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Sample records for biosynthesis induces matrix

  1. Regulation of extracellular matrix biosynthesis by matrix components

    SciTech Connect

    Holderbaum, D.; Ehrhart, L.A.

    1986-03-01

    The authors have previously shown that smooth muscle cells derived from healthy rabbit aortic media synthesize less collagen and fibronectin when grown on culture dishes coated with rabbit plasma fibronectin. In these cultures noncollagen protein synthesis was not affected, suggesting a specific regulatory mechanism. Their current studies expand this observation by examining the ability of proteolytically derived, specific domains of plasma fibronectin to effect decreases in collagen and fibronectin synthesis by cultured arterial smooth muscle. Rabbit plasma fibronectin was digested with bovine ..cap alpha..-chymotrypsin by the method of Hahn and Yamada. The resultant proteolytic fragments were separated by their ability to bind to gelatin-agarose. Culture dishes were coated with either (1) cell binding fragment of fibronectin, (2) gelatin binding fragment, (3) intact fibronectin, (4) type I collagen derived from lathyritic rat skin or (5) bovine serum albumin. Preconfluent cultures were labeled with /sup 3/H-Pro for 24 hr. Fibronectin synthesis was determined by immunoprecipitation of /sup 3/H-fibronectin. Collagen synthesis was measured by monitoring /sup 3/H-Hyp formation. Decreased collagen and fibronectin synthesis was evident in cells grown on intact fibronectin, cell binding fragment of fibronectin and type I collagen. Cells plated on gelatin binding fragment synthesized both collagen and fibronectin at levels comparable to cells on albumin coated dishes. They conclude that the regulatory activity of fibronectin on matrix biosynthesis resides on the cell binding domain of the molecule and that type I collagen can exert a similar effect.

  2. Biosynthesis of rat enamel matrix components in vivo

    SciTech Connect

    Sasaki, S.; Shimokawa, H.; Tanaka, K.

    1982-12-01

    The biosynthesis of enamel matrix components of developing rat incisors was investigated by measuring the incorporation of /sup 3/H-proline, /sup 32/P-phosphate, and /sup 35/S-sulfate in vivo. /sup 3/H- and /sup 32/P-radioactivity was found in what seemed to be a prototype of enamel proteins. Subsequent shifts in other protein and peptide fractions were observed. /sup 35/S was also incorporated into components other than the above-mentioned proteins.

  3. 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.

  4. 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

  5. Cuticular wax biosynthesis as a way of inducing drought resistance.

    PubMed

    Seo, Pil Joon; Park, Chung-Mo

    2011-07-01

    Plants have evolved diverse adaptive strategies to cope with drought or water deficit conditions, such as stomatal closure, maintenance of root growth and water uptake, and biosynthesis of osmoprotectants. Accumulation of cuticular waxes also contributes to drought resistance. However, it is still unclear how cuticular wax biosynthesis is regulated in response to drought and how it is associated with plant responses to drought at the molecular level. The abscisic acid (ABA)-inducible MYB96 transcription factor plays a role in drought resistance. Notably, it also regulates cuticular wax biosynthesis by binding directly to the promoters of genes encoding fatty acid elongating enzymes, such as KCS, KCR and ECR that constitute a rate-limiting step in cuticular wax biosynthesis. In the myb96-1D mutant that constitutively express the MYB96 gene, many of genes involved in cuticular wax biosynthesis are upregulated and accordingly, cuticular wax accumulation is greatly elevated. In contrast, cuticular wax accumulation is reduced in the myb96-1 mutant, linking drought with cuticular wax biosynthesis. It is evident that the MYB96 transcription factor incorporates drought stress signals into a gene regulatory network that modulates cuticular wax biosynthesis under drought stress conditions, providing a first molecular mechanism by which cuticular wax biosynthesis contributes to drought resistance and protection from pathogenic and mechanical damages as well.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. Tumorigenic Potential of Extracellular Matrix Metalloproteinase Inducer

    PubMed Central

    Zucker, Stanley; Hymowitz, Michelle; Rollo, Ellen E.; Mann, Richard; Conner, Cathleen E.; Cao, Jian; Foda, Hussein D.; Tompkins, David C.; Toole, Bryan P.

    2001-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs. PMID:11395366

  13. Arsenic Induced Decreases in the Vascular Matrix

    PubMed Central

    Hays, Allison M.; Lantz, R. Clark; Rodgers, Laurel S.; Sollome, James J.; Vaillancourt, Richard R.; Andrew, Angeline S.; Hamilton, Joshua W.; Camenisch, Todd D.

    2008-01-01

    Chronic ingestion of arsenic is associated with increased incidence of respiratory and cardiovascular diseases. To investigate the role of arsenic in early events in vascular pathology, C57BL/6 mice ingested drinking water with or without 50 ppb sodium arsenite (AsIII) for four, five or eight weeks. At five and eight weeks, RNA from the lungs of control and AsIII exposed animals was processed for microarray. Sixty-five genes were significantly and differentially expressed. Differential expression of extracellular matrix (ECM) gene transcripts was particularly compelling as 91% of genes in this category, including elastin and collagen, were significantly decreased. In additional experiments, real time RT-PCR showed an AsIII induced decrease in many of these ECM gene transcripts in the heart and NIH3T3 fibroblast cells. Histological stains for collagen and elastin show a distinct disruption in the ECM surrounding small arteries in the heart and lung of AsIII exposed mice. Immunohistochemical detection of a-smooth muscle actin in blood vessel walls was decreased in the AsIII exposed animals. These data reveal a functional link between AsIII exposure and disruption in the vascular ECM. These AsIII induced early pathological events may predispose humans to respiratory and cardiovascular diseases linked to chronic low dose AsIII exposure. PMID:18812580

  14. Is tartrazine-induced asthma related to inhibition of prostaglandin biosynthesis?

    PubMed

    Vargaftig, B B; Bessot, J C; Pauli, G

    1980-01-01

    Since it has been suggested that aspirin-induced asthma is due to inhibition of prostaglandin (PG) biosynthesis, the present investigation was undertaken to determine whether tartrazine would display a similar profile. Use was made of the exquisite sensitivity of blood platelets, both in humans and in rats, to aggregating substances generated from arachidonic acid during PG biosynthesis and of the ability of aspirin to inhibit aggregation and generation of those substances. Failure to affect aggregation by arachidonic acid as well as the accompanying formation of thromboxane A2, demonstrates that neither tartrazine nor its metabolite sulfanilic acid inhibit platelet PG synthesis. It seems unlikely that tartrazine-induced asthma results from inhibition of PG biosynthesis.

  15. 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.

  16. 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.

  17. Mycolic Acid-Containing Bacteria Induce Natural-Product Biosynthesis in Streptomyces Species▿ †

    PubMed Central

    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. PMID:21097597

  18. 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.

  19. 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.

  20. Ageratum enation virus Infection Induces Programmed Cell Death and Alters Metabolite Biosynthesis in Papaver somniferum.

    PubMed

    Srivastava, Ashish; Agrawal, Lalit; Raj, Rashmi; Jaidi, Meraj; Raj, Shri K; Gupta, Swati; Dixit, Ritu; Singh, Poonam C; Tripathi, Tusha; Sidhu, Om P; Singh, Brahma N; Shukla, Sudhir; Chauhan, Puneet S; Kumar, Susheel

    2017-01-01

    A previously unknown disease which causes severe vein thickening and inward leaf curl was observed in a number of opium poppy (Papaver somniferum L.) plants. The sequence analysis of full-length viral genome and associated betasatellite reveals the occurrence of Ageratum enation virus (AEV) and Ageratum leaf curl betasatellite (ALCB), respectively. Co-infiltration of cloned agroinfectious DNAs of AEV and ALCB induces the leaf curl and vein thickening symptoms as were observed naturally. Infectivity assay confirmed this complex as the cause of disease and also satisfied the Koch's postulates. Comprehensive microscopic analysis of infiltrated plants reveals severe structural anomalies in leaf and stem tissues represented by unorganized cell architecture and vascular bundles. Moreover, the characteristic blebs and membranous vesicles formed due to the virus-induced disintegration of the plasma membrane and intracellular organelles were also present. An accelerated nuclear DNA fragmentation was observed by Comet assay and confirmed by TUNEL and Hoechst dye staining assays suggesting virus-induced programmed cell death. Virus-infection altered the biosynthesis of several important metabolites. The biosynthesis potential of morphine, thebaine, codeine, and papaverine alkaloids reduced significantly in infected plants except for noscapine whose biosynthesis was comparatively enhanced. The expression analysis of corresponding alkaloid pathway genes by real time-PCR corroborated well with the results of HPLC analysis for alkaloid perturbations. The changes in the metabolite and alkaloid contents affect the commercial value of the poppy plants.

  1. Function of Hevea brasiliensis NAC1 in dehydration-induced laticifer differentiation and latex biosynthesis.

    PubMed

    Cao, Yuxin; Zhai, Jinling; Wang, Qichao; Yuan, Hongmei; Huang, Xi

    2017-01-01

    HbNAC1 is a transcription factor in rubber plants whose expression is induced by dehydration, leading to latex biosynthesis. Laticifer is a special tissue in Hevea brasiliensis where natural rubber is biosynthesized and accumulated. In young stems of epicormic shoots, the differentiation of secondary laticifers can be induced by wounding, which can be prevented when the wounding site is wrapped. Using this system, differentially expressed genes were screened by suppression subtractive hybridization (SSH) and macroarray analyses. This led to the identification of several dehydration-related genes that could be involved in laticifer differentiation and/or latex biosynthesis, including a NAC transcription factor (termed as HbNAC1). Tissue sections confirmed that local tissue dehydration was a key signal for laticifer differentiation. HbNAC1 was localized at the nucleus and showed strong transcriptional activity in yeast, suggesting that HbNAC1 is a transcription factor. Furthermore, HbNAC1 was found to bind to the cis-element CACG in the promoter region of the gene encoding the small rubber particle protein (SRPP). Transgenic experiments also confirmed that HbNAC1 interacted with the SRPP promoter when co-expressed, and enhanced expression of the reporter gene β-glucuronidase occurred in planta. In addition, overexpression of HbNAC1 in tobacco plants conferred drought tolerance. Together, the data suggest that HbNAC1 might be involved in dehydration-induced laticifer differentiation and latex biosynthesis.

  2. Ageratum enation virus Infection Induces Programmed Cell Death and Alters Metabolite Biosynthesis in Papaver somniferum

    PubMed Central

    Srivastava, Ashish; Agrawal, Lalit; Raj, Rashmi; Jaidi, Meraj; Raj, Shri K.; Gupta, Swati; Dixit, Ritu; Singh, Poonam C.; Tripathi, Tusha; Sidhu, Om P.; Singh, Brahma N.; Shukla, Sudhir; Chauhan, Puneet S.; Kumar, Susheel

    2017-01-01

    A previously unknown disease which causes severe vein thickening and inward leaf curl was observed in a number of opium poppy (Papaver somniferum L.) plants. The sequence analysis of full-length viral genome and associated betasatellite reveals the occurrence of Ageratum enation virus (AEV) and Ageratum leaf curl betasatellite (ALCB), respectively. Co-infiltration of cloned agroinfectious DNAs of AEV and ALCB induces the leaf curl and vein thickening symptoms as were observed naturally. Infectivity assay confirmed this complex as the cause of disease and also satisfied the Koch’s postulates. Comprehensive microscopic analysis of infiltrated plants reveals severe structural anomalies in leaf and stem tissues represented by unorganized cell architecture and vascular bundles. Moreover, the characteristic blebs and membranous vesicles formed due to the virus-induced disintegration of the plasma membrane and intracellular organelles were also present. An accelerated nuclear DNA fragmentation was observed by Comet assay and confirmed by TUNEL and Hoechst dye staining assays suggesting virus-induced programmed cell death. Virus-infection altered the biosynthesis of several important metabolites. The biosynthesis potential of morphine, thebaine, codeine, and papaverine alkaloids reduced significantly in infected plants except for noscapine whose biosynthesis was comparatively enhanced. The expression analysis of corresponding alkaloid pathway genes by real time-PCR corroborated well with the results of HPLC analysis for alkaloid perturbations. The changes in the metabolite and alkaloid contents affect the commercial value of the poppy plants. PMID:28729873

  3. 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.

  4. 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

  5. Transcriptomic analyses reveal species-specific light-induced anthocyanin biosynthesis in chrysanthemum.

    PubMed

    Hong, Yan; Tang, Xingjiao; Huang, He; Zhang, Yuan; Dai, Silan

    2015-03-17

    The flower colour of agricultural products is very important for their commercial value, which is mainly attributed to the accumulation of anthocyanins. Light is one of the key environmental factors that affect the anthocyanin biosynthesis. However, the deep molecular mechanism remains elusive, and many problems regarding the phenotypic change and the corresponding gene regulation are still unclear. In the present study, Chrysanthemum × morifolium 'Purple Reagan', a light-responding pigmentation cultivar, was selected to investigate the mechanism of light-induced anthocyanin biosynthesis using transcriptomic analyses. Only cyanidin derivatives were identified based on the analyses of the pigmentation in ray florets. Shading experiments revealed that the capitulum was the key organ and that its bud stage was the key phase responding to light. These results were used to design five libraries for transcriptomic analyses, including three capitulum developmental stages and two light conditions. RNA sequences were de novo assembled into 103,517 unigenes, of which 60,712 were annotated against four public protein databases. As many as 2,135 unigenes were differentially expressed between the light and dark libraries with 923 up-regulated and 1,212 down-regulated unigenes in response to shading. Next, interactive pathway analysis showed that the anthocyanin biosynthetic pathway was the only complete metabolic pathway both modulated in response to light and related to capitulum development. Following the shading treatment, nearly all structural genes involved in the anthocyanin biosynthetic pathway were down-regulated. Moreover, three CmMYB genes and one CmbHLH gene were identified as key transcription factors that might participate in the regulation of anthocyanin biosynthesis under light conditions based on clustering analysis and validation by RT-qPCR. Finally, a light-induced anthocyanin biosynthesis pathway in chrysanthemums was inferred. The pigmentation of the ray

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

    PubMed

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

    2015-03-01

    Hydrogen sulphide (H2S) is a gaseous mediator strongly involved in cardiovascular homeostasis, where it provokes vasodilatation. Having previously shown that H2 S contributes to testosterone-induced vasorelaxation, here we aim to uncover the mechanisms underlying this effect. H2 S 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. 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. 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. © 2014 The British Pharmacological Society.

  7. Glycerol-3-phosphate O-acyltransferase is required for PBAN-induced sex pheromone biosynthesis in Bombyx mori.

    PubMed

    Du, Mengfang; Liu, Xiaoguang; Liu, Xiaoming; Yin, Xinming; Han, Shuangyin; Song, Qisheng; An, Shiheng

    2015-01-29

    Female moths employ their own pheromone blends as a communicational medium in mating behavior. The biosynthesis and release of sex pheromone in female moths are regulated by pheromone biosynthesis activating neuropeptide (PBAN) and the corresponding action of PBAN has been well elucidated in Bombyx mori. However, very little is known about the molecular mechanism regarding the biosynthesis of sex pheromone precursor. In this study, quantitative proteomics was utilized to comprehensively elucidate the expression dynamics of pheromone glands (PGs) during development. Proteomic analysis revealed a serial of differentially expressed sex pheromone biosynthesis-associated proteins at the different time points of B. mori development. Most interestingly B. mori glycerol-3-phosphate O-acyltransferase (BmGPAT) was found to be expressed during the key periods of sex pheromone biosynthesis. RNAi knockdown of BmGPAT confirmed the important function of this protein in the biosynthesis of sex pheromone precursor, triacylglcerol (TAG), and subsequently PBAN-induced production of sex pheromone, bombykol. Behavioral analysis showed that RNAi knockdown of GPAT significantly impaired the ability of females to attract males. Our findings indicate that GPAT acts to regulate the biosynthesis of sex pheromone precursor, TAG, thus influencing PBAN-induced sex pheromone production and subsequent mating behavior.

  8. Glycerol-3-phosphate O-acyltransferase is required for PBAN-induced sex pheromone biosynthesis in Bombyx mori

    PubMed Central

    Du, Mengfang; Liu, Xiaoguang; Liu, Xiaoming; Yin, Xinming; Han, Shuangyin; Song, Qisheng; An, Shiheng

    2015-01-01

    Female moths employ their own pheromone blends as a communicational medium in mating behavior. The biosynthesis and release of sex pheromone in female moths are regulated by pheromone biosynthesis activating neuropeptide (PBAN) and the corresponding action of PBAN has been well elucidated in Bombyx mori. However, very little is known about the molecular mechanism regarding the biosynthesis of sex pheromone precursor. In this study, quantitative proteomics was utilized to comprehensively elucidate the expression dynamics of pheromone glands (PGs) during development. Proteomic analysis revealed a serial of differentially expressed sex pheromone biosynthesis-associated proteins at the different time points of B. mori development. Most interestingly B. mori glycerol-3-phosphate O-acyltransferase (BmGPAT) was found to be expressed during the key periods of sex pheromone biosynthesis. RNAi knockdown of BmGPAT confirmed the important function of this protein in the biosynthesis of sex pheromone precursor, triacylglcerol (TAG), and subsequently PBAN-induced production of sex pheromone, bombykol. Behavioral analysis showed that RNAi knockdown of GPAT significantly impaired the ability of females to attract males. Our findings indicate that GPAT acts to regulate the biosynthesis of sex pheromone precursor, TAG, thus influencing PBAN-induced sex pheromone production and subsequent mating behavior. PMID:25630665

  9. 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

  10. 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

  11. 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.

  12. Role of allopregnanolone biosynthesis in acute stress-induced anxiety-like behaviors in mice.

    PubMed

    Yoshizawa, Kazumi; Okumura, Ayano; Nakashima, Kozue; Sato, Tomoyo; Higashi, Tatsuya

    2017-08-01

    The neurosteroid allopregnanolone (3α, 5α-tetra-hydroprogesterone: ALLO) elicits anxiolytic, anticonvulsant, and hypnotic anesthetic effects in vivo similar to those induced by other positive allosteric modulators of the GABAA receptor. Endogenous ALLO has been shown to be rapidly elevated in the brain by acute stress paradigms, such as immobilization, in animal models. The present study was designed to ascertain the role of neurosteroid biosynthesis in the anxiety-like behavior induced by immobilization stress. Mice were exposed to an immobilization stressor for 2 h. After 24 h, the mice that had been immobilized did not behave significantly differently in the elevated plus maze (EPM) test and in the elevated open platform (EOP) test than the mice that had not been immobilized. In contrast, finasteride-pretreated immobilization stressed mice did behave significantly differently in the EPM and EOP tests. These findings suggest that ALLO biosynthesis contributes to stress resistance. Furthermore, the ALLO mimetic drug alfaxalone appeared to antagonize the effects of finasteride by significantly changing the behavior in the EPM test or in the EOP test in finasteride (10 mg kg(-1) )-pretreated immobilized mice. In addition, alfaxalone, unlike diazepam, did not affect the muscle tone of the mice, as measured by the grip strength test. These results suggest that alfaxalone is a promising anxiolytic candidate lacking benzodiazepine-like muscle-relaxant effects. © 2017 Wiley Periodicals, Inc.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. Starch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-Induced Stomatal Closing.

    PubMed

    Azoulay-Shemer, Tamar; Bagheri, Andisheh; Wang, Cun; Palomares, Axxell; Stephan, Aaron B; Kunz, Hans-Henning; Schroeder, Julian I

    2016-06-01

    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. © 2016 American Society of Plant Biologists. All Rights Reserved.

  20. Autologous Matrix-Induced Chondrogenesis in the Knee: A Review.

    PubMed

    Lee, Yee Han Dave; Suzer, Ferzan; Thermann, Hajo

    2014-07-01

    Autologous matrix-induced chondrogenesis (AMIC) is a 1-step cartilage restoration technique that combines microfracture with the use of an exogenous scaffold. This matrix covers and mechanically stabilizes the clot. There have been an increasing number of studies performed related to the AMIC technique and an update of its use and results is warranted. Using the PubMed database, a literature search was performed using the terms "AMIC" or "Autologous Matrix Induced Chondrogenesis." A total of 19 basic science and clinical articles were identified. Ten studies that were published on the use of AMIC for knee chondral defects were identified and the results of 219 patients were analyzed. The improvements in Knee Injury and Osteoarthritis Outcome Score, International Knee Documentation Committee Subjective, Lysholm and Tegner scores at 2 years were comparable to the published results from autologous chondrocyte implantation (ACI) and matrix ACI techniques for cartilage repair. Our systematic review of the current state of the AMIC technique suggests that it is a promising 1-stage cartilage repair technique. The short-term clinical outcomes and magnetic resonance imaging results are comparable to other cell-based methods. Further studies with AMIC in randomized studies versus other repair techniques such as ACI are needed in the future.

  1. Combined transcriptomic and proteomic analysis constructs a new model for light-induced anthocyanin biosynthesis in eggplant (Solanum melongena L.).

    PubMed

    Li, Jing; Ren, Li; Gao, Zhen; Jiang, Mingmin; Liu, Yang; Zhou, Lu; He, Yongjun; Chen, Huoying

    2017-09-20

    Light is a key environmental factor affecting anthocyanin biosynthesis. Our previous study demonstrated that 'Lanshan Hexian' is a light-sensitive eggplant cultivar, but its regulatory mechanism is unknown. Here, delphinidin-3-[4-(cis-p-coumaroyl)-rhamnosyl-glucopyranoside]-5-glucopyranoside and delphinidin-3-[4-(trans-p-coumaroyl)-rhamnosyl-glucopyranoside]-5-glucopyranoside were identified as the main anthocyanin components in 'Lanshan Hexian' by ultra-performance liquid chromatography-tandem mass spectrometry. Three time points of anthocyanin accumulation, including the start point (0 d), fastest point (5 d) and highest point (12 d), were investigated by using RNA-seq and iTRAQ technology. The corresponding correlation coefficients of differentially expressed genes and differentially expressed proteins were 0.6936, 0.2332 and 0.6672. Anthocyanin biosynthesis was a significantly enriched pathway, and CHI, F3H, 3GT, 5GT and HY5 were regulated at both transcriptional and translational levels. Moreover, some transcription factors and photoreceptors may participate in light-induced anthocyanin biosynthesis like the known transcription factors MYB113 and TT8. The transient expression assay indicated that SmMYB35, SmMYB44 and a SmMYB86 isoform might involve in the light-induced anthocyanin biosynthesis pathway. Finally, a regulatory model for light-induced anthocyanin biosynthesis in eggplant was constructed. Our work provides a new direction for the study of the molecular mechanisms of light-induced anthocyanin biosynthesis in eggplant. This article is protected by copyright. All rights reserved.

  2. 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.

  3. 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).

  4. LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation.

    PubMed

    Asai, Nobuyuki; Ohkawara, Bisei; Ito, Mikako; Masuda, Akio; Ishiguro, Naoki; Ohno, Kinji

    2014-08-22

    Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Leishmania spp: Delta-aminolevulinate-inducible neogenesis of porphyria by genetic complementation of incomplete heme biosynthesis pathway

    PubMed Central

    Dutta, Sujoy; Furuyama, Kazumichi; Sassa, Shigeru; Chang, Kwang-Poo Chang

    2008-01-01

    To further develop the Leishmania model for porphyria based on their deficiencies in heme biosynthesis, three Old World species were doubly transfected as before for Leishmania amazonensis with cDNAs, encoding the 2nd and 3rd enzymes in the pathway. Expression of the transgenes was verified immunologically at the protein level and functionally by uroporphyrin neogenesis that occurs only after exposure of the double-transfectants to delta-aminolevulinate. All species examined were equally deficient in heme biosynthesis, as indicated by the accumulation of uroporphyrin as the sole porphyrin and the production of coproporphyrin upon further transfection of one representative species with the downstream gene. The results obtained thus demonstrate that at least the first five enzymes for heme biosynthesis are absent in all species examined, rendering their transfectants inducible with aminolevulinate to accumulate porphyrins and thus useful as cellular models for human porphyrias. PMID:18164705

  6. Biosynthesis of cellular and secreted proteins during follicle-stimulating hormone-induced granulosa cell differentiation.

    PubMed

    Knecht, M; Shinohara, O; Catt, K J

    1986-09-01

    The synthesis of cellular and secreted proteins by differentiating granulosa cells from diethylstilbestrol-treated immature rats was studied by one- and two-dimensional polyacrylamide gel electrophoresis. In cultured granulosa cells, FSH altered the relative biosynthesis of specific cellular and secreted proteins in a concentration- and time-dependent manner. The incorporation of [35S]methionine into cellular proteins of Mr 42,000, 48,000, and 58,000 was enhanced by increasing amounts of the gonadotropin, whereas the labeling of a 44,000 Mr protein was reduced. Similarly, FSH increased the labeling of secreted proteins with relative Mr of 16,000, 17,000, 20,000, 25,000, 36,000, 41,000, 46,000, 111,000, and 153,000, and decreased that of proteins with Mr of 38,000, 48,000, 191,000, and 250,000. The expression of specific proteins was related to the degree of cellular maturation, since some proteins were newly synthesized during the early stages of granulosa cell development (less than 6 h), whereas others were more evident in the middle (24 h) or later (48 h) phases of culture. Also, the level of specific protein synthesis was variable since certain proteins were progressively produced during culture, and the biosynthesis of others fluctuated or was reduced during development. The effects of FSH on protein synthesis were mimicked by other cAMP-inducing ligands, including cholera toxin, forskolin, and 8-bromo-cAMP. Removal of FSH at 24 h of culture was followed by reversion of the protein biosynthetic pattern at 48 h to that of control cells, indicating that continued exposure to the gonadotropin is required during development. Cells cultured in the absence of ligands for 24 h synthesized proteins characteristic of differentiated cells when subsequently cultured with forskolin. These results indicate that FSH selectively alters the biosynthesis of cell-associated and secreted proteins during granulosa cell maturation. The characterization of these gene products and

  7. Engineered drought-induced biosynthesis of α-tocopherol alleviates stress-induced leaf damage in tobacco.

    PubMed

    Espinoza, Analía; San Martín, Alex; López-Climent, María; Ruiz-Lara, Simón; Gómez-Cadenas, Aurelio; Casaretto, José A

    2013-09-15

    Tocopherols are members of the vitamin E complex and essential antioxidant compounds synthesized in chloroplasts that protect photosynthetic membranes against oxidative damage triggered by most environmental stresses. Tocopherol deficiency has been shown to affect germination, retard growth and change responses to abiotic stress, suggesting that tocopherols may be involved in a number of diverse physiological processes in plants. Instead of seeking constitutive synthesis of tocopherols to improve stress tolerance, we followed an inducible approach of enhancing α-tocopherol accumulation under dehydration conditions in tobacco. Two uncharacterized stress inducible promoters isolated from Arabidopsis and the VTE2.1 gene from Solanum chilense were used in this work. VTE2.1 encodes the enzyme homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol biosynthesis. Transgenic tobacco plants expressing ScVTE2.1 under the control of stress-inducible promoters showed increased levels of α-tocopherol when exposed to drought conditions. The accumulation of α-tocopherol correlated with higher water content and increased photosynthetic performance and less oxidative stress damage as evidenced by reduced lipid peroxidation and delayed leaf senescence. Our results indicate that stress-induced expression of VTE2.1 can be used to increase the vitamin E content and to diminish detrimental effects of environmental stress in plants. The stress-inducible promoters introduced in this work may prove valuable to future biotechnological approaches in improving abiotic stress resistance in plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

  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. 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.

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

    PubMed Central

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

    2009-01-01

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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. Trade-offs between biomass growth and inducible biosynthesis of polyhydroxybutyrate in transgenic poplar.

    PubMed

    Dalton, David A; Ma, Cathleen; Shrestha, Shreya; Kitin, Peter; Strauss, Steven H

    2011-09-01

    Polyhydroxybutyrate (PHB) is a bioplastic that can be produced in transgenic plants by the coexpression of three bacterial genes for its biosynthesis. PHB yields from plants have been constrained by the negative impacts on plant health that result from diversion of resources into PHB production; thus, we employed an ecdysone analogue-based system for induced gene expression. We characterized 49 insertion events in hybrid transgenic poplar (Populus tremula x alba) that were produced using Agrobacterium transformation and studied two high-producing events in detail. Regenerated plants contained up to 1-2% PHB (dry weight) in leaves after 6-8 weeks of induction. Strong induction was observed with 1-10 mm Intrepid and limited direct toxicity observed. Confocal fluorescence microscopy was used to visualize PHB granules in chloroplasts after chemical treatment to reduce autofluorescence. A greenhouse study indicated that there were no negative consequences of PHB production on growth unless the PHB content exceeded 1% of leaf weight; at PHB levels above 1%, growth (height, diameter and total mass) decreased by 10%-34%. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  16. AibA/AibB Induces an Intramolecular Decarboxylation in Isovalerate Biosynthesis by Myxococcus xanthus.

    PubMed

    Bock, Tobias; Luxenburger, Eva; Hoffmann, Judith; Schütza, Vlad; Feiler, Christian; Müller, Rolf; Blankenfeldt, Wulf

    2017-08-07

    Isovaleryl coenzyme A (IV-CoA) is an important precursor for iso-fatty acids and lipids. It acts in the development of myxobacteria, which can produce this compound from acetyl-CoA through alternative IV-CoA biosynthesis (aib). A central reaction of aib is catalyzed by AibA/AibB, which acts as a cofactor-free decarboxylase despite belonging to the family of CoA-transferases. We developed an efficient expression system for AibA/AibB that allowed the determination of high-resolution crystal structures in complex with different ligands. Through mutational studies, we show that an active-site cysteine previously proposed to be involved in decarboxylation is not required for activity. Instead, AibA/AibB seems to induce an intramolecular decarboxylation by binding its substrate in a hydrophobic cavity and forcing it into a bent conformation. Our study opens opportunities for synthetic biology studies, since AibA/AibB may be suitable for the production of isobutene, a precursor of biofuels and chemicals. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Fenretinide prevents lipid-induced insulin resistance by blocking ceramide biosynthesis.

    PubMed

    Bikman, Benjamin T; Guan, Yuguang; Shui, Guanghou; Siddique, M Mobin; Holland, William L; Kim, Ji Yun; Fabriàs, Gemma; Wenk, Markus R; Summers, Scott A

    2012-05-18

    Fenretinide is a synthetic retinoid that is being tested in clinical trials for the treatment of breast cancer and insulin resistance, but its mechanism of action has been elusive. Recent in vitro data indicate that fenretinide inhibits dihydroceramide desaturase, an enzyme involved in the biosynthesis of lipotoxic ceramides that antagonize insulin action. Because of this finding, we assessed whether fenretinide could improve insulin sensitivity and glucose homeostasis in vitro and in vivo by controlling ceramide production. The effect of fenretinide on insulin action and the cellular lipidome was assessed in a number of lipid-challenged models including cultured myotubes and isolated muscles strips incubated with exogenous fatty acids and mice fed a high-fat diet. Insulin action was evaluated in the various models by measuring glucose uptake or disposal and the activation of Akt/PKB, a serine/threonine kinase that is obligate for insulin-stimulated anabolism. The effects of fenretinide on cellular lipid levels were assessed by LC-MS/MS. Fenretinide negated lipid-induced insulin resistance in each of the model systems assayed. Simultaneously, the drug depleted cells of ceramide, while promoting the accumulation of the precursor dihydroceramide, a substrate for the reaction catalyzed by Des1. These data suggest that fenretinide improves insulin sensitivity, at least in part, by inhibiting Des1 and suggest that therapeutics targeting this enzyme may be a viable therapeutic means for normalizing glucose homeostasis in the overweight and diabetic.

  18. Fenretinide Prevents Lipid-induced Insulin Resistance by Blocking Ceramide Biosynthesis*

    PubMed Central

    Bikman, Benjamin T.; Guan, Yuguang; Shui, Guanghou; Siddique, M. Mobin; Holland, William L.; Kim, Ji Yun; Fabriàs, Gemma; Wenk, Markus R.; Summers, Scott A.

    2012-01-01

    Fenretinide is a synthetic retinoid that is being tested in clinical trials for the treatment of breast cancer and insulin resistance, but its mechanism of action has been elusive. Recent in vitro data indicate that fenretinide inhibits dihydroceramide desaturase, an enzyme involved in the biosynthesis of lipotoxic ceramides that antagonize insulin action. Because of this finding, we assessed whether fenretinide could improve insulin sensitivity and glucose homeostasis in vitro and in vivo by controlling ceramide production. The effect of fenretinide on insulin action and the cellular lipidome was assessed in a number of lipid-challenged models including cultured myotubes and isolated muscles strips incubated with exogenous fatty acids and mice fed a high-fat diet. Insulin action was evaluated in the various models by measuring glucose uptake or disposal and the activation of Akt/PKB, a serine/threonine kinase that is obligate for insulin-stimulated anabolism. The effects of fenretinide on cellular lipid levels were assessed by LC-MS/MS. Fenretinide negated lipid-induced insulin resistance in each of the model systems assayed. Simultaneously, the drug depleted cells of ceramide, while promoting the accumulation of the precursor dihydroceramide, a substrate for the reaction catalyzed by Des1. These data suggest that fenretinide improves insulin sensitivity, at least in part, by inhibiting Des1 and suggest that therapeutics targeting this enzyme may be a viable therapeutic means for normalizing glucose homeostasis in the overweight and diabetic. PMID:22474281

  19. 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.

  20. 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

  1. Auxin-Induced Ethylene Triggers Abscisic Acid Biosynthesis and Growth Inhibition1

    PubMed Central

    Hansen, Hauke; Grossmann, Klaus

    2000-01-01

    The growth-inhibiting effects of indole-3-acetic acid (IAA) at high concentration and the synthetic auxins 7-chloro-3-methyl-8-quinolinecarboxylic acid (quinmerac), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), 4-amino-3,6,6-trichloropicolinic acid (picloram), and naphthalene acetic acid, were investigated in cleavers (Galium aparine). When plants were root treated with 0.5 mm IAA, shoot epinasty and inhibition of root and shoot growth developed during 24 h. Concomitantly, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, and ACC and ethylene production were transiently stimulated in the shoot tissue within 2 h, followed by increases in immunoreactive (+)-abscisic acid (ABA) and its precursor xanthoxal (xanthoxin) after 5 h. After 24 h of treatment, levels of xanthoxal and ABA were elevated up to 2- and 24-fold, relative to control, respectively. In plants treated with IAA, 7-chloro-3-methyl-8-quinolinecarboxylic acid, naphthalene acetic acid, 2-methoxy-3,6-dichlorobenzoic acid, and 4-amino-3,6,6-trichloropicolinic acid, levels of ethylene, ACC, and ABA increased in close correlation with inhibition of shoot growth. Aminoethoxyvinyl-glycine and cobalt ions, which inhibit ethylene synthesis, decreased ABA accumulation and growth inhibition, whereas the ethylene-releasing ethephon promoted ABA levels and growth inhibition. In accordance, tomato mutants defective in ethylene perception (never ripe) did not produce the xanthoxal and ABA increases and growth inhibition induced by auxins in wild-type plants. This suggests that auxin-stimulated ethylene triggers ABA accumulation and the consequent growth inhibition. Reduced catabolism most probably did not contribute to ABA increase, as indicated by immunoanalyses of ABA degradation and conjugation products in shoot tissue and by pulse experiments with [3H]-ABA in cell suspensions of G. aparine. In contrast, studies using inhibitors of ABA biosynthesis (fluridone, naproxen, and tungstate), ABA

  2. TGF-beta3-induced palatogenesis requires matrix metalloproteinases.

    PubMed

    Blavier, L; Lazaryev, A; Groffen, J; Heisterkamp, N; DeClerck, Y A; Kaartinen, V

    2001-05-01

    Cleft lip and palate syndromes are among the most common congenital malformations in humans. Mammalian palatogenesis is a complex process involving highly regulated interactions between epithelial and mesenchymal cells of the palate to permit correct positioning of the palatal shelves, the remodeling of the extracellular matrix (ECM), and subsequent fusion of the palatal shelves. Here we show that several matrix metalloproteinases (MMPs), including a cell membrane-associated MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) were highly expressed by the medial edge epithelium (MEE). MMP-13 was expressed both in MEE and in adjacent mesenchyme, whereas gelatinase A (MMP-2) was expressed by mesenchymal cells neighboring the MEE. Transforming growth factor (TGF)-beta3-deficient mice, which suffer from clefting of the secondary palate, showed complete absence of TIMP-2 in the midline and expressed significantly lower levels of MMP-13 and slightly reduced levels of MMP-2. In concordance with these findings, MMP-13 expression was strongly induced by TGF-beta3 in palatal fibroblasts. Finally, palatal shelves from prefusion wild-type mouse embryos cultured in the presence of a synthetic inhibitor of MMPs or excess of TIMP-2 failed to fuse and MEE cells did not transdifferentiate, phenocopying the defect of the TGF-beta3-deficient mice. Our observations indicate for the first time that the proteolytic degradation of the ECM by MMPs is a necessary step for palatal fusion.

  3. Nestin depletion induces melanoma matrix metalloproteinases and invasion

    PubMed Central

    Lee, Chung-Wei; Zhan, Qian; Lezcano, Cecilia; Frank, Markus H.; Huang, John; Larson, Allison; Lin, Jennifer Y.; Wan, Marilyn T.; Lin, Ping-I; Ma, Jie; Kleffel, Sonja; Schatton, Tobias; Lian, Christine G.; Murphy, George F.

    2015-01-01

    Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression. Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood. Because cytoskeletal changes are known to accompany MMP expression, we sought to examine the potential role of the poorly understood cytoskeletal protein, nestin, in modulating melanoma MMPs. Nestin knockdown (KD) upregulated expression of specific MMPs and MMP-dependent invasion both through extracellular matrix barriers in vitro and in peritumoral connective tissue of xenografts in vivo. Development of 3-dimensionsal melanospheres that in vitro partially recapitulate non-invasive tumorigenic melanoma growth was inhibited by nestin KD, although ECM invasion by aberrant melanospheres that did form was enhanced. Mechanistically, nestin KD-dependent melanoma invasion was associated with intracellular redistribution of phosphorylated focal adhesion kinase (pFAK) and increased melanoma cell responsiveness to transforming growth factor-beta (TGF-β), both implicated in pathways of melanoma invasion. The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence. PMID:25365206

  4. 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.

  5. 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.

  6. 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.

  7. Folate rescues vitamin B12 depletion-induced inhibition of nuclear thymidylate biosynthesis and genome instability.

    PubMed

    Palmer, Ashley M; Kamynina, Elena; Field, Martha S; Stover, Patrick J

    2017-05-16

    Clinical vitamin B12 deficiency can result in megaloblastic anemia, which results from the inhibition of DNA synthesis by trapping folate cofactors in the form of 5-methyltetrahydrofolate (5-methylTHF) and subsequent inhibition of de novo thymidylate (dTMP) biosynthesis. In the cytosol, vitamin B12 functions in the remethylation of homocysteine to methionine, which regenerates THF from 5-methylTHF. In the nucleus, THF is required for de novo dTMP biosynthesis, but it is not understood how 5-methylTHF accumulation in the cytosol impairs nuclear dTMP biosynthesis. The impact of vitamin B12 depletion on nuclear de novo dTMP biosynthesis was investigated in methionine synthase-null human fibroblast and nitrous oxide-treated HeLa cell models. The nucleus was the most sensitive cellular compartment to 5-methylTHF accumulation, with levels increasing greater than fourfold. Vitamin B12 depletion decreased de novo dTMP biosynthesis capacity by 5-35%, whereas de novo purine synthesis, which occurs in the cytosol, was not affected. Phosphorylated histone H2AX (γH2AX), a marker of DNA double-strand breaks, was increased in vitamin B12 depletion, and this effect was exacerbated by folate depletion. These studies also revealed that 5-formylTHF, a slow, tight-binding inhibitor of serine hydroxymethyltransferase (SHMT), was enriched in nuclei, accounting for 35% of folate cofactors, explaining previous observations that nuclear SHMT is not a robust source of one-carbons for de novo dTMP biosynthesis. These findings indicate that a nuclear 5-methylTHF trap occurs in vitamin B12 depletion, which suppresses de novo dTMP biosynthesis and causes DNA damage, accounting for the pathophysiology of megaloblastic anemia observed in vitamin B12 and folate deficiency.

  8. Characteristics of ethylene biosynthesis-inducing xylanase movement in tobacco leaves.

    PubMed

    Sharon, A; Bailey, B A; McMurtry, J P; Taylor, R; Anderson, J D

    1992-12-01

    (125)I-Labeled ethylene biosynthesis-inducing xylanase (EIX) was used to study the movement of this protein in tobacco (Nicotiana tabacum) tissues. A biologically active (125)I-labeled EIX was obtained using chloramine-T as the oxidizing agent. Labeled EIX was detected in the far most edges of the leaf 5 min after it was applied to the petiole of a detached leaf. EIX was distributed uniformly throughout the leaf, including the mesophyll area within 5 to 15 min, after which there was only little change in the distribution of radioactivity in the leaf. (125)I-Labeled EIX was extracted from treated leaves, and EIX translocation in the leaf was blocked by preincubation of labeled EIX with anti-EIX antibodies, indicating that the intact peptide moves in the leaf. Injection of anti-EIX antibodies into the intercellular spaces of the leaf mesophyll prevented induction of necrosis by EIX, suggesting the mesophyll as the site of EIX action. EIX was translocated both to upper and lower parts of the plant when applied to a whole plant through the petiole of a cut leaf. Radioactivity was found in all leaves and in the stem, although some leaves accumulated much more EIX than others; EIX was not found in the roots. There was no difference between the accumulation pattern of EIX in fresh and ethylene-treated leaves or between sensitive (Xanthi) and insensitive (Hicks) tobacco cultivars. These data support the hypothesis that intact EIX protein is translocated to the leaf mesophyll, where it directly elicits plant defense responses.

  9. 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.

  10. 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.

  11. Role of Tomato Lipoxygenase D in Wound-Induced Jasmonate Biosynthesis and Plant Immunity to Insect Herbivores

    PubMed Central

    Li, Shuyu; Wang, Bao; Huang, Tingting; Du, Minmin; Sun, Jiaqiang; Kang, Le; Li, Chang-Bao; Li, Chuanyou

    2013-01-01

    In response to insect attack and mechanical wounding, plants activate the expression of genes involved in various defense-related processes. A fascinating feature of these inducible defenses is their occurrence both locally at the wounding site and systemically in undamaged leaves throughout the plant. Wound-inducible proteinase inhibitors (PIs) in tomato (Solanum lycopersicum) provide an attractive model to understand the signal transduction events leading from localized injury to the systemic expression of defense-related genes. Among the identified intercellular molecules in regulating systemic wound response of tomato are the peptide signal systemin and the oxylipin signal jasmonic acid (JA). The systemin/JA signaling pathway provides a unique opportunity to investigate, in a single experimental system, the mechanism by which peptide and oxylipin signals interact to coordinate plant systemic immunity. Here we describe the characterization of the tomato suppressor of prosystemin-mediated responses8 (spr8) mutant, which was isolated as a suppressor of (pro)systemin-mediated signaling. spr8 plants exhibit a series of JA-dependent immune deficiencies, including the inability to express wound-responsive genes, abnormal development of glandular trichomes, and severely compromised resistance to cotton bollworm (Helicoverpa armigera) and Botrytis cinerea. Map-based cloning studies demonstrate that the spr8 mutant phenotype results from a point mutation in the catalytic domain of TomLoxD, a chloroplast-localized lipoxygenase involved in JA biosynthesis. We present evidence that overexpression of TomLoxD leads to elevated wound-induced JA biosynthesis, increased expression of wound-responsive genes and, therefore, enhanced resistance to insect herbivory attack and necrotrophic pathogen infection. These results indicate that TomLoxD is involved in wound-induced JA biosynthesis and highlight the application potential of this gene for crop protection against insects and

  12. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. 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

  14. 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.

  15. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  18. Peptostreptococcus anaerobius Induces Intracellular Cholesterol Biosynthesis in Colon Cells to Induce Proliferation and Causes Dysplasia in Mice.

    PubMed

    Tsoi, Ho; Chu, Eagle S H; Zhang, Xiang; Sheng, Jianqiu; Nakatsu, Geicho; Ng, Siew C; Chan, Anthony W H; Chan, Francis K L; Sung, Joseph J Y; Yu, Jun

    2017-05-01

    Stool samples from patients with colorectal cancer (CRC) have a higher abundance of Peptostreptococcus anaerobius than stool from individuals without CRC, based on metagenome sequencing. We investigated whether P anaerobius contributes to colon tumor formation in mice and its possible mechanisms of carcinogenesis. We performed quantitative polymerase chain reaction analyses to measure P anaerobius in 112 stool samples and 255 colon biopsies from patients with CRC or advanced adenoma and from healthy individuals (controls) undergoing colonoscopy examination at hospitals in Hong Kong and Beijing. C57BL/6 mice were given broad-spectrum antibiotics, followed by a single dose of azoxymethane, to induce colon tumor formation. Three days later, mice were given P anaerobius or Esherichia coli MG1655 (control bacteria), via gavage, for 6 weeks. Some mice were also given the nicotinamide adenine dinucleotide phosphate oxidase inhibitor apocynin. Intestine tissues were collected and analyzed histologically. The colon epithelial cell line NCM460 and colon cancer cell lines HT-29 and Caco-2 were exposed to P anaerobius or control bacteria; cells were analyzed by immunoblot, proliferation, and bacterial attachment analyses and compared in gene expression profiling studies. Gene expression was knocked down in these cell lines with small interfering RNAs. P anaerobius was significantly enriched in stool samples from patients with CRC and in biopsies from patients with colorectal adenoma or CRC compared with controls. Mice depleted of bacteria and exposed to azoxymethane and P anaerobius had a higher incidence of intestinal dysplasia (63%) compared with mice not given the bacteria (8.3%; P < .01). P anaerobius mainly colonized the colon compared with the rest of the intestine. Colon cells exposed to P anaerobius had significantly higher levels of proliferation than control cells. We found genes that regulate cholesterol biosynthesis, Toll-like receptor (TLR) signaling, and AMP

  19. 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.

  20. Ca(2+)-regulated cyclic electron flow supplies ATP for nitrogen starvation-induced lipid biosynthesis in green alga.

    PubMed

    Chen, Hui; Hu, Jinlu; Qiao, Yaqin; Chen, Weixian; Rong, Junfeng; Zhang, Yunming; He, Chenliu; Wang, Qiang

    2015-10-09

    We previously showed that both the linear photosynthetic electron transportation rate and the respiration rate dropped significantly during N starvation-induced neutral lipid accumulation in an oil-producing microalga, Chlorella sorokiniana, and proposed a possible role for cyclic electron flow (CEF) in ATP supply. In this study, we further exploited this hypothesis in both Chlorella sorokiniana C3 and the model green alga Chlamydomonas. We found that both the rate of CEF around photosystem I and the activity of thylakoid membrane-located ATP synthetase increased significantly during N starvation to drive ATP production. Furthermore, we demonstrated that the Chlamydomonas mutant pgrl1, which is deficient in PGRL1-mediated CEF, accumulated less neutral lipids and had reduced rates of CEF under N starvation. Further analysis revealed that Ca(2+) signaling regulates N starvation-induced neutral lipid biosynthesis in Chlamydomonas by increasing calmodulin activity and boosting the expression of the calcium sensor protein that regulates Pgrl1-mediated CEF. Thus, Ca(2+)-regulated CEF supplies ATP for N starvation-induced lipid biosynthesis in green alga. The increased CEF may re-equilibrate the ATP/NADPH balance and recycle excess light energy in photosystems to prevent photooxidative damage, suggesting Ca(2+)-regulated CEF also played a key role in protecting and sustaining photosystems.

  1. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26404260

  3. 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.

  4. The Mechanism of Ethylene Signaling Induced by Endophytic Fungus Gilmaniella sp. AL12 Mediating Sesquiterpenoids Biosynthesis in Atractylodes lancea.

    PubMed

    Yuan, Jie; Sun, Kai; Deng-Wang, Meng-Yao; Dai, Chuan-Chao

    2016-01-01

    Ethylene, the first known gaseous phytohormone, is involved in plant growth, development as well as responses to environmental signals. However, limited information is available on the role of ethylene in endophytic fungi induced secondary metabolites biosynthesis. Atractylodes lancea is a traditional Chinese herb, and its quality depends on the main active compounds sesquiterpenoids. This work showed that the endophytic fungus Gilmaniella sp. AL12 induced ethylene production in Atractylodes lancea. Pre-treatment of plantlets with ethylene inhibiter aminooxyacetic acid (AOA) suppressed endophytic fungi induced accumulation of ethylene and sesquiterpenoids. Plantlets were further treated with AOA, salicylic acid (SA) biosynthesis inhibitor paclobutrazol (PAC), jasmonic acid inhibitor ibuprofen (IBU), hydrogen peroxide (H2O2) scavenger catalase (CAT), nitric oxide (NO)-specific scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO). With endophytic fungi inoculation, IBU or PAC did not inhibit ethylene production, and JA and SA generation were suppressed by AOA, showing that ethylene may act as an upstream signal of JA and SA pathway. With endophytic fungi inoculation, CAT or cPTIO suppressed ethylene production, and H2O2 or NO generation was not affected by 1-aminocyclopropane-1-carboxylic acid (ACC), showing that ethylene may act as a downstream signal of H2O2 and NO pathway. Then, plantlets were treated with ethylene donor ACC, JA, SA, H2O2, NO donor sodium nitroprusside (SNP). Exogenous ACC could trigger JA and SA generation, whereas exogenous JA or SA did not affect ethylene production, and the induced sesquiterpenoids accumulation triggered by ACC was partly suppressed by IBU and PAC, showing that ethylene acted as an upstream signal of JA and SA pathway. Exogenous ACC did not affect H2O2 or NO generation, whereas exogenous H2O2 and SNP induced ethylene production, and the induced sesquiterpenoids accumulation

  5. The Mechanism of Ethylene Signaling Induced by Endophytic Fungus Gilmaniella sp. AL12 Mediating Sesquiterpenoids Biosynthesis in Atractylodes lancea

    PubMed Central

    Yuan, Jie; Sun, Kai; Deng-Wang, Meng-Yao; Dai, Chuan-Chao

    2016-01-01

    Ethylene, the first known gaseous phytohormone, is involved in plant growth, development as well as responses to environmental signals. However, limited information is available on the role of ethylene in endophytic fungi induced secondary metabolites biosynthesis. Atractylodes lancea is a traditional Chinese herb, and its quality depends on the main active compounds sesquiterpenoids. This work showed that the endophytic fungus Gilmaniella sp. AL12 induced ethylene production in Atractylodes lancea. Pre-treatment of plantlets with ethylene inhibiter aminooxyacetic acid (AOA) suppressed endophytic fungi induced accumulation of ethylene and sesquiterpenoids. Plantlets were further treated with AOA, salicylic acid (SA) biosynthesis inhibitor paclobutrazol (PAC), jasmonic acid inhibitor ibuprofen (IBU), hydrogen peroxide (H2O2) scavenger catalase (CAT), nitric oxide (NO)-specific scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO). With endophytic fungi inoculation, IBU or PAC did not inhibit ethylene production, and JA and SA generation were suppressed by AOA, showing that ethylene may act as an upstream signal of JA and SA pathway. With endophytic fungi inoculation, CAT or cPTIO suppressed ethylene production, and H2O2 or NO generation was not affected by 1-aminocyclopropane-1-carboxylic acid (ACC), showing that ethylene may act as a downstream signal of H2O2 and NO pathway. Then, plantlets were treated with ethylene donor ACC, JA, SA, H2O2, NO donor sodium nitroprusside (SNP). Exogenous ACC could trigger JA and SA generation, whereas exogenous JA or SA did not affect ethylene production, and the induced sesquiterpenoids accumulation triggered by ACC was partly suppressed by IBU and PAC, showing that ethylene acted as an upstream signal of JA and SA pathway. Exogenous ACC did not affect H2O2 or NO generation, whereas exogenous H2O2 and SNP induced ethylene production, and the induced sesquiterpenoids accumulation

  6. 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

  7. 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

  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. beta-Carotene accumulation induced by the cauliflower Or gene is not due to an increased capacity of biosynthesis.

    PubMed

    Li, Li; Lu, Shan; Cosman, Kelly M; Earle, Elizabeth D; Garvin, David F; O'Neill, Jennifer

    2006-06-01

    The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a rare carotenoid gene mutation that confers a high level of beta-carotene accumulation in various tissues of the plant, turning them orange. To investigate the biochemical basis of Or-induced carotenogenesis, we examined the carotenoid biosynthesis by evaluating phytoene accumulation in the presence of norflurazon, an effective inhibitor of phytoene desaturase. Calli were generated from young seedlings of wild type and Or mutant plants. While the calli derived from wild type seedlings showed a pale green color, the calli derived from Or seedlings exhibited intense orange color, showing the Or mutant phenotype. Concomitantly, the Or calli accumulated significantly more carotenoids than the wild type controls. Upon treatment with norflurazon, both the wild type and Or calli synthesized significant amounts of phytoene. The phytoene accumulated at comparable levels and no major differences in carotenogenic gene expression were observed between the wild type and Or calli. These results suggest that Or-induced beta-carotene accumulation does not result from an increased capacity of carotenoid biosynthesis.

  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. 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.

  13. 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

  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. TAA1-Regulated Local Auxin Biosynthesis in the Root-Apex Transition Zone Mediates the Aluminum-Induced Inhibition of Root Growth in Arabidopsis[C][W][OPEN

    PubMed Central

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

    2014-01-01

    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. PMID:25052716

  16. Matrix imbalance by inducing expression of metalloproteinase and oxidative stress in cochlea of hyperhomocysteinemic mice

    PubMed Central

    Kundu, Soumi; Tyagi, Neetu; Sen, Utpal

    2010-01-01

    Clinical study reports hearing loss in patients with low folic acid (FA) and elevated homocysteine (Hcy). We hypothesize that elevated Hcy induces imbalance in matrix turnover and oxidative stress in cochlea. Cystathione β-synthase heterozygous knockout mice were used as model for hyperhomocysteinemia. Matrix remodeling induced by Hcy resulted from elevated MMP-2, -9, and -14. MMP-2 and -9 showed elevated gelatinase activity in CBS (±) cochlea. Tissue inhibitors of matrix metalloproteinase were significantly lower in CBS (±) cochlea. The expression analyses for MMPs and TIMPs were equally represented at protein and mRNA levels. Cochlea of CBS mice showed following structural changes; (1) detachment of tectorial membrane lying on hair cells (2) thinner s. vascularis (3) large fibroblast in spiral ligament. Hcy induced higher protein nitrotyrosination and cytosolic NADPHoxidase subunit p22phox in cochlea. It is thus suggested that Hcy induced matrix imbalance, structural changes and oxidative stress in cochlea. PMID:19590937

  17. 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.

  18. Streptomyces scabiei and its toxin thaxtomin A induce scopoletin biosynthesis in tobacco and Arabidopsis thaliana.

    PubMed

    Lerat, Sylvain; Babana, Amadou H; El Oirdi, Mohamed; El Hadrami, Abdelbassed; Daayf, Fouad; Beaudoin, Nathalie; Bouarab, Kamal; Beaulieu, Carole

    2009-12-01

    Streptomyces scabiei is the predominant causal agent of common scab of potato in North America. The virulence of common scab-causing streptomycetes relies on their capacity to synthesize thaxtomins. In this study, the effects of S. scabiei infection and of thaxtomin A, the main toxin produced by S. scabiei, were tested for the elicitation of plant defense molecules in the model plants tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Tobacco leaves infected with spores of S. scabiei strain EF-35 or infiltrated with purified thaxtomin A produced a blue fluorescent compound that was not detected in leaves infiltrated with spores of a S. scabiei mutant deficient in thaxtomin A biosynthesis. Thin layer chromatography and high performance liquid chromatography identified this fluorescent compound as scopoletin, a plant defense phytoalexin. Arabidopsis seedlings grown in liquid medium also excreted scopoletin as a reaction to S. scabiei and thaxtomin A. The effects of the presence of scopoletin on S. scabiei were also investigated. The phytoalexin scopoletin caused a slight reduction of bacterial growth and a severe decrease of thaxtomin A production. Scopoletin was shown to inhibit thaxtomin A production by repression of a gene involved in the toxin biosynthesis.

  19. 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.

  20. 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.

  1. Integrin-linked kinase is involved in matrix-induced hepatocyte differentiation

    SciTech Connect

    Gkretsi, Vasiliki; Bowen, William C.; Yang, Yu; Wu, Chuanyue; Michalopoulos, George K. . E-mail: michalopoulosgk@UPMC.edu

    2007-02-16

    Hepatocytes have restricted proliferative capacity in culture and when cultured without matrix, lose the hepatocyte-specific gene expression and characteristic cellular micro-architecture. Overlay of matrix-preparations on de-differentiated hepatocytes restores differentiation. Integrin-linked kinase (ILK) is a cell-matrix-adhesion protein crucial in fundamental processes such as differentiation and survival. In this study, we investigated the role of ILK, and its binding partners PINCH, {alpha}-parvin, and Mig-2 in matrix-induced hepatocyte differentiation. We report here that ILK is present in the liver and localizes at cell-matrix adhesions of cultured hepatocytes. We also show that ILK, PINCH, {alpha}-parvin, and Mig-2 expression level is dramatically reduced in the re-differentiated hepatocytes. Interestingly, hepatocytes lacking ILK undergo matrix-induced differentiation but their differentiation is incomplete, as judged by monitoring cell morphology and production of albumin. Our results show that ILK and cell-matrix adhesion proteins play an important role in the process of matrix-induced hepatocyte differentiation.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. Impairment of cobalt-induced riboflavin biosynthesis in a Debaryomyces hansenii mutant.

    PubMed

    Seda-Miró, Jasmine M; Arroyo-González, Nancy; Pérez-Matos, Ana; Govind, Nadathur S

    2007-11-01

    Flavinogenic yeasts such as Debaryomyces hansenii overproduce riboflavin (RF) in the presence of heavy metals. Growth and RF production were compared between wild-type D. hansenii and a RF production-impaired metal-tolerant ura3 mutant in the presence of sublethal cobalt(II) concentrations. Debaryomyces hansenii (wild type) exhibits an extended lag phase with an increase in RF synthesis. Supplementation of exogenous uracil shortened the lag phase at the highest concentration of cobalt(II) used, suggesting that uracil has a possible role in metal acclimation. The D. hansenii ura3 mutant isolated by chemical mutagenesis exhibited a higher level of metal tolerance, no extended lag phase, and no marked increase in RF synthesis. Transformation of the mutant with the URA3 gene isolated from Saccharyomyces cerevisiae or D. hansenii did not restore wild-type characteristics, suggesting a second mutation that impairs RF oversynthesis. Our results demonstrate that growth, metal sensitivity, and RF biosynthesis are linked.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. Matrix metalloproteinases with gelatinolytic activity induced by Paracoccidioides brasiliensis infection

    PubMed Central

    Nishikaku, Angela Satie; Ribeiro, Luciana Cristina; Molina, Raphael Fagnani Sanchez; Albe, Bernardo Paulo; Cunha, Cláudia da Silva; Burger, Eva

    2009-01-01

    Matrix metalloproteinases (MMPs) modulate extracellular matrix turnover, inflammation and immunity. We studied MMP-9 and MMP-2 in experimental paracoccidioidomycosis. At 15 and 120 days after infection (DAI) with virulent Paracoccidioides brasiliensis, MMP-9 was positive by immunohistochemistry in multinucleated giant cells, in mononuclear cells with macrophage and lymphocyte morphologies and also in fungal cells in the lesions of susceptible and resistant mice. Using gelatin zymography, pro- and active MMP-9 and active MMP-2 were detected in all infected mice, but not in controls. Gelatinolytic activity was not observed in P. brasiliensis extracts. Semiquantitative analysis of gelatinolytic activities revealed weak or absent MMP-2 and strong MMP-9 activity in both mouse strains at 15 DAI, declining at 120 DAI. Avirulent P. brasiliensis-infected mice had residual lesions with MMP-9-positive pseudoxantomatous macrophages, but no gelatinase activity at 120 DAI. Our findings demonstrate the induction of MMPs, particularly MMP-9, in experimental paracoccidioidomycosis, suggesting a possible influence in the pattern of granulomas and in fungal dissemination. PMID:19765107

  12. Long-Term Submergence-Induced Elongation in Rumex palustris Requires Abscisic Acid-Dependent Biosynthesis of Gibberellin11

    PubMed Central

    Benschop, Joris J.; Bou, Jordi; Peeters, Anton J.M.; Wagemaker, Niels; Gühl, Kerstin; Ward, Dennis; Hedden, Peter; Moritz, Thomas; Voesenek, Laurentius A.C.J.

    2006-01-01

    Rumex palustris (polygonceae) responds to complete submergence with enhanced elongation of its youngest petioles. This process requires the presence of gibberellin (GA) and is associated with an increase in the concentration of GA1 in elongating petioles. We have examined how GA biosynthesis was regulated in submerged plants. Therefore, cDNAs encoding GA-biosynthetic enzymes GA 20-oxidase and GA 3-oxidase, and the GA-deactivating enzyme GA 2-oxidase were cloned from R. palustris and the kinetics of transcription of the corresponding genes was determined during a 24 h submergence period. The submergence-induced elongation response could be separated into several phases: (1) during the first phase of 4 h, petiole elongation was insensitive to GA; (2) from 4 to 6 h onward growth was limited by GA; and (3) from 15 h onward underwater elongation was dependent, but not limited by GA. Submergence induced an increase of GA1 concentration, as well as enhanced transcript levels of RpGA3ox1. Exogenous abscisic acid repressed the transcript levels of RpGA20ox1 and RpGA3ox1 and thus inhibited the submergence-induced increase in GA1. Abscisic acid had no effect on the tissue responsiveness to GA. PMID:16766669

  13. 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.

  14. 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.

  15. Exogenously induced expression of ethylene biosynthesis, ethylene perception, phospholipase D, and Rboh-oxidase genes in broccoli seedlings

    PubMed Central

    Jakubowicz, Małgorzata; Gałgańska, Hanna; Nowak, Witold; Sadowski, Jan

    2010-01-01

    In higher plants, copper ions, hydrogen peroxide, and cycloheximide have been recognized as very effective inducers of the transcriptional activity of genes encoding the enzymes of the ethylene biosynthesis pathway. In this report, the transcriptional patterns of genes encoding the 1-aminocyclopropane-1-carboxylate synthases (ACSs), 1-aminocyclopropane-1-carboxylate oxidases (ACOs), ETR1, ETR2, and ERS1 ethylene receptors, phospholipase D (PLD)-α1, -α2, -γ1, and -δ, and respiratory burst oxidase homologue (Rboh)-NADPH oxidase-D and -F in response to these inducers in Brassica oleracea etiolated seedlings are shown. ACS1, ACO1, ETR2, PLD-γ1, and RbohD represent genes whose expression was considerably affected by all of the inducers used. The investigations were performed on the seedlings with (i) ethylene insensitivity and (ii) a reduced level of the PLD-derived phosphatidic acid (PA). The general conclusion is that the expression of ACS1, -3, -4, -5, -7, and -11, ACO1, ETR1, ERS1, and ETR2, PLD-γ 1, and RbohD and F genes is undoubtedly under the reciprocal cross-talk of the ethylene and PAPLD signalling routes; both signals affect it in concerted or opposite ways depending on the gene or the type of stimuli. The results of these studies on broccoli seedlings are in agreement with the hypothesis that PA may directly affect the ethylene signal transduction pathway via an inhibitory effect on CTR1 (constitutive triple response 1) activity. PMID:20581125

  16. 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

  17. 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.

  18. Arsenic, chromium and NaCl induced artemisinin biosynthesis in Artemisia annua L.: a valuable antimalarial plant.

    PubMed

    Paul, Shilpi; Shakya, Kanika

    2013-12-01

    Effect of As(III), Cr(VI) and NaCl on plant growth, antioxidant enzymes, SOD, TBRAS, protein, cDNA amplification of key genes of artemisinin pathway and artemisinin biosynthesis have been investigated to explore the actual changes in total herb and artemisinin yield in a crop cycle of Artemisia annua. Enhanced TBARS and SOD activity (4 U mg⁻¹), decreased catalase activity and total cholorophyll content were observed under metal(loid) and NaCl stress. Accumulation of As (III; µg mg⁻¹ DW) was higher in roots (10.75±0.00) than shoot (0.43±0.00) at 10 µg ml⁻¹. While Cr(VI; µg ml⁻¹ DW) accumulated more in shoots (37±9.6, 41.1±7.2 and 52.71±19.6). cDNA template of these treated plants along with control were amplified with HMGR, ADS and CYP71AV1 genes (artemisinin biosynthetic pathway genes); showed very low expression with Cr(VI) while As(III) (5 and 7.5 µg ml⁻¹) showed higher expression than control. The results obtained from this study suggest that A. annua can grow well with favoring artemisinin biosynthesis with treatment of As(III) 5, 7.5 µg ml⁻¹ and NaCl, while 10 µg ml⁻¹ As(III) and all doses of Cr(VI) affect artemisinin synthesis. Finally some evidence also suggests that As(III), Cr(VI) and NaCl induces stress affects on total herb yield of plant.

  19. 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.

  20. IR induced photochemistry of glycolaldehyde in nitrogen matrix

    NASA Astrophysics Data System (ADS)

    Duvernay, Fabrice; Butscher, Teddy; Chiavassa, Thierry; Coussan, Stephane

    2017-10-01

    The conformational equilibria and IR isomerisation processes of glycolaldehyde (HOCH2CHO) conformers have been studied at low temperature in nitrogen cryogenic matrix. The effects of selective IR irradiation, in the νOH and νCH domains, have been followed by FTIR spectroscopy. After deposition the main form is the most stable one, CC (Cis-Cis), which features an intramolecular hydrogen bond. Upon selective IR irradiation, this latter form is able to interconvert into two open forms, those called TT (Trans-Trans), and TG (Trans-Gauche). From TT and TG forms, no recovering (by tunneling, dark process, irradiation, annealing, etc.) of the initial state (i.e. CC regeneration) has been observed. However, a TT ⇌ TG thermal equilibrium is observed with a standard reaction enthalpy about 0.37 kJ mol-1.

  1. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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 marke...

  3. 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

  4. 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.

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

    PubMed Central

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

    2013-01-01

    SUMMARY 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. PMID:24072791

  6. Comparative metagenomic analyses reveal viral-induced shifts of host metabolism towards nucleotide biosynthesis

    PubMed Central

    2014-01-01

    Background Viral genomes often contain metabolic genes that were acquired from host genomes (auxiliary genes). It is assumed that these genes are fixed in viral genomes as a result of a selective force, favoring viruses that acquire specific metabolic functions. While many individual auxiliary genes were observed in viral genomes and metagenomes, there is great importance in investigating the abundance of auxiliary genes and metabolic functions in the marine environment towards a better understanding of their role in promoting viral reproduction. Results In this study, we searched for enriched viral auxiliary genes and mapped them to metabolic pathways. To initially identify enriched auxiliary genes, we analyzed metagenomic microbial reads from the Global Ocean Survey (GOS) dataset that were characterized as viral, as well as marine virome and microbiome datasets from the Line Islands. Viral-enriched genes were mapped to a “global metabolism network” that comprises all KEGG metabolic pathways. Our analysis of the viral-enriched pathways revealed that purine and pyrimidine metabolism pathways are among the most enriched pathways. Moreover, many other viral-enriched metabolic pathways were found to be closely associated with the purine and pyrimidine metabolism pathways. Furthermore, we observed that sequential reactions are promoted in pathways having a high proportion of enriched genes. In addition, these enriched genes were found to be of modular nature, participating in several pathways. Conclusions Our naïve metagenomic analyses strongly support the well-established notion that viral auxiliary genes promote viral replication via both degradation of host DNA and RNA as well as a shift of the host metabolism towards nucleotide biosynthesis, clearly indicating that comparative metagenomics can be used to understand different environments and systems without prior knowledge of pathways involved. PMID:24666644

  7. 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.

  8. 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

  9. Matrix Metalloproteinase-9 Protects Islets from Amyloid-induced Toxicity*

    PubMed Central

    Meier, Daniel T.; Tu, Ling-Hsien; Zraika, Sakeneh; Hogan, Meghan F.; Templin, Andrew T.; Hull, Rebecca L.; Raleigh, Daniel P.; Kahn, Steven E.

    2015-01-01

    Deposition of human islet amyloid polypeptide (hIAPP, also known as amylin) as islet amyloid is a characteristic feature of the pancreas in type 2 diabetes, contributing to increased β-cell apoptosis and reduced β-cell mass. Matrix metalloproteinase-9 (MMP-9) is active in islets and cleaves hIAPP. We investigated whether hIAPP fragments arising from MMP-9 cleavage retain the potential to aggregate and cause toxicity, and whether overexpressing MMP-9 in amyloid-prone islets reduces amyloid burden and the resulting β-cell toxicity. Synthetic hIAPP was incubated with MMP-9 and the major hIAPP fragments observed by MS comprised residues 1–15, 1–25, 16–37, 16–25, and 26–37. The fragments 1–15, 1–25, and 26–37 did not form amyloid fibrils in vitro and they were not cytotoxic when incubated with β cells. Mixtures of these fragments with full-length hIAPP did not modulate the kinetics of fibril formation by full-length hIAPP. In contrast, the 16–37 fragment formed fibrils more rapidly than full-length hIAPP but was less cytotoxic. Co-incubation of MMP-9 and fragment 16–37 ablated amyloidogenicity, suggesting that MMP-9 cleaves hIAPP 16–37 into non-amyloidogenic fragments. Consistent with MMP-9 cleavage resulting in largely non-amyloidogenic degradation products, adenoviral overexpression of MMP-9 in amyloid-prone islets reduced amyloid deposition and β-cell apoptosis. These findings suggest that increasing islet MMP-9 activity might be a strategy to limit β-cell loss in type 2 diabetes. PMID:26483547

  10. Transgenic expression of matrix metalloproteinase-2 induces coronary artery ectasia

    PubMed Central

    Dahi, Sia; Karliner, Joel S; Sarkar, Rajabrata; Lovett, David H

    2011-01-01

    Coronary artery ectasia (CAE) is generally diagnosed in patients undergoing arteriography for presumptive atherosclerotic coronary artery disease. CAE is commonly considered as a variant of atherosclerotic disease; however, recent studies suggest that CAE is the result of a systemic vascular disorder. There is increasing evidence that aneurysmal vascular disease is a systemic disorder characterized by enhanced expression of pro-inflammatory cytokines and increased synthesis of enzymes capable of degrading elastin and other components of the vascular wall. Matrix metalloproteinase-2 degrades a number of extracellular substrates, including elastin and has been shown to play a critical role in the development of abdominal aortic aneurysms. This study characterizes the development of CAE in a unique murine transgenic model with cardiac-specific expression of active MMP-2. Transgenic mice were engineered to express an active form of MMP-2 under control of the α-myosin heavy chain promoter. Coronary artery diameters were quantified, along with studies of arterial structure, elastin integrity and vascular expression of the MMP-2 transgene. Latex casts quantified total coronary artery volumes and arterial branching. Mid-ventricular coronary luminal areas were increased in the MMP-2 transgenics, coupled with foci of aneurysmal dilation, ectasia and perivascular fibrosis. There was no evidence for atherogenesis. Coronary vascular elastin integrity was compromised and coupled with inflammatory cell infiltration. Latex casts of the coronary arteries displayed ectasia with fusiform dilatation. The MMP-2 transgenic closely replicates human CAE and supports a critical and initiating role for this enzyme in the pathogenesis of this disorder. PMID:21039989

  11. 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

  12. Mechanism of the radiation-induced transformations of fluoroform in solid noble gas matrixes

    NASA Astrophysics Data System (ADS)

    Sosulin, Ilya S.; Shiryaeva, Ekaterina S.; Feldman, Vladimir I.

    2017-09-01

    The X-ray induced transformations in the CHF3/Ng systems (Ng=Ne, Ar, Kr or Xe) at 6 K were studied by FTIR spectroscopy. The radiation-induced decomposition of CHF3 was found to be rather inefficient in solid xenon with low ionization energy, which suggests primary significance of the positive hole transfer from matrix to the fluoroform molecule. CF3•, :CF2, CHF2• and CF4 were identified as the products of low-temperature radiolysis in all the noble gas matrixes. In addition, the anionic complex HF ⋯ CF2- was detected in Ne and Ar matrixes. The radiolysis also resulted in formation of noble gas compounds (HArF in argon, HKrF in krypton, and XeF2 in xenon). While XeF2 and HArF were essentially formed directly after irradiation (presumably due to reactions of 'hot' fluorine atoms), HKrF mainly resulted from annealing of irradiated samples below 20 K due to thermally induced mobility of trapped fluorine atoms. In both krypton and xenon matrixes, the thermally induced reactions of F atoms occur at lower temperatures than those of H atoms, while the opposite situation is observed in argon. The mechanisms of the radiation-induced processes and their implications are discussed.

  13. 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.

  14. Matrix-M™ adjuvation broadens protection induced by seasonal trivalent virosomal influenza vaccine.

    PubMed

    Cox, Freek; Saeland, Eirikur; Baart, Matthijs; Koldijk, Martin; Tolboom, Jeroen; Dekking, Liesbeth; Koudstaal, Wouter; Lövgren Bengtsson, Karin; Goudsmit, Jaap; Radošević, Katarina

    2015-12-08

    Influenza virus infections are responsible for significant morbidity worldwide and therefore it remains a high priority to develop more broadly protective vaccines. Adjuvation of current seasonal influenza vaccines has the potential to achieve this goal. To assess the immune potentiating properties of Matrix-M™, mice were immunized with virosomal trivalent seasonal vaccine adjuvated with Matrix-M™. Serum samples were isolated to determine the hemagglutination inhibiting (HAI) antibody titers against vaccine homologous and heterologous strains. Furthermore, we assess whether adjuvation with Matrix-M™ broadens the protective efficacy of the virosomal trivalent seasonal vaccine against vaccine homologous and heterologous influenza viruses. Matrix-M™ adjuvation enhanced HAI antibody titers and protection against vaccine homologous strains. Interestingly, Matrix-M™ adjuvation also resulted in HAI antibody titers against heterologous influenza B strains, but not against the tested influenza A strains. Even though the protection against heterologous influenza A was induced by the adjuvated vaccine, in the absence of HAI titers the protection was accompanied by severe clinical scores and body weight loss. In contrast, in the presence of heterologous HAI titers full protection against the heterologous influenza B strain without any disease symptoms was obtained. The results of this study emphasize the promising potential of a Matrix-M™-adjuvated seasonal trivalent virosomal influenza vaccine. Adjuvation of trivalent virosomal vaccine does not only enhance homologous protection, but in addition induces protection against heterologous strains and thus provides overall more potent and broad protective immunity.

  15. Glucose-induced delay of seed germination in rice is mediated by the suppression of ABA catabolism rather than an enhancement of ABA biosynthesis.

    PubMed

    Zhu, Guohui; Ye, Nenghui; Zhang, Jianhua

    2009-03-01

    Both glucose and ABA play crucial roles in the regulation of seed germination and post-germination development. In Arabidopsis thaliana, up-regulation of ABA biosynthesis is suggested as one of the possible mechanisms mediating the glucose-induced delay in seed germination. Since the endogenous ABA level is controlled by the equilibrium between ABA biosynthesis and catabolism, we investigated how this equilibrium is related to the regulation of seed germination by glucose in rice. When ABA biosynthesis was inhibited by nordihydroguaiaretic acid (NDGA), an inhibitor of the ABA anabolic enzyme 9-cis-epoxycarotenoid dioxygenase (NCED), rice seed germination showed no response. In contrast, inhibition of ABA catabolism by diniconazole significantly arrested seed germination, suggesting that the regulation of ABA catabolism plays a major role. Further experiments indicated that the expression of OsABA8ox3, a key gene in ABA catabolism and encoding ABA 8'-hydroxylase in rice, was significantly increased during the first 6 h of imbibition, which was consistent with the decline of ABA content in the imbibed seeds. Expression of OsABA8ox genes, especially OsABA8ox2 and OsABA8ox3, was sensitively suppressed in the presence of exogenously supplied glucose. In contrast, the expression profiles of OsNCED genes that control the limiting step of ABA biosynthesis showed no significant changes in response to low levels of glucose. Our results demonstrated that the glucose-induced delay of seed germination is a result of the suppression of ABA catabolism rather than any enhancement of ABA biosynthesis during rice seed germination.

  16. 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.

  17. [The study on the enamel remineralization by enamel matrix proteins' inducing].

    PubMed

    Wang, Zhi-wei; Zhao, Yue-ping; Zhou, Chang-ren; Li, Hong

    2008-07-01

    To find the enamel matrix proteins on the impact of enamel mineralization through experiments. A combination of protein and beneficial carboxyl groups was grafted on the surface of enamel defects of rats through UV radiation then put into the enamel matrix proteins of calcium phosphate agar acetate solution systems, through scanning enamel surface with the electron microscopy to observe the morphological changes of enamel then analyse the regulation that enamel matrix proteins have done to the white hydroxyapatite crystals on the composition and morphology. In the enamel matrix protein added gel system, we can see the growth of hydroxyapatite crystals, and crystal showed a good degree of crystallinity and contained a small amount of CO3(2-) substituted hydroxyapatite crystals. The temperature at 37 degrees C water bath, after adding the enamel matrix proteins to gel system, the new hydroxyapatite crystals were numerous which proved that enamel matrix proteins played an important role in nucleation and growth of hydroxyapatite crystal, so it could be indicated that enamel matrix proteins could induce the enamel remineralization.

  18. 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.

  19. 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

  20. The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis1[OPEN

    PubMed Central

    Wang, Zhen-Yu; Gehring, Chris; Zhu, Jianhua; Li, Feng-Min; Zhu, Jian-Kang; Xiong, Liming

    2015-01-01

    Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1. PMID:25416474

  1. 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

  2. Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation 1

    PubMed Central

    Viola, Roberto; Davies, Howard V.

    1991-01-01

    Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of 13C-labeling [NMR] in sucrose when tissue was supplied with [2-13C]glucose). Fluoride inhibited the incorporation of [U-14C] glucose, [U-14C]sucrose, [U-14C]glucose 1-phosphate, and [U-14C] glycerol into starch. The incorporation of [U-14C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of 14C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions. PMID:16668446

  3. 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.

  4. Inhibition of CDKS by roscovitine suppressed LPS-induced *NO production through inhibiting NFkappaB activation and BH4 biosynthesis in macrophages.

    PubMed

    Du, Jianhai; Wei, Na; Guan, Tongju; Xu, Hao; An, Jianzhong; Pritchard, Kirkwood A; Shi, Yang

    2009-09-01

    In inflammatory diseases, tissue damage is critically associated with nitric oxide ((*)NO) and cytokines, which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on (*)NO production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of (*)NO induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IkappaB kinase beta (IKKbeta), IkappaB, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun NH(2)-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1beta, and IL-6 but not tumor necrosis factor (TNF)-alpha. Tetrahydrobiopterin (BH(4)), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (BH(2)). Roscovitine significantly inhibited LPS-induced BH(4) biosynthesis and decreased BH(4)-to-BH(2) ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for BH(4) biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced (*)NO production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited (*)NO production, iNOS, and COX-2 upregulation, activation of NFkappaB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced (*)NO production in macrophages by suppressing nuclear factor-kappaB activation and BH(4) biosynthesis, which might be mediated by CDK1, CDK5, and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which

  5. Roles of matrix metalloproteinases in flow-induced outward vascular remodeling

    PubMed Central

    Ota, Ryo; Kurihara, Chie; Tsou, Tsung-Ling; Young, William L.; Yeghiazarians, Yerem; Chang, Mayland; Mobashery, Shahriar; Sakamoto, Atsuhiro; Hashimoto, Tomoki

    2009-01-01

    Sustained hemodynamic stresses, especially high blood flow, result in flow-induced outward vascular remodeling. Our previous study showed that macrophage depletion reduced flow-induced outward remodeling of the rat common carotid artery, indicating that macrophages play critical roles in flow-induced outward vascular remodeling. Macrophage is known to release proteinases, including matrix metalloproteinases (MMPs). Degradation and loosening of extracellular matrix by MMPs may facilitate vascular remodeling. Therefore, we assessed the roles of MMPs in flow-induced outward vascular remodeling by using the flow-augmented common carotid artery model in mice. We validated that ligation of the left common carotid artery increased blood flow and luminal diameter of the right common carotid artery without significant change in blood pressure of mice. To assess the roles of MMPs in flow-induced outward vascular remodeling, we used doxycycline (broad-spectrum MMP inhibitor), SB-3CT (selective MMP inhibitor), MMP-9 knockout mice, and MMP-12 knockout mice. Although there was only a trend for doxycycline treatment to reduce flow-induced outward vascular remodeling, SB-3CT treatment significantly reduced flow-induced outward vascular remodeling. In addition, flow-induced outward vascular remodeling was significantly reduced in MMP-9 knockout mice, but not in MMP-12 knockout mice. These data revealed that MMPs, especially MMP-9, play critical roles in flow-induced outward vascular remodeling. PMID:19513084

  6. 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

  7. Physiological price of an induced chemical defense: photosynthesis, respiration, biosynthesis, and growth.

    PubMed

    Zangerl, A R; Arntz, A Michele; Berenbaum, May R

    1997-02-01

    A recurring theme in defense allocation theories is that defenses are costly. Most studies that attempt to quantify a cost of defense seek to establish a trade-off between a component of plant fitness and the level of a constitutive defense. Such estimates are ambiguous because they cannot discount the cost of traits that are correlated with defense but are not themselves defensive. We examined the effects of damage-induced synthesis of furanocoumarins, known defense compounds, on the growth of wild parsnip. Plants that had 2% of their leaf area removed accumulated 8.6% less total biomass and 14% less root biomass than intact plants over a 4-week period. We also found that this small amount of leaf damage significantly reduced net photosynthetic rates 0.5 h after damage; the effect was temporary, as photosynthetic rates were no longer significantly different after 48 h. Lastly, we found that increases in respiration rates associated with damage coincided spatially and temporally with increases in furanocoumarin production, and that respiration increases were phenotypically correlated with furanocoumarin production. When damage-induced changes in furanocoumarin content and respiration rates were expressed in glucose equivalents and compared, the energetic cost of furanocoumarin production (12.6 μg glucose cm(-2)) accounted for all of the increase in respiration (12.0 μg glucose cm(-2)). A comparison of other secondary compounds in damaged and intact leaflets revealed that myristicin, a furanocoumarin synergist, is the only other compound aside from furanocoumarins that is inducible. The inducible defense system of wild parsnip thus appears to involve a small subset of secondary compounds. Synthesis of these compounds is tightly linked to damage-induced rates of respiration. Because the negative impact that damage had on the rate of net photosynthesis was short-lived, the impact of damage on growth observed in this study was likely due to the cost of

  8. 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.

  9. Differential expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in avian tibial dyschondroplasia.

    PubMed

    Shahzad, Muhammad; Liu, Jingying; Gao, Jianfeng; Wang, Zhi; Zhang, Ding; Nabi, Fazul; Li, Kun; Li, Jiakui

    2015-01-01

    Tibial dyschondroplasia (TD) is an avian bone disorder of different aetiologies that may be associated with lameness. The disorder is characterized by focal disruption of endochondral bone formation, with a lack of matrix proteolysis and an accumulation of non-mineralized avascular cartilage. The aim of this study was to determine the expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in normal, thiram-induced TD lesions and in the process of recovery from TD in broiler chickens. An extracellular matrix (ECM) degrading enzyme, matrix metalloproteinase-9 (MMP-9), was selected to investigate the effects of CD147 in the degradation of ECM. Gene expression was analysed by quantitative real-time polymerase chain reaction and protein levels by immunohistochemistry and western blotting. The birds were divided into three groups: thiram fed; recovery; and controls. Genes encoding CD147 and MMP-9 were down-regulated during the development of the disease, and were up-regulated during recovery. Western blotting also showed lower protein levels of CD147 in TD, which increased during the recovery phase associated with ECM degradation and growth plate repair. The findings of this study suggest that ECM has a crucial role in the occurrence of TD and that CD147 appears to play a pivotal role in matrix proteolysis in the chicken, similar to that in other species.

  10. 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.

  11. 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

  12. 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

  13. 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.

  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. 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.

  16. Vitamin B12 insufficiency induces cholesterol biosynthesis by limiting s-adenosylmethionine and modulating the methylation of SREBF1 and LDLR genes.

    PubMed

    Adaikalakoteswari, Antonysunil; Finer, Sarah; Voyias, Philip D; McCarthy, Ciara M; Vatish, Manu; Moore, Jonathan; Smart-Halajko, Melissa; Bawazeer, Nahla; Al-Daghri, Nasser M; McTernan, Philip G; Kumar, Sudhesh; Hitman, Graham A; Saravanan, Ponnusamy; Tripathi, Gyanendra

    2015-01-01

    The dietary supply of methyl donors such as folate, vitamin B12, betaine, methionine, and choline is essential for normal growth, development, and physiological functions through the life course. Both human and animal studies have shown that vitamin B12 deficiency is associated with altered lipid profile and play an important role in the prediction of metabolic risk, however, as of yet, no direct mechanism has been investigated to confirm this. Three independent clinical studies of women (i) non-pregnant at child-bearing age, (ii) in early pregnancy, and (iii) at delivery showed that low vitamin B12 status was associated with higher total cholesterol, LDL cholesterol, and cholesterol-to-HDL ratio. These results guided the investigation into the cellular mechanisms of induced cholesterol biosynthesis due to vitamin B12 deficiency, using human adipocytes as a model system. Adipocytes cultured in low or no vitamin B12 conditions had increased cholesterol and homocysteine levels compared to control. The induction of cholesterol biosynthesis was associated with reduced s-adenosylmethionine (AdoMet)-to-s-adenosylhomocysteine (AdoHcy) ratio, also known as methylation potential (MP). We therefore studied whether reduced MP could lead to hypomethylation of genes involved in the regulation of cholesterol biosynthesis. Genome-wide and targeted DNA methylation analysis identified that the promoter regions of SREBF1 and LDLR, two key regulators of cholesterol biosynthesis, were hypomethylated under vitamin B12-deficient conditions, and as a result, their expressions and cholesterol biosynthesis were also significantly increased. This finding was further confirmed by the addition of the methylation inhibitor, 5-aza-2'-deoxycytidine, which resulted in increased SREBF1 and LDLR expressions and cholesterol accumulation in vitamin B12-sufficient conditions. Finally, we observed that the expression of SREBF1, LDLR, and cholesterol biosynthesis genes were increased in adipose tissue

  17. 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.

  18. [Expression of various matrix metalloproteinases in mice with hyperoxia-induced acute lung injury].

    PubMed

    Zhang, Xiang-feng; Ding, Shao-fang; Gao, Yuan-ming; Liang, Ying; Foda, Hussein D

    2006-08-01

    To investigate the role of matrix metalloproteinases (MMPs) and extracellular matrix metalloproteinase inducer (EMMPRIN) in the pathogenesis of acute lung injury induced by hyperoxia. Fifty four mice were exposed in sealed cages to >98% oxygen (for 24-72 hours), and another 18 mice to room air. The severity of lung injury was assessed, and the expression of mRNA and protein of MMP-2, MMP-9 and EMMPRIN in lung tissue, after exposure for 24, 48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Hyperoxia caused acute lung injury; this was accompanied by increased expression of an upregulation of MMP-2, MMP-9 and EMMPRIN mRNA and protein in lung tissues. Hyperoxia causes acute lung injury in mice; increases in MMP-2, MMP-9 and EMMPRIN may play an important role in the development of hyperoxia induced lung injury in mice.

  19. Biosynthesis in vitro of tryptophanase by polyribosomes from induced cultures of Escherichia coli

    PubMed Central

    Bashar, S. A. M. Khairul; Parish, J. H.; Brown, Marjorie

    1971-01-01

    1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues). PMID:4942304

  20. 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.

  1. Potassium deficiency induces the biosynthesis of oxylipins and glucosinolates in Arabidopsis thaliana

    PubMed Central

    2010-01-01

    Background Mineral fertilization and pest control are essential and costly requirements for modern crop production. The two measures go hand in hand because plant mineral status affects plant susceptibility to pests and vice versa. Nutrient deficiency triggers specific responses in plants that optimize nutrient acquisition and reprogram metabolism. K-deficient plants illustrate these strategies by inducing high-affinity K-uptake and adjusting primary metabolism. Whether and how K deficient plants also alter their secondary metabolism for nutrient management and defense is not known. Results Here we show that K-deficient plants contain higher levels of the phytohormone jasmonic acid (JA), hydroxy-12-oxo-octadecadienoic acids (HODs) and 12-oxo-phytodienoic acid (OPDA) than K-sufficient plants. Up-regulation of the 13-LOX pathway in response to low K was evident in increased transcript levels of several biosynthetic enzymes. Indole and aliphatic glucosinolates accumulated in response to K-deficiency in a manner that was respectively dependent or independent on signaling through Coronatine-Insensitive 1 (COI1). Transcript and glucosinolate profiles of K-deficient plants resembled those of herbivore attacked plants. Conclusions Based on our results we propose that under K-deficiency plants produce oxylipins and glucosinolates to enhance their defense potential against herbivorous insects and create reversible storage for excess S and N. PMID:20701801

  2. 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

  3. 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.

  4. Peroxiredoxin 4 Improves Insulin Biosynthesis and Glucose-induced Insulin Secretion in Insulin-secreting INS-1E Cells*

    PubMed Central

    Mehmeti, Ilir; Lortz, Stephan; Elsner, Matthias; Lenzen, Sigurd

    2014-01-01

    Oxidative folding of (pro)insulin is crucial for its assembly and biological function. This process takes place in the endoplasmic reticulum (ER) and is accomplished by protein disulfide isomerase and ER oxidoreductin 1β, generating stoichiometric amounts of hydrogen peroxide (H2O2) as byproduct. During insulin resistance in the prediabetic state, increased insulin biosynthesis can overwhelm the ER antioxidative and folding capacity, causing an imbalance in the ER redox homeostasis and oxidative stress. Peroxiredoxin 4 (Prdx4), an ER-specific antioxidative peroxidase can utilize luminal H2O2 as driving force for reoxidizing protein disulfide isomerase family members, thus efficiently contributing to disulfide bond formation. Here, we examined the functional significance of Prdx4 on β-cell function with emphasis on insulin content and secretion during stimulation with nutrient secretagogues. Overexpression of Prdx4 in glucose-responsive insulin-secreting INS-1E cells significantly metabolized luminal H2O2 and improved the glucose-induced insulin secretion, which was accompanied by the enhanced proinsulin mRNA transcription and insulin content. This β-cell beneficial effect was also observed upon stimulation with the nutrient insulin secretagogue combination of leucine plus glutamine, indicating that the effect is not restricted to glucose. However, knockdown of Prdx4 had no impact on H2O2 metabolism or β-cell function due to the fact that Prdx4 expression is negligibly low in pancreatic β-cells. Moreover, we provide evidence that the constitutively low expression of Prdx4 is highly susceptible to hyperoxidation in the presence of high glucose. Overall, these data suggest an important role of Prdx4 in maintaining insulin levels and improving the ER folding capacity also under conditions of a high insulin requirement. PMID:25122762

  5. 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.

  6. α-dl-Difluoromethylornithine, a Specific, Irreversible Inhibitor of Putrescine Biosynthesis, Induces a Phenotype in Tobacco Similar to That Ascribed to the Root-Inducing, Left-Hand Transferred DNA of Agrobacterium rhizogenes

    PubMed Central

    Burtin, D.; Martin-Tanguy, J.; Tepfer, D.

    1991-01-01

    α-dl-Difluoromethylarginine (DFMA) and α-dl-difluoromethylornithine (DFMO), specific irreversible inhibitors of putrescine biosynthesis were applied to Nicotiana tabacum var. Xanthi nc during floral induction. DFMO, but not DFMA, induced a phenotype in tobacco that resembles the transformed phenotype attributed to the root-inducing, left-hand, transferred DNA of Agrobacterium rhizogenes, including wrinkled leaves, shortened internodes, reduced apical dominance, and retarded flowering. Similar treatment of transformed plants (T phenotype) accentuated their phenotypic abnormalities. Cyclohexylammonium and methylglyoxal bis (guanylhydrazone), inhibitors of spermidine and spermine biosynthesis, produced reproductive abnormalities, but did not clearly mimic the transformed phenotype. This work strengthens the previously reported correlation between the degree of expression of the transformed phenotype due to the root-inducing, left-hand, transferred DNA and inhibition of polyamine accumulation, strongly suggesting that genes carried by the root-inducing, transferred DNA may act through interference with polyamine production via the ornithine pathway. Images Figure 1 PMID:16668006

  7. alpha-dl-Difluoromethylornithine, a Specific, Irreversible Inhibitor of Putrescine Biosynthesis, Induces a Phenotype in Tobacco Similar to That Ascribed to the Root-Inducing, Left-Hand Transferred DNA of Agrobacterium rhizogenes.

    PubMed

    Burtin, D; Martin-Tanguy, J; Tepfer, D

    1991-02-01

    alpha-dl-Difluoromethylarginine (DFMA) and alpha-dl-difluoromethylornithine (DFMO), specific irreversible inhibitors of putrescine biosynthesis were applied to Nicotiana tabacum var. Xanthi nc during floral induction. DFMO, but not DFMA, induced a phenotype in tobacco that resembles the transformed phenotype attributed to the root-inducing, left-hand, transferred DNA of Agrobacterium rhizogenes, including wrinkled leaves, shortened internodes, reduced apical dominance, and retarded flowering. Similar treatment of transformed plants (T phenotype) accentuated their phenotypic abnormalities. Cyclohexylammonium and methylglyoxal bis (guanylhydrazone), inhibitors of spermidine and spermine biosynthesis, produced reproductive abnormalities, but did not clearly mimic the transformed phenotype. This work strengthens the previously reported correlation between the degree of expression of the transformed phenotype due to the root-inducing, left-hand, transferred DNA and inhibition of polyamine accumulation, strongly suggesting that genes carried by the root-inducing, transferred DNA may act through interference with polyamine production via the ornithine pathway.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Tilapia (Oreochromis mossambicus) brain cells respond to hyperosmotic challenge by inducing myo-inositol biosynthesis.

    PubMed

    Gardell, Alison M; Yang, Jun; Sacchi, Romina; Fangue, Nann A; Hammock, Bruce D; Kültz, Dietmar

    2013-12-15

    This study aimed to determine the regulation of the de novo myo-inositol biosynthetic (MIB) pathway in Mozambique tilapia (Oreochromis mossambicus) brain following acute (25 ppt) and chronic (30, 60 and 90 ppt) salinity acclimations. The MIB pathway plays an important role in accumulating the compatible osmolyte, myo-inositol, in cells in response to hyperosmotic challenge and consists of two enzymes, myo-inositol phosphate synthase and inositol monophosphatase. In tilapia brain, MIB enzyme transcriptional regulation was found to robustly increase in a time (acute acclimation) or dose (chronic acclimation) dependent manner. Blood plasma osmolality and Na(+) and Cl(-) concentrations were also measured and significantly increased in response to both acute and chronic salinity challenges. Interestingly, highly significant positive correlations were found between MIB enzyme mRNA and blood plasma osmolality in both acute and chronic salinity acclimations. Additionally, a mass spectrometry assay was established and used to quantify total myo-inositol concentration in tilapia brain, which closely mirrored the hyperosmotic MIB pathway induction. Thus, myo-inositol is a major compatible osmolyte that is accumulated in brain cells when exposed to acute and chronic hyperosmotic challenge. These data show that the MIB pathway is highly induced in response to environmental salinity challenge in tilapia brain and that this induction is likely prompted by increases in blood plasma osmolality. Because the MIB pathway uses glucose-6-phosphate as a substrate and large amounts of myo-inositol are being synthesized, our data also illustrate that the MIB pathway likely contributes to the high energetic demand posed by salinity challenge.

  13. Tilapia (Oreochromis mossambicus) brain cells respond to hyperosmotic challenge by inducing myo-inositol biosynthesis

    PubMed Central

    Gardell, Alison M.; Yang, Jun; Sacchi, Romina; Fangue, Nann A.; Hammock, Bruce D.; Kültz, Dietmar

    2013-01-01

    SUMMARY This study aimed to determine the regulation of the de novo myo-inositol biosynthetic (MIB) pathway in Mozambique tilapia (Oreochromis mossambicus) brain following acute (25 ppt) and chronic (30, 60 and 90 ppt) salinity acclimations. The MIB pathway plays an important role in accumulating the compatible osmolyte, myo-inositol, in cells in response to hyperosmotic challenge and consists of two enzymes, myo-inositol phosphate synthase and inositol monophosphatase. In tilapia brain, MIB enzyme transcriptional regulation was found to robustly increase in a time (acute acclimation) or dose (chronic acclimation) dependent manner. Blood plasma osmolality and Na+ and Cl− concentrations were also measured and significantly increased in response to both acute and chronic salinity challenges. Interestingly, highly significant positive correlations were found between MIB enzyme mRNA and blood plasma osmolality in both acute and chronic salinity acclimations. Additionally, a mass spectrometry assay was established and used to quantify total myo-inositol concentration in tilapia brain, which closely mirrored the hyperosmotic MIB pathway induction. Thus, myo-inositol is a major compatible osmolyte that is accumulated in brain cells when exposed to acute and chronic hyperosmotic challenge. These data show that the MIB pathway is highly induced in response to environmental salinity challenge in tilapia brain and that this induction is likely prompted by increases in blood plasma osmolality. Because the MIB pathway uses glucose-6-phosphate as a substrate and large amounts of myo-inositol are being synthesized, our data also illustrate that the MIB pathway likely contributes to the high energetic demand posed by salinity challenge. PMID:24072790

  14. Light-Induced Expression of a MYB Gene Regulates Anthocyanin Biosynthesis in Red Apples1

    PubMed Central

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

    2006-01-01

    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. PMID:17012405

  15. 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

  16. 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

  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. 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.

  19. 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.

  20. Induced biosynthesis of resveratrol and the prenylated stilbenoids arachidin-1 and arachidin-3 in hairy root cultures of peanut: Effects of culture medium and growth stage.

    PubMed

    Condori, Jose; Sivakumar, Ganapathy; Hubstenberger, John; Dolan, Maureen C; Sobolev, Victor S; Medina-Bolivar, Fabricio

    2010-05-01

    Previously, we have shown that hairy root cultures of peanut provide a controlled, sustainable and scalable production system that can be induced to produce stilbenoids. However to leverage peanut hairy roots to study the biosynthesis of this polyphenolic biosynthetic pathway, growing conditions and elicitation kinetics of these tissue cultures must be defined and understood. To this end, a new peanut cv. Hull hairy root (line 3) that produces resveratrol and its prenylated analogues arachidin-1 and arachidin-3 upon sodium acetate-mediated elicitation was established. Two culture media were compared for impact on root growth and stilbenoid biosynthesis/secretion. The levels of ammonium, nitrate, phosphate and residual sugars were monitored along growth and elicitation period. A modified MS (MSV) medium resulted in higher root biomass when compared to B5 medium. The stilbenoid profile after elicitation varied depending on the age of the culture (6, 9, 12, and 15-day old). After elicitation at day 9 (exponential growth in MSV medium), over 90% of the total resveratrol, arachidin-1 and arachidin-3 accumulated in the medium. Our studies demonstrate the benefits of the hairy root culture system to study the biosynthesis of stilbenoids including valuable prenylated polyphenolic compounds.

  1. 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.

  2. 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

  3. Passive strain-induced matrix synthesis and organization in shape-specific, cartilaginous neotissues.

    PubMed

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

    2014-12-01

    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

  4. 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

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

    USDA-ARS?s Scientific Manuscript database

    The effects of physical wounding on ABA biosynthesis and catabolism and expression of genes encoding key ABA metabolic enzymes were determined in potato (Solanum tuberosum L.) tubers. An increase in ABA and ABA metabolite content was observed 48 h after wounding and remained elevated through 96 h. ...

  6. Rho-mediated Contractility Exposes a Cryptic Site in Fibronectin and Induces Fibronectin Matrix Assembly

    PubMed Central

    Zhong, Cuiling; Chrzanowska-Wodnicka, Magdalena; Brown, James; Shaub, Amy; Belkin, Alexey M.; Burridge, Keith

    1998-01-01

    Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447–1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987

  7. 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

  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. Influence of whole body irradiation and local shielding on matrix-induced endochondral bone differentiation

    SciTech Connect

    Wientroub, S.; Weiss, J.F.; Catravas, G.N.; Reddi, A.H. )

    1990-01-01

    Subcutaneous implantation of demineralized bone matrix into allogeneic rats induces endochondral bone formation. We have investigated the effects of irradiation on the sequelae of the interaction of collagenous matrix and mesenchymal cells and on cartilage and bone differentiation. Rats were irradiated in a vertical direction with a midline dose of 850 rad. Radiation entered the rats ventrally while a small area of the upper thorax was locally shielded. After irradiation, bone matrix was implanted in shielded and nonshielded sites, and the implants were studied at various stages. On day 3, (3H)thymidine incorporation, an index of cell proliferation, was inhibited by 70% in the nonshielded sites compared to nonirradiated control rats. The degree of inhibition (35%) was less pronounced in shielded sites. Furthermore, there was recovery of cell proliferation in the shielded sites as opposed to the nonshielded contralateral site. A similar pattern was observed on day 7 as assessed by 35SO4 incorporation into proteoglycans during chondrogenesis. Bone formation and mineralization were quantified on day 11 by alkaline phosphatase activity and 45Ca incorporation. In nonshielded sites, there was a 73% inhibition of alkaline phosphatase activity. In conclusion, radiation impaired progenitor cell proliferation which resulted in decreased cartilage and bone differentiation. These findings imply that local mesenchymal cells proliferate and differentiate into bone in response to implanted collagenous matrix.

  10. Changes in eggshell mechanical properties, crystallographic texture and in matrix proteins induced by moult in hens.

    PubMed

    Ahmed, A M H; Rodriguez-Navarro, A B; Vidal, M L; Gautron, J; García-Ruiz, J M; Nys, Y

    2005-06-01

    The effect of moult on eggshell mechanical properties, on composition and concentrations of organic matrix components and on eggshell microstructure was investigated. The observed changes were studied to understand the role of organic matrix and eggshell microstructure in eggshell strength. Moult was induced by zinc oxide (20 g zinc/kg diet) in 53 ISA Brown laying hens at 78 weeks of age. No difference was observed for egg or eggshell weights after moult. In contrast, moult improved the shell breaking strength (28.09 vs 33.71 N). After moult, there was a decrease in the average size of calcite crystals composing the eggshell and in their heterogeneity, whereas crystal orientation remained basically the same. After moulting, the total protein concentration in eggshell increased slightly. The comparisons of SDS-PAGE profiles of the organic matrix constituents extracted before and after moulting showed changes in staining intensity of certain bands. After moult, bands associated with main proteins specific to eggshell formation (OC-116 and OC-17) showed higher staining intensity, while the intensity of the egg white proteins (ovotransferrin, ovalbumin and lysozyme) decreased. ELISA confirmed the decrease in ovotransferrin after moult. Its concentration was inversely correlated with breaking strength before moult. These observations suggest that changes in eggshell crystal size could be due to changes in organic matrix composition. These changes may provide a mechanism for the improvement in shell solidity after moulting.

  11. Inhibition of CDKS by roscovitine suppressed LPS-induced ·NO production through inhibiting NFκB activation and BH4 biosynthesis in macrophages

    PubMed Central

    Wei, Na; Guan, Tongju; Xu, Hao; An, Jianzhong; Pritchard, Kirkwood A.

    2009-01-01

    In inflammatory diseases, tissue damage is critically associated with nitric oxide (·NO) and cytokines, which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on ·NO production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of ·NO induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IκB kinase β (IKKβ), IκB, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun NH2-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1β, and IL-6 but not tumor necrosis factor (TNF)-α. Tetrahydrobiopterin (BH4), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (BH2). Roscovitine significantly inhibited LPS-induced BH4 biosynthesis and decreased BH4-to-BH2 ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for BH4 biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced ·NO production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited ·NO production, iNOS, and COX-2 upregulation, activation of NFκB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced ·NO production in macrophages by suppressing nuclear factor-κB activation and BH4 biosynthesis, which might be mediated by CDK1, CDK5, and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation. PMID:19553566

  12. 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.

  13. Calcipotriol counteracts betamethasone-induced decrease in extracellular matrix components related to skin atrophy.

    PubMed

    Norsgaard, Hanne; Kurdykowski, Sandrine; Descargues, Pascal; Gonzalez, Tatiana; Marstrand, Troels; Dünstl, Georg; Røpke, Mads

    2014-10-01

    The calcipotriol/betamethasone dipropionate fixed-combination gel is widely used for topical treatment of psoriasis vulgaris. It has been hypothesized that calcipotriol counteracts glucocorticoid-induced skin atrophy which is associated with changes in the extracellular matrix (ECM). To elucidate the combined effects of calcipotriol and betamethasone on key ECM components, a comparative study to the respective mono-treatments was carried out. The effect on collagen I synthesis, matrix metalloproteinase (MMP) secretion, and hyaluronic acid (HA) production was investigated in primary human fibroblast and keratinocyte cultures as well as in a human skin explant model. We show that calcipotriol counteracts betamethasone-induced suppression of collagen I synthesis. Similarly, calcipotriol and betamethasone have opposing effects on MMP expression in both fibroblasts and keratinocytes. Moreover, calcipotriol is able to restore betamethasone-impaired HA synthesis in keratinocytes and prevent betamethasone-induced epidermal thinning in minipigs upon treatment with the calcipotriol/betamethasone gel. In summary, our results show for the first time in primary human skin cultures that calcipotriol reduces early signs of betamethasone-induced skin atrophy by modulation of key ECM components. These results indicate that the calcipotriol component of the fixed-combination gel counteracts the atrophogenic effects of betamethasone on the skin.

  14. 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.

  15. 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.

  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. 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

  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. 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.

  20. Thermomechanical analysis of freezing-induced cell-fluid-matrix interactions in engineered tissues

    PubMed Central

    Han, Bumsoo; Teo, Ka Yaw; Ghosh, Soham; Dutton, J. Craig; Grinnell, Frederick

    2012-01-01

    Successful cryopreservation of functional engineered tissues (ETs) is significant to tissue engineering and regenerative medicine, but it is extremely challenging to develop a successful protocol because the effects of cryopreservation parameters on the post-thaw functionality of ETs are not well understood. Particularly, the effects on the microstructure of their extracellular matrix (ECM) have not been well studied, which determines many functional properties of the ETs. In this study, we investigated the effects of two key cryopreservation parameters – i) freezing temperature and corresponding cooling rate; and ii) the concentration of cryoprotective agent (CPA) on the ECM microstructure as well as the cellular viability. Using dermal equivalent as a model ET and DMSO as a model CPA, freezing-induced spatiotemporal deformation and post-thaw ECM microstructure of ETs was characterized while varying the freezing temperature and DMSO concentrations. The spatial distribution of cellular viability and the cellular actin cytoskeleton was also examined. The results showed that the tissue dilatation increased significantly with reduced freezing temperature (i.e., rapid freezing). A maximum limit of tissue deformation was observed for preservation of ECM microstructure, cell viability and cell-matrix adhesion. The dilatation decreased with the use of DMSO, and a freezing temperature dependent threshold concentration of DMSO was observed. The threshold DMSO concentration increased with lowering freezing temperature. In addition, an analysis was performed to delineate thermodynamic and mechanical components of freezing-induced tissue deformation. The results are discussed to establish a mechanistic understanding of freezing-induced cell-fluid-matrix interaction and phase change behavior within ETs in order to improve cryopreservation of ETs. PMID:23246556

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  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. Increased Potassium Conductance of Brain Mitochondria Induces Resistance to Permeability Transition by Enhancing Matrix Volume*

    PubMed Central

    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 Ca2+ 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 mitoKATP 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 ΔpH. The beneficial effect of valinomycin on CRC was not mediated by H2O2-induced protein kinase Cϵ activation. Rather, increased K+ conductance reduced H2O2 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. PMID:19880514

  8. RAGE-mediated extracellular matrix proteins accumulation exacerbates HySu-induced pulmonary hypertension.

    PubMed

    Jia, Daile; He, Yuhu; Zhu, Qian; Liu, Huan; Zuo, Caojian; Chen, Guilin; Yu, Ying; Lu, Ankang

    2017-05-01

    Extracellular matrix (ECM) proteins accumulation contributes to the progression of pulmonary arterial hypertension (PAH), a rare and fatal cardiovascular condition defined by high pulmonary arterial pressure, whether primary, idiopathic, or secondary to other causes. The receptor for advanced glycation end products (RAGE) is constitutively expressed in the lungs and plays an important role in ECM deposition. Nonetheless, the mechanisms by which RAGE mediates ECM deposition/formation in pulmonary arteries and its roles in PAH progression remain unclear. Expression of RAGE and its activating ligands, S100/calgranulins and high mobility group box 1 (HMGB1), were increased in both human and mouse pulmonary arterial smooth muscle cells (PASMCs) under hypoxic conditions and were also strikingly upregulated in pulmonary arteries in hypoxia plus SU5416 (HySu)-induced PAH in mice. RAGE deletion alleviated pulmonary arterial pressure and restrained extracellular matrix accumulation in pulmonary arteries in HySu-induced PAH murine model. Moreover, blocking RAGE activity with a neutralizing antibody in human PASMCs, or RAGE deficiency in mouse PASMCs exposed to hypoxia, suppressed the expression of fibrotic proteins by reducing TGF-β1 expression. RAGE reconstitution in deficient mouse PASMCs restored hypoxia-stimulated TGF-β1 production via ERK1/2 and p38 MAPK pathway activation and subsequently increased ECM protein expression. Interestingly, HMGB1 acting on RAGE, not toll-like receptor 4 (TLR4), induced ECM deposition in PASMCs. Finally, in both idiopathic PAH patients and HySu-induced PAH mice, soluble RAGE (sRAGE) levels in serum were significantly elevated compared to those in controls. Activation of RAGE facilitates the development of hypoxia-induced pulmonary hypertension by increase of ECM deposition in pulmonary arteries. Our results indicate that sRAGE may be a potential biomarker for PAH diagnosis and disease severity, and that RAGE may be a promising target for

  9. 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.

  10. Effects of short-term glucocorticoid treatment on changes in cartilage matrix degradation and chondrocyte gene expression induced by mechanical injury and inflammatory cytokines

    PubMed Central

    2011-01-01

    Introduction Traumatic joint injury damages cartilage and causes adjacent joint tissues to release inflammatory cytokines, increasing the risk of developing osteoarthritis. The main objective of this study was to determine whether the combined catabolic effects of mechanical injury, tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R) on cartilage could be abolished by short-term treatment with glucocorticoids such as dexamethasone. Methods In an initial dexamethasone-dose-response study, bovine cartilage explants were treated with TNFα and increasing concentrations of dexamethasone. Bovine and human cartilage explants were then subjected to individual and combined treatments with TNFα, IL-6/sIL-6R and injury in the presence or absence of dexamethasone. Treatment effects were assessed by measuring glycosaminoglycans (GAG) release to the medium and synthesis of proteoglycans. Additional experiments tested whether pre-exposure of cartilage to dexamethasone could prevent GAG loss and inhibition of biosynthesis induced by cytokines, and whether post-treatment with dexamethasone could diminish the effects of pre-established cytokine insult. Messenger ribonucleic acid (mRNA) levels for genes involved in cartilage homeostasis (proteases, matrix molecules, cytokines, growth and transcription factors) were measured in explants subjected to combined treatments with injury, TNFα and dexamethasone. To investigate mechanisms associated with dexamethasone regulation of chondrocyte metabolic response, glucocorticoid receptor (GR) antagonist (RU486) and proprotein convertase inhibitor (RVKR-CMK) were used. Results Dexamethasone dose-dependently inhibited GAG loss and the reduction in biosynthesis caused by TNFα. The combination of mechanical injury, TNFα and IL-6/sIL-6R caused the most severe GAG loss; dexamethasone reduced this GAG loss to control levels in bovine and human cartilage. Additionally, dexamethasone pre-treatment or post

  11. 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.

  12. Titanium implants induce expression of matrix metalloproteinases in bone during osseointegration.

    PubMed

    Shubayev, Veronica I; Brånemark, Rickard; Steinauer, Joanne; Myers, Robert R

    2004-01-01

    Implanted pure titanium fixtures are able to completely integrate with bone, in part because of the formation of a strong extracellular matrix (ECM) bond at the titanium-bone interface. In this study, we used a rodent femur model of intramedullary osseointegration to analyze the changes in immunoreactivity of ECM-controlling matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinase-3 (TIMP-3), and tumor necrosis factor alpha (TNF-alpha) during osseointegration. We observed dramatic increases in MMP-2, MMP-9, MMP-7, TIMP-3, and TNF-alpha in osteocytes, osteoclasts, haversian canals, and the interface matrix in bone ipsilateral to the titanium implant. An increase in TIMP-3, MMP-9, and MMP-7 in hypertrophied chondrocytes and the vascular component of the epiphysial growth plate was also observed in experimental bone. These findings were not seen in contralateral or sham-operated bone, where the titanium fixtures were threaded into the femur and immediately removed. Our data link titanium-induced bone remodeling to changes in expression and distribution of MMPs.

  13. Matrix metalloproteinases contribute to endotoxin and interleukin-1β induced vascular dysfunction

    PubMed Central

    Lalu, M M; Cena, J; Chowdhury, R; Lam, A; Schulz, R

    2006-01-01

    Background and purpose: The acute vascular inflammatory dysfunction associated with endotoxaemia may reflect an imbalance between matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), induced by the endotoxin. This possibility was tested in rat aortic tissue. Experimental approaches: Tone induced by phenylephrine in aortic rings was measured after exposure in vitro to ambient lipopolysaccharide (LPS) or the proinflammatory cytokine interleukin-1β (IL-1β) for 6h, with or without MMP inhibitors (doxycycline or GM6001). Gelatinase and MMP activities, TIMP proteins and contractility were measured in aortae taken from rats 6h after receiving LPS in vivo. Key results: Inhibition of MMP prevented the loss of phenylephrine–induced tone in aortic rings after LPS or IL-1β. IL-1β also increased release of MMP-2 activity from aortic tissue. In aortae exposed in vivo to LPS, net gelatinase, MMP-9 activities and TIMP-1 protein levels were increased, whereas TIMP-4 was reduced. These aortae were hypocontractile to both phenylephrine and KCl. Hypocontractility was partially reversed by doxycycline ex vivo. Conclusions and Implications: MMP inhibitors ameliorate vascular hyporeactivity induced by either LPS or IL-1β in vitro. LPS in vivo alters the balance between MMPs and TIMPs, contributing to vascular dysfunction which is partially reversed by MMP inhibitors. Vascular MMPs are activated as a result of LPS or IL-1β-induced stress and contribute to the hyporeactivity of blood vessels to vasoconstrictors. PMID:16880766

  14. Hemosomegenesis and hemoglobin biosynthesis in vertebrates.

    PubMed

    Brunner Júnior, A; de Rizzo, E; Morena, D D; Cianciarullo, A M; Jared, C; Morena, P

    1992-08-01

    1. Ultrastructural observations on maturing rabbit embryo erythroid cells led to the finding of hemoglobinized organelles distinguishable from mitochondria due to their highly dense matrix, two or three longitudinally arranged double lamellae, and smaller diameters. Intraorganellar 50-60 A particles identical to those contained in the hemoglobinized cytoplasm were found. 2. Their hemoglobin (Hb) content was demonstrated by electrophoresis of the concentrated supernatant from the isolated, washed, and osmotically lysed organellar fraction. We have proposed that these organelles are the sites for heme integration into the globin (G) polypeptide chains and subunits assembly. The term hemosome has been suggested for such entities. 3. This hypothesis has been sustained by several analytical and experimental works based on the postulation that hemosomes should be found at higher frequencies where the Hb biosynthesis rate is more intensive, or where the induction of this biosynthesis is always dependent on the formation of hemosomes. 4. Maturing erythroid cells of the circulating embryo blood contain hemosomes in higher frequency than in liver erythroid cells, coinciding with the higher Hb biosynthesis rate in peripheral blood than in the liver. In bleeding anemia, the decay of Hb concentration parallels the reduction of the mean number of hemosomes per reticulocyte, in comparison with normal reticulocytes. 5. In HeLa cells and epithelial cultured cells induced to synthesize Hb, it was shown that this biosynthesis is ever concomitant with the formation of hemosomes and depends on the presence of erythropoietin, as occurs in erythroid cells. 6. Studies on hemosomegenesis and Hb biosynthesis experimentally effected in epithelial cultured cells, allowed the interpretation of the sequence of events leading to hemosome formation in maturing erythroid cells. Simultaneously with iron uptake, mitochondria differentiate to lamellated bodies and, successively, expansions rise for

  15. 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.

  16. 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

  17. Phase field modelling of strain induced crystal growth in an elastic matrix.

    PubMed

    Laghmach, Rabia; Candau, Nicolas; Chazeau, Laurent; Munch, Etienne; Biben, Thierry

    2015-06-28

    When a crystal phase grows in an amorphous matrix, such as a crystallisable elastomer, containing cross-links and/or entanglements, these "topological constraints" need to be pushed away from the crystal phase to allow further crystallization. The accumulation of these topological constraints in the vicinity of the crystal interface may store elastic energy and affect the phase transition. To evaluate the consequences of such mechanism, we introduce a phase field model based on the Flory theory of entropic elasticity. We show that the growth process is indeed sensibly affected, in particular, an exponential increase of the surface energy with the displacement of the interface is induced. This explains the formation of stable nano-crystallites as it is observed in the Strain Induced Crystallization (SIC) of natural rubber. Although simple, the model developed here is able to account for many interesting features of SIC, for instance, the crystallite shapes and their sizes which depend on the applied deformation.

  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. Calcium channel blockade reduces mechanical strain-induced extracellular matrix gene response in lamina cribrosa cells.

    PubMed

    Quill, B; Irnaten, M; Docherty, N G; McElnea, E M; Wallace, D M; Clark, A F; O'Brien, C J

    2015-07-01

    This study examines the effect of the L-type calcium channel blocker verapamil on mechanical strain-induced extracellular matrix genes in optic nerve head lamina cribrosa (LC) cells. Changes in LC cell intracellular calcium [Ca(2+)]i following hypotonic cell membrane stretch were measured with the fluorescent probe fura-2/AM. Fluorescence intensity was measured, after labelling, by calcium (Ca2+) imaging confocal microscopy. Confluent human LC cell cultures were serum starved for 24 h prior to exposure to cyclical mechanical strain (1 Hz, 15%) for 24 h in the presence or absence of verapamil (10 mm). Transforming growth factor-β 1 (TGF-β1), collagen 6A3 (COL6A3) and chondroitin sulfate proteoglycan 2 (CSPG2) mRNA expression levels were assessed by quantitative RT-PCR. Hypotonic cell membrane stretch of LC cells from normal donors significantly increased [Ca2+]i (p<0.05). Exposure to cyclical mechanical strain (15% strain) produced a statistically significant increase in the three matrix genes that were examined (TGF-β1, COL6A3 and CSPG2). This response in both cyclical and mechanical stretch was significantly reduced by pretreating LC cells with the L-type calcium channel blocker verapamil (p<0.05). This study provides evidence of a novel mechanotransduction pathway linking mechanical strain, cation channel function and the induction of LC cell matrix gene transcription. This highlights the potential involvement of calcium influx in the activation of matrix remodelling responses in the optic nerve head and supports the rationale that calcium channel blockers may attenuate disease progression in glaucoma. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  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. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

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

    PubMed

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

    2011-08-01

    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. © 2011 The Author(s).

  3. 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.

  4. Induction of autoantibodies against lung matrix proteins and smoke-induced inflammation in mice.

    PubMed

    Brandsma, Corry-Anke; Timens, Wim; Geerlings, Marie; Jekel, Henrike; Postma, Dirkje S; Hylkema, Machteld N; Kerstjens, Huib A M

    2010-12-13

    Smoking is the major etiologic factor in COPD, yet the exact underlying pathogenetic mechanisms have not been elucidated. Since a few years, there is mounting evidence that a specific immune response, partly present as an autoimmune response, contributes to the pathogenesis of COPD. Increased levels of anti-Hep-2 epithelial cell and anti-elastin autoantibodies as well as antibodies against airway epithelial and endothelial cells have been observed in COPD patients. Whether the presence of these autoantibodies contributes to the pathogenesis of COPD is unclear. To test whether induction of autoantibodies against lung matrix proteins can augment the smoke-induced inflammatory response, we immunized mice with a mixture of the lung extracellular matrix (ECM) proteins elastin, collagen, and decorin and exposed them to cigarette smoke for 3 or 6 months. To evaluate whether the immunization was successful, the presence of specific antibodies was assessed in serum, and presence of specific antibody producing cells in spleen and lung homogenates. In addition, the presence of inflammatory cells and cytokines was assessed in lung tissue and emphysema development was evaluated by measuring the mean linear intercept. We demonstrated that both ECM immunization and smoke exposure induced a humoral immune response against ECM proteins and that ECM immunization itself resulted in increased macrophage numbers in the lung. The specific immune response against ECM proteins did not augment the smoke-induced inflammatory response in our model. By demonstrating that smoke exposure itself can result in a specific immune response and that presence of this specific immune response is accompanied by an influx of macrophages, we provide support for the involvement of a specific immune response in the smoke-induced inflammatory response as can be seen in patients with COPD.

  5. 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

  6. 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

  7. 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

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

    NASA Technical Reports Server (NTRS)

    Liaw, Benjamin; Zeichner, Glenn; Liu, Yanxiong; Bowles, Kenneth J. (Technical Monitor)

    2000-01-01

    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's missions and current interests, such as ballistic impact testing of composite fan containment and high strain rate deformation modeling of polymer matrix composites. Currently, impact-induced delamination and fracture in 6061-T6 aluminum/cast acrylic sandwich plates adhered by epoxy were generated in an instrumented drop-weight impact machine. Although only a small dent was produced on the aluminum side when a hemispherical penetrator tup was dropped onto it from a couple of inches, a large ring of delamination at the interface was observed. The delamination damage was often accompanied by severe shattering in the acrylic substratum. Damage patterns in the acrylic layer include radial and ring cracks and, together with delamination at the interface, may cause peeling-off of acrylic material from the sandwich plate. Theory of stress-wave propagation can be used to explain these damage patterns. The impact tests were conducted at various temperatures. The results also show clearly that temperature effect is very important in impact damage. For pure cast acrylic nil-ductile transition (NDT) occurs between 185-195 F Excessive impact energy was dissipated into fracture energy when tested at temperature below this range or through plastic deformation when tested at temperature above the NDT temperature. Results from this study will be used as baseline data for studying fiber-metal laminates, such as GLARE and ARALL for advanced aeronautical and astronautical applications.

  9. 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

  10. Extracellular matrix metalloproteinase inducer is increased in smokers' bronchoalveolar lavage fluid.

    PubMed

    Betsuyaku, Tomoko; Tanino, Mishie; Nagai, Katsura; Nasuhara, Yasuyuki; Nishimura, Masaharu; Senior, Robert M

    2003-07-15

    Extracellular matrix metalloproteinase inducer (EMMPRIN), also called basigin, is present in the lung during development, but its expression in normal adult lung is minimal. Increases of EMMPRIN have been found in various forms of experimental lung injury. To determine whether EMMPRIN might be involved in alveolar injury/repair associated with smoking, we developed an ELISA for EMMPRIN and applied it to bronchoalveolar lavage fluids from never-smokers (n = 7), former smokers (n = 16), and current smokers (n = 58). The smoker groups included subjects with emphysema, as determined by high-resolution chest computed tomography. EMMPRIN levels were significantly elevated in current and former smokers (315 +/- 20 and 175 +/- 15 pg/ml SEM, respectively, compared with 31 +/- 7 pg/ml in never-smokers), but the EMMPRIN levels of smokers with emphysema were not different from smokers without emphysema. Immunohistochemistry of smokers' lung tissue showed EMMPRIN in bronchiolar epithelium and alveolar macrophages, but EMMPRIN mRNA in alveolar macrophages was not different between current and never-smokers. Matrix metalloproteinase-1 was also detectable in the bronchoalveolar lavage fluid from some smokers but not in never-smokers. These findings indicate that smoking is associated with increased intrapulmonary EMMPRIN. Whether EMMPRIN is involved in smoking-induced lung pathology remains to be determined.

  11. 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.

  12. Ectopic Expression of KNOTTED1-Like Homeobox Protein Induces Expression of Cytokinin Biosynthesis Genes in Rice1[W

    PubMed Central

    Sakamoto, Tomoaki; Sakakibara, Hitoshi; Kojima, Mikiko; Yamamoto, Yuko; Nagasaki, Hiroshi; Inukai, Yoshiaki; Sato, Yutaka; Matsuoka, Makoto

    2006-01-01

    Some phytohormones such as gibberellins (GAs) and cytokinins (CKs) are potential targets of the KNOTTED1-like homeobox (KNOX) protein. To enhance our understanding of KNOX protein function in plant development, we identified rice (Oryza sativa) genes for adenosine phosphate isopentenyltransferase (IPT), which catalyzes the rate-limiting step of CK biosynthesis. Molecular and biochemical studies revealed that there are eight IPT genes, OsIPT1 to OsIPT8, in the rice genome, including a pseudogene, OsIPT6. Overexpression of OsIPTs in transgenic rice inhibited root development and promoted axillary bud growth, indicating that OsIPTs are functional in vivo. Phenotypes of OsIPT overexpressers resembled those of KNOX-overproducing transgenic rice, although OsIPT overexpressers did not form roots or ectopic meristems, both of which are observed in KNOX overproducers. Expression of two OsIPT genes, OsIPT2 and OsIPT3, was up-regulated in response to the induction of KNOX protein function with similar kinetics to those of down-regulation of GA 20-oxidase genes, target genes of KNOX proteins in dicots. However, expression of these two OsIPT genes was not regulated in a feedback manner. These results suggest that OsIPT2 and OsIPT3 have unique roles in the developmental process, which is controlled by KNOX proteins, rather than in the maintenance of bioactive CK levels in rice. On the basis of these findings, we concluded that KNOX protein simultaneously decreases GA biosynthesis and increases de novo CK biosynthesis through the induction of OsIPT2 and OsIPT3 expression, and the resulting high-CK and low-GA condition is required for formation and maintenance of the meristem. PMID:16861569

  13. 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.

  14. 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.

  15. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

    PubMed

    Hasaneen, Nadia A; Cao, Jian; Pulkoski-Gross, Ashleigh; Zucker, Stanley; Foda, Hussein D

    2016-02-17

    Idiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial-stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown. To determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α-smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- β1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the β-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated β-catenin. Human lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-β1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an

  16. 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.

  17. 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

  18. 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

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

    PubMed

    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

    2016-05-03

    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.

  20. 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.

  1. 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.

  2. Virus-induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid-7-hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis.

    PubMed

    Salim, Vonny; Yu, Fang; Altarejos, Joaquín; De Luca, Vincenzo

    2013-12-01

    Iridoids are a major group of biologically active molecules that are present in thousands of plant species, and one versatile iridoid, secologanin, is a precursor for the assembly of thousands of monoterpenoid indole alkaloids (MIAs) as well as a number of quinoline alkaloids. This study uses bioinformatics to screen large databases of annotated transcripts from various MIA-producing plant species to select candidate genes that may be involved in iridoid biosynthesis. Virus-induced gene silencing of the selected genes combined with metabolite analyses of silenced plants was then used to identify the 7-deoxyloganic acid 7-hydroxylase (CrDL7H) that is involved in the 3rd to last step in secologanin biosynthesis. Silencing of CrDL7H reduced secologanin levels by at least 70%, and increased the levels of 7-deoxyloganic acid to over 4 mg g(-1) fresh leaf weight compared to control plants in which this iridoid is not detected. Functional expression of this CrDL7H in yeast confirmed its biochemical activity, and substrate specificity studies showed its preference for 7-deoxyloganic acid over other closely related substrates. Together, these results suggest that hydroxylation precedes carboxy-O-methylation in the secologanin pathway in Catharanthus roseus. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  3. 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

  4. In Planta Variation of Volatile Biosynthesis: An Alternative Biosynthetic Route to the Formation of the Pathogen-Induced Volatile Homoterpene DMNT via Triterpene Degradation in Arabidopsis Roots

    PubMed Central

    Sohrabi, Reza; Huh, Jung-Hyun; Badieyan, Somayesadat; Rakotondraibe, Liva Harinantenaina; Kliebenstein, Daniel J.; Sobrado, Pablo; Tholl, Dorothea

    2015-01-01

    Plant-derived volatile compounds such as terpenes exhibit substantial structural variation and serve multiple ecological functions. Despite their structural diversity, volatile terpenes are generally produced from a small number of core 5- to 20-carbon intermediates. Here, we present unexpected plasticity in volatile terpene biosynthesis by showing that irregular homo/norterpenes can arise from different biosynthetic routes in a tissue specific manner. While Arabidopsis thaliana and other angiosperms are known to produce the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) or its C16-analog (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene by the breakdown of sesquiterpene and diterpene tertiary alcohols in aboveground tissues, we demonstrate that Arabidopsis roots biosynthesize DMNT by the degradation of the C30 triterpene diol, arabidiol. The reaction is catalyzed by the Brassicaceae-specific cytochrome P450 monooxygenase CYP705A1 and is transiently induced in a jasmonate-dependent manner by infection with the root-rot pathogen Pythium irregulare. CYP705A1 clusters with the arabidiol synthase gene ABDS, and both genes are coexpressed constitutively in the root stele and meristematic tissue. We further provide in vitro and in vivo evidence for the role of the DMNT biosynthetic pathway in resistance against P. irregulare. Our results show biosynthetic plasticity in DMNT biosynthesis in land plants via the assembly of triterpene gene clusters and present biochemical and genetic evidence for volatile compound formation via triterpene degradation in plants. PMID:25724638

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. Glibenclamide prevents increased extracellular matrix formation induced by high glucose concentration in mesangial cells.

    PubMed

    Giannico, Giovanna; Cortes, Pedro; Baccora, Mohammed H; Hassett, Clare; Taube, David W; Yee, Jerry

    2007-01-01

    Other than stimulation of cell contractility, little is known about the potential metabolic effects induced by sulfonylureas, independently of insulin action. Previous studies from our laboratory demonstrated complete abrogation of glomerulosclerosis in an experimental model of type 1 diabetes chronically (9 mo) treated with low-dose sulfonylureas (Biederman JI, Vera E, Pankhaniya R, Hassett C, Giannico G, Yee J, Cortes P. Kidney Int 67: 554-565, 2005). Therefore, the effects of glibenclamide (Glib) on net collagen I, collagen IV, and fibronectin medium net secretion and cell layer collagen I deposition were investigated in mesangial cells continuously exposed to 25 mM glucose for 8 wk and treated with predetermined increasing concentrations of Glib for the same period. Clinically relevant concentrations (0.01 microM) of Glib fully suppressed the high glucose-enhanced accumulation of collagen I, collagen IV, and fibronectin in the medium and inhibited collagen I deposition in the cell layer. These effects occurred while transforming growth factor (TGF)-beta1 medium concentration remained elevated and glucose uptake was increased to levels above those in 25 mM glucose-incubated cultures. The decreased collagen I accumulation occurred simultaneously with enhanced collagen I mRNA expression in concert with marked suppression of plasminogen inhibitor type-1 (PAI-1) mRNA and protein expression. This strongly suggests an accelerated matrix turnover favoring breakdown. Glib-induced effects demonstrated a biphasic pattern, being absent or reversed in cells treated with higher Glib concentrations (0.1 or 1 microM). Therefore, chronic Glib treatment at low concentrations markedly diminishes the high glucose-induced enhanced accumulation of extracellular matrix components by suppression of steady-state PAI-1 transcriptional activity. These results and those previously reported in vivo suggest that long-term Glib treatment may prevent glomerulosclerosis in insulin

  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. 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

  12. Cadherin-11 Overexpression Induces Extracellular Matrix Remodeling and Calcification in Mature Aortic Valves.

    PubMed

    Sung, Derek C; Bowen, Caitlin J; Vaidya, Kiran A; Zhou, Jingjing; Chapurin, Nikita; Recknagel, Andrew; Zhou, Bin; Chen, Jonathan; Kotlikoff, Michael; Butcher, Jonathan T

    2016-08-01

    Calcific aortic valve (AoV) disease is a significant clinical problem for which the regulatory mechanisms are poorly understood. Enhanced cell-cell adhesion is a common mechanism of cellular aggregation, but its role in calcific lesion formation is not known. Cadherin-11 (Cad-11) has been associated with lesion formation in vitro, but its function during adult valve homeostasis and pathogenesis is not known. This study aims to elucidate the specific functions of Cad-11 and its downstream targets, RhoA and Sox9, in extracellular matrix remodeling and AoV calcification. We conditionally overexpressed Cad-11 in murine heart valves using a novel double-transgenic Nfatc1(Cre);R26-Cad11(TglTg) mouse model. These mice developed hemodynamically significant aortic stenosis with prominent calcific lesions in the AoV leaflets. Cad-11 overexpression upregulated downstream targets, RhoA and Sox9, in the valve interstitial cells, causing calcification and extensive pathogenic extracellular matrix remodeling. AoV interstitial cells overexpressing Cad-11 in an osteogenic environment in vitro rapidly form calcific nodules analogous to in vivo lesions. Molecular analyses revealed upregulation of osteoblastic and myofibroblastic markers. Treatment with a Rho-associated protein kinase inhibitor attenuated nodule formation, further supporting that Cad-11-driven calcification acts through the small GTPase RhoA/Rho-associated protein kinase signaling pathway. This study identifies one of the underlying molecular mechanisms of heart valve calcification and demonstrates that overexpression of Cad-11 upregulates RhoA and Sox9 to induce calcification and extracellular matrix remodeling in adult AoV pathogenesis. The findings provide a potential molecular target for clinical treatment. © 2016 American Heart Association, Inc.

  13. Inhibitory effects of arbutin on melanin biosynthesis of alpha-melanocyte stimulating hormone-induced hyperpigmentation in cultured brownish guinea pig skin tissues.

    PubMed

    Lim, Yu-Ji; Lee, Eunjoo H; Kang, Tong Ho; Ha, Sang Keun; Oh, Myung Sook; Kim, Seong Min; Yoon, Tae-Jin; Kang, Chulhun; Park, Ji-Ho; Kim, Sun Yeou

    2009-03-01

    Arbutin has been used as a whitening agent in cosmetic products. Melanin, the major pigment that gives color to skin, may be over-produced with sun exposure or in conditions such as melasma or hyperpigmentary diseases. Tyrosinase is a key enzyme that catalyzes melanin synthesis in melanocytes; therefore, inhibitors of the tyrosinase enzyme could be used for cosmetic skin whitening. A recent study has reported that arbutin decreases melanin biosynthesis through the inhibition of tyrosinase activity. However, this inhibitory mechanism of arbutin was not sufficiently demonstrated in skin tissue models. We found that arbutin both inhibits melanin production in B16 cells induced with alpha-MSH and decreases tyrosinase activity in a cell-free system. Furthermore, the hyperpigmentation effects of alpha-MSH were abrogated by the addition of arbutin to brownish guinea pig and human skin tissues. These results suggest that arbutin may be a useful agent for skin whitening.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Twinning-induced plasticity (TWIP) and work hardening in Ti-based metallic glass matrix composites.

    PubMed

    Fan, J; Qiao, J W; Wang, Z H; Rao, W; Kang, G Z

    2017-05-12

    The present study demonstrates that Ti-based metallic glass matrix composites (MGMCs) with a normal composition of Ti43Zr32Ni6Ta5Be14 containing ductile dendrites dispersed in the glass matrix has been developed, and deformation mechanisms about the tensile property have been investigated by focusing on twinning-induced plasticity (TWIP) effect. The Ti-based MGMC has excellent tensile properties and pronounced tensile work-hardening capacity, with a yield strength of 1100 MPa and homogeneous elongation of 4%. The distinguished strain hardening is ascribed to the formation of deformation twinning within the dendrites. Twinning generated in the dendrites works as an obstacle for the rapid propagation of shear bands, and then, the localized necking is avoided, which ensures the ductility of such kinds of composites. Besides, a finite-element model (FEM) has been established to explain the TWIP effect which brings out a work-hardening behavior in the present MGMC instead of a localized strain concentration. According to the plasticity theory of traditional crystal materials and some new alloys, TWIP effect is mainly controlled by stacking fault energy (SFE), which has been analyzed intensively in the present MGMC.

  19. 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.

  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. Force-induced elastic matrix-mediated interactions in the presence of a rigid wall

    NASA Astrophysics Data System (ADS)

    Menzel, Andreas M.

    We consider an elastic composite material containing particulate inclusions in a soft elastic matrix that is bounded by a rigid wall, e.g., the substrate. If such a composite serves as a soft actuator, forces are imposed on or induced between the embedded particles. We investigate how the presence of the rigid wall affects the interactions between the inclusions in the elastic matrix. For no-slip boundary conditions, we transfer Blake's derivation of a corresponding Green's function from low-Reynolds-number hydrodynamics to the linearly elastic case. Results for no-slip and free-slip surface conditions are compared to each other and to the bulk behavior. Our results suggest that walls with free-slip surface conditions are preferred when they serve as substrates for soft actuators made from elastic composite materials. As we further demonstrate, the presence of a rigid wall can qualitatively change the interactions between the inclusions. In effect, it can switch attractive interactions into repulsive ones (and vice versa). It should be straightforward to observe the effects in future experiments and to combine our results, e.g., with the modeling of biological cells and tissue on rigid surfaces.

  2. 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

  3. Featured Article: Cardioprotective effects of lysyl oxidase inhibition against volume overload-induced extracellular matrix remodeling

    PubMed Central

    El Hajj, Elia C; El Hajj, Milad C; Ninh, Van K

    2015-01-01

    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. PMID:26582054

  4. Irradiation of skin with visible light induces reactive oxygen species and matrix-degrading enzymes.

    PubMed

    Liebel, Frank; Kaur, Simarna; Ruvolo, Eduardo; Kollias, Nikiforos; Southall, Michael D

    2012-07-01

    Daily skin exposure to solar radiation causes cells to produce reactive oxygen species (ROS), which are a primary factor in skin damage. Although the contribution of the UV component to skin damage has been established, few studies have examined the effects of non-UV solar radiation on skin physiology. Solar radiation comprises <10% of UV, and thus the purpose of this study was to examine the physiological response of skin to visible light (400-700 nm). Irradiation of human skin equivalents with visible light induced production of ROS, proinflammatory cytokines, and matrix metalloproteinase (MMP)-1 expression. Commercially available sunscreens were found to have minimal effects on reducing visible light-induced ROS, suggesting that UVA/UVB sunscreens do not protect the skin from visible light-induced responses. Using clinical models to assess the generation of free radicals from oxidative stress, higher levels of free radical activity were found after visible light exposure. Pretreatment with a photostable UVA/UVB sunscreen containing an antioxidant combination significantly reduced the production of ROS, cytokines, and MMP expression in vitro, and decreased oxidative stress in human subjects after visible light irradiation. Taken together, these findings suggest that other portions of the solar spectrum aside from UV, particularly visible light, may also contribute to signs of premature photoaging in skin.

  5. 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.

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

    PubMed

    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.

  7. Quercetin Attenuates Warfarin-induced Vascular Calcification in Vitro Independently from Matrix Gla Protein*

    PubMed Central

    Beazley, Kelly E.; Eghtesad, Saman; Nurminskaya, Maria V.

    2013-01-01

    Warfarin can stimulate vascular calcification in vitro via activation of β-catenin signaling and/or inhibition of matrix Gla protein (MGP) carboxylation. Calcification was induced in vascular smooth muscle cells (VSMCs) with therapeutic levels of warfarin in normal calcium and clinically acceptable phosphate levels. Although TGF/BMP and PKA pathways are activated in calcifying VSMCs, pharmacologic analysis reveals that their activation is not contributory. However, β-catenin activity is important because inhibition of β-catenin with shRNA or bioflavonoid quercetin prevents calcification in primary human VSMCs, rodent aortic rings, and rat A10 VSMC line. In the presence of quercetin, reactivation of β-catenin using the glycogen synthase kinase-3β (GSK-3β) inhibitor LiCl restores calcium accumulation, confirming that quercetin mechanism of action hinges on inhibition of the β-catenin pathway. Calcification in VSMCs induced by 10 μm warfarin does not associate with reduced levels of carboxylated MGP, and inhibitory effects of quercetin do not involve induction of MGP carboxylation. Further, down-regulation of MGP by shRNA does not alter the effect of quercetin. These results suggest a new β-catenin-targeting strategy to prevent vascular calcification induced by warfarin and identify quercetin as a potential therapeutic in this pathology. PMID:23223575

  8. Pioglitazone inhibits TGFβ induced keratocyte transformation to myofibroblast and extracellular matrix production.

    PubMed

    Pan, Hong-Wei; Xu, Jin-Tang; Chen, Jian-Su

    2011-10-01

    Phenotype transformation of corneal keratocyte to myofibroblast plays an important role in the wound healing process of cornea and TGFβ is considered to be the most important mediator to induce myofibroblast trans-differentiation. Peroxisome proliferator-activated receptors-γ (PPAR-γ) activation has been proved to exert anti-fibrotic effect in many tissues. In this study, we investigated the effect of PPAR-γ agonist, pioglitazone, on myofibroblast transformation, extracellular matrix production and cell proliferation. The results showed pioglitazone inhibited the TGFβ-driven myofibroblast differentiation, as determined by F-actin fluorescence staining, α-smooth muscle actin-specific immunocytochemistry and western blot analysis. Pioglitazone also potently attenuated TGFβ induced type I collagen and fibronectin mRNA and protein production. Moreover, pioglitazone showed inhibitory effect on TGFβ induced cell proliferation. The irreversible PPAR-γ antagonist GW9662, partially reversed the inhibition of collagen I and fibronectin expression but not myofibroblast transformation, suggesting both PPAR-γ dependent and PPAR-γ independent mechanisms were involved in the action of pioglitazone. Therefore, our study indicates pioglitazone has a potential application in therapy of corneal fibrosis and PPAR-γ might be a promising therapy target.

  9. Decreased Biosynthesis of Jasmonic Acid via Lipoxygenase Pathway Compromised Caffeine-Induced Resistance to Colletotrichum gloeosporioides Under Elevated CO2 in Tea Seedlings.

    PubMed

    Li, Xin; Ahammed, Golam Jalal; Li, Zhixin; Tang, Meijun; Yan, Peng; Han, Wenyan

    2016-11-01

    Caffeine, the major purine alkaloid in tea has long been known for its role in plant defense. However, its effect on Colletotrichum gloeosporioides that causes brown blight disease in tea is largely unknown especially under elevated CO2. Here we show that elevated CO2 reduced endogenous caffeine content in tea leaves, but sharply increased susceptibility of tea to C. gloeosporioides. The expression of C. gloeosporioides actin gene was gradually increased during the postinoculation period. In contrast, foliar application of caffeine decreased the C. gloeosporioides-induced necrotic lesions and the expression of C. gloeosporioides actin. Analysis of endogenous jasmonic acid (JA) content revealed that exogenous caffeine could induce JA content under both CO2 conditions in absence of fungal infection; however, in presence of fungal infection, caffeine increased JA content only under elevated CO2. Furthermore, exogenous caffeine enhanced lipoxygenase (LOX) activity and its biosynthetic gene expression under both CO2 conditions, indicating that increased JA biosynthesis via LOX pathway by caffeine might strengthen plant defense only under elevated CO2, while caffeine-induced defense under ambient CO2 might be associated with JA-independent LOX pathway in tea. These results provide novel insights into caffeine-induced plant defense mechanisms that might help to develop an eco-friendly approach for disease control.

  10. 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.

  11. Ethanol inhibits methionine adenosyltransferase II activity and S-adenosylmethionine biosynthesis and enhances caspase-3-dependent cell death in T lymphocytes: relevance to alcohol-induced immunosuppression.

    PubMed

    Hote, Prachi T; Sahoo, Rashmita; Jani, Tanvi S; Ghare, Smita S; Chen, Theresa; Joshi-Barve, Swati; McClain, Craig J; Barve, Shirish S

    2008-06-01

    An important aspect in alcohol abuse-associated immune suppression is the loss of T helper CD4(+) lymphocytes, leading to impairment of multiple immune functions. Our work has shown that ethanol can sensitize CD4(+) T lymphocytes to caspase-3-dependent activation-induced cell death (AICD). It has been demonstrated that the formation of S-adenosylmethionine (SAMe) catalyzed by methionine adenosyltransferase (MAT) II is essential for CD4(+) T-cell activation and proliferation. Since ethanol is known to affect SAMe metabolism in hepatocytes, we investigated the effect of ethanol on MAT II activity/expression, SAMe biosynthesis and cell survival in CD4(+) T lymphocytes. We demonstrate for the first time that ethanol at a physiologically relevant concentration (25 mM) substantially decreased the enzymatic activity of MAT II in T lymphocytes. Ethanol was observed to decrease the transcription of MAT2A, which encodes the catalytic subunit of MAT II and is vital for MAT II activity and SAMe biosynthesis. Furthermore, correspondent to its effect on MAT II, ethanol decreased intracellular SAMe levels and enhanced caspase-3-dependent AICD. Importantly, restoration of intracellular SAMe levels by exogenous SAMe supplementation considerably decreased both caspase-3 activity and apoptotic death in T lymphocytes. In conclusion, our data show that MAT II and SAMe are critical molecular components essential for CD4(+) T-cell survival that are affected by ethanol, leading to enhanced AICD. Furthermore, these studies provide a clinical paradigm for the development of much needed therapy using SAMe supplementation in the treatment of immune dysfunction induced by alcohol abuse.

  12. Dynamics of electric field induced particle alignment in nonpolar polymer matrix

    NASA Astrophysics Data System (ADS)

    Tai, Xiangyang; Wu, Guozhang; Yui, Hiroshi; Asai, Shigeo; Sumita, Masao

    2003-11-01

    The dynamics of electric field induced particle alignment in nonpolar polymer matrix to build one-dimensional conductive materials was investigated. The influence of electric field on particle alignment was real-time traced by dynamic percolation measurement using carbon black (CB) filled polyethylene as a model system. The activation energy of the continuous CB path formation was calculated and found to be unchanged with CB alignment. The critical percolation concentration at thermodynamic equilibrium state φc* was deduced to characterize the anisotropism of network structure, by which the thermodynamic prerequisite electric field E* for the transition from three-dimensional isotropic network to one-dimensional chain could be easily found out.

  13. 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.

  14. Laser induced fluorescence imaging of thermal damage in polymer matrix composites

    SciTech Connect

    Wachter, E.A.; Fisher, W.G.; Meyer, K.E.

    1996-12-31

    A simple, fluorescence-based imaging system was developed for identifying regions of thermal damage in polymer-matrix composites (PMCs). PMCs have important applications where low weight and high mechanical strength are needed. One concern in the aerospace industry is the tendency of some PMC materials to become irreversibly damaged when exposed to high temperatures. Traditional nondestructive evaluation (NDE) techniques are capable of detecting physical flaws, such as cracks and delaminations, but have not proven effective for detecting initial heat damage, which occurs on a molecular scale. Spectroscopic techniques such as laser-induced fluorescence provide an attractive means for detecting thermal damage on large, irregularly shaped surfaces. This paper describes instrumentation capable of rapidly detecting thermal damage in graphite/epoxy components.

  15. 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.

  16. Cerebral ischemia induces transcription of inflammatory and extracellular-matrix-related genes in rat cerebral arteries.

    PubMed

    Vikman, Petter; Ansar, Saema; Henriksson, Marie; Stenman, Emelie; Edvinsson, Lars

    2007-12-01

    Cerebral ischemia results in a local inflammatory response that contributes to the size of the lesion, however, the involvement of the cerebral vasculature is unknown. We hypothesise that the expression of inflammatory genes (Il6, iNOS, cxcl2, TNF-alpha and Il-1beta) and extracellular-matrix-related genes (MMP9, MMP13) is induced in cerebral arteries following cerebral ischemia via activation of mitogen activated kinases (MAPKs). This hypothesis was tested in vivo by experimental subarachnoid haemorrhage (SAH) and temporal middle cerebral artery occlusion (MCAO), and by organ culture of isolated cerebral arteries with quantitative real time PCR (mRNA expression) and immunohistochemistry (localization of protein expression). The gene promoters were investigated in silica with computer analysis. The mRNA analysis revealed that the ischemic models, SAH and MCAO, as well as organ culture of isolated cerebral arteries resulted in transcriptional upregulation of the abovementioned genes. The protein expression involved phosphorylation of three different MAPKs signalling pathways (p38, ERK 1/2 and SAPK/JNK) and the downstream transcription factors (ATF-2, Elk-1, c-Jun) shown by immunohistochemistry and quantified by image analysis. All three models revealed the same pattern of activation in the cerebrovascular smooth muscle cells. The in silica analysis demonstrated binding sites for said transcription factors. The results suggest that cerebral ischemia and organ culture induce activation of p38, ERK 1/2 and SAPK/JNK in cerebral arteries which in turn activate the transcription factors ATF-2, Elk-1 and c-Jun and the expression of inflammatory and extracellular-matrix-related genes in the wall of cerebral arteries.

  17. Matrix-encapsulation cell-seeding technique to prevent cell detachment during arthroscopic implantation of matrix-induced autologous chondrocytes.

    PubMed

    Masri, Maria; Lombardero, Germán; Velasquillo, Cristina; Martínez, Valentín; Neri, Rosario; Villegas, Hilda; Ibarra, Clemente

    2007-08-01

    The goal of this study is to evaluate the efficiency of obtaining a large number of viable cells within a construct that will not be detached by high fluid flow during arthroscopic implantation. Arthroscopic osteochondral biopsy specimens were obtained from the medial femoral trochlea of 8 horses. Chondrocytes were isolated by collagenase digestion and expanded in M199 media until confluency. After 10 to 12 days, cultures were trypsinized and cells resuspended in culture media. Then, 5 x 10(6) cells x mL(-1) were seeded on a culture dish and the same amount in a flask. Once extracellular matrix was formed, a polyglycolic/polylactic acid disk was placed in the culture dish. Cells obtained from the culture flasks (2 x 10(7) cells) were seeded onto the polymer and encapsulated by lifting the monolayer of cells and matrix from the bottom of the dish with surgical forceps. On days 1, 3, 5, 7, and 9, viability was evaluated by calcein fluorescence. Fiber cell attachment was evaluated before implantation by environmental scanning electron microscopy. Six horses were implanted with naive cell-polymer constructs, and two horses were implanted with adenoviral vector with green fluorescent protein (AdGFP)-transduced cells. Biopsy specimens of repair tissue were evaluated at 8 weeks in 6 horses and at 4 weeks in the 2 horses implanted with AdGFP-transduced cells by second-look arthroscopy and biopsy, histochemistry, and confocal laser scanning microscopy via MitoTracker Red 580 (Invitrogen [Molecular Probes], Gibco, Carlsbad, CA) to assess cell viability. Viability and attachment of cells to polymer were confirmed by calcein fluorescence microscopy and environmental scanning electron microscopy. Consistency of the construct was ideal for implantation between 7 and 9 days. Repair tissue with AdGFP chondrocytes after 4 weeks showed fluorescent cells also positive to MitoTracker probe by confocal laser scanning microscopy. Repair tissue after 8 weeks showed very cellular new

  18. 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

  19. An intracellular matrix metalloproteinase-2 isoform induces tubular regulated necrosis: implications for acute kidney injury.

    PubMed

    Ceron, Carla S; Baligand, Celine; Joshi, Sunil; Wanga, Shaynah; Cowley, Patrick M; Walker, Joy P; Song, Sang Heon; Mahimkar, Rajeev; Baker, Anthony J; Raffai, Robert L; Wang, Zhen J; Lovett, David H

    2017-06-01

    Acute kidney injury (AKI) causes severe morbidity, mortality, and chronic kidney disease (CKD). Mortality is particularly marked in the elderly and with preexisting CKD. Oxidative stress is a common theme in models of AKI induced by ischemia-reperfusion (I-R) injury. We recently characterized an intracellular isoform of matrix metalloproteinase-2 (MMP-2) induced by oxidative stress-mediated activation of an alternate promoter in the first intron of the MMP-2 gene. This generates an NH2-terminal truncated MMP-2 (NTT-MMP-2) isoform that is intracellular and associated with mitochondria. The NTT-MMP-2 isoform is expressed in kidneys of 14-mo-old mice and in a mouse model of coronary atherosclerosis and heart failure with CKD. We recently determined that NTT-MMP-2 is induced in human renal transplants with delayed graft function and correlated with tubular cell necrosis. To determine mechanism(s) of action, we generated proximal tubule cell-specific NTT-MMP-2 transgenic mice. Although morphologically normal at the light microscopic level at 4 mo, ultrastructural studies revealed foci of tubular epithelial cell necrosis, the mitochondrial permeability transition, and mitophagy. To determine whether NTT-MMP-2 expression enhances sensitivity to I-R injury, we performed unilateral I-R to induce mild tubular injury in wild-type mice. In contrast, expression of the NTT-MMP-2 isoform resulted in a dramatic increase in tubular cell necrosis, inflammation, and fibrosis. NTT-MMP-2 mice had enhanced expression of innate immunity genes and release of danger-associated molecular pattern molecules. We conclude that NTT-MMP-2 "primes" the kidney to enhanced susceptibility to I-R injury via induction of mitochondrial dysfunction. NTT-MMP-2 may be a novel AKI treatment target.

  20. 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.

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

    PubMed

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

    2010-03-01

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

  2. 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).

  3. HIV-1 matrix protein p17 misfolding forms toxic amyloidogenic assemblies that induce neurocognitive disorders.

    PubMed

    Zeinolabediny, Yasmin; Caccuri, Francesca; Colombo, Laura; Morelli, Federica; Romeo, Margherita; Rossi, Alessandro; Schiarea, Silvia; Ciaramelli, Carlotta; Airoldi, Cristina; Weston, Ria; Donghui, Liu; Krupinski, Jerzy; Corpas, Rubén; García-Lara, Elisa; Sarroca, Sara; Sanfeliu, Coral; Slevin, Mark; Caruso, Arnaldo; Salmona, Mario; Diomede, Luisa

    2017-09-04

    Human immunodeficiency virus type-1 (HIV-1)-associated neurocognitive disorder (HAND) remains an important neurological manifestation that adversely affects a patient's quality of life. HIV-1 matrix protein p17 (p17) has been detected in autoptic brain tissue of HAND individuals who presented early with severe AIDS encephalopathy. We hypothesised that the ability of p17 to misfold may result in the generation of toxic assemblies in the brain and may be relevant for HAND pathogenesis. A multidisciplinary integrated approach has been applied to determine the ability of p17 to form soluble amyloidogenic assemblies in vitro. To provide new information into the potential pathogenic role of soluble p17 species in HAND, their toxicological capability was evaluated in vivo. In C. elegans, capable of recognising toxic assemblies of amyloidogenic proteins, p17 induces a specific toxic effect which can be counteracted by tetracyclines, drugs able to hinder the formation of large oligomers and consequently amyloid fibrils. The intrahippocampal injection of p17 in mice reduces their cognitive function and induces behavioral deficiencies. These findings offer a new way of thinking about the possible cause of neurodegeneration in HIV-1-seropositive patients, which engages the ability of p17 to form soluble toxic assemblies.

  4. Induced sputum-retrieved matrix metalloproteinase 9 and tissue metalloproteinase inhibitor 1 in granulomatous diseases

    PubMed Central

    Fireman, E; Kraiem, Z; Sade, O; Greif, J; Fireman, Z

    2002-01-01

    Matrix metalloproteinases (MMPs) capable of degrading various components of connective tissue matrices, and tissue inhibitor metalloproteinases (TIMPs) are considered important in lung parenchymal remodeling and repair processes in pulmonary diseases. Induced sputum (IS) is a reliable noninvasive method to investigate pathogenesis, pathophysiology and treatment of lung disease. This study was designed to determine whether IS-MMP9/TIMP1 levels demonstrate lung parenchymal remodeling in sarcoidosis (SA) and Crohn's disease (CRD) patients. Sputum was induced and processed conventionally in 13 SA patients, 18 CRD patients and 9 controls. Two-hundred cells were counted on Giemsa-stained cytopreps, and T lymphocytes subsets (CD4 = T helper and CD8 = T suppressor cytotoxic cells) were analysed by FACS using monoclonal antibodies.MMP-9 and TIMP-1 were measured using commercial ELISA kits. MMP-9 concentrations, but not those of TIMP-1, were significantly greater in the sputum supernatant in SA and CRD patients compared to controls (P = 0·018 and P = 0·0019, respectively). The molar ratio, MMP-9/TIMP-1, was significantly higher in SA and CRD patients compared to controls (P = 0·008 and P = 0·024, respectively). Gelatinase species having a molecular weight similar to that of MMP-9 were demonstrated by zymographic analysis. MMP-9 levels were highly correlated with the CD4/CD8 ratio and DLCO capacity in SA but less in CRD patients. MMP-9 levels in IS provide a sensitive marker for pulmonary damage. PMID:12390324

  5. 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

  6. Release of matrix metalloproteinase-8 during physiological trafficking and induced mobilization of human hematopoietic stem cells.

    PubMed

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

    2013-05-01

    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.

  7. CBFβ enhances de novo protein biosynthesis of its binding partners HIV-1 Vif and RUNX1 and potentiates the Vif-induced degradation of APOBEC3G.

    PubMed

    Miyagi, Eri; Kao, Sandra; Yedavalli, Venkat; Strebel, Klaus

    2014-05-01

    Vif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBFβ was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBFβ remains unclear. Several studies noted reduced Vif expression in CBFβ knockdown cells while others saw no significant impact of CBFβ on Vif stability. Here, we confirmed that CBFβ increases Vif steady-state levels. CBFβ affected expression of neither viral Gag nor Vpu protein, indicating that CBFβ regulates Vif expression posttranscriptionally. Kinetic studies revealed effects of CBFβ on both metabolic stability and the rate of Vif biosynthesis. These effects were dependent on the ability of CBFβ to interact with Vif. Importantly, at comparable Vif levels, CBFβ further enhanced A3G degradation, suggesting that CBFβ facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBFβ also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBFβ had no detectable effect on RUNX1 metabolic stability. We propose that CBFβ acts as a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G. In this study, we show that CBFβ has a profound effect on the expression of the HIV-1 infectivity factor Vif and the cellular transcription factor RUNX1, two proteins that physically interact with CBFβ. Kinetic studies revealed that CBFβ increases the rate of Vif and RUNX1 biosynthesis at the level of translation. Mutants of Vif unable to physically interact with CBFβ were nonresponsive to CBFβ. Our data suggest that CBFβ exerts a chaperone-like activity (i) to minimize the production of defective ribosomal products (DRiPs) by binding to nascent protein to prevent premature termination and (ii) to stabilize mature

  8. 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

  9. 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.

  10. 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. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  11. 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 Central

    Brouwer, Bastiaan; Gardeström, Per; Keech, Olivier

    2014-01-01

    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. PMID:24604733

  12. Wolbachia-induced loss of male fertility is likely related to branch chain amino acid biosynthesis and iLvE in Laodelphax striatellus.

    PubMed

    Ju, Jia-Fei; Hoffmann, Ary A; Zhang, Yan-Kai; Duan, Xing-Zhi; Guo, Yan; Gong, Jun-Tao; Zhu, Wen-Chao; Hong, Xiao-Yue

    2017-06-01

    Wolbachia are endosymbionts that infect many species of arthropods and nematodes. Wolbachia-induced cytoplasmic incompatibility (CI) is the most common phenotype in affected hosts, involving embryonic lethality in crosses between Wolbachia-infected males and uninfected females. The molecular mechanisms underlying this phenomenon are currently unclear. Here we examine the molecular correlates of the Wolbachia infection in Laodelphax striatellus (Fallén), an important rice pest, where embryonic lethality is strong and almost complete. We compared the gene expression of 4-day-old Wolbachia-infected and uninfected L. striatellus testes to identify candidate genes for paternal-effect embryonic lethality induction. Based on microarray analysis, iLvE was the most down-regulated gene; this gene mediates branched-chain amino acid (BCAA) biosynthesis and participates in many processes related to reproductive performance. After knocking down iLvE by RNAi in uninfected male L. striatellus, male fertility was reduced, leading to a decrease in embryo hatching rates, but fertility was rescued in crosses between these males and Wolbachia-infected females. Removal of BCAA in chemically-defined diets of uninfected males also led to a loss of male fertility. Low amino acid nutrition may enhance exposure time of sperm to Wolbachia in the testes to affect adult reproduction in L. striatellus by reducing the number of sperm transferred per mating by males. These results indicate that Wolbachia may decrease male fertility in L. striatellus by acting on iLvE, a key factor of BCAA biosynthesis, and delaying sperm maturation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  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. Hypoxia induces connexin 43 dysregulation by modulating matrix metalloproteinases via MAPK signaling.

    PubMed

    Wu, Xianghong; Huang, Wen; Luo, Gang; Alain, Laval Andy

    2013-12-01

    Connexin 43 (Cx43) is a major structural protein found in the gap junctions of the ventricular myocardium and a major determinant of its electrical properties. The effects of matrix metalloproteinases (MMPs), the mitogen-activated protein kinase (MAPK) signaling pathway, transcription factor NF-kB, and activator protein-1 (AP-1)/c-Jun on the regulation of Cx43 gene expression in H9c2 cardiomyocytes were assessed. The MAPK signaling pathway (MEK/ERK1/2 and PI3K) and transcription factors NF-kB and AP-1/c-Jun were inhibited, then Cx43 expression was assessed using Western blot analysis, and MMP-9 activity was assessed using gelatin zymography. Hypoxia decreased the Cx43 protein level by approximately 30-50 %. Doxycycline (10 μg/mL), an inhibitor of MMP, markedly attenuated the hypoxia-induced downregulation of Cx43 protein expression at 6 h. The hypoxia-induced decrease in Cx43 protein expression was significantly reversed by U0126 (10 μM), a MEK/ERK1/2 inhibitor, at 6 and 12 h; LY294002 (30 μM), a PI3K inhibitor, downregulated Cx43 expression. Hypoxia-induced MMP-9 activation was inhibited by treatment with LY294002, U0126, and, most especially, U0126. JSH-23 (30 μM), an NF-kB inhibitor, and SP600125 (10 μM), an AP-1/c-Jun inhibitor, attenuated the loss of Cx43. These results suggest that MAPK signaling and the activities NF-kB and MMPs play an important roles in the regulation of Cx43 expression.

  16. Topographical extracellular matrix cues on anticancer drug-induced cytotoxicity in stem cells.

    PubMed

    Kim, Jangho; Kim, Yeon Ju; Bae, Won-Gyu; Jang, Kyung-Jin; Lim, Ki Taek; Choung, Pill-Hoon; Choung, Yun-Hoon; Chung, Jong Hoon

    2015-08-01

    In recent years, cell chip-based platforms have begun to show promise as a means of corroborating the findings of in vivo animal tests for cytotoxicity, and perhaps in the future partially replacing the need for such animal models. In contrast to the conventional culture methods, micro- and nanofabrication techniques can be utilized to provide a set of mechanostimulatory signals to the cells that mimic the context of extracellular matrix (ECM) of the tissue in which a particular cell line resides. Here, we report periodic lateral topographic striations, with a pitch ranging approximately from 200 to 800 nm with an intention to mimic a common geometry of fibrils in the ECM such as collagen or elastin, as a platform for investigating anticancer drug-induced cytotoxicity in stem cells. The ECM cues could facilitate perimeter, elongation, and gap junction formation of mesenchymal stem cells (MSCs), which eventually influenced the fate of cells in terms of death and survival against the common chemotherapeutic agent cisplatin. Interestingly, the appropriate inhibition of gap junctions of MSCs on the ECM mimicking substrates could prevent the cisplatin-induced cytotoxicity through the inhibition of the cisplatin-induced 'death signal communication' as compared to that on the flat substrates. Our results imply that nanoscale topography is an important consideration for chip-based cytotoxicity assays, which uniquely enable the consideration and rational design of ECM-like topographic features, and furthermore, that the natural topography of the ECM in the context of stem cell niches may serve as an important indicator for chemotherapeutic agent sensitivity. © 2014 Wiley Periodicals, Inc.

  17. The role of matrix metalloproteinase-9 in cigarette smoke-induced emphysema.

    PubMed

    Atkinson, Jeffrey J; Lutey, Barbara A; Suzuki, Yoko; Toennies, Holly M; Kelley, Diane G; Kobayashi, Dale K; Ijem, Whitney G; Deslee, Gaetan; Moore, Carla H; Jacobs, M Eileen; Conradi, Susan H; Gierada, David S; Pierce, Richard A; Betsuyaku, Tomoko; Senior, Robert M

    2011-04-01

    Matrix metalloprotease (MMP)-9 is an elastolytic endopeptidase produced by activated macrophages that may be involved in the development of human pulmonary emphysema and could be inhibited with existing compounds. Mouse models have demonstrated that excess MMP-9 production can result in permanent alveolar destruction. To determine if MMP-9 causes cigarette smoke-induced emphysema using MMP-9 knockout mice and human samples. Mouse lungs were analyzed for inflammation and airspace enlargement using a mainstream smoke-exposure model. Human macrophage mRNA was isolated from subjects with emphysema by laser capture microdissection. Human blood monocyte mRNA was isolated from subjects with greater than 30 pack-year smoking history. Human gene expression was determined by quantitative polymerase chain reaction and compared with emphysema severity determined by automated computed tomography analysis. Plasma Clara cell secretory protein and surfactant protein-D were quantified to measure ongoing lung injury. Mice deficient in MMP-9 develop the same degree of cigarette smoke-induced inflammation and airspace enlargement as strain-matched controls. Macrophages are the predominant source of MMP-9 production in human emphysema specimens and similar quantities of macrophage MMP-9 mRNA is present in areas of lung with and without emphysema. Circulating monocytes produce more MMP-9 in individuals with advanced emphysema severity despite no correlation of MMP-9 with markers of ongoing lung damage. These results suggest that MMP-9 in humans who smoke is similar to smoke-exposed mice, where MMP-9 is present in emphysematous lung but not correlated with the emphysema. To the degree that the mechanisms of emphysema in humans who smoke resemble the mouse model, these data suggest specific inhibition of MMP-9 is unlikely to be an effective therapy for cigarette smoke-induced emphysema. Clinical trial registered with www.clinicaltrials.gov (NCT 00757120).

  18. Hormone-induced 14-3-3γ Adaptor Protein Regulates Steroidogenic Acute Regulatory Protein Activity and Steroid Biosynthesis in MA-10 Leydig Cells*

    PubMed Central

    Aghazadeh, Yasaman; Rone, Malena B.; Blonder, Josip; Ye, Xiaoying; Veenstra, Timothy D.; Hales, D. Buck; Culty, Martine; Papadopoulos, Vassilios

    2012-01-01

    Cholesterol is the sole precursor of steroid hormones in the body. The import of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis, relies on the formation of a protein complex that assembles at the outer mitochondrial membrane called the transduceosome. The transduceosome contains several mitochondrial and cytosolic components, including the steroidogenic acute regulatory protein (STAR). Human chorionic gonadotropin (hCG) induces de novo synthesis of STAR, a process shown to parallel maximal steroid production. In the hCG-dependent steroidogenic MA-10 mouse Leydig cell line, the 14-3-3γ protein was identified in native mitochondrial complexes by mass spectrometry and immunoblotting, and its levels increased in response to hCG treatment. The 14-3-3 proteins bind and regulate the activity of many proteins, acting via target protein activation, modification and localization. In MA-10 cells, cAMP induces 14-3-3γ expression parallel to STAR expression. Silencing of 14-3-3γ expression potentiates hormone-induced steroidogenesis. Binding motifs of 14-3-3γ were identified in components of the transduceosome, including STAR. Immunoprecipitation studies demonstrate a hormone-dependent interaction between 14-3-3γ and STAR that coincides with reduced 14-3-3γ homodimerization. The binding site of 14-3-3γ on STAR was identified to be Ser-194 in the STAR-related sterol binding lipid transfer (START) domain, the site phosphorylated in response to hCG. Taken together, these results demonstrate that 14-3-3γ negatively regulates steroidogenesis by binding to Ser-194 of STAR, thus keeping STAR in an unfolded state, unable to induce maximal steroidogenesis. Over time 14-3-3γ homodimerizes and dissociates from STAR, allowing this protein to induce maximal mitochondrial steroid formation. PMID:22427666

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. Endoplasmic reticulum stress modulates nicotine-induced extracellular matrix degradation in human periodontal ligament cells.

    PubMed

    Lee, S-I; Kang, K-L; Shin, S-I; Herr, Y; Lee, Y-M; Kim, E-C

    2012-06-01

    Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine-induced cytotoxicity and ECM degradation. Cytotoxicity and cell death were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT-PCR and western blot analysis. Treatment with nicotine reduced cell viability and increased the proportion of annexin V-negative, propidium iodide-positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2α and glucose-regulated protein-78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP-1, MMP-2, MMP-8 and MMP-9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine-induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small

  5. 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

  6. Laser-induced breakdown spectroscopy for the quantitative analysis of metals in sediments using natural zeolite matrix

    NASA Astrophysics Data System (ADS)

    Austria, E. S.; Fuentes, E. M.; Nuesca, G. M.; Lamorena, R. B.

    2017-10-01

    The dependence of laser-induced breakdown spectroscopy (LIBS) to the matrix of the sample remains an important consideration in performing quantitative analysis. In this study, a new matrix was introduced in the preparation of solid powder calibration curves. Heat-treated natural zeolite and KBr were mixed separately into high purity metal powders to generate calibration curves using a univariate approach. A LIBS technique was used in the detection and quantitative analysis of Cr, Cu and Pb in river sediment samples. The relative percent difference (RPD) was calculated to describe the variability measurements made using ICP/OES and LIBS as well as to evaluate the accuracy of the method. Calculated limits of detection in the matrices prepared were comparable with literature values and ranged from 0.41 to 6.1 ppm. The resulting metal concentrations indicate that the natural zeolite matrix were closer to the reference values compared to the KBr matrix. By employing principal component analysis (PCA), heat treated zeolite was shown as a potential diluent or binder for generating calibration curves and could provide matrix-matched standards in identification of target metals from unknown sediment samples. The natural zeolite appeared to closely simulate the ablation behavior and property of the samples, and it is found to be a potential suitable matrix for the quantitative LIBS analysis of sediments.

  7. 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

  8. 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

  9. Transgenic expression of human matrix metalloproteinase-9 augments monocrotaline-induced pulmonary arterial hypertension in mice

    PubMed Central

    George, Joseph; D’Armiento, Jeanine

    2013-01-01

    Objectives Pulmonary arterial hypertension (PAH) is characterized by intimal lesions, right ventricular hypertrophy, and adventitial thickening of pulmonary arteries with progressive pulmonary hypertension. This investigation was aimed to examine the effects of transgenic expression of human matrix metalloproteinase-9 (MMP-9) in the pathogenesis of PAH. Methods PAH was induced using serial subcutaneous administration of monocrotaline (MCT). Right ventricular pressure was measured through the right jugular vein using a 1.4F Millar Mikro-tip catheter-transducer. Zymography, western blotting, and quantitative reverse transcription PCR (qRT-PCR) were carried out for MMP-9. Immunohistochemistry was performed for α-smooth muscle actin (α-SMA) and Mac-3 antigen. Results Measurement of right ventricular pressure demonstrated 2.5-fold and 3.7-fold elevation after the administration of MCT in wild-type and MMP-9 transgenic mice, respectively. Zymography, western blotting, and qRT-PCR depicted increased activity and expression of MMP-9 after treatment with MCT, which were augmented in transgenic mice. There was marked pulmonary inflammation with extensive infiltration of mononuclear cells, which was more intense in MMP-9 transgenic mice. SMA and Mac-3 staining demonstrated hypertrophy of pulmonary arteries with occlusion of precapillary vessels and extensive infiltration of macrophages, respectively. All these changes were aggravated in MCT-treated MMP-9 transgenic mice when compared to normal littermates. Conclusion Our study demonstrated that the MCT-induced PAH in mouse is a reproducible and potentially valuable animal model for the human disease. Our results further demonstrated that MMP-9 plays a significant role in the pathogenesis of PAH and effective blocking of MMP-9 could provide an option in the therapeutic intervention of human PAH. PMID:21063214

  10. Transgenic expression of human matrix metalloproteinase-9 augments monocrotaline-induced pulmonary arterial hypertension in mice.

    PubMed

    George, Joseph; D'Armiento, Jeanine

    2011-02-01

    Pulmonary arterial hypertension (PAH) is characterized by intimal lesions, right ventricular hypertrophy, and adventitial thickening of pulmonary arteries with progressive pulmonary hypertension. This investigation was aimed to examine the effects of transgenic expression of human matrix metalloproteinase-9 (MMP-9) in the pathogenesis of PAH. PAH was induced using serial subcutaneous administration of monocrotaline (MCT). Right ventricular pressure was measured through the right jugular vein using a 1.4F Millar Mikro-tip catheter-transducer. Zymography, western blotting, and quantitative reverse transcription PCR (qRT-PCR) were carried out for MMP-9. Immunohistochemistry was performed for α-smooth muscle actin (α-SMA) and Mac-3 antigen. Measurement of right ventricular pressure demonstrated 2.5-fold and 3.7-fold elevation after the administration of MCT in wild-type and MMP-9 transgenic mice, respectively. Zymography, western blotting, and qRT-PCR depicted increased activity and expression of MMP-9 after treatment with MCT, which were augmented in transgenic mice. There was marked pulmonary inflammation with extensive infiltration of mononuclear cells, which was more intense in MMP-9 transgenic mice. SMA and Mac-3 staining demonstrated hypertrophy of pulmonary arteries with occlusion of precapillary vessels and extensive infiltration of macrophages, respectively. All these changes were aggravated in MCT-treated MMP-9 transgenic mice when compared to normal littermates. Our study demonstrated that the MCT-induced PAH in mouse is a reproducible and potentially valuable animal model for the human disease. Our results further demonstrated that MMP-9 plays a significant role in the pathogenesis of PAH and effective blocking of MMP-9 could provide an option in the therapeutic intervention of human PAH.

  11. Curcumin inhibits paraquat induced lung inflammation and fibrosis by extracellular matrix modifications in mouse model.

    PubMed

    Tyagi, Namitosh; Dash, D; Singh, Rashmi

    2016-12-01

    Paraquat (PQ), a potent herbicide can cause severe toxicity. We report here that fibroproliferation phase of acute lung injury (ALI) is initiated much earlier (within 48 h) after PQ intoxication than previously reported (after 2 weeks) and we aimed to study the protective effects of intranasal curcumin as new therapeutic strategy in mouse model. Mice (Park's strain) were divided into five experimental groups (I) control, received only saline (0.9 % NaCl) (II) PQ, mice intoxicated with PQ (50 mg/kg, i.p., single dose); (III) curcumin, treated with curcumin (5 mg/kg, i.n) an hour before PQ administration; (IV)Veh, DMSO (equal volume to curcumin) given an hour before PQ exposure; (V) DEXA, mice treated with dexamethasone (1 mg/kg, i.p) before an hour of PQ intoxication. After 48 h of the PQ exposure, all mice were sacrificed and samples were analyzed. Pretreatment with intranasal curcumin (5 mg/kg) could modify the PQ-intoxication (50 mg/kg, i.p) induced structural remodeling of lung parenchyma at an early phase of acute lung injury. Significant increase in inflammatory cell count, reactive oxygen species and hydroxyproline levels were decreased after curcumin pretreatment (all p < 0.05). Histological examination and zymography results were also found consistent. Our results show that curcumin pretreatment decreased the expression of alpha smooth muscle actin (α-SMA), matrix metalloproteinases-9 (MMP-9) and changed the expression of tissue inhibitors of metalloproteinase (TIMP-1) after PQ intoxication. Single toxic dose of PQ has initiated fibroproliferation within 48 h and intranasal curcumin may prove as new therapeutic strategy for PQ induced ALI and fibroproliferation.

  12. Vascular lysyl oxidase over-expression alters extracellular matrix structure and induces oxidative stress.

    PubMed

    Varona, Saray; García-Redondo, Ana B; Martínez-González, Jose; Salaices, Mercedes; Briones, Ana M; Rodríguez, Cristina

    Lysyl oxidase (LOX) participates in the assembly of collagen and elastin fibres. The impact of vascular LOX over-expression on extracellular matrix (ECM) structure and its contribution to oxidative stress has been analysed. Studies were conducted on mice over-expressing LOX (Tg), specifically in smooth muscle cells (VSMC). Gene expression was assessed by real-time PCR analysis. Sirius Red staining, H2O2 production and NADPH oxidase activity were analysed in different vascular beds. The size and number of fenestra of the internal elastic lamina were determined by confocal microscopy. LOX activity was up-regulated in VSMC of transgenic mice compared with cells from control animals. At the same time, transgenic cells deposited more organised elastin fibres and their supernatants induced a stronger collagen assembly in in vitro assays. Vascular collagen cross-linking was also higher in Tg mice, which showed a decrease in the size of fenestrae and an enhanced expression of Fibulin-5. Interestingly, higher H2O2 production and NADPH oxidase activity was detected in the vascular wall from transgenic mice. The H2O2 scavenger catalase attenuated the stronger deposition of mature elastin fibres induced by LOX transgenesis. LOX over-expression in VSMC was associated with a change in the structure of collagen and elastin fibres. LOX could constitute a novel source of oxidative stress that might participate in elastin changes and contribute to vascular remodelling. Copyright © 2017 Sociedad Española de Arteriosclerosis. Publicado por Elsevier España, S.L.U. All rights reserved.

  13. Attenuation of dextran sodium sulphate induced colitis in matrix metalloproteinase-9 deficient mice

    PubMed Central

    Santana, Alfredo; Medina, Carlos; Paz-Cabrera, Maria Cristina; Díaz-Gonzalez, Federico; Farré, Esther; Salas, Antonio; Radomski, Marek W; Quintero, Enrique

    2006-01-01

    AIM: To study whether matrix metalloproteinase-9 (MMP-9) is a key factor in epithelial damage in the dextran sodium sulphate (DSS) model of colitis in mice. METHODS: MMP-9-deficient and wild-type (wt) mice were given 5% DSS in drinking water for 5 d followed by recovery up to 7 d. On d 5 and 12 after induction of colitis, gelatinases, MMP-2 and MMP-9, were measured in homogenates of colonic tissue by zymography and Western blot, whereas tissue inhibitor of metalloproteinases (TIMPs) were measured by reverse zymography. The gelatinolytic activity was also determined in supernatants of polymorphonuclear leukocytes (PMN) isolated from mice blood. Moreover, intestinal epithelial cells were stimulated with TNF-α to study whether these cells were able to produce MMPs. Finally, colonic mucosal lesions were measured by microscopic examination. RESULTS: On d 5 of colitis, the activity of MMP-9 was increased in homogenates of colonic tissues (0.24 ± 0.1 vs 21.3 ± 6.4, P < 0.05) and PMN from peripheral blood in wt (0.5 ± 0.1 vs 10.4 ± 0.7, P < 0.05), but not in MMP-9-deficient animals. The MMP-9 activity was also up-regulated by TNF-α in epithelial intestinal cells (2.5 ± 0.5 vs 14.7 ± 3.0, P < 0.05). Although colitis also led to increase of TIMP-1 activity, the MMP-9/TIMP-1 balance remained elevated. Finally, in the MMP-9-deficient colitic mice both the extent and severity of intestinal epithelial injury were significantly attenuated when compared with wt mice. CONCLUSION: We conclude that DSS induced colitis is markedly attenuated in animals lacking MMP-9. This suggests that intestinal injury induced by DSS is modulated by MMP-9 and that inhibition of this gelatinase may reduce inflammation. PMID:17072979

  14. 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.

  15. p120 and Kaiso Regulate Helicobacter pylori-induced Expression of Matrix Metalloproteinase-7

    PubMed Central

    Ogden, Seth R.; Wroblewski, Lydia E.; Weydig, Christiane; Romero-Gallo, Judith; O'Brien, Daniel P.; Israel, Dawn A.; Krishna, Uma S.; Fingleton, Barbara; Reynolds, Albert B.; Wessler, Silja

    2008-01-01

    Helicobacter pylori is the strongest known risk factor for gastric adenocarcinoma, yet only a fraction of infected persons develop cancer. One H. pylori constituent that augments disease risk is the cytotoxin-associated gene (cag) pathogenicity island, which encodes a secretion system that translocates bacterial effector molecules into host cells. Matrix metalloproteinase (MMP)-7, a member of a family of enzymes with tumor-initiating properties, is overexpressed in premalignant and malignant gastric lesions, and H. pylori cag+ strains selectively increase MMP-7 protein levels in gastric epithelial cells in vitro and in vivo. We now report that H. pylori-mediated mmp-7 induction is transcriptionally regulated via aberrant activation of p120-catenin (p120), a component of adherens junctions. H. pylori increases mmp-7 mRNA levels in a cag- and p120-dependent manner and induces translocation of p120 to the nucleus in vitro and in a novel ex vivo gastric gland culture system. Nuclear translocation of p120 in response to H. pylori relieves Kaiso-mediated transcriptional repression of mmp-7, which is implicated in tumorigenesis. These results indicate that selective and coordinated induction of mmp-7 expression by H. pylori cag+ isolates may explain in part the augmentation in gastric cancer risk associated with these strains. PMID:18653469

  16. 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.

  17. 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.

  18. 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.

  19. Analysis of gold in rock samples using laser-induced breakdown spectroscopy: Matrix and heterogeneity effects

    NASA Astrophysics Data System (ADS)

    Rifai, Kheireddine; Laflamme, Marcel; Constantin, Marc; Vidal, François; Sabsabi, Mohamad; Blouin, Alain; Bouchard, Paul; Fytas, Konstantinos; Castello, Maryline; Kamwa, Blandine Nguegang

    2017-08-01

    We used the laser-induced breakdown spectroscopy (LIBS) technique to determine the concentration of gold in rock samples. 44 reference materials (mostly compressed fine powders) of various chemical compositions, with a quasi-homogeneous concentration of gold varying from 0 to 1000 ppm, were used to establish the calibration curve for the Au I 267.59 nm line. A chemometric study based on the principal component analysis (PCA) showed that 83% of the LIBS spectra variations are attributable to the presence of iron in the samples. Two distinct branches were obtained in the calibration curve: one for Si-rich samples (< 5% of iron) and one for Fe-rich samples (> 13% of iron) with limits of detection of 0.8 ppm and 1.5 ppm, respectively. Different normalization schemes of the gold signal were tested in order to reduce the matrix effects. The LIBS analysis was performed on various mineral samples of practical interest, namely two Si-rich uncompressed ore powders, fine and granular, and three bulk drill cores. The fluctuations in the gold concentration measurements appear to be about two times greater in the granular powder (5-10%) than in the fine one (2-5%). A detailed mapping of the gold concentration on a solid drill core was also performed, revealing multiscale heterogeneity of the gold distribution on the surface of the sample.

  20. 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

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

    PubMed Central

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

    2014-01-01

    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. PMID:24550310

  2. Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) as a novel regulator of myogenic cell differentiation.

    PubMed

    Attia, Mohamed; Mohamed, Attia; Huet, Eric; Eric, Huet; Delbé, Jean; Jean, Delbé; Ledoux, Dominique; Dominique, Ledoux; Menashi, Suzanne; Suzanne, Menashi; Martelly, Isabelle; Isabelle, Martelly

    2011-01-01

    Matrix metalloproteinases (MMPs) are thought to play an important role in skeletal muscle cell growth and differentiation. In view of the MMP inducing function of EMMPRIN/CD147, its role in myogenic cell differentiation was investigated. EMMPRIN level increased during differentiation of both rat primary myoblasts derived from satellite cells and mouse C2.7 myogenic cells and was associated with an alteration in its molecular forms. In parallel, expression of pro-MMP-9 gradually decreased and that of pro-MMP-2 and active MMP-2 increased. While small interfering RNA (siRNA) inhibition of EMMPRIN expression accelerated cell differentiation, exogenously added recombinant EMMPRIN inhibited differentiation by an MMP-mediated mechanism, as the MMP inhibitor marimastat abrogated EMMPRIN's effect. Our results further suggest that EMMPRIN regulates differentiation through an MMP activation of transforming growth factor beta (TGFβ), a known inhibitor of myoblast's differentiation, as the increased activation and signaling of TGFβ by EMMPRIN was attenuated in the presence of marimastat. EMMPRIN inhibition may thus represent a novel strategy in the treatment of muscular degenerative disorders.

  3. 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

  4. 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.

  5. 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.

  6. Microstructures induced by excimer laser surface melting of the SiCp/Al metal matrix composite

    NASA Astrophysics Data System (ADS)

    Qian, D. S.; Zhong, X. L.; Yan, Y. Z.; Hashimoto, T.; Liu, Z.

    2017-08-01

    Laser surface melting (LSM) was carried out on the SiCp/Al metal matrix composite (MMC) using a KrF excimer laser with a fluence of 7 J/cm2. The re-solidification microstructure was characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM) equipped with energy dispersive X-ray detector, and X-ray diffraction (XRD) analysis. It was found that a 2.5 μm thick melted layer was formed in the near-surface region, in which dissolution of the intermetallics and removal of the SiC particles occurred. The thermal and material response upon laser irradiation was simulated using three models, i.e. analytical model, finite element model (FEM) and smoothed-particle hydrodynamics (SPH) model. The effect of SiC particles on the LSM process, the mechanism of the SiC removal and the re-solidification microstructures in the melted layer were discussed. The simulation results were in good agreement with the experimental results and contributed to the generic understanding of the re-solidification microstructures induced by ns-pulsed lasers.

  7. Complex graph matrix representations and characterizations of proteomic maps and chemically induced changes to proteomes.

    PubMed

    Balasubramanian, Krishnan; Khokhani, Kanan; Basak, Subhash C

    2006-05-01

    We have presented a complex graph matrix representation to characterize proteomics maps obtained from 2D-gel electrophoresis. In this method, each bubble in a 2D-gel proteomics map is represented by a complex number with components which are charge and mass. Then, a graph with complex weights is constructed by connecting the vertices in the relative order of abundance. This yields adjacency matrices and distance matrices of the proteomics graph with complex weights. We have computed the spectra, eigenvectors, and other properties of complex graphs and the Euclidian/graph distance obtained from the complex graphs. The leading eigenvalues and eigenvectors and, likewise, the smallest eigenvalues and eigenvectors, and the entire graph spectral patterns of the complex matrices derived from them yield novel weighted biodescriptors that characterize proteomics maps with information of charge and masses of proteins. We have also applied these eigenvector and eigenvalue maps to contrast the normal cells and cells exposed to four peroxisome proliferators, namely, clofibrate, diethylhexyl phthalate (DEHP), perfluorodecanoic acid (PFDA), and perfluoroctanoic acid (PFOA). Our complex eigenspectra show that the proteomic response induced by DEHP differs from the corresponding responses of other three chemicals consistent with their chemical structures and properties.

  8. 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

  9. Force-induced elastic matrix-mediated interactions in the presence of a rigid wall.

    PubMed

    Menzel, Andreas M

    2017-05-14

    We consider a soft elastic matrix that contains particulate inclusions and is bounded by a rigid wall, e.g., the substrate. Such a situation arises in elastic composite materials. They may serve as soft actuators when forces are imposed on or induced between the embedded particles. We investigate how the presence of the rigid wall affects the interactions between the inclusions. First, for no-slip boundary conditions, we transfer Blake's derivation of a corresponding Green's function from low-Reynolds-number hydrodynamics to the linearly elastic case. Then, we list the general expressions to describe the situation for point-like particles in the presence of no-slip and free-slip surface conditions. To compare the effect of the different surface conditions to each other and to the bulk behavior, we address the example situation of pairwise interactions between two embedded particles. The axis through both particle centers is either aligned parallel or perpendicular to the surface. Our results suggest that walls with free-slip surface conditions are preferred when they serve as substrates for soft actuators made from elastic composite materials. As we further demonstrate, the presence of a rigid wall can qualitatively change the interactions between the inclusions. In effect, it can switch attractive interactions into repulsive ones (and vice versa). It should be straightforward to observe the effects in future experiments and to combine our results, e.g., with the modeling of biological cells and tissue on rigid surfaces.

  10. 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. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  11. 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. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Extracellular matrix elasticity modulates TGF-β-induced p38 activation and myofibroblast transdifferentiation in human tenon fibroblasts.

    PubMed

    Meyer-ter-Vehn, Tobias; Han, Hong; Grehn, Franz; Schlunck, Günther

    2011-11-25

    Extracellular matrix and the cytokine TGF-β influence scar formation in an interdependent fashion. In this study, the impact of extracellular matrix elasticity on TGF-β-induced signal transduction and myofibroblast transdifferentiation was examined. Primary human tenon fibroblasts were seeded on collagen-coated glass coverslips (rigid environment) or collagen or polyacrylamide gels (elastic environment) of different compliance and stimulated with TGF-β. Myofibroblast transdifferentiation was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis for the marker gene α-smooth muscle actin (SMA), and SMA incorporation into stress fibers was determined by confocal immunofluorescence microscopy. CTGF transcription was assessed by RT-qPCR. Signaling pathways were examined by Western blot using phosphospecific antibodies and by immunofluorescence microscopy. TGF-β-dependent myofibroblast transdifferentiation was enhanced in a stiff environment. Increasing matrix elasticity attenuated TGF-β-induced myofibroblast transdifferentiation and the associated CTGF expression. TGF-β-induced p38 activation was reduced on elastic substrates. The results suggest that matrix elasticity influences TGF-β-dependent activation of p38 signaling and subsequent myofibroblast transdifferentiation. Biomechanical cues represent an important determinant of scarring processes. Therefore, cellular signals elicited by mechanotransduction deserve consideration in the design of novel antifibrotic strategies.

  13. 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

  14. 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.

  15. A Candida Biofilm-Induced Pathway for Matrix Glucan Delivery: Implications for Drug Resistance

    PubMed Central

    Taff, Heather T.; Nett, Jeniel E.; Zarnowski, Robert; Ross, Kelly M.; Sanchez, Hiram; Cain, Mike T.; Hamaker, Jessica; Mitchell, Aaron P.; Andes, David R.

    2012-01-01

    Extracellular polysaccharides are key constituents of the biofilm matrix of many microorganisms. One critical carbohydrate component of Candida albicans biofilms, β-1,3 glucan, has been linked to biofilm protection from antifungal agents. In this study, we identify three glucan modification enzymes that function to deliver glucan from the cell to the extracellular matrix. These enzymes include two predicted glucan transferases and an exo-glucanase, encoded by BGL2, PHR1, and XOG1, respectively. We show that the enzymes are crucial for both delivery of β-1,3 glucan to the biofilm matrix and for accumulation of mature matrix biomass. The enzymes do not appear to impact cell wall glucan content of biofilm cells, nor are they necessary for filamentation or biofilm formation. We demonstrate that mutants lacking these genes exhibit enhanced susceptibility to the commonly used antifungal, fluconazole, during biofilm growth only. Transcriptional analysis and biofilm phenotypes of strains with multiple mutations suggest that these enzymes act in a complementary fashion to distribute matrix downstream of the primary β-1,3 glucan synthase encoded by FKS1. Furthermore, our observations suggest that this matrix delivery pathway works independently from the C. albicans ZAP1 matrix formation regulatory pathway. These glucan modification enzymes appear to play a biofilm-specific role in mediating the delivery and organization of mature biofilm matrix. We propose that the discovery of inhibitors for these enzymes would provide promising anti-biofilm therapeutics. PMID:22876186

  16. 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.

  17. 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.

  18. 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

  19. A stress-inducible gene for 9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis under water stress in drought-tolerant cowpea.

    PubMed

    Iuchi, S; Kobayashi, M; Yamaguchi-Shinozaki, K; Shinozaki, K

    2000-06-01

    Four cDNA clones named CPRD (cowpea responsive to dehydration) corresponding to genes that are responsive to dehydration were isolated using differential screening of a cDNA library prepared from 10-h dehydrated drought-tolerant cowpea (Vigna unguiculata) plants. One of the cDNA clones has a homology to 9-cis-epoxycarotenoid dioxygenase (named VuNCED1), which is supposed to be involved in abscisic acid (ABA) biosynthesis. The GST (glutathione S-transferase)-fused protein indicates a 9-cis-epoxycarotenoid dioxygenase activity, which catalyzes the cleavage of 9-cis-epoxycarotenoid. The N-terminal region of the VuNCED1 protein directed the fused sGFP (synthetic green-fluorescent protein) into the plastids of the protoplasts, indicating that the N-terminal sequence acts as a transit peptide. Both the accumulation of ABA and expression of VuNCED1 were strongly induced by drought stress in the 8-d-old cowpea plant, whereas drought stress did not trigger the expression of VuABA1 (accession no. AB030295) gene that encodes zeaxanthin epoxidase. These results indicate that the VuNCED1 cDNA encodes a 9-cis-epoxycarotenoid dioxygenase and that its product has a key role in the synthesis of ABA under drought stress.

  20. 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.

  1. A Stress-Inducible Gene for 9-cis-Epoxycarotenoid Dioxygenase Involved in Abscisic Acid Biosynthesis under Water Stress in Drought-Tolerant Cowpea1

    PubMed Central

    Iuchi, Satoshi; Kobayashi, Masatomo; Yamaguchi-Shinozaki, Kazuko; Shinozaki, Kazuo

    2000-01-01

    Four cDNA clones named CPRD (cowpea responsive to dehydration) corresponding to genes that are responsive to dehydration were isolated using differential screening of a cDNA library prepared from 10-h dehydrated drought-tolerant cowpea (Vigna unguiculata) plants. One of the cDNA clones has a homology to 9-cis-epoxycarotenoid dioxygenase (named VuNCED1), which is supposed to be involved in abscisic acid (ABA) biosynthesis. The GST (glutathione S-transferase)-fused protein indicates a 9-cis-epoxycarotenoid dioxygenase activity, which catalyzes the cleavage of 9-cis-epoxycarotenoid. The N-terminal region of the VuNCED1 protein directed the fused sGFP (synthetic green-fluorescent protein) into the plastids of the protoplasts, indicating that the N-terminal sequence acts as a transit peptide. Both the accumulation of ABA and expression of VuNCED1 were strongly induced by drought stress in the 8-d-old cowpea plant, whereas drought stress did not trigger the expression of VuABA1 (accession no. AB030295) gene that encodes zeaxanthin epoxidase. These results indicate that the VuNCED1 cDNA encodes a 9-cis-epoxycarotenoid dioxygenase and that its product has a key role in the synthesis of ABA under drought stress. PMID:10859185

  2. 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.

  3. 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.

  4. Differential expression of lactic acid isomers, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-8 in vaginal fluid from women with vaginal disorders.

    PubMed

    Beghini, J; Linhares, I M; Giraldo, P C; Ledger, W J; Witkin, S S

    2015-11-01

    Do metabolites in vaginal samples vary between women with different vaginal disorders. Cross-sectional study. Campinas, Brazil. Seventy-seven women (39.9%) with no vaginal disorder, 52 women (26.9%) with vulvovaginal candidiasis (VVC), 43 women (22.3%) with bacterial vaginosis (BV), and 21 women (10.9%) with cytolytic vaginosis (CTV). Concentrations of D- and L-lactic acid, extracellular matrix metalloproteinase inducer (EMMPRIN), and matrix metalloproteinase-8 (MMP-8), and the influence of Candida albicans on EMMPRIN production by cultured vaginal epithelial cells, were determined by enzyme-linked immunosorbent assay (ELISA). Associations were determined by the Mann-Whitney U-test and by Spearman's rank correlation test. Metabolite levels and their correlation with diagnoses. Vaginal concentrations of D- and L-lactic acid were reduced from control levels in BV (P < 0.0001); L-lactic acid levels were elevated in CTV (P = 0.0116). EMMPRIN and MMP-8 concentrations were elevated in VVC (P < 0.0001). EMMPRIN and L-lactic acid concentrations (P ≤ 0.008), but not EMMPRIN and D-lactic acid, were correlated in all groups. EMMPRIN also increased in proportion with the ratio of L- to D-lactic acid in controls and in women with BV (P ≤ 0.009). Concentrations of EMMPRIN and MMP-8 were correlated in controls and women with VVC (P ≤ 0.0002). Candida albicans induced EMMPRIN release from vaginal epithelial cells. Vaginal secretions from women with BV are deficient in D- and L-lactic acid, women with VVC have elevated EMMPRIN and MMP-8 levels, and women with CTV have elevated L-lactic acid levels. These deviations may contribute to the clinical signs, symptoms, and sequelae that are characteristic of these disorders. © 2014 Royal College of Obstetricians and Gynaecologists.

  5. Mitigation of gamma-radiation induced abasic sites in genomic DNA by dietary nicotinamide supplementation: metabolic up-regulation of NAD(+) biosynthesis.

    PubMed

    Batra, Vipen; Kislay, Binita

    2013-09-01

    The search for non-toxic radio-protective drugs has yielded many potential agents but most of these compounds have certain amount of toxicity. The objective of the present study was to investigate dietary nicotinamide enrichment dependent adaptive response to potential cytotoxic effect of (60)Co γ-radiation. To elucidate the possible underlying mechanism(s), male Swiss mice were maintained on control diet (CD) and nicotinamide supplemented diet (NSD). After 6 weeks of CD and NSD dietary regimen, we exposed the animals to γ-radiation (2, 4 and 6Gy) and investigated the profile of downstream metabolites and activities of enzymes involved in NAD(+) biosynthesis. Increased activities of nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase (NMNAT) were observed up to 48h post-irradiation in NSD fed irradiated mice. Concomitant with increase in liver NAMPT and NMNAT activities, NAD(+) levels were replenished in NSD fed and irradiated animals. However, NAMPT and NMNAT-mediated NAD(+) biosynthesis and ATP levels were severely compromised in liver of CD fed irradiated mice. Another major finding of these studies revealed that under γ-radiation stress, dietary nicotinamide supplementation might induce higher and long-lasting poly(ADP)-ribose polymerase 1 (PARP1) and poly(ADP-ribose) glycohydrolase (PARG) activities in NSD fed animals compared to CD fed animals. To investigate liver DNA damage, number of apurinic/apyrimidinic sites (AP sites) and level of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) residues were quantified. A significant increase in liver DNA AP sites and 8-oxo-dG levels with concomitant increase in caspase-3 was observed in CD fed and irradiated animals compared to NSD fed and irradiated mice. In conclusion present studies show that under γ-radiation stress conditions, dietary nicotinamide supplementation restores DNA excision repair activity via prolonged activation of PARP1 and PARG activities. Present results

  6. 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

  7. An extracellular matrix culture system for induced pluripotent stem cells derived from human dental pulp cells.

    PubMed

    Chen, Y; Zheng, Y-L; Qiu, D-B; Sun, Y-P; Kuang, S-J; Xu, Y; He, F; Gong, Y-H; Zhang, Z-G

    2015-11-01

    Induced pluripotent stem cells (iPSCs) have emerged as a promising tool for treating incurable diseases. The current challenges are to avoid potential xenopathogenic transmission and immune rejection potentially caused by exposure of iPSCs to animal-derived products. In addition, an efficient feeder cell-free culture condition will be required for minimizing batch-to-batch variation and facilitating scale-up. Therefore, establishing an efficient extracellular matrix (ECM) culture system is considered as a prerequisite for the future clinical application of iPSC-based cell therapies. In this study, we evaluated the feasibility of culturing iPSCs in ECM derived from human dental pulp cells (hDCP). iPSCs growing in Matrigel were transferred to ECM or Matrigel and cultured in mTeSRTM1 medium. The number of adherent cells in the ECM group was higher than that in the Matrigel group after incubation for 8, 12, and 24 h, indicating that the ECM could enhance cell adherence. The adhesion of cells to ECM not only depends on simple physical attachment with ECM, but also mediated by fibronectin in the ECM. The hDPC-iPSCs showed orderly growth in the ECM, suggesting that the ECM could promote the growth and proliferation of hDPC-iPSCs. We also observed that stem cells growed along to avoid contact inhibition. The iPSCs maintained undifferentiated state when cultured in ECM when the iPSCs and ECM are of the same cell origin. ECM and mTeSRTM1, can both be used as new culture medium for iPSCs that facilitates the clinical application of iPSC-based cell therapies in the future.

  8. Matrix metalloproteinase activity is required for activity-induced angiogenesis in rat skeletal muscle.

    PubMed

    Haas, T L; Milkiewicz, M; Davis, S J; Zhou, A L; Egginton, S; Brown, M D; Madri, J A; Hudlicka, O

    2000-10-01

    Proteolysis of the capillary basement membrane is a hallmark of inflammation-mediated angiogenesis, but it is undetermined whether proteolysis plays a critical role in the process of activity-induced angiogenesis. Matrix metalloproteinases (MMPs) constitute the major class of proteases responsible for degradation of basement membrane proteins. We observed significant elevations of mRNA and protein levels of both MMP-2 and membrane type 1 (MT1)-MMP (2.9 +/- 0.7- and 1.5 +/- 0.1-fold above control, respectively) after 3 days of chronic electrical stimulation of rat skeletal muscle. Inhibition of MMP activity via the inhibitor GM-6001 prevented the growth of new capillaries as assessed by the capillary-to-fiber ratio (1.34 +/- 0.08 in GM-6001-treated muscles compared with 1.69 +/- 0.03 in control 7-day-stimulated muscles). This inhibition correlated with a significant reduction in the number of capillaries with observable breaks in the basement membrane, as assessed by electron microscopy (0.27 +/- 0.27% in GM-6001-treated muscles compared with 3.72 +/- 0.65% in control stimulated muscles). Proliferation of capillary-associated cells was significantly elevated by 2 days and remained elevated throughout 14 days of stimulation. Capillary-associated cell proliferation during muscle stimulation was not affected by MMP inhibition (80.3 +/- 9.3 nuclei in control and 63.5 +/- 8.5 nuclei in GM-6001-treated animals). We conclude that MMP proteolysis of capillary basement membrane proteins is a critical component of physiological angiogenesis, and we postulate that capillary-associated proliferation precedes and occurs independently of endothelial cell sprout formation.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. Polydatin alleviates alcohol-induced acute liver injury in mice: Relevance of matrix metalloproteinases (MMPs) and hepatic antioxidants.

    PubMed

    Koneru, Meghana; Sahu, Bidya Dhar; Gudem, Sagarika; Kuncha, Madhusudana; Ravuri, Halley Gora; Kumar, Jerald Mahesh; Kilari, Eswar Kumar; Sistla, Ramakrishna

    2017-04-15

    Alcohol, a most commonly consumed beverage, is the foremost cause of liver injury throughout the world. Polydatin, a stilbenoid glucoside, was known to possess antioxidant and anti-inflammatory properties and is being investigated for use in various disorders. The present study was intended at investigating the hepatoprotective efficacy of polydatin against acute-alcohol induced liver injury model in mice. C57BL/6 mice were fed with five doses of 50% ethyl alcohol (10ml/kg body weight) to induce acute liver injury. Effect of polydatin against alcohol induced hepatic injury was investigated by giving 50 or 100mg/kg polydatin, orally, for 8 days. Serum markers of liver injury, morphology, histology and fibrosis of liver tissue, levels of enzymatic and non-enzymatic antioxidants and the mitochondrial respiratory enzyme activities in liver tissue were investigated. The activities and the protein expression of matrix metalloproteinases (MMP-2 and -9), the expression of NF-κB in the liver tissue were also studied. Polydatin pre-treatment significantly alleviated the alcohol induced hepatic injury by reducing the serum liver injury markers, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), attenuating oxidative stress and restoring antioxidant balance in the hepatic tissue. Simultaneously, polydatin pre-treatment also prevented alcohol induced mitochondrial damage and refurbished the matrix metalloproteinases levels of the hepatic tissue. The findings of the present study suggest that polydatin may have a potential benefit in preventing alcohol-induced acute hepatic injury. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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).

  1. Ethanol impairs muscarinic receptor-induced neuritogenesis in rat hippocampal slices: role of astrocytes and extracellular matrix proteins

    PubMed Central

    Giordano, Gennaro; Guizzetti, Marina; Dao, Khoi; Mattison, Hayley A.; Costa, Lucio G.

    2011-01-01

    In an in vitro co-culture system of astrocytes and neurons, stimulation of cholinergic muscarinic receptors in astrocytes had been shown to cause neuritogenesis in hippocampal neurons, and this effect was inhibited by ethanol. The present study sought to confirm these earlier findings in a more complex system, in vitro rat hippocampal slices in culture. Exposure of hippocampal slices to the cholinergic agonist carbachol (1 mM for 24 h) induced neurite outgrowth in hippocampal pyramidal neurons, which was mediated by activation of muscarinic M3 receptors. Specifically, carbachol induced a >4-fold increase in the length of the longest neurite, and a 4-fold increase in the length of minor neurites and in the number of branches. Co-incubation of carbachol with ethanol (50 mM) resulted in significant inhibition of the effects induced by carbachol on all parameters measured. Neurite outgrowth in CNS neurons is dependent on various permissive factors that are produced and released by glial cells. In hippocampal slices carbachol increased the levels of two extracellular matrix protein, fibronectin and laminin-1, by 1.6-fold, as measured by Western blot. Co-incubation of carbachol with ethanol significantly inhibited these increases. Carbachol-induced increases in levels of extracellular matrix proteins were antagonized by a M3 muscarinic receptor antagonist. Furthermore, function-blocking fibronectin or laminin-1 antibodies antagonized the effect of carbachol on neurite outgrowth. These results indicate that in hippocampal slices stimulation of muscarinic M3 receptors induces neurite outgrowth, which is mediated by fibronectin and laminin-1, two extracellular matrix proteins released by astrocytes. By decreasing fibronectin and laminin levels ethanol prevents carbachol-induced neuritogenesis. These findings highlight the importance of glial-neuronal interactions as important targets in the developmental neurotoxicity of alcohol. PMID:21884684

  2. 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.

  3. 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.

  4. Laser Induced Fluorescence (LIF) Nondestructive Evaluation of Incipient Heat Damage in Polymer Matrix Composites, A2476

    DTIC Science & Technology

    2017-02-15

    Composites , A2476 By D. W. Merdes, C. M. Bowie, C. A. Moose Approved for public release; distribution unlimited...Nondestructive Evaluation of Incipient Heat Damage in Polymer Matrix Composites 5a. CONTRACT NUMBER All listed in Block 13 5b. GRANT NUMBER N/A 5c...against four sets of polymer matrix composite (PMC) specimens that had been heated under controlled conditions of time and temperature at the Naval Air

  5. 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

  6. 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.

  7. 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.

  8. Sodium butyrate blocks interferon-gamma (IFN-γ)-induced biosynthesis of MHC class III gene products (complement C4 and factor B) in human fetal intestinal epithelial cells

    PubMed Central

    Kitamura, K; Andoh, A; Inoue, T; Amakata, Y; Hodohara, K; Fujiyama, Y; Bamba, T

    1999-01-01

    Human intestinal epithelial cells have been established as local sites for complement biosynthesis. In this study, we investigated the effects of IFN-γ and sodium butyrate on biosynthesis of MHC class III gene products (complement C4 and factor B) in the human fetal intestinal epithelial cell line INT-407. IFN-γ induced a dose- and time-dependent increase in C4 and factor B secretion. However, sodium butyrate dose-dependently inhibited IFN-γ-induced C4 and factor B secretion. These effects were also observed at the mRNA level. Immunoblotting indicated that IFN-γ induced a rapid activation of Stat1α, and fluorescence immunohistochemistry detected a translocation of Stat1α into the nucleus within 1 h. However, the translocation of Stat1α was not affected by the addition of sodium butyrate. Nuclear run-on assay indicated that IFN-γ induced a weak increase in the transcription rate of factor B gene, and sodium butyrate did not affect this response. IFN-γ and sodium butyrate induced a counter-regulatory effect on C4 and factor B secretion: IFN-γ acted as a potent inducer, but sodium butyrate potently abrogated these responses. These are mainly regulated through the post-transcriptional mechanism. PMID:10540154

  9. 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.

  10. Extracellular matrix-induced synthesis of a low molecular weight collagen by fetal calf ligament fibroblasts.

    PubMed

    Sage, H; Mecham, R

    1987-01-01

    Fetal calf ligamentum nuchae fibroblasts, cultured from animals of different gestational age, synthesize a unique, low molecular weight collagen termed FCL-1 (Sage, H., Mecham, R., Johnson, C., and Bornstein, P., 1983, J. Cell Biol. 97:1933-1938). Previous studies on the elastogenic differentiation of these cells in vitro demonstrated that the extracellular matrix (ECM) protein elastin was specifically induced in undifferentiated fibroblasts when they were grown on ligament ECM isolated from animals at later stages of development (Mecham, R.P., Madaras, J.G., and Senior, R.M., 1984. J. Cell Biol. 98:1804-1812). To investigate the expression of FCL-1 as a function of developmental age, we grew fetal calf ligament fibroblasts from an 85 d (first trimester) animal (FCL 85d) on three different substrata: ligament from a 120 d (second trimester) animal, ligament from a 270 d (term) animal, and unmodified plastic tissue culture dishes. FCL 270d fibroblasts were grown on plastic substrata and served as a differentiated cellular control. Analysis of metabolically radiolabeled proteins from both the culture media and the cell layers showed that the synthesis of FCL-1 was selectively increased in those cells cultured on ligament ECM. For FCL 85d fibroblasts grown on 120 d and 270 d ligaments, FCL-1 comprised 17% and 22%, respectively, of the culture medium proteins that precipitated at concentrations of ammonium sulfate from 20-50%. FCL 85d and 270d fibroblasts grown on plastic substrata yielded values of 2.5% and 1.0%, respectively. This effect appeared to be specific for this collagen and did not reflect a general increase in the synthesis of connective tissue proteins of the ECM (e.g., types I and III procollagen). As percent of total newly-synthesized cellular protein, the output of FCL-1 was 10-fold higher by FCL 85d cells grown on 270d ligament ECM (5.8%) as compared to that of the same cellular population grown on a plastic surface (0.56%). The presence of the

  11. 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.

  12. 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

  13. 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.

  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 Stems1

    PubMed Central

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

    2002-01-01

    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. PMID:12114556

  16. 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.

  17. 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.

  18. NaCl Induces Flavonoid Biosynthesis through a Putative Novel Pathway in Post-harvest Ginkgo Leaves.

    PubMed

    Ni, Jun; Hao, Juan; Jiang, Zhifang; Zhan, Xiaori; Dong, Lixiang; Yang, Xiuli; Sun, Zhehang; Xu, Wenya; Wang, Zhikun; Xu, Maojun

    2017-01-01

    The flavonoids in the extracts of Ginkgo leaves have been shown to have great medical value: thus, a method to increase the flavonoid contents in these extracts is of significant importance for human health. In the present study, we investigated the changes in flavonoid contents and the corresponding gene expression levels in post-harvest Ginkgo leaves after various treatments. We found that both ultraviolet-B and NaCl treatment induced flavonoid accumulation. However, gene expression analysis showed that the increases in flavonoid contents were achieved by different pathways. Furthermore, post-harvest Ginkgo leaves responded differently to NaCl treatment compared with naturally grown leaves in both flavonoid contents and corresponding gene expression. In addition, combined treatment with ultraviolet-B and NaCl did not further increase the flavonoid contents compared with ultraviolet-B or NaCl treatment alone. Our results indicate the existence of a novel mechanism in response to NaCl treatment in post-harvest Ginkgo leaves, and provide a technique to increase flavonoid content in the pharmaceutical industry.

  19. NaCl Induces Flavonoid Biosynthesis through a Putative Novel Pathway in Post-harvest Ginkgo Leaves

    PubMed Central

    Ni, Jun; Hao, Juan; Jiang, Zhifang; Zhan, Xiaori; Dong, Lixiang; Yang, Xiuli; Sun, Zhehang; Xu, Wenya; Wang, Zhikun; Xu, Maojun

    2017-01-01

    The flavonoids in the extracts of Ginkgo leaves have been shown to have great medical value: thus, a method to increase the flavonoid contents in these extracts is of significant importance for human health. In the present study, we investigated the changes in flavonoid contents and the corresponding gene expression levels in post-harvest Ginkgo leaves after various treatments. We found that both ultraviolet-B and NaCl treatment induced flavonoid accumulation. However, gene expression analysis showed that the increases in flavonoid contents were achieved by different pathways. Furthermore, post-harvest Ginkgo leaves responded differently to NaCl treatment compared with naturally grown leaves in both flavonoid contents and corresponding gene expression. In addition, combined treatment with ultraviolet-B and NaCl did not further increase the flavonoid contents compared with ultraviolet-B or NaCl treatment alone. Our results indicate the existence of a novel mechanism in response to NaCl treatment in post-harvest Ginkgo leaves, and provide a technique to increase flavonoid content in the pharmaceutical industry. PMID:28659935

  20. The MAT1-2-1 mating-type gene upregulates photo-inducible carotenoid biosynthesis in Fusarium verticillioides.

    PubMed

    Adám, Attila L; García-Martínez, Jorge; Szucs, Endre P; Avalos, Javier; Hornok, László

    2011-05-01

    Filamentous ascomycetes, including mitotic holomorphs, have constitutively transcribed MAT (mating type) genes. These genes encode transcription factors considered to be the major regulators of sexual communication. The proven targets of the MAT transcription factors are pheromone precursor and pheromone receptor genes. However, recent studies demonstrated that MAT proteins may also affect other genes not involved directly in the mating process. When grown in the light, Fusarium verticillioides produces the acidic xanthophyll neurosporoxanthin and lower amounts of nonpolar precursor carotenes, such as phytoene, torulene, β-carotene, and γ-carotene. Depending on the illumination conditions, a drastic decrease or the absence of light-inducible carotenoid accumulation was detected in three independent ΔFvMAT1-2-1 knockout mutants of F. verticillioides as compared with the parental wild-type strain. Transcript levels of the carB, carRA, and carT genes, encoding key enzymes of the carotenoid biosynthetic pathway, were also significantly reduced in the mutants. The downregulation of these genes in the ΔFvMAT1-2-1 mutant indicates that MAT genes play a role in the control of carotenogenesis in Fusarium. The finding that mating-type genes regulate important processes unrelated to sex helps to understand the presence of functional MAT genes in asexually reproducing fungus populations. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  1. All-Arthroscopic Autologous Matrix-Induced Chondrogenesis for the Treatment of Osteochondral Lesions of the Talus.

    PubMed

    Usuelli, Federico Giuseppe; de Girolamo, Laura; Grassi, Miriam; D'Ambrosi, Riccardo; Montrasio, Umberto Alfieri; Boga, Michele

    2015-06-01

    Several surgical techniques have been described for the treatment of talar chondral lesions. Among them, microfracture is well established. Autologous matrix-induced chondrogenesis (AMIC), using microfracture and biomaterials, has shown promising results for the treatment of knee osteochondral lesions and has been proposed for the ankle as an open technique. We describe an all-arthroscopic AMIC technique. The benefits of an all-arthroscopic procedure include smaller incisions with less soft-tissue dissection, better visualization of the joint, and a quicker recovery compared with open surgery. The use of matrix to support cartilage regeneration promotes good-quality cartilage tissue with satisfactory long-term outcomes. Our all-arthroscopic AMIC technique uses a type I-type III porcine collagen matrix (Chondro-Gide; Geistlich Pharma, Wolhusen, Switzerland) and is characterized by 2 different arthroscopic surgical phases. First, adequate exposure is achieved through use of a Hintermann spreader (Integra LifeSciences, Plainsboro, NJ) with sufficient joint distraction and wet lesion preparation. The second surgical step is performed dry, involving matrix placement and fixation. The all-arthroscopic AMIC technique for the treatment of osteochondral lesions of the talus allows a very precise reconstruction in the case of cartilage defects and avoids the need for a more invasive operation associated with higher morbidity and a longer surgical time.

  2. All-Arthroscopic Autologous Matrix-Induced Chondrogenesis for the Treatment of Osteochondral Lesions of the Talus

    PubMed Central

    Usuelli, Federico Giuseppe; de Girolamo, Laura; Grassi, Miriam; D'Ambrosi, Riccardo; Montrasio, Umberto Alfieri; Boga, Michele

    2015-01-01

    Several surgical techniques have been described for the treatment of talar chondral lesions. Among them, microfracture is well established. Autologous matrix-induced chondrogenesis (AMIC), using microfracture and biomaterials, has shown promising results for the treatment of knee osteochondral lesions and has been proposed for the ankle as an open technique. We describe an all-arthroscopic AMIC technique. The benefits of an all-arthroscopic procedure include smaller incisions with less soft-tissue dissection, better visualization of the joint, and a quicker recovery compared with open surgery. The use of matrix to support cartilage regeneration promotes good-quality cartilage tissue with satisfactory long-term outcomes. Our all-arthroscopic AMIC technique uses a type I–type III porcine collagen matrix (Chondro-Gide; Geistlich Pharma, Wolhusen, Switzerland) and is characterized by 2 different arthroscopic surgical phases. First, adequate exposure is achieved through use of a Hintermann spreader (Integra LifeSciences, Plainsboro, NJ) with sufficient joint distraction and wet lesion preparation. The second surgical step is performed dry, involving matrix placement and fixation. The all-arthroscopic AMIC technique for the treatment of osteochondral lesions of the talus allows a very precise reconstruction in the case of cartilage defects and avoids the need for a more invasive operation associated with higher morbidity and a longer surgical time. PMID:26258040

  3. 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.

  4. 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

  5. Rho-ROCK-Myosin Signaling Meditates Membrane Type 1 Matrix Metalloproteinase-induced Cellular Aggregation of Keratinocytes*

    PubMed Central

    Dangi-Garimella, Surabhi; Redig, Amanda J.; Shields, Mario A.; Siddiqui, Mohammed A.; Munshi, Hidayatullah G.

    2010-01-01

    Membrane type 1-matrix metalloproteinase (MT1-MMP, MMP14), which is associated with extracellular matrix (ECM) breakdown in squamous cell carcinoma (SCC), promotes tumor formation and epithelial-mesenchymal transition. However, in this report we demonstrate that MT1-MMP, by cleaving the underlying ECM, causes cellular aggregation of keratinocytes and SCC cells. Treatment with an MMP inhibitor abrogated MT1-MMP-induced phenotypic changes, but decreasing E-cadherin expression did not affect MT1-MMP-induced cellular aggregation. As ROCK1/2 can regulate cell-cell and cell-ECM interaction, we examined its role in mediating MT1-MMP-induced phenotypic changes. Blocking ROCK1/2 expression or activity abrogated the cellular aggregation resulting from MT1-MMP expression. Additionally, blocking Rho and non-muscle myosin attenuated MT1-MMP-induced phenotypic changes. Moreover, SCC cells expressing only the catalytically active MT1-MMP protein demonstrated increased cellular aggregation and increased myosin II activity in vivo when injected subcutaneously into nude mice. Together, these results demonstrate that expression of MT1-MMP may be anti-tumorigenic in keratinocytes by promoting cellular aggregation. PMID:20605790

  6. 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

  7. 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

  8. 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.

  9. Light-induced and sensing capabilities of SI-ATRP modified graphene oxide particles in elastomeric matrix

    NASA Astrophysics Data System (ADS)

    Osicka, Josef; Cvek, Martin; Mrlik, Miroslav; Ilcikova, Marketa; Pavlinek, Vladimir; Mosnacek, Jaroslav

    2017-04-01

    Photoactuators can concern light stimuli in appropriate wavelength into mechanical response. Such reversible changes in the material shape are highly promising in their applications as remote controllers, or safety sensors. In this work we were focused on light-induced actuation and sensing performance of the prepared materials. In this case poly(dimethyl siloxane) PDMS with various amounts of silicone oil and curing agent was used as matrix. Graphene oxide (GO) as filler in its neat form as well as its modified analogue were used in concentration of 0.1 vol. %. Modified GO particles were controllably coated with poly(methyl methacrylate) polymer chains using surface-initiated atom transfer radical polymerization (SI-ATRP) approach in order improve interactions between the filler and matrix which consequently lead to the enhanced light-induced actuation performance. Generally, the both, GO particles as well as modified ones were characterized using FTIR, Raman spectroscopy and finally conductivity measurement to confirm the controllable coating and simultaneously proceeded reduction. By studying of dielectric properties (activation energies), viscoelastic properties, which were investigated using dynamic mechanical analysis, the interactions between the filler and matrix were evaluated with connection to their light-responsive and sensing capabilities.

  10. 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.

  11. 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.

  12. Local serotonin mediates cyclic strain-induced phenotype transformation, matrix degradation, and glycosaminoglycan synthesis in cultured sheep mitral valves.

    PubMed

    Lacerda, Carla M R; Kisiday, John; Johnson, Brennan; Orton, E Christopher

    2012-05-15

    This study addressed the following questions: 1) Does cyclic tensile strain induce protein expression patterns consistent with myxomatous degeneration in mitral valves? 2) Does cyclic strain induce local serotonin synthesis in mitral valves? 3) Are cyclic strain-induced myxomatous protein expression patterns in mitral valves dependent on local serotonin? Cultured sheep mitral valve leaflets were subjected to 0, 10, 20, and 30% cyclic strain for 24 and 72 h. Protein levels of activated myofibroblast phenotype markers, α-smooth muscle actin (α-SMA) and nonmuscle embryonic myosin (SMemb); matrix catabolic enzymes, matrix metalloprotease (MMP) 1 and 13, and cathepsin K; and sulfated glycosaminoglycan (GAG) content in mitral valves increased with increased cyclic strain. Serotonin was present in the serum-free media of cultured mitral valves and concentrations increased with cyclic strain. Expression of the serotonin synthetic enzyme tryptophan hydroxylase 1 (TPH1) increased in strained mitral valves. Pharmacologic inhibition of the serotonin 2B/2C receptor or TPH1 diminished expression of phenotype markers (α-SMA and SMemb) and matrix catabolic enzyme (MMP1, MMP13, and cathepsin K) expression in 10- and 30%-strained mitral valves. These results provide first evidence that mitral valves synthesize serotonin locally. The results further demonstrate that tensile loading modulates local serotonin synthesis, expression of effector proteins associated with mitral valve degeneration, and GAG synthesis. Inhibition of serotonin diminishes strain-mediated protein expression patterns. These findings implicate serotonin and tensile loading in mitral degeneration, functionally link the pathogeneses of serotoninergic (carcinoid, drug-induced) and degenerative mitral valve disease, and have therapeutic implications.

  13. 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.

  14. Near-infrared laser-induced structural changes of glycine·water complexes in an Ar matrix.

    PubMed

    Kócs, Lenke; Najbauer, Eszter E; Bazsó, Gábor; Magyarfalvi, Gábor; Tarczay, György

    2015-03-19

    The structures of glycine·H2O complexes have been reinvestigated in low-temperature inert matrices. To go beyond the former matrix-isolation IR studies, NIR laser irradiation was used to change the relative abundances of the different complexes in the matrix. It is shown that the irradiation of the first overtone of the OH stretching mode of glycine as well as of the first overtone of the OH stretching mode of the water molecule in the complex can induce structural changes. Comparison of the experimental IR spectra with the IR spectra computed for different structures resulted in more reliable assignments of spectral patterns and identification of more structures than in former studies.

  15. 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.

  16. 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].

  17. 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.

  18. 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.

  19. Periodic Mechanical Stress INDUCES Chondrocyte Proliferation and Matrix Synthesis via CaMKII-Mediated Pyk2 Signaling.

    PubMed

    Liang, Wenwei; Li, Zeng; Wang, Zhen; Zhou, Jinchun; Song, Huanghe; Xu, Shun; Cui, Weiding; Wang, Qing; Chen, Zhefeng; Liu, Feng; Fan, Weimin

    2017-01-01

    Periodic mechanical stress can promote chondrocyte proliferation and matrix synthesis to improve the quality of tissue-engineered cartilage. Although the integrin β1-ERK1/2 signal cascade has been implicated in periodic mechanical stress-induced mitogenic effects in chondrocytes, the precise mechanisms have not been fully established. The current study was designed to probe the roles of CaMKII and Pyk2 signaling in periodic mechanical stress-mediated chondrocyte proliferation and matrix synthesis. Chondrocytes were subjected to periodic mechanical stress, proliferation was assessed by direct cell counting and CCK-8 assay; gene expressions were analyzed using quantitative real-time PCR, protein abundance by Western blotting. Mechanical stress, markedly enhanced the phosphorylation levels of Pyk2 at Tyr402 and CaMKII at Thr286. Both suppression of Pyk2 with Pyk2 inhibitor PF431396 or Pyk2 shRNA and suppression of CaMKII with CaMKII inhibitor KN-93 or CaMKII shRNA blocked periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. Additionally, either pretreatment with KN-93 or shRNA targeted to CaMKII prevented the activation of ERK1/2 and Pyk2 under conditions of periodic mechanical stress. Interestingly, in relation to periodic mechanical stress, in the context of Pyk2 inhibition with PF431396 or its targeted shRNA, only the phosphorylation levels of ERK1/2 were abrogated, while CaMKII signal activation was not affected. Moreover, the phosphorylation levels of CaMKII- Thr286 and Pyk2- Tyr402 were abolished after pretreatment with blocking antibody against integrinβ1 exposed to periodic mechanical stress. Our results collectively indicate that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis through the integrinβ1-CaMKII-Pyk2-ERK1/2 signaling cascade. © 2017 The Author(s). Published by S. Karger AG, Basel.

  20. 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

  1. Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin.

    PubMed

    Bae, Jung-Soo; Han, Mira; Shin, Hee Soon; Kim, Min-Kyoung; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

    2017-01-04

    Perilla frutescens (L.) Britt. (Lamiaceae) is a traditional herb that is consumed in East Asian countries as a traditional medicine. This traditional herb has been documented for centuries to treat various diseases such as depression, allergies, inflammation and asthma. However, the effect of Perilla frutescens on skin has not been characterized well. The present study aimed to investigate the effect of Perilla frutescens leaves extract (PLE) on ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin. Human dermal fibroblasts and Skh-1 hairless mice were irradiated with UV and treated with PLE. Protein and mRNA levels of various target molecules were analyzed by western blotting and quantitative RT-PCR, respectively. Histological changes of mouse skin were analyzed by H&E staining. To elucidate underlying mechanism of PLE, activator protein-1 (AP-1) DNA binding assay and the measurement of reactive oxygen species (ROS) were performed. PLE significantly inhibited basal and UV-induced MMP-1 and MMP-3 expression dose-dependently, and also decreased UV-induced phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinases. This inhibitory effects of PLE on MMP-1 and MMP-3 were mediated by reduction of ROS generation and AP-1 DNA binding activity induced by UV. Furthermore, PLE promoted type I procollagen production irrespective of UV irradiation. In the UV-irradiated animal model, PLE significantly reduced epidermal skin thickness and MMP-13 expression induced by UV. Our results demonstrate that PLE has the protective effect against UV-induced dermal matrix damage. Therefore, we suggest that PLE can be a potential agent for prevention of skin aging. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development.

    PubMed

    Dalman, Kerstin; Wind, Julia Johanna; Nemesio-Gorriz, Miguel; Hammerbacher, Almuth; Lundén, Karl; Ezcurra, Ines; Elfstrand, Malin

    2017-01-06

    The NAC family of transcription factors is one of the largest gene families of transcription factors in plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis. These transcription factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress induced processes has received relatively little attention. This study investigates NAC family transcription factors involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato. The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities, several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426:183-196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03 exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3'H and PaLAR3 were consistently down regulated in the overexpression lines. In accordance, the overexpression lines showed reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by directly interacting with the promoter of the repressed genes. However, transactivation studies of PaNAC03 and PaLAR3 in Nicotiana benthamiana showed that PaNAC03 activated PaLAR3A, suggesting that PaNAC03 does not act as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid biosynthetic genes. PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated

  3. Matrix fibronectin disruption and altered endothelial cell adhesion induced by activated leukocytes

    SciTech Connect

    Vincent, P.; Richards, P.; Saba, T.; DelVecchio, P.

    1986-03-01

    Sequestration of activated leukocytes (PMN) within the lung may contribute to pulmonary vascular injury following trauma, sepsis, or intravascular coagulation. Monolayers of cultured rat endothelial cells were utilized to evaluate the effect of activated PMNs on endothelial cell attachment and the extracellular fibronectin matrix over a 4 hr incubation interval. Rat endothelial cells were identified by immunofluorescent staining of Factor VIII R:Ag. Endothelial cells were labeled with /sup 51/Cr in order to establish a cell injury assay in which the release of pelletable (cell associated) or non-pelletable activity was measured in the media. PMN activation was verified by chemiluminescence activity. Following phorbol myristate acetate (PMA) the leukocytes aggregated, chemiluminesced, and caused detachment of /sup 51/Cr endothelial cells. Endothelial detachment increased as a function of time with a plateau by 3 hrs. Immunofluorescent analysis of extracellular fibronectin in endothelial cell cultures revealed disruption of the fibrillar matrix fibronectin in association with endothelial cell disadhesion. Matrix fibronectin disruption was not seen with PMNs or PMA alone. Thus, disruption of the fibronectin matrix by released proteases may contribute to endothelial cell detachment.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. Platelet-rich plasma induces annulus fibrosus cell proliferation and matrix production.

    PubMed

    Pirvu, T N; Schroeder, J E; Peroglio, M; Verrier, S; Kaplan, L; Richards, R G; Alini, M; Grad, S

    2014-04-01

    Platelet-rich plasma (PRP) contains growth factors and creates a 3D structure upon clotting; PRP or platelet lysate (PL) might be considered for annulus fibrosus (AF) repair. Bovine AF cells were cultured with 25% PRP, 50% PRP, 25% PL, 50% PL, or 10% FBS. After 2 and 4 days, DNA, glycosaminoglycan (GAG), and mRNA levels were analyzed. Histology was performed after injection of PRP into an AF defect in a whole disc ex vivo. By day 4, significant increases in DNA content were observed in all treatment groups. All groups also showed elevated GAG synthesis, with highest amounts at 50% PL. Collagen I and II expression was similar between groups; aggrecan, decorin, and versican expression was highest at 25% PL. Injection of PRP into the AF defect resulted in an increased matrix synthesis. Platelet-rich preparations increased the matrix production and cell number and may therefore be considered to promote AF repair.

  13. 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.

  14. 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.

  15. 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

  16. Matrix metalloproteinase 12 modulates high-fat-diet induced glomerular fibrogenesis and inflammation in a mouse model of obesity.

    PubMed

    Niu, Honglin; Li, Ying; Li, Haibin; Chi, Yanqing; Zhuang, Minghui; Zhang, Tao; Liu, Maodong; Nie, Lei

    2016-01-29

    Obesity-induced kidney injury contributes to albuminuria, which is characterized by a progressive decline in renal function leading to glomerulosclerosis and renal fibrosis. Matrix metalloproteinases (MMPs) modulate inflammation and fibrosis by degrading a variety of extracellular matrix and regulating the activities of effector proteins. Abnormal regulation of MMP-12 expression has been implicated in abdominal aortic aneurysm, atherosclerosis, and emphysema, but the underlying mechanisms remain unclear. The present study examined the function of MMP-12 in glomerular fibrogenesis and inflammation using apo E(-/-) or apo E(-/-)MMP-12(-/-) mice and maintained on a high-fat-diet (HFD) for 3, 6, or 9 months. MMP-12 deletion reduced glomerular matrix accumulation, and downregulated the expression of NADPH oxidase 4 and the subunit-p67(phox), indicating the inhibition of renal oxidative stress. In addition, the expression of the inflammation-associated molecule MCP-1 and macrophage marker-CD11b was decreased in glomeruli of apo E(-/-)MMP-12(-/-) mice fed HFD. MMP-12 produced by macrophages infiltrating into glomeruli contributed to the degradation of collagen type IV and fibronectin. Crescent formation due to renal oxidative stress in Bowman's space was a major factor in the development of fibrogenesis and inflammation. These results suggest that regulating MMP-12 activity could be a therapeutic strategy for the treatment of crescentic glomerulonephritis and fibrogenesis.

  17. Release of Tensile Strain on Engineered Human Tendon Tissue Disturbs Cell Adhesions, Changes Matrix Architecture, and Induces an Inflammatory Phenotype

    PubMed Central

    Bayer, Monika L.; Schjerling, Peter; Herchenhan, Andreas; Zeltz, Cedric; Heinemeier, Katja M.; Christensen, Lise; Krogsgaard, Michael; Gullberg, Donald; Kjaer, Michael

    2014-01-01

    Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro-inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors showed a contrasting response with a clear drop in integrin subunit α11 mRNA and protein expression, and an increase in α2 integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules were upregulated. Stimulation with the cytokine TGF-β1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads to a decrease in phenotypical markers for tendon, while expression of pro-inflammatory mediators is induced. PMID:24465881

  18. 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. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  19. 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

  20. The Arabidopsis 14-3-3 Protein RARE COLD INDUCIBLE 1A Links Low-Temperature Response and Ethylene Biosynthesis to Regulate Freezing Tolerance and Cold Acclimation[C][W

    PubMed Central

    Catalá, Rafael; López-Cobollo, Rosa; Mar Castellano, M.; Angosto, Trinidad; Alonso, José M.; Ecker, Joseph R.; Salinas, Julio

    2014-01-01

    In plants, the expression of 14-3-3 genes reacts to various adverse environmental conditions, including cold, high salt, and drought. Although these results suggest that 14-3-3 proteins have the potential to regulate plant responses to abiotic stresses, their role in such responses remains poorly understood. Previously, we showed that the RARE COLD INDUCIBLE 1A (RCI1A) gene encodes the 14-3-3 psi isoform. Here, we present genetic and molecular evidence implicating RCI1A in the response to low temperature. Our results demonstrate that RCI1A functions as a negative regulator of constitutive freezing tolerance and cold acclimation in Arabidopsis thaliana by controlling cold-induced gene expression. Interestingly, this control is partially performed through an ethylene (ET)-dependent pathway involving physical interaction with different ACC SYNTHASE (ACS) isoforms and a decreased ACS stability. We show that, consequently, RCI1A restrains ET biosynthesis, contributing to establish adequate levels of this hormone in Arabidopsis under both standard and low-temperature conditions. We further show that these levels are required to promote proper cold-induced gene expression and freezing tolerance before and after cold acclimation. All these data indicate that RCI1A connects the low-temperature response with ET biosynthesis to modulate constitutive freezing tolerance and cold acclimation in Arabidopsis. PMID:25122152

  1. Lon protease: a novel mitochondrial matrix protein in the interconnection between drug-induced mitochondrial dysfunction and ER stress.

    PubMed

    Polo, Miriam; Alegre, Fernando; Moragrega, Angela B; Gibellini, Lara; Marti-Rodrigo, Alberto; Blas-Garcia, Ana; Esplugues, Juan V; Apostolova, Nadezda

    2017-09-23

    Mitochondria-associated membranes (MAMs) are specific ER-domains that enable direct interaction with mitochondria and mediate metabolic flow and Ca(2+) transfer. A growing list of proteins have been identified as MAMs components, but how they are recruited and function during complex cell stress situations is still not understood while the participation of mitochondrial matrix proteins is largely undervalued. This work compares mitochondrial/ER contact during combined ER stress/mitochondrial dysfunction using a model of human hepatoma cells (Hep3B cell line) treated for 24h with classic pharmacological inducers of ER stress (thapsigargin), mitochondrial dysfunction (CCCP or rotenone) or both (the antiretroviral drug Efavirenz used at clinically relevant concentrations). Markers of mitochondrial dynamics (Drp1, OPA1, Mfn2) were expressed differentially with these stimuli, pointing to a specificity of combined ER/mitochondrial stress. Lon, a matrix protease involved in protein and mtDNA quality control, was upregulated at mRNA and protein levels under all conditions. However, only with efavirenz, the mitochondrial content of Lon was diminished while its extramitochondrial presence was increased, as did its localization to MAMs, where it enhanced mitochondria/ER interaction as shown by co-immunoprecipitation experiments of MAM protein partners and confocal microscopy imaging. A specific dual drug-induced mitochondria-ER effect enhances MAMs content and extramitochondrial Lon expression. This is the first report of this phenomenon and suggests a novel MAM-linked function of Lon protease. This article is protected by copyright. All rights reserved.

  2. 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.

  3. 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.

  4. 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