. Actinomycin D abolished FGF2-induced stimulation, strongly suggesting that FGF2 modulated MGP gene transcription. We transiently transfected chondrocytes with a construct containing the mouse MGP promoter from -5000 to -168 base pairs, relative to the transcription start site of the gene linked to the luciferase gene (MGP-Luc). In transfected cells, FGF2 stimulated luciferase activity up to sevenfold while IGF1 had no effect. Hence, FGF2 induces transcription of the MGP gene via the 5'-flanking region of the gene. Using a series of deleted MGP-Luc constructs, we identified a sequence of 748 base pairs which was sufficient for transcriptional activation by FGF2. These results led us to postulate that the inhibitory chondrogenic action of FGF2 involves a mechanism whereby MGP gene transcription and protein are induced. PMID:12230429

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

    PubMed

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

    2017-03-07

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

  13. Keratocytes are induced to produce collagen type II: A new strategy for in vivo corneal matrix regeneration.

    PubMed

    Greene, Carol Ann; Green, Colin R; Dickinson, Michelle E; Johnson, Virginia; Sherwin, Trevor

    2016-09-10

    The stroma, the middle layer of the cornea, is a connective tissue making up most of the corneal thickness. The stromal extracellular matrix (ECM) consists of highly organised lamellae which are made up of tightly packed fibrils primarily composed of collagens type I and V. This layer is interspersed with keratocytes, mesenchymal cells of neural crest origin. We have previously shown that adult corneal keratocytes exhibit phenotypic plasticity and can be induced into a neuronal phenotype. In the current study we evaluated the potential of keratocytes to produce collagen type II via phenotypic reprogramming with exogenous chondrogenic factors. The cornea presents a challenge to tissue engineers owing to its high level of organisation and the phenotypic instability of keratocytes. Traditional approaches based on a scar model do not support the engineering of functional stromal tissue. Type II collagen is not found in the adult cornea but is reported to be expressed during corneal development, raising the possibility of using such an approach to regenerate the corneal ECM. Keratocytes in culture and within intact normal and diseased tissue were induced to produce collagen type II upon treatment with transforming growth factor Beta3 (TGFβ3) and dexamethasone. In vivo treatment of rat corneas also resulted in collagen type II deposition and a threefold increase in corneal hardness and elasticity. Furthermore, the treatment of corneas and subsequent deposition of collagen type II did not cause opacity, fibrosis or scarring. The induction of keratocytes with specific exogenous factors and resulting deposition of type II collagen in the stroma can potentially be controlled by withdrawal of the factors. This might be a promising new approach for in vivo corneal regeneration strategies aimed at increasing corneal integrity in diseases associated with weakened ectatic corneal tissue such as keratoconus.

  14. Synapse loss regulated by matrix metalloproteinases in traumatic brain injury is associated with hypoxia inducible factor-1alpha expression.

    PubMed

    Ding, Jamie Y; Kreipke, Christian W; Schafer, Patrick; Schafer, Steven; Speirs, Susan L; Rafols, José A

    2009-05-01

    The present study assessed the role of matrix metalloproteinase-2 (MMP-2) and -9 in synapse loss after traumatic brain injury (TBI) and the role of hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factor up-regulated during hypoxia, in the regulation of MMP-2 and -9 expression post-TBI. Adult male Sprague-Dawley rats (n=6 per group, 400 g-425 g) were injured using Marmarou's closed-head acceleration impact model and allowed to survive for 1, 4, 24 and 48 h. In another set of experiments, 30 min after TBI, animals were treated with Minocycline (inhibitor of MMPs), or 2-Methoxyestradiol (2ME2, inhibitor of HIF-1alpha) and sacrificed at 4 h after injury. Relative amounts of synaptophysin, a presynaptic vesicular protein, HIF-1alpha, as well as MMP-2 and -9 were assessed by real-time PCR and Western blotting. Activity levels of MMP-2 and -9 were determined by zymography. Synaptophysin expression was significantly (p<0.05) decreased at 1 h through 48 h after TBI. A significant increase in gene and protein expressions of HIF-1alpha, MMP-2 and -9, as well as enzyme activity of MMP-2 and -9 at the same time points was also detected. Inhibition of either MMPs or HIF-1alpha significantly reversed the TBI-induced decrease in synaptophysin. Inhibition of HIF-1alpha reduced expression of MMP-2 and -9. This study showed an early detection of a correlation between synaptic loss and MMP expression after TBI. The data also supports a role for HIF-1alpha in the MMP regulatory cascade in synapse loss after TBI, suggesting potential targets for reducing loss of synaptic terminals.

  15. Synapse Loss Regulated by Matrix Metalloproteinases in Traumatic Brain Injury Is Associated with Hypoxia-Inducible Factor-1α Expression

    PubMed Central

    Ding, Jamie Y.; Kreipke, Christian W.; Schafer, Patrick; Schafer, Steven; Speirs, Susan L.; Rafols, José A.

    2009-01-01

    The present study assessed the role of matrix metalloproteinase-2 (MMP-2) and -9 in synapse loss after traumatic brain injury (TBI) and the role of hypoxia inducible factor-1α (HIF-1α a transcription factor upregulated during hypoxia, in the regulation of MMP-2 and -9 expression post TBI. Adult male Sprague-Dawley rats (n=6 per group, 400g-425g) were injured using Marmarou's closed head acceleration impact model and allowed to survive for 1, 4, 24 and 48 hours. In another set of experiments, 30 minutes after TBI, animals were treated with Minocycline (inhibitor of MMPs), or 2-Methoxyestradiol (2ME2, inhibitor of HIF-1α) and sacrificed at 4 hours after injury. Relative amounts of synaptophysin, a presynaptic vesicular protein, HIF-1α, as well as MMP-2 and -9 were assessed by real-time PCR and Western blotting. Activity levels of MMP-2 and -9 were determined by zymography. Synaptophysin expression was significantly (p<0.05) decreased at 1 hour through 48 hours after TBI. A significant increase in gene and protein expressions of HIF-1α, MMP-2 and -9, as well as enzyme activity of MMP-2 and -9 at the same time points was also detected. Inhibition of either MMPs or HIF-1α significantly reversed the TBI-induced decrease in synaptophysin. Inhibition of HIF-1α reduced expression of MMP-2 and -9. This study showed an early detection of a correlation between synaptic loss and MMP expression after TBI. The data also supports a role for HIF-1α in the MMP regulatory cascade in synapse loss after TBI, suggesting potential targets for reducing loss of synaptic terminals. PMID:19285046

  16. Fetal fibronectin signaling induces matrix metalloproteases and cyclooxygenase-2 (COX-2) in amnion cells and preterm birth in mice.

    PubMed

    Mogami, Haruta; Kishore, Annavarapu Hari; Shi, Haolin; Keller, Patrick W; Akgul, Yucel; Word, R Ann

    2013-01-18

    Fetal fibronectin (fFN) in cervical and vaginal secretions has been used as a predictor of preterm delivery. Here, we clarified the pathological function of fFN on cell type-specific matrix metalloproteinases (MMPs) and prostaglandin synthesis in fetal membranes. Treatment of amnion mesenchymal cells with fFN resulted in dramatic increases in MMP-1 and MMP-9 mRNA and enzymatic activity as well as COX-2 mRNA and prostaglandin E(2) synthesis, activating both NFκB and ERK1/2 signaling. Fetal FN-induced increases in MMPs and COX-2 were mediated through its extra domain A and Toll-like receptor 4 expressed in mesenchymal cells. Lipopolysaccharide and TNF-α increased the release of free FN in medium of amnion epithelial cells in culture. Finally, injection of fFN in pregnant mice resulted in preterm birth. Collectively, these results indicate that fFN is not only a marker of preterm delivery but also plays a significant role in the pathogenesis of preterm labor and premature rupture of fetal membranes.

  17. Matrix Metalloproteinases Regulate the Formation of Dendritic Spine Head Protrusions during Chemically Induced Long-Term Potentiation

    PubMed Central

    Szepesi, Zsuzsanna; Bijata, Monika; Ruszczycki, Blazej; Kaczmarek, Leszek; Wlodarczyk, Jakub

    2013-01-01

    Dendritic spines are are small membranous protrusions that extend from neuronal dendrites and harbor the majority of excitatory synapses. Increasing evidence has shown that matrix metalloproteinases (MMPs), a family of extracellularly acting and Zn2+-dependent endopeptidases, are able to rapidly modulate dendritic spine morphology. Spine head protrusions (SHPs) are filopodia-like processes that extend from the dendritic spine head, representing a form of postsynaptic structural remodeling in response to altered neuronal activity. Herein, we show that chemically induced long-term potentiation (cLTP) in dissociated hippocampal cultures upregulates MMP-9 activity that controls the formation of SHPs. Blocking of MMPs activity or microtubule dynamics abolishes the emergence of SHPs. In addition, autoactive recombinant MMP-9, promotes the formation of SHPs in organotypic hippocampal slices. Furthermore, spines with SHPs gained postsynaptic α-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) receptors upon cLTP and the synaptic delivery of AMPA receptors was controlled by MMPs. The present results strongly imply that MMP-9 is functionally involved in the formation of SHPs and the control of postsynaptic receptor distribution upon cLTP. PMID:23696812

  18. Shotgun proteomics implicates extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation

    PubMed Central

    Moore, Nadia H.; Costa, Lucio G.; Shaffer, Scott A; Goodlett, David R.; Guizzetti, Marina

    2009-01-01

    Astrocytes play an important role in neuronal development through the release of soluble factors that affect neuronal maturation. Shotgun proteomics followed by Gene Ontology analysis was used in this study to identify proteins present in the conditioned medium of primary rat astrocytes. 133 secreted proteins were identified, the majority of which were never before reported to be produced by astrocytes. Extracellular proteins were classified based on their biological and molecular functions; most of the identified proteins were involved in neuronal development. Semi-quantitative proteomic analysis was carried out to identify changes in the levels of proteins released by astrocytes after stimulation with the cholinergic agonist carbachol, as we have previously reported that carbachol-treated astrocytes elicit neuritogenesis in hippocampal neurons through the release of soluble factors. Carbachol up-regulated the secretion of 15 proteins and down-regulated the release of 17 proteins. Changes in the levels of four proteins involved in neuronal differentiation (thrombospondin-1, fibronectin, plasminogen activator inhibitor-1, and plasminogen activator urokinase) were verified by Western blot or ELISA. In conclusion, this study identified a large number of proteins involved in neuronal development in the astrocyte secretome and implicated extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation. PMID:19077055

  19. Shotgun proteomics implicates extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation.

    PubMed

    Moore, Nadia H; Costa, Lucio G; Shaffer, Scott A; Goodlett, David R; Guizzetti, Marina

    2009-02-01

    Astrocytes play an important role in neuronal development through the release of soluble factors that affect neuronal maturation. Shotgun proteomics followed by gene ontology analysis was used in this study to identify proteins present in the conditioned medium of primary rat astrocytes. One hundred and thirty three secreted proteins were identified, the majority of which were never before reported to be produced by astrocytes. Extracellular proteins were classified based on their biological and molecular functions; most of the identified proteins were involved in neuronal development. Semi-quantitative proteomic analysis was carried out to identify changes in the levels of proteins released by astrocytes after stimulation with the cholinergic agonist carbachol, as we have previously reported that carbachol-treated astrocytes elicit neuritogenesis in hippocampal neurons through the release of soluble factors. Carbachol up-regulated secretion of 15 proteins and down-regulated the release of 17 proteins. Changes in the levels of four proteins involved in neuronal differentiation (thrombospondin-1, fibronectin, plasminogen activator inhibitor-1, and plasminogen activator urokinase) were verified by western blot or ELISA. In conclusion, this study identified a large number of proteins involved in neuronal development in the astrocyte secretome and implicated extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation.

  20. Boswellia frereana (frankincense) suppresses cytokine-induced matrix metalloproteinase expression and production of pro-inflammatory molecules in articular cartilage.

    PubMed

    Blain, Emma J; Ali, Ahmed Y; Duance, Victor C

    2010-06-01

    The aim of this study was to assess the anti-inflammatory efficacy of Boswellia frereana extracts in an in vitro model of cartilage degeneration and determine its potential as a therapy for treating osteoarthritis. Cartilage degradation was induced in vitro by treating explants with 5 ng/ml interleukin1alpha (IL-1alpha) and 10 ng/ml oncostatin M (OSM) over a 28-day period, in the presence or absence of 100 microg/ml B. frereana. Treatment of IL-1alpha/OSM stimulated cartilage explants with B. frereana inhibited the breakdown of the collagenous matrix. B. frereana reduced MMP9 and MMP13 mRNA levels, inhibited MMP9 expression and activation, and significantly reduced the production of nitrite (stable end product of nitric oxide), prostaglandin E2 and cycloxygenase-2. Epi-lupeol was identified as the principal constituent of B. frereana. This is the first report on the novel anti-inflammatory properties of Boswellia frereana in an in vitro model of cartilage degradation. We have demonstrated that B. frereana prevents collagen degradation, and inhibits the production of pro-inflammatory mediators and MMPs. Due to its efficacy we propose that B. frereana should be examined further as a potential therapeutic agent for treating inflammatory symptoms associated with arthritis.

  1. Designing Solvent Exchange-Induced In Situ Forming Gel from Aqueous Insoluble Polymers as Matrix Base for Periodontitis Treatment.

    PubMed

    Srichan, Tharatree; Phaechamud, Thawatchai

    2017-01-01

    An in situ forming gel is a dosage form which is promised for site-specific therapy such as periodontal pocket of periodontitis treatment. Ethylcellulose, bleached shellac, and Eudragit RS were applied in this study as a polymeric matrix for in situ forming gel employing N-methyl pyrrolidone (NMP) as solvent. Solutions comprising ethylcellulose, bleached shellac, and Eudragit RS in NMP were evaluated for viscosity, rheology, and rate of water penetration. Ease of administration by injection was determined as the force required to expel polymeric solutions through a needle using texture analyzer. In vitro gel formation and in vitro gel degradation were conducted after injection into phosphate buffer solution pH 6.8. Ethylcellulose, bleached shellac, and Eudragit RS could form the in situ gel, in vitro. Gel viscosity and pH value depended on percentage amount of the polymer, whereas the water diffusion at early period likely relied on types of polymer. Furthermore, the solutions containing higher polymer concentration exhibited the lower degree of degradation. All the preparations were acceptable as injectable dosage forms because the applied force was lower than 50 N. All of them inhibited Staphylococcus aureus, Escherichia coli, Candida albicans, Streptococcus mutans, and Porphyrommonas gingivalis growth owing to antimicrobial activity of NMP which exhibited a potential use for periodontitis treatment. Moreover, the developed systems presented as the solvent exchange induced in situ forming gel and showed capability to be incorporated with the suitable antimicrobial active compounds for periodontitis treatment which should be further studied.

  2. Melatonin inhibits TPA-induced oral cancer cell migration by suppressing matrix metalloproteinase-9 activation through the histone acetylation

    PubMed Central

    Yeh, Chia-Ming; Lin, Chiao-Wen; Yang, Jia-Sin; Yang, Wei-En; Su, Shih-Chi; Yang, Shun-Fa

    2016-01-01

    Melatonin exerts antimetastatic effects on liver and breast cancer and also inhibits matrix metalloproteinase (MMP) activity. However, the detailed impacts and underlying mechanisms of melatonin on oral cancer cell metastasis are still unclear. This study showed that melatonin attenuated the 12-O-tetradecanoylphorbol-13-acetate-induced migration of oral cancer cell lines, HSC-3 and OECM-1. Zymography, quantitative real-time PCR, and Western blotting analyses revealed that melatonin lessened MMP-9 enzyme activity as well as the expression of MMP-9 mRNA and protein. Furthermore, melatonin suppressed the phosphorylation of the ERK1/2 signalling pathway, which dampened MMP-9 gene transcription by affecting the expression of transcriptional coactivators, such as CREB-binding protein (CREBBP) and E1A binding protein p300 (EP300), and decreasing histone acetylation in HSC-3 and OECM-1 cells. Examinations on clinical samples exhibited that MMP-9, CREBBP, and EP300 were significantly increased in oral cancer tissues. Moreover, the relative level of CREBBP was positively correlated with the expression of MMP-9 and EP300. In conclusion, we demonstrated that melatonin inhibits the motility of HSC-3 and OECM-1 cells in vitro through a molecular mechanism that involves attenuation of MMP-9 expression and activity mediated by decreased histone acetylation. PMID:26980735

  3. Hyperglycemia diverts dividing osteoblastic precursor cells to an adipogenic pathway and induces synthesis of a hyaluronan matrix that is adhesive for monocytes.

    PubMed

    Wang, Aimin; Midura, Ronald J; Vasanji, Amit; Wang, Andrew J; Hascall, Vincent C

    2014-04-18

    Isolated rat bone marrow stromal cells cultured in osteogenic medium in which the normal 5.6 mm glucose is changed to hyperglycemic 25.6 mm glucose greatly increase lipid formation between 21-31 days of culture that is associated with decreased biomineralization, up-regulate expression of cyclin D3 and two adipogenic markers (CCAAT/enhancer binding protein α and peroxisome proliferator-activated receptor γ) within 5 days of culture, increase neutral and polar lipid synthesis within 5 days of culture, and form a monocyte-adhesive hyaluronan matrix through an endoplasmic reticulum stress-induced autophagic mechanism. Evidence is also provided that, by 4 weeks after diabetes onset in the streptozotocin-induced diabetic rat model, there is a large loss of trabecular bone mineral density without apparent proportional changes in underlying collagen matrices, a large accumulation of a hyaluronan matrix within the trabecular bone marrow, and adipocytes and macrophages embedded in this hyaluronan matrix. These results support the hypothesis that hyperglycemia in bone marrow diverts dividing osteoblastic precursor cells (bone marrow stromal cells) to a metabolically stressed adipogenic pathway that induces synthesis of a hyaluronan matrix that recruits inflammatory cells and establishes a chronic inflammatory process that demineralizes trabecular cancellous bone.

  4. A Cannabinoid Receptor 2 Agonist Prevents Thrombin-Induced Blood-Brain Barrier Damage via the Inhibition of Microglial Activation and Matrix Metalloproteinase Expression in Rats.

    PubMed

    Li, Lin; Tao, Yihao; Tang, Jun; Chen, Qianwei; Yang, Yang; Feng, Zhou; Chen, Yujie; Yang, Liming; Yang, Yunfeng; Zhu, Gang; Feng, Hua; Chen, Zhi

    2015-12-01

    Thrombin mediates the life-threatening cerebral edema and blood-brain barrier (BBB) damage that occurs after intracerebral hemorrhage (ICH). We previously found that the selective cannabinoid receptor 2 (CB2R) agonist JWH-133 reduced brain edema and neurological deficits following germinal matrix hemorrhage (GMH). We explored whether CB2R stimulation ameliorated thrombin-induced brain edema and BBB permeability as well as the possible molecular mechanism involved. A total of 144 Sprague-Dawley (S-D) rats received a thrombin (20 U) injection in the right basal ganglia. JWH-133 (1.5 mg/kg) or SR-144528 (3.0 mg/kg) and vehicle were intraperitoneally (i.p.) injected 1 h after surgery. Brain water content measurement, Evans blue (EB) extravasation, Western blot, and immunofluorescence were used to study the effects of a CB2R agonist 24 h after surgery. The results demonstrated that JWH-133 administration significantly decreased thrombin-induced brain edema and reduced the number of Iba-1-positive microglia. JWH-133 also decreased the number of P44/P42(+)/Iba-1(+) microglia, lowered Evans blue extravasation, and inhibited the elevated matrix metallopeptidase (MMP)-9 and matrix metallopeptidase (MMP)-12 activities. However, a selective CB2R antagonist (SR-144528) reversed these effects. We demonstrated that CB2R stimulation reduced thrombin-induced brain edema and alleviated BBB damage. We also found that matrix metalloproteinase suppression may be partially involved in these processes.

  5. The dineolignan from Saururus chinensis, manassantin B, inhibits tumor-induced angiogenesis via downregulation of matrix metalloproteinases 9 in human endothelial cells.

    PubMed

    Liu, Zhaojie; Lu, Hong; Liu, Rong; Chen, Bin; Wang, Shan; Ma, Junchao; Fu, Jianjiang

    2014-08-01

    Manassantin B (MB) is a neolignan isolated from Saururus chinensis that exhibits a range of activities, including anti-inflammatory, antiseptic and antitumor activity. MB was recently found to affect cell adhesion and expression of several adhesion molecules. Based on the important roles of these adhesion molecules in angiogenesis, we evaluated a possible role for MB in tumor-induced angiogenesis in endothelial cells (ECs). In the present study, we found that MB blocked tumor-induced tube formation of ECs and significantly inhibited the invasion of ECs through the reconstituted basement membrane. MB suppressed the activity of matrix metalloproteinases (MMPs) and downregulated the expression of matrix metalloproteinases 9. Western blotting showed reduction of RUNX2 activation by MB. RUNX2 transcription factor assay and chromatin immunoprecipitation assay showed that the interaction between RUNX2 and target sequences in the matrix metalloproteinases 9 promoters was inhibited by MB. Our findings suggested that the inhibitory effects of MB on tumor-induced angiogenesis were caused by matrix metalloproteinases 9 inhibition, which was associated with the downregulation of RUNX2 transcriptional activity.

  6. Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L. ) cell suspension cultures

    SciTech Connect

    Kuehnl, T.K.; Koch, U.; Heller, W.; Wellmann, E.

    1987-10-01

    Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsible for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.

  7. Understanding the Constitutive and Induced Biosynthesis of Mono- and Sesquiterpenes in Grapes (Vitis vinifera): A Key to Unlocking the Biochemical Secrets of Unique Grape Aroma Profiles.

    PubMed

    Schwab, Wilfried; Wüst, Matthias

    2015-12-16

    The present review integrates current knowledge on mono- and sesquiterpenes in grapes with a special focus on biochemical and physiological aspects. Recent research has impressively shown the prominence of terpenoid metabolism in grapevine (Vitis sp). The 69 putatively functional mono- and sesquiterpene synthases that were identified by the analysis of the updated 12-fold sequencing and assembly of the grapevine genome deliver the scaffolds for structural diversity and display a surprising expansion of the terpene synthase (TPS) gene family in grapevine when compared to other plants like Arabidopsis thaliana (32 TPS). While monoterpenes occur as highly functionalized compounds and are stored as their corresponding glycoconjugates in berry tissues, sesquiterpenes are mainly present as unsaturated hydrocarbons and accumulate in the epicuticular wax layer of intact berries. Interestingly, both groups of terpenes appear to be involved as volatile organic compounds in plant defense and their biosynthesis is enhanced via the jasmonic acid signaling pathway. These novel aspects will help to understand how environmental cues affect the genes and enzymes of various metabolic pathways of relevant wine aroma compounds with numerous links to enology and wine flavor chemistry.

  8. Salicylic acid-induced changes in physiological parameters and genes of the flavonoid biosynthesis pathway in Artemisia vulgaris and Dendranthema nankingense during aphid feeding.

    PubMed

    Sun, Y; Xia, X L; Jiang, J F; Chen, S M; Chen, F D; Lv, G S

    2016-02-19

    Phloem-feeding aphids cause serious damage to plants. The mechanisms of plant-aphid interactions are only partially understood and involve multiple pathways, including phytohormones. In order to investigate whether salicylic acid (SA) is involved and how it plays a part in the defense response to the aphid Macrosiphoniella sanbourni, physiological changes and gene expression profiles in response to aphid inoculation with or without SA pretreatment were compared between the aphid-resistant Artemisia vulgaris 'Variegata' and the susceptible chrysanthemum, Dendranthema nankingense. Changes in levels of reactive oxygen species, malondialdehyde (MDA), and flavonoids, and in the expression of genes involved in flavonoid biosynthesis, including PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), CHI (chalcone isomerase), F3H (flavanone 3-hydroxylase), F3'H (flavanone 3'-hydroxylase), and DFR (dihydroflavonol reductase), were investigated. Levels of hydrogen peroxide, superoxide anions, MDA, and flavonoids, and their related gene expression, increased after aphid infestation and SA pretreatment followed by aphid infestation; the aphid-resistant A. vulgaris exhibited a more rapid response than the aphid-susceptible D. nankingense to SA treatment and aphid infestation. Taken together, our results suggest that SA could be used to increase aphid resistance in the chrysanthemum.

  9. The inhibition of 2-arachidonoyl-glycerol (2-AG) biosynthesis, rather than enhancing striatal damage, protects striatal neurons from malonate-induced death: a potential role of cyclooxygenase-2-dependent metabolism of 2-AG.

    PubMed

    Valdeolivas, S; Pazos, M R; Bisogno, T; Piscitelli, F; Iannotti, F A; Allarà, M; Sagredo, O; Di Marzo, V; Fernández-Ruiz, J

    2013-10-17

    The cannabinoid CB2 receptor, which is activated by the endocannabinoid 2-arachidonoyl-glycerol (2-AG), protects striatal neurons from apoptotic death caused by the local administration of malonate, a rat model of Huntington's disease (HD). In the present study, we investigated whether endocannabinoids provide tonic neuroprotection in this HD model, by examining the effect of O-3841, an inhibitor of diacylglycerol lipases, the enzymes that catalyse 2-AG biosynthesis, and JZL184 or OMDM169, two inhibitors of 2-AG inactivation by monoacylglycerol lipase (MAGL). The inhibitors were injected in rats with the striatum lesioned with malonate, and several biochemical and morphological parameters were measured in this brain area. Similar experiments were also conducted in vitro in cultured M-213 cells, which have the phenotypic characteristics of striatal neurons. O-3841 produced a significant reduction in the striatal levels of 2-AG in animals lesioned with malonate. However, surprisingly, the inhibitor attenuated malonate-induced GABA and BDNF deficiencies and the reduction in Nissl staining, as well as the increase in GFAP immunostaining. In contrast, JZL184 exacerbated malonate-induced striatal damage. Cyclooxygenase-2 (COX-2) was induced in the striatum 24 h after the lesion simultaneously with other pro-inflammatory responses. The COX-2-derived 2-AG metabolite, prostaglandin E2 glyceryl ester (PGE2-G), exacerbated neurotoxicity, and this effect was antagonized by the blockade of PGE2-G action with AGN220675. In M-213 cells exposed to malonate, in which COX-2 was also upregulated, JZL184 worsened neurotoxicity, and this effect was attenuated by the COX-2 inhibitor celecoxib or AGN220675. OMDM169 also worsened neurotoxicity and produced measurable levels of PGE2-G. In conclusion, the inhibition of 2-AG biosynthesis is neuroprotective in rats lesioned with malonate, possibly through the counteraction of the formation of pro-neuroinflammatory PGE2-G, formed from COX-2

  10. Chirality and protein biosynthesis.

    PubMed

    Banik, Sindrila Dutta; Nandi, Nilashis

    2013-01-01

    Chirality is present at all levels of structural hierarchy of protein and plays a significant role in protein biosynthesis. The macromolecules involved in protein biosynthesis such as aminoacyl tRNA synthetase and ribosome have chiral subunits. Despite the omnipresence of chirality in the biosynthetic pathway, its origin, role in current pathway, and importance is far from understood. In this review we first present an introduction to biochirality and its relevance to protein biosynthesis. Major propositions about the prebiotic origin of biomolecules are presented with particular reference to proteins and nucleic acids. The problem of the origin of homochirality is unresolved at present. The chiral discrimination by enzymes involved in protein synthesis is essential for keeping the life process going. However, questions remained pertaining to the mechanism of chiral discrimination and concomitant retention of biochirality. We discuss the experimental evidence which shows that it is virtually impossible to incorporate D-amino acids in protein structures in present biosynthetic pathways via any of the two major steps of protein synthesis, namely aminoacylation and peptide bond formation reactions. Molecular level explanations of the stringent chiral specificity in each step are extended based on computational analysis. A detailed account of the current state of understanding of the mechanism of chiral discrimination during aminoacylation in the active site of aminoacyl tRNA synthetase and peptide bond formation in ribosomal peptidyl transferase center is presented. Finally, it is pointed out that the understanding of the mechanism of retention of enantiopurity has implications in developing novel enzyme mimetic systems and biocatalysts and might be useful in chiral drug design.

  11. 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

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

    PubMed

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

    2013-06-19

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

  13. 82-kDa choline acetyltransferase and SATB1 localize to β-amyloid induced matrix attachment regions

    PubMed Central

    Winick-Ng, Warren; Caetano, Fabiana A.; Winick-Ng, Jennifer; Morey, Trevor M.; Heit, Bryan; Rylett, R. Jane

    2016-01-01

    The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-β1–42 (Aβ1–42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1–42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1–42 -exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1–42 -induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1–42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ -exposure. PMID:27052102

  14. Thin-Layer Matrix Sublimation with Vapor-Sorption Induced Co-Crystallization for Sensitive and Reproducible SAMDI-TOF MS Analysis of Protein Biosensors

    NASA Astrophysics Data System (ADS)

    Roth, Michael J.; Kim, Jaekuk; Maresh, Erica M.; Plymire, Daniel A.; Corbett, John R.; Zhang, Junmei; Patrie, Steven M.

    2012-10-01

    Coupling immunoassays on self-assembled monolayers (SAMs) to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) provides improved assay selectivity compared with traditional photometric detection techniques. We show that thin-layer-transfer (TLT) of α-cyano-4-hydroxycinnaminic acid (CHCA) MALDI matrix via vacuum sublimation followed by organic solvent-based vapor-sorption induced co-crystallization (VIC) results in unique matrix/analyte co-crystallization tendencies that optimizes assay reproducibility and sensitivity. Unique matrix crystal morphologies resulted from VIC solvent vapors, indicating nucleation and crystal growth characteristics depend upon VIC parameters. We observed that CHCA microcrystals generated by methanol VIC resulted in >10× better sensitivity, increased analyte charging, and improved precision compared with dried droplet measurements. The uniformity of matrix/analyte co-crystallization across planar immunoassays directed at intact proteins yielded low spectral variation for single shot replicates (18.5 % relative standard deviation, RSD) and signal averaged spectra (<10 % RSD). We envision that TLT and VIC for MALDI-TOF will enable high-throughput, reproducible array-based immunoassays for protein molecular diagnostic assays in diverse biochemical and clinical applications.

  15. Matrix metalloproteinase 7 restrains Helicobacter pylori-induced gastric inflammation and premalignant lesions in the stomach by altering macrophage polarization.

    PubMed

    Krakowiak, M S; Noto, J M; Piazuelo, M B; Hardbower, D M; Romero-Gallo, J; Delgado, A; Chaturvedi, R; Correa, P; Wilson, K T; Peek, R M

    2015-04-02

    Helicobacter pylori is the strongest risk factor for the development of gastric cancer. Although the specific mechanisms by which this pathogen induces carcinogenesis have not been fully elucidated, high-expression interleukin (IL)-1β alleles are associated with increased gastric cancer risk among H. pylori-infected persons. In addition, loss of matrix metalloproteinase 7 (MMP7) increases mucosal inflammation in mouse models of epithelial injury, and we have shown that gastric inflammation is increased in H. pylori-infected MMP7(-/-) C57BL/6 mice. In this report, we define mechanisms that underpin such responses and extend these results into a genetic model of MMP7 deficiency and gastric cancer. Wild-type (WT) or MMP7(-/-) C57BL/6 mice were challenged with broth alone as an uninfected control or the H. pylori strain PMSS1. All H. pylori-challenged mice were successfully colonized. As expected, H. pylori-infected MMP7(-/-) C57BL/6 mice exhibited a significant increase in gastric inflammation compared with uninfected or infected WT C57BL/6 animals. Loss of MMP7 resulted in M1 macrophage polarization within H. pylori-infected stomachs, as assessed by Luminex technology and immunohistochemistry, and macrophages isolated from infected MMP7-deficient mice expressed significantly higher levels of the M1 macrophage marker IL-1β compared with macrophages isolated from WT mice. To extend these findings into a model of gastric cancer, hypergastrinemic WT INS-GAS or MMP7(-/-) INS-GAS mice were challenged with H. pylori strain PMSS1. Consistent with findings in the C57BL/6 model, H. pylori-infected MMP7-deficient INS-GAS mice exhibited a significant increase in gastric inflammation compared with either uninfected or infected WT INS-GAS mice. In addition, the incidence of gastric hyperplasia and dysplasia was significantly increased in H. pylori-infected MMP7(-/-) INS-GAS mice compared with infected WT INS-GAS mice, and loss of MMP7 promoted M1 macrophage polarization. These

  16. Upstream regulation of mycotoxin biosynthesis.

    PubMed

    Alkhayyat, Fahad; Yu, Jae-Hyuk

    2014-01-01

    Mycotoxins are natural contaminants of food and feed products, posing a substantial health risk to humans and animals throughout the world. A plethora of filamentous fungi has been identified as mycotoxin producers and most of these fungal species belong to the genera Aspergillus, Fusarium, and Penicillium. A number of studies have been conducted to better understand the molecular mechanisms of biosynthesis of key mycotoxins and the regulatory cascades controlling toxigenesis. In many cases, the mycotoxin biosynthetic genes are clustered and regulated by one or more pathway-specific transcription factor(s). In addition, as biosynthesis of many secondary metabolites is coordinated with fungal growth and development, there are a number of upstream regulators affecting biosynthesis of mycotoxins in fungi. This review presents a concise summary of the regulation of mycotoxin biosynthesis, focusing on the roles of the upstream regulatory elements governing biosynthesis of aflatoxin and sterigmatocystin in Aspergillus.

  17. The effect of matrix microstructure on thermally induced residual stresses in SiC/titanium aluminide composites

    SciTech Connect

    Pindera, M.J. . Civil Engineering Applied Mechanics Dept.); Freed, A.D. . Lewis Research Center)

    1994-04-01

    This paper examines the effect of varying the microstructural composition of titanium aluminide on the evolution of residual stresses in titanium aluminide matrix composites. An analytical model is developed to determine residual stresses in fiber and matrix phases of unidirectional, SiC/Ti-Al composites subjected to axisymmetric thermal loading. The model uses elements of the concentric cylinder model and the method of cells to calculate residual thermal stresses in the presence of temperature-dependent and inelastic behavior of the fiber and matrix phases. The concentric cylinder model is employed as a geometric model for the unidirectional composite, whereas the method of cells is employed in modeling the microstructure of the titanium aluminide matrix phase. The titanium aluminide matrix consists of distinct brittle and ductile [alpha] and [beta] phases whose volume content is varied in the present scheme to understand how the resulting residual stresses can be altered. Both spatially uniform and nonuniform variations of the [alpha] and [beta] phases are considered. The results explain the occurrence of radial microcracks in SiC/Ti-Al composites in the presence of a [beta]-depleted region at the fiber/matrix interface, and validate the potential of engineering the matrix phase to reduce residual stresses in these composites.

  18. Factor Xa releases matrix metalloproteinase-2 (MMP-2) from human vascular smooth muscle cells and stimulates the conversion of pro-MMP-2 to MMP-2: role of MMP-2 in factor Xa-induced DNA synthesis and matrix invasion.

    PubMed

    Rauch, Bernhard H; Bretschneider, Ellen; Braun, Marina; Schrör, Karsten

    2002-05-31

    Pro-matrix metalloproteinase-2 (pro-MMP-2) is expressed in vascular smooth muscle cells (SMCs). We report that activated coagulation factor X (FXa) induces the release of MMP-2 (65 kDa) from human SMCs. In addition, FXa cleaves pro-MMP-2 (72 kDa) into MMP-2. Pro-MMP-2 and MMP-2 were determined by gelatin zymography. MMP-2 was generated in conditioned medium containing pro-MMP-2 in a concentration-dependent fashion by FXa (3 to 100 nmol/L). FX at concentrations up to 300 nmol/L was ineffective. The conversion of pro-MMP-2 to MMP-2 was inhibited by a selective FXa inhibitor (DX-9065a) at 3 to 10 micromol/L. There was a concentration-dependent induction of an intermediate MMP-2 form (68 kDa) in lysates of FXa-treated cells. This indicates that cellular mechanisms are involved in FXa-induced conversion of pro-MMP-2. As a possible biological consequence of MMP-2 activation by FXa, DNA synthesis and matrix invasion of SMCs were determined. Both were stimulated by FXa and inhibited by the selective FXa inhibitor DX-9065a and the MMP inhibitor GM 6001 but not by hirudin or aprotinin. It is concluded that stimulation of SMCs by FXa increases the levels of MMP-2 in the extracellular space and that two different mechanisms are involved: release of active MMP-2 and cleavage of secreted pro-MMP-2. Both might contribute to the mitogenic potency of FXa and FXa-stimulated matrix invasion of SMCs.

  19. Amphetamine and cocaine induce drug-specific activation of the c-fos gene in striosome-matrix compartments and limbic subdivisions of the striatum.

    PubMed Central

    Graybiel, A M; Moratalla, R; Robertson, H A

    1990-01-01

    Amphetamine and cocaine are stimulant drugs that act on central monoaminergic neurons to produce both acute psychomotor activation and long-lasting behavioral effects including addiction and psychosis. Here we report that single doses of these drugs induce rapid expression of the nuclear proto-oncogene c-fos in the forebrain and particularly in the striatum, an extrapyramidal structure implicated in addiction and in long-term drug-induced changes in motor function. The two drugs induce strikingly different patterns of c-fos expression in the striosome-matrix compartments and limbic subdivisions of the striatum, and their effects are pharmacologically distinct, although both are sensitive to dopamine receptor blockade. We propose that differential activation of immediate-early genes by psychostimulants may be an early step in drug-specific molecular cascades contributing to acute and long-lasting psychostimulant-induced changes in behavior. Images PMID:2118661

  20. Designer microbes for biosynthesis

    PubMed Central

    Quin, Maureen B.; Schmidt-Dannert, Claudia

    2014-01-01

    Microbes have long been adapted for the biosynthetic production of useful compounds. There is increasing demand for the rapid and cheap microbial production of diverse molecules in an industrial setting. Microbes can now be designed and engineered for a particular biosynthetic purpose, thanks to recent developments in genome sequencing, metabolic engineering, and synthetic biology. Advanced tools exist for the genetic manipulation of microbes to create novel metabolic circuits, making new products accessible. Metabolic processes can be optimized to increase yield and balance pathway flux. Progress is being made towards the design and creation of fully synthetic microbes for biosynthetic purposes. Together, these emerging technologies will facilitate the production of designer microbes for biosynthesis. PMID:24646570

  1. Microbial biosynthesis of alkanes.

    PubMed

    Schirmer, Andreas; Rude, Mathew A; Li, Xuezhi; Popova, Emanuela; del Cardayre, Stephen B

    2010-07-30

    Alkanes, the major constituents of gasoline, diesel, and jet fuel, are naturally produced by diverse species; however, the genetics and biochemistry behind this biology have remained elusive. Here we describe the discovery of an alkane biosynthesis pathway from cyanobacteria. The pathway consists of an acyl-acyl carrier protein reductase and an aldehyde decarbonylase, which together convert intermediates of fatty acid metabolism to alkanes and alkenes. The aldehyde decarbonylase is related to the broadly functional nonheme diiron enzymes. Heterologous expression of the alkane operon in Escherichia coli leads to the production and secretion of C13 to C17 mixtures of alkanes and alkenes. These genes and enzymes can now be leveraged for the simple and direct conversion of renewable raw materials to fungible hydrocarbon fuels.

  2. Terpenoid biosynthesis in prokaryotes.

    PubMed

    Boronat, Albert; Rodríguez-Concepción, Manuel

    2015-01-01

    Prokaryotic organisms (archaea and eubacteria) are found in all habitats where life exists on our planet. This would not be possible without the astounding biochemical plasticity developed by such organisms. Part of the metabolic diversity of prokaryotes was transferred to eukaryotic cells when endosymbiotic prokaryotes became mitochondria and plastids but also in a large number of horizontal gene transfer episodes. A group of metabolites produced by all free-living organisms is terpenoids (also known as isoprenoids). In prokaryotes, terpenoids play an indispensable role in cell-wall and membrane biosynthesis (bactoprenol, hopanoids), electron transport (ubiquinone, menaquinone), or conversion of light into chemical energy (chlorophylls, bacteriochlorophylls, rhodopsins, carotenoids), among other processes. But despite their remarkable structural and functional diversity, they all derive from the same metabolic precursors. Here we describe the metabolic pathways producing these universal terpenoid units and provide a complete picture of the main terpenoid compounds found in prokaryotic organisms.

  3. Biosynthesis of methanopterin

    SciTech Connect

    White, R.H. )

    1990-06-05

    The biosynthetic pathway for the generation of the methylated pterin in methanopterins was determined for the methanogenic bacteria Methanococcus volta and Methanobacterium formicicum. Extracts of M. volta were found to readily cleave L-7,8-dihydroneopterin to 7,8-dihydro-6-(hydroxymethyl)pterin, which was confirmed to be a precursor of the pterin portion of the methanopterin. (methylene{sup 2}H)-6-(hydroxymethyl)pterin was incorporated into methanopterin by growing cells of M. volta to an extent of 30%. Both the C-11 and C-12 methyl groups of methanopterin originate from (methyl-{sup 2}H{sub 3})methionine. Cells grown in the presence of (methylene-{sup 2}H)-6-(hydroxymethyl)pterin, (ethyl-{sup 2}H{sub 4})-6-(1 (RS)-hydroxyethyl)pterin, (methyl-{sup 2}H{sub 3})-6-(hydroxymethyl)-7-methylpterin, (ethyl-{sup 2}H{sub 4}, methyl-{sup 2}H{sub 3})-6-(1 (RS)-hydroxyethyl)-7-methylpterin, and (1-ethyl-{sup 3}H)-6-(1 (RS)-hydroxyethyl)-7-methylpterin showed that only the non-7-methylated pterins were incorporated into methanopterin. Cells extracts of M. formicicum readily condensed synthetic (methylene-{sup 3}H)-7,8-H{sub 2}-6-(hydroxymethyl)pterin-PP with methaniline to generate demethylated methanopterin, which is then methylated to methanopterin by the cell extract in the presence of S-adenosylmethionine. These observations indicate that the pterin portion of methanopterin is biosynthetically derived from 7,8-H{sub 2}-6-(hydroxymethyl)pterin, which is coupled to methaniline by a pathway analogous to the biosynthesis of folic acid. This pathway for the biosynthesis of methanopterin represents the first example of the modification of the specificity of a coenzyme through a methylation reaction.

  4. Methionine Biosynthesis in Lemna

    PubMed Central

    Thompson, Gregory A.; Datko, Anne H.; Mudd, S. Harvey; Giovanelli, John

    1982-01-01

    Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O-phosphohomoserine-dependent cystathionine γ-synthase, O-phosphohomoserine sulfhydrylase, and O-acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine γ-synthase, it was observed that (a) adding external methionine (2 μm) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 μml-aminoethoxyvinylglycine or with 36 μm lysine plus 4 μm threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S-adenosylmethionine, or S-methylmethionine sulfonium to the assay mixture. Parallel studies of O-phosphohomoserine sulfhydrylase and O-acetylserine sulfhydrylase showed that O-phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine γ-synthase activity, whereas O-acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O-acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O-acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O-phosphohomoserine sulfhydrylase. The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine γ-synthase in Lemna, (b) O-phosphohomoserine sulfhydrylase and cystathionine γ-synthase are probably activities of one enzyme that has low specificity for its sulfur-containing substrate, and (c) O-acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of

  5. Salicylic Acid Biosynthesis and Metabolism

    PubMed Central

    Dempsey, D'Maris Amick; Vlot, A. Corina; Wildermuth, Mary C.; Klessig, Daniel F.

    2011-01-01

    Salicylic acid (SA) has been shown to regulate various aspects of growth and development; it also serves as a critical signal for activating disease resistance in Arabidopsis thaliana and other plant species. This review surveys the mechanisms involved in the biosynthesis and metabolism of this critical plant hormone. While a complete biosynthetic route has yet to be established, stressed Arabidopsis appear to synthesize SA primarily via an isochorismate-utilizing pathway in the chloroplast. A distinct pathway utilizing phenylalanine as the substrate also may contribute to SA accumulation, although to a much lesser extent. Once synthesized, free SA levels can be regulated by a variety of chemical modifications. Many of these modifications inactivate SA; however, some confer novel properties that may aid in long distance SA transport or the activation of stress responses complementary to those induced by free SA. In addition, a number of factors that directly or indirectly regulate the expression of SA biosynthetic genes or that influence the rate of SA catabolism have been identified. An integrated model, encompassing current knowledge of SA metabolism in Arabidopsis, as well as the influence other plant hormones exert on SA metabolism, is presented. PMID:22303280

  6. Assessment of glenoid chondral healing: comparison of microfracture to autologous matrix-induced chondrogenesis in a novel rabbit shoulder model

    PubMed Central

    Wang, Vincent M.; Karas, Vasili; Lee, Andrew S.; Yin, Ziying; Van Thiel, Geoffrey S.; Hussey, Kristen; Sumner, D. Rick; Chubinskaya, Susan; Magin, Richard L.; Verma, Nikhil N.; Romeo, Anthony A.; Cole, Brian J.

    2015-01-01

    Background Management of glenohumeral arthrosis in young patients is a considerable challenge, with a growing need for non-arthroplasty alternatives. The objectives of this study were to develop an animal model to study glenoid cartilage repair and to compare surgical repair strategies to promote glenoid chondral healing. Methods Forty-five rabbits underwent unilateral removal of the entire glenoid articular surface and were divided into 3 groups—duntreated defect (UD), microfracture (MFx), and MFx plus type I/III collagen scaffold (autologous matrix-induced chondrogenesis [AMIC])—for the evaluation of healing at 8 weeks (12 rabbits) and 32 weeks (33 rabbits) after injury. Contralateral shoulders served as unoperated controls. Tissue assessments included 11.7-T magnetic resonance imaging (long-term healing group only), Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-μCT), and histologic investigation (grades on International Cartilage Repair Society II scoring system). Results At 8 weeks, x-ray attenuation, thickness, and volume did not differ by treatment group. At 32 weeks, the T2 index (ratio of T2 values of healing to intact glenoids) was significantly lower for the MFx group relative to the AMIC group (P = .01) whereas the T1ρ index was significantly lower for AMIC relative to MFx (P = .01). The micro–computed tomography–derived repair tissue volume was significantly higher for MFx than for UD. Histologic investigation generally suggested inferior healing in the AMIC and UD groups relative to the MFx group, which exhibited improvements in both integration of repair tissue with subchondral bone and tidemark formation over time. Discussion Improvements conferred by AMIC were limited to magnetic resonance imaging outcomes, whereas MFx appeared to promote increased fibrous tissue deposition via micro–computed tomography and more hyaline-like repair histologically. The findings from this novel model suggest that

  7. Nicotine-induced retardation of chondrogenesis through down-regulation of IGF-1 signaling pathway to inhibit matrix synthesis of growth plate chondrocytes in fetal rats

    SciTech Connect

    Deng, Yu; Cao, Hong; Cu, Fenglong; Xu, Dan; Lei, Youying; Tan, Yang; Magdalou, Jacques; Wang, Hui; Chen, Liaobin

    2013-05-15

    Previous studies have confirmed that maternal tobacco smoking causes intrauterine growth retardation (IUGR) and skeletal growth retardation. Among a multitude of chemicals associated with cigarette smoking, nicotine is one of the leading candidates for causing low birth weights. However, the possible mechanism of delayed chondrogenesis by prenatal nicotine exposure remains unclear. We investigated the effects of nicotine on fetal growth plate chondrocytes in vivo and in vitro. Rats were given 2.0 mg/kg·d of nicotine subcutaneously from gestational days 11 to 20. Prenatal nicotine exposure increased the levels of fetal blood corticosterone and resulted in fetal skeletal growth retardation. Moreover, nicotine exposure induced the inhibition of matrix synthesis and down-regulation of insulin-like growth factor 1 (IGF-1) signaling in fetal growth plates. The effects of nicotine on growth plates were studied in vitro by exposing fetal growth plate chondrocytes to 0, 1, 10, or 100 μM of nicotine for 10 days. Nicotine inhibited matrix synthesis and down-regulated IGF-1 signaling in chondrocytes in a concentration-dependent manner. These results suggest that prenatal nicotine exposure induces delayed chondrogenesis and that the mechanism may involve the down-regulation of IGF-1 signaling and the inhibition of matrix synthesis by growth plate chondrocytes. The present study aids in the characterization of delayed chondrogenesis caused by prenatal nicotine exposure, which might suggest a candidate mechanism for intrauterine origins of osteoporosis and osteoarthritis. - Highlights: ► Prenatal nicotine-exposure could induce delayed chondrogenesis in fetal rats. ► Nicotine inhibits matrix synthesis of fetal growth plate chondrocytes. ► Nicotine inhibits IGF-1 signaling pathway in fetal growth plate chondrocytes.

  8. Effect of intrafollicular indomethacin injection on gonadotropin surge-induced expression of select extracellular matrix degrading enzymes and their inhibitors in bovine preovulatory follicles.

    PubMed

    Li, Qinglei; Jimenez-Krassel, Fermin; Kobayashi, Yasuhiro; Ireland, James J; Smith, George W

    2006-03-01

    A growing body of evidence supports an obligatory role for intrafollicular prostanoids in the mechanism of ovulation. However, the prostanoid-dependent mediators of the follicular extracellular matrix degradation required for ovulation are unknown. The objectives of this study were to determine the cellular compartment(s) in which the gonadotropin surge-induced regulation of select extracellular matrix degrading enzymes and their cognate inhibitors occurs in bovine preovulatory follicles, and to test whether such regulation is blocked by intrafollicular administration of the prostanoid synthesis and ovulation inhibitor, indomethacin (INDO). Follicular fluid prostaglandin E2 concentrations were elevated in diluent-treated follicles before ovulation (24 h after GnRH injection), but the increase was blocked in INDO-treated follicles. Real-time PCR analysis revealed the specific follicular cell types where gonadotropin surge-induced increases in mRNA abundance for members of the matrix metalloproteinase/tissue inhibitor of metalloproteinase and plasminogen activator families occurred. INDO treatment increased thecal cell mRNA for tissue inhibitor of metalloproteinase-4 and its protein abundance in the apex of preovulatory follicles before ovulation, but suppressed granulosal cell mRNA and activity for tissue plasminogen activator in follicular fluid and the follicle apex. Plasmin activity was also suppressed in the follicular fluid of INDO-treated follicles. Effects of INDO injection on select matrix metalloproteinases were not observed. The results suggest that gonadotropin surge-induced regulation of tissue inhibitor of metalloproteinase-4 and tissue plasminogen activator may be prostanoid dependent, and support a potential role for increased tissue plasminogen activator expression and decreased tissue inhibitor of metalloproteinase-4 expression in the mechanism of ovulation.

  9. The regulation of ascorbate biosynthesis.

    PubMed

    Bulley, Sean; Laing, William

    2016-10-01

    We review the regulation of ascorbate (vitamin C) biosynthesis, focusing on the l-galactose pathway. We discuss the regulation of ascorbate biosynthesis at the level of gene transcription (both repression and enhancement) and translation (feedback inhibition of translation by ascorbate concentration) and discuss the eight proteins that have been demonstrated to date to affect ascorbate concentration in plant tissues. GDP-galactose phosphorylase (GGP) and GDP-mannose epimerase are critical steps that regulate ascorbate biosynthesis. These and other biosynthetic genes are controlled at the transcriptional level, while GGP is also controlled at the translational level. Ascorbate feedback on enzyme activity has not been observed unequivocally.

  10. PEP-1-SIRT2-induced matrix metalloproteinase-1 and -13 modulates type II collagen expression via ERK signaling in rabbit articular chondrocytes.

    PubMed

    Eo, Seong-Hui; Choi, Soo Young; Kim, Song Ja

    2016-11-01

    Matrix metalloproteinases (MMPs) are critical for the degradation of the extracellular matrix (ECM), which includes cartilage-specific collagen types I, II and XI. We previously found that PEP-1-sirtuin (SIRT)2 could induce dedifferentiation of articular chondrocytes; however, the underlying mechanisms remains unclear. We addressed this in the present study by examining the association between PEP-1-SIRT2 and the expression of MMP-1 and MMP-13 and type II collagen in rabbit articular chondrocytes. We found that PEP-1-SIRT2 increased MMP-1 and -13 expression in a dose- and time-dependent manner, as determined by western blotting. A similar trend in MMP-1 and -13 levels was observed in cultures during expansion to four passages. Pharmacological inhibition of MMP-1 and -13 blocked the PEP-1-SIRT2-induced decrease in type II collagen level. Phosphorylation of extracellular regulated kinase (ERK) was increased by PEP-1-SIRT2; however, treatment with the mitogen-activated protein kinase inhibitor PD98059 suppressed PEP-1-SIRT2-induced MMP-1 and -13 expression and dedifferentiation while restoring type II collagen expression in passage 2 cells. These results suggest that PEP-1-SIRT2 promotes MMP-induced dedifferentiation via ERK signaling in articular chondrocytes.

  11. p-Coumaric Acid Attenuates UVB-Induced Release of Stratifin from Keratinocytes and Indirectly Regulates Matrix Metalloproteinase 1 Release from Fibroblasts.

    PubMed

    Seok, Jin Kyung; Boo, Yong Chool

    2015-05-01

    Ultraviolet (UV) radiation-induced loss of dermal extracellular matrix is associated with skin photoaging. Recent studies demonstrated that keratinocyte-releasable stratifin (SFN) plays a critical role in skin collagen metabolism by inducing matrix metalloproteinase 1 (MMP1) expression in target fibroblasts. In the present study, we examined whether SFN released from UVB-irradiated epidermal keratinocytes increases MMP1 release from dermal fibroblasts, and whether these events are affected by p-coumaric acid (p-CA), a natural phenolic compound with UVB-shielding and antioxidant properties. HaCaT cells were exposed to UVB in the absence and presence of p-CA, and the conditioned medium was used to stimulate fibroblasts in medium transfer experiments. The cells and media were analyzed to determine the expressions/releases of SFN and MMP1. UVB exposure increased SFN release from keratinocytes into the medium. The conditioned medium of UVB-irradiated keratinocytes increased MMP1 release from fibroblasts. The depletion of SFN using a siRNA rendered the conditioned medium of UVB-irradiated keratinocytes ineffective at stimulating fibroblasts to release MMP1. p-CA mitigated UVB-induced SFN expression in keratinocytes, and attenuated the MMP1 release by fibroblasts in medium transfer experiments. In conclusion, the present study demonstrated that the use of UV absorbers such as p-CA would reduce UV-induced SFN-centered signaling events involved in skin photoaging.

  12. Investigation of the Matrix Effect on the Accuracy of Quantitative Analysis of Trace Metals in Liquids Using Laser-Induced Breakdown Spectroscopy with Solid Substrates.

    PubMed

    Xiu, Junshan; Dong, Lili; Qin, Hua; Liu, Yunyan; Yu, Jin

    2016-12-01

    The detection limit of trace metals in liquids has been improved greatly by laser-induced breakdown spectroscopy (LIBS) using solid substrate. A paper substrate and a metallic substrate were used as a solid substrate for the detection of trace metals in aqueous solutions and viscous liquids (lubricating oils) respectively. The matrix effect on quantitative analysis of trace metals in two types of liquids was investigated. For trace metals in aqueous solutions using paper substrate, the calibration curves established for pure solutions and mixed solutions samples presented large variation on both the slope and the intercept for the Cu, Cd, and Cr. The matrix effects among the different elements in mixed solutions were observed. However, good agreement was obtained between the measured and known values in real wastewater. For trace metals in lubricating oils, the matrix effect between the different oils is relatively small and reasonably negligible under the conditions of our experiment. A universal calibration curve can be established for trace metals in different types of oils. The two approaches are verified that it is possible to develop a feasible and sensitive method with accuracy results for rapid detection of trace metals in industrial wastewater and viscous liquids by laser-induced breakdown spectroscopy.

  13. Characterization of an autoinducer of penicillin biosynthesis in Penicillium chrysogenum.

    PubMed

    Martín, Jorge; García-Estrada, Carlos; Rumbero, Angel; Recio, Eliseo; Albillos, Silvia M; Ullán, Ricardo V; Martín, Juan-Francisco

    2011-08-15

    Filamentous fungi produce an impressive variety of secondary metabolites; many of them have important biological activities. The biosynthesis of these secondary metabolites is frequently induced by plant-derived external elicitors and appears to also be regulated by internal inducers, which may work in a way similar to that of bacterial autoinducers. The biosynthesis of penicillin in Penicillium chrysogenum is an excellent model for studying the molecular mechanisms of control of gene expression due to a good knowledge of the biochemistry and molecular genetics of β-lactam antibiotics and to the availability of its genome sequence and proteome. In this work, we first developed a plate bioassay that allows direct testing of inducers of penicillin biosynthesis using single colonies of P. chrysogenum. Using this bioassay, we have found an inducer substance in the conditioned culture broths of P. chrysogenum and Acremonium chrysogenum. No inducing effect was exerted by γ-butyrolactones, jasmonic acid, or the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine. The conditioned broth induced penicillin biosynthesis and transcription of the pcbAB, pcbC, and penDE genes when added at inoculation time, but its effect was smaller if added at 12 h and it had no effect when added at 24 h, as shown by Northern analysis and lacZ reporter studies. The inducer molecule was purified and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as 1,3-diaminopropane. Addition of pure 1,3-diaminopropane stimulated the production of penicillin by about 100% compared to results for the control cultures. Genes for the biosynthesis of 1,3-diaminopropane have been identified in the P. chrysogenum genome.

  14. Mueller-matrix of laser-induced autofluorescence of polycrystalline films of dried peritoneal fluid in diagnostics of endometriosis

    NASA Astrophysics Data System (ADS)

    Ushenko, Yuriy A.; Koval, Galina D.; Ushenko, Alexander G.; Dubolazov, Olexander V.; Ushenko, Vladimir A.; Novakovskaia, Olga Yu.

    2016-07-01

    This research presents investigation results of the diagnostic efficiency of an azimuthally stable Mueller-matrix method of analysis of laser autofluorescence of polycrystalline films of dried uterine cavity peritoneal fluid. A model of the generalized optical anisotropy of films of dried peritoneal fluid is proposed in order to define the processes of laser autofluorescence. The influence of complex mechanisms of both phase (linear and circular birefringence) and amplitude (linear and circular dichroism) anisotropies is taken into consideration. The interconnections between the azimuthally stable Mueller-matrix elements characterizing laser autofluorescence and different mechanisms of optical anisotropy are determined. The statistical analysis of coordinate distributions of such Mueller-matrix rotation invariants is proposed. Thereupon the quantitative criteria (statistic moments of the first to the fourth order) of differentiation of polycrystalline films of dried peritoneal fluid, group 1 (healthy donors) and group 2 (uterus endometriosis patients), are determined.

  15. Neural regulation of sex pheromone biosynthesis in Heliothis moths

    PubMed Central

    Teal, P. E. A.; Tumlinson, J. H.; Oberlander, H.

    1989-01-01

    Pheromone biosynthesis in females of Heliothis zea is regulated endogenously by a neuropeptide produced in the subesophageal ganglion. We have found that the ventral nerve cord must be intact for normal induction of pheromone biosynthesis and that pheromonotropic activity is associated with extracts of the abdominal nerve cord, but only during the period when pheromone is produced. We did not find evidence of pheromonotropic activity in hemolymph obtained from females that were producing pheromone. Extracts of the brain—subesophageal ganglion complex, which contain pheromone biosynthesis activating neuropeptide (PBAN), induced pheromone biosynthesis when applied to the terminal abdominal ganglion only if nerves from this ganglion to the pheromone gland were intact. Brain—subesophageal ganglion extracts did not induce biosynthesis when applied directly to the pheromone glands in vitro. From our results, we conclude that the target site of PBAN is not the pheromone gland but the terminal abdominal ganglion, and we hypothesize that the abdominal nerve cord transports PBAN to the terminal abdominal ganglion. PMID:16594023

  16. Roles of lignin biosynthesis and regulatory genes in plant development

    PubMed Central

    Yoon, Jinmi; Choi, Heebak

    2015-01-01

    Abstract Lignin is an important factor affecting agricultural traits, biofuel production, and the pulping industry. Most lignin biosynthesis genes and their regulatory genes are expressed mainly in the vascular bundles of stems and leaves, preferentially in tissues undergoing lignification. Other genes are poorly expressed during normal stages of development, but are strongly induced by abiotic or biotic stresses. Some are expressed in non‐lignifying tissues such as the shoot apical meristem. Alterations in lignin levels affect plant development. Suppression of lignin biosynthesis genes causes abnormal phenotypes such as collapsed xylem, bending stems, and growth retardation. The loss of expression by genes that function early in the lignin biosynthesis pathway results in more severe developmental phenotypes when compared with plants that have mutations in later genes. Defective lignin deposition is also associated with phenotypes of seed shattering or brittle culm. MYB and NAC transcriptional factors function as switches, and some homeobox proteins negatively control lignin biosynthesis genes. Ectopic deposition caused by overexpression of lignin biosynthesis genes or master switch genes induces curly leaf formation and dwarfism. PMID:26297385

  17. Roles of lignin biosynthesis and regulatory genes in plant development.

    PubMed

    Yoon, Jinmi; Choi, Heebak; An, Gynheung

    2015-11-01

    Lignin is an important factor affecting agricultural traits, biofuel production, and the pulping industry. Most lignin biosynthesis genes and their regulatory genes are expressed mainly in the vascular bundles of stems and leaves, preferentially in tissues undergoing lignification. Other genes are poorly expressed during normal stages of development, but are strongly induced by abiotic or biotic stresses. Some are expressed in non-lignifying tissues such as the shoot apical meristem. Alterations in lignin levels affect plant development. Suppression of lignin biosynthesis genes causes abnormal phenotypes such as collapsed xylem, bending stems, and growth retardation. The loss of expression by genes that function early in the lignin biosynthesis pathway results in more severe developmental phenotypes when compared with plants that have mutations in later genes. Defective lignin deposition is also associated with phenotypes of seed shattering or brittle culm. MYB and NAC transcriptional factors function as switches, and some homeobox proteins negatively control lignin biosynthesis genes. Ectopic deposition caused by overexpression of lignin biosynthesis genes or master switch genes induces curly leaf formation and dwarfism.

  18. Genetic engineering of the biosynthesis of glycinebetaine leads to alleviate salt-induced potassium efflux and enhances salt tolerance in tomato plants.

    PubMed

    Wei, Dandan; Zhang, Wen; Wang, Cuicui; Meng, Qingwei; Li, Gang; Chen, Tony H H; Yang, Xinghong

    2017-04-01

    Tomato (Solanum lycopersicum cv. 'Moneymaker') was transformed with the choline oxidase gene codA from Arthrobacter globiformis, which was modified to allow for targeting to both chloroplasts and the cytosol. Glycine betaine (GB) was accumulated in transformed plants, while no detectable GB was found in wild-type (WT) plants. Compared to WT plants, transgenic lines showed significantly higher photosynthetic rates (Pn) and antioxidant enzyme activities and lower reactive oxygen species (ROS) accumulation in the leaves when exposed to salt stress. Furthermore, compared with WT plants, K(+) efflux decreased and Na(+) efflux increased in roots of transgenic plants under salt stress; resulted in lower Na(+)/K(+) ratios in transgenic lines. The exogenous application of GB also significantly reduced NaCl-induced K(+) efflux and increased Na(+) efflux in WT plants. A qRT-PCR assay indicated that GB enhanced NaCl-induced expression of genes encoding the K(+) transporter, Na(+)/H(+) antiporter, and H(+)-ATPase. These results suggest that the enhanced salt tolerance conferred by codA in transgenic tomato plants might be due to the regulation of ion channel and transporters by GB, which would allow high potassium levels and low sodium levels to be maintained in transgenic plants under salt stress condition.

  19. Stereoselectivity in Polyphenol Biosynthesis

    NASA Technical Reports Server (NTRS)

    Lewis, Norman G.; Davin, Laurence B.

    1992-01-01

    Stereoselectivity plays an important role in the late stages of phenyl-propanoid metabolism, affording lignins, lignans, and neolignans. Stereoselectivity is manifested during monolignol (glucoside) synthesis, e.g., where the geometry (E or Z) of the pendant double bond affects the specificity of UDPG:coniferyl alcohol glucosyltransferases in different species. Such findings are viewed to have important ramifications in monolignol transport and storage processes, with roles for both E- and Z-monolignols and their glucosides in lignin/lignan biosynthesis being envisaged. Stereoselectivity is also of great importance in enantiose-lective enzymatic processes affording optically active lignans. Thus, cell-free extracts from Forsythia species were demonstrated to synthesize the enantiomerically pure lignans, (-)-secoisolariciresinol, and (-)-pinoresinol, when NAD(P)H, H2O2 and E-coniferyl alcohol were added. Progress toward elucidating the enzymatic steps involved in such highly stereoselective processes is discussed. Also described are preliminary studies aimed at developing methodologies to determine the subcellular location of late-stage phenylpropanoid metabolites (e.g., coniferyl alcohol) and key enzymes thereof, in intact tissue or cells. This knowledge is essential if questions regarding lignin and lignan tissue specificity and regulation of these processes are to be deciphered.

  20. Parallels in lignin biosynthesis

    PubMed Central

    Weng, Jing-Ke; Banks, Jo Ann

    2008-01-01

    A hallmark of vascular plants is the development of a complex water-conducting system, which is physically reinforced by the heterogeneous aromatic polymer lignin. Syringyl lignin, a major building block of lignin, is often thought to be uniquely characteristic of angiosperms; however, it was demonstrated over fifty years ago that that syringyl lignin is found in another group of plants, known as the lycophytes, the ancestors of which diverged from all the other vascular plant lineages 400 million years ago.1 To determine the biochemical basis for this common biosynthetic ability, we isolated and characterized cytochrome P450-dependent monooxygenases (P450s) from the lycophyte Selaginella moellendorffii and compared them to the enzyme that is required for syringyl lignin synthesis in angiosperms. Our results showed that one of these P450s encodes an enzyme that is functionally analogous to but phylogenetically independent from its angiosperm counterpart. Here, we discuss the evolution of lignin biosynthesis in vascular plants and the role of Selaginella moellendorffii in plant comparative biology and genomics. PMID:19704782

  1. Sexual pheromone induces diffusion of the pheromone-homologous polypeptide in the extracellular matrix of Volvox carteri.

    PubMed

    Ishida, Koichi

    2007-11-01

    The C-terminal domain of pherophorin II is homologous to the sexual pheromone of Volvox carteri and is released from other domains during sexual induction. Green fluorescent protein fused to the C terminus of pherophorin II was located at the extracellular matrix directly surrounding the gonidium, the final target of the sexual-induction signal.

  2. Genome-Wide Analysis of Hydrogen Peroxide-Regulated Gene Expression in Arabidopsis Reveals a High Light-Induced Transcriptional Cluster Involved in Anthocyanin Biosynthesis1[w

    PubMed Central

    Vanderauwera, Sandy; Zimmermann, Philip; Rombauts, Stéphane; Vandenabeele, Steven; Langebartels, Christian; Gruissem, Wilhelm; Inzé, Dirk; Van Breusegem, Frank

    2005-01-01

    In plants, reactive oxygen species and, more particularly, hydrogen peroxide (H2O2) play a dual role as toxic by-products of normal cell metabolism and as regulatory molecules in stress perception and signal transduction. Peroxisomal catalases are an important sink for photorespiratory H2O2. Using ATH1 Affymetrix microarrays, expression profiles were compared between control and catalase-deficient Arabidopsis (Arabidopsis thaliana) plants. Reduced catalase levels already provoked differences in nuclear gene expression under ambient growth conditions, and these effects were amplified by high light exposure in a sun simulator for 3 and 8 h. This genome-wide expression analysis allowed us to reveal the expression characteristics of complete pathways and functional categories during H2O2 stress. In total, 349 transcripts were significantly up-regulated by high light in catalase-deficient plants and 88 were down-regulated. From this data set, H2O2 was inferred to play a key role in the transcriptional up-regulation of small heat shock proteins during high light stress. In addition, several transcription factors and candidate regulatory genes involved in H2O2 transcriptional gene networks were identified. Comparisons with other publicly available transcriptome data sets of abiotically stressed Arabidopsis revealed an important intersection with H2O2-deregulated genes, positioning elevated H2O2 levels as an important signal within abiotic stress-induced gene expression. Finally, analysis of transcriptional changes in a combination of a genetic (catalase deficiency) and an environmental (high light) perturbation identified a transcriptional cluster that was strongly and rapidly induced by high light in control plants, but impaired in catalase-deficient plants. This cluster comprises the complete known anthocyanin regulatory and biosynthetic pathway, together with genes encoding unknown proteins. PMID:16183842

  3. Interleukin-1β activates focal adhesion kinase and Src to induce matrix metalloproteinase-9 production and invasion of MCF-7 breast cancer cells

    PubMed Central

    Mon, Naing Naing; Senga, Takeshi; Ito, Satoko

    2017-01-01

    Interleukin-1β (IL-1b) is a pleiotropic cytokine that is important in tumor progression and invasion. Matrix metalloproteinase-9 (MMP-9), which is a secreted matrix-degrading enzyme, is one of the key regulators of tumor invasion and metastasis. The current report indicated that IL-1b promotes MMP-9 production and cell invasion in non-metastatic MCF-7 breast cancer cells. IL-1b activated focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (Src). Moreover, inhibiting the Src/FAK pathway reduced the IL-1b-induced production of MMP-9 and cell invasion. To investigate the functional role of FAK in MMP-9 production cell lines expressing mutant FAK in FAK knock-out mouse fibroblasts were generated. In wild-type FAK-expressing cells, MMP-9 production was induced by IL-1b stimulation. By contrast, IL-1b-induced MMP-9 production was abrogated in FAK knock-out, FAK Y397F, FAK Y925F, and kinase dead mutant-expressing cells. Therefore the results of the current study indicate that FAK and Src kinases are activated by IL-1b and play a critical role in MMP-9 production and tumor cell invasion. PMID:28356984

  4. Targeting of Proteoglycan Synthesis Pathway: A New Strategy to Counteract Excessive Matrix Proteoglycan Deposition and Transforming Growth Factor-β1-Induced Fibrotic Phenotype in Lung Fibroblasts.

    PubMed

    Shaukat, Irfan; Barré, Lydia; Venkatesan, Narayanan; Li, Dong; Jaquinet, Jean-Claude; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2016-01-01

    Stimulation of proteoglycan (PG) synthesis and deposition plays an important role in the pathophysiology of fibrosis and is an early and dominant feature of pulmonary fibrosis. Transforming growth factor-β1 (TGF-β1) is a major cytokine associated with fibrosis that induces excessive synthesis of matrix proteins, particularly PGs. Owing to the importance of PGs in matrix assembly and in mediating cytokine and growth factor signaling, a strategy based on the inhibition of PG synthesis may prevent excessive matrix PG deposition and attenuates profibrotic effects of TGF-β1 in lung fibroblasts. Here, we showed that 4-MU4-deoxy-β-D-xylopyranoside, a competitive inhibitor of β4-galactosyltransferase7, inhibited PG synthesis and secretion in a dose-dependent manner by decreasing the level of both chondroitin/dermatan- and heparin-sulfate PG in primary lung fibroblasts. Importantly, 4-MU4-deoxy-xyloside was able to counteract TGF-β1-induced synthesis of PGs, activation of fibroblast proliferation and fibroblast-myofibroblast differentiation. Mechanistically, 4-MU4-deoxy-xyloside treatment inhibited TGF-β1-induced activation of canonical Smads2/3 signaling pathway in lung primary fibroblasts. The knockdown of β4-galactosyltransferase7 mimicked 4-MU4-deoxy-xyloside effects, indicating selective inhibition of β4-galactosyltransferase7 by this compound. Collectively, this study reveals the anti-fibrotic activity of 4-MU4-deoxy-xyloside and indicates that inhibition of PG synthesis represents a novel strategy for the treatment of lung fibrosis.

  5. Targeting of Proteoglycan Synthesis Pathway: A New Strategy to Counteract Excessive Matrix Proteoglycan Deposition and Transforming Growth Factor-β1-Induced Fibrotic Phenotype in Lung Fibroblasts

    PubMed Central

    Shaukat, Irfan; Barré, Lydia; Venkatesan, Narayanan; Li, Dong; Jaquinet, Jean-Claude; Fournel-Gigleux, Sylvie; Ouzzine, Mohamed

    2016-01-01

    Stimulation of proteoglycan (PG) synthesis and deposition plays an important role in the pathophysiology of fibrosis and is an early and dominant feature of pulmonary fibrosis. Transforming growth factor-β1 (TGF-β1) is a major cytokine associated with fibrosis that induces excessive synthesis of matrix proteins, particularly PGs. Owing to the importance of PGs in matrix assembly and in mediating cytokine and growth factor signaling, a strategy based on the inhibition of PG synthesis may prevent excessive matrix PG deposition and attenuates profibrotic effects of TGF-β1 in lung fibroblasts. Here, we showed that 4-MU4-deoxy-β-D-xylopyranoside, a competitive inhibitor of β4-galactosyltransferase7, inhibited PG synthesis and secretion in a dose-dependent manner by decreasing the level of both chondroitin/dermatan- and heparin-sulfate PG in primary lung fibroblasts. Importantly, 4-MU4-deoxy-xyloside was able to counteract TGF-β1-induced synthesis of PGs, activation of fibroblast proliferation and fibroblast-myofibroblast differentiation. Mechanistically, 4-MU4-deoxy-xyloside treatment inhibited TGF-β1-induced activation of canonical Smads2/3 signaling pathway in lung primary fibroblasts. The knockdown of β4-galactosyltransferase7 mimicked 4-MU4-deoxy-xyloside effects, indicating selective inhibition of β4-galactosyltransferase7 by this compound. Collectively, this study reveals the anti-fibrotic activity of 4-MU4-deoxy-xyloside and indicates that inhibition of PG synthesis represents a novel strategy for the treatment of lung fibrosis. PMID:26751072

  6. Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin

    PubMed Central

    Fang, Chien-Liang; Huang, Ling-Hung; Tsai, Hung-Yueh; Chang, Hsin-I

    2016-01-01

    Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts. PMID:27428951

  7. Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin.

    PubMed

    Fang, Chien-Liang; Huang, Ling-Hung; Tsai, Hung-Yueh; Chang, Hsin-I

    2016-07-14

    Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts.

  8. Complexity of the transcriptional network controlling secondary wall biosynthesis.

    PubMed

    Zhong, Ruiqin; Ye, Zheng-Hua

    2014-12-01

    Secondary walls in the form of wood and fibers are the most abundant biomass produced by vascular plants, and are important raw materials for many industrial uses. Understanding how secondary walls are constructed is of significance in basic plant biology and also has far-reaching implications in genetic engineering of plant biomass better suited for various end uses, such as biofuel production. Secondary walls are composed of three major biopolymers, i.e., cellulose, hemicelluloses and lignin, the biosynthesis of which requires the coordinated transcriptional regulation of all their biosynthesis genes. Genomic and molecular studies have identified a number of transcription factors, whose expression is associated with secondary wall biosynthesis. We comprehensively review how these secondary wall-associated transcription factors function together to turn on the secondary wall biosynthetic program, which leads to secondary wall deposition in vascular plants. The transcriptional network regulating secondary wall biosynthesis employs a multi-leveled feed-forward loop regulatory structure, in which the top-level secondary wall NAC (NAM, ATAF1/2 and CUC2) master switches activate the second-level MYB master switches and they together induce the expression of downstream transcription factors and secondary wall biosynthesis genes. Secondary wall NAC master switches and secondary wall MYB master switches bind to and activate the SNBE (secondary wall NAC binding element) and SMRE (secondary wall MYB-responsive element) sites, respectively, in their target gene promoters. Further investigation of what and how developmental signals trigger the transcriptional network to regulate secondary wall biosynthesis and how different secondary wall-associated transcription factors function cooperatively in activating secondary wall biosynthetic pathways will lead to a better understanding of the molecular mechanisms underlying the transcriptional control of secondary wall biosynthesis.

  9. Curcumin induces glutathione biosynthesis and inhibits NF-kappaB activation and interleukin-8 release in alveolar epithelial cells: mechanism of free radical scavenging activity.

    PubMed

    Biswas, Saibal K; McClure, Danny; Jimenez, Luis A; Megson, Ian L; Rahman, Irfan

    2005-01-01

    Oxidants and tumor necrosis factor-alpha (TNF-alpha) activate transcription factors such as nuclear factor-kappaB (NF-kappaB), which is involved in the transcription of proinflammatory mediators, including interleukin-8 (IL-8). Curcumin (diferuloylmethane) is a naturally occurring flavonoid present in the spice turmeric, which has a long traditional use as a chemotherapeutic agent for many diseases. We hypothesize that curcumin may possess both antioxidant and antiinflammatory properties by increasing the glutathione levels and inhibiting oxidant- and cytokine-induced NF-kappaB activation and IL-8 release from cultured alveolar epithelial cells (A549). Treatment of A549 cells with hydrogen peroxide (H2O2; 100 microM) and TNF-alpha (10 ng/ml) significantly increased NF-kappaB and activator protein-1 (AP-1) activation, as well as IL-8 release. Curcumin inhibited both H2O2- and TNF-alpha-mediated activation of NF-kappaB and AP-1, and IL-8 release. Furthermore, an increased level of GSH and glutamylcysteine ligase catalytic subunit mRNA expression was observed in curcumin-treated cells as compared with untreated cells. Curcumin interacted directly with superoxide anion (O2*-) and hydroxyl radical (*OH) as shown by electron paramagnetic resonance, quenching the interaction of the radicals with the spin trap, Tempone-H. This suggests that curcumin has multiple properties: as an oxygen radical scavenger, antioxidant through modulation of glutathione levels, and antiinflammatory agent through inhibition of IL-8 release in lung cells.

  10. Enzymes in the extracellular matrix of Volvox: an inducible, calcium-dependent phosphatase with a modular composition.

    PubMed

    Hallmann, A

    1999-01-15

    The volvocine algae provide the unique opportunity for exploring development of an extracellular matrix. Volvox is the most advanced member of this family and represents the simplest multicellular organism, with differentiated cells, a complete division of labor, and a complex extracellular matrix, which serves structural and enzymatic functions. In Volvox carteri a glycosylated extracellular phosphatase was identified, which is partially released from the extracellular matrix into the growth medium. The phosphatase is synthesized in response to inorganic phosphate starvation and is strictly calcium-dependent. The metalloenzyme has been purified to homogeneity and characterized. Its gene and cDNA have been cloned. Comparisons of genomic and cDNA sequences revealed an extremely intron-rich gene (32 introns). With an apparent molecular mass of 160 kDa the Volvox extracellular phosphatase is the largest phosphatase cloned, with no sequence similarity to any other phosphatase. This enzyme exhibits a modular composition. There are two large domains and a small one. The large domains are highly homologous to each other and therefore most likely originated from gene duplication and fusion. At least one EF-hand motif for calcium binding was identified in this extracellular protein. Volvox extracellular phosphatase is the first calcium-dependent extracellular phosphatase to be cloned.

  11. Root-induced hydraulic changes of soil matrix - an in-situ experimental study in the wetland of Poyang Lake

    NASA Astrophysics Data System (ADS)

    Zhao, Z.; Jin, G.; Hua, G.; Chen, B.; Tao, X.; Li, L.

    2013-12-01

    Plant roots may change the structure of the surface soil layer and hence the groundwater flow in wetlands. Such modifications would affect the soil condition, which in turn may influence the growth of the plants and the structure of the natural vegetation community. In order to determine the relationship between the distribution of the roots and the hydraulic property of the soil matrix, we conducted a series of in-situ experiments based on falling head test at a field site in the wetland of Poyang Lake. Areas dominated by three typical plants in the wetland, Artemisia selengensis, Phalaris arundinacea and Carex tristachya, were examined. Comparisons were also made between areas with and without plants covered. The results show that the roots improved the flow conditions of soil matrix, especially for Carex tristachya, which had a great plant and root density. The volume fraction of the roots and the grain composition of the soil were also analyzed. The regression analyses show that the root density played an important role in affecting the saturated hydraulic conductivity near the surface where the soil matrix has a larger root volume fraction. Deep into soil (after 15cm from the surface), the root volume fraction decreased and the influence of the grain composition on changes of the saturated hydraulic conductivity became more significant. These results may improve the understandings of the relationship between hydrological conditions and the growth of vegetation in the wetland.

  12. Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo

    SciTech Connect

    Franco, Gilson C.N.; Nakanishi, Tadashi; Ohta, Kouji; Rosalen, Pedro L.; Groppo, Francisco C.; Bartlett, John D.; Stashenko, Philip; Taubman, Martin A.; Kawai, Toshihisa

    2011-06-10

    Tetracycline antibiotics, including doxycycli/e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.

  13. PPARγ-induced upregulation of CD36 enhances hematoma resolution and attenuates long-term neurological deficits after germinal matrix hemorrhage in neonatal rats.

    PubMed

    Flores, Jerry J; Klebe, Damon; Rolland, William B; Lekic, Tim; Krafft, Paul R; Zhang, John H

    2016-03-01

    Germinal matrix hemorrhage remains the leading cause of morbidity and mortality in preterm infants in the United States with little progress made in its clinical management. Survivors are often afflicted with long-term neurological sequelae, including cerebral palsy, mental retardation, hydrocephalus, and psychiatric disorders. Blood clots disrupting normal cerebrospinal fluid circulation and absorption after germinal matrix hemorrhage are thought to be important contributors towards post-hemorrhagic hydrocephalus development. We evaluated if upregulating CD36 scavenger receptor expression in microglia and macrophages through PPARγ stimulation, which was effective in experimental adult cerebral hemorrhage models and is being evaluated clinically, will enhance hematoma resolution and ameliorate long-term brain sequelae using a neonatal rat germinal matrix hemorrhage model. PPARγ stimulation (15d-PGJ2) increased short-term PPARγ and CD36 expression levels as well as enhanced hematoma resolution, which was reversed by a PPARγ antagonist (GW9662) and CD36 siRNA. PPARγ stimulation (15d-PGJ2) also reduced long-term white matter loss and post-hemorrhagic ventricular dilation as well as improved neurofunctional outcomes, which were reversed by a PPARγ antagonist (GW9662). PPARγ-induced upregulation of CD36 in macrophages and microglia is, therefore, critical for enhancing hematoma resolution and ameliorating long-term brain sequelae.

  14. PPARγ-induced Upregulation of CD36 Enhances Hematoma Resolution and Attenuates Long-term Neurological Deficits after Germinal Matrix Hemorrhage in Neonatal Rats

    PubMed Central

    Flores, Jerry J.; Klebe, Damon; Rolland, William B.; Lekic, Tim; Krafft, Paul R.; Zhang, John H.

    2016-01-01

    Germinal matrix hemorrhage remains the leading cause of morbidity and mortality in preterm infants in the United States with little progress made in its clinical management. Survivors are often afflicted with long-term neurological sequelae, including cerebral palsy, mental retardation, hydrocephalus, and psychiatric disorders. Blood clots disrupting normal cerebrospinal fluid circulation and absorption after germinal matrix hemorrhage are thought to be important contributors towards post-hemorrhagic hydrocephalus development. We evaluated if upregulating CD36 scavenger receptor expression in microglia and macrophages through PPARγ stimulation, which was effective in experimental adult cerebral hemorrhage models and is being evaluated clinically, will enhance hematoma resolution and ameliorate long-term brain sequelae using a neonatal rat germinal matrix hemorrhage model. PPARγ stimulation (15d-PGJ2) increased short-term PPARγ and CD36 expression levels as well as enhanced hematoma resolution, which was reversed by a PPARγ antagonist (GW9662) and CD36 siRNA. PPARγ stimulation (15d-PGJ2) also reduced long-term white matter loss and post-hemorrhagic ventricular dilation as well as improved neurofunctional outcomes, which were reversed by a PPARγ antagonist (GW9662). PPARγ-induced upregulation of CD36 in macrophages and microglia is, therefore, critical for enhancing hematoma resolution and ameliorating long-term brain sequelae. PMID:26739391

  15. Betulin suppressed interleukin-1β-induced gene expression, secretion and proteolytic activity of matrix metalloproteinase in cultured articular chondrocytes and production of matrix metalloproteinase in the knee joint of rat

    PubMed Central

    Ra, Ho Jong; Lee, Hyun Jae; Jo, Ho Seung; Nam, Dae Cheol; Lee, Young Bok; Kang, Byeong Hun; Moon, Dong Kyu; Kim, Dong Hee

    2017-01-01

    We investigated whether betulin affects the gene expression, secretion and proteolytic activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective effect of betulin. Rabbit articular chondrocytes were cultured and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of betulin on IL-1β-induced secretion and proteolytic activity of MMP-3 was investigated using western blot analysis and casein zymography, respectively. Effect of betulin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) betulin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) betulin inhibited the secretion and proteolytic activity of MMP-3; (3) betulin suppressed the production of MMP-3 protein in vivo. These results suggest that betulin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes. PMID:28066137

  16. Betulin suppressed interleukin-1β-induced gene expression, secretion and proteolytic activity of matrix metalloproteinase in cultured articular chondrocytes and production of matrix metalloproteinase in the knee joint of rat.

    PubMed

    Ra, Ho Jong; Lee, Hyun Jae; Jo, Ho Seung; Nam, Dae Cheol; Lee, Young Bok; Kang, Byeong Hun; Moon, Dong Kyu; Kim, Dong Hee; Lee, Choong Jae; Hwang, Sun-Chul

    2017-01-01

    We investigated whether betulin affects the gene expression, secretion and proteolytic activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective effect of betulin. Rabbit articular chondrocytes were cultured and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of betulin on IL-1β-induced secretion and proteolytic activity of MMP-3 was investigated using western blot analysis and casein zymography, respectively. Effect of betulin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) betulin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) betulin inhibited the secretion and proteolytic activity of MMP-3; (3) betulin suppressed the production of MMP-3 protein in vivo. These results suggest that betulin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes.

  17. Charge retention characteristics of silicide-induced crystallized polycrystalline silicon floating gate thin-film transistors for active matrix organic light-emitting diode.

    PubMed

    Park, Jae Hyo; Son, Se Wan; Byun, Chang Woo; Kim, Hyung Yoon; Joo, So Na; Lee, Yong Woo; Yun, Seung Jae; Joo, Seung Ki

    2013-10-01

    In this work, non-volatile memory thin-film transistor (NVM-TFT) was fabricated by nickel silicide-induced laterally crystallized (SILC) polycrystalline silicon (poly-Si) as the active layer. The nickel seed silicide-induced crystallized (SIC) poly-Si was used as storage layer which is embedded in the gate insulator. The novel unit pixel of active matrix organic light-emitting diode (AMOLED) using NVM-TFT is proposed and investigated the electrical and optical performance. The threshold voltage shift showed 17.2 V and the high reliability of retention characteristic was demonstrated until 10 years. The retention time can modulate the recharge refresh time of the unit pixel of AMOLED up to 5000 sec.

  18. Galangin and kaempferol suppress phorbol-12-myristate-13-acetate-induced matrix metalloproteinase-9 expression in human fibrosarcoma HT-1080 cells.

    PubMed

    Choi, Yu Jung; Lee, Young Hun; Lee, Seung-Taek

    2015-01-01

    Matrix metalloproteinase (MMP)-9 degrades type IV collagen in the basement membrane and plays crucial roles in several pathological implications, including tumorigenesis and inflammation. In this study, we analyzed the effect of flavonols on MMP-9 expression in phorbol-12-myristate-13-acetate (PMA)-induced human fibrosarcoma HT-1080 cells. Galangin and kaempferol efficiently decreased MMP-9 secretion, whereas fisetin only weakly decreased its secretion. Galangin and kaempferol did not affect cell viability at concentrations up to 30 μM. Luciferase reporter assays showed that galangin and kaempferol decrease transcription of MMP-9 mRNA. Moreover, galangin and kaempferol strongly reduce IκBα phosphorylation and significantly decrease JNK phosphorylation. These results indicate that galangin and kaempferol suppress PMA-induced MMP-9 expression by blocking activation of NF-κB and AP-1. Therefore, these flavonols could be used as chemopreventive agents to lower the risk of diseases involving MMP-9.

  19. CopA3 peptide prevents ultraviolet-induced inhibition of type-I procollagen and induction of matrix metalloproteinase-1 in human skin fibroblasts.

    PubMed

    Kim, Dong-Hee; Kim, Han-Hyuk; Kim, Hyeon-Jeong; Jung, Hyun-Gug; Yu, Jae-Myo; Lee, Eun-Su; Cho, Yong-Hun; Kim, Dong-In; An, Bong-Jeun

    2014-05-20

    Ultraviolet (UV) exposure is well-known to induce premature aging, which is mediated by matrix metalloproteinase-1 (MMP-1) activity. A 9-mer peptide, CopA3 (CopA3) was synthesized from a natural peptide, coprisin, which is isolated from the dung beetle Copris tripartitus. As part of our continuing search for novel bioactive natural products, CopA3 was investigated for its in vitro anti-skin photoaging activity. UV-induced inhibition of type-I procollagen and induction of MMP-1 were partially prevented in human skin fibroblasts by CopA3 peptide in a dose-dependent manner. At a concentration of 25 μM, CopA3 nearly completely inhibited MMP-1 expression. These results suggest that CopA3, an insect peptide, is a potential candidate for the prevention and treatment of skin aging.

  20. Biosynthesis of the phytoalexin pisatin. [Pisum sativum

    SciTech Connect

    Preisig, C.L.; Bell, J.N.; Matthews, D.E.; VanEtten, H.D. ); Sun, Yuejin; Hrazdina, G. )

    1990-11-01

    NADPH-dependent reduction of 2{prime},7-dihydroxy-4{prime},5{prime}-methylenedioxyisoflavone to the isoflavanone sophorol, a proposed intermediate step in pisatin biosynthesis, was detected in extracts of Pisum sativum. This isoflavone reductase activity was inducible by treatment of pea seedlings with CuCl{sub 2}. The timing of induction coincided with that of the 6a-hydroxymaackiain 3-O-methyltransferase, which catalyzes the terminal biosynthetic step. Neither enzyme was light inducible. Further NADPH-dependent metabolism of sophorol by extracts of CuCl{sub 2}-treated seedlings was also observed; three products were radiolabeled when ({sup 3}H)sophorol was the substrate, one of which is tentatively identified as maackiain.

  1. Beta-adrenoceptor Activation by Norepinephrine Enhances Lipopolysaccharide-induced Matrix Metalloproteinase-9 Expression Through the ERK/JNK-c-Fos Pathway in Human THP-1 Cells

    PubMed Central

    Yin, Xiang; Zhou, Linli; Han, Fei; Han, Jie; Zhang, Yuanyuan; Sun, Zewei; Zhao, Wenting; Wang, Zhen

    2017-01-01

    Aim: Atherosclerosis is a chronic inflammatory disease, which leads to thrombosis and acute coronary syndrome. Matrix metalloproteinase-9 (MMP-9) is involved in the stability of the extracellular matrix (ECM) and atherosclerosis plaque. Until now, it is established that lipopolysaccharide (LPS) and norepinephrine (NE) are associated with the pathological process of atherosclerosis. However, the combined effect of LPS and NE on MMP-9 is unclear. We investigated the combined effect of LPS and NE on MMP-9 expression in human monocytes and the mechanism involved in the process. Methods: THP-1 cells were cultured and treated with LPS and/or NE. MMP-9 and TIMP-1 gene and protein expression were detected by real time PCR and ELISA, respectively. MMP-9 activity was detected by gelatin zymography. Adrenoceptor antagonists and MAPKs inhibitors were used to clarify the mechanism. Pathway-related proteins were detected by Western blot. Results: We found that NE enhances LPS-induced MMP-9 and TIMP-1 expression as well as MMP-9 activity in THP-1 cells. This effect is reversed by the beta (β)-adrenoceptor antagonist propranolol, extracellular signal-regulated kinases (ERK) inhibitor U0126, and c-Jun N-terminal kinase (JNK) inhibitor SP600125. NE enhances LPS-induced ERK/JNK phosphorylation. NE up-regulates LPS-induced c-Fos expression, which is counteracted by propranolol, U0126, and SP600125. Furthermore, c-Fos silence reverses the effect of NE on MMP-9 activity. Conclusions: Our results suggest that NE enhances LPS-induced MMP-9 expression through β-adrenergic receptor and downstream ERK/JNK-c-Fos pathway. This study may help us to understand the combined effect and mechanism of NE/LPS on MMP-9 expression. PMID:27237101

  2. Extracellular matrix metalloproteinase inducer (CD147) is a novel receptor on platelets, activates platelets, and augments nuclear factor kappaB-dependent inflammation in monocytes.

    PubMed

    Schmidt, Roland; Bültmann, Andreas; Fischel, Sina; Gillitzer, Angelika; Cullen, Paul; Walch, Axel; Jost, Philipp; Ungerer, Martin; Tolley, Neal D; Lindemann, Stephan; Gawaz, Meinrad; Schömig, Albert; May, Andreas E

    2008-02-15

    In atherosclerosis, circulating platelets interact with endothelial cells and monocytes, leading to cell activation and enhanced recruitment of leukocytes into the vascular wall. The invasion of monocytes is accompanied by overexpression of matrix metalloproteinases (MMPs), which are thought to promote atherosclerosis and trigger plaque rupture. Following interaction with itself, the extracellular matrix metalloproteinase inducer (EMMPRIN) induces MMP synthesis via a little-known intracellular pathway. Recently, we showed upregulation of EMMPRIN on monocytes during acute myocardial infarction. EMMPRIN also stimulates secretion of MMP-9 by monocytes and of MMP-2 by smooth muscle cells, indicating that it may be an important regulator of MMP activity. Expression of EMMPRIN on platelets has not been described until now. Here, we demonstrate that resting platelets show low surface expression of EMMPRIN, which is upregulated by various platelet stimulators (flow cytometry). EMMPRIN is located in the open canalicular system and in alpha granules of platelets (according to electron microscopy and sucrose gradient ultracentrifugation). Platelet stimulation with recombinant EMMPRIN-Fc induced surface expression of CD40L and P-selectin (according to flow cytometry), suggesting that EMMPRIN-EMMPRIN interaction activates platelets. Coincubation of platelets with monocytes induced EMMPRIN-mediated nuclear factor kappaB activation (according to Western blot) in monocytes with increased MMP-9 (zymography), interleukin-6, and tumor necrosis factor-alpha secretion (according to ELISA) by monocytes. In conclusion, EMMPRIN displays a new platelet receptor that is upregulated on activated platelets. Binding of EMMPRIN to platelets fosters platelet degranulation. Platelet-monocyte interactions via EMMPRIN stimulate nuclear factor kappaB-driven inflammatory pathways in monocytes, such as MMP and cytokine induction. Thus, EMMPRIN may represent a novel target to diminish the burden of

  3. Reduced Peripheral PGE2 Biosynthesis in Plasmodium falciparum Malaria Occurs through Hemozoin-Induced Suppression of Blood Mononuclear Cell Cyclooxygenase-2 Gene Expression via an Interleukin-10–Independent Mechanism

    PubMed Central

    Keller, Christopher C; Hittner, James B; Nti, Benjamin K; Weinberg, J Brice; Kremsner, Peter G; Perkins, Douglas J

    2004-01-01

    Molecular immunologic determinants of disease severity during Plasmodium falciparum malaria are largely undetermined. Our recent investigations showed that peripheral blood mononuclear cell (PBMC) cyclooxygenase-2 (COX-2) gene expression and plasma prostaglandin E2 (PGE2) production are suppressed in children with falciparum malaria relative to healthy, malaria-exposed children with partial immunity. Furthermore, decreased COX-2/PGE2 levels were significantly associated with increased plasma interleukin-10 (IL-10), an anti-inflammatory cytokine that inhibits the expression of COX-2 gene products. To determine the mechanism(s) responsible for COX-2–derived PGE2 suppression, PBMCs were cultured from children with falciparum malaria. PGE2 production was suppressed under baseline and COX-2–promoting conditions (stimulation with lipopolysaccharide [LPS] and interferon [IFN]-γ) over prolonged periods, suggesting that an in vivo–derived product(s) was responsible for reduced PGE2 biosynthesis. Ingestion of hemozoin (malarial pigment) by PBMC was investigated as a source of COX-2/PGE2 suppression in PBMCs from healthy, malaria-naive adults. In addition, synthetically prepared hemozoin, β-hematin, was used to investigate the effects of the core iron component of hemozoin, ferriprotoporphyrin-IX (FPIX). Physiologic concentrations of hemozoin or β-hematin suppressed LPS- and IFN-γ–induced COX-2 mRNA in a time- and dose-dependent manner, resulting in decreased COX-2 protein and PGE2 production. Suppression of COX-2/PGE2 by hemozoin was not due to decreased cell viability as evidenced by examination of mitochondrial bioactivity. These data illustrate that ingestion of FPIX by blood mononuclear cells is responsible for suppression of COX-2/PGE2. Although hemozoin induced overproduction of IL-10, neutralizing IL-10 antibodies failed to restore PGE2 production. Thus, acquisition of hemozoin by blood mononuclear cells is responsible for suppression of PGE2 in malaria

  4. Heme and heme biosynthesis intermediates induce heme oxygenase-1 and cytochrome P450 2A5, enzymes with putative sequential roles in heme and bilirubin metabolism: different requirement for transcription factor nuclear factor erythroid- derived 2-like 2.

    PubMed

    Lämsä, Virpi; Levonen, Anna-Liisa; Sormunen, Raija; Yamamoto, Masayuki; Hakkola, Jukka

    2012-11-01

    Cytochrome P450 2A5 (CYP2A5) oxidizes bilirubin to biliverdin and represents a putative candidate for maintaining bilirubin at safe but adequate antioxidant levels. Curiously, CYP2A5 is induced by both excessive heme and chemicals that inhibit heme synthesis. We hypothesized that heme homeostasis is a key modifier of Cyp2a5 expression via transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2) and characterized the coordination of CYP2A5 and heme oxygenase-1 (HMOX1) responses using wild-type and Nrf2(-/-) primary mouse hepatocytes. HMOX1 was rapidly elevated by exogenous hemin, thereby limiting the transactivation of Cyp2a5 until high heme (> 5µM) exposure. Nrf2 was mandatory for CYP2A5 but not for HMOX1 induction by heme. CYP2A5 was intensively and HMOX1 moderately elevated in heme synthesis blockades by succinylacetone and N-methyl protoporphyrin IX, and Nrf2 partially mediated the induction of CYP2A5. Immunoelectron microscopy revealed that CYP2A5 is targeted Nrf2 dependently both to the endoplasmic reticulum (ER) and mitochondria. However, excessive heme increased CYP2A5 predominantly in the ER. Phenobarbital, dibutyryl-cAMP, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) overexpression stimulate heme biosynthesis and induce CYP2A5. Acute but not chronic CYP2A5 induction by phenobarbital required Nrf2, whereas CYP2A5 induction by dibutyryl-cAMP and PGC-1α was potentiated by Nrf2 knockout. Collectively, heme homeostasis is established as a crucial regulator of hepatic Cyp2a5 expression mediated via Nrf2 activation, whereas Nrf2 is redundant for Hmox1 induction by heme. Similar subcellular targeting and coordination of CYP2A5 and HMOX1 responses suggest favorable conditions for enhanced CYP2A5-mediated bilirubin maintenance in altered heme homeostasis that predisposes to oxidative stress.

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

    PubMed

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

    2012-02-01

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

  6. IL-1β-induced, matrix metalloproteinase-3-regulated proliferation of embryonic stem cell-derived odontoblastic cells is mediated by the Wnt5 signaling pathway

    SciTech Connect

    Ozeki, Nobuaki; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kawai, Rie; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2014-10-15

    We previously established a method for differentiating induced pluripotent stem cells and embryonic stem (ES) cells into α2 integrin-positive odontoblast-like cells. We also reported that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation and suppresses apoptosis in these cells, suggesting that MMP-3 plays a potentially unique physiological role in the regeneration of odontoblast-like cells. Here, we examined whether up-regulation of MMP-3 activity by IL-1β was mediated by Wnt signaling and led to increased proliferation of odontoblast-like cells. IL-1β increased mRNA and protein levels of Wnt5a, Wnt5b and the Wnt receptor Lrp5. Exogenous Wnt5a and Wnt5b were found to increase MMP-3 mRNA, protein and activity, and interestingly the rate of proliferation in these cells. Treatment with siRNAs against Wnt5a, Wnt5b and Lrp5 suppressed the IL-1β-induced increase in MMP-3 expression and suppressed cell proliferation, an effect rescued by application of exogenous Wnt5. These results demonstrate the sequential involvement of Wnt5, Lrp5 and MMP-3 in effecting IL-1β-induced proliferation of ES cell-derived odontoblast-like cells. - Highlights: • IL-1β induces Wnt5, Lrp5/Fzd9 and MMP-3 in ES cell-derived odontoblast-like cells. • IL-1β-induced Wnt5 expression results in increased cell proliferation. • Exogenous Wnt5 increases MMP-3 activity and cell proliferation. • Exogenous Wnt5 rescues IL-1β-driven proliferation with anti-Wnt5 siRNA suppression. • IL-1β-induced cell proliferation involves Wnt5, Lrp5, and MMP-3 sequentially.

  7. Auxin biosynthesis and storage forms.

    PubMed

    Korasick, David A; Enders, Tara A; Strader, Lucia C

    2013-06-01

    The plant hormone auxin drives plant growth and morphogenesis. The levels and distribution of the active auxin indole-3-acetic acid (IAA) are tightly controlled through synthesis, inactivation, and transport. Many auxin precursors and modified auxin forms, used to regulate auxin homeostasis, have been identified; however, very little is known about the integration of multiple auxin biosynthesis and inactivation pathways. This review discusses the many ways auxin levels are regulated through biosynthesis, storage forms, and inactivation, and the potential roles modified auxins play in regulating the bioactive pool of auxin to affect plant growth and development.

  8. Auxin biosynthesis and storage forms

    PubMed Central

    Strader, Lucia C.

    2013-01-01

    The plant hormone auxin drives plant growth and morphogenesis. The levels and distribution of the active auxin indole-3-acetic acid (IAA) are tightly controlled through synthesis, inactivation, and transport. Many auxin precursors and modified auxin forms, used to regulate auxin homeostasis, have been identified; however, very little is known about the integration of multiple auxin biosynthesis and inactivation pathways. This review discusses the many ways auxin levels are regulated through biosynthesis, storage forms, and inactivation, and the potential roles modified auxins play in regulating the bioactive pool of auxin to affect plant growth and development. PMID:23580748

  9. Overexpression of Arabidopsis ECERIFERUM1 Promotes Wax Very-Long-Chain Alkane Biosynthesis and Influences Plant Response to Biotic and Abiotic Stresses1[W

    PubMed Central

    Bourdenx, Brice; Bernard, Amélie; Domergue, Frédéric; Pascal, Stéphanie; Léger, Amandine; Roby, Dominique; Pervent, Marjorie; Vile, Denis; Haslam, Richard P.; Napier, Johnathan A.; Lessire, René; Joubès, Jérôme

    2011-01-01

    Land plant aerial organs are covered by a hydrophobic layer called the cuticle that serves as a waterproof barrier protecting plants against desiccation, ultraviolet radiation, and pathogens. Cuticle consists of a cutin matrix as well as cuticular waxes in which very-long-chain (VLC) alkanes are the major components, representing up to 70% of the total wax content in Arabidopsis (Arabidopsis thaliana) leaves. However, despite its major involvement in cuticle formation, the alkane-forming pathway is still largely unknown. To address this deficiency, we report here the characterization of the Arabidopsis ECERIFERUM1 (CER1) gene predicted to encode an enzyme involved in alkane biosynthesis. Analysis of CER1 expression showed that CER1 is specifically expressed in the epidermis of aerial organs and coexpressed with other genes of the alkane-forming pathway. Modification of CER1 expression in transgenic plants specifically affects VLC alkane biosynthesis: waxes of TDNA insertional mutant alleles are devoid of VLC alkanes and derivatives, whereas CER1 overexpression dramatically increases the production of the odd-carbon-numbered alkanes together with a substantial accumulation of iso-branched alkanes. We also showed that CER1 expression is induced by osmotic stresses and regulated by abscisic acid. Furthermore, CER1-overexpressing plants showed reduced cuticle permeability together with reduced soil water deficit susceptibility. However, CER1 overexpression increased susceptibility to bacterial and fungal pathogens. Taken together, these results demonstrate that CER1 controls alkane biosynthesis and is highly linked to responses to biotic and abiotic stresses. PMID:21386033

  10. Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses.

    PubMed

    Bourdenx, Brice; Bernard, Amélie; Domergue, Frédéric; Pascal, Stéphanie; Léger, Amandine; Roby, Dominique; Pervent, Marjorie; Vile, Denis; Haslam, Richard P; Napier, Johnathan A; Lessire, René; Joubès, Jérôme

    2011-05-01

    Land plant aerial organs are covered by a hydrophobic layer called the cuticle that serves as a waterproof barrier protecting plants against desiccation, ultraviolet radiation, and pathogens. Cuticle consists of a cutin matrix as well as cuticular waxes in which very-long-chain (VLC) alkanes are the major components, representing up to 70% of the total wax content in Arabidopsis (Arabidopsis thaliana) leaves. However, despite its major involvement in cuticle formation, the alkane-forming pathway is still largely unknown. To address this deficiency, we report here the characterization of the Arabidopsis ECERIFERUM1 (CER1) gene predicted to encode an enzyme involved in alkane biosynthesis. Analysis of CER1 expression showed that CER1 is specifically expressed in the epidermis of aerial organs and coexpressed with other genes of the alkane-forming pathway. Modification of CER1 expression in transgenic plants specifically affects VLC alkane biosynthesis: waxes of TDNA insertional mutant alleles are devoid of VLC alkanes and derivatives, whereas CER1 overexpression dramatically increases the production of the odd-carbon-numbered alkanes together with a substantial accumulation of iso-branched alkanes. We also showed that CER1 expression is induced by osmotic stresses and regulated by abscisic acid. Furthermore, CER1-overexpressing plants showed reduced cuticle permeability together with reduced soil water deficit susceptibility. However, CER1 overexpression increased susceptibility to bacterial and fungal pathogens. Taken together, these results demonstrate that CER1 controls alkane biosynthesis and is highly linked to responses to biotic and abiotic stresses.

  11. Increased calvaria cell differentiation and bone matrix formation induced by fibroblast growth factor receptor 2 mutations in Apert syndrome.

    PubMed

    Lomri, A; Lemonnier, J; Hott, M; de Parseval, N; Lajeunie, E; Munnich, A; Renier, D; Marie, P J

    1998-03-15

    Apert syndrome, associated with fibroblast growth factor receptor (FGFR) 2 mutations, is characterized by premature fusion of cranial sutures. We analyzed proliferation and differentiation of calvaria cells derived from Apert infants and fetuses with FGFR-2 mutations. Histological analysis revealed premature ossification, increased extent of subperiosteal bone formation, and alkaline phosphatase- positive preosteoblastic cells in Apert fetal calvaria compared with age-matched controls. Preosteoblastic calvaria cells isolated from Apert infants and fetuses showed normal cell growth in basal conditions or in response to exogenous FGF-2. In contrast, the number of alkaline phosphatase- positive calvaria cells was fourfold higher than normal in mutant fetal calvaria cells with the most frequent Apert FGFR-2 mutation (Ser252Trp), suggesting increased maturation rate of cells in the osteoblastic lineage. Biochemical and Northern blot analyses also showed that the expression of alkaline phosphatase and type 1 collagen were 2-10-fold greater than normal in mutant fetal calvaria cells. The in vitro production of mineralized matrix formed by immortalized mutant fetal calvaria cells cultured in aggregates was also increased markedly compared with control immortalized fetal calvaria cells. The results show that Apert FGFR-2 mutations lead to an increase in the number of precursor cells that enter the osteogenic pathway, leading ultimately to increased subperiosteal bone matrix formation and premature calvaria ossification during fetal development, which establishes a connection between the altered genotype and cellular phenotype in Apert syndromic craniosynostosis.

  12. A qualitative tool combining an interaction matrix and a GIS to map vulnerability to traffic induced air pollution.

    PubMed

    Mavroulidou, Maria; Hughes, Susan J; Hellawell, Emma E

    2004-04-01

    Local authorities and transport planners need fast and straightforward tools to perform their preliminary air quality assessments. Such tools are required to provide an initial impression of the local air quality and to highlight areas requiring a more rigorous investigation. This paper presents a technique to develop such a tool, for performing an initial assessment of air quality due to traffic in an urban area. The technique combines an interaction matrix methodology as developed for rock engineering systems, with Geographical Information System (GIS) map overlaying. This interaction matrix methodology incorporates a total system approach, which identifies the main parameters and quantifies the interactions between them. Weighting values for these parameters are obtained either through parametric studies, using numerical modelling, or from engineering judgement. These weightings are applied to spatial datasets for a study area using a GIS. The GIS results are presented in the form of a vulnerability map, which highlights the areas susceptible to poor air quality. This visual interpretation of the results is ideal for local authorities, who have to report to a wide range of non-specialists in the field, for example, planners, councillors and the public. The vulnerability map compares favourably with pollutant concentration patterns, obtained from an advanced dispersion model.

  13. CIL-102 induces matrix metalloproteinase-2 (MMP-2)/MMP-9 down-regulation via simultaneous suppression of genetic transcription and mRNA stability.

    PubMed

    Liu, Wen-Hsin; Chen, Yeh-Long; Chang, Long-Sen

    2012-12-01

    This study explores the CIL-102 suppression mechanism on matrix metalloproteinase-2 (MMP-2) and MMP-9 expression in human leukemia K562 cells. CIL-102 attenuated K562 cell invasion with decreased MMP-2/MMP-9 protein expression and mRNA levels. Moreover, CIL-102 reduced luciferase activity of MMP-2/MMP-9 promoter constructs and MMP-2/MMP-9 mRNA stability. CIL-102 treatment induced JNK and p38 MAPK activation but reduced the phospho-ERK level. Transfection of constitutively active MEK1 restored MMP-2 and MMP-9 promoter activity in CIL-102-treated cells, while suppression of p38 MAPK/JNK activation abolished CIL-102-induced MMP-2/MMP-9 mRNA decay. CIL-102-induced p38 MAPK/JNK activation led to protein phosphatase 2A-mediated tristetraprolin (TTP) down-regulation. The reduction in TTP-KH-type splicing regulatory protein (KSRP) complexes formation promoted KSRP-mediated MMP-2/MMP-9 mRNA decay in CIL-102-treated K562 cells. Moreover, CIL-102 reduced invasion and MMP-2/MMP-9 expression in breast and liver cancer cells. Taken together, our data indicate that CIL-102 induces MMP-2/MMP-2 down-regulation via simultaneous suppression of genetic transcription and mRNA stability, and suggest a potential utility for CIL-102 in reducing MMP-2/MMP-9-mediated cancer progression.

  14. Ethanol extract of Cordyceps militaris grown on germinated soybeans attenuates dextran-sodium-sulfate- (DSS-) induced colitis by suppressing the expression of matrix metalloproteinases and inflammatory mediators.

    PubMed

    Park, Dong Ki; Park, Hye-Jin

    2013-01-01

    The effect of Cordyceps militaris (CM) grown on germinated soybeans (GSC) in the inflammatory bowel disease (IBD) model was studied. To demonstrate the preventive effect of GSC extract in a dextran-sodium-sulfate- (DSS-) induced acute colitis mouse model, GSC was administered 2 days before DSS coadministration. GSC significantly suppressed DSS-induced disease activity index (DAI) as well as histopathological scores, compared to control or CM-treated group. To elucidate the anti-IBD activity of GSC, we checked the level of matrix metalloproteinases (MMPs) and inflammatory mediators. GSC extract decreased the level of MMP-3 and -9 mRNAs and p53 proteins. The level and activity of LPS-induced MMP-9 were reduced in GSC-treated RAW264.7 cells. It also attenuated the level of inducible nitric oxide synthase (iNOS) and tumor necrosis factor- (TNF-) α mRNAs both in colon tissue and in macrophage cells. These results suggest that GSC can be applied as a protective agent against IBDs.

  15. Effects of gangliosides from deer bone extract on the gene expressions of matrix metalloproteinases and collagen type II in interleukin-1β-induced osteoarthritic chondrocytes

    PubMed Central

    Suh, Hyung Joo; Lee, Hyunji; Min, Byung Jung; Jung, Sung Ug

    2016-01-01

    BACKGROUND/OBJECTIVES We investigated the anti-osteoarthritic effects of deer bone extract on the gene expressions of matrix metalloproteinases (MMPs) and collagen type II (COL2) in interleukin-1β-induced osteoarthritis (OA) chondrocytes. MATERIALS/METHODS Primary rabbit chondrocytes were treated as follows: CON (PBS treatment), NC (IL-1β treatment), PC (IL-1β + 100 µg/mL glucosamine sulphate/chondroitin sulphate mixture), and DB (IL-1β + 100 µg/mL deer bone extract). RESULTS The results of the cell viability assay indicated that deer bone extract at doses ranging from 100 to 500 µg/mL inhibits cell death in chondrocytes induced by IL-1β. Deer bone extract was able to significantly recover the mRNA expression of COL2 that was down-regulated by IL-1β (NC: 0.79 vs. DB: 0.87, P < 0.05) and significantly decrease the mRNA expression of MMP-3 (NC: 2.24 vs. DB: 1.75) and -13 (NC: 1.28 vs. DB: 0.89) in OA chondrocytes (P < 0.05). CONCLUSIONS We concluded that deer bone extract induces accumulation of COL2 through the down-regulation of MMPs in IL-1β-induced OA chondrocytes. Our results suggest that deer bone extract, which contains various components related to OA, including chondroitin sulphate, may possess anti-osteoarthritic properties and be of value in inhibiting the pathogenesis of OA. PMID:27909553

  16. Inhibitory effect of Chinese green tea on cigarette smoke-induced up-regulation of airway neutrophil elastase and matrix metalloproteinase-12 via antioxidant activity.

    PubMed

    Chan, Ka Ho; Chan, Stanley Chi Hang; Yeung, Sze Chun; Man, Ricky Ying Keung; Ip, Mary Sau Man; Mak, Judith Choi Wo

    2012-09-01

    Our recent study has indicated that Chinese green tea (Lung Chen), in which epigallocatechin-3-gallate (EGCG) accounts for 60% of catechins, protected cigarette smoke-induced lung injury. We now hypothesized that Lung Chen tea may also have potential effect on lung oxidative stress and proteases/anti-proteases in a smoking rat model. Sprague-Dawley rats were exposed to either sham air (SA) or 4% cigarette smoke (CS) plus 2% Lung Chen tea or water by oral gavage. Serine proteases, matrix metalloproteinases (MMPs) and their respective endogenous inhibitors were determined in bronchoalveolar lavage (BAL) and lung tissues by gelatin/casein zymography and biochemical assays. Green tea consumption significantly decreased CS-induced elevation of lung lipid peroxidation marker, malondialdehyde (MDA), and CS-induced up-regulation of neutrophil elastase (NE) concentration and activity along with that of α(1)-antitrypsin (α(1)-AT) and secretory leukoproteinase inhibitor (SLPI) in BAL and lung. In parallel, significant elevation of MMP-12 activity was found in BAL and lung of the CS-exposed group, which returned to the levels of SA-exposed group after green tea consumption but not CS-induced reduction of tissue inhibitor of metalloproteinase (TIMP)-1 activity, which was not reversed by green tea consumption. Taken together, our data supported the presence of local oxidative stress and protease/anti-protease imbalance in the airways after CS exposure, which might be alleviated by green tea consumption through its biological antioxidant activity.

  17. Ethanol Extract of Cordyceps militaris Grown on Germinated Soybeans Attenuates Dextran-Sodium-Sulfate- (DSS-) Induced Colitis by Suppressing the Expression of Matrix Metalloproteinases and Inflammatory Mediators

    PubMed Central

    Park, Dong Ki; Park, Hye-Jin

    2013-01-01

    The effect of Cordyceps militaris (CM) grown on germinated soybeans (GSC) in the inflammatory bowel disease (IBD) model was studied. To demonstrate the preventive effect of GSC extract in a dextran-sodium-sulfate- (DSS-) induced acute colitis mouse model, GSC was administered 2 days before DSS coadministration. GSC significantly suppressed DSS-induced disease activity index (DAI) as well as histopathological scores, compared to control or CM-treated group. To elucidate the anti-IBD activity of GSC, we checked the level of matrix metalloproteinases (MMPs) and inflammatory mediators. GSC extract decreased the level of MMP-3 and -9 mRNAs and p53 proteins. The level and activity of LPS-induced MMP-9 were reduced in GSC-treated RAW264.7 cells. It also attenuated the level of inducible nitric oxide synthase (iNOS) and tumor necrosis factor- (TNF-) α mRNAs both in colon tissue and in macrophage cells. These results suggest that GSC can be applied as a protective agent against IBDs. PMID:23841050

  18. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    SciTech Connect

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kinoshita, Katsue; Hase, Naoko; Nakata, Kazuhiko; Kondo, Ayami; Mogi, Makio; Nakamura, Hiroshi

    2014-04-15

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7{sup +}hSMSC)-derived osteoblast-like (α7{sup +}hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7{sup +}hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7{sup +}hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7{sup +}hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form via

  19. Matrix metalloproteinase-9, -10, and -12, MDM2 and p53 expression in mouse liver during dimethylnitrosamine-induced oxidative stress and genomic injury.

    PubMed

    Syed, Ismail; Rathod, Jasmine; Parmar, Mayur; Corcoran, George B; Ray, Sidhartha D

    2012-06-01

    Treatment during early tumor development has greater success because tissue growth remains largely confined to its original locus. At later stages, malignant cells migrate from their original location, invade surrounding normal areas, and can disseminate widely throughout the body. Remodeling of the extracellular matrix (ECM) serves as a key facilitator of this dissemination. Proteolytic enzymes including plasmin and matrix metalloproteinases (MMPs) play an integral role in degrading the surrounding ECM proteins and clearing a path for tumor cell migration. Specific MMPs are highly expressed late during malignant tumor invasion. It is not understood whether early changes in MMPs influence apoptotic and necrotic cell death, processes known to govern the early stages of carcinogenesis. Similarly, the interaction between MDM2 and p53 is tightly controlled by a complex array of post-translational modifications, which in turn dictates the stability and activity of both p53 and MDM2. The present studies examine the hypothesis that model hepatotoxin dimethylnitrosamine (DMN), which is also a model carcinogen, will induce the MMP family of proteins after administration in hepatotoxic doses. Doses of 25, 50, and 100 mg/kg DMN were administered i.p. to male C3H mice. Changes in parameters associated with apoptotic and necrotic cell death, DNA damage, cell proliferation, and extracellular proteinases were examined in liver at 24 h. Serum ALT activity, oxidative stress [malondialdehyde], and caspase-activated DNAse mediated DNA laddering increased in a dose-dependent manner, as did the level of MDM2 protein. MMP-9, -10 and -12 (gelatinase-B, stromelysin-2, macrophage elastase), and p53 protein levels increased following 25 mg/kg DMN, but were successively decreased after higher DMN doses. The results of this study demonstrate changes in MDM2 and MMPs during DMN-induced acute liver injury and provide a plausible linkage between DMN-induced oxidative stress-mediated genomic

  20. Membrane Type 1 Matrix Metalloproteinase induces an epithelial to mesenchymal transition and cancer stem cell-like properties in SCC9 cells

    PubMed Central

    2013-01-01

    Background Tissue invasion and metastasis are acquired abilities of cancer and related to the death in oral squamous cell carcinoma (OSCC). Emerging observations indicate that the epithelial-to-mesenchymal transition (EMT) is associated with tumor progression and the generation of cells with cancer stem cells (CSCs) properties. Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) is a cell surface proteinase, which is involved in degrading extracellular matrix components that can promote tumor invasion and cell migration. Methods In the current study, we utilized SCC9 cells stably transfected with an empty vector (SCC9-N) or a vector encoding human MT1-MMP (SCC9-M) to study the role of MT1-MMP in EMT development. Results Upon up-regulation of MT1-MMP, SCC9-M cells underwent EMT, in which they presented a fibroblast-like phenotype and had a decreased expression of epithelial markers (E-cadherin, cytokeratin18 and β-catenin) and an increased expression of mesenchymal markers (vimentin and fibronectin). We further demonstrated that MT1-MMP-induced morphologic changes increased the level of Twist and ZEB, and were dependent on repressing the transcription of E-cadherin. These activities resulted in low adhesive, high invasive abilities of the SCC9-M cells. Furthermore, MT1-MMP-induced transformed cells exhibited cancer stem cell (CSC)-like characteristics, such as low proliferation, self-renewal ability, resistance to chemotherapeutic drugs and apoptosis, and expression of CSCs surface markers. Conclusions In conclusion, our study indicates that overexpression of MT1-MMP induces EMT and results in the acquisition of CSC-like properties in SCC9 cells. Our growing understanding of the mechanism regulating EMT may provide new targets against invasion and metastasis in OSCC. PMID:23548172

  1. TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    SciTech Connect

    Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin; Yao, Jin

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  2. Biosynthesis of the Caenorhabditis elegans dauer pheromone.

    PubMed

    Butcher, Rebecca A; Ragains, Justin R; Li, Weiqing; Ruvkun, Gary; Clardy, Jon; Mak, Ho Yi

    2009-02-10

    To sense its population density and to trigger entry into the stress-resistant dauer larval stage, Caenorhabditis elegans uses the dauer pheromone, which consists of ascaroside derivatives with short, fatty acid-like side chains. Although the dauer pheromone has been studied for 25 years, its biosynthesis is completely uncharacterized. The daf-22 mutant is the only known mutant defective in dauer pheromone production. Here, we show that daf-22 encodes a homolog of human sterol carrier protein SCPx, which catalyzes the final step in peroxisomal fatty acid beta-oxidation. We also show that dhs-28, which encodes a homolog of the human d-bifunctional protein that acts just upstream of SCPx, is also required for pheromone production. Long-term daf-22 and dhs-28 cultures develop dauer-inducing activity by accumulating less active, long-chain fatty acid ascaroside derivatives. Thus, daf-22 and dhs-28 are required for the biosynthesis of the short-chain fatty acid-derived side chains of the dauer pheromone and link dauer pheromone production to metabolic state.

  3. Disorders of carnitine biosynthesis and transport.

    PubMed

    El-Hattab, Ayman W; Scaglia, Fernando

    2015-11-01

    Carnitine is a hydrophilic quaternary amine that plays a number of essential roles in metabolism with the main function being the transport of long-chain fatty acids from the cytosol to the mitochondrial matrix for β-oxidation. Carnitine can be endogenously synthesized. However, only a small fraction of carnitine is obtained endogenously while the majority is obtained from diet, mainly animal products. Carnitine is not metabolized and is excreted in urine. Carnitine homeostasis is regulated by efficient renal reabsorption that maintains carnitine levels within the normal range despite variabilities in dietary intake. Diseases occurring due to primary defects in carnitine metabolism and homeostasis are comprised in two groups: disorders of carnitine biosynthesis and carnitine transport defect. While the hallmark of carnitine transport defect is profound carnitine depletion, disorders of carnitine biosynthesis do not cause carnitine deficiency due to the fact that both carnitine obtained from diet and efficient renal carnitine reabsorption can maintain normal carnitine levels with the absence of endogenously synthesized carnitine. Carnitine transport defect phenotype encompasses a broad clinical spectrum including metabolic decompensation in infancy, cardiomyopathy in childhood, fatigability in adulthood, or absence of symptoms. The phenotypes associated with the carnitine transport defect result from the unavailability of enough carnitine to perform its functions particularly in fatty acid β-oxidation. Carnitine biosynthetic defects have been recently described and the phenotypic consequences of these defects are still emerging. Although these defects do not result in carnitine deficiency, they still could be associated with pathological phenotypes due to excess or deficiency of intermediate metabolites in the carnitine biosynthetic pathway and potential carnitine deficiency in early stages of life when brain and other organs develop. In addition to these two

  4. Transcription factors in alkaloid biosynthesis.

    PubMed

    Yamada, Yasuyuki; Sato, Fumihiko

    2013-01-01

    Higher plants produce a large variety of low-molecular weight secondary compounds. Among them, nitrogen-containing alkaloids are the most biologically active and are often used pharmaceutically. Whereas alkaloid chemistry has been intensively investigated, alkaloid biosynthesis, including the relevant biosynthetic enzymes, genes and their regulation, and especially transcription factors, is largely unknown, as only a limited number of plant species produce certain types of alkaloids and they are difficult to study. Recently, however, several groups have succeeded in isolating the transcription factors that are involved in the biosynthesis of several types of alkaloids, including bHLH, ERF, and WRKY. Most of them show Jasmonate (JA) responsiveness, which suggests that the JA signaling cascade plays an important role in alkaloid biosynthesis. Here, we summarize the types and functions of transcription factors that have been isolated in alkaloid biosynthesis, and characterize their similarities and differences compared to those in other secondary metabolite pathways, such as phenylpropanoid and terpenoid biosyntheses. The evolution of this biosynthetic pathway and regulatory network, as well as the application of these transcription factors to metabolic engineering, is discussed.

  5. Hydrogen sulfide induces systemic tolerance to salinity and non-ionic osmotic stress in strawberry plants through modification of reactive species biosynthesis and transcriptional regulation of multiple defence pathways.

    PubMed

    Christou, Anastasis; Manganaris, George A; Papadopoulos, Ioannis; Fotopoulos, Vasileios

    2013-04-01

    Hydrogen sulfide (H2S) has been recently found to act as a potent priming agent. This study explored the hypothesis that hydroponic pretreatment of strawberry (Fragaria × ananassa cv. Camarosa) roots with a H2S donor, sodium hydrosulfide (NaHS; 100 μM for 48 h), could induce long-lasting priming effects and tolerance to subsequent exposure to 100mM NaCI or 10% (w/v) PEG-6000 for 7 d. Hydrogen sulfide pretreatment of roots resulted in increased leaf chlorophyll fluorescence, stomatal conductance and leaf relative water content as well as lower lipid peroxidation levels in comparison with plants directly subjected to salt and non-ionic osmotic stress, thus suggesting a systemic mitigating effect of H2S pretreatment to cellular damage derived from abiotic stress factors. In addition, root pretreatment with NaHS resulted in the minimization of oxidative and nitrosative stress in strawberry plants, manifested via lower levels of synthesis of NO and H(2)O(2) in leaves and the maintenance of high ascorbate and glutathione redox states, following subsequent salt and non-ionic osmotic stresses. Quantitative real-time RT-PCR gene expression analysis of key antioxidant (cAPX, CAT, MnSOD, GR), ascorbate and glutathione biosynthesis (GCS, GDH, GS), transcription factor (DREB), and salt overly sensitive (SOS) pathway (SOS2-like, SOS3-like, SOS4) genes suggests that H2S plays a pivotal role in the coordinated regulation of multiple transcriptional pathways. The ameliorative effects of H2S were more pronounced in strawberry plants subjected to both stress conditions immediately after NaHS root pretreatment, rather than in plants subjected to stress conditions 3 d after root pretreatment. Overall, H2S-pretreated plants managed to overcome the deleterious effects of salt and non-ionic osmotic stress by controlling oxidative and nitrosative cellular damage through increased performance of antioxidant mechanisms and the coordinated regulation of the SOS pathway, thus proposing a

  6. Transdermal Delivery of Insulin by Amidated Pectin Hydrogel Matrix Patch in Streptozotocin-Induced Diabetic Rats: Effects on Some Selected Metabolic Parameters

    PubMed Central

    Hadebe, Silindile I.; Ngubane, Phikelelani S.; Serumula, Metse R.; Musabayane, Cephas T.

    2014-01-01

    Purpose Studies in our laboratory are concerned with developing optional insulin delivery routes based on amidated pectin hydrogel matrix gel. We therefore investigated whether the application of pectin insulin (PI)-containing dermal patches of different insulin concentrations sustain controlled release of insulin into the bloodstream of streptozotocin (STZ)-induced diabetic rats with concomitant alleviation of diabetic symptoms in target tissues, most importantly, muscle and liver. Methods Oral glucose test (OGT) responses to PI dermal matrix patches (2.47, 3.99, 9.57, 16.80 µg/kg) prepared by dissolving pectin/insulin in deionised water and solidified with CaCl2 were monitored in diabetic rats given a glucose load after an 18-h fast. Short-term (5 weeks) metabolic effects were assessed in animals treated thrice daily with PI patches 8 hours apart. Animals treated with drug-free pectin and insulin (175 µg/kg, sc) acted as untreated and treated positive controls, respectively. Blood, muscle and liver samples were collected for measurements of selected biochemical parameters. Results After 5 weeks, untreated diabetic rats exhibited hyperglycaemia and depleted hepatic and muscle glycogen concentrations. Compared to untreated STZ-induced diabetic animals, OGT responses of diabetic rats transdermally applied PI patches exhibited lower blood glucose levels whilst short-term treatments restored hepatic and muscle glycogen concentrations. Plasma insulin concentrations of untreated diabetic rats were low compared with control non-diabetic rats. All PI treatments elevated plasma insulin concentrations of diabetic rats although the levels induced by high doses (9.57 and 16.80 µg/kg) were greater than those caused by low doses (2.47 and 3.99 µg/kg) but comparable to those in sc insulin treated animals. Conclusions The data suggest that the PI hydrogel matrix patch can deliver physiologically relevant amounts of pharmacologically active insulin. Novelty of the Work A new

  7. Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

    SciTech Connect

    Eum, Sung Yong Jaraki, Dima; András, Ibolya E.; Toborek, Michal

    2015-09-15

    Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft-associated PP

  8. (-)-Menthol biosynthesis and molecular genetics

    NASA Astrophysics Data System (ADS)

    Croteau, Rodney B.; Davis, Edward M.; Ringer, Kerry L.; Wildung, Mark R.

    2005-12-01

    (-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint ( Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4 S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general “allylic oxidation-conjugate reduction” scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1 R, 3 R, 4 S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

  9. Effects of rosuvastatin on the production and activation of matrix metalloproteinase-2 and migration of cultured rat vascular smooth muscle cells induced by homocysteine*

    PubMed Central

    Shi, Ya-fei; Chi, Ju-fang; Tang, Wei-liang; Xu, Fu-kang; Liu, Long-bin; Ji, Zheng; Lv, Hai-tao; Guo, Hang-yuan

    2013-01-01

    Objective: To test the influence of homocysteine on the production and activation of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) and on cell migration of cultured rat vascular smooth muscle cells (VSMCs). Also, to explore whether rosuvastatin can alter the abnormal secretion and activation of MMP-2 and TIMP-2 and migration of VSMCs induced by homocysteine. Methods: Rat VSMCs were incubated with different concentrations of homocysteine (50–5 000 μmol/L). Western blotting and gelatin zymography were used to investigate the expressions and activities of MMP-2 and TIMP-2 in VSMCs in culture medium when induced with homocysteine for 24, 48, and 72 h. Transwell chambers were employed to test the migratory ability of VSMCs when incubated with homocysteine for 48 h. Different concentrations of rosuvastatin (10−9–10−5 mol/L) were added when VSMCs were induced with 1 000 μmol/L homocysteine. The expressions and activities of MMP-2 and TIMP-2 were examined after incubating for 24, 48, and 72 h, and the migration of VSMCs was also examined after incubating for 48 h. Results: Homocysteine (50–1 000 μmol/L) increased the production and activation of MMP-2 and expression of TIMP-2 in a dose-dependent manner. However, when incubated with 5 000 μmol/L homocysteine, the expression of MMP-2 was up-regulated, but its activity was down-regulated. Increased homocysteine-induced production and activation of MMP-2 were reduced by rosuvastatin in a dose-dependent manner whereas secretion of TIMP-2 was not significantly altered by rosuvastatin. Homocysteine (50–5 000 μmol/L) stimulated the migration of VSMCs in a dose-dependent manner, but this effect was eliminated by rosuvastatin. Conclusions: Homocysteine (50–1 000 μmol/L) significantly increased the production and activation of MMP-2, the expression of TIMP-2, and the migration of VSMCs in a dose-dependent manner. Additional extracellular rosuvastatin can decrease

  10. Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2.

    PubMed

    Eum, Sung Yong; Jaraki, Dima; András, Ibolya E; Toborek, Michal

    2015-09-15

    Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs.

  11. Low-level laser therapy stimulates tissue repair and reduces the extracellular matrix degradation in rats with induced arthritis in the temporomandibular joint.

    PubMed

    Lemos, George Azevedo; Rissi, Renato; de Souza Pires, Ivan Luiz; de Oliveira, Letícia Prado; de Aro, Andrea Aparecida; Pimentel, Edson Rosa; Palomari, Evanisi Teresa

    2016-08-01

    The objective of this study was to characterize morphological and biochemistry action of low-level laser therapy (LLLT) on induced arthritis in the temporomandibular joint (TMJ) of rats. Twenty-four male Wistar rats were randomly divided into groups with 12 animals each: (AG) group with arthritis induced in the left TMJ and (LG) group with arthritis induced in the left TMJ and treated with LLLT (830 nm, 30 mW, 3 J/cm(2)). Right TMJs in the AG group were used as noninjected control group (CG). Arthritis was induced by intra-articular injection of 50 μl Complete Freund's Adjuvant (CFA) and LLLT began 1 week after arthritis induction. Histopathological analysis was performed using sections stained with hematoxylin-eosin, Toluidine Blue, and picrosirius. Biochemical analysis was determined by the total concentration of sulfated glycosaminoglycans (GAGs) and evaluation of matrix metalloproteinases (MMP-2 and MMP-9). Statistical analysis was performed using paired and unpaired t tests, with p < 0.05. Compared to AG, LG had minor histopathological changes in the TMJ, smaller thickness of the articular disc in the anterior (p < 0.0001), middle (p < 0.0001) and posterior regions (p < 0.0001), high birefringence of collagen fibers in the anterior (p < 0.0001), middle (p < 0.0001) and posterior regions (p < 0.0001) on the articular disc, and statistically lower activity of MMP-2 latent (p < 0.0001), MMP-2 active (P = 0.02), MMP-9 latent (p < 0.0001), and MMP-9 active (p < 0.0001). These results suggest that LLLT can increase the remodeling and enhancing tissue repair in TMJ with induced arthritis.

  12. Wnt16 Signaling Is Required for IL-1β-Induced Matrix Metalloproteinase-13-Regulated Proliferation of Human Stem Cell-Derived Osteoblastic Cells.

    PubMed

    Ozeki, Nobuaki; Mogi, Makio; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kawai, Rie; Kondo, Ayami; Nakata, Kazuhiko

    2016-02-06

    We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7⁺hSMSC)-derived osteoblast-like (α7⁺hSMSC-OB) cells, and found that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-13-regulated proliferation of these cells. These data suggest that MMP-13 plays a potentially unique physiological role in the regeneration of osteoblast-like cells. Here, we examined whether up-regulation of MMP-13 activity by IL-1β was mediated by Wingless/int1 (Wnt) signaling and increased the proliferation of osteoblast-like cells. IL-1β increased the mRNA and protein levels of Wnt16 and the Wnt receptor Lrp5/Fzd2. Exogenous Wnt16 was found to increase MMP-13 mRNA, protein and activity, and interestingly, the proliferation rate of these cells. Treatment with small interfering RNAs against Wnt16 and Lrp5 suppressed the IL-1β-induced increase in cell proliferation. We revealed that a unique signaling cascade IL-1β→Wnt16→Lrp5→MMP-13, was intimately involved in the proliferation of osteoblast-like cells, and suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation are regulated by Wnt16.

  13. The Effector Protein BPE005 from Brucella abortus Induces Collagen Deposition and Matrix Metalloproteinase 9 Downmodulation via Transforming Growth Factor β1 in Hepatic Stellate Cells

    PubMed Central

    Arriola Benitez, Paula Constanza; Rey Serantes, Diego; Herrmann, Claudia Karina; Pesce Viglietti, Ayelén Ivana; Vanzulli, Silvia; Giambartolomei, Guillermo Hernán; Comerci, Diego José

    2015-01-01

    The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway. PMID:26667834

  14. Wnt16 Signaling Is Required for IL-1β-Induced Matrix Metalloproteinase-13-Regulated Proliferation of Human Stem Cell-Derived Osteoblastic Cells

    PubMed Central

    Ozeki, Nobuaki; Mogi, Makio; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kawai, Rie; Kondo, Ayami; Nakata, Kazuhiko

    2016-01-01

    We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7+hSMSC)-derived osteoblast-like (α7+hSMSC-OB) cells, and found that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-13-regulated proliferation of these cells. These data suggest that MMP-13 plays a potentially unique physiological role in the regeneration of osteoblast-like cells. Here, we examined whether up-regulation of MMP-13 activity by IL-1β was mediated by Wingless/int1 (Wnt) signaling and increased the proliferation of osteoblast-like cells. IL-1β increased the mRNA and protein levels of Wnt16 and the Wnt receptor Lrp5/Fzd2. Exogenous Wnt16 was found to increase MMP-13 mRNA, protein and activity, and interestingly, the proliferation rate of these cells. Treatment with small interfering RNAs against Wnt16 and Lrp5 suppressed the IL-1β-induced increase in cell proliferation. We revealed that a unique signaling cascade IL-1β→Wnt16→Lrp5→MMP-13, was intimately involved in the proliferation of osteoblast-like cells, and suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation are regulated by Wnt16. PMID:26861315

  15. Stereoselective suppressive effects of protopanaxadiol epimers on UV-B-induced reactive oxygen species and matrix metalloproteinase-2 in human dermal keratinocytes.

    PubMed

    Oh, Sun-Joo; Lee, Sihyeong; Kho, Ye Eun; Kim, Kyunghoon; Jin, Chang Duck; Lim, Chang-Jin

    2015-01-01

    This study aimed to assess the skin-related anti-photoaging activities of the 2 epimeric forms of protopanaxadiol (PPD), 20(S)-PPD and 20(R)-PPD, in cultured human keratinocytes (HaCaT cells). The anti-photoaging activity was evaluated by analyzing the levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), as well as cell viability for HaCaT cells under UV-B irradiation. The activities for MMP-2 and -1 in conditioned medium were determined using gelatin zymography, and MMP-2 protein in the conditioned medium was detected using Western blot analysis. 20(S)-PPD, but not 20(R)-PPD, suppressed UV-B-induced ROS elevation. Neither of the epimers, at the concentrations used, exhibited cytotoxicity, irrespective of UV-B irradiation. 20(S)-PPD, but not 20(R)-PPD, exhibited an inhibitory effect on UV-B-induced MMP-2 activity and expression in HaCaT cells. In brief, only 20(S)-PPD, a major metabolic product of PPD-type ginsenosides, inhibits UV-B-induced ROS and MMP-2 elevation, implying its stereospecific anti-photoaging activity on the skin.

  16. The Effector Protein BPE005 from Brucella abortus Induces Collagen Deposition and Matrix Metalloproteinase 9 Downmodulation via Transforming Growth Factor β1 in Hepatic Stellate Cells.

    PubMed

    Arriola Benitez, Paula Constanza; Rey Serantes, Diego; Herrmann, Claudia Karina; Pesce Viglietti, Ayelén Ivana; Vanzulli, Silvia; Giambartolomei, Guillermo Hernán; Comerci, Diego José; Delpino, María Victoria

    2015-12-14

    The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway.

  17. A critical role for suppressors of cytokine signaling 3 in regulating LPS-induced transcriptional activation of matrix metalloproteinase-13 in osteoblasts

    PubMed Central

    Gao, Anqi; Kantarci, Alpdogan; Herrera, Bruno Schneider; Gao, Hongwei

    2013-01-01

    Suppressor of cytokine signaling 3 (SOCS3) is a key regulator of cytokine signaling in macrophages and T cells. Although SOCS3 seems to contribute to the balance between the pro-inflammatory actions of IL-6 family of cytokines and anti-inflammatory signaling of IL-10 by negatively regulating gp130/Jak/Stat3 signal transduction, how and the molecular mechanisms whereby SOCS3 controls the downstream impact of TLR4 are largely unknown and current data are controversial. Furthermore, very little is known regarding SOCS3 function in cells other than myeloid cells and T cells. Our previous study demonstrates that SOCS3 is expressed in osteoblasts and functions as a critical inhibitor of LPS-induced IL-6 expression. However, the function of SOCS3 in osteoblasts remains largely unknown. In the current study, we report for the first time that LPS stimulation of osteoblasts induces the transcriptional activation of matrix metalloproteinase (MMP)-13, a central regulator of bone resorption. Importantly, we demonstrate that SOCS3 overexpression leads to a significant decrease of LPS-induced MMP-13 expression in both primary murine calvariae osteoblasts and a mouse osteoblast-like cell line, MC3T3-E1. Our findings implicate SOCS3 as an important regulatory mediator in bone inflammatory diseases by targeting MMP-13. PMID:23638389

  18. Paracrine Effects of Adipose-Derived Stem Cells on Matrix Stiffness-Induced Cardiac Myofibroblast Differentiation via Angiotensin II Type 1 Receptor and Smad7.

    PubMed

    Yong, Kar Wey; Li, Yuhui; Liu, Fusheng; Bin Gao; Lu, Tian Jian; Wan Abas, Wan Abu Bakar; Wan Safwani, Wan Kamarul Zaman; Pingguan-Murphy, Belinda; Ma, Yufei; Xu, Feng; Huang, Guoyou

    2016-10-05

    Human mesenchymal stem cells (hMSCs) hold great promise in cardiac fibrosis therapy, due to their potential ability of inhibiting cardiac myofibroblast differentiation (a hallmark of cardiac fibrosis). However, the mechanism involved in their effects remains elusive. To explore this, it is necessary to develop an in vitro cardiac fibrosis model that incorporates pore size and native tissue-mimicking matrix stiffness, which may regulate cardiac myofibroblast differentiation. In the present study, collagen coated polyacrylamide hydrogel substrates were fabricated, in which the pore size was adjusted without altering the matrix stiffness. Stiffness is shown to regulate cardiac myofibroblast differentiation independently of pore size. Substrate at a stiffness of 30 kPa, which mimics the stiffness of native fibrotic cardiac tissue, was found to induce cardiac myofibroblast differentiation to create in vitro cardiac fibrosis model. Conditioned medium of hMSCs was applied to the model to determine its role and inhibitory mechanism on cardiac myofibroblast differentiation. It was found that hMSCs secrete hepatocyte growth factor (HGF) to inhibit cardiac myofibroblast differentiation via downregulation of angiotensin II type 1 receptor (AT1R) and upregulation of Smad7. These findings would aid in establishment of the therapeutic use of hMSCs in cardiac fibrosis therapy in future.

  19. Evaluation of Stress-Induced Microbial Siderophore from Pseudomonas aeruginosa Strain S1 as a Potential Matrix Metalloproteinase Inhibitor in Wound Healing Applications.

    PubMed

    Thyagarajan, Sita Lakshmi; Kandhasamy, S; Ramanathan, Giriprasath; Sivagnanam, Uma Tiruchirapalli; Perumal, P T

    2016-05-01

    Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes capable of causing various inflammatory and various degenerative diseases if over-expressed. The active site of these enzymes is a zinc binding motif which binds to the specific site on the substrate and induce degradation. Hence an inhibitor is required to form a complex with zinc motif which hampers the binding ability of MMPs. To obtain novel MMPs inhibitor for wound healing, the chelating activity of siderophore from the microbial source was focused. During screening for siderophore production, strain S1 produced the highest amount of siderophore in the minimal salts medium. The isolate was confirmed as Pseudomonas aeruginosa strain S1 based on 16S rRNA gene sequencing and phylogenetic analysis. The activity of the siderophore was assayed using chrome azurol sulphonate and purified by the chromatographic techniques. The structural evidence through Fourier transform infrared and nuclear magnetic resonance spectra revealed that the isolated siderophore is a catecholate type with the distinctive characters. The positive results of calcein and fluozin-3 assays indicate that siderophore could bind to divalent metal ions, namely Fe(2+) and Zn(2+). As the siderophore compound focused on wound healing property, the in vitro studies revealed the viability of NH3T3 fibroblast cells and its efficiency in matrix modulating was confirmed through gelatin zymogram.

  20. Hybrid Gel Composed of Native Heart Matrix and Collagen Induces Cardiac Differentiation of Human Embryonic Stem Cells without Supplemental Growth Factors

    PubMed Central

    Duan, Yi; Liu, Zen; O'Neill, John; Wan, Leo Q.; Freytes, Donald O.; Vunjak-Novakovic, Gordana

    2011-01-01

    Our goal was to assess the ability of native heart extracellular matrix (ECM) to direct cardiac differentiation of human embryonic stem cells (hESCs) in vitro. In order to probe the effects of cardiac matrix on hESC differentiation, a series of hydrogels was prepared from decellularized ECM from porcine hearts by mixing ECM and collagen type I at varying ratios. Maturation of cardiac function in embryoid bodies formed from hESCs was documented in terms of spontaneous contractile behavior and the mRNA and protein expression of cardiac markers. Hydrogel with high ECM content (75% ECM, 25% collagen, no supplemental soluble factors) increased the fraction of cells expressing cardiac marker troponin T, when compared with either hydrogel with low ECM content (25% ECM, 75% collagen, no supplemental soluble factors) or collagen hydrogel (100% collagen, with supplemental soluble factors). Furthermore, cardiac maturation was promoted in high-ECM content hydrogels, as evidenced by the striation patterns of cardiac troponin I and by upregulation of Cx43 gene. Consistently, high-ECM content hydrogels improved the contractile function of cardiac cells, as evidenced by increased numbers of contracting cells and increased contraction amplitudes. The ability of native ECM hydrogel to induce cardiac differentiation of hESCs without the addition of soluble factors makes it an attractive biomaterial system for basic studies of cardiac development and potentially for the delivery of therapeutic cells into the heart. PMID:21744185

  1. Paracrine Effects of Adipose-Derived Stem Cells on Matrix Stiffness-Induced Cardiac Myofibroblast Differentiation via Angiotensin II Type 1 Receptor and Smad7

    PubMed Central

    Yong, Kar Wey; Li, Yuhui; Liu, Fusheng; Bin Gao; Lu, Tian Jian; Wan Abas, Wan Abu Bakar; Wan Safwani, Wan Kamarul Zaman; Pingguan-Murphy, Belinda; Ma, Yufei; Xu, Feng; Huang, Guoyou

    2016-01-01

    Human mesenchymal stem cells (hMSCs) hold great promise in cardiac fibrosis therapy, due to their potential ability of inhibiting cardiac myofibroblast differentiation (a hallmark of cardiac fibrosis). However, the mechanism involved in their effects remains elusive. To explore this, it is necessary to develop an in vitro cardiac fibrosis model that incorporates pore size and native tissue-mimicking matrix stiffness, which may regulate cardiac myofibroblast differentiation. In the present study, collagen coated polyacrylamide hydrogel substrates were fabricated, in which the pore size was adjusted without altering the matrix stiffness. Stiffness is shown to regulate cardiac myofibroblast differentiation independently of pore size. Substrate at a stiffness of 30 kPa, which mimics the stiffness of native fibrotic cardiac tissue, was found to induce cardiac myofibroblast differentiation to create in vitro cardiac fibrosis model. Conditioned medium of hMSCs was applied to the model to determine its role and inhibitory mechanism on cardiac myofibroblast differentiation. It was found that hMSCs secrete hepatocyte growth factor (HGF) to inhibit cardiac myofibroblast differentiation via downregulation of angiotensin II type 1 receptor (AT1R) and upregulation of Smad7. These findings would aid in establishment of the therapeutic use of hMSCs in cardiac fibrosis therapy in future. PMID:27703175

  2. Balance between apoptosis or survival induced by changes in extracellular-matrix composition in human mesangial cells: a key role for ILK-NFκB pathway.

    PubMed

    del Nogal, María; Luengo, Alicia; Olmos, Gemma; Lasa, Marina; Rodriguez-Puyol, Diego; Rodriguez-Puyol, Manuel; Calleros, Laura

    2012-12-01

    Renal fibrosis is the final outcome of many clinical conditions that lead to chronic renal failure, characterized by a progressive substitution of cellular elements by extracellular-matrix proteins, in particular collagen type I. The aim of this study was to identify the mechanisms responsible for human mesangial cell survival, conditioned by changes in extracellular-matrix composition. Our results indicate that collagen I induces apoptosis in cells but only after inactivation of the pro-survival factor NFκB by either the super-repressor IκBα or the PDTC inhibitor. Collagen I activates a death pathway, through ILK/GSK-3β-dependent Bim expression. Moreover, collagen I significantly increases NFκB-dependent transcription, IκBα degradation and p65/NFκB translocation to the nucleus; it activates β1 integrin and this is accompanied by increased activity of ILK which leads to AKT activation. Knockdown of ILK or AKT with small interfering RNA suppresses the increase in NFκB activity. NFκB mediates cell survival through the antiapoptotic protein Bcl-xL. Our data suggest that human mesangial cells exposed to abnormal collagen I are protected against apoptosis by a complex mechanism involving integrin β1/ILK/AKT-dependent NFκB activation with consequent Bcl-xL overexpression, that opposes a simultaneously activated ILK/GSK-3β-dependent Bim expression and this dual mechanism may play a role in the progression of glomerular dysfunction.

  3. Strain hardening of fascia: static stretching of dense fibrous connective tissues can induce a temporary stiffness increase accompanied by enhanced matrix hydration.

    PubMed

    Schleip, Robert; Duerselen, Lutz; Vleeming, Andry; Naylor, Ian L; Lehmann-Horn, Frank; Zorn, Adjo; Jaeger, Heike; Klingler, Werner

    2012-01-01

    This study examined a potential cellular basis for strain hardening of fascial tissues: an increase in stiffness induced by stretch and subsequent rest. Mice lumbodorsal fascia were isometrically stretched for 15 min followed by 30 min rest (n=16). An increase in stiffness was observed in the majority of samples, including the nonviable control samples. Investigations with porcine lumbar fascia explored hydration changes as an explanation (n=24). Subject to similar loading procedures, tissues showed decreases in fluid content immediately post-stretch and increases during rest phases. When allowed sufficient resting time, a super-compensation phenomenon was observed, characterised by matrix hydration higher than initial levels and increases in tissue stiffness. Therefore, fascial strain hardening does not seem to rely on cellular contraction, but rather on this super-compensation. Given a comparable occurrence of this behaviour in vivo, clinical application of routines for injury prevention merit exploration.

  4. Laser induced fluorescence and phosphorescence of matrix isolated glyoxal - Evidence for exciplex formation in the A 1Au and a 3Au states

    NASA Technical Reports Server (NTRS)

    Van Ijzendoorn, L. J.; Baas, F.; Koernig, S.; Greenberg, J. M.; Allamandola, L. J.

    1986-01-01

    Laser-induced fluorescence and phosphorescence as well as infrared and visible absorption spectra of glyoxal in Ar, N2, and CO matrices are presented and analyzed. Glyoxal in its first excited electronic state is shown to form an exciplex with its nearest neighbors in all three matrices, and transitions normally forbidden dominate the emission spectra. The spectral characteristics of these complexes are similar to those of the Ar-glyoxal complex found in supersonic beam experiments. Due to the matrix cage effect, no vibrational predissociation is observed. The phosphorescence lifetime is determined and an upper limit is given for the fluorescence lifetime. This, in combination with the relative intensities of fluorescence and phosphorescence, can be used to place limits on the quantum yields of the various relaxation processes.

  5. Tantalum coating on TiO2 nanotubes induces superior rate of matrix mineralization and osteofunctionality in human osteoblasts.

    PubMed

    Frandsen, Christine J; Brammer, Karla S; Noh, Kunbae; Johnston, Gary; Jin, Sungho

    2014-04-01

    Nanostructured surface geometries have been the focus of a multitude of recent biomaterial research, and exciting findings have been published. However, only a few publications have directly compared nanostructures of various surface chemistries. The work herein directly compares the response of human osteoblast cells to surfaces of identical nanotube geometries with two well-known orthopedic biomaterials: titanium oxide (TiO2) and tantalum (Ta). The results reveal that the Ta surface chemistry on the nanotube architecture enhances alkaline phosphatase activity, and promotes a ~30% faster rate of matrix mineralization and bone-nodule formation when compared to results on bare TiO2 nanotubes. This study implies that unique combinations of surface chemistry and nanostructure may influence cell behavior due to distinctive physico-chemical properties. These findings are of paramount importance to the orthopedics field for understanding cell behavior in response to subtle alterations in nanostructure and surface chemistry, and will enable further insight into the complex manipulation of biomaterial surfaces. With increased focus in the field of orthopedic materials research on nanostructured surfaces, this study emphasizes the need for careful and systematic review of variations in surface chemistry in concurrence with nanotopographical changes.

  6. Matrix metalloproteinase-9 expression is enhanced in renal parietal epithelial cells of zucker diabetic Fatty rats and is induced by albumin in in vitro primary parietal cell culture.

    PubMed

    Zhang, Yuanyuan; George, Jasmine; Li, Yun; Olufade, Rebecca; Zhao, Xueying

    2015-01-01

    As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy

  7. Matrix isolation model studies on the radiation-induced transformations of small molecules of astrochemical and atmospheric interest

    NASA Astrophysics Data System (ADS)

    Feldman, Vladimir I.; Ryazantsev, Sergey V.; Saenko, Elizaveta V.; Kameneva, Svetlana V.; Shiryaeva, Ekaterina S.

    2016-07-01

    The radiation-induced transformations of small molecules at low temperatures play an important role in the interstellar, planetary and atmospheric chemistry. This work presents a review of our recent model studies on the radiation chemistry of relevant molecules in solid noble gas (Ng) matrices, including some preliminary new results. Among the triatomic molecules, water and carbon dioxide were studied in detail. The radiation-induced degradation of isolated H2O yields hydrogen atoms and OH radicals, while oxygen atoms are produced at higher doses. Isolated CO2 molecules are decomposed to yield CO and trapped oxygen atoms. Upon annealing the trapped O and H atoms are mobilized selectively at different temperatures and react with other trapped species. The formation of HCO and HOCO radicals was observed in the mixed H2O/CO2/Ng systems. Other studies were concerned with the radiation-induced degradation of simple organic molecules (methanol, formic acid) and chlorofluorocarbons (CFCl3, CF2Cl2). Preliminary results for methanol revealed deep dehydrogenation yielding HCO and CO, whereas CO2, CO and HOCO were detected as primary products for formic acid. In the case of chlorofluorocarbons, significance of ionic channels was demonstrated. The implications of the results for modeling the processes in astrochemical ices and atmosphere are discussed.

  8. Biosynthesis of Phytosiderophores 1

    PubMed Central

    Shojima, Shinsuke; Nishizawa, Naoko-Kishi; Fushiya, Shinji; Nozoe, Shigeo; Irifune, Tomohiro; Mori, Satoshi

    1990-01-01

    2′ -Deoxymugineic acid (DMA), one of mugineic acid-family phytosiderophores (MAs), was synthesized in vitro both from l-methionine and from nicotianamine (NA) with a cell-free system derived from root tips of iron-deficient barley (Hordeum vulgare L.). The reactions producing DMA from NA needed an amino group acceptor (i.e. 2-oxoglutarate, pyruvate, or oxalacetic acid) and a reductant (i.e. NADH or NADPH). The activity of the enzymes to produce NA from l-methionine was the highest at about pH 9. This biosynthetic activity was markedly induced by iron-deficiency stress. The synthesis of NA from S-adenosyl-l-methionine was more efficient than from l-methionine. From the results with the cell-free system reported here, we propose a revised biosynthetic pathway of MAs. Images Figure 5 PMID:16667646

  9. Biosynthesis of anti-HCV compounds using thermophilic microorganisms.

    PubMed

    Rivero, Cintia W; De Benedetti, Eliana C; Sambeth, Jorge E; Lozano, Mario E; Trelles, Jorge A

    2012-10-01

    This work describes the application of thermophilic microorganisms for obtaining 6-halogenated purine nucleosides. Biosynthesis of 6-chloropurine-2'-deoxyriboside and 6-chloropurine riboside was achieved by Geobacillus stearothermophilus CECT 43 with a conversion of 90% and 68%, respectively. Furthermore, the selected microorganism was satisfactorily stabilized by immobilization in an agarose matrix. This biocatalyst can be reused at least 70 times without significant loss of activity, obtaining 379mg/L of 6-chloropurine-2'-deoxyriboside. The obtained compounds can be used as antiviral agents.

  10. N-(2-Aminoethyl) Ethanolamine-Induced Morphological, Biochemical, and Biophysical Alterations in Vascular Matrix Associated With Dissecting Aortic Aneurysm

    PubMed Central

    Chen, Zhenping; Xu, Ya; Bujalowski, Paul; Oberhauser, Andres F.; Boor, Paul J.

    2015-01-01

    Dissecting aortic aneurysm (DAA) is an extended tear in the wall of the aorta along the plane of the vascular media. Our previous studies indicated in a developmental animal model, that DAA was related to pathological alteration in collagen, especially collagen type III. Accordingly, in the present studies, neonatal aortic vascular smooth muscle cells (VSMC) and timed pregnant Sprague-Dawley rat dams were treated with N-(2-aminoethyl) ethanolamine (AEEA), which, as shown previously, causes DAA in offspring. Morphological changes in extracellular matrix (ECM) produced by VSMC in vitro were detailed with scanning electron microscopy (SEM), and biochemical changes in cells and ECM produced by VSMCs were defined by Western blotting. Biophysical changes of the collagen extracted from both the ECM produced by VSMC and extracted from fetal rat aortas were studied with atomic force microscopy (AFM). ECM disruption and irregularities were observed in VSMCs treated with AEEA by SEM. Western blotting showed that collagen type I was much more extractable, accompanied by a decrease of the pellet size after urea buffer extraction in the AEEA-treated VSMC when compared with the control. AFM found that collagen samples extracted from the fetal rat aortas of the AEEA-treated dam, and in the in vitro formed ECM prepared by decellularization, became stiffer, or more brittle, indicating that the 3D organization associated with elasticity was altered by AEEA exposure. Our results show that AEEA causes significant morphological, biochemical, and biomechanical alterations in the ECM. These in vitro and in vivo strategies are advantageous in elucidating the underlying mechanisms of DAA. PMID:26443843

  11. Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation.

    PubMed

    Li, Bojun; Lin, Zhe; Mitsi, Maria; Zhang, Yang; Vogel, Viola

    2015-01-01

    An increasing body of evidence suggests important roles of extracellular matrix (ECM) in regulating stem cell fate. This knowledge can be exploited in tissue engineering applications for the design of ECM scaffolds appropriate to direct stem cell differentiation. By probing the conformation of fibronectin (Fn) using fluorescence resonance energy transfer (FRET), we show here that heparin treatment of the fibroblast-derived ECM scaffolds resulted in more extended conformations of fibrillar Fn in ECM. Since heparin is a highly negatively charged molecule while fibronectin contains segments of positively charged modules, including FnIII13, electrostatic interactions between Fn and heparin might interfere with residual quaternary structure in relaxed fibronectin fibers thereby opening up buried sites. The conformation of modules FnIII12-14 in particular, which contain one of the heparin binding sites as well as binding sites for many growth factors, may be activated by heparin, resulting in alterations in growth factor binding to Fn. Indeed, upregulated osteogenic differentiation was observed when hMSCs were seeded on ECM scaffolds that had been treated with heparin and were subsequently chemically fixed. In contrast, either rigidifying relaxed fibers by fixation alone, or heparin treatment without fixation had no effect. We hypothesize that fibronectin's conformations within the ECM are activated by heparin such as to coordinate with other factors to upregulate hMSC osteogenic differentiation. Thus, the conformational changes of fibronectin within the ECM could serve as a 'converter' to tune hMSC differentiation in extracellular matrices. This knowledge could also be exploited to promote osteogenic stem cell differentiation on biomedical surfaces.

  12. Connective tissue growth factor modulates podocyte actin cytoskeleton and extracellular matrix synthesis and is induced in podocytes upon injury.

    PubMed

    Fuchshofer, Rudolf; Ullmann, Sabrina; Zeilbeck, Ludwig F; Baumann, Matti; Junglas, Benjamin; Tamm, Ernst R

    2011-09-01

    Structural changes of podocytes and retraction of their foot processes are a critical factor in the pathogenesis of minimal change nephritis and glomerulosclerosis. Here we tested, if connective tissue growth factor (CTGF) is involved in podocyte injury during acute and chronic puromycin aminonucleoside nephrosis (PAN) as animal models of minimal change nephritis, and focal segmental glomerulosclerosis, respectively. Rats were treated once (acute PAN) or for 13 weeks (chronic PAN). In both experimental conditions, CTGF and its mRNA were found to be highly upregulated in podocytes. The upregulation correlated with onset and duration of proteinuria in acute PAN, and glomerulosclerosis and high expression of glomerular fibronectin, and collagens I, III, and IV in chronic PAN. In vitro, treatment of podocytes with recombinant CTGF increased amount and density of actin stress fibers, the expression of actin-associated molecules such as podocalyxin, synaptopodin, ezrin, and actinin-4, and activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Moreover, we observed increased podocyte expression of mRNA for transforming growth factor (TGF)-β2, TGF-β receptor II, fibronectin, and collagens I, III, and IV. Treatment of cultured podocytes with puromycin aminonucleoside resulted in loss of actin stress fibers and cell death, effects that were partially prevented when CTGF was added to the culture medium. Depletion of CTGF mRNA in cultured podocytes by RNA interference reduced both the number of actin stress fibers and the expression of actin-associated molecules. We propose that the expression of CTGF is acutely upregulated in podocytes as part of a cellular attempt to repair structural changes of the actin cytoskeleton. When the damaging effects on podocyte structure and function persist chronically, continuous CTGF expression in podocytes is a critical factor that promotes progressive accumulation of glomerular extracellular matrix and

  13. Mechanical characterization of matrix-induced autologous chondrocyte implantation (MACI®) grafts in an equine model at 53 weeks.

    PubMed

    Griffin, Darvin J; Bonnevie, Edward D; Lachowsky, Devin J; Hart, James C A; Sparks, Holly D; Moran, Nance; Matthews, Gloria; Nixon, Alan J; Cohen, Itai; Bonassar, Lawrence J

    2015-07-16

    There has been much interest in using autologous chondrocytes in combination with scaffold materials to aid in cartilage repair. In the present study, a total of 27 animals were used to compare the performance of matrix-assisted chondrocyte implantation (MACI®) using a collagen sponge as a chondrocyte delivery vehicle, the sponge membrane alone, and empty controls. A total of three distinct types of mechanical analyses were performed on repaired cartilage harvested from horses after 53 weeks of implantation: (1) compressive behavior of samples to measure aggregate modulus (HA) and hydraulic permeability (k) in confined compression; (2) local and global shear modulus using confocal strain mapping; and (3) boundary friction coefficient using a custom-built tribometer. Cartilage defects receiving MACI® implants had equilibrium modulus values that were 70% of normal cartilage, and were not statistically different than normal tissue. Defects filled with Maix™ membrane alone or left empty were only 46% and 51-63% of control, respectively. The shear modulus of tissue from all groups of cartilage defects were between 4 and 10 times lower than control tissue, and range from 0.2 to 0.4 MPa. The average values of boundary mode friction coefficients of control tissue from all groups ranged from 0.42 to 0.52. This study represents an extensive characterization of the mechanical performance of the MACI® grafts implant in a large animal model at 53 weeks. Collectively, these data demonstrate a range of implant performance, revealing similar compressive and frictional properties to native tissue, with inferior shear properties.

  14. Sesamin inhibits macrophage-induced vascular endothelial growth factor and matrix metalloproteinase-9 expression and proangiogenic activity in breast cancer cells.

    PubMed

    Lee, Chun-Chung; Liu, Ko-Jiunn; Wu, Yu-Chen; Lin, Sue-Jane; Chang, Ching-Chun; Huang, Tze-Sing

    2011-06-01

    Sesamin is a sesame component with antihypertensive and antioxidative activities and has recently aroused much interest in studying its potential anticancer application. Macrophage is one of the infiltrating inflammatory cells in solid tumor and may promote tumor progression via enhancement of tumor angiogenesis. In this study, we investigated whether sesamin inhibited macrophage-enhanced proangiogenic activity of breast cancer cell lines MCF-7 and MDA-MB-231. Using vascular endothelial cell capillary tube and network formation assays, both breast cancer cell lines exhibited elevated proangiogenic activities after coculture with macrophages or pretreatment with macrophage-conditioned medium. This elevation of proangiogenic activity was drastically suppressed by sesamin. Vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) induced by macrophages in both cell lines were also inhibited by sesamin. Nuclear levels of HIF-1α and NF-κB, important transcription factors for VEGF and MMP-9 expression, respectively, were obviously reduced by sesamin. VEGF induction by macrophage in MCF-7 cells was shown to be via ERK, JNK, phosphatidylinositol 3-kinase, and NF-κB-mediated pathways. These signaling molecules and additional p38(MAPK) were also involved in macrophage-induced MMP-9 expression. Despite such diverse pathways were induced by macrophage, only Akt and p38(MAPK) activities were potently inhibited by sesamin. Expression of interleukin (IL)-6, IL-8, and tumor necrosis factor-α were substantially increased and involved in macrophage-induced VEGF and MMP-9 mRNA expression in MCF-7 cells. Sesamin effectively inhibited the expression of these cytokines to avoid the reinforced induction of VEGF and MMP-9. In conclusion, sesamin potently inhibited macrophage-enhanced proangiogenic activity of breast cancer cells via inhibition of VEGF and MMP-9 induction.

  15. Matrix-isolation studies on the radiation-induced chemistry in H₂O/CO₂ systems: reactions of oxygen atoms and formation of HOCO radical.

    PubMed

    Ryazantsev, Sergey V; Feldman, Vladimir I

    2015-03-19

    The radiation-induced transformations occurring upon X-ray irradiation of solid CO2/H2O/Ng systems (Ng = Ar, Kr, Xe) at 8-10 K and subsequent annealing up to 45 K were studied by Fourier transform infrared spectroscopy. The infrared (IR) spectra of deposited matrices revealed the presence of isolated monomers, dimers, and intermolecular H2O···CO2 complexes. Irradiation resulted in effective decomposition of matrix-isolated carbon dioxide and water yielding CO molecules and OH radicals, respectively. Annealing of the irradiated samples led to formation of O3, HO2, and a number of xenon hydrides of HXeY type (in the case of xenon matrices). The formation of these species was used for monitoring of the postirradiation thermally induced chemical reactions involving O and H atoms generated by radiolysis. It was shown that the radiolysis of CO2 in noble-gas matrices produced high yields of stabilized oxygen atoms. In all cases, the temperatures at which O atoms become mobile and react are lower than those of H atoms. Dynamics and reactivity of oxygen atoms was found to be independent of the precursor nature. In addition, the formation of HOCO radicals was observed in all the noble-gas matrices at remarkably low temperatures. The IR spectra of HOCO and DOCO were first characterized in krypton and xenon matrices. It was concluded that the formation of HOCO was mainly due to the radiation-induced evolution of the weakly bound H2O···CO2 complexes. This result indicates the significance of weak intermolecular interactions in the radiation-induced chemical processes in inert low-temperature media.

  16. Potential Role of Fibroblast-Like Synoviocytes in Joint Damage Induced by Brucella abortus Infection through Production and Induction of Matrix Metalloproteinases ▿

    PubMed Central

    Scian, Romina; Barrionuevo, Paula; Giambartolomei, Guillermo H.; De Simone, E